r/EPA
United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park
NC 27711
EPA-600/9-78-026
September 1978
Research and Development
Viral Pesticides:
Present Knowledge and
Potential Effects on Public
and Environmental Health
s
^tP
'*T i
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Cover: Polyhedra of the Heliothis zea nuclear polyhedrosis virus.
This virus is the active ingredient of the first commercial viral
pesticide registered by the U.S. Environmental Protection Agency.
Original electron micrograph by Susan W. Bell.
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EPA-600/9-78-026
September 1978
Viral Pesticides:
Present Knowledge and
Potential Effects on Public
and Environmental Health
Symposium Proceedings
Edited by
Max D. Summers, Ph.D.
Department of Entomology
Texas A&M University
College Station, Texas 77843
and
Clinton Y. Kawanishi, Ph.D.
Health Effects Research Laboratory
Toxic Effects Branch
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
" "~ion Agency
l.-J:j; 1670
Health Effects Research Laboratory
Office of Health and Ecological Effects
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
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Foreword
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance
the quality of our environment in order to promote the public health and
welfare and the productive capacity of our nation's population.
The Health Effects Research Laboratory, Research Triangle Park, conducts
a coordinated environmental health research program in toxicology, epide-
miology, and clinical studies using human volunteer subjects. These studies
address problems in air pollution, non-ionizing radiation, environmental
carcinogenesis, and the toxicology of pesticides as well as other chemical
pollutants. The Laboratory develops and revises air quality criteria
documents on pollutants for which national ambient air quality standards
exist or are proposed, provides the data for registration of new pesticides
or proposed suspension of those already in use, conducts research on hazar-
dous and toxic materials, and is preparing the health basis for non-ionizing
radiation standards. Direct support to the regulatory function of the Agency
is provided in the form of expert testimony and preparation of affidavits
as well as expert advice to the Administrator to assure the adequacy of
health care and surveillance of persons having suffered imminent and sub-
stantial endangerment of their health.
in
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The majority of the registered pesticides are chemical agents. A few,
however, are biological in nature because the active ingredients are micro-
bial. Of these microorganisms, viruses are perhaps the most unique in
structure, biology, and the intimacy of their parasitic relationship with
their hosts. These proceedings consider whether potential biohazards to
human health and other biological components of the environment exist when
insect viruses are used as pesticides and whether such potentials have been
adequately assessed in view of our current knowledge of these agents.
Gordon Hueter, Ph.D.
Director
Health Effects Research Laboratory
IV
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Preface
The time is appropriate for the development and implementation of alterna-
tive and/or supplemental methods to chemical pest control, and a variety of
strategies are being considered in integrated pest management programs. A
series of conferences since 1970 have documented that insect pathogenic viruses
such as baculoviruses represent one of the most promising approaches in pest
control strategies. A joint FAO/WHO meeting in 1972 on "The Use of Viruses for
the Control of Insect Pests and Disease Vectors" evaluated those insect viruses
with the best potential for safe use. From that conference a test protocol was
prepared pertinent for safety testing of promising viral pesticide agents.
Some of the general conclusions of the FAO/WHO conference were: 1) Adequate
safety tests and precautions were essential not only to safeguard vertebrates
and other nontarget animals, but to minimize the risks of unforeseen accidents
that might prejudice future development and use. 2) Identification of insect
viruses was required to determine the pattern of specificity. 3) There was
need for routine and sensitive methods for assaying all stages of virus infec-
tion and for monitoring and precisely identifying the virus or viral agents
responsible. 4) Research was needed on the specificity of infection and the
defense mechanisms of invertebrates. 5) Safety testing should detect and
determine whether or not a baculovirus could enter vertebrate cells. 6) Since
genetic changes could occur not only in susceptible hosts but the virus, a
means of evaluating mutability was needed. 7) Consideration was given to the
potential risks for nontarget invertebrates and whether such risks may increase
depending upon the host range of the viral agent and the extent to which it
was used outside of its natural geographical distribution.
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The major conclusions and recommendations went essentially unnoticed
until 1974 when a joint EPA-USDA Working Symposium was convened on "Baculo-
viruses for Insect Pest Control, Safety Considerations." Selected scien-
tists, including a few vertebrate virologists, were convened with insect
virologists and scientists involved in applied studies to critically evaluate
the problems related to the development and safe use of baculoviruses. The
discussions and major recommendations of that conference echoed those pub-
lished in the WHO/FAO publication of 1973. However, at that time the mole-
cular biology of baculovirus structure, genetics, and infection processes had
not advanced much relative to the state of the art in 1972. The reports and
recommendations of that conference are particularly pertinent within the
context of the discussions and recommendations of this symposium (M.D.
Summers, R. Engler, L.A. Falcon, P. Vail, Baculoviruses for Insect Pest
Control: Safety Considerations. American Society for Microbiology, 1975).
As a result of the joint EPA-USDA conference, a Baculovirus Research, Regis-
tration and Development Program was organized, formally recognized, and
accepted by the Environmental Protection Agency in 1975; because of a variety
of problems it did not realize functional organization. The program was
designed to identify and clarify research priorities and set up a mechanism
for the development and safe application of baculoviruses as pesticides.
We convened again at Myrtle Beach, South Carolina, in 1977, under the
auspices of the US EPA, Health Effects Research Laboratory at Research Trian-
gle Park, to continue our dialogue of many of the same questions and problems
related to this unique approach to pest control. However, this symposium dif-
fers from previous efforts in the focus and nature of the background and ex-
pertise of the invited participants. The primary intent of this symposium was
the evaluation of insect viruses by plant, insect, and vertebrate virologists
with expertise in epidemiology and molecular biology from basic and applied
perspectives to ascertain whether, based on this knowledge, a pragmatic assess-
ment can be made of the safety of these agents for use as pesticides.
We attempted to juxtapose the objective opinions of the plant and verte-
brate virologists with those of insect virologists to scientifically and
comprehensively evaluate the benefits, potential risks, and problems of such
an approach. It is hoped that these proceedings will be a source of infor-
mation and sound base to facilitate productive, organized activity on basic
and applied research for the development and safe use of viral pesticides.
Max D. Summers, Ph.D.
Clinton Y. Kawanishi, Ph.D.
VI
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Abstract
A select panel of invertebrate, vertebrate, and plant virologists, each
a recognized authority and representing specialized areas in the field of
virology, was convened to review the progress and status of the development
and use of insect viruses, in particular baculoviruses, as biological pest-
icides. In integrated pest management practices the concept and approach
of using certain insect viruses as specific biological pest control agents
have been shown to be effective and provide a promising alternative and/or
supplemental approach for pest control practices. In fact, two such viruses
have been registered by the Environmental Protection Agency for control of
important insect pests of forest and cotton ecosystems.
Available data have not revealed any deleterious effects of virological
pesticides on other invertebrates, plants, and vertebrates, including man,
in the ecosystem. The Environmental Protection Agency has registered viral
pesticides based on protocols for chemical pesticidal agents and on the as-
sumption that by using viruses which occur naturally biohazardous situations
will not arise. However, it was also recognized that with commercial develop-
ment and/or the selective development of more effective and virulent viruses
and some of the problems associated with patenting natural agents, such
approaches will likely bring this group of viruses into the realm of genetic
manipulation.
Also, the use of in vitro production systems to mass produce viral
pesticides without appropriate quality control could result in undesirable
VII
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consequences. It is recognized that there is worldwide development of
microbial agents for use as pesticides and consequently it may be necessary
to implement more stringent supervision and regulatory practices in order
to monitor for potentially hazardous combinations, e.g., the contamination
of a viral pesticide by a vertebrate virus that may arise as a result of
in vitro and in vivo production.
Safety testing and safety testing criteria were evaluated relative to
the state of the art and knowledge of factors responsible for baculovirus
specificity and infection of susceptible and nonsusceptible hosts. It was
emphasized that knowledge of the molecular biology, pathogenesis, and
genetics of insect viruses is not presently comparable to that in other
areas of vertebrate virology. Therefore, the technology to use or assess
invasion and infection of susceptible and nonsusceptible hosts needs to be
improved. It was the general opinion that some of the most sensitive
methods for detecting virus entry and replication in exposed cells or hosts
should be incorporated into Environmental Protection Agency safety testing
guidelines. Of major importance was the development and application of
more sensitive and refined technology for identification and detection of
virus or viral genomes. Consideration of the question of persistence or
expression of viral genomes or part of genomes in permissive and non-permis-
sive systems indicated that DNA probe technology and sensitive serological
assays should be applied to a spectrum of baculovirus exposed cell types
that is representative of organisms in the treated environment.
Because many specific and sensitive detection techniques to monitor
insect viral activities in the physical or biological environment are
presently available, it was recommended that the Environmental Protection
Agency hasten implementation of specific recommendations for evaluating
safety with improved virus identification procedures and sensitive methods
for detecting virus invasion and/or infection of nontarget systems. The
majority agreed that data most pertinent or relevant to evaluating the
safe use of viral pesticides be reconfirmed by the Environmental Protection
Agency. To provide a more effective and comprehensive mechanism for the
continued evaluation of the progress and development of viral pesticides,
it was emphasized that more basic research in the area of understanding
the factors responsible for replication and pathogenesis of insect viruses
in their natural host systems be studied. Basic to this are the study of
the genetics of these viruses and the identification of specific genetic
and phenotypic markers which can be used for more precise studies of the
molecular biology and ecology. It was emphasized that the use of specific
DNA probes for genetic markers and the identification of specific phenotypic
viii
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markers which can be detected by such techniques as radioimmunoassay be
given high priority.
It was specifically emphasized that procedures for safety evaluation
of chemical pesticides are not adequate for biological agents such as
viruses. The lack of a clear procedure and policy for the development and
registration of viral agents relative to safety and regulatory acceptability
causes considerable uncertainty. It was also noted, however, that flexible
guidelines are needed because the area and its application could possibly
develop so rapidly that it would be inappropriate to require specific safety
testing procedures. Consequently, the use of viruses as alternative control
agents is wrought with misunderstanding, misinterpretation, and so forth.
Such an atmosphere discourages long-term planning and development. Further-
more, viruses are only one class of a large variety of potential biological
pesticide agents and compounds which may be rapidly forthcoming for use in
integrated pest management. The Environmental Protection Agency's scope
and vision must encompass these as well.
It was recommended that an advisory committee of expert vertebrate and
invertebrate virologists be immediately appointed, initially as an ad hoc
committee and later as a permanent panel to provide a creditable base of
opinion and information to evaluate present needs and provide an objective
forum for continued considerations of potential biohazard relative to other
living systems in the environment. The value of such a committee would be
in situations where during the course of research virus-host interactions
are discovered that may appear alarming but perhaps are placed out of con-
text relative to risk and public safety. Such reports could have profound
influence on public opinion. Much of this could be moderated or more care-
fully evaluated by having the appropriate advisory group to deal with such
situations.
The development and use of biological agents for regulation and pest
control may presently not be of such magnitude as to appear to be of major
immediate importance. However, because of the problems associated with
environmental pollution, persistence, toxicity, and the general welfare of
public health, large-scale use of many major chemical pesticides may be
curbed one or two decades hence. The use of biological agents or products
within the framework of integrated pest management strategies is being
considered in agriculture in the United States and in underdeveloped coun-
tries. Information presented at this conference indicates a significant
increase of this kind of effort on a worldwide basis.
IX
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Program Committee
AUGUST CURLEY CLINTON Y. KAWANISHI, PH.D.
Chief, Toxic Effects Branch U.S. Environmental Protection Agency
U.S. Environmental Protection Agency Toxic Effects Branch
Health Effects Research Laboratory Health Effects Research Laboratory
Environmental Toxicology Division Environmental Toxicology Division
(MD 66) (MD 67)
Research Triangle Park, North Research Triangle Park, North
Carolina 27711 Carolina 27711
ROBERT R. GRANADOS, PH.D. MAX D. SUMMERS, PH.D.
Virologist Department of Entomology
Boyce Thompson Institute Texas A&M University
Cornell University College Station, Texas 77843
Ithaca, New York 14853
VICTOR STOLLAR, M.D.
W.K. JOKLIK, PH.D. Professor of Microbiology
Department of Microbiology and College of Medicine and Dentistry
Immunology of New Jersey
Duke University Medical Center Rutgers Medical School
Durham, North Carolina 27710 Piscataway, New Jersey 08854
xi
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Virology Panel
JEFFREY J. COLLINS, PH.D.
Assistant Professor of Surgery and
of Microbiology and Immunology
Duke University Medical Center
Post Office Box 2926
Durham, North Carolina 27710
RICHARD A. DICAPUA, PH.D.
Associate Professor of Immunology
School of Pharmacy
University of Connecticut
Storrs, Connecticut 06268
RETO ENGLER, PH.D.
U.S. Environmental Protection Agency
Toxicology Branch
Registration Division (WH-567)
401 M Street, S.W.
Washington, D.C. 20460
LOUIS A. FALCON, PH.D.
Associate Insect Pathologist
Department of Entomological Sciences
333 Hilgard Hall
University of California
Berkeley, California 94530
BERNARD N. FIELDS, M.D.
Professor of Microbiology and
Molecular Genetics
Chief of Infectious Diseases
Harvard University Medical School
Peter Bent Brigham Hospital
25 Shattuck Street
Boston, Massachusetts 02115
ROBERT R. GRANADOS, PH.D.
Virologist
Boyce Thompson Institute
Cornell University
Ithaca, New York 14853
KEITH A. HARRAP, PH.D.
NERC, Unit of Invertebrate Virology
5 South Parks Road
Oxford, 0X1 3VB
UNITED KINGDOM
JOHN A. HOLOWCZAK, PH.D.
Associate Professor of Microbiology
Department of Microbiology
College of Medicine and Dentistry
of New Jersey
Rutgers Medical School
Piscataway, New Jersey 08854
XIII
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PEPPER HOOPS
Department of Entomology
Texas A&M University
College Station, Texas 77843
CARLO M. IGNOFFO, PH.D.
Laboratory Director
U.S. Department of Agriculture, ARS
Biological Control of Insects
Research Laboratory
Post Office Box A
Columbia, Missouri 65201
W.K. JOKLIK, PH.D.
Chairman
Department of Microbiology and
Immunology
Duke University Medical Center
Durham, North Carolina 27710
CLINTON Y. KAWANISHI, PH.D.
U.S. Environmental Protection Agency
Toxic Effects Branch
Health Effects Research Laboratory
Environmental Toxicology Division
(MD 67)
Research Triangle Park, North
Carolina 27711
DENNIS L. KNUDSON, PH.D.
Yale Arbovirus Research Unit
Yale University School of Medicine
60 College Street
New Haven, Connecticut 06510
JOHN F. LONGWORTH , PH.D.
Insect Virology Section
DSIR Entomology Division
Private Bag
Auckland, NEW ZEALAND
WILLIAM J. MCCARTHY, PH.D.
Associate Research Virologist
Pesticide Research Laboratory and
Graduate Center
Pennsylvania State University
University Park, Pennsylvania 16802
WILLIAM MEINKE, PH.D.
Virologist
Department of Microbiology
College of Medicine
University of Arizona
Tucson, Arizona 85724
JOSEPH S. PAGANO, M.D.
Director, Cancer Research Center
University of North Carolina
Box 3, Swing Building 217H
Chapel Hill, North Carolina 27514
FRED RAPP, PH.D.
Professor and Chairman
Department of Microbiology
Pennsylvania State University
College of Medicine
500 University Drive
Hershey, Pennsylvania 17033
ROBERT E. SHOPE, M.D.
Professor of Epidemiology
Yale Arbovirus Research Unit
Yale University School of Medicine
60 College Street
New Haven, Connecticut 06510
RALPH SMITH, PH.D.
Department of Microbiology and
Immunology
Post Office Box 3020
Duke University Medical Center
Durham, North Carolina 27710
VICTOR STOLLAR, M.D.
Professor of Microbiology
College of Medicine and Dentistry
of New Jersey
Rutgers Medical School
Piscataway, New Jersey 08854
MAX D. SUMMERS, PH.D.
Department of Entomology
Texas A&M University
College Station, Texas 77843
XIV
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LOY E. VOLKMAN, PH.D. MILTON ZAITLIN, PH.D.
Microbiologist Professor of Plant Pathology
Department of Microbiology Department of Plant Pathology
Lovelace-Bataan Medical Center Cornell University
5200 Gibson Boulevard, Southeast Plant Science Building
Albuquerque, New Mexico 87108 Ithaca, New York 14853
xv
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Contents
Foreword
Preface v
Abstract vii
Program Committee xi
Virology Panel xiii
Recommendations 1
Introduction
Max D. Summers, Ph.D 3
Frode Ulvedal, Ph.D 6
Victor Stollar, M.D 7
PART I. CURRENT USE OF VIRUSES
AS ALTERNATIVES TO CHEMICAL PESTICIDES
Viruses as Alternatives to Chemical Pesticides
in the Western Hemisphere
Louis A. Falcon, Ph.D 11
Discussion 24
The International Scope of Invertebrate
Virus Research in Controlling Pests
Keith A. Harrap, Ph.D., and T.W. Tinsley, Ph.D 27
Discussion 41
xvii
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PART II. VIRUSES: BIOLOGY AND IDENTIFICATION
Baculoviruses
Max D. Summers, Ph.D 45
Discussion 71
Identification of Nonoccluded Viruses
of Invertebrates
John F. Longworth, Ph.D., and Paul D. Scotti, Ph.D 75
Discussion 86
Biology of Cytoplasmic Polyhedrosis
Viruses and Entomopoxviruses
Robert R. Granados, Ph.D 89
Discussion 102
PART III. VIRUSES: RECENT ADVANCES
Recent Advances in the Antigenic Characterization
of Nuclear Polyhedrosis Viruses
Richard A. DiCapua, Ph.D., James E. Peters,
and Philip W. Norton 105
Discussion 113
Recent Advances in Baculovirus Serology:
Radioimmunoassay and Immunoperoxidase Assay
Pepper Hoops and Max D. Summers, Ph.D 115
Discussion 133
Cell Culture Studies; Standardization
of Biological Activity
Loy E. Volkman, Ph.D 135
Discussion 149
Pathogenic-Invertebrate Viruses:
In Vitro Specificity
Dennis L. Knudson, Ph.D 151
Discussion 164
XVIII
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PART IV. PANEL DISCUSSION
Viral Pesticides: Implementation and Safety
Victor Stollar, M.D., moderator 169
PART V. SAFETY: A CRITIQUE
Review of Safety Tests and Methods
of Evaluating Infectivity
Clinton Y. Kawanishi, Ph.D 199
Discussion 211
Methods of Evaluating the Presence
of Viruses and Virus Components
William Meinke, Ph.D 215
Discussion 223
Hazard Evaluation for Viral Pesticides:
Test Data Requirements
Reto Engler, Ph.D., and Martin H. Rogoff, Ph.D 225
Discussion 232
PART VI. PANEL DISCUSSION
Safety Procedures and Future Recommendations
W.K. Joklik, Ph.D., moderator 241
PART VII. PANEL DISCUSSION
Discussion of Preliminary Draft of
Panel Recommendations
Robert E. Shope, M.D., presenter 283
Conference Attendees 309
XIX
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Recommendations
EPA Symposium: Viral Pesticides: Present Knowledge and Potential Effects
on Public and Environmental Health. Myrtle Beach, South
Carolina, March 21-23, 1977.
Baculoviruses provide a promising alternative approach to pest control.
Current evaluation methods suggest that the viruses are effective. Further-
more, available data have not revealed any deleterious effects for other
components of the ecosystem, i.e., other invertebrates, plants, and verte-
brates including man.
Safety testing criteria should continually respond to improved technology.
We draw attention to the "Guidance for Safety Testing of Baculoviruses"
section B-l, b-2.* It is important that the most sensitive methods for detec-
ting virus replication are incorporated into the EPA guidelines for safety
testing of baculoviruses. Recent developments in molecular biology have
provided more sensitive and refined tools and offer the potential for testing
at improved levels of specificity. By their implementation, the opportunity
can be taken to improve further the safety tests for baculovirus pesticides.
We draw attention again to the identification criteria listed on page 179 in
"Guidance for Safety Testing of Baculoviruses."* The recent development of
restriction fragment analysis of a variety of DNA-containing viruses has
provided a powerful new tool which should be included among the identification
techniques.
*Baculoviruses for Insect Pest Control: Safety Considerations. M.D. Summers,
R. Engler, L.A. Falcon and P.V. Vail, eds. American Society for Microbiology,
Washington, B.C., 1975. 186 pp.
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A fundamental concern for safety requires investigation of the question
of persistence or expression of the viral genome or parts of the viral genome
in non-target systems. Such tests should include at least two human cell types,
rodent, avian, and fish cells. The development of such tests should embrace
a variety of techniques such as molecular hybridization and radioimmune assay.
Before a biologic agent is registered as a viral pesticide, the data most
relevant to the safety tests should be reconfirmed by EPA.
We strongly advise that EPA move with all speed to implement the above
recommendations for safety involving identification, baculovirus genome tests,
and sensitive methods for detecting virus replication, at least by January
1979.
Research Program
To provide a continuing basis for improvement and an evaluation of new
products, it is imperative that certain areas bearing on the safety of insect
viruses, particularly baculoviruses, should be fully understood.
1. A better understanding is needed of the replication and
pathogenesis of the viruses in their natural hosts and cell
culture systems.
2. The genetics of the viruses and the development of specific
genetic markers are vital for precise ecological study.
For example, the possible range of interaction between the
viral pesticide and other viruses in the biologic environment
needs to be explored.
These general recommendations of the invited panel are intended to focus
attention on areas urgently in need of research. They represent only certain
elements of major issues raised in the panel discussion, and they are put
forward as the basis for the formulation of a specific program at an early
date. We feel that this could best be achieved by a permanent and independent
advisory panel.
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Introduction
Max D. Summers, Ph.D.
Texas A&M University
College Station, Texas
Historically, the major effort in the field of viral pesticides has
been in agriculture. However, the application of viral pesticides has a
potential effect on human welfare as well. Pest control in this context of
using insect pathogens is not new. Viral pesticides have been used in both
artificial and natural environments since the evolution of insect viruses.
Various components of agriculture have merely attempted to exploit viruses
in a pragmatic way for economic benefit. Biological agents and, in parti-
cular, viruses and fungi have been and currently are considered as alter-
natives or aids in pest management strategies for several reasons:
The development of resistance and cross resistance to chemical
pesticides
The rising cost of chemicals and application technology
Environmental pollution, which on a long- and short-term basis,
has undesirable side effects related to public health.
Because of this, there is a justified interest in developing more natural or
biodegradable compounds such as insect viruses.
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Those of us working with viral pesticides have been through this subject
many times before. I would like to cite a few conferences that have taken
place over the past few years dealing with this area of interest. In 1973,
there was a joint publication as a result of a 1972 WHO-FAO (Food and Agri-
culture Organization) conference on insect viruses and their use in biologi-
cal control. In 1974, a joint EPA-USDA (U.S. Department of Agriculture)
conference on baculovirus safety was convened in Washington. Also in 1974,
there was a conference sponsored by the EPA on the impact of the use of
microorganisms on the aquatic environment (Pensacola, Florida). In 1975, the
United States. National Academy of Sciences published a five-volume compre-
hensive evaluation entitled Pest Control; An Assessment of Present and
Alternative Technologies. In addition, there have been numerous meetings
with and within the Society for Invertebrate Pathology. In 1976, the USDA
and the Agricultural Research Service (ARS) met as a working group to prepare
a working document on biological control agents, and organized activity will
develop from that. The result is that the problems and benefits of using
viral pesticides have already been identified, and recommendations have been
made by capable scientists.
Yet, as is always the case in a developing science, there is concern
about whether adequate precautions have been made to predict or assure the
safe use of viral pesticides. There are those who are confident that safety
is not a problem, those who advise caution, and those who demand absolute
criteria and procedures to measure and/or predict safe use of viral agents.
We have learned from experience that despite all available knowledge of safe
application for chemical pesticides, we can make serious, irreversible mis-
takes. These mistakes are usually unintentional, but they can endanger
public health for generations to come. Therefore, it is not so unrealistic
to give some organized thought to the safe use of viral agents, placing in
perspective our knowledge of mechanisms of infection relative to available
technology.
We approach our task at this conference with an awareness of the problems
and potential problems that may or may not occur to endanger public health.
Present estimates from all previous conferences and published information
suggest that viral pesticides are safe to use, and that the potential appli-
cation of viral agents as insect control agents is quite good. Historically,
there has been more effort in this area than one might think. Presently,
an intensified effort is directed toward the increased use of viral pesti-
cides, and it is likely that much more effort will be expended in future
programs.
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I feel that this meeting is unique as compared to previous meetings.
Here, the focus is on the virologist's perspective, relative to the applica-
tion of viruses in their proposed role. We have been fortunate to assemble
a few select individuals working in insect virology. But that group has met
many times before and because of that, some may feel that this conference
is a waste of time. Previously, however, the virological content of our
conference has been included with discussions of applied and field studies.
Although that area is represented at this conference, that emphasis is not
the primary focus of this meeting. Our attention will be directed toward
the basic science of invertebrate virology and whether we are adequately
prepared with the proper tools and knowledge to assure the safe use of viral
pesticides, or to predict possible situations that could cause undesirable
consequences. This is our most important obligation because the practical
use of viruses in pest management schemes is really going to evolve during
the next two decades.
One of the major factors that makes this conference unique is that we
have been able to include as participants several prominent vertebrate and
plant virologists. I would like to thank those participants and also add
that of the original 12 virologists Drs. Joklik and Schlessinger recommended,
only two declined.
I would like everyone to be frank and straightforward in identifying
any problems or benefits of viral pesticides research, taking a scientific
attitude, and keeping politics at a minimum. By the end of this conference,
we would like to identify some major points that will aid in the development
of the basic science and application of viral pesticides and provide a list
of recommendations and priorities. This should be accented from the viro-
logist's perspective. With that as an introduction, I would like to turn
the program over to the Environmental Protection Agency and Dr. Frode Ulvedal,
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Frode Ulvedal, Ph.D.
Environmental Protection Agency
Washington, D.C.
As many of you are aware, viruses are just one taxonomic group of bio-
logical agents that is being developed, registered, and used as a pesticide.
According to the philosophy propounded in the 1972 amendment of the Federal
Pesticide, Fungicide, and Rodenticide Act, which states in Section 20 that
the administrator of EPA will undertake research to grant or contract with
other Federal agencies, universities, and others to carry out the purposes
of the Act, and in the context of this symposium, to give priority to research
to develop biologically integrated alternatives for pest controls our
agency is encouraging the development of effective pest control strategies
while charged with the responsibility to ascertain that the remedy is not
worse than the original problem.
Although the biological agents we will discuss in this symposium are
present as normal components of the environment, when they are used as pesti-
cides they are comparatively new to the agency, and our knowledge in this
area is still developing. Currently, according to my knowledge, only two
baculoviruses are registered for use as pesticides, but many others are being
considered. Consequently, we must continually update our knowledge of bio-
logical agents as information about their physical properties becomes avail-
able through our efforts to protect humans against their adverse effects on
health. We hope this symposium will contribute to this objective.
Gathered here as speakers and panel members are scientists knowledgeable
about invertebrate viruses. This is, therefore, a unique opportunity for
the speakers to impart to their distinguished medical and plant virology
colleagues on the virology advisory panel the present knowledge of inverte-
brate viruses, and the status of accumulated knowledge concerning the pro-
perties of medically and agriculturally important viruses. We seek their
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advice on whether they see potential problems associated with the use of
viruses as pesticides, and their aid in determining the necessary research.
By providing information on the unique properties of viruses, the pro-
ceedings of this symposium should assist the Environmental Protection Agency
in its efforts to develop more rational and relevant approaches to safety
testing and registration. Additionally, the symposium should facilitate
development of the viral agents to their full potential as a unique class
of alternative pesticides that are effective, less disruptive to the environ-
ment, and hopefully, not detrimental to human health. I hope we shall all
gain knowledge and a perspective of viruses as potential pesticidal agents
from the ensuing presentations and discussions.
Victor Stollar, M.D.
Rutgers Medical School
Piscataway, New Jersey
I would like to make a few brief remarks before getting into the content
of the program. Some of us feel ill at ease dealing with a large group of
viruses that are largely unfamiliar to us. I hope this uneasiness will be
dispelled shortly. I realize also that we should keep politics out of our
discussions. Nevertheless, I was struck by a front page newspaper article
several months ago that quoted a Senate staff report describing the govern-
ment program for assuring the safety of pesticides as chaotic. This article
referred, fortunately, to chemical pesticides. I assume that part of our
task at this conference is to see that viral pesticides will be used in the
best possible way.
I think Dr. Summers has already formulated the basic problems we wish
to address. Just to run through them again, the questions we want to keep
-------
in our minds over the next few days are: "Is the use of viral pesticides
feasible? If so, which viruses can be used?" We want to keep in mind what
we know about these viruses and what we still have to learn about them. We
need solid, basic, and theoretical information, as well as which techniques
in the laboratory and in the field need further development. We want to come
out of this meeting with recommendations, both general and specific. I think
these recommendations will come out of the discussion panels. We also want
to keep in mind the potential hazards of using viral pesticides.
The presentations during the first part of the day are going to be for-
mal and informative; we would prefer minimal discussion or questions follow-
ing them. I think the most appropriate kinds of questions would be those
that are strictly informational or questions to clarify unclear material.
Let us leave the pertinent discussion for the latter part of the afternoon.
Not all of the participants are listed on the formal program, but cer-
tainly, we want and need the advice, suggestions, and criticisms of all the
participants. We are going to hear first about the present status of viral
pesticide programs, both in this hemisphere and in international programs.
These are large-scale programs.
I do not want to steal attention from the first two speakers, but I
thought it might be interesting to draw to your attention a magazine article.
I like to work in the garden on weekends, and for several years, I have had
a subscription to a journal called Organic Gardening. I was quite struck
last October to see an article called "An Insect Control Method Too Good
To Be True." The article describes, basically, a very simple method of
insect control for the do-it-yourself gardener, using insect viruses. The
article describes a process of finding dead and sick insects, homogenizing
them, diluting them, and spreading them in the garden. So, certain elements
in the field of viral pesticides may, in a sense, be already out of control.
-------
PARTI
CURRENT USE OF VIRUSES AS
ALTERNATIVES TO CHEMICAL PESTICIDES
-------
Viruses as Alternatives to Chemical
Pesticides in the Western Hemisphere
Louis A. Falcon, Ph.D.
Division of Entomology and Parasitology
University of California, Berkeley
Chemical pesticides are overpromoted and overused throughout most of
the Western World. From personal experiences, I would say that more than
half the pesticide used in agriculture is not necessary and is wasted. Recog-
nizing that indiscriminate use of pesticides is a contributing factor in
environmental pollution, more people are becoming interested in creating and
maintaining a healthy environment with little or no chemical pesticides. As
an agricultural entomologist by training and an environmentalist out of
necessity, I am working with others in an effort to find and develop satis-
factory alternatives to chemical pesticides. Several directions are being
followed, one of which is the development of microbial agents for pest con-
trol. In this conference we deal specifically with the development of one
group of microbial agents, the insect viruses.
Of the known insect viruses, the nuclear polyhedrosis (NPV) and granulo-
sis viruses (GV) of the genus Baculovirus continue to offer the greatest
potential for insect pest control in agriculture and forestry. They appear
particularly well suited for this purpose because candidate viruses tested
thus far have demonstrated:
1. Efficacy and usefulness in the control of target insect pests.
2. A high degree of specificity, which makes them especially
valuable for use in integrated pest management programs.
11
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3. Safety, as they have never been shown to be hazardous to
humans or other warm-blooded animals.
Although this conference will examine various types of insect viruses,
my comments apply specifically to the baculoviruses.
TO WHAT EXTENT ARE INSECT VIRUSES BEING USED AS ALTERNATIVES TO CHEMICAL
PESTICIDES IN THE WESTERN HEMISPHERE
There has been and continues to be little direct use of insect viruses
for pest control in the Western Hemisphere. In the United States only two
insect viruses have been registered with the EPA and are available for com-
mercial use. One is the product Elcarฎ, which contains the NPV of the boll-
worm, Heliothis zea. It was registered by Sandoz, Inc. The other insect
virus product was registered by the U.S. Forest Service, and contains the
NPV of the Douglas fir tussock moth (Orgyia pseudotsugata). A third bacu-
lovirus, the gypsy moth (Porthetria dispar) NPV, is close to registration.
Only one other baculovirus is receiving sufficient attention at this time
to be considered within the range of registration and that is an NPV origin-
ally isolated from the alfalfa looper, Autographa californica. Over the past
two years the Agricultural Research Service (ARS) of the U.S. Department of
Agriculture has made efforts to develop a petition for an experimental use
permit and an exemption from a tolerance for this NPV from the EPA (A.M.
Heimpel, personal communication). I am not aware of efforts to register
insect viruses in other countries of the Western Hemisphere with the exception
of Canada where scientists are working closely with their U.S. counterparts
on the same viruses.
In 1976, Sandoz, Inc., began marketing Elcar in several cotton growing
states of the United States. While only small quantities were reported sold
during the first year, the market is expected to increase as growers become
aware of the availability of this selective microbial control agent. Research
is continuing in an effort to improve the Elcar product with much attention
being given to formulation and methods of application. This particular pro-
duct has provided a model for the development and commercialization of other
baculovirus pest control products.
So far the Douglas fir tussock moth NPV product appears to be little
more than a curiosity on the shelf. There is a very real need for an educa-
tional program to train forest workers on the large-scale use of this baculo-
12
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virus (1). Ostensibly, potential users would rather spend their energies
trying to restore DDT instead of learning to use the Douglas fir tussock
moth NPV.
During the last three decades, several baculoviruses have been employed
for insect pest control, including the alfalfa caterpillar (Colias eurytheme)
NPV for control of caterpillars on alfalfa in California during the 1950's (2);
the NPV of the cabbage looper (Trichoplusia ni) which has been employed with
good success by vegetable, cotton, and soybean growers in the U.S., Canada,
Mexico, Nicaragua, and Colombia; the NPV of the beet armyworm (Spodoptera
exigua) used in California and Arizona on cotton, alfalfa, and vegetables;
and NPVs isolated from various species of sawflies (Neodlprion and Diprion
spp.) for the protection of Christmas tree plantations and forests in the U.S.
and Canada.
WHAT IS THE POTENTIAL FOR USING INSECT VIRUSES AS PEST CONTROL AGENTS
One way to examine the potential for using insect viruses as pest con-
trol agents is to look at agricultural crop loss figures for various insect
pests which can be controlled by these agents. Let us look at pest losses
in California (Annex A). This is the leading agricultural state in the U.S.,
growing nearly 50% of the fruits and vegetables consumed in the nation. In
addition, it is the top cotton producing state, and this industry is the major
consumer of chemical pesticides in the U.S. In 1974, agricultural returns
for California amounted to $8.5 billion, or about 11% of the total agriculture
q
product for the United States, which was $95 x 10 .
In Central America, Nicaragua imported over US$ 10 x 10 in chemical
pesticides to protect a cotton crop worth US$ 60 x 10 in 1973. The results
of a 3-year study showed that biological control agents including the use of
insect viruses could reduce expenditures for pesticides by 50%. That is a
large financial saving for a developing country with an annual total govern-
ment budget of about US$ 85 x 106 (3).
Taking the Western Hemisphere as a whole, it appears that about 30% of
the current insect pest problems in agricultural crop production are suscep-
tible to control by insect viruses, specifically the baculoviruses. The
potential economic benefits from their development and use are significant.
13
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WHAT IS THE CURRENT INPUT FOR DEVELOPING INSECT VIRUSES FOR MICROBIAL CONTROL
Overall, the current yearly expenditure devoted to the research and
development of insect viruses appears to be circa US$ 5 x 10 for the entire
Western Hemisphere. This is equivalent to about 2.5% of the estimated over-
all crop loss caused by virus susceptible pest species for California alone.
On a hemisphere basis it is probably less than .0025%. Most of the research
and development activity is in the U.S. and Canada with scattered activity
in a few South American countries, namely Colombia, Venezuela, and Nicaragua.
Less than 60 scientific man years are currently going into viral pesticide
research and development each year.
For the United States, the data I compiled indicated that the current
yearly investment is about $1.6 million at the state level, i.e., for agri-
cultural experimental stations and universities. Only eight states are
involved in insect virus pest control research, and California appears to
have the biggest commitment with about a $300,000 yearly investment (Annex B).
At the Federal level, the USDA is investing about $2.0 million yearly. Six
ARS and two FS stations are doing the bulk of the research and development
work. Recently it was reported that 75% of the USDA's pest control research
budget is for support of nonchemical pesticide research. If this is true,
apparently less than 1% of this money is used for insect virus pesticide
research and development. In the commercial arena, industry appears to be
investing less than $400,000 per annum. In recent years only Sandoz, Inc.,
and Nutrilite Products, Inc., have been actively involved in developing insect
viruses for pest control. The problems associated with the development and
industrialization of insect viruses were fully discussed by Falcon (4).
WHAT IS THE EFFORT NEEDED
In 1973, the Food and Agriculture Organization (FAO) of the United
Nations published a list of the NPVs and GVs (Annex C) that would be useful
for integrated pest control (5). While this list was developed on a global
basis, it applies equally well to the Western Hemisphere. Under the category
of most promising insect viruses needed for integrated pest control, a maxi-
mum of 12 NPVs and a minimum of two NPVs are indicated. The former figure
assumes one NPV per insect species listed in categories 1, 2, and 3 and one
for category 4, while the latter takes into account the broader host range
of some NPVs such as Autographa NPV, which can infect several species of
lepidoptera. In addition, four GVs were listed. The list encompasses the
major lepidopterous pest species of food, fiber, and oil crops in the world.
14
-------
Along with the above, another category "... viruses that may have good
potential for commercial production for integrated pest control" was also
included. Five NPVs and two GVs were listed here. In conclusion, according
to the FAO list, a maximum of 16 NPVs but possibly as few as seven NPVs plus
six GVs are all that are needed to achieve high utilization of insect viruses
for pest control in the Western Hemisphere and, for that matter, on a worldwide
scale.
As demonstrated with the Heliothis and Douglas fir tussock moth NPVs,
the technology is available to produce, formulate, and store sufficient quan-
tities of each insect virus for small- and large-scale field testing purposes.
A protocol for safety testing has been published (6). Also, an EPA publica-
tion providing guidelines to aid in determining the efficacy and usefulness
of candidate baculoviruses is being printed and should be available in 1978.
The case I have attempted to develop shows that the total effort needed
to develop insect viruses for pest control is not large, yet the rewards
can be plentiful. I calculated that an additional US$ 1.6 million per year
for five years is required to undertake the basic research and development
required to make the 16 insect viruses listed in category 1 (Annex C) avail-
able for pest control. Ideally the monies should be coordinated through a
central agency, such as the National Institutes of Health (NIH) or National
Science Foundation (NSF), and made available to qualified and interested
researchers through competitive grants subjected to peer review. The bulk
of the monies should be allocated to efficacy testing, as this activity will
select the useful insect viruses which can then be subjected to complete
safety testing for registration purposes.
A model to follow for developing the program described above is a forest
pest program involving the U.S. Forest Service and several universities (7).
It deals with three pests: the Douglas fir tussock moth, the southern pine
beetle, and the gypsy moth. The Federal funds that were made available
through this program have permitted the development and registration of the
Douglas fir tussock moth and the gypsy moth NPVs. There has never been a
comparable program for agriculture.
WHY HAS SUPPORT NOT DEVELOPED
Support for the research and development of insect viruses has not
developed mainly because naturally occurring arthropod viruses are not patent-
able (4). Thus there is little incentive for private industry to invest
15
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money for research and development as is done for chemical insecticides which
are patentable. Furthermore, insect viruses do not have the market potential
of chemical pesticides. Since they are selective and do not disrupt the
environment, the use of insect viruses does not create the "treadmill effect"
which often occurs with the use of chemical pesticides. The more chemical
pesticides are used, the more they are needed, whereas insect viruses are
needed less with increased use. Consequently, not only is there little
incentive for private industry to develop insect viruses, they will do every-
thing possible to prevent the development because they fear the competition.
Within USDA-ARS, the development of insect viruses has been hampered by
competition from research on other control methods. For the past decade,
the bulk of the pest control research dollar has been invested in the research
and development of the sterile-male technique, an approach which failed to
develop as anticipated.
Another reason why insect viruses have not developed rapidly is due to
the rather hard position taken first by the Food and Drug Administration (FDA)
and later by EPA. The policy is that insect viruses must be registered as
pesticides in the same way as chemicals. The primary impact of this ruling
was to stop the use of insect viruses for pest control in those areas where
they were being successfully utilized. In actuality, insect viruses are
naturally occurring components of the environment, the same as parasites and
predators, pest insects, nematodes, bacteria, etc. Recently the EPA Registra-
tion Division agreed to permit the use of a wood-rotting fungus that when used
as an inoculum on freshly cut tree stumps would not require registration
because it is a "naturally occurring fungus." This has occurred with other
agents, and demonstrates that the registration system is capable of some
flexibility. However, everything possible must be done to ensure that this
approach is applied correctly and fairly in all situations dealing with natur-
ally occurring agents.
Another problem interfering with the development of baculoviruses has
been and continues to be the "hazard monger." Hazard mongers are research
workers who are also opportunists and see this as an area which can be
developed to their advantage. They employ scare tactics about the hazards
associated with viruses. They lump the viruses associated with insects and
make broad generalizations, forgetting that the baculoviruses are really
quite different from other insect viruses. What is needed is an objective,
coordinated effort to demonstrate the safety of each virus to be used and to
show why the baculoviruses are not hazardous (6).
16
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A strong education effort is required to place the insect viruses in
their proper perspective at all levels (i.e., lawmakers, regulatory agencies,
researchers, users, and society in general). At the government regulation
level, protocols and policies for registration and use must be established on
a permanent basis, so these will remain, regardless of the "people-exchange"
that goes on in Washington. Some progress has been made in this area as
exemplified by the guidance for safety testing published by the EPA (6) and
the EPA guidelines for establishing the efficacy and usefulness of microbial
agents for pest control (in press).
Finally, the most difficult problem facing the development of insect
viruses for pest control continues to be insufficient funding. Although a
few research programs include insect viruses as part of their activities,
the total effort is very small. Microbial control has never had full-fledged
representation at decision-making levels of Federal programs. For example,
there is no one representing this area on the National Program Staff of USDA
on a full-time basis. Traditionally, it is an area assigned to others who
are experts on something else.
In summary, I have attempted to show that the insect viruses, particu-
larly baculoviruses, have potential as pest control agents and are sorely
needed for this purpose. Only a very few, 16 or less, are needed to help
combat about 30% of the insect pests in the field. The technology to develop
them is available, but current efforts are minimal and haphazard at best. A
strong national program with adequate funding is required to stimulate
research and development. The capacity, technology, ability, and justifica-
tion are there. At this point, it requires the right political moves to
bring it about.
If this area is allowed to develop to its effective maximum level, the
potential effects on public and environmental health will be beneficial. The
possible hazards that may exist will be identified, and we will begin to under-
stand why baculoviruses behave as they do. Most important of all could be
the development of effective, useful, environmentally safe products which
can replace the hazardous, environmentally unsuitable products that are in use
today.
17
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ANNEX A
ESTIMATED CROP LOSSES CAUSED BY INSECT SPECIES
SUSCEPTIBLE TO CONTROL WITH BACULOVIRUSES IN CALIFORNIA DURING 1974*
Insects Estimated overall loss
Alfalfa caterpillar - Colias eurytheme $ 4,423,807
Alfalfa semilooper - Autographa californica 1,985,700
Armyworms - Pseudaletia unipuncta
Prodenia "praefica 47,862,147
Spodoptera exigua 11,236,695
Cankerwonns - Alsophila pometaria
Palecrita vernata 400,900
Codling moth - Laspeyresia pomonella 2,788,490
Corn earworm - Heliothis zea 69,695,996
Cutworms complex - Agrotes ipsilon (plus 4 other spp.) 10,615,424
Fruit-tree leafroller - Archips argyrospilus 3,708,708
Imported cabbageworm - Pieris rapae 966,775
Loopers - Cabbage - Trichoplusia ni 12,198,875
Other 1,149,902
Diamond back moth - Plutella xylostella 533,399
Oriental fruit moth - Grapholitha molesta 4,255,290
Pink bollworm - Pectinophora gossypiella 19,016,839
Potato tuberworm - Phthorimaea operculella 1,359,360
Tent caterpillars - Malacosoma spp. 296,900
Total $192,495,207
*Source: Hawthorne, R.M. (8).
18
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ANNEX B
ESTIMATED EXPENDITURES FOR BACULOVIRUS PEST CONTROL
ORIENTED RESEARCH AND DEVELOPMENT AT PRINCIPAL LOCATIONS
IN THE WESTERN HEMISPHERE FOR 1975-76
Location
U.S.
States - Agricultural Experiment
Alabama
Arkansas
California
Florida
Mississippi
Ohio
Pennsylvania
Texas
Federal - USDA
ARS
Beltsville, MD
Brownsville, TX
Columbia, MO
Fresno , CA
Phoenix, AZ
Tifton, GA
FS
Corvallis, OR
Hamden, CT
Private Industry
Sandoz
Nutrilite
Other Groups
Boyce Thompson Institute
TOTALS - U.S.
Principal
scientific
man years*
Stations
1
2
3
2
1
2
2
2
2.5
1
2
1
1
1
6
6
3
.5
3
42
Approximate
expenditures!
$100,000
200,000
300,000
200,000
100,000
200,000
200,000
200,000
1,500,
250,000
100,000
200,000
100,000
100,000
100,000
600,000
600,000
2,050,
300,000
50,000
300,000
650,
4,200,
000
000
000
000
(Continued)
19
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ANNEX B (CONTINUED)
Location
Principal
scientific Approximate
man years* expenditurest
Canada
Sault Ste. Marie
Forestry Service
Dept . of Agriculture
3
3
1
300,000
300,000
100,000
Colombia, S.A.
Federacion Nacional de Algodoneros
Venezuela, S.A.
Venezuelan Institute of Scientific
Research
.5
700,000
50,000
100,000
150,000
TOTALS - Western Hemisphere
50.5
US$5,050,000
*Calculations based on percent time allocated by each individual and rounded
to nearest whole number in most cases.
TEstimated expenditures based on salaries, supplies and expense and equipment
and facilities with $100,000 per scientist used as a mean figure.
20
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ANNEX C
I. Most promising insect viruses needed for integrated pest control
A. Nuclear polyhedrosis viruses (NPV)
1. Viruses of the Spodoptera complex (includes most species for-
merly in Prodenia, e.g., littoralis, exigua, frugiperda,
litura, and exempta). At present includes a large complex of
viruses. Hopefully, one microbial product would be effective
against the entire complex.
2. Viruses of the Heliothis^ complex, e.g., armigera, zea, and
virescens. Hopefully, one product would be effective against
the entire complex.
3. Viruses of Plusiinae, e.g., Trichoplusia, Plusia, and
Pseudoplusia.
4. Autographa californica virus, which is cross-infective to
genera in several lepidopterous families, e.g., Pectinophora,
Bucculatrix, Heliothis, Trichoplusia, Spodoptera, Estigmene,
and Plutella.
B. Granulosis viruses (GV)
5. Laspeyresia pomonella (codling moth).
6. Phthorimaea operculella (potato tuberworm).
7. Pieris (Mamestra) spp. (crucifer caterpillars).
8. Argyrotaenia velutinana (redbanded leafroller).
II. Other insect groups with viruses that may have good potential for
commercial production for integrated pest control
9. Neodiprion complex (NPV).
10. Malacosoma complex (NPV).
11. Agrotis, Peridroma, and related cutworms (NPV).
12. Porthetria dispar (NPV).
(Continued)
21
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ANNEX C (CONTINUED)
13. Chilo suppressalis (GV).
14. Ephestia cautella (NPV + GV).
15. Mamestra brassicae (NPV).
Source: FAO (5).
22
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REFERENCES
1. Neisess, J., and H.B. Hubbard. Application of Microbial Insecticides in
Forests. Proceedings of a Symposium of the Entomological Society of
America, Hawaii, 1976.
2. Steinhaus, E.A., and C.G. Thompson. Preliminary Field Tests Using a
Polyhedrosis Virus to Control the Alfalfa Caterpillar. Econ. Entomology,
42:301-305, 1949.
3. Falcon, L.A., and R. Daxl. Report to the Government of Nicaragua on the
Integrated Control of Cotton Pests Project NIC/70/002/AFP. Food and
Agriculture Organization of the United Nations, Rome, 1973. 60 pp.
4. Falcon, L.A. Problems Associated With the Use of Arthropod Viruses in
Pest Control. Annual Review of Entomology, 21:305-324, 1976.
5. Food and Agriculture Organization of the United Nations. Use of Insect
Viruses in Integrated Pest Control. Plant Prot. Bull, 5:142-143, 1973.
6. Summers, M., R. Engler, L.A. Falcon, and P. Vail, eds. Baculoviruses
for Insect Pest Control: Safety Considerations. American Society for
Microbiology, U.S. Environmental Protection Agency, Washington, D.C.,
1975. 186 pp.
7. United States Department of Agriculture. Combined Forest Pest Programs
Special Report. USDA, Washington, 1974. 9 pp.
8. Hawthorne, R.M. Estimated Damage and Crop Loss Caused by Insect/Mite
Pests 1974. E-82-14. State of California Department of Food and Agri-
culture, Sacramento, 1975. 14 pp.
23
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DISCUSSION
SHOPE: Dr. Falcon, you covered very clearly the Insects of agricultural
importance, but you did not mention the need for baculovirus pesticides to
control insects or arthropods of medical importance. Was this left out
purposely, or do you see a need there also?
FALCON: I left this out purposely because the information I have received
from people working in this area indicates there appears to be little poten-
tial in the area at this time. So far, viruses that have been isolated from
insects of medical importance such as mosquitoes do not appear to be very
virulent or effective. In general the application of insect pathogens
against arthropods in this group has not been too successful. As I s^aw it,
my responsibility was to review current programs. To my knowledge, there are
no programs in this particular area. Possibly other people here might be
able to comment further on this matter.
RAPP: You mentioned a 200 million dollar loss to agriculture in California
due to insects susceptible to control with baculoviruses. How accurate are
these figures?
FALCON: This type of information has been accumulating in California for
about two decades. In 1970, for example, the same group of insects was esti-
mated to have cost approximately 70 million dollars, so the increase has more
than quadrupled in four years. Of course these types of data are susceptible
to many variables and sometimes may be misleading, but they are the best we
24
-------
have. Also I wish to indicate that California is an ideal area for agricul-
ture, and generally speaking, there is not the magnitude of pest problems
that exists in other areas of the U.S. or, for example, in Central America.
California is principally a desert-type agricultural situation and has fewer
pest problems than many other areas. So the crop loss figures cannot be
applied across the United States.
25
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The International Scope of Invertebrate
Virus Research in Controlling Pests
Keith A. Harrap, Ph.D., and T. W. Tinsley, Ph.D.
NERC, Unit of Invertebrate Virology
Oxford, United Kingdom
For convenience this survey has been divided into three sections:
1. Past, present, and future control programs.
2. The trend of research toward the goal of producing a viral
pesticide and using it responsibly.
3. The problems associated with the irresponsible use of
viruses as pesticidal agents.
The preceding paper was concerned with the situation in North America
and the Western Hemisphere generally, and these geographical areas therefore
are excluded from this presentation. Our comments here are limited to con-
sidering mainly Baculoviruses for pest control purposes.
For convenience, past and present control programs have been summarized
by geographical area in Table 1 (Europe and Africa) and Table 2 (the Far East
and South Pacific). Table 3 summarizes possible future virus control pro-
grams. These lists should not be considered exhaustive, and it is quite
likely that there are or have been a number of small virus control projects
against pest insects that are not represented. Let us now examine the various
programs itemized in the tables in more detail.
27
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Two viruses have been used in the United Kingdom both against saw-
flies. With Neodiprion sertifer a number of ad hoc trials were made in
Eastern England and Scotland around 1960 with crude preparations of larval
cadavers (1, 2). Little quantification was possible as no virus purification
was attempted, and the properties or even content of the spray application
were not known in detail. This ad hoc type of spray application then ceased
until characterization of the virus could be undertaken. This work is now
nearing completion in our laboratory at Oxford. Application trials of puri-
fied virus start this year on a selected area of pine sawfly infestation in
Scotland. _N. sertifer virus is produced commercially in Finland and has also
been used there for control of pine sawfly.
In Wales, areas of spruce were attacked by another sawfly, Gilpinia her-
cyniae, and a virus epizootic became established naturally. Only point
sources of inoculum were used for further virus release so that virus spread
could be monitored. A period of four years' observation of this epizootic
by P.F. Entwistle and his colleagues from our laboratory has produced valuable
data on the factors influencing the progress of a virus epizootic in a field
situation.
Certain biochemical characteristics of nun moth (Lymantria monacha) and
gypsy moth (Porthetria dispar) NPVs have been studied in Oxford by 0. Zethner
while he was a visiting worker in our laboratory. The nun moth NPV has been
used in Denmark by Zethner in the Silkeborg North Forest in control projects.
An area of about 15 hectares of forest was treated using virus purified by
centrifugation and applied at 10 polyhedra per ml, 80 liters per hectare;
0.4% pinolene was used as a sticking agent. Approximately 90% mortality
occurred as a result of NPV infection and half the larvae died as pupae.
Damage in 1973 could not be avoided in the NPV-treated area, but during 1974
the population of the nun moth, as well as the damage caused, was very much
reduced in the virus treatment areas in comparison with other areas of the
forest both untreated and chemically treated (0. Zethner, personal communi-
cation). Detailed description of this work is presently in the course of
publication.
Some very promising trials have also been done in Denmark by Zethner on
cutworms (Agrotis segetum) using granulosis virus (GV). This work is in pro-
gress at present. Virus was produced in larvae reared on synthetic diet,
purified by centrifugation, and applied in suspension. Approximately 80%
reduction in damage to red beets and carrots was observed when compared with
untreated control plots. There is some evidence that the virus remains
effective one year or more after application.
31
-------
In Africa Baculoviruses have been used against two species of Spodoptera.
The properties of these viruses (and JJ. frugiperda and to a lesser degree _ง_.
exigua virus) have been fully investigated in Oxford, and the data are pre-
sently either in press or published (20, 21). There is also a considerable
effort being made in our laboratory to understand the replication of these
viruses in cell culture systems. Both viruses have been toxicity-tested
(following the procedure recommended by the Insect Pathology Laboratory, USDA,
as published in WHO Tech. Rep. No. 531) at the Microbiological Research Esta-
blishment, Porton Down, Salisbury, U.K. This work was contracted from the
U.K. Ministry of Overseas Development, Centre for Overseas Pest Research, and
this organization intends to evaluate these viruses in control programs in
Africa and the Mediterranean. Purified virus is supplied by our laboratory
for this work. In addition, tests for virus replication in those animals used
in the toxicity testing procedures are now being undertaken by Oxford staff
working at Porton Down. Briefly, the protocol here involves the inoculation
of virus-sensitive cell cultures with tissue homogenates from animals sub-
jected to the various routes of baculovirus exposure (e.g., oral, dermal,
pulmonary). Preliminary results appear to demonstrate the ability of the
cell culture systems to yield positive CPE with polyhedra formation from such
types of inoculum. These viruses then are fairly well characterized and
safety-tested. Application in the field is now the responsibility of the
Ministry of Overseas Development, and they are also undertaking larval bio-
assay tests in preparation for field control projects. Under similar arrange-
ments characterization work is in progress at Oxford for the baculovirus of
the bollworm, Heliothis armigera, and Heliothis zea virus has been included
in the program for comparison.
Table 2 shows similar information for the Far East and South Pacific.
The first example, control of the oil and coconut palm pest Darna trima, will
be considered in detail below. The CPV used in Japan apparently has been
toxicity safety-tested but we have no details at present of the procedures
used (22). We are not aware of safety testing or published data on the pro-
perties of the P^. rapae granulosis viruses used in Australia and New Zealand.
Oryctes virus has been used very successfully to control the beetle Oryctes
rhinoceros in Samoa, the Philippines, and Malaysia and could possibly be used
in the Seychelles to control Oryctes monoceros. This virus was characterized
at Oxford at the request of FAO and we understand that it has been safety-
tested in France but are not aware of the procedures used (23). Possible
future control programs using viruses are given in Table 3. Several are con-
tinuations of programs already discussed and listed in Tables 1 and 2.
32
-------
There have been many other pesticidal virus release programs in the USSR
and Eastern Europe of which we have only scant information so a tabulation has
not been attempted. However, a list of past and present preparations of
viruses in or near commercial use (Table 4) shows three products from the
USSR in addition to the more familiar North American products.
Clearly, then, there is a fair range of activity. Unfortunately, the
state of the art is very different in the different examples cited. An
evaluation of the trend*of the research effort and responsible use of virus
pesticides can therefore form a useful second part of this article.
In some cases (e.g., _S_. littoralis; j^. exempta; N. sertifer; _H. armigera;
H. zea; L. monacha; _L. dispar), a logical development is discernible toward
the goal of using a Baculovirus as a pesticide. In other cases only certain
parts of the work which we consider to be necessary are completed or even con-
templated.
First, one should have knowledge of the physical, chemical, and biologi-
cal properties of any virus that might ultimately find itself on the market
place in the hands of the inexperienced operator. One can argue how complete
such knowledge has to be, but certainly sufficient data on the virus should
be available for it to be recognized; that is, identified as a specific can-
didate virus. Table 5 summarizes certain properties of Spodoptera NPVs which
have enabled us to achieve this with these viruses at Oxford. Second, some
safety testing of the purified and characterized candidate virus should be
done for toxicity and more importantly for replicative events in nontarget
cells and tissues. Accurate detection and recognition of the virus is a neces-
sary prerequisite for assessing such tests. One can, of course, argue about
the extent of such tests for fundamentally similar viruses. For example, how
different in properties do the viruses have to be to justify individual safety
tests? Third, knowledge of the replicative processes of Baculoviruses using
established laboratory model systems could indicate any potential risk of a
more subtle nature which large-scale application of these viruses might create.
At some stage before safety testing has proceeded very far, or indeed even
started, but after knowledge of the virus properties is available, one needs
to demonstrate the effectiveness of the purified virus as a control agent in
small field tests. In the U.K., registration under the Ministry of Agricul-
ture, Fisheries and Food, Pesticide Safety and Precaution scheme is required
to allow a trial of this type under strict conditions. This procedure is to
be used this year by our laboratory in Scotland in a control trial using N_.
sertifer NPV. The virus characterization is virtually completed, control
33
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efficacy will now be demonstrated in the field. This is the philosophy advo-
cated and followed in Oxford, and other agencies involved with us as research
contractors or seeking our opinion have been given this advice.
If such procedures are not followed, there are real dangers of using these
viruses irresponsibly. This can be illustrated with an example from a pest
control project in Sabah, Southeast Asia, with which we are familiar and which
is well known to us in Oxford through material sent in for diagnosis. I
referred to it earlier. This analysis forms the third part of this article
the irresponsible use of pesticidal viruses.
Oil and coconut palms are liable to attack by the limacodid Darna trima.
This insect causes devastating damage to foliage up to 2,000 larvae per
frond have been recorded and insecticidal control has not always been success-
ful. However, natural epizootics have been observed to reduce the population
rapidly. When they occur, dead larvae can be collected, ground up, strained
through cheesecloth and the filtrate used as a control spray. This method
was used in Sabah using 4 ounces of such insect material per 20 gallons of
water and spray applications with mistblowers or knapsack sprayers at 20
gallons per acre. The application method depended on the economic threshold
of the pest, e.g., 1-50 larvae per total palm, spray every tenth row; 50-100
every fifth; 100-200 every second row, and so on. Using this method, control
was dramatically successful, virtually 100% in 5-6 days with one spray
application at a cost only 40% that of chemical pesticides (13).
Our laboratory in Oxford had identified two sizes of small spherical
virus in D_. trima insects from Sarawak in 1972. Last year material was
received from Sabah for diagnosis material equivalent to that in use for
the control program outlined above. We found three viruses: a granulosis
virus (Baculovirus), a 27 nm RNA spherical virus (enterovirus-like) having
four structural polypeptides, and a 34 nm RNA spherical virus (calici-like)
having one polypeptide.
We do not know which virus was most effective in the pest control pro-
gram or indeed whether the mortality achieved indicated a combined effect of
the three viruses isolated. What this example clearly indicates is that
insects can support replication of several viruses, often simultaneously,
and without proper purification and identification one simply does not know
how the control is being achieved, how to calibrate it, or what the hazards
might be. In this instance, two of the viruses isolated have features in
common with viruses of vertebrates. Indeed, work by Dr. F.O. MacCallum in
37
-------
our laboratory has shown positive antigen-antibody reactions with one or other
of the two RNA spherical viruses in human sera from Malaysia. Previous work
has indicated positive antigen-antibody reaction with such viruses and sera
of several species of wild and domestic animals in which antibody has presu-
mably been elicited with a related virus to which the animal had been exposed.
This example, then, shows both the dramatic success possible using a virus
to control a pest insect and underlines the dangers which might be inherent
in the "bug juice" do-it-yourself approach a method that has even been
advocated in the U.S. by well-meaning publications. The argument of a "safe,"
"natural" method of pest control which finds favor with ecologists and con-
servationists might well hold other dangers for our environment. What is
more and relevant to those of us at this conference disillusion with
the rate of progress in developing conventional biological control methods
could encourage the do-it-yourself approach, thereby creating the very hazards
we are trying to avoid.
38
-------
REFERENCES
1. Rivers, C.F., and M. Crooke. Proc. Vth World For. Congr., 2:951-952, 1960.
2. Rivers, C.F. Entomophaga, Mem. Hors. Ser., 2:477-480, 1960.
3. Magnoler, A. Entomophaga, 12:199-207, 1967.
4. Magnoler, A.J. Invert. Path., 11:326-328, 1968.
5. Biliotti, E. Revue Path. Veg. Ent. Agric., 38:149-155, 1959.
6. Brown, E.S., and G. Swalne. E. Afr. Agric. For. J., 35:237-245, 1965.
7. Brown, E.S. Rept. Xllth Specialist Entomological and Insecticides
Committee, Mombasa, Appendix 16, 1966.
8. Abul-Nasr, S. Bull. Soc. Fouad I. Ent., 40:321-332, 1956.
9. Abul-Nasr, S. J. Insect Path., 1:112-120, 1959.
10. Coaker, T.H. Ann. Appl. Biol., 46:536-541, 1958.
11. Ossowski, L.L.J. Ann. Appl. Biol., 45:81-89, 1957.
12. Ossowski, L.L.J. Ann. Appl. Biol., 48:299-313, 1960.
13. Tiong, R.H.C., and D.D. Munro. Malay. Int. Agric. Oil Palm Conf., 1976.
14. Koyama, R., and K. Katagiri. Bull. Gort. Forest Expt. Stn. No. 207,
Tokyo, 1967.
15. Koyama, R. J. Jap. Forest Soc., 43:91-96, 1961.
16. Wilson, F. Aust. J. Agric. Res., 11:485-497, 1960.
17. Kelsey, J.M. N.Z. J. Agric. Res., 1:778-782, 1958.
18. Marschall, K.J. Nature (London), 225:288-289, 1970.
19. Zelazny, B. J. Invert. Path., 27:221-227, 1976.
20. Kelly, D.C. Virology, 76:468-471, 1977.
39
-------
21. Harrap, K.A., C.C. Payne, and J.S. Robertson. Virology, 78:92-109, 1977.
22. Aizawa, K. Proc. 1st Int. Coll. Invert. Path., Kingston, Ontario, 1976.
pp. 59-63.
23. Payne, C.C. J. Gen. Virol., 25:105-116, 1974.
24. Harper, J.D. Proc. 1st Int. Coll. Invert. Path., Kingston, Ontario,
1976. pp. 69-73.
25. Ignoffo, C.M. Envir. Letters, 8:23-40, 1975.
40
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DISCUSSION
LONGWORTH: I would like to make a comment regarding the Darna trima story.
None of us would condone such an approach, but isn't it true, Dr. Harrap,
that there were no fatalities that we know of, in fact, there were far, far
less than was the case with the conventional insecticides that had been used
before?
HARRAP: That is true. There have been no reports of adverse clinical symp-
toms or pathologies in the plantation workers. The only evidence we have
of anything possibly occurring, and this does not come from the same planta-
tion, is the antigen-antibody reactions. In fact, there were more problems
on that plantation relative to reactions to defoliants and chemical pesti-
cides than to viruses. This does not, of course, mean that something more
insidious or more subtle might be occurring.
ZAITLIN: Of course, the ultimate recipients of these sprays are plants. In
your testing program, do you test the effect of viral pesticides on plant
species?
HARRAP: We are not set up for such tests on plants in our laboratory.
STOLLAR: Could you clarify the terms "baculovirus" and "nuclear polyhedrosis"?
HARRAP: The preferred term suggested by the International Committee is
"baculovirus." This term covers both granulosis and nuclear polyhedrosis
41
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viruses. The difference is that the nuclear polyhedrosis virus has a large
polyhedron-like inclusion body with many virus particles located in it. The
granulosis virus has a granule, capsule-like inclusion body with one virus
particle in it. To all other intents and purposes, they are very similar and
have been combined as baculoviruses.
42
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PART II
VIRUSES: BIOLOGY AND IDENTIFICATION
-------
Baculoviruses
Max D. Summers, Ph.D.
Texas A&M University
College Station, Texas
INTRODUCTION
Nuclear polyhedrosis (NPV) and granulosis viruses (GV) contain DNA
genomes, are classified in the family Baculoviridae (1), and are presently
identified on the basis of morphology and host specificity (2). The char-
acteristic structure associated with the virus is a proteinic inclusion
ranging in size from 1-15 microns in which rod-shaped enveloped nucleocapsids
are randomly embedded. The NPVs exhibit two major structural relationships:
inclusions which contain enveloped single nucleocapsids, or those containing
multiples of nucleocapsids common to a single envelope (3). All granulosis
viruses are similar in structure: the typical inclusion body is ovicylindri-
cal with sizes from different species of approximately 250-300 x 450-550 nm,
and they usually contain only one virus per inclusion. The structure of the
enveloped nucleocapsid, as observed by electron microscopy, is similar to
that of the NPVs; an enveloped rod-shaped particle with dimensions of 40-60
nm x 350-400 nm.
Initially, baculoviruses were considered specific for insect arthropod
hosts. The discovery of the pink shrimp Penaeus duorarum NPV (4, 5) has
altered this concept. Nonoccluded particles with baculovirus structure have
been observed in nuclei of hemocytes and connective tissue elements of the
European crab, Carcinus maneas (6), and in the epithelium of hepatopancreatic
cells of the blue crab, Callinectis sapidus (7). Observations of this nature
45
-------
led to curiosity and speculation about biological and structural relatedness
of these arthropod viruses. This will ultimately require comparisons of
insect baculoviruses with structurally similar viruses of noninsect inverte-
brates .
Polyhedron Structure
Biochemical studies on the crystal inclusion of NPVs and GVs have identi-
fied as the major component only one polypeptide (3, 8-14). After alkali
solubilization and purification, this protein is referred to as granulin for
GVs and polyhedrin for NPVs (12).
The crystal structure dissociates under dilute alkaline saline conditions
to give components sedimenting at approximately 12S (9, 12, 15-17). Depen-
dent upon the virus studied, molecular weights for the subunit based upon
sedimentation properties are estimated to range from 180,000 to 378,000 (Table
1). For some purified granulins and polyhedrins, the isolated subunits are
apparently capable of aggregation into higher order polymers (9, 13, 18).
Treatment of the solubilized granulin or polyhedrin with SDS and electrophore-
sis in SDS-polyacrylamide gels shows that the basic unit of structure is a
smaller molecule which can vary slightly in size with species (3, 14, 17, 19,
20). Using vertical slab polyacrylamide gel electrophoresis, mobility dif-
ferences can be shown for granulins and polyhedrins ranging from 25,500 to
31,000 'daltons (Figure 1, Table 1).
Various protein denaturants and solubilization procedures in addition to
alkali have been successfully employed to dissociate the crystal of Tricho-
plusia ni GV at neutrality (21) and the activity of the denaturing agents on
crystal structure correlated with ultrastructural alterations (22). Each
denaturating agent produced characteristic alterations in crystal structure;
however, enveloped nucleocapsids were only obtained by carbonate (weak alkali)
solubilization. Ultrastructural comparisons of crystal dissolution in the
midgut lumen of Trichoplusia ni larvae showed that carbonate solubilization
in vitro approximated that observed to occur in vivo.
Barrap et al. (25) studied the serological and structural properties
of Spodoptera littoralis, Spodoptera exempta, and Spodoptera frugiperda NPVs.
All three viruses were morphologically similar (MNPVs) and by electron micro-
scopy indistinguishable from each other. As compared by SDS-polyacrylamide
gel electrophoresis there was no significant difference in size among poly-
hedrins. There were two bands; a major band of 28-29,000 and a smaller band
46 -
-------
ABCDEFGH I
_ 160,000
~~ 150,000
_ 94,000
~ 90,000
68,000
53,000
40,000
3 1,000
25,000
17,200
t t I t t 1 It
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Figure 1. Molecular weights and relative mobilities of granulins and
polyhedrins disrupted in 2% SDS and 5% mercaptoethanol. 1.5 Mg of each was
electrophoresed in 11% polyacrylamide slab gels in the presence of 0.1% SDS.
SfGV and TnGV granulin, and HzSNPV, TnSNPV, HaMNPV, AgMNPV, RoMIPV, and Ac^DJPV
polyhedrin .are in A through H, respectively. The molecular weights of the
standard protein markers are: RNA polymerase subunits, 160,000, 150,000,
90,000 and 40,000; phosphorylase-a, 94,000; bovine serum albumin, 68,000;
L-glutamic dehydrogenase, 53,000; DNA nuclease, 31,000; chymotrypsinogen,
25,000; and myoglobin, 17,200. Relative migrations are indicated adjacent to
the virus polypeptides.
47
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TABLE 1. GRANULINS AND POLYHEDRINS
Virus
B. mori SNPV
T. ni SNPV
P. dispar MNPV
P. brassicae GV
ฃ. ni GV
T. ni SNPV
AcMNPV
RoMNPV
AgMNPV
HaMNPV
HzjSNPV
TnGV
SfGV
Pdj5NPV
B. mori SNPV
G. mellonella NPV
S. frugiperda NPV
S. exempt a NPV
S. littoralis NPV
0. pseudotsugata SNPV
0. pseudotsugata MNPV
Porthetria dispar NPV
Prothetria dispar NPV
6.26
13.6
29.7
8.76
11.0
11.3
12.5
2 to
7.25
3.45
15.8
2.0
2.0
12
12
*
s2Q,w MWt
S (pH 11) 120,000*
ฃ 19.7, j[
S^ 38.0 S^ (pH 7)
J5, 3.35 j[ (pH 8.5)
S (pH 11)
S, 11.5 S (pH 11)
S, 16.0 S
4 S^ (pH 11)
*
(pH 11) 180,000
ฃ (minor)
S (minor)
31,000*
30,000*
30,000*
29,000*
28,000*
27,000*
25,000*
26,000*
50,000*
S 28,000*
S 28,000*
28-29,000*
28-29,000*
28-29,000*
S 26,000*
S 26,000*
30,350
29,000
References
(23)
(23)
(23)
(24)
(16)
(9)
(12)
(20)
(20)
(12)
(12)
(12)
(12)
(13)
(13)
(12)
(17)
(17)
(25)
(25)
(25)
(18)
(18)
(26)
(27)
t
Sedimentation.
Polyacrylamide gel electrophoresis in the presence of 10% SDS.
48
-------
suspected to be a product derived by proteolytic cleavage. Production of
the lower molecular weight component was time dependent and could be inhi-
bited by heat and excessively high pH.
During alkali-solubilization of polyhedra purified from infected insects
a protease can be activated (3, 13). Although the protease appears to be
present in occluded virus extracted from infected insects, conformation of
similar activity from occluded virus purified from cell culture is yet to
be established. If the enzyme is not inactivated by the appropriate inhibitor
or heat treatment procedure, hydrolytic activity can produce several lower
molecular weight products.
Comparisons of the primary structure of purified granulins and polyhe-
drins (undegraded) has been conducted by two dimensional high voltage electro-
phoresis which can discriminate single amino acid differences among major
peptides (14). All polyhedrins and granulins studied [Trichoplusia ni SNPV
(TnSNPV); Rachiplusia ou MNPV (RoMNPV); Autographa californica MNPV
(AcMNPV); Trichoplusia ni GV (TnGV); and Spodoptera frugiperda (SfGV)] had
similar primary structures; yet each virus had different peptides, indicating
regions of nonsimilarity. TnGV and SfGV granulins showed the closest degree
of similarity as compared to polyhedrins. Each NPV or GV studied to date by
peptide mapping has shown distinct polyhedrin or granulin structure (28, 29).
The evidence indicates that these are virus specified but group related pro-
teins . The primary sequence for Bombyx mori polyhedrin has been determined
(11).
The most thoroughly characterized granulin or TnGV has chemical and phy-
sical properties which should be investigated and confirmed as properties
consistent with other granulins and polyhedrins. The acidic protein is appar-
ently phosphorylated, phenol soluble, and contains a high concentration of
hydrophobic amino acids. Phosphorylated proteins are routinely identified as
structural components of RNA and DNA vertebrate viruses, although the deter-
mination of a biological function for phosphorylated proteins has been diffi-
cult. Certain reports suggest a regulatory relationship with a viral kinase
and/or possible relationships to regulatory processes involving transcriptase
activity (30). A class of phenol soluble, nuclear acidic proteins in eukar-
yotic systems has been the object of investigation relative to possible
regulatory role(s) during growth and differentiation (31).
49
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Virus Structure
By use of the standard technique (adding dilute alkaline saline to
purified polyhedra at a pH of 10.9) the enveloped nucleocapsid can be
released and purified (16, 32-36). In a GV of SNPV, the enveloped nucleo-
capsid will usually occur as a single band upon sedimentation or isopycnic
centrifugation. If it is of the multiple type (MNPV), several bands will
be observed in the gradient after rate zonal, or centrifugation to equili-
brium. In CsCl each band will differ in density according to the number of
nucleocapsids per envelope with densities ranging from approximately 1.20-
1.25 g/ml to 1.32 g/ml (20, 25, 36).
Studies have shown that the qualitative and quantitative nature of
multiple nucleocapsid banding profiles may be strain or species specific.
Each of the three Spodoptera NPVs reported by Harrap et al. (25) had a char-
acteristic banding profile after centrifugation to quasi-equilibrium in
sucrose gradients, j^. exempta consistently showed a small number of virus
bands, five as compared to ^. frugiperda and ^. littoralis; each of the
latter had eight. There was no similarity or matching in position or quan-
tity in each of the enveloped nucleocapsid peaks among the viruses studied.
Removal of the envelope by the appropriate detergent treatment produces
a nucleocapsid with a characteristic structure (8, 37-39). The nucleocapsid
has a density of about 1.48 g/ml after isopycnic banding sucrose or in CsCl
gradients (20, 25). Electron microscope observations suggest that one end
structure of the nucleocapsid is involved in a nuclear pore interaction to
uncoat the DNA genome early during the infection process (40, 41).
Virus Structural Polypeptides
For serological studies it is important to establish and utilize repro-
ducible criteria for virus or antigen purity (20, 32, 42). Polypeptide pro-
files derived from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are one
such criterion.
Baculovirus enveloped nucleocapsids have several structural proteins
ranging in size from 8,000 to more than 100,000. Using SDS-PAGE comparison
of polypeptides for NPVs and GVs shows that each virus has, qualitatively
and quantitatively, a different polypeptide composition. SDS-PAGE of the
NPV structural polypeptides of Porthetria dispar (PdMNPV) (27) and Tricho-
plusia ni NPV (43) shows the presence of 14 and 12 major polypeptides,
50
-------
respectively. A preliminary report by McCarthy and Liu (26) shows 25 pro-
teins present in the PdMNPV enveloped nucleocapsid. Eleven bands were
observed present in enveloped nucleocapsids of j^. exempta (25). After NP-40
treatment and envelope removal, two major proteins of j>. littoralis (L32
and L14) were observed to be nucleocapsid proteins, and E39 for J3. exempta
and F36 and F33 for _S_. frugiperda.
Summers and Smith (20) studied the relative mobilities of SDS-treated
structural polypeptides of eight baculoviruses (Figure 2). Depending on the
baculovirus, a range of 15-25 bands was resolved. Some bands exhibited
similar mobility for different viruses, for example AcMNPV and RoMNPV
appeared closely related; however, each virus had a distinct composition
when comparing relative mobility and the number of bands.
After centrifugation to quasi-equilibrium in sucrose gradients, the
least dense band (1.20-1.21 g/ml) of enveloped single nucleocapsids from
AcMNPV, RoMNPV, Anticarsa gemmitalis (AgMNPV) and Heliothis armigera (HaMNPV)
were separated from the multiples (usually 7-8 additional bands ranging
in density from 1.22 to 1.25 g/ml), and compared by SDS-PAGE (Figures 3-5).
Qualitative and quantitative differences exist between multiples and singles
for each virus. For example, VP30, VP28, VP19, VP18.5, and VP16 of AcMNPV
singles were observed to be in relatively lesser concentration when compared
to the same bands in multiples. VP58, VP36, and VP23 are apparently absent
or diminished in relative concentration to a level not detectable in the
gels (Figure 5). The polypeptide composition of HaMNPV singles shows that
VP64, VP47, VP28, and VP20 are present in increased concentration relative
to the same protein in multiples, VP89, VP75, VP62, and VP51 were not ob-
served in multiples. In contrast, there is more of HaMNPV VP45 in multiples
as compared to singles. Similar relationships were observed for the other
MNPVs as well (Figure 3).
The envelopes of AcMNPV, RoMNPV, and TnGV were removed by treatment
with NP-40. The other five viruses were resistant to this chemical treat-
ment and yielded mostly intact virus (20). The buoyant densities of AcMNPV,
RoMNPV, and TnGV nucleocapsids and capsids, after banding in cesium chloride
gradients, were approximately 1.48 g/ml and 1.33 g/ml, respectively. AcMNPV
capsids were composed of two major peptides, VP37 and VP18.5, and trace
quantities of VP30.5 and VP30 (Figure 6). RoMNPV capsids had several major
polypeptides; VP16, VP17, VP18, VP30, and VP36, with detectable quantities
of VP30.5. The TnGV capsid was composed of two major polypeptides VP31 and
VP17, with trace quantities of VP29 and VP26. Each capsid preparation con-
sistently contained several other minor polypeptides which could be components
51
-------
AcMN RoMN AgMN HoMN TnSN HzงN TnGV SfGV
ABC DEFGH
160,000
150,000
94,000
90,000
68,000
53,000
40,000
17,200
Figure 2. Polyacrylamide slab gel electrophoresis of enveloped nucleo-
capsid polypeptides. The viruses were purified by equilibrium banding in
sucrose (1.17 to 1.25 g/ml) after being liberated from the polyhedra with 0.1 M
Na2C03, 0.01 M EDTA, 0.17 M NaCl at 0ฐ to 4ฐC for 60 min. The band containing"
only one nucleocapsid per envelope (singles) was removed from the gradient,
prepared for electrophoresis by disruption in 2.0% SDS and 5.0% mercaptoethanol
for 3 min at 100ฐC at a protein concentration of 1 mg/ml; a total of 25 Mg
protein was electrophoresed in 11% polyacrylamide. The virus polypeptides (VP)
of AcMNPV, RoMNPV, AgMNPV, HaMNPV, Ts^NPV, HzSNPV, TnGV, and SfGV are shown in
A, B, C, D, E, F, G, and H, respectively.
52
-------
AcMNPV RoMNPV HaMNPV AgMNPV
ABCDEFGH
160,000 _
150,000 ~~
94,000
90,000
68,000
53,000 -
40,000 -
31,000
25,000
17,200
Figure !3. Comparison on a 11% polyacrylamide slab gel of polypeptides
from enveloped single nucleocapsids and multiples of nucleocapsids. Each of
the four MNPV viruses used in this study was liberated from the polyhedra and
gel electrophoresis conducted as described in Figure 2; except for collecting
the top virus band (singles) from the sucrose gradient, the rest of the virus
bands (multiples) were collected and analyzed. The VPs of singles and multi-
ples, respectively, of AcMNPV are observed in A and B, RoMNPV in C and D,
HaMNPV in E and F, and AgMNPV in G and H. The positions of the molecular
weight standards are indicated next to the VPs.
53
-------
to
ee
o
1 0
.2
1.0
Figure 4. Densitometer tracings (600 nm) from transparencies of the
stained envelope nucleocapsid polypeptides from (A) HaMNPV bundles and (B)
HaMNPV singles that were electrophoresed as described in Figure 2.
54
-------
1.0
.8
E
.2
(D 0
1.0
.8
.6
I II I III! I I III II I
S ?? SSS!
X. ~
II II l\
sa 23 ta
ง:5 J >>
Figure 5. Densitometer tracing (600 nm) from transparencies of stained
enveloped nucleocapsid polypeptides of (A) AcMNPV bundles and (B) AcMNPV sin-
gles that were electrophoresed as described in Figure 2.
55
-------
of the viral capsid; however, further study will be needed to identify
those proteins. The differences between capsid and enveloped nucleocapsids
shown in Figure 6 suggest that the envelope composition of baculoviruses
is complex (25). For example, AcMNPV contains approximately 18 bands of
which only 3 are capsid proteins.
Payne (44) isolated nucleocapsids from the Oryctes baculovirus using
NP-40. The Oryctes baculovirus has certain structural features similar to
baculoviruses of Lepidoptera and Hymenoptera. The DNAs are of comparable size
and conformation, and serological cross-reactions have been shown to occur
with Spodoptera NPVs. However, the Oryctes baculovirus possesses a tail-like
structure which is not similar to that of other insect baculoviruses. This
structure is similar to that observed on parasitoid particles (40) which have
been observed to interact with the nuclear pore of infected cells during the
uncoating process. Twelve structural proteins were resolved in the Oryctes
baculovirus enveloped nucleocapsid. After treatment with NP-40 eight appeared
to be components of the nucleocapsid.
Extracellular or Nonoccluded Virus
The nonoccluded or extracellular virus has different biological and
physical properties when compared to those of enveloped nucleocapsids which
are occluded and require alkali treatment for liberation from the crystal.
During cell infection, at least three and possibly four infectious forms of
virus can be present (45): one is the occluded virus which obtains an
envelope from probable de novo synthesis in the nucleus and then becomes
occluded (46-48); particles which bud from the inner nuclear membrane into
the cytoplasm (46, 47, 49); and nucleocapsids which bud through from plas-
ma membrane (36, 46, 47, 49) into the cell culture medium or insect blood.
The occluded, the budded, and the intracellular viruses are all reported
as infectious, although it has not been verified whether the nucleocapsid
is an intracellular infectious form.
A number of studies have reported on the properties of the nonoccluded
infectious forms occurring in hemolymph and cell culture medium (36, 50-55).
However, in most in vitro studies the extracellular virus was collected
from the cell culture medium after extensive cell lysis. It is probable
that the intracellular enveloped, unenveloped, and extracellular infectious
forms were all included under the somewhat broad classification of non-
occluded virus (47, 51). Therefore, confusion existed as to the nature
and identity of the nonoccluded infectious forms.
56
-------
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a.
in
a.
s
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si
II
o
o
0
60
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8
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tO ^ fl CU
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57
-------
Summers and Volkman (36) compared the properties of culture-derived,
plasma membrane-budded jnonoccluded jvirus (PMB-NOV) from RoMNPV and AcMNPV
to those of alkali-liberated enveloped nucleocapsids. If collected at 24 h
PMB-NOV were loosely enveloped single nucleocapsids with a distinct surface
modification. Infectious virions obtained from insect hemolymph had physi-
cal and biological properties similar to particles obtained from culture
medium. Both the PMB-NOV and the virus obtained from hemolymph banded at
a density of 1.17-1.18 g/ml. The alkali-released virions were for the
most part multiple nucleocapsids which banded at various densities. The
buoyant density of each depended on the number of nucleocapsids per envelope
and ranged from 1.20 g/ml for enveloped single nucleocapsids to 1.25 g/ml
for multiples. The envelopes from alkali-liberated virus were relatively
tight fitting with the nucleocapsid and were devoid of surface projections.
Volkman et al. (45) showed that alkali-liberated and nonoccluded
forms were antigenically different by neutralization studies. Antisera
raised against purified AcMNPV alkali-liberated virus (which we described
as LOVAL: Larval j>ccluded virus Alkali-liberated) did not neutralize the
AcMNPV nonoccluded virus from infectious hemolymph, alkali-treated and un-
treated NOV from TN-368-10 cells or infectious virus liberated from fat
body cells. It did neutralize both AcMNPV and RoMNPV LOVAL. Antiserum to
RoMNPV LOVAL did not neutralize RoMNPV NOV but did neutralize both RoMNPV
and AcMNPV LOVAL. Antisera raised against AcMNPV PMB-NOV neutralized not
only all nonoccluded forms of both AcMNPV and RoMNPV tested, but the alkali-
liberated infectious virus forms as well. Absorption of the PMB-NOV anti-
sera with AcMNPV and RoMNPV LOVAL removed all of the anti-LOVAL neutralization
activity, but did not diminish anti-NOV neutralization titers. This shows
that whereas the alkali-liberated and nonoccluded forms of the virus share
some antigenic determinants, those involved in the neutralization of the
two different infectious forms are different.
Physical-infectious particle calculations showed that PMB-NOV AcMNPV
from the culture medium of TN-368-13 cells is 1,700-fold more infectious
than LOVAL multiples and 1,900-fold more infectious than LOVAL singles as
titrated in vitro (Table 2).
58
-------
TABLE 2. AcMNPV PHYSICAL-INFECTIOUS PARTICLE RATIOS
AS ASSAYED IN VITRO (45)
Virus
prepn
PMB-NOV
LOVAL I
LOVAL II-X
Mg/Mg of
protein
0.19
0.20
0.30
DNA
g/Mg of
protein
(x 10~7)
1.9
2.0
3.0
Particle/
Mg of
protein
(x 109)
1.1
1.2
1.8
PFU/Mg of
protein
8.9 x 106
5.0 x 103
8.4 x 103
Particles/
PFU
1.28 x 102
2.4 x 105
2.2 x 105
t
Single nucleocapsid per envelope
Many nucleocapsids per envelope
Baculovirus DNAs
Baculoviruses contain double-stranded DNA of high molecular weight
which is covalently closed (56). The properties of a few baculovirus DNAs
have been investigated by comparing extraction procedures, sedimentation,
electron microscopy, and renaturation kinetics.
Summers and Anderson (57-59) studied GV and NPV DNAs using sedimenta-
tion relative to that of bacteriophage T4 DNA. The molecular weight for
Trichoplusia ni GV was estimated at 99-119 x 10 ; Spodoptera frugiperda GV,
95-114 x 10b; Trichoplusia ni SNPV 99-119 x 106; Rachiplusia ou MNPV, 95-114
x 10 ; Spodoptera frugiperda MNPV, 95-114 x 10 . Sedimentation coefficients
were 60^, 59j>, 60JJ, 59ฃ, and 59^, respectively.
Centrifugation to equilibrium in ethidium bromide-CsCl gradients
revealed the presence of two bands of DNA at 1.54 g/ml and 1.58 g/ml which
were double-stranded linear, and relaxed circular DNA (light band), and
covalently closed, double-stranded DNA (heavy band). The covalently closed
and relaxed circular DNA forms co-sedimented in sucrose gradients with an
_3
ionic strength greater than 10 . By using gradients with the appropriate
ionic strength, three sedimenting forms could be detected: super-coiled
DNA (ccDNA or Form I), relaxed circular DNA (rcDNA or Form II), and double-
stranded, linear DNA (dlDNA or Form III). Using the classical technique of
59
-------
nicking with low concentrations of pancreatic deoxyribonuclease, a discon-
tinuous conversion of circular (Form II) to double-stranded linear DNA (Form
III) occurred, and a continuous conversion from double-stranded linear to
lower molecular weight fragments of random sizes.
Summers and Anderson (57) observed that lyophilization of occluded
virus preparations resulted in the inability to recover appreciable quanti-
ties of covalently closed DNA. This suggested that the lyophilization
process induced a single strand sisson into the phosphodiester linkage.
The reasons for this were not clear; however, it was suspected that an endo-
nuclease may have been responsible.
It was noted that ccDNA sedimented approximately 3 to 4 times faster
in alkaline sucrose gradients as compared to denatured linear and circular
DNAs; this was behavior typical of denatured, covalently closed DNA. Co-
sedimentation of GV and NPV DNAs showed that TnGV and SfGV were slightly
different, although denatured GV DNAs sedimented faster than the NPV DNAs.
Denatured NPV DNAs could not be differentiated from each other by sedimen-
tation in alkaline sucrose gradients compared by relative sedimentation
with denatured T4 DNA. Denatured TnGV and SfGV DNAs had sedimentation co-
efficients of 59_S and estimated molecular weights of 84 x 10 , a value which
is closer to more accurate measurements on the sizes of NPV DNAs (60).
Harrap et al. (25) studied the chemical and physical properties of
three Spodoptera NPVs. Two bands of labeled DNA were obtained after banding
in ethidium-bromide-CsCl gradients separated by a density of 0.04 g/ml. On
neutral sucrose gradients the viral DNA preparations sedimented as three
bands: the fastest (68.4jp was ccDNA; the major sedimenting band at 57.5
to 61.BS_ was rcDNA; and the 50.3 to 54.98^ band was dlDNA. Molecular
weight estimates for the three NPV DNAs were: S_. littoralis, 71.7 to 83.7
x 106; _S. exempt a, 79.8 to 92.3 x 106; and S_. frugiperda, 62.8 to 73.3 x 106.
Differences in sedimentation between ^. littoralis and ^. exempta could not
be detected by co-sedimentation. All three DNAs had different densities of
1.7056, 1.7010, and 1.7005 g/ml for _S. littoralis, J5. exempta, and S_. fru-
giperda, respectively. Comparison of thermal denaturation profiles showed
that values for GC content calculated by melting profiles and those derived
from densities did not agree closely; for example, 46.5% GC estimated from
density, and 52-53% G+C determined by T for S. littoralis DNA.
m
Kelly (61) studied the DNAs of four closely related Spodoptera NPVs
(Spodoptera frugiperda, S. exempta, S. exigua, and S. littoralis) assessing
60
-------
sizes and degree of homologies by DNA reassociation kinetics. Molecular
weights (relative to bacteriophage T4, 126 x 10 ) determined by reassociation
kinetics were 66.8 x 106, 82.5 x 106, 65.4 x 106, and 62.4 x 106, respec-
tively. The extent of sequence homology between viral DNAs was determined
125
by allowing I-labeled DNA fragments to reassociate in the presence of a
vast excess of homologous or heterologous DNA. The data revealed that the
four viruses were genetically related to some degree, j^. exigua and j^.
exempta genomes were more closely related (approximately 60%) than the ge-
nome of j[. frugiperda and S^. littoralis (approximately 15%).
Two NPVs (MNPV and JJNPV) have been isolated from the Douglas fir tus-
sock moth, Orgyia pseudotsugata (62). The DNAs were analyzed by reassocia-
tion kinetics using both optical renaturation and S. nuclease assay. Genome
size was also analyzed by analytical ultracentrifugation, and base composi-
tion by melting properties.
The OpMNPV had a T of 76.9 and an estimated G+C content of 54%. Rena-
m
turation kinetics using optical methods and Escherichia coli (IS. coli) DNA
as a standard gave an estimated genome size of 86 x 10 . This calculation
125
was made after correction for G+C content. Reassociation using I-labeled
DNA was examined and compared with lambda phage and E_. coli DNAs with the
extent of annealing determined by digestion with S. nuclease. DNA reasso-
1 ฃ
ciated at a rate of an equivalent genome size of 70-80 x 10 daltons.
Reassociation of the OpMNPV DNA followed second order reaction kinetics.
The data indicated that the viral DNA is composed mainly of unique sequences
although the presence of any small repeated portion could not be excluded.
DNA size was also determined by sedimentation. The DNA preparation
analyzed by equilibrium banding in an ethidium bromide-CsCl gradient was
shown to be in the linear form. However, documentation of the presence or
absence of circular molecules was not made using rate-zonal sedimentation.
The estimate derived by sedimentation for each NPV DNA was 96 x 10 daltons.
Most molecular weights of baculovirus DNAs have been derived by sedimen-
tation in neutral sucrose gradients relative to bacteriophage DNA standards
and/or renaturation kinetics. Better confirmation of size using more accu-
rate means of measurement has been provided by Burgess (60). Molecular
weight estimates were made on ccDNAs extracted from seven NPVs and three GVs.
By a comparison of contour length with that of the replicative form of Fl
bacteriophage DNA, the molecular weights ranged from 58 x 10 to 94 x 10 for
ten baculoviruses: Spodoptera frugiperda, 80 x 10 ; Spodoptera littoralis,
61
-------
80 x 10 ; Epiphyas postvittana, 58 x 10 ; Pseudaletia separata, 94 x 10 ;
Heliothis armigera, 77 x 10 ; Chrysodeixis eriosoma, 77 x 10 ; Laspeyresia
pomonella, 71 x 10 ; and Wiseana cervinata GV, 69 x 10 .
Skuratovskaya et al. (63) studied the size and infectivity of super-
coiled DNA purified from the NPV of Galleria mellonella. Separation of
covalently closed DNA from linear and open molecules (contour lengths 50-
52 microns, 96-100 x 10 ) was achieved by centrifugation to equilibrium in
ethidium bromide-CsCl. Both bands were infectious as tested by injection.
Scharnhorst et al. (64) observed DNA molecules from Heliothis zea
SNPV by the spreading technique and detected a heterogenous population of
molecules with contour lengths ranging from 15-45 microns. The most pro-
minent sizes observed were of approximately 20-25 microns. Thirty-six per-
cent of the molecules existed as a supercoiled form. An internal standard
was not utilized for a direct comparison and measurement. The molecules
with lengths of 20-25 microns had molecular weights of 40-50 x 10 . This
corresponded to their unpublished estimates derived from DNA reassociation
kinetics.
Baculovirus Infection and Maturation
A comparison of cellular infection in vivo (insects) with that shown
to occur in vitro (cell cultures) reveals that some aspects of the infection
process are similar, yet there are important differences. In nature the
primary infection is accomplished by the occluded form (polyhedra) of the
virus. However, secondary infection occurs by the nonoccluded or extra-
cellular form of the virus, a process analogous to subsequent infection of
cells in continuous culture. The biological, structural, physical, and
serological properties of these two forms of enveloped nucleocapsids, as
discussed earlier, are significantly different (45). A summary of the
infection processes as it occurs in vivo and in vitro should be appropriate
in an attempt to search for biological correlations to the structural pro-
perties and sources of the virus.
During invasion and infection, the polyhedron crystal dissociates in
the gut lumen. The enveloped nucleocapsid enters the gut cell by viral
envelope fusion with the microvillar membrane (38). The nucleocapsid has
been observed in cytoplasm where it may possibly uncoat by unknown mechanisms
(53) or interact with the nuclear pore and release the genome into the
nucleus (40, 41). The latent period before the appearance of infectious
62
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virus has been approximated to be six to eight hours for AcMNPV (45, 46).
When assembly of the nucleocapsid occurs in the nucleus, it may have two
fates: one possibility is that it may escape the nucleus, enter the cyto-
plasm, and bud from the surface of the cell into insect hemolymph or into
the cell culture medium. This form of the virus has been referred to as
a nonoccluded virus (NOV), a plasma membrane budded virus (PMB-NOV), or
an extracellular virus. It is also possible that the nucleocapsid may
obtain an envelope by de novo synthesis (48) in the nucleus and become
occluded. Large numbers of polyhedra accumulate in the nucleus finally
disrupting the cell.
The basic factors controlling occlusion of enveloped nucleocapsids in
the nucleus or budding from the cellular surface are not understood. It
has been suggested that the source and/or information in the different forms
of the viral envelope may be responsible (41). Release of extracellular
virus and occlusion appears to be a time and tissue dependent relationship
(45). Nucleocapsids which are assembled before the synthesis of some virus
specific protein, perhaps the polyhedrin required for occlusion, are able
to escape the nucleus. Those assembled after the synthesis or presence of
polyhedrin are occluded.
There are major differences when comparing the biological properties
of the two forms of virus. The nonoccluded virus is more highly infectious.
For AcMNPV it has an infectious:physical particle ratio of 1:128. The
alkali-liberated virus as titrated in TN-368 cells is less infectious, one
particle in 200,000 to 240,000 (Table 2).
A preliminary study (65) used comparative bioassays in insects and
the polyhedral plaque assay in cell culture for measuring the relative
infectivity of extracellular and alkali-liberated virus. The assays were
compared both by oral (per os) and hemocoelic injection. By oral injection
PMB-NOV was 1 x 10 less infectious when compared to infectivity by poly-
hedral plaque assay. Injection into the hemocoel showed that PMB-NOV is
highly infectious and comparable to the levels of infectivity obtained by
in vitro assay. Since the nonoccluded form of the virus was significantly
more infectious by hemocoelic injection than by infection via the midgut
(natural route), this result is consistent with the role of NOV or extra-
cellular virus in the nature infection cycle, that is, the secondary in-
fection of tissue.
A comparison of LOVAL (alkali-liberated-virus) infection by per os
and hemocoelic injection does not support previous assumptions that the
63
-------
alkali-liberated form of the virus is more infectious by injection than
feeding. LOVAL was only 9-fold more infectious by per os infection as
compared to polyhedral plaque assay. Infectivity by oral injection was
only 3-fold more efficient when compared to infection achieved by injection
into the hemocoel.
SUMMARY
The focus of this conference is to evaluate the molecular virology of
baculoviruses relative to some of the more theoretical concerns of their
safe use in environmental applications. Some of the ultimate questions for
which we are attempting to provide realistic assessments are: can baculo-
viruses and/or their genomes enter nontarget cells, persist or integrate,
or undergo recombination with host DNA or other viral DNA genomes to expand
their host range so as to pose any undesirable or unpredictable consequences
when used as biological pesticides. As can be seen from the preceding,
basic information on baculovirus structure and activity is presently too
limited to evaluate, predict, or estimate any potential interaction. There-
fore, it is clear that the safety testing required for registration of a
baculovirus formulation also does not provide the pertinent information
for such assessments.
In a reasonably short period of time, available technology could be
employed to investigate the potential interactions identified above in both
susceptible and unsusceptible host cells. It is recommended that high
priority be given to the study of the molecular aspects of baculovirus
structure, biology, and specificity of the infection process. The genetics
of baculovirus genomes should be investigated relative to their gene pro-
ducts. Genotypic and phenotypic markers should be identified as those
important for the development and application of diagnostic detection probes
for surveying the fate of baculovirus activity in any physical or biological
environment. Since baculoviruses are genetically and phenotypically com-
plex, this approach will be necessary in order to provide for a more realis-
tic safe use and risk assessment of baculoviruses in their proposed role
as biological pesticides.
64
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Sidorova, and S.B. Serebryanyi. A Determination of the Molecular
Weight of the Inclusion Body Protein of the Nuclear Polyhedrosis Virus
of the Mulberry Silkworm Bombyx mori. Biokhimiya., 38:1015-1019, 1973.
18. Rohrmann, G.F. Characterization of N-polyhedrin of Two Baculovirus
Strains Pathogenic for Orgyia pseudotsugata. Biochemistry, 16:1631-
1634, 1977.
19. Eppstein, D.A., and J.A. Thoma. Alkaline Protease Associated with the
Matrix Protein of a Virus Infecting the Cabbage Looper. Biochem.
Biophys. Res. Commun., 62:478-484, 1975.
20. Summers, M.D., and G.E. Smith. Baculovirus Structural Polypeptides.
Virology, 84:390-402, 1978.
21. Egawa, K., and M.D. Summers. Solubilization of Trichoplusia ni Granulo-
sis Virus Proteinic Crystal. I. Kinetics. J. Invertebr. Pathol.,
19:395-404, 1972.
22. Kawanishi, C.Y., K. Egawa, and M.D. Summers. Solubilization of Tricho-
plusia ni Granulosis Virus Proteinic Crystal. II. Ultrastructure. J.
Invertebr. Pathol., 20:95-100, 1972.
23. Shigematsu, H., and S. Suzuki. Relationship of Crystallization to the
Nature of the Polyhedron Protein with Reference to a Nuclear-polyhedro-
sis Virus of the Silkworm, Bombyx mori. J. Invertebr. Pathol., 17:375-
382, 1971.
24. Scott, H.A., S.Y. Young III, and J. McMasters. Isolation and Some Pro-
perties of Components of Nuclear Polyhedra from the Cabbage Looper,
Trichoplusia ni. J. Invertebr. Pathol., 18:177-182, 1975.
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25. Harrap, K.A., C.C. Payne, and J.S. Robertson. The Properties of
Three Baculoviruses from Closely Related Hosts. Virology, 79:14-31,
1977.
26. McCarthy, W.J., and S.Y. Liu. The Structural Proteins of Porthetria
dispar Nuclear Polyhedrosis Virus. In: Proceedings of the First
International Colloquium on Invertebrate Pathology and Fourth Annual
Meeting, Society for Invertebrate Pathology, Queen's University, Kings-
ton, Canada, 1976. pp. 330-331.
27. Padhi, S.B., E.E. Eikenberry, and T. Chase. Electrophoresis of the
Proteins of the Nuclear Polyhedrosis Virus of Porthetria dispar. Inter-
virology, 4:333-345, 1975.
28. Cilbulsky, R.J., J.D. Harper, and R.T. Guadauskas. Biochemical Com-
parison of Virion Proteins from Five Nuclear Polyhedrosis Viruses
Infecting Plusiine Larvae (Lepidoptera;Noctuidae). J. Invertebr.
Pathol., 30:314-317, 1977.
29. Kozlov, E.A., T.L. Levitina, N.M. Sidorova, Y.L. Radavskii, and S.B.
Serebryanyi. Comparative Chemical Studies of the Polyhedral Proteins
of the Nuclear Polyhedrosis Viruses of Bombyx mori and Galleria mellon-
ella. J. Invertebr. Pathol., 25:103-107, 1975.
30. Pal, B.K., and P.R. Burman. Phosphoproteins: Structural Components
of Oncornaviruses. J. Virol., 15:540-549, 1975.
31. Stein, G.S., T.C. Spellberg, and L.J. Kleinschmidt. Nonhistone Chromo-
somal Proteins and Gene Regulations. Science, 183:817-824, 1974.
32. Bell, C.D., and G.B. Orlob. Serological Studies on Virions and Poly-
hedron Protein of a Nuclear Polyhedrosis Virus of the Cabbage Looper,
Trichoplusia ni. Virology, 78:162-172, 1977.
33. Harrap, K.A. The Structure of Nuclear Polyhedrosis Viruses. II. The
Virus Particle. Virology, 50:124-132, 1972.
34. Harrap, K.A. The Structure of Nuclear Polyhedrosis Viruses. III.
Virus Assembly. 50:133-139, 1972.
35. Summers, M.D., and J.D. Paschke. Alkali-liberated Granulosis Virus
of Trichoplusia ni Granulosis Virus of Trichoplusia ni. I. Density
Gradient Purification of Virus Components and Some of Their _Iii Vitro
Chemical and Physical Properties. J. Invertebr. Pathol., 16:227-240,
1970.
36. Summers, M.D., and L.E. Volkman. Comparison of Biophysical and Mor-
phological Properties of Occluded and Extracellular-Nonoccluded Bacu-
lovirus from In Vivo and In Vitro Host Systems. J. Virol., 17:962-
972, 1976.
67
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37. Kawanishi, C.Y., and J.D. Paschke. The Relationship of Buffer pH and
Ionic Strength on the Yield of Virions and Nucleocapsids Obtained by
the Dissolution of Rachiplusia ou Nuclear Polyhedra. In: Proceed-
ings of the Fourth International Colloquium on Insect Pathology with
the Society for Invertebrate Pathology, College Park, Maryland, 1970.
pp. 127-146.
38. Summers, M.D. Baculoviruses (Baculoviridae). In: The Atlas of
Insect and Plant Viruses, Maramorosch, K., ed. Academic Press, New
York, 1977. pp. 3-27.
39. Teakle, R.E. A Nuclear-polyhedrosis Virus of Anthela varia (Lepidop-
tera:Anthelidae). J. Invertebr. Pathol., 14:18-27, 1969.
40. Stoltz, D., and S.B. Vinson. Baculovirus-like Particles in the Repro-
ductive Tracts of Female Parasitoid Wasps. II: The Genus Apanteles.
Canadian J. Microbiol., 23:28-37, 1977.
41. Summers, M.D. Electron Microscopic Observations on Granulosis Virus
Entry, Uncoating, and Replication Processes During Infection of the
Midgut Cells of Trichoplusia ni. J. Ultrastructure Res., 35:606-625,
1971.
42. Summers, M.D., and P. Hoops. Radioimmunoassay of Baculovirus Granulins
and Polyhedrins. J. Virol., 1978.
43. Young, S.Y., and J.S. Lovell. Virion Proteins of the Trichoplusia ni
Nuclear Polyhedrosis Virus. J. Invertebr. Pathol., 22:471-472, 1973.
44. Payne, C.C. The Isolation and Characterization of a Virus from
Oryctes rhinoceros. J. Gen. Virol., 25:105-116, 1974.
45. Volkman, L.E., M.D. Summers, and C. Hsieh. Occluded and Nonoccluded
Nuclear Polyhedrosis Virus Grown in Trichoplusia ni: Comparative Neu-
tralization, Comparative Infectivity, and In Vitro Growth Studies. J.
Virol., 19:820-832, 1976.
46. Hirumi, H., K. Hirumi, and A.H. Mclntosh. Morphogenesis of a Nuclear
Polyhedrosis Virus of the Alfalfa Looper in a Continuous Cabbage Looper
Cell Line. Ann. N.Y. Acad. Sci., 266:302-326, 1975.
47. Knudson, D.L., and K.A. Harrap. Replication of a Nuclear Polyhedrosis
Virus in a Continuous Cell Culture of Spodoptera frugiperda: Micro-
scopy Study of the Sequence of Events of the Virus Infection. J.
Virol., 17:254-268, 1976.
48. Stoltz, D., C. Pavan, and A.B. DaCunha. Nuclear Polyhedrosis Virus:
a Possible Example of De Novo Intranuclear Membrane Morphogenesis. J.
Gen. Virol., 19:145-150, 1973.
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49. MacKinnon, E.A., J.F. Henderson, D.B. Stoltz, and P. Faulkner. Morpho-
genesis of a Nuclear Polyhedrosis Virus under Conditions of Prolonged
Passage In Vitro. J. Ultrastructure Res., 49:419-435, 1974.
50. Doughtery, E.D. , J.L. Vaughn, and C.F. Relchelderfer. Characteristics
of the Nonoccluded Form of a Nuclear Polyhedrosis Virus. Intervirology,
5:109-121, 1975.
51. Henderson, J.F. , P. Faulkner, and E.A. MacKinnon. Some Biophysical
Properties of Virus Present in Tissue Cultures Infected with the Nuclear
Polyhedrosis Virus of Trichoplusia ni. J. Gen. Virol., 22:143-146, 1974.
52. Kawarabata, T. Highly Infectious Free Virions in the Hemolymph of the
Silkworm (Bombyx mori) Infected with a Nuclear Polyhedrosis Virus.
J. Invertebr. Pathol., 24:196-200, 1974.
53. Knudson, D.L., and K.A. Harrap. Replication of a Nuclear Polyhedrosis
Virus in a Continuous Cell Culture of Spodoptera frugiperda; Purifica-
tion, Assay of Infectivity, and Growth Characteristics of the Virus.
J. Virol., 14:934-944, 1976.
54. Stairs, G.R., and B.J. Ellis. Electron Microscope and Microfilter
Studies on Infectious Nuclear Polyhedrosis Virus in Galleria mellonella
Larvae. J. Invertebr. Pathol., 17:350-353, 1971.
55. Zherebtsova, E.N., L.I. Strokovskaya and A.P. Gudz-Gorban. Subviral
Infectivity in Nuclear Polyhedrosis of the Great Wax Moth (Galleria
mellonella L.) Acta Virol., 16:427-431, 1972.
56. Summers, M.D. Deoxyribonucleic Acids of Baculoviruses. In: Virology
in Agriculture, Romberger, J.A., ed. Allanheld, Osmun and Co. Pub-
lishers, Inc., 1977. pp. 233-246.
57. Summers, M.D., and D.L. Anderson. Characterization of DNA Isolated
from the Granulosis Viruses of the Cabbage Looper, Trichoplusia ni
and the Fall Armyworm, Spodoptera frugiperda. Virology, 50:459-471,
1972.
58. Summers, M.D., and D.L. Anderson. Granulosis Virus Deoxyribonucleic
Acid: A Closed, Double-stranded Molecule. J. Virol., 9:710-713, 1972.
59. Summers, M.D., and D.L. Anderson. Characterization of Nuclear Polyhe-
drosis Virus DNAs. J. Virol., 12:1336-1346, 1973.
60. Burgess, S. Molecular Weights of Lepidopteran Baculovirus DNAs:
Derivation by Electron Microscopy. J. Gen. Virol., 37:1-10, 1977.
61. Kelly, D.C. The DNA Contained by Nuclear Polyhedrosis Viruses Isolated
from Four Spodoptera sp. (Lepidoptera, Noctuidate): Genome Size and
Homology Assessed by DNA Reassociation Kinetics. Virology, 76:468-
471, 1977.
69
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62. Rohrmann, G.F., J.W. Carnegie, M.E. Martignoni, and G.S. Beaudreau.
Characterization of the Genome of the Nucleopolyhedrosis Bundle Virus
Pathogenic for Orgyia pseudotsugata. Virology, 80:421-425, 1977.
63. Skuratovskaya, I.N., L.I. Strokovskaya, E.N. Zherebtsova, and A.P.
Gudz-Gorban. Supercoiled DNA of Nuclear Polyhedrosis Virus of Calleria
mellonella L. Arch. Virol., 53:79-86, 1977.
64. Scharnhorst, D.W., K.L. Saving, S.B. Vuturo, P.M. Cooke, and R.F. Weaver,
Structural Studies on the Polyhedral Inclusion Bodies, Virions and DNA
of the Nuclear Polyhedrosis Virus of the Cotton Bollworm Heliothis zea.
J. Virol., 21:292-300, 1977.
65. Volkman, L.E., and M.D. Summers. Autographa californica Nuclear Poly-
hedrosis Virus: Comparative Infectivity of the Occluded, Alkali-liber-
ated, and Nonoccluded Forms. J. Invertebr. Pathol., 30:102-103, 1977.
70
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DISCUSSION
SMITH: Have these various viruses been tested for the serological related-
ness of these granulins?
SUMMERS: Yes. I have discussed only the structural relatedness using pep-
tide mapping techniques. Later, DiCapua and Hoops will review the serologi-
cal properties.
SMITH: Are granulins and polyhedrins virus-specified?
SUMMERS: I think you can deduce from the peptide mapping studies that they
are.
SMITH: When a host makes an antibody to the proteins of the virus, is this
a major component of the antibody specificity?
SUMMERS: Against the virus?
SMITH: Yes. Let's say you inject the virus as an antigen into a mouse or
rat.
SUMMERS: It will be more appropriate to discuss this subject later.
RAPP: I have a question about the crystalline polypeptide that you say is
between 25 and 30 thousand. Is that polypeptide also found in the virion
capsid structure? In other words, is it viral-specified?
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SUMMERS: There is a protein in the enveloped nucleocapsid with similar
mobility to that we described as polyhedrin. We have not determined whether
polyhedrin protein is a structural component of the enveloped nucleocapsid.
ULVEDAL: This morning we heard that an insect may harbor several viruses.
Do you think with present techniques we could separate these viruses before
we use them on a field trial?
SUMMERS: Yes. I am confident that there are a few laboratories that have
the ability to evaluate them with respect to their purity before they are
used.
ULVEDAL: How pure? One hundred percent pure? What range of purity are we
talking about?
SUMMERS: With the techniques we employ, as I have presented to you here, we
can identify baculoviruses.
STOLLAR: Is the virus you used to study the DNA and proteins obtained from
whole insects?
SUMMERS: Yes, all the structural protein studies that we have done have been
with occluded viruses purified from infected insects.
HOLOWCZAK: You seem to indicate that if these viruses are going to be use-
ful as pesticides, they have to be presented as occluded particles. Is there
a difference between occluded and nonoccluded viruses in terms of survival?
SUMMERS: The nonoccluded virus does not survive well.
HOLOWCZAK: Purification would be a difficulty if more than one kind of
virus could be occluded probably impossible.
SUMMERS: Especially if there were mixed populations or strains.
HOLOWCZAK: Have you infected subjects with two related viruses?
SUMMERS: I have decided not to do this until our detection technology is
capable of differentiating between the two with techniques to screen for
mixed populations.
72
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HOLOWCZAK: Are all these pathways of morphogenesis due to one kind of virus?
SUMMERS: The biological behavior of a baculovirus in vivo and in vitro is
complex. Observations on the structure and biology of baculovirus infection
suggest we are working with mixed populations of viruses. At the moment,
we do not have sufficient data to answer your question.
RAPP: When you perform a bioassay, you release occluded virus and NOVs
IB. v:i-vo' Do yฐu do that by injection or per os?
SUMMERS: By per os injection and hemocoelic injection. We compared both
per os. We did not put the virus on any diet; we injected it directly into
the gut.
RAPP: Since identification of viruses is going to be critical, especially
after field trials, you gave us your favorite scheme for identifying viruses,
by their polypeptides. Does DNA hybridization technology correlate with
your method of identification?
SUMMERS: I did not give you my favorite scheme for identification. I gave
a "most difficult" scheme for identification. I discussed an idealistic
approach to evaluate whether serological techniques can be specifically and
effectively used with a few highly purified and characterized proteins. We
want to test how good our serology can be. Certain DNA studies can be very
definitive and can get very dependable results, but very few people are doing
restriction-enzyme fragmentation physical mapping. 1 do not believe we
have enough data accumulated to begin to make informed statements.
RAPP: I know there is cross hybridization between baculovirus DNA, and you
demonstrated similarities among polyhedrins. What extent of cross hybridi-
zation is mirrored by similarities in your tests?
SUMMERS: We are not presently making those kinds of comparisons. We are
concentrating on proteins. Perhaps Dr. Harrap can reply to your question.
HARRAP: The extent of cross hybridization we see among Spodoptera baculo-
virus system is mirrored by the sort of serological relatedness among the
various isolates we have used.
73
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Identification of Nonoccluded Viruses
of Invertebrates
John F. Longworth, Ph.D., and Paul D. Scotti, Ph.D.
DSIR, Entomology Division
Auckland, New Zealand
The term "nonoccluded" is still used to describe those viruses other
than NPV, GV, CPV, and entomopoxviruses. The latter viruses become occluded
within a proteinaceous crystal at the end of the infection process. The
term "nonoccluded" therefore is of little definitive value since it includes
such distinct groups as:
iridoviruses,
parvoviruses,
small isometric RNA viruses.
Its use should be discouraged, in favor of using appropriate group names.
The comparative virology of iridoviruses, parvoviruses, and small RNA
viruses has been discussed in recent reviews (1-4) and the small RNA viruses
affecting bees have been separately reviewed (5).
THE IRIDOVIRUSES
The iridoviruses, together with African swine fever virus, frog virus
3, lymphocystis virus, etc., comprise the icosahedral cytoplasmic DNA virus
group. The iridoviruses are commonly associated with invertebrates in an
aquatic or a soil environment. The group includes isolates affecting blue-
green algae, fungi, and fresh water crustacea. They are large particles,
75
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up to 180 run in diameter, containing linear, double-stranded DNA of greater
than 100 x 10 mol. wt. Over 20 isolates are known from insects and while
many have a wide host range within the class Insecta, none are known to
infect vertebrates or vertebrate cell lines except that it has been reported
that Chilo iridescent virus (CIV) multiplies in cultured viper spleen
cells (6). Within field populations of an insect species, infections tend
to develop slowly and insect to insect transfer most commonly results from
cannibalism; large doses of virus are necessary for successful per os in-
fection. Epizootics, leading to marked reduction in insect populations,
are rarely observed with iridoviruses and they are not regarded as suitable
candidates for applied control.
Several laboratories in the United Kingdom, United States, New Zealand,
and Europe have been or are involved with biochemical and serological char-
acterization of these viruses. Their polypeptide, lipid, and nucleic acid
compositions are fairly well understood, and studies of the biochemical
events in virus replication have also been conducted for some iridoviruses
(7).
THE PARVOVIRUSES
Only two invertebrate parvoviruses have been adequately described.
These are closely related serologically, their polypeptide composition is
identical (2) but there are marked differences in host range. Unlike verte-
brate parvoviruses which have three major polypeptides, the invertebrate
isolates have four. The linear, single-stranded DNA occurs as 4- and - forms
in separate particles (8). Two principal investigators are working with
these viruses, E. Kurstak at Montreal, and B.C. Kelly at Oxford.
Purified densonucleosis virus, originally isolated from the greater
wax moth, Galleria mellonella, replicated in mouse L cells. Some mouse L
cells produced DNV virions, although in lower quantities than can be obtained
from insects. The L cell-derived virions caused an infection in _G. mellon-
ella larvae typical of DNV (9). The majority of the cells, however, were
apparently transformed by the Galleria virus, and viral antigens were de-
tected by immunofluorescence. Infective virus could not be recovered from
these cells.
76
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SMALL ISOMETRIC RNA VIRUSES
Of most interest to this meeting, however, is the rapidly expanding
group of known small RNA viruses affecting invertebrates. At the First
International Colloquium for Invertebrate Pathology held in September 1976,
we listed the properties of nearly 30 such viruses (4) and commented that
apart from one or two very obvious similarities between some viruses it was
premature to attempt a definitive subgrouping. In the light of new compara-
tive information, particularly that obtained by C. Reinganum when working
at T.W. Tinsley's laboratory, at least four logical groupings are now possi-
ble. Other individual viruses are clearly distinct, one from another, and
are probably the first representatives to be described of other groups.
The groupings in Table 1 will be described more fully in a forthcoming paper.
The first group includes Nodamura virus, isolated from mosquitoes in
Japan, and Heteronychus arator virus from the New Zealand black beetle.
These viruses have closely similar biochemical and biophysical properties
(10). In particular, both have a divided, single-stranded RNA genome of
1.0 and 0.5 x 10 mol. wt.; with Nodamura virus, both RNA species are neces-
sary for infection. At present, it is not clear whether the RNA species
are separately encapsidated; if they are, a quantitative assay would exhibit
a two-hit dose-response curve. Both isolates have a wide host range within
insects, but Nodamura virus will also infect cultured vertebrate cells and
mice. Indeed, this is the only authenticated instance of replication of an
insect pathogenic virus in vertebrates. Because of the divided genome,
there are very real possibilities of genetic reassortment, not only in con-
trived laboratory situations, but also in the field, if two such viruses
were to infect the same host. Nodamura virus replicates without CPE in
Aedes aegypti cells and _A. albopictus cells and can be titrated in mice.
The H.. arator virus will replicate in Drosophila cells and causes a cyto-
pathic effect (CPE) after 9 or 10 days, and a titration system is being
developed. The 11. arator virus does not replicate in A. albopictus cells,
and does not appear to be infective for BHK cells or mouse L cells.
The cricket paralysis virus (CrPV) group includes two serologically
related isolates, Drosophila C virus and cricket paralysis virus. The Dro-
sophila C virus is widely distributed in France, Europe, and the Mediterranean
region, and it most commonly occurs as an inapparent infection. Cricket
paralysis virus was first isolated from Teleogryllus commodus in Australia
and is serologically related to Drosophila C virus. There are, however, dis-
tinct host range differences between the two isolates (N. Plus, personal
77
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communication). Each virus appears to replicate best in its specific host
and only CrPV multiplies in the cricket Gryllus bimaculatus (N. Plus, per-
sonal communication). CrPV is normally present in T[. commodus as an inap-
parent infection and has also been isolated from other cricket species, as
well as from Heteroptera and Lepidoptera in several countries. A tissue
culture assay method for CrPV is now available (11) and, using a 50% tissue
culture infective dose (TCID,^) assay method, we have begun a survey of the
incidence and development of CrPV in cricket populations in New Zealand.
Additionally, we will be examining the incidence of this virus in other in-
vertebrates in the same area.
Cricket paralysis virus will replicate in Drosophila cell lines 1 and
2, and in Aedes aegypti and A., albopictus cells. It also replicates in
cultured cells of Porthetria dispar (N. Plus, personal communication). We
have examined the replication of cricket paralysis virus in Drosophila cells;
the viral RNA apparently directs the synthesis of large precursor polypep-
tides which are then cleaved into smaller stable proteins. The largest
species detected in pulse experiments was around 120,000 daltons, and this
peak disappeared during the subsequent chase. The overall pattern of poly-
peptide synthesis directed by CrPV RNA closely resembles that of poliovirus.
It seems logical, therefore, to consider members of the cricket paralysis
virus group as true picornaviruses.
The Gonometa virus group includes three serologically related viruses
(Plus, Reinganum, personal communication): one from bees, one from flies,
one from mothsfrom three different orders of insects in fact. Size, den-
sity, sedimentation coefficient and, for the Gonometa isolate, polypeptide
composition, are all similar to the vertebrate enterovirus group. The Gono-
meta isolate (from East Africa) is one of the few invertebrate small RNA
viruses to have been used in insect control with success and without ob-
vious effects on vertebrates. Longworth et al. (12) demonstrated that IgM
antibodies to Gonometa virus were present in a range of domestic and wild
animals in the United Kingdom and postulated repeated exposure to low
dosages of a virus at that time undescribed in the United Kingdom. The
more recent discovery of viruses related to the Gonometa isolate in bees
and Drosophila flies in Europe lends further support to this hypothesis.
No IgG antibodies were detected in the test animals which reacted with Gono-
meta virus and the route of exposure to the virus remains open to conjecture.
The Nudaurelia 6 virus group includes serologically related isolates
from a range of countries affecting Saturniid and Limacodid moths. These
viruses are 35 nm in diameter, 1.30 density, 210 S, and have one major
79
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polypeptide. The RNA content (11.0%) is low by comparison with most other
small RNA viruses, and the capsid exhibits T, symmetry (13); the 240 pro-
tein subunits are arranged in trimers with four trimers per icosahedral
face. Nudaurelia 6 virus will replicate in Nudaurelia and Bombyx mori
cell cultures (14).
Ecology of Small Isometric RNA Viruses
The presence of antibodies in vertebrates to invertebrate pathogenic
viruses needs careful study. The work with Gonometa virus first drew
attention to the problem, but has not proceeded further than defining the
antibody class involved. Lack of virus hindered this work, but we know that
similar studies are in progress at Oxford with other small RNA viruses of
invertebrates. The phenomenon is not an isolated one, for in the same study,
antibodies were also demonstrated in United Kingdom vertebrates to an RNA
virus from the Nudaurelia 6 group.
In New Zealand, we have examined cattle sera from the site where the
RNA virus of II. arator was isolated and failed to find antibodies to that
virus using the immunodiffusion technique. However, low levels of precipi-
tating and neutralizing antibodies to CrPV were detected, and this virus
had not been recorded in New Zealand at that time. Subsequently, with tissue
culture assay methods, we have detected cricket paralysis virus in field
crickets from the same site. There may be a purely casual association
between the virus present in crickets and antibodies in serum from cattle
in the same area, as the cattle must inhale or ingest insect debris fre-
quently, but we plan to explore this further, and to determine the nature of
the neutralizing antibodies.
Nodamura virus has only been isolated in the field from mosquitoes, but
high levels of neutralizing antibodies were found in pigs and, to a much
lower extent, in herons. The infections of the mosquitoes were inapparent,
but the virus can cause fatal paralysis in bees, ticks, moth larvae, and
mice and is transmissible to mice by infected Aedes aegypti. It replicates
in cultured vertebrate and invertebrate cells without CPE (15).
Thus, there is some evidence that there may be an association between
some insect small RNA viruses and vertebrates, and this warrants closer
study. There is direct evidence that at least one virus the Nodamura
isolate is pathogenic to vertebrates. It is possible that genetic reassort-
ment could occur between divided genome viruses of this type, if they in-
80 l
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feet the same host. With this in mind it would seem unnecessary to point
out the potential hazards of deliberately using such viruses in a spray
program to control insects, yet this has happened against Gonometa in East
Africa and against Darna trima in Borneo and Sabah. No ill effects have
been reported, but then no survey of other invertebrates and vertebrates in
the environment was made.
Small Isometric Viruses as Possible Contaminants in Baculovirus Preparations
It is appropriate to consider the possible contamination of baculovirus
preparations with a small isometric virus. It has been shown recently (16)
that at least one such virus will coinfect Autographa californica together
with its specific baculovirus. In fact, one 40 nm virus replicated in nuclei
in A. californica larvae or in Trichoplusia ni cells equally well whether
or not the baculovirus was replicating in the same nucleus. This baculo-
virus of course is currently a candidate for development for insect control.
In Heliothis zea, a parvovirus and a picornavirus have only recently been
isolated from laboratory cultures in the United Kingdom (Tinsley, personal
communication); the parvovirus is serologically related to Galleria parvo-
virus, and the picornavirus is related to cricket paralysis virus.
Small isometric RNA viruses are more common in insects than has been
supposed, and may pose real problems in baculovirus preparations. However,
where the properties of the likely contaminant are known, it is possible to
devise rapid and sensitive assays to monitor for its presence, and, if neces-
sary, it is not difficult to eliminate such contamination from baculovirus
preparations by relatively simple purification procedures.
It is, of course, desirable to propagate the candidate virus in a
disease-free laboratory colony of the specific host, but it is pertinent
to stress here that while most of the invertebrate picorna-like viruses
can kill their hosts, many of them occur in nature as inapparent infections.
Nodamura virus does not kill its Culex mosquito host (17); Kelp-fly virus
(18) was only isolated by passaging through moth larvae, extracts of appar-
ently healthy Kelp-flies which did not contain detectable viruses. Many bee
viruses only kill their host when injected in high concentration (5). We
have detected an average of less than 100 infectious particles of CrPV
per cricket from field samplesa level which would defy detection by stan-
dard serological techniques. One cannot assume that absence of mortality in
a laboratory insect colony necessarily means absence of a small RNA virus.
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It is practicable to reduce the level of a contaminating small isome-
tric RNA virus drastically during purification of baculovirus polyhedra;
the S?~ of polyhedra is very high, and they rapidly reach their isopycnic
point at around 54% w/w sucrose. Though the RNA virus has a higher density,
its Sป0 is so low that it is well separated on the gradient. We have per-
formed experiments to show that contamination by CrPV can be drastically
reduced in a baculovirus preparation by simple extraction procedures and
reduced below the level of detection of our tissue culture assay by one
cycle of zonal centrifugation (19).
Future Research Needs with Small Isometric Viruses of Invertebrates
1. The small isometric viruses which have been described should
be fully characterized as a basis for reliable comparative
studies. This applies particularly to the small RNA viruses.
2. The in vitro and in vivo host range of small isometric viruses
should be established to allow an assessment of the likelihood
of hazard not only to vertebrates, but to beneficial inverte-
brates .
3. The significance of antibody responses in domestic and wild
animals to small RNA viruses of invertebrates needs to be
determined. Do all the instances involve IgM only, and
does this imply limited casual exposure to antigen, rather
than infection?
4. Can mosquitoes acquire infective doses of invertebrate
small isometric viruses and transmit these to vertebrates?
5. Can the divided genome RNA viruses exchange genetic infor-
mation? If so, how common are these viruses; do they
present a hazard?
6. We need quantitative assays, particularly for small
isometric viruses which do not cause CPE.
7. Does the possible presence of small isometric viruses in
commercial baculovirus preparations constitute a hazard which
merits including additional safeguards in the production
process? There appear to be three alternatives:
a. Assuming that present bioassay checks in the
development and production process, including batch
tests, give sufficient protection already.
b. Adding specific tests such as radioimmunoassay or
tissue culture assays for viruses which have been
isolated previously from the candidate insect.
- J
82
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c. Instituting simple purification procedures in the
production process which would exclude possible
contaminant viruses.
Finally, it should be pointed out that there are less than ten people
who are engaged in research on small isometric viruses of invertebrates on
a full-time basis; these workers are all in the United Kingdom, Canada,
France, Australia, and New Zealand.
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REFERENCES
1. Kelly, B.C., and J.S. Robertson. Icosahedral Cytoplasmic Deoxyribo-
viruses. J. Gen. Virol., 20:17-41, 1973.
2. Tinsley, T.W., and J.F. Longworth. Parvoviruses. J. Gen. Virol.,
20:71-75, 1973.
3. Brown, F., and R. Hull. Comparative Virology of the Small RNA Viruses.
J. Gen. Virol., 20:43-60, 1973.
4. Longworth, J.F., and P.D. Scotti. Properties and Comparative Aspects
of Small Isometric Viruses of Invertebrates. In: Proceedings of the
First International Colloquium on Invertebrate Pathology, Queens Uni-
versity, Kingston, Canada, 1976. pp. 30-35.
5. Bailey, L. Viruses Attacking the Honeybee. Adv. Virus Res., 20:271-
304, 1976.
6. Mclntosh, A.H., and M. Kimura. Replication of the Insect Chilo Iri-
descent Virus (CIV) in a Poikilothermic Vertebrate Cell Line. Inter-
virology, 4:257-267, 1974.
7. Kelly, D.C., and T.W. Tinsley. Iridescent Virus Replication: Patterns
of Nucleic Acid Synthesis in Insect Cells Infected with Iridescent
Virus Types 2 and 6. J. Invertebr. Pathol., 24:169-178, 1974.
8. Kurstak, E., J-P. Vernoux, A. Niveleau, and P.A. Onji. Visualisation
du DNA du Virus de la Densonucleose (VDN) a Chaines Monocatenaires
Complementaires de Polarites Inverses Plus et Moins. C.R. Acad. Sci.,
Ser. 0272:762-765, 1971.
9. Kurstak, E., S. Belloncik, and C. Brailovsky. Transmission de Cellules
L de Souris par un Virus d'Invertebres: Le Virus de la Densonucleose
(VDN). C.R. Acad. Sci., Ser. 0.8:1716-1719, 1969.
10. Longworth, J.F., and G.P. Carey. A Small RNA Virus with a Divided
Genome from Heteronychus arator (F.) [Coleoptera:Scarabaeidae]. J.
Gen. Virol., 33:31-40, 1976.
11. Scotti, P.D. End-point Dilution and Plaque Assay Methods for Titration
of Cricket Paralysis Virus in Cultured Drosophila Cells. J. Gen. Virol.,
(in press).
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12. Longworth, J.F., J.S. Robertson, T.W. Tinsley, D.J. Rowlands, and F.
Brown. Reactions Between an Insect Picornavirus and Naturally Occur-
ring IgM Antibodies in Several Mammalian Species. Nature (London),
242:314-316, 1973.
13. Finch, J.T., R.A. Crowther, D.A. Hendry, and J.K. Struthers. The
Structure of Nudaurelia capensis 6 Virus: the First Example of a
Capsid with Icosahedral Surface Symmetry T=4. J. Gen. Virol., 24:191-
200, 1974.
14. Tripconey, D. Studies on a Nonoccluded Virus of the Pine Tree Emperor
Moth. J. Invert. Pathol., 15:268-275, 1970.
15. Bailey, L., J.F.E. Newman, and J.S. Porterfield. The Multiplication of
Nodamura Virus in Insect and Mammalian Cell Cultures. J. Gen. Virol.,
26:15-20, 1975.
16. Hess, R., M.D. Summers, L.A. Falcon, and D.B. Stoltz. Characteristics
of a Virus Like Icosahedral Particle in Mixed Infections with Autographa
californica Nuclear Polyhedrosis Virus, 1976.
17. Bailey, L. and H.A. Scott. The Pathogenicity of Nodamura Virus for
Insects. Nature, 241:545, 1973.
18. Scotti, P.O., A.J. Gibbs, and N.G. Wrigley. Kelp-fly Virus. J. Gen.
Virol., 30:1-9, 1976.
19. Longworth, J.F., and P.D. Scotti. J. Invertebr. Pathol., 1977.
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DISCUSSION
LONGWORTH: Do you have additional information on the 40 ran isometric virus-
like particle observed in a mixed infection with Autographa NPV?
SUMMERS: We know it has one structural polypeptide, but the density of
1.24 gm/cc is a mystery to us. We have not yet confirmed whether it is a
DNA or RNA virus.
STOLLAR: These small RNA viruses look like agents with which care will have
to be exercised if they are to be used as contaminants in any viral pesti-
cides. Does Nodamura kill suckling mice?
LONGWORTH: Perhaps Dr. Shop'e can answer this question.
SHOPE: Nodamura virus kills baby mice by intracerebral and intraperitoneal
inoculation, but it would not be transmissible to adult mice, or at least
it does not make them very sick. I am not certain whether antibody studies
have been conducted using adult mice.
STOLLAR: What did you say about replication in vertebrate cells in culture?
LONGWORTH: There was no CPE in invertebrate or vertebrate cells Jji vitro.
STOLLAR: There are different size RNAs. Is there any suggestion of dif-
ferent size particles?
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LONGWORTH: No, the size is homogenous in all respects.
SMITH: I am encouraged by your approach of looking at those animals in the
field that are probably most directly exposed to these agents. Have insect-
ivorous species such as bats been monitored at all? Have you found evi-
dence of replication of these viruses?
LONGWORTH: As far as I know there is no evidence of replication at all.
Using a radioimmunoassay, we looked for antibodies to New Zealand NPV in
birds that prey on caterpillars. The bird feces contained 18 percent by
weight of inclusion bodies, having gone through unaffected. Those 26 birds
have no antibodies even though it was a major epizootic.
SUMMERS: What if you used a nonoccluded virus?
LONGWORTH: This work was not done. The sera are available, as I understand
it. Kalmakoff, who is the principal investigator, is continuing the work.
HARRAP: Quite a bit of work is being done at Oxford on the antigen-antibody
reactions with two of these viruses, Nudaurelia 6 and Darna trima, but it
has not really appeared in a published form. I will summarize using Darna
trima virus (34 nm in size) and the Nudaurelia 6 virus. We had reactions to
one or the other, and sometimes to both sheep, cats, pigs, humans, and
with some African animals, for example, lions, wart hogs, water buffalo, and
two zebras. All Antarctic sera that we tested from the British Antarctic
survey have been negative. We are now looking at birds, whose sera we have
not yet examined. The aim of the project is not to screen vast numbers of
sera, but to identify for certain what the antibody type is or, more import-
antly, try to identify the agent in the animal that elicits the antibody
response that will react with our insect virus. We have detected particles
in sera by electron microscopy, especially with sera from a particular flock
of sheep in Oxfordshire. The particles are the right size for picorna
viruses. We are hopeful that we can make isolations of virus from the sera.
The antibody level seems to increase and the particles disappear in indivi-
dual sheep as we bleed them over a period of time. So, it is a matter of
choosing the right stage for attempts at virus isolation.
PAGANO: After the Nodamura virus replicates in suckling mice, has it been
repassed in mice? Does it gain virulence?
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LONGWORTH: I cannot recall whether this has been done. I can refer you to
the publications of Newman or Brown.
SHOPE: I worked with Nodamura virus in 1959 and we passaged it in baby mice,
and it maintained virulence on passage, 10 or 12 passages. As far as I
know, it did not gain virulence, as injected by the intraperitoneal route.
It did not kill adult mice because we immunized our mice with high passage
level virus, nor did it kill adult mice by intracerebral inoculation.
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Biology of Cytoplasmic Polyhedrosis Viruses and
Entomopoxviruses
Robert R. Granados, Ph.D.
Boyce Thompson Institute
Cornell University
Ithaca, New York
Although the major attention during this symposium will be focused on
the baculoviruses, it was felt that some time should be devoted to the
discussion of cytoplasmic polyhedrosis viruses (CPVs) and entomopoxviruses
(EPVs). Indeed, there is not only interest in some countries in developing
these viruses as viral pesticides, but at least one CPV has been registered
in Japan for use against a forest insect pest.
Unlike baculoviruses, CPVs and EPVs should be more familiar to most
virologists since these viruses have many properties in common with plant
and/or vertebrate viruses. My discussion will be limited to presenting a
brief overview of the current knowledge of these two insect virus groups.
For the purpose of this discussion, I will use the term "occlusion
body" (OB) to designate the proteinaceous, virus-occluding structures com-
monly referred to as polyhedral inclusion bodies, polyhedra, inclusion
bodies, spheroids, virus-containing inclusions, granules, capsules, and
so on. The term "OB" was coined by Dr. Brian Federici (1) a few years ago
in order to avoid confusion with the term "inclusion body" which is used by
vertebrate virologists to denote a very different structure.
The international committee on taxonomy of viruses recently recommended
that a genus comprising CPVs be established within the family Reoviridae
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(2). Viruses in this family possess genomes consisting of several (10-12)
segments of double-stranded (ds) RNA with molecular weights ranging from
0.3-3 x 10 , all ds-RNA pieces are encapsidated within a single virus particle,
and the virion has an isometric capsid (double protein shell) with icosahe-
dral symmetry, 60-80 nm in diameter (Table 1). CPVs differ from other mem-
bers of the Reoviridae family in that arthropods (primarily insects) are the
only known hosts, and virions may be occluded in proteinaceous OBs. Recently,
a CPV was found in a freshwater daphnid (Crustacea) (3).
TABLE 1. CYTOPLASMIC POLYHEDROSIS VIRUS
(FAMILY REOVIRIDAE)
Cryptogram:
[R/2 : 13- 18/25-30 : So/S : I/O]
Main characteristics:
1. Genome has about 10 pieces of double-stranded RNA.
2. Total genome molecular weight of 13-18 x 10 daltons.
3. Virions are 50-60 nm in diameter with projections
at vertices of icosahedron. Two-layered capsids.
4. RNA-dependent RNA polymerase present in virion.
5. Particles may be occluded in crystalline protein
occlusion bodies.
6. Initially they infect only the cells of the midgut
but may spread to other tissues.
7. Host range: Lepidoptera, Diptera, Hymenoptera,
Neuroptera, and Crustacea.
The CPV of the silkworm, Bombyx mori, is the most thoroughly studied
virus of this group. The particles consist of two concentric icosahedral
capsids. The outer capsid has a diameter of 65 nm and the inner shell a
diameter of approximately 45 nm. The outer shell of the virion consists of
12 pentagonal capsomeres, localized at the vertices of the icosahedron.
The capsomeres are in the shape of hollow pentagonal disks with an outer
diameter of 20 nm and inner diameter of 5 nm. Each capsomere has a hollow
segmented projection. At the end of these projections, structures measuring
12 nm in diameter have been observed. Tubular structures connect the outer
capsid shell to the inner shell at the 12 vertices.
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The replication cycle of CPVs has not been critically examined in syn-
chronously infected cells. Following larval ingestion of OBs, these cry-
stals are dissolved by the gut juices in the lumen, thereby releasing
numerous virus particles. Kobayashi (4), in a study designed to determine
the mechanism of _B. mori CPV penetration into host intestinal cells main-
tained in vitro, observed that the first stage of entry was the attachment
of viral projections to the cell surface. Within 10 minutes postinoculation,
the attached virions released viral core material into the cells. This was
suggested by the presence of empty capsid shells on the cell surface and by
the presence of dense material in the cytoplasm directly beneath empty par-
ticles. Kobayashi concluded that the viral core substance was released as
a filament and injected in much the same manner as a bacteriophage. Phago-
cytosis did not appear to play a part in virus penetration. Several inves-
tigations have indicated that after internalization of the viral genome,
transcription of the parental RNA occurs in the nucleus. Virus transcrip-
tion in the nucleus is subsequently followed by protein and ds-RNA synthesis,
virus assembly, and occlusion in the cytoplasm.
Nonoccluded virions may represent more than 70% of the total number of
virus particles in an infected insect (5). Preliminary studies (6) suggest
that the biochemical properties of occluded and nonoccluded JB. mori CPV
particles are similar. B. mori particles extracted from OBs (occluded vir-
2
ions) have a density of 1.43-1.48 g/cm in CsCl and contain five structural
polypeptides, as determined by polyacrylamide gel electrophoresis. Non-
occluded virus particles have the same RNA components, structural proteins,
and density as occluded virions. It has not been determined if the biologi-
cal properties of these two "forms" of CPV are similar. Insofar as the OB
is concerned, _B. mori CPV has a major polypeptide with a molecular weight
of 27,100 daltons and contains a covalently attached carbohydrate moiety.
Electrophoretic analysis of the proteins of virus particles and OBs
has been shown to be a useful tool in differentiating between CPVs. How-
ever, a more powerful tool for the identification of CPVs is an analysis of
the number and/or size of the RNA genome segments following fractionation
on 3% polyacrylamide gels (7). For example, Payne (7) reported that when
the ds-RNAs of 33 CPV isolates were examined by gel electrophoresis, some
viruses had identical profiles, and he was able to group 29 of the isolates
into 11 virus "types" which were identified by major differences in the
mobilities of the RNA segments. Four of the CPV isolates contained more
than 10 RNA segments, and these were interpreted as mixtures of two viruses.
Gel analysis of viral RNAs can be made without difficulty and from impure
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samples if necessary (7). It would appear that adequate tools for the identi-
fication of CPVs are currently available.
Most of the research on CPVs has been conducted with virus produced in
living insects. Only in the last 2 years has there been some effort in
developing tissue culture techniques (8, 9). In our laboratory, we have been
able to infect four insect cell lines with CPV the fall armyworm, Spodoptera
frugiperda; the cabbage looper, Trichoplusia ni; the salt marsh caterpillar,
Estigmene acrea; and the gypsy moth, Porthetria dispar. An immunofluorescence
assay for CPV in cell cultures was developed, and the ultrastructure of the
replication cycle in vitro was studied (8). However, much more work is still
needed in this area before the routine use of insect cell cultures in CPV
research can be realized.
In vivo specificity studies have shown that CPVs have a wider host range
than EPVs or baculoviruses. Approximately 175 insect species (mainly Lepidop-
tera) are known to have a CPV infection (10), and recently a CPV-type virus
was reported in the freshwater daphnid, Dimocephalus expinosus (3). This
crustacean was also infected with an iridovirus-type virus. At present, at
least three types of viruses commonly found in insects (two baculoviruses, one
CPV, and one iridovirus) have now been reported in the Crustacea. These recent
findings reemphasize the need for more research directed toward determining
the kinds of viruses which occur in Crustacea and other invertebrates.
There is little information on the effect of CPVs on other invertebrates
or vertebrates. No apparent infection resulted following inoculation of tur-
key embryos (11) or four mammalian cell lines (14-HeLa, human amnion cells,
porcine kidney, and mouse sarcoma) with ]3. mori CPV. In our laboratory, we
were unable to infect (based on CPE using light and electron microscopy) four
mosquito cell lines and two vertebrate cell lines (HeLa and L-929 mouse fibro-
blast) with a CPV from T_. ni^ (8).
The potential of CPVs as viral pesticides is good. There are several
CPVs that have given reasonably good control of their hosts (Table 2). At
least one has been registered for use against the pine caterpillar (Dendrolimus
spectabilis) and is commercially produced under the trade name Matsukemin (12).
The safety of this CPV was tested using mice, rabbits, chick embryos, hamsters,
and fish. No pathogenicity was observed in any test (13). A major obstacle
in the development of CPVs as viral insecticides is their taxonomic relatedness
to Reovirus and other vertebrate viruses. If safety testing continues to yield
negative results (i.e., no adverse effects), there may very well be a renewed
interest in the study of these viruses.
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TABLE 2. SOME CYTOPLASMIC POLYHEDROSIS VIRUSES (CPVs)
WHICH HAVE BEEN TESTED AS BIOLOGICAL CONTROL AGENTS
Virus Target insect Country
Lymantria CPV Lymantria fumida Japan
Trichoplusia CPV Trichoplusia ni^ United States
Thaumetopoea CPV Thaumetopoea pityocampa France
Dendrolimus CPV* Dendrolimus spectabilis Japan
Lymantria CPV Lymantria dispar Japan
*Registered in April 1974; commercial name: Matsukemin
I would now like to turn my attention to a discussion of entomopox-
viruses. These viruses are members of the genus Entomopoxvirus (EPV) in
the family Poxviridae (2). Viruses in this family are large, brick-shaped,
or ovoid virions 250 x 350 nm in dimension (Table 3). The virions have a
lipoprotein envelope with tubular or globular protein structures on the
outer surface, enclosing one or two lateral bodies and a core, containing
the genome. The genome consists of a single molecule of double-stranded
DNA of molecular weight 130-240 x 10 daltons. Virions contain more than
30 structural proteins and several viral enzymes. Insects are the only
known hosts of insect poxviruses although some vertebrate poxviruses are
known to be vectored by insects in a nonpersistent manner. Four orders of
insects, Lepidoptera, Coleoptera, Diptera, and Orthoptera, are affected,
with most viruses isolated from Lepidoptera and Coleoptera.
Unlike other members of the Poxviridae, EPVs can be subdivided into
three subgenera based on the morphology of the virion (14) (see Figure 1).
Subgroup 1 (= subgenus C) has brick-shaped virions with two lateral bodies
and a biconcave core. These viruses affect species in the order Diptera
(e.g., mosquitoes and chironomids), and morphologically the virions are
very similar to vertebrate viruses. Subgroup 2 (= subgenus A) has ovoid
virions with one lateral body and a unilateral concave core. These viruses
affect the coleopteran (beetles) species. Subgroup 3 (= subgenus B) has
ovoid virions with a sleeve-shaped lateral body and cylindrical core.
Lepidopterans (moths) and orthopterans (grasshoppers) are hosts for this
virus subgroup.
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VACCINIA VIRUS
SUBGROUP I
DIPTERA
TRANSVERSE
SUBGROUP 2
COLEOPTERA
SUBGROUP 3
LEPIDOPTERA
ORTHOPTERA
Figure 1. Diagrammatic representation of 3 entomopoxviruses and vaccinia
virus in 3 planes of symmetry.
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TABLE 3. INSECT POXVIRUS SUBGROUP (FAMILY POXVIRIDAE)
Approved name:
Entomopoxvirus
Cryptogram:
[D/2 : 140-240/5-6 : Xo/* : I/O]
Main characteristics:
1. Genome consists of a single molecule of double-stranded
DNA. Molecular weight 140-240 x 106 daltons.
2. Brick-shaped or ovoid virion, 170-250 x 300-450 nm.
3. Virions contain at least four enzymes found in
vertebrate poxviruses.
4. Particles may be occluded in crystalline protein
occlusion bodies.
5. Three subgenera based on morphology of virions.
6. Host range: Lepidoptera, Coleoptera, Diptera,
and Orthoptera.
During the course of viral replication, many EPVs of all three sub-
groups produce OBs as well as antigenically distinct spindle-shaped bodies.
These "spindles" are always virus-free and are proteinaceous in nature.
A somewhat analogous situation occurs with some, but not all, vertebrate
poxviruses. These poxviruses (e.g., strains of cowpox) produce A-type
inclusions that are distinctly different from the so-called B-type inclu-
sions (viroplasms), the site of viral multiplication. Depending on the
virus type and strain, these A-type inclusions have distinctly different pro-
perties and may either contain virions or be completely free of them.
The replication cycle of at least two EPVs (Melolontha melolontha and
Amsacta moorei) has been carefully studied in living insects, and A. moorei
EPV is currently being studied in detail in BTI-EAA cell cultures. The
entry of an EPV (_A. moorei) into a host insect has been studied in_ vivo and
in vitro (15, 16). After ingestion of OBs, the gut juices dissolve the
protein crystal, thereby releasing virion into the lumen of the midintestine
(15). Within 2 hours postinoculation, fusion of the viral envelope and
microvillus membrane occurs and virus cores plus lateral bodies enter into
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the microvilli. The viral cores then descend down the microvilli, enter
the cell cytoplasm, and initiate an infection. Under cell culture condi-
tions, virus uptake occurs by viropexis (phagocytosis). Virus synthesis
and assembly occur in the cytoplasm in viroplasmic areas (or B-type inclu-
sions). EPV undergo a maturation process from immature forms to mature
forms, and these virions may be released from infected cells or occluded
within OBs. Except for minor details, the replicative cycle of EPVs and
vertebrate poxviruses is similar. This was further confirmed by recent
studies which showed that rifampicin (an inhibitor of vertebrate poxvirus
multiplication) was also effective against _A. moorei EPV in cell cultures
(17). In cultures treated with 100 pg/ml of rifampicin, virion formation
was blocked, and viral membranes with irregular contours accumulated around
"viroplasmic areas." This inhibition was reversible.
EPVs and vertebrate poxviruses have numerous properties in common
(Table 4). Some notable differences include the following:
1. Host range.
2. Length of the replication cycle is much longer for EPVs.
3. Insect viruses do not appear to reactivate as do vertebrate
viruses (D. Roberts, personal communication).
4. The DNA G-HC ratios of EPVs appear to be 30 to 40% lower
than G+C values for vaccinia DNA (18).
Recent studies (19) have been aimed at characterizing and identifying
several EPVs from all three subgroups. The molecular weight of DNA from
five EPVs was determined by electron microscopy and compared with vertebrate
virus DNA (vaccinia). Lepidopteran EPV-DNA (135 x 10 ) was approximately
equal to vaccinia DNA (132 x 10 ) in molecular weight. The molecular weight
of dipteran and coleopteran EPV-DNA (200 x 10 ) was approximately 50%
greater than vaccinia DNA. The orthopteran EPV-DNA (123 x 10 ) molecular
weight was less but similar to the lepidopteran viruses. The dipteran
and coleopteran EPV genomes were similar to those reported for fowlpox DNA.
Therefore, the two DNA size classes found in EPVs are also found in verte-
brate poxviruses.
Langridge (personal communication) has also examined the structural
polypeptides of virus particles of two lepidopteran EPVs, one orthopteran
EPV, and vaccinia, by SDS acrylamide gel electrophoresis. Each EPV contained
approximately 30 peptides and differences in peptide distribution in the
gels (based primarily on molecular weight) indicated that EPVs are consi-
derably different from each other and vaccinia.
96
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TABLE 4. PROPERTIES OF INVERTEBRATE AND VERTEBRATE POXVIRUSES
Property
Host
Invertebrate
Vertebrate
Size (nm) 350 x 250
Host Range Insects
Replication Cycle (hrs) 18-24
Buoyant Density 1.262
(Virus, gm/cc)
Inclusions (= occlusion bodies) +
RNA Polymerase 2
DNAse 2
Nucleotide Phosphohydrolase 1
Nucleic Acid DNA (ds)
DNA Molecular Weight 135-200
(Daltons x 106)
Buoyant density 1.678
(DNA, gm/cc)
TM (ฐC) 77-78
G + C (Mole %) 18-22
Rifampicin Sensitive +
325 x 200
Vertebrates
6-8
1.279
2
2
1
DNA (ds)
132-200
1.692
83
34
There are several insect cell lines susceptible to A^ moorei EPV
Heliothis zea, IPLB-1075; several Porthetria dispar lines; and Estigmene
acrea, BTI-EAA). The best system is the BTI-EAA cell line which was derived
from _E. acrea hemocytes and grows in suspension (16). Only _A. moorei EPV
has been grown in cultured cells, and there is an obvious need for more work
in this area of EPV research. BTI-EAA cells are highly susceptible to
AMEPV and a plaque assay system was recently established which is similar
to the agar overlay assay developed by Dr. H.A. Wood for A^ californica
NPV. Two AMEPV virus clones have been isolated using the plaque assay tech-
nique, and these will be compared to the "wild" virus type.
Synthesis of AMEPV proteins during infection of BTI-EAA cells, by fol-
lowing incorporation of S labeled methionine into viral peptides, is cur-
rently being investigated (W.H.R. Langridge, personal communication).
Separation and identification of viral peptides by PAGE autoradiography
allowed the identification of time postinoculation when viral peptides were
synthesized, cleaved, or degraded.
97
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As indicated earlier, insects are the only known hosts of EPV. In
vivo transmission tests have shown that EPV are only cross transmitted to
related hosts and current information indicates that these viruses have an
in vivo specificity similar to the baculoviruses. As is the case with
CPVs, the effect of EPVs on vertebrate animals has received little atten-
tion. At least three EPVs (M. melolontha EPV, AMEPV, and Choristoneura
fumiferana EPV) have been tested on rats and mice (20). Virus and/or OBs
were administered by feeding or inoculation. No adverse effects were
observed in any test with these EPVs. Caged wild mammals (Microtus
pennsylvanicus, Peromyscus maniculatus, Clethrionomys gapperi) and labora-
tory mice exposed to aerial spray of field-applied C^. fumiferana EPV showed
no adverse effect (20).
In vitro studies have shown that EPVs will not readily infect insect
cell lines. Only _A. moorei EPV can be grown in vitro. All attempts to
infect various insect cell cultures with _E. auxiliaris and Goeldichironomus
holoprasinus EPVs have failed. We have inoculated HeLa and mouse L-929
cells with jA. moorei EPV, and no cytopathic effect (CPE) was observed by
phase or electron microscopy. Most of the virus did not become cell-asso-
ciated; however, occasionally a few virions were observed within the phago-
cytic vacuoles but never in the cytoplasm per se.
Studies by Roberts and Campbell (21) have shown that centrifugation
(1000 x G - 1 hr) of _A. moorei EPV onto various vertebrate and insect cell
lines will cause enhancement of cell fusion and/or death of cells. This
effect will not occur if cultures are inoculated without centrifugation or
if centrifugation without virus is carried out. Studies to resolve this
phenomenon are in progress.
Several EPVs have been used experimentally in small-scale microbial
control programs (Table 5). Reasonable success has been achieved with some
of these viruses, and some researchers feel that EPVs should have the same
potential for use in biological control as the baculoviruses.
In summary, viruses are generally considered among the safest of all
microbial agents proposed for insect control. However, work on CPVs and
EPVs has been hampered by fears of their safety since morphological and
biochemical similarities to vertebrate viruses exist. Although current data
suggest that CPVs and EPVs are safe, much more research is needed in order
to develop and use these viruses in a safe and effective manner.
98
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TABLE 5. ENTOMOPOXVIRUSES (EPVs) WHICH HAVE BEEN TESTED
AS BIOLOGICAL CONTROL AGENTS
Virus Target insect Country
Wiseana EPV Wiseana spp. New Zealand
Melolontha EPV Melolontha melolontha France
Choristoneura EPV Choristoneura fumiferana Canada
Anomala EPV Anomala cuprea Japan
99
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REFERENCES
1. Federici, B. Occlusion Body: An Alternative Term for "Inclusion Body"
and Similar Terms Used in the Invertebrate Virus Literature. Soc. for
Invert. Pathol. Newsletter, 6:6, 1974.
2. Fenner, F. Classification and Nomenclature of Viruses. Intervirology,
7:1-116, 1976.
3. Federici, B., and E.I. Hazard. Iridovirus and Cytoplasmic Polyhedrosis
Virus in the Freshwater Daphnid Dimocephalus expinosus. Nature, 254:
327-328, 1975.
4. Kobayashi, M. Replication Cycle of Cytoplasmic-Polyhedrosis Virus as
Observed with the Electron Microscope. In: The Cytoplasmic-Polyhedro-
sis Virus of the Silkworm, H. Aruga and Y. Tanada, eds. University of
Tokyo Press, 1971. pp. 103.
5. Hayashi, Y. Occluded and Free Virions in Midgut Cells of Malacosoma
disstria Infected with Cytoplasmic-Polyhedrosis Virus (CPV). J. Invert.
Path., 16:442-450, 1970.
6. Payne, C.C., and J. Kalmakoff. Biochemical Properties of Polyhedra
and Virus Particles of the Cytoplasmic Polyhedrosis Virus of Bombyx
mori. Intervirology, 4:354-364, 1974.
7. Payne, C.C. Insect Virus Characterization; Essential or Irrelevant to
Identification and Diagnosis. Proceedings of the First International
Colloquium on Invertebrate Pathology, Queens University, Kingston,
Ontario, 1976. pp. 2-6.
8. Granados, R.R. Multiplication of a Cytoplasmic Polyhedrosis Virus (CPV)
in Insect Tissue Cultures. Abstracts of the Third International Con-
gress for Virology, Madrid, Spain, 1975. pp. 98.
9. Granados, R.R. Infection and Replication of Insect Pathogenic Viruses
in Tissue Culture. Advances in Virus Res., 20:189-236, 1976.
10. David, W.A.L. The Status of Viruses Pathogenic for Insects and Mites.
Annual Rev. Entomol., 20:97-117, 1975.
11. Cantwell, G.E., R.M. Faust, and H.K. Poole. Attempts to Cultivate
Insect Viruses in Avian Eggs. J. Invert. Pathol., 10:161-162, 1968.
100
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12. Aizawa, K. Recent Development in the Production and Utilization of
Microbial Insecticides in Japan. Proceedings of the First International
Colloquium on Invertebrate Pathology, Queens University, Kingston, On-
tario, 1976. pp. 59-63.
13. Katagiri, K. Use of Viruses for Control of Some Forest Insects in Japan.
Rev. Plant Prot. Res., 2:31-41, 1969.
14. Granados, R.R. Insect Poxviruses: Pathology, Morphology, and Develop-
ment. Misc. Publ. Entomol. Soc. Amer., 9:73-94, 1973.
15. Granados, R.R. Entry of an Insect Poxvirus by Fusion of the Virus
Envelope with the Host Cell Membrane. Virology, 52:305-309, 1973.
16. Granados, R.R., and M. Naughton. Replication of Amsacta moorei Ento-
mopoxvirus and Autographa californica Nuclear Polyhedrosis Virus in
Hemocyte Cell Lines from Estigmene acrea. In: Invertebrate Tissue
Culture. Applications in Medicine, Biology, and Agriculture, K. Mara-
morosch and E. Kurstak, eds. Academic Press, New York, 1976. pp. 379-389.
17. Granados, R.R., and M. Naughton. Effect of Rifampicin on Amsacta ento-
mopoxvirus Morphogenesis. Abstracts of the Third International Congress
for Virology, Madrid, Spain, 1975. pp. 96.
18. Langridge, W.H.R., R.F. Bozarth, and D.W. Roberts. The Base Composi-
tion of Entomopoxvirus DNA. Virology, 76:616-620, 1977.
19. Langridge, W.H.R., and D.W. Roberts. Molecular Weight of DNA from Four
Entomopoxviruses Determined by Electron Microscopy. J. Virology, 21:
301-308, 1977.
20. Ignoffo, C.M. Effects of Entomopathogens on Vertebrates. Ann. N.Y.
Acad. Sci., 217:141-164, 1973.
21. Roberts, D.W., and A.S. Campbell. Effect of Entomopoxviruses on Verte-
brate Cells: Cytotoxicity and Reduction of Vaccinia Plaque Numbers
in Mouse L Cells. Proceedings of the First International Colloquium
on Invertebrate Pathology, Queens University, Kingston, Ontario, 1976.
pp. 403-404.
101
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DISCUSSION
COLLINS: You did not mention specifically whether both the CPVs and the
EPVs have been tested in vitro for their ability to affect animal cells.
GRANADOS: I mentioned in the early part of my talk that Dr. Kawanishi is
going to include this in his talk tomorrow morning.
COLLINS: With the radioimmunoassay that is now available with CPVs, has
anyone yet tried to define group or type antigens for individual CPVs?
GRANADOS: Most of the work in this particular area has been conducted by
Dr. Payne at Oxford. Perhaps Dr. Harrap could elaborate on this.
HARRAP: Dr. Payne has done some radioimmunoassay work; however, we were
so pleased with RNA profile identification that we did not pursue the sero-
logy too much.
STOLLAR: Do you have any knowledge of the occlusion protein of the pox
viruses? Is it a simple one, like that of baculoviruses?
GRANADOS: The major polypeptide is in the range of 27 to 30 thousand in
its molecular weight, similar in size to that found in baculoviruses. In
addition, there is an alkaline protease; however, this work is still not
complete.
102
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PART III
VIRUSES: RECENT ADVANCES
-------
Recent Advances in the Antigenic Characterization of
Nuclear Polyhedrosis Viruses*
Richard A. DiCapua, Ph.D., James E. Peters,
and Philip W. Norton
University of Connecticut, Storrs
With the current interest in nuclear polyhedrosis viruses as biological
insecticides, the need for specific and appropriate serological assays for
the identification and quantification of these viruses has become increas-
ingly important. Specific and reproducible serological data concerning
these viruses will be required for environmentally sound decisions about
their utilization.
The first comprehensive serological characterization of the nuclear
polyhedrosis viruses (NPV), following the introduction of serological assess-
ment of insect viruses by Aoki and Chigasaki in 1921, was initiated in the
laboratories of Krywienczyk and Bergold (1, 2). Through their work, utili-
zing complement fixation and immunodiffusion assays, three distinct serolo-
gical categories were proposed: 1) the capsule viruses of Lepidoptera, 2)
the polyhedrosis viruses of Lepidoptera and 3) the polyhedrosis viruses of
Hymenoptera (1, 2). More recently, Norton and DiCapua (3) and Tignor et al.
(4), utilizing complement fixation, hemagglutination-inhibition, and immuno-
dif fusion assays, have independently demonstrated a serological relationship
between the polyhedrosis viruses of Lepidoptera and Hymenoptera (i.e.,
shared antigenicity between _L. dispar and N^. sertifer NPV polyhedrins).
*
Supported by the U.S. Department of Agriculture, Forest Service Grants No.
FS-NE-14, FS-NE-28, and FS-NE-35. This work was funded by the USDA Gypsy
Moth Research, Development and Applications Program.
105 /
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More exhaustively, DiCapua and Norton (3) have identified, as indicated in
Table 1, at least one common baculovirus polyhedrin group-specific antigen
(BP-gs) in 15 different nuclear polyhedrosis and granulosis viruses (in press),
TABLE 1. NUCLEAR POLYHEDROSIS AND GRANULOSIS
(BACULO)VIRUSES CONTAINING BP-gs ANTIGEN(S)
NPV GV
A* caJa N.ซ teadea m. disstria
_A. californica (). leucostigma _P. brassicae
B^. mori _P. idaeusalis
II. pseudotsugata _P. includens
- ea '
Jj. dispar j5. frugiperda
N. sertifer
Our laboratory is currently directing its efforts toward the isolation
of type-specific antigens (ts) from several nuclear polyhedrosis viruses,
with particular interest in _L. dispar NPV. To date, neither the identifi-
cation nor isolation of a type-specific antigen(s) (i.e., an antigen elici-
ting an antibody [monospecific] which will react only with the homologous
nuclear polyhedrosis virus) has been reported. It is, therefore, the pur-
pose of this communication to report the presence of several type-specific
antigens of JL. dispar NPV (B-Ld-ts) and the methods used to identify these
antigens.
The isolation of NPV proteins (i.e., containing both group- and type-
specific antigens) continues to involve polyhedron dissolution by the method
of Bergold or modifications thereof. Prior to dissolution, we pretreat
the inclusion bodies with 1% SDS, 3M urea and utilize sucrose gradient
centrifugation to remove nonviral components [modifications of Hedlund (5),
McCarthy and Liu (6)]. The alkaline dissolution is carried out by suspend-
ing 5 mg/ml of polyhedral inclusion bodies in 0.008M or 0.05M Na2C03 at 37ฐC
with variations in dissolution duration of 3, 5, 30, or 60 minutes. The
dissolution period is terminated by the addition of an equal volume of
106
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0.05M TRIS buffer (pH 8.5). The suspension of virus and polyhedrin is then
centrifuged twice to provide a final supernatant devoid of intact virions.
Evidence from our experiments and those of Krywienczyk and Bergold (1, 2),
Beaton and Filshie (7), and Summers (8) indicates this alkaline dissolute
contains solubilized virion components as well as polyhedrin.
The 0.05M Na2C03, 37ฐC, 5-minute alkaline dissolute of L. dispar NPV
was then subjected to isolectric precipitation (IP) for the purpose of
obtaining aliquots of different proteins which could then be utilized as
immunogens and assay antigens. The IP procedure, a modification of that
developed by Dr. Charles Reichelderfer, University of Maryland (personal
communication), employs sequential pH adjustments utilizing HC1 to reduce
the pH stepwise. The precipitate produced after each pH adjustment was
collected, washed thoroughly, and recollected by centrifugation. These pH
fractions were then used as the immunogen or test antigen reagent identified
above.
Immune sera were produced against these immunogens by a modification
of the method of Vaitukaitus (9). Inocula were administered, in complete
Freund's adjuvant, by multiple-site intradermal route in the shaved backs
of New Zealand white rabbits. The rabbits were boosted at 2-week intervals
and bled 8-10 weeks post-initiation of the inoculation sequence. The
resulting immune sera were then used for various assays, including immuno-
diffusion, immunoelectrophoresis, crossed immunoelectrophoresis, and mixed
hemagglutination. Control rabbit and guinea pig antisera, with specificity
for noninfected hemolymph, hemocytes, ovarian culture cells, and Bacillus
thuriniensis, do not produce, in these assays, cross reactivity to polyhedrin
or virions released from the "clean" polyhedra.
In our earlier antigenic studies of L^. dispar and A. californica NPV
polyhedrins, we utilized the 60-minute dissolution period to acquire
immunogen for antiserum production in rabbits, guinea pigs and chickens
(2, 10). In collaboration with Dr. William McCarthy, Penn. State University,
we were able to demonstrate, by PAGE analysis, 1) the presence of alkaline
protease degraded polyhedrin components which are eliminated by decreasing
dissolution times to 3-5 minutes with an immediate pH adjustment to 8.5,
and 2) which are absent from L_. dispar and A_. californica polyhedrins of
cell culture derived polyhedra, independent of dissolution time or carbonate
concentration (McCarthy and DiCapua, submitted for publication, Intervirology),
Thus our studies on the extent of the presence of BP-gs include antisera
raised against homologous and heterologous "degraded" and nondegraded host
and nondegraded tissue culture derived polyhedrins (Table 1). These anti-
107
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Tn
Figure 1. Immunoelectrophoresis. L.d. - Lymantria dispar NPV poly-
hedrin; T.n. - Trichoplusla ni NPV polyhedrin; 1 - Rabbit antisera to L,.
disPar NpV polyhedrin. The polyhedrins used as assay antigen and immunogen
were collected after 30 minutes dissolution in 0.05 M Na CCU.
W&
> * '
Figure 2. Indirect mixed hemadsorption. Arrows indicate rosette for-
mation absent in assays utilizing noninfected hemocytes (not shown).
108
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sera, produced via an immunization schedule previously published by Norton
and DiCapua (2), as well as by the Vaitukaitus modification (9), produce
quantitative but not qualitative BP-gs results.
Through the use of immunoelectrophoresis [modification of Grabar and
Williams (11)] and crossed immunoelectrophoresis (12), we are able to identify
at least five distinct antigens associated with L,. dispar NPV, four of which,
by comparative immunoelectrophoresis, are type specific (i.e., B-Ld-ts). In
addition, three of these L^. dispar NPV type specific have electrophoretic
mobilities slower and the fourth, faster than the group antigen (i.e., BP-gs).
This has been determined by comparison with alkaline dissolution preparations
of T_. ni (Figure 1), (). leucostigma, N_. sertifer, J5. frugiperda, and E_.
zea NPVs. We have also identified differences in the electrophoretic mobi-
lity of the groups antigen from these viruses. Adsorption of _L. dispar
polyhedrin preparations with antisera produced against S_. frugiperda and/or
S^. exempta polyhedrins produces, by immunoelectrophoresis, precipitates
with mobilities analogous to the type-specific antigens of, but not the group-
specific antigen of, _L. dispar. This is in absolute accordance with the
literal definition of type-specific antigenicity.
Utilizing sucrose gradient isolated virion preparations, we can demon-
strate that several of the slow mobility type-specific antigens of the poly-
hedrin of ~L_. dispar NPV are associated with the virion and, probably, the
nucleocapsid. However, since we can also demonstrate group antigenicity in
the virion preparations, this may be due to polyhedrin contamination as
observed by Summers in his experiments. Consequently, the question of true
shared antigenicity between virions and polyhedrin vs. contamination remains
unresolved.
The last technique we wish to discuss is that of mixed hemadsorption
(MHA) chosen because of its sensitivity for the detection of nanogram levels
of antibody and its application to cell culture systems (13, 14). The
indirect MHA combines infected hemocytes, taken from 2nd instar L^. dispar
48 hours post-per os infection, with the gamma-globulin fraction of the rabbit
anti L^. dispar NPV polyhedrin. This mixture is incubated at 37ฐC for 3 hours
(to produce a potential agglutinate) and then mixed with guinea pig ery-
throcytes covalently linked to goat anti-rabbit globulin to produce the
rosette formation (Figure 2) which is indicative of the presence of infected
hemocytes. The direct MHA directly combines infected hemocytes with
guinea pig erythrocytes covalently linked to the rabbit anti L^. dispar NPV
polyhedrin. The major control for either assay (i.e., absence of rosette
formation) substitutes noninfected for infected hemocytes.
109
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SUMMARY
1. Although a comprehensive model of baculovirus antigenic relation-
ships is not yet available, it is clear from the presence of
BP-gs in numerous nuclear polyhedrosis (and granulosis) viruses
that these viruses cannot be classified by host phylogeny.
2. Appropriate controls for anti-insect and/or insect nonbaculovirus
predators are negative.
3. We have identified a BP-gs antigen in 15 NPV and two GV polyhedrins.
Its concentration is, but its antigenicity is not, dependent on
carbonate concentration or dissolution time within the limits
discussed (i.e., the BP-gs determinant) and appears to be a repeti-
tive polyhedrin structure independent of the unit size of the poly-
hedrin employed as assay antigen or immunogen.
4. We have identified four L^. dispar type-specific (B-Ld-ts) antigens.
Their concentration and possibly their antigenic structure are
dependent on carbonate concentration, dissolution time, and alkaline
protease activity. The origin of these type-specific antigens
(polyhedrin vs. virion) has not been clarified. However, the
appearance of these B-Ld-ts antigens in polyhedrins released
during longer dissolution times leads us to believe they are of
virion origin.
5. Immunoelectrophoresis and immunoadsorption electrophoresis assays
clearly provide for the possibility of separating the B-Ld-ts
and other NPV type-specific antigens for the production of mono-
specific reagents.
6. Crossed immunoelectrophoresis and immunoelectrophoresis assays indi-
cate that there are several precipitogens in L^. dispar polyhedrin
whose type-specific antigens cannot^ be resolved by the immunodif-
fusion, complement fixation, and fluorescent antibody assays used
for verification of the BP-gs antigen.
110
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REFERENCES
1. Krywienczyk, J., and G.H. Bergold. Serologic Relationships Between
Inclusion Body Proteins of Some Lepidoptera and Hymenoptera. J. Immuno-
logy, 84:404-408, 1960.
2. Krywienczyk, J., and G.H. Bergold. Serological Relationships Between
Insect Viruses and Their Inclusion Body Proteins. J. Insect. Path., 2:
118-123, 1960.
3. Norton, P.W., and R.A. DiCapua. Group Specific Antigenicity of Nuclear
Polyhedrosis Virus Proteins. Abstract of the Eighth Annual Society on
Invertebrate Pathology Meeting, Corvallis, Oregon, 1975.
4. Tignor, G.H., H.M. Mazzone, and R.E. Shope. Serological Studies with
Baculoviruses of P^. dispar and _N. sertifer. Proceedings of the First
International Colloquium on Invertebrate Pathology, Queen's University
Press, Kingston, Canada, 1976. pp. 13.
5. Hedlund, R.C. Field and Laboratory Investigations of a Nuclear Poly-
hedrosis Virus of the Gypsy Moth Porthetria dispar (L). D. Phil. Thesis,
Department of Entomology. Pennsylvania State University, University
Park, 1974.
6. McCarthy, W.J., and S.Y. Liu. Electrophoretic and Serological Charac-
terization of Porthetria dispar Polyhedron Protein. J. Invert. Path.,
28:57-65, 1976.
7. Beaton, C.D., and B.K. Filshie. Comparative Ultrastructural Studies of
Insect Granulosis and Nuclear Polyhedrosis Viruses. J. Gen. Virol.,
31:151-161, 1976.
8. Summers, M. Biophysical and Biochemical Properties of Baculoviruses.
EPA-USDA Working Symposium, Bethesda, Maryland, 1974, American Society
of Microbiology, Washington, D.C., 1975.
9. Vaitukaitus, F., J.B. Robbins, E. Nieschlag, and G.R. Ross. A Method
for Producing Specific Antisera With Small Doses of Immunogen. J.
Chim. Endocrinol., 33:998-999, 1971.
10. DiCapua, R.A., P.W. Norton, and W.J. McCarthy. Comparison of Tissue
Culture and Host Derived Porthetria (Lymantria) dispar NPV Proteins.
Abstracts of the Eighth Annual Society of the Invertebrate Pathology
Meeting, Corvallis, Oregon, 1975.
111
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11. Grabar, P., and C.A. Williams. Method Permettant L'Etude Conjugee des
Proprietes Electrophoretiques et Immunochimiques d'un Melange de Pro-
teines. Application au Serum Sanquin. Biochim. Biophys. Acta, 10:
193-94, 1953.
12. Lundahl, P., and L. Liljas. Crossed Immunoelectrophoresis. Polyacryl-
amide Gel Electrophoresis Followed by Electrophoresis into Agarose
Gel Containing Antibodies. Analytical Biochemistry, 65:50-59, 1975.
13. Fagraeus, A., and A. Espmark. Use of Mixed Haemadsorption Method in
Virus-Infected Tissue Culture. Nature, 190:370-71, 1961.
14. Tachibana, T., P. Worst, E. Klein. Detection of Cell Surface Antigens
on Monolayer Cells. II. Application of Mixed Haemadsorption on a
Micro Scale. Immunology, 19:809-16, 1970.
112
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DISCUSSION
COLLINS: I am unclear as to what you think the derivation of the types of
the specific antigens are. Are you saying that these are proteolytic cleav-
age products of a molecule that has a group determinant, or is there any
relation? Have you tried to process your polyhedrin material in the presence
of protease inhibitors and use this as your immunogen?
DICAPUA: No.
COLLINS: Also, this may be premature, but has anyone looked into those
situations in which one finds immunity in animals? Suppose they are in the
same area as some of these insects in which it has been recorded that a
certain human population in Malaysia has antibodies against some of the
baculoviruses. Is that immunity group specific or type specific?
HARRAP: We have approached this problem in another way. We have tried to
separate the components of the viruses first, making antisera independently
of polyhedrin, enveloped nucleocapsids, and nucleocapsids. We found, using
the Spodoptera model, that antisera to enveloped virus particles have a num-
ber of common or cross-reacting antigens in immunodiffusion, but one or
maybe two specific ones. When we examined nucleocapsid antisera, we could
discriminate between two of the viruses. The antiserum to the third was
lost. Certainly two of them have a specific immunodiffusion line. This was
surprising because we expected type specific responses to be on the envelope.
113
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Now both these antisera will specifically recognize virus infection in
cell culture with the appropriate virus, and you can detect viral antigen
by 20-24 hours post-infection in individually infected cells. There is no
cross reactivity using the nucleocapsid antiserum. In fact, in immuno-
fluorescence, cross reactivity with the virus particle's antiserum is very
slight. Using immunodiffusion, we can detect cross reaction.
COLLINS: I would not be too surprised about the type specificity in capsid
proteins. There is a lot of precedence in the C-type viruses which have
type-specific determinants, both on glycoproteins and envelope major capsid
protein.
IGNOFFO: How sensitive is the HA assay?
DICAPUA: We can detect about 50 mg material; it is not very sensitive in
terms of HA technique.
114
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Recent Advances in Baculovirus Serology:
Radioimmunoassay and Immunoperoxidase Assay
Pepper Hoops and Max D. Summers, Ph.D.
Texas A&M University
College Station, Texas
INTRODUCTION
Although baculovirus serology has become an area of expanding interest
in recent years, little progress has been achieved in delineating the species-
specific relationships desirable for the development of immunological identi-
fication and detection techniques capable of evaluating viral interactions
in biological systems. Furthermore, many of the results of past serological
investigations are difficult to interpret because of a variety of purifica-
tion procedures that have been utilized for the preparation of antigens for
antisera production. Along with this, few workers have documented the purity
and stability of antigen preparations through biophysical and biochemical
methods. Also, highly sensitive and quantitative serological techniques
presently used for the antigenic characterization of viruses and detection
of viral activity with vertebrate and plant viruses have not been routinely
applied in baculovirus serological research.
ANTIGEN PREPARATION AND EVALUATION
Advances in the biochemical characterization of baculoviruses emphasize
the necessity for employment of antigen preparations of known purity and
stability. Recent reports have documented the presence of an alkaline acti-
vated protease associated with granulosis and nuclear polyhedrosis viruses,
115
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which under normal isolation procedures routinely used by insect virologists,
is capable of the degradation of polyhedrin and granulin (1, 2, 3). Antigen
heterogeneity, as a result of enzyme degradation and the effect upon subse-
quent serological evaluation, can be demonstrated with immunodiffusion
studies.
Figure 1 illustrates the immunodiffusion patterns obtained when an anti-
serum, raised against Trichoplusia ni (TnGV) granulin (26,000 mol. wt.)>
isolated under conditions designed to inhibit protease activity, and subse-
quently further purified by polyacrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate (3), was used to compare the antigenic
relationships of eight baculovirus polyhedrins and granulins. Trichoplusia
ni granulin forms three precipitin lines with homologous antiserum. Spodop-
tera frugiperda (SfGV) granulin, structurally very similar as determined by
two-dimensional peptide mapping (4), shows a reaction of identity with two
of the precipitin lines (Figure 1A). Autographa californica (AcMNPV),
Rachiplusia ou (RoMNPV), Heliothis armigera (HaMNPV), Anticarsa gemmatalis
(AgMNPV), and Trichoplusia r^i (TnSNPV) polyhedrins demonstrate reactions
of identity with two precipitin lines (Figure IB, C). Heliothis zea (Hz^NPV)
polyhedrin shows a reaction of identity with one precipitin line (Figure 1C).
However, the TnGV granulin antiserum also recognized additional antigenic
determinants on AcMNPV, RoMNPV, HaMNPV, AgMNPV, and TnSNPV polyhedrins
reflected as either nonidentity or partial identity reactions (Figure IB, C).
When the same antiserum was reacted with eight polyhedrins and granulins
demonstrating considerable degradation believed to be induced by enzyme
activity, the diffusion patterns in Figure 2 resulted. The reaction of
identity seen in Figure 1A between TnGV and SfGV granulins now appears as a
reaction of partial identity, as indicated by spur formation. Also, the
location of the cross-reacting precipitin lines has shifted toward the anti-
gen well. The reactions of identity between TnGV granulin and AcMNPV,
RoMNPV, HaMNPV, AgMNPV, HzSNPV, and TnSNPV polyhedrins observed in Figures
IB and C can no longer be detected. Again, only partial identity reactions
appear for AcMNPV, RoMNPV, HaMNPV, and TnSNPV polyhedrins. Degraded AgMNPV
and HzjJNPV polyhedrins do not react with this antiserum.
Thus, the ability of TnGV granulin antiserum to discern serological
relationships among these proteins has been altered as a result of the phy-
sical integrity of the antigen preparations used. Past attempts at serolo-
gical characterizations of polyhedrins and granulins may have suffered from
similar artifacts. Therefore, efforts involving production of antisera
116
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with discriminating properties must be related to standardized antigen pre-
parations routinely monitored for purity and stability.
RADIOIMMUNOASSAY
Many of the standard serological methodologies have been employed in
the antigenic characterization of baculoviruses, including immunodiffusion
(5-15), immunoelectrophoresis (16, 17), immunofluorescenee (18-20), comple-
ment fixation (21-26), hemagglutination (27-30), and viral neutralization
(20, 31). However, improved serological techniques widely used in other
virological systems have not been routinely applied to baculovirus studies.
Radioimmunoassay (RIA), the most sensitive immunological assay for
quantitating biological substances, has just recently been applied to the
measurement of baculovirus antigens. Kalmakoff et al. (32) report a radio-
l^
immunoassay for polyhedrin of Wiseana cervinata which can detect 3 x 10
polyhedra per ml, approximately 1 pg protein. Furthermore, this assay can
distinguish VJ. cervinata polyhedrin from _B. mori NPV polyhedrin.
Realizing that more quantitative applications utilizing serological
techniques with both improved sensitivity and specificity are desirable, the
following preliminary data on the application of radioimmunoassay (RIA),
immunoradiometric assay (IMRA), and immunoperoxidase are presented.
A microtiter solid-phase RIA and IMRA based upon the assay for hepatitis
B viral antigens described by Purcell et al. (33) is being investigated in
our laboratory for the detection and antigenic comparison of baculovirus
granulins, polyhedrins, and enveloped nucleocapsids. Figure 3 shows the
ability of log dilutions of AcMNPV polyhedrin ranging from 10 pg to 10 pg
125 ~~
to compete with I-AcMNPV polyhedrin for antibody binding. Using 10%
displacement as the lowest limit of significant detection, this assay can
detect 75-100 pg of homologous antigen. This represents approximately 40
polyhedra. Immunoradiometric assay, a modification of RIA employing I-
labeled AcMNPV polyhedrin antibodies} displays a similar sensitivity as
indicated in Figure 4. Of particular note in this assay is the specificity
of binding of AcMNPV polyhedrin antiserum compared to homologous viruses.
Neither AcMNPV LOVAL (Larval derived Occluded Virus that has been Alkali
Liberated) nor PMB-NOV (Plasma Membrane Budded Nonoccluded Virus) shows
binding of I-AcMNPV polyhedrin antiserum at the sensitive levels of
detection displayed in this experiment, i.e., below 200 ng antigen concentra-
tion.
119
-------
-a
c:
=3
o
CO
20*
-2
-1
0 1234
Nanograms Competing Antigen (log)
Figure 3. Radioimmunoassay of AcMNPV polyhedrin. The assay measured
AcMNPV polyhedrin () by the ability of unlabeled polyhedrin to compete
125
with a limiting dilution of AcMNPV polyhedrin antiserum for binding I-
125
AcMNPV polyhedrin. The results are expressed as the total bound I-poly-
hedrin standardized to 100% in the absence of competing antigen.
120
-------
in
CM
I=*CA
-00 -1
0
1
ANTIGEN CONCENTRATION (log|0 ng )
Figure A. Immunoradiometric assay of AcMNPV polyhedrin. Log dilutions
AcMNPV polyhedrin (o), AcMNPV LOVAL (), and AcMNPV PMB-NOV (A) were incu-
bated in an excess of AcMNPV polyhedrin antiserum and measured by the ability
125
of bound antigen to bind I-lgG from polyhedrin antiserum. Preimmune
serum binding of AcMNPV polyhedrin is indicated by (A).
121
-------
In comparative immunodiffusion tests, we have found that AcMNPV poly-
hedrin antiserum does not react with either SfGV or TnGV granulins at equi-
valent antigen concentrations. When SfGV and TnGV granulins were compared
with AcMNPV polyhedrin by competitive RIA, the results in Figure 5 were
obtained. Both SfGV and TnGV granulins were shown by RIA to possess similar
antigenic determinants to AcMNPV polyhedrin, as evidenced by the ability to
compete for antibody binding. However, while 0.5 ng of AcMNPV polyhedrin
125
was necessary to displace 50% of the homologous I-AcMNPV polyhedrin, 20
and 25 ng representing 40- and 50-fold increases in relative antigen con-
centrations of TnGV and SfGV were required for equal displacement. Upon
utilization of appropriate antigen ratios, cross reactions of TnGV and SfGV
granulins with AcMNPV polyhedrin have been recently confirmed in immunodif-
fusion tests. In this comparison, the advantages of a more sensitive and
quantitative immunological assay are evident.
In order to test the RIA for the detection of polyhedrin in vivo and
thereby evaluate polyhedrin antisera against a naturally produced polyhedrin,
the measurement of polyhedrin in AcMNPV infected TN-368 cell cultures was
undertaken. Initial aliquots of 10 cells which had been infected for 18
hours were disrupted, and 10-fold dilutions of the cell supernatants were
tested by RIA. Based upon a standard curve of uninfected cells with known
concentrations of purified AcMNPV polyhedrin added, polyhedrin could be
detected in as few as 100 cells with a sensitivity of 1 ng polyhedrin.
A kinetic experiment was then performed to ascertain when polyhedrin
synthesis could first be detected during the infection cycle. The results
indicate that polyhedrin synthesis can first be detected between 8 and 12
hours post-infection, which correlates with immunoperoxidase studies to be
described later.
Competitive RIA of polyhedrins and granulins from eight baculoviruses
have now been conducted utilizing AcMNPV polyhedrin and TnGV granulin anti-
sera. The numerous cross reactions seen in immunodiffusion studies have
been confirmed and furthermore quantified by RIA. The results indicate
that many antisera would have to be prepared and screened to find possible
discriminating reagents for the identification of baculoviruses using poly-
hedrin and granulin as a standard.
The detection of enveloped nucleocapsids has also been evaluated by RIA.
In a competitive RIA of purified enveloped nucleocapsids from six nuclear
125
polyhedrosis and two granulosis viruses using an I-AcMNPV-anti-AcMNPV
122
-------
o
CO
40-
20-
-2
-1
0 1234
Nanograms Competing Antigen (log)
Figure 5. Immunological comparison of AcMNPV polyhedrin, SfGV, and
TnGV granulins by radioimmunoassay. The assay measured the ability of un-
labeled antigen to compete with a limiting dilution of AcMNPV polyhedrin
antiserum for I-AcMNPV polyhedrin binding. () AcMNPV polyhedrin; (o)
SfGV granulin; and (X) TnGV granulin.
123
-------
,0
CM
o
X
Z
D
O
OL
u
9
8
7
6
D Q-
I
00 I
ANTIGEN
1 2
CONCENTRATION (log|O ng)
Figure 6. Immunological comparison of AcMNPV LOVAL, PMB-NOV, and
polyhedrin by immunoradiometric assay as described in Figure 4. (A) AcMNPV
125
LOVAL antiserum and I-IgG binding were employed to measure AcMNPV LOVAL
(), AcMNPV PMB-NOV (), and AcMNPV polyhedrin (o). (B) AcMNPV PMB-NOV
12S
antiserum and I-IgG were used to measure AcMNPV PMB-NOV (o), AcMNPV LOVAL
(A), and AcMNPV polyhedrin ().
124
-------
in
01
-00-1 0 1 2 3
ANTIGEN CONCENTRATION ( loglo ng )
Figure 6B
125
-------
assay system, AcMNPV and RoMNPV demonstrate no significant difference in
their ability to compete for antibody. Heliothis armigera, Heliothis zea,
Anticarsa gemmatalis, and Trichoplusia ni NPVs and Spodoptera frugiperda
and Trichoplusia ni GVs did not compete at 10,000-fold greater antigen con-
centrations. Antisera against RoMNPV purified enveloped nucleocapsids also
could not distinguish homologous virus from AcMNPV in a similar experiment.
Thus, AcMNPV and RoMNPV antisera recognize common antigenic determinants
between AcMNPV and RoMNPV not present or accessible in the other viruses
tested. Furthermore, comparative neutralization studies demonstrated AcMNPV
antiserum could neutralize these two viruses with equal efficiency, support-
ing the close relatedness of these viruses (31).
Polyacrylamide gel analysis in the presence of sodium dodecyl sulfate
indicates that AcMNPV and RoMNPV purified enveloped nucleocapsids contain a
number of polypeptides with similar electrophoretic mobilities. However,
proteins or unique mobilities for each virus were also observed by Summers
and Smith, whose findings are now in press. Therefore, the production of
identifying antisera for closely related viruses such as AcMNPV and RoMNPV
may ultimately require isolation of unique structural proteins for use in
immunization.
Immunoradiometric assays were also performed to determine whether the
multiple viral forms characteristic of many baculovirus infections were sero-
us
logically distinguishable (31). JI-labeled antisera to AcMNPV LOVAL and
AcMNPV PMB-NOV were used in binding studies with each form of virus. Figure
6A shows that AcMNPV LOVAL antiserum can discriminate between LOVAL and
PMB-NOV forms of AcMNPV. The reciprocal binding assay using PMB-NOV anti-
serum gave similar results (Figure 6B). However, cross reactions did occur
with heterologous forms at higher antigen concentrations. Neutralization
studies conducted with the same antisera indicate that while PMB-NOV anti-
sera will neutralize LOVAL, LOVAL antiserum did not neutralize the PMB-NOV
form under the experimental conditions employed. This reveals a complex
serological relationship from the standpoint of neutralization antigens (31).
Again, the identification of viruses at this level will require either more
discriminating antisera or antisera raised against isolated virus-specific
antigens.
126
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IMMUNOPEROXIDASE
Plaque formation, which is the result of polyhedra formation and current-
ly the routine method for measurement of virus infectivity in vitro, is now
being questioned in light of increasing evidence of host or tissue cell
influence upon the synthesis and assembly of this protein. Imtnunological
techniques employing specific viral antigens offer alternative means for the
detection of viral activity. To this end, immunoperoxidase has been employed
in screening infected cells for the detection of enveloped nucleocapsid
replication relative to polyhedrin synthesis and polyhedra production.
When TN-368-10 cell lines were infected with AcMNPV and monitored for
the presence of viral antigens with respect to time using AcMNPV enveloped
nucleocapsid and polyhedrin antisera, the typical brown coloration observed
in positive reactions appeared at 8-10 hours post-infection with AcMNPV
enveloped nucleocapsid antiserum. This corresponds to the appearance of
intracellular and extracellular infectious virus as assayed by the conven-
tional polyhedral plaque assay (34, 35). The substitution of preimmune serum
for immune serum abrogated the reaction, indicating the specificity of the
immunoperoxidase assay. AcMNPV polyhedrin antiserum first detected polyhedrin
at 12 hours post-infection, preceding detectable polyhedra formation by 2
hours. Again, preimmune sera showed no reaction.
The infection of several other insect cell lines with AcMNPV at the
level of polyhedrin synthesis and enveloped nucleocapsid replication were
also monitored using immunoperoxidase. Significantly, it was found that
Bombyx mori cells were infected with AcMNPV as indicated by the positive
reactions obtained with AcMNPV enveloped nucleocapsid antiserum. However,
AcMNPV polyhedrin antiserum gave negative reactions. Infection of JJ. mori
cells has been confirmed in supporting studies through back titration of
infected cell supernatants in TN-368-10 cell lines, yielding titers of 10
PFU/ml. Thus, the presence of polyhedrin, and as a result polyhedra for-
mation, may not be an indication of infection by baculoviruses in new
systems that have not been characterized. This confirms the need to develop
detection techniques specific for enveloped nucleocapsid replication in
addition to polyhedra production.
127
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SUMMARY
Relative to the results of early and even more recent reports on the
serology of baculoviruses, some of which are difficult to interpret, it is
obvious that one of the major problems was, and is, that of antigen purity
and specific criteria utilized to establish such. It is easily observed
that techniques are now available to routinely monitor and identify the
composition of baculoviruses and prepare the basis for developing reprodu-
cible and standardized antigens as reagents. Until we are more confident
of the sensitivity and specificity of our serological techniques and results,
it will be important to reference those results relative to, for example,
SDS-PAGE profiles of virus and/or viral proteins.
The example of the apparent group-related baculovirus antigens of
granulins and polyhedrin sets the precedent for this observation. Indivi-
dual baculoviruses have been shown to have a unique polyhedrin or granulin
associated with them. However, these proteins have also been shown to have
similar or related primary structures by two-dimensional peptide mapping
(4). The serological data presented confirm this. Based upon what we know
about the structure and chemical and physical properties of this class of
proteins, it should be theoretically possible to develop agent-specific
antisera to each one. However, we have to consider the need for it because
of the potential problems of relying on the presence of polyhedrin or granu-
lin as a reliable indicator of infection.
It seems more reasonable to concentrate more attention toward the
serological and structural characterization and comparisons of baculovirus
enveloped nucleocapsids. However, the complex structure of these viruses
reveals that this task may not be an easy one. Within the virion we need
to probe for group-related and virus-specific proteins and develop antisera
accordingly.
The preliminary results, herein with a limited array of baculovirus
antisera which are likely not of desirable RIA quality, appear promising.
As in vertebrate and plant virology, serological probes can be developed
for kinetic studies which are both specific and sensitive to record the fate
of baculoviruses and baculovirus proteins. Once the appropriate proteins
have been identified, highly specific antisera can be developed against
purified viral antigens.
The area of baculovirus serology and immunology does have historical
precedence, but relative to the present discussion, knowledge of baculo-
128
-------
virus structure and activity in cellular systems, baculovirus serology is
entering a new dimension of use and application. It is not likely going
to be an easy area of investigation because of the structural and genetic
complexity of baculoviruses. At the moment we must focus our attention
toward simple, yet reliable techniques for identification, but most of all,
techniques must be used which work, regardless of simplicity. We must
refine serological techniques to the ultimate obtainable in terms of
detection and sensitivity relative to retaining the specificity of our
antisera. Finally, from this we will develop routine, standardized proce-
dures and reagents which are more reliable than is presently the situation.
129
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34. Hink, W.F., and P.V. Vail. A Plaque Assay for Titration of Alfalfa
Looper Nuclear Polyhedrosis Virus in a Cabbage Looper (TN-368) Cell
Line. J. Invertebr. Pathol., 22:168-174, 1973.
35. Volkman, L.E., and M.D. Summers. Nuclear Polyhedrosis Virus Detection:
Relative Capabilities of Clones Developed from Trichoplusia ni Ovarian
Cell Line TN-368 To Serve as Indicator Cells in Plaque Assay. J. Virol.,
16:1630-1637, 1975.
132
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DISCUSSION
HEIMPEL: What are your sources of viruses? Do you get them from one source,
or is a conglomerate of resources combined to get them?
HOOPS: Which viruses?
HEIMPEL: For example, the Rachiplusia ou and the Autographa californica
viruses. Do you have one source or do you obtain them from many sources?
HOOPS: Our source of Rachiplusia ou is from Dr. Kawanishi. We have various
sources of Autographa californica, but we used just one source for these
studies.
GRANADOS: How can you explain the apparent absence of the Autographa virus
from the nucleus of Bombyx mori cells?
HOOPS: If you will remember, by using Autographa californica NPV antiserum,
you saw a reaction in Bombyx mori cells. It appeared predominantly in the
cytoplasm. This, we think, points to the fact that even though nucleocapsid
assembly occurs in the nucleus, our antisera recognize predominantly en-
velope nucleocapsid antigen associated with those particles that are occluded.
So, the large amount of staining is not visible that is normally seen in
infected TN-368 cells, where an assembly occurs of envelope nucleocapsids
in the nucleus.
133
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GRANADOS: This is good evidence then that the envelope nucleocapsid within
the nucleoplasm itself is distinct, and that it acquires the membrane during
the budding through the nuclear membrane.
HOOPS: We do not have enough information to make that kind of statement
based upon present evidence.
GRANADOS: Your assay does not detect envelope virions in the ]J. mori
nucleus, and yet, we know there is envelope material.
HOOPS: Previous studies have shown considerable numbers of nucleocapsids
in the virogenic stroma. We know that in most systems the envelope mate-
rial is assembled in the nucleus; however, we do not yet know that for
Autographa californica in Bombyx mori.
134
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Cell Culture Studies: Standardization
of Biological Activity
Loy E. Volkman, Ph.D.
Lovelace-Bataan Medical Center
Albuquerque, New Mexico
In considering the topic of standardization of biological activity in
cell culture, I would like to discuss some aspects of the nature of both the
host lepidopteran cells and the parasitic nuclear polyhedrosis viruses,
since the recognition of infection is based on some change due to the inter-
action of both components of the host/parasite system. I would like to
examine the basis of the assays that are now used for determining MNPV bio-
logical activity in vitro and for quantitating biological activity, and
discuss possible risks involved in relying on these assays.
First, I'll briefly review some characteristics of the MNPVs as they
behave in cell culture (M refers to the multiple-nucleocapsids-per-envelope
type of NPV). So far as I know, attempts to replicate jJNPVs (single-nucleo-
capsids-per-envelope) and granulosis viruses in cell culture have been un-
successful.
Figure 1 is a schematic diagram of the many different forms that can
occur in an MNPV infected TN-368 cell. The occluded form, of course,
occurs in the nucleus, but there also are nonoccluded forms in the nucleus,
only some of which are destined to become occluded. What fundamental fac-
tors are involved in determining which nucleocapsids become occluded and
which do not are not known at this time, but it does seem that being
enveloped is a prerequisite for nucleocapsid occlusion (1). There are
135
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PMB NOV
Figure 1. A schematic diagram illustrating the many different pheno-
typic forms of virus that occur in an Autographa californica MNPV infected
TN-368 cell.
groups of nucleocapsids as well as single nucleocapsids in the nucleus that
are unenveloped. As Hirumi et al. (2), Knudson and Harrap (3), and Mac
Kinnon et al. (4) have described, some of these nucleocapsids bud from the
inner nuclear membrane into the cisternae of the endoplasmic reticulum.
The enveloping membranes do not appear to be stable, however, because fre-
quently nucleocapsids have been seen being released into the cytoplasm
through breaks in the membranes. Perhaps this then is at least one of the
sources of the single nucleocapsids that bud from the plasma membrane
(Figure 2). In the Autographa californica MNPV/TN-368 system, the viruses
that bud from the plasma membrane before 48 hours post-infection are pri-
marily in the single-nucleocapsid-per-envelope form with occasional double
nucleocapsids per envelope (5). The envelope is very loosely arranged about
the nucleocapsid (5). Thus, it is evident that this virus system entails
136
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several variables that should be considered in the standardization of biolo-
gical activity. The occluded particles are said not to be infectious in vitro
until they are released from the polyhedra (6). That still leaves intra-
nuclear unenveloped nucleocapsids, various sizes of intranuclear bundles of
nucleocapsids whose envelopes were derived by probable de novo synthesis (7),
various sizes of intracytoplasmic bundles of nucleocapsids whose envelopes
were derived by budding through the inner nuclear membrane into cisternae
of the endoplasmic reticulum, various stages of decomposition of the mem-
branes of the latter, unenveloped intracytoplasmic nucleocapsids, and, fin-
ally, what we call a plasma membrane budded nonoccluded virus, which is,
again, primarily single nucleocapsid per envelope.
What about the infectivity of all these various forms of particles?
Does the source of the envelope, for instance, or the size of the bundle
have an effect on the infectivity? It is not unreasonable to expect that
the source of the envelope might have a bearing on the host range, or at
least the tissue range, and that the number of genome equivalents in the
infecting unit might have an effect on the resulting cytopathic effect, or
at least the time course of that effect. Another difficulty encountered
with this system is in the concept of "multiplicity of infection" because
one is dealing with a variable number multicapsid virus. As long as multi-
ple genome equivalents are bundled together as a group, they can meet all
the experimental requirements of single hit kinetics (and thus effect a
Poisson distribution), and yet it is possible that multiple complements of
genomes are required to induce certain types of cytopathic effects. These
are possibilities that need to be examined. To do this one needs to purify
each of these different forms and determine what its potential is. So far
the best characterized form of in vitro produced virus is the plasma mem-
brane budded nonoccluded virus (PMB-NOV). This is so, simply because it is
the most easily purified.
Figure 3 demonstrates the effect of infection by MNPV on the multipli-
cation of TN-368 cells. TN-368-13 and TN-368-10 are sublines started from
single cells isolated from Fred Hink's parent cell line of Trichoplusia ni
virgin moth ovarian cells (TN-368) (8). In this particular experiment, the
infected cultures were exposed to the PMB-NOV form of Autographa californica
NPV at an MOI of 10 to 20 to assure 100% infection of the cells. It is
important to note not only the inhibition of cellular division by the
infection, but the absence of significant cytolysis before 48 hours post-
infection. This means that virus collected from the cell culture medium
prior to 48 hours post-infection approaches a pure preparation of PMB-NOV.
138
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24 48
Hours
72
Figure 3. Growth curves of infected ( ) and uninfected ( ) TN-368-
10 (*) and TN-368-13 () cells. Cell samples from infected and uninfected
cultures were removed periodically and viable cell counts were made. For
the infected cultures, the zero hour sample was taken at the end of the
adsorption period.
Since PMB-NOV is composed of single enveloped nucleocapsids, "multiplicity
of infection" for these particles can be regarded as is standard.
Several things have been learned from studying this form of the virus.
We have found that this form can induce polyhedra formation, and when
assayed by the polyhedra plaque method on our most sensitive indicator
line, TN-368-10, one in 128 particles is infectious (9). We know that in
the course of virus production in cell culture, the budding of PMB-NOV pre-
cedes the occluding of intranuclear particles, and that as the occlusion
process begins, the budding is shut down. This was determined in an experi-
ment in which we attempted to correlate the various morphological stages of
cytopathic effect, or CPE, as shown in Figure 4, with a PMB-NOV growth
curve. We designated three classes of cytopathic effect: No cytopathic
effect, as shown in section A; prepolyhedral cytopathic effect, as shown
in B and C (these are early and late, respectively); and polyhedral cyto-
pathic effect, wherein the cells obviously contain one or more occluded
virus as shown in D. To do this study we simultaneously infected two TN-368
139
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* J:
Figure 4. Progression of GPL induced in TN-368 cells by Autographa
californica MNPV. (A) Uninfected TN-368-13 cells (x3,200). (B) First
stage of CPE. The cell has ruunded up, the nucleus is enlarged, all nucleoli
have disappeared, and the virogenir stroma has formed (xSOOO). (C) Viro-
genic stroma seems to have become nore compact. Often there is Brownian
motion around the internal periphery of the nuclear membrane during this
stage (x8000). (D) Cells contain polyhedra (xSOOO).
140
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sublines, TN-368-10 and TN-368-13, with PMB-NOV at an MOI of 10 to 20, and
at various times after infection we removed a sample of the homogeneously
suspended cells, titered the supernatant fluid by the plaque assay, and
visually inspected at least 300 of the pelleted cells to determine the
percentage of cells in each specific stage of cytopathic effect.
Figure 5 shows the results of that study. The open squares represent
percent cells showing prepolyhedral cytopathic effect, which begins at just
before 10 hours post-infection; the encircled stars represent the percent cells
with polyhedral cytopathic effect, which increases, of course, as the pre-
polyhedral cytopathic effect falls off. The closed circles represent the
titer of PMB-NOV in the culture fluid. The virus begins to bud a little
before 10 hours post-infection, and continues until about 35 hours post-infec-
tion. The histogram represents the change in PFU concentration with time.
We see that the budding of the virus corresponds with the prepolyhedral
stage of cytopathic effect and that budding is shut down with the onset of
polyhedral formation. The results suggest that the budded virus is completed
before intranuclear virus becomes occluded.
Further, we know from electron micrographs of preparations of negatively
stained gradient purified PMB-NOV in comparison with micrographs of parti-
cles that were alkali-liberated from insect grown polyhedra, PMB-NOV have
modified surface structures that the latter lack (5). (See Figures 6 and
7A and B.) The infectivity of the isolated single-nucleocapsid-per-enve-
lope alkali-liberated particles from an MNPV preparation is much less than
that of PMB-NOV, as assessed by the plaque assay. About 1 in 2.4 x 10
particles are infectious, as compared with the PMB-NOVs 1 in 128 (9).
Further, there are antigenic differences between the two forms as indicated
by their infectivity being neutralized by different populations of anti-
bodies (9). These bits of information indicate that there are significant
differences between PMB-NOV and the alkali-released occluded virus. We do
not know how the intracellular forms of NOV compare, however. We do know
that at least some of the intracellular forms are infectious, but we don't
know which ones. It is yet to be definitely established whether or not the
unenveloped nucleocapsids are infectious, for example.
The assays most commonly used in evaluating MNPV infectivity in vitro,
the plaque assay and the dilution end point assay, use the production of
polyhedra as the sign of infectivity (4, 6, 10). The polyhedra produced
are not always infectious, however, and they may contain only a few single
enveloped nucleocapsids, if any at all. In addition, there may be only 10
or less polyhedra per nucleus. It is this type of polyhedron that appears
141
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Figure 6. Preparation of Autographa californica PMB-NOV negatively
stained with ammonium molybdate, showing the presence of viral envelope in
loose and/or variable association and the presence of surface projections or
peplomers on the viral envelope (x!72,000).
143
-------
Figure 7. Preparation of alkali-liberated and gradient-purified Auto-
grapha californica MNPV. negatively stained with ammonium molybdate. (A)
Enveloped single nucleocapsids with envelope intact or partially disrupted.
(B) Many nucleocapsids common to a viral envelope with envelope intact or
partially disrupted.
144
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in the so-called FP plaques (11, 12). The alternative plaque type is the
MP type which is composed of many polyhedra per nucleus and many bundles of
enveloped virus per polyhedron. Studies by Potter et al. (13) have shown
that if virus picked from an MP plaque is passed serially in cell culture,
the MP plaque type is the predominant type in early passages, but by passage
level 25, FP is the predominant type. If the FP plaque is picked and passed
likewise, it remains homogeneous. It was postulated that NPV undergoes
mutation and selection to the advantage of the FP phenotype. This hypothesis
may indeed be correct, but perhaps in addition, it would be wise to consi-
der whether or not the phenotype of the infecting virion plays a role in
the FP/MP phenomenon. Because of the multiple forms commonly produced in
a single infected cell, the technique of picking a plaque could very well
yield mixed phenotypic forms of the "purified" virus.
Consideration of the FP plaque phenomenon touches upon a very worrisome
aspect of our method of assaying for infectivity by polyhedra production.
Ramoska and Hink (14) reported that when they examined FP plaques using
electron microscopy they found that the majority of infected cells contained
no polyhedra whatsoever. We should consider the possibility of virus pro-
duction without concomitant polyhedra production and what that implies for
our in vitro handle on measuring infectivity. In 1971, Ignoffo, Shapiro,
and Hink (15) reported that an established line of Heleothis zea ovarian
cells could be infected by a Heleothis zea NPV and produce infectious virus
but no polyhedra. Rnudson and Tinsley (6) have suggested that Spodoptera
frugiperda cells grown to confluency are incapable of producing cytopathic
effect upon NPV infection. Further, James Vaughn observed that a special
cell line of Porthetria dispar infected with _P. dispar NPV showed no sign
of CPE, yet when those cells were examined with an electron microscope,
numerous intranuclear enveloped viral nucleocapsids were seen (personal
communication). More recently, studies in Max Summer's lab have revealed
that less than 0.5% of a culture of Bombyx mori cells infected with Autographa
californica NPV contained polyhedra at 48 hours post-infection, yet when the
culture medium was assayed for infectivity on TN-368 cells, about 10 PFU
per ml were found. Further, when these cells were examined using an immuno-
peroxidase technique, less than 0.5% reacted with anti-polyhedrin, while over
90% reacted with anti-alkali-liberated MNPV.
Results such as these indicate that polyhedra production is not always
a reliable indication of NPV infection and replication. The recognition
of this leads to the realization that granulosis virus and SNPV may be
infectious in cell culture after all, but thus far have gone undetected
145
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because of the lack of polyhedra production. Similarly, since polyhedra
production differs with the line and subline of host cell, as well as with
the infecting NPV, we may not be assessing our host ranges accurately. It
is recognized that some investigators have used electron microscopy to
assess GV and J5NPV infectivity in some in vitro systems, but the complexity,
cost, and time-consuming nature of this technique make it of limited use
as a screening tool for many viruses in many different cell lines. In short,
we urgently need to establish an assay that reveals the replication of the
nonoccluded as well as the occluded virus. Without such an assay the stan-
dardization of biological activity in cell culture is equivocal and very
limited. Perhaps the best indicator system we currently have is the Hink-
Vail plaque assay (10) using TN-368 cells, and it was shown that sublines
of these cells vary at least 75% in their capabilities of revealing infect-
ious virus (16).
In summary, in order to improve the level of standardization of NPV
biological activity, we need to take a harder look at the infectious capa-
bilities of the different nonoccluded forms of virus. We need to be cogni-
zant of the fact that we are working with a mixed form virus, and we need
to establish an assay that is more closely tied in with virus replication
than is polyhedra formation.
146
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REFERENCES
1. Summers, M.D., and H.J. Arnott. Ultrastructural Studies of Inclusion
Formation and Virus Occlusion in Nuclear Polyhedrosis and Granulosis
Virus Infected Cells of Trichoplusia ni (Huber). J. Ultrastruct. Res.,
28:462-480, 1969.
2. Hirumi, H., K. Hirumi, and A.H. Mclntosh. Morphogenesis of a Nuclear
Polyhedrosis Virus of the Alfalfa Looper in a Continuous Cabbage Looper
Cell Line. Ann. N.Y. Acad. Sci., 266:302-326, 1975.
3. Knudson, D.L., and K.A. Harrap. Replication of a Nuclear Polyhedrosis
Virus in a Continuous Cell Culture of Spodoptera frugiperda: Microscopy
Study of the Sequence of Events of the Virus Infection. J. Virol.,
17:254-268, 1976.
4. MacKinnon, E.A., J.F. Henderson, D.B. Stoltz, and P. Faulkner. Morpho-
genesis of Nuclear Polyhedrosis Virus under Conditions of Prolonged
Passage In Vitro. J. Ultrastruct. Res., 49:419-435, 1974.
5. Summers, M.D., and L.E. Volkman. Comparison of Biophysical and Morpho-
logical Properties of Occluded and Extracellular Nonoccluded Baculovirus
from In Vivo and In Vitro Host Systems. J. Virol., 17:962-972, 1976.
6. Knudson, D.L. , and T.W. Tinsley. Replication of a Nuclear Polyhedrosis
Virus in a Continuous Cell Culture of Spodoptera frugiperda; Purification,
Assay of Infectivity, and Growth Characteristics of the Virus. J. Virol.,
14:934-944, 1974.
7. Stoltz, D.B., C. Pavan, and A.B. DaCunha. Nuclear Polyhedrosis Virus:
A Possible Example of De Novo Intranuclear Membrane Morphogenesis. J.
Gen. Virol., 19:145-150, 1973.
8. Hink, W.F. Established Insect Cell Line from the Cabbage Looper,
Trichoplusia ni. Nature (London), 226:466-467, 1970.
9. Volkman, L.E., M.D. Summers, and C-H. Hsieh. Occluded and Non-
occluded Nuclear Polyhedrosis Virus Grown in Trichoplusia ni; Compara-
tive Neutralization, Comparative Infectivity, and In Vitro Growth
Studies. J. Virol., 19:820-832, 1976.
10. Hink, W.F., and P.V. Vail. A Plaque Assay for Titration of Alfalfa
Looper Nuclear Polyhedrosis Virus in a Cabbage Looper (TN-368) Cell
LI-,.;. J. Invertebr. Pathol., 22:168-174, 1973.
147
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11. Hink, W.F., and E. Strauss. Replication and Passage of Alfalfa Looper
Nuclear Polyhedrosis Virus Plaque Variants in Cloned Cell Cultures and
Larval Stages of Four Host Species. J. Invertebr. Pathol., 27:49-55,
1976.
12. Brown, M., and P. Faulkner. Factors Affecting the Yield of Virus in
a Cloned Cell Line of Trichoplusia ni Infected with a Nuclear Polyhe-
drosis Virus. J. Invertebr. Pathol., 26:251-257, 1974.
13. Potter, K.N., P. Faulkner, and E.A. MacKinnon. Strain Selection During
Serial Passage of Trichoplusia ni Nuclear Polyhedrosis Virus. J. Virol.,
18:1040-1050, 1976.
14. Ramoska, W.A., and W.F. Hink. Electron Microscope Examination of Two
Plaque Variants from a Nuclear Polyhedrosis Virus of the Alfalfa Looper,
Autographa californica. J. Invertebr. Pathol., 23:197-201, 1974.
15. Ignoffo, C.M., M. Shapiro, and W.F. Hink. Replication and Serial Pas-
sage of Infectious Heliothis Nuclear Polyhedrosis Virus in an Estab-
lished Line of Heliothis zea Cells. J. Invertebr. Pathol., 18:131-134,
1971.
16. Volkman, L.E., and M.D. Summers. Nuclear Polyhedrosis Virus Detection:
Relative Capabilities of Clones Developed from Trichoplusia ni^ Ovarian
Cell Line TN-368 to Serve as Indicator Cells in a Plaque Assay. J.
Virol., 16:1630-1637, 1975.
148
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DISCUSSION
SMITH: During the process of budding does the particle pick up any of the
host nuclear or cytoplasmic membrane? Has anyone examined the structure and
composition of the membrane?
VOLKMAN: Not yet. To answer your first question, we assume that the parti-
cle probably does.
SMITH: And this membrane is or is not present in the occluded virus?
VOLKMAN: There is an envelope, but it is not the same. The envelope of
the occluded viruses is obtained intranuclearly.
RAPP: The major difference between the occluded and the nonoccluded virus
is the fact that they occur singly or in pairs. Is there any other differ-
ence? Is there an antigenic difference? Is there some kind of biochemical
difference that you can pinpoint for us? That alone would make them geneti-
cally mixed. Is there any evidence that suggests that they are genetically
different?
VOLKMAN: There is no evidence that I know of that suggests they are gene-
tically different, although I do not believe anyone has taken a critical
look at that possibility. I think most people assume they are genetically
the same, but they are phenotypically different.
HARRAP: I think we have one assay already, other than plaque assay. We can
use virus particle antiserum on infected cell cultures. As I mentioned
149
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before, we can detect the infected cells at 24 hours; so, if you tested
again in 48 hours, you could show the numbers increasing, etc. That is one
alternative for looking at the presence of virus actually in the infected
cell. I wondered if you had looked at possible different levels of infecti-
vity with the different degrees of envelopment of the nucleocapsids. This
is something we have been thinking about.
VOLKMAN: Yes, we have done only one study. We compared two nucleocapsids
or more vs. single nucleocapsids from alkali-liberated preparations. We
found there is vety little difference in the level of infectivity between
those two groups as assayed both by the plaque assay in cell culture, and
in vivo by per os and intrahemocoelic administration.
(Tape change: some discussion missed.)
SUMMERS: For occluded enveloped nucleocapsids, the envelope is obtained in
the nucleus and not by budding through the nuclear or plasma membrane. Is
there a similar process that occurs with vertebrate viruses? The source of
the virus envelope is obviously fundamental to differences in the physical,
biological, and serological properties of the two forms of virus-occluded
enveloped nucleocapsids and nonoccluded particles. This can be referred to
your earlier question which you persistently bring to our attention what
are we working with?
VOLKMAN: I would like to emphasize again that there seem to be at least
three sources of envelopes: intranuclear de novo synthesis, budding through
the intranuclear membrane into the cytoplasm, and budding through plasma
membrane.
RAPP: There are viral systems with large amounts of proteins and crystalline
arrays but no envelope material. The adenoviruses are an example. The
question is whether insect cells normally have that type of nuclear material,
as it is not normally present in the mammalian nucleus. Has anyone examined
that?
SUMMERS: Not definitively.
150
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Pathogenic-Invertebrate Viruses:
In Vitro Specificity
Dennis L. Knudson, Ph.D.
Yale University School of Medicine
New Haven, Connecticut
Invertebrate-associated animal viruses have been recently defined as
being comprised of two distinct groups based on a criterion of pathologic
effect (1). One group, arthropod-borne viruses (arboviruses), uses inverte-
brate vectors for biological transmission, replicates in invertebrates with
little deleterious effect, and replicates in vertebrates with varying
degrees of pathology. The second group, pathogenic-invertebrate viruses, is
characterized by replication in invertebrates with pathologic effect and by
the absence of any demonstrable pathology in vertebrates. Invertebrate-
associated animal viruses such as Baculoviridae, cytoplasmic polyhedrosis
viruses of Reoviridae, entomopoxviruses of Poxviridae, iridoviruses of Iri-
doviridae, and picorna-like viruses of Picornaviridae constitute the patho-
genic-invertebrate virus group. This discussion of in vitro specificity
will be restricted to this latter group of viruses, with particular emphasis
on the Baculoviridae.
Both invertebrate and vertebrate continuous cell lines have been employed
in assessments of in vitro specificity of pathogenic-invertebrate viruses,
and the specificity of these viruses in vertebrate cell lines has been
reviewed previously (2-4). The general consensus indicates that there is
little evidence for the replication of pathogenic-invertebrate viruses in
vertebrate cell lines with the possible exceptions of some of the icosahe-
dral viruses. In contrast, pathogenic-invertebrate viruses do replicate in
151
-------
many of the invertebrate cell lines (1, 5). The existing data on the host
range of pathogenic-invertebrate viruses in invertebrate cell lines are
compiled in Tables 1 to 6. Both the positive demonstrations of virus repli-
cation and the negative results are recorded in the tables versus the appro-
priate invertebrate cell line.
It is tempting to speculate as to the significance of these data on
the apparent in vitro specificity of pathogenic-invertebrate viruses. For
example, the picorna-like virus seems to be specific for dipteran cell lines,
and the entomopoxviruses and baculoviruses seem to be specific for lepidop-
teran cell lines. In contrast, the cytoplasmic polyhedrosis viruses and
iridoviruses seem to lack in vitro specificity because they may replicate
in both dipteran and lepidopteran cell lines. The data on Baculoviridae
reveal that the in vitro replication of granulosis viruses has not been
demonstrated. Furthermore, a few of the nuclear polyhedrosis viruses seem
to exhibit an in vitro host range with a specificity which may reside at a
familic or generic level. Autographa californica nuclear polyhedrosis virus
(NPV) and Trichoplusia ni NPV appear to be two exceptions because they
replicate in cell lines derived from lepidopterans of several different
families.
Unfortunately, these speculations are premature and based on too little
data. The "not tested" or zeroes in the tables outnumber the tested
accounts, and furthermore, some of the tested accounts are recorded as per-
sonal communications or unpublished reports. This latter point becomes
particularly apparent when examining the literature of the pathogenic-inver-
tebrate virus specificity in vertebrate in vitro systems. Additionally, it
is difficult to assess the significance of an unquantified negative result.
In assessments of in vitro specificity and in the interpretation of
such data there are several parameters that must be clearly defined or, at
least, considered. A methodology, for example, should be employed by which
both the positive and negative results can be quantified, that is, relative
to an internal standard such as a homologous system. The nature of the
inoculum should be stated, and its biological activity should be quantified.
The conditions of the test must be noted, particularly when there is devia-
tion from the homologous system. Pertinent conditions to be considered
and reported would include temperature, media, pH, and the growth phase of
the test cells, such as log growth or stationary phase. The level of sen-
sitivity in the detection of a viral effect on the cells must be clearly
stated.
152
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154
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TABLE 3. PATHOGENIC-INVERTEBRATE VIRUSES:
HOST RANGE IN INVERTEBRATE CELL LINESt
Cell line*
Lepidoptera
H. zea S. frugiperda T. ni
Baculovirus
Granulosis virus (GV)
Heliothis zea GV
Spodoptera frugiperda GV
Trichoplusia ni GV
- = no virus replication demonstrated.
R.H. Goodwin (personal communication).
TABLE 4. PATHOGENIC-INVERTEBRATE VIRUSES:
HOST RANGE IN INVERTEBRATE CELL LINES
Cell line*
Lepidoptera
H. zea S. frugiperda T. ni Ref.
Baculovirus
Nuclear polyhedrosis virus (NPV)
single embedded type
Heliothis zea NPV
Trichoplusia ni NPV
(14, 15)t
(14, 15)t
+ = virus replication demonstrated; - = no virus replication demonstrated;
and 0 = not tested.
t
R.H. Goodwin (personal communication).
155
-------
TABLE 5. PATHOGENIC-INVERTEBRATE VIRUSES:
HOST RANGE IN INVERTEBRATE CELL LINES
Cell line*
Dlptera
A.
A. _A. tritaeni-
albopictus Stephens! orhynchus Ref,
Baculovirus
Nuclear polyhedrosis virus (NPV)
multiple embedded type
Autographa californica NPV - - - t
Spodoptera frugiperda NPV - 0 0 t
Trichoplusia ni^ NPV - t
*
+ = virus replication demonstrated; - = no virus replication demonstrated;
and 0 = not tested.
R.H. Goodwin (personal communication).
156
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157
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I would like to present an example of a methodology by which both the
positive and negative results can be quantified in assessments of in vitro
specificity of baculoviruses. The in vitro host range of six NPVs isolated
from insects of the family Noctuidae was examined in five lepidopteran
continuous cell lines. Three of the cell lines were derived from tissues
of insects from the family Noctuidae. While the remaining two, the
Lymantria dispar cell line (IPLB-PD-65Z) and the Choristoneura fumiferana
cell line (IPRI-CF124), were derived from tissues of insects from the
families Lymantriidae and Tortricidae, respectively. The cell lines were
all grown in the same medium, TC100 (22), and the viral inocula were all
low-speed clarification supernatants of infected cell cultures (23).
The test simply consists of an endpoint dilution titration of a given
NPV in the various cell lines. A dilution series of the virus was made,
and 0.1 ml of each dilution was introduced in the test system containing a
specific number of cells which came from log growth phase cell cultures.
The same dilution series was also titrated into the homologous cell system.
The homologous system was operationally defined as the cell line in which
the viral inoculum was grown. After an incubation period of five days,
the titration was scored for the characteristic baculovirus cytopathic
effect; that is, the formation of polyhedra in the nuclei of the cells (22).
The 50% tissue culture infective dose (TCID ) per milliliter of inoculum
was calculated from the homologous system and the test cell line or hetero-
logous system. The efficiency of the virus titration or assay in different
cell lines was expressed relative to the homologous system. Table 7
represents the data on the in vitro host range of the six baculoviruses in
the five lepidopteran cell lines presented as the efficiency of assay, the
heterologous TCID,-^ per ml exponent over the homologous TCID^ per ml ex-
ponent. The expression equal to or less than (<) 1.5 in the table is indi-
cative of the lower level of quantification of these tests. A tenth of a
ml of a 10 dilution gave no cytopathic effect in the test system, and
therefore, the TCID n per ml would be 10 * . These data allow comparisons
to be made of the virus titer between a given heterologous cell line and
homologous cell line. A simple normalization step, however, allows com-
parisons to be made between the cell lines and the different viruses. The
data from Table 7 which have been normalized and expressed as log,n func-
tion are shown in Table 8. The values noted in Table 8 represent an index
of the efficiency of the virus assay in the heterologous cell line relative
to the homologous cell line, or simply, an efficiency index. The efficiency
indices are useful for several reasons. They allow direct comparisons to
be made because the indices represent constants which should only vary by
158
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160
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the standard error of the titration. When the columns are examined, a com-
parison of the indices is indicative of the susceptibility of the cell line
to the various NPVs that were tested, and the level of the susceptibility
is quantified. When the rows are examined, a comparison of the indices
reflects the replication potential of a virus in the cell lines that were
tested, and likewise, this function is also quantified. For example, the
low level of replication of S_. exempt a NPV-S/SF2 in the ฃ. fumiferana cell
line may not have been detected if the virus inoculum had a lower initial
titer.
The data in Table 8 do indicate the degree of in vitro specificity of
baculoviruses in invertebrate cell lines using the noted levels of input
test virus and using the detection system of cytopathic effect. Clearly,
this approach only represents the first step in the assessment of the in
vitro specificity of baculoviruses. Undoubtedly, tests of increased sensi-
tivity in the detection of viral replicative events in cells will represent
the second and further steps in such specificity assessments.
These data are preliminary observations which will be reported else-
where in greater detail.
Acknowledgment s
The expert technical assistance of C.A. Mongillo and the grant support
from the Rockefeller Foundation, the World Health Organization, and the
USPHS 1 R01 AI 13727 are acknowledged.
161
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REFERENCES
1. Knudson, D.L., and S.M. Buckley. Invertebrate Cell Culture Methods for
the Study of Invertebrate-Associated Animal Viruses. In: Methods in
Virology, K. Maramorosch and H. Koprowski, eds. Academic Press, New
York, 1977. pp. 323-391.
2. Ignoffo, C.M. Specificity of Insect Viruses. Bull. Entomol. Soc.
Amer., 14:265-276, 1968.
3. Ignoffo, C.M. Effects of Entomopathogens on Vertebrates. Ann. N.Y.
Acad. Sci., 217:141-172, 1973.
4. Mclntosh, A.H. In Vitro Specificity and Mechanism of Infection. In:
Baculoviruses for Insect Pest Control: Safety Considerations, M.
Summers, R. Engler, L.A. Falcon, and P.V. Vail, eds, American Society
for Microbiology, Washington, B.C., 1975. pp. 63-69.
5. Granados, R.R. Infection and Replication of Insect Pathogenic Viruses
in Tissue Culture. Adv. Virus. Res., 20:189-236, 1976.
6. Kawarabata, T., and Y. Hayashi. Development of a Cytoplasmic Polyhe-
drosis Virus in an Insect Cell Line. J. Invertebr. Pathol., 19:414-
415, 1972.
7. Granados, R.R., W.J. McCarthy, and M. Naughton. Replication of a Cyto-
plasmic Polyhedrosis Virus in an Established Cell Line of Trichoplusia
n^ Cells. Virology, 59:584-586, 1974.
8. Kelly, D.C., and T.W. Tinsley. Iridescent virus replication: A micro-
scope study of Aedes aegypti and Antherea eucalypti Cells in Culture
Infected with Iridescent Virus Types 2 and 6. Microbios., 9:75-93,
1974.
9. Kelly, D.C., and T.W. Tinsley. Iridescent Virus Replication: Patterns
of Nucleic Acid Synthesis in Insect Cells Infected with Iridescent
Virus Types 2 and 6. J. Invertebr. Pathol., 24:169-178, 1974.
10. Mclntosh, A.H., and M. Kimura. Replication of the Insect Chilo Iri-
descent Virus (CIV) in a Poikilotheraic Vertebrate Cell Line. Inter-
virology, 4:257-267, 1974.
11. Webb, S.R., J.D. Paschke, G.W. Wagner, and W.R. Campbell. Bioassay of
mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of
Aedes aegypti. J. Invertebr. Pathol., 26:205-212, 1975.
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12. Hink, W.F. A Compilation of Invertebrate Cell Lines and Culture Media.
In: Invertebrate Tissue Culture: Research Applications, K. Maramorosch,
ed. Academic Press, New York, 1976. pp. 319-369.
13. Hukuhara, T., and Y. Hashimoto. Multiplication of Tipula and Chilo
Iridescent Viruses in the Cells of Antherea eucalypti. J. Invertebr.
Pathol., 9:278-281, 1967.
14. Goodwin, R.H., J.L. Vaughn, J.R. Adams, and S.J. Louloudes. The Influ-
ence of Insect Cell Lines and Tissue-Culture Media on Baculovirus Poly-
hedra Production. Misc. Publ. Entomol. Soc. Am., 9:66-72, 1973.
15. Ignoffo, C.M., M. Shapiro, and W.F. Hink. Replication and Serial Pas-
sage of Infectious Heliothis Nucleopolyhedrosis Virus in an Established
Line of Heliothis zea Cells. J. Invertebr. Pathol., 18:131-134, 1971.
16. Vail, P.V., D.L. Jay, and W.F. Hink. Replication and Infectivity of
the Nuclear Polyhedrosis Virus of the Alfalfa Looper, Autographa cali-
fornica, Produced in Cells Grown In Vitro. J. Invertebr. Pathol.,
22:231-237, 1973.
17. Raghow, R., and T.D.C. Grace. Studies on a Nuclear Polyhedrosis Virus
in Bombyx mori Cells In Vitro. I. Multiplication Kinetics and Ultra-
structural Studies. J. Ultrastruct. Res., 47:384-399, 1974.
18. Sohi, S.S., and J.C. Cunningham. Replication of a Nuclear Polyhedrosis
Virus in Serially Transferred Insect Hemocyte Cultures. J. Invertebr.
Pathol., 19:51-61, 1972.
19. Knudson, D.L. Replication of Nuclear Polyhedrosis Viruses in Cell Cul-
tures. Ph.D. Thesis, University of Oxford, Oxford, United Kingdom, 1975.
20. Goodwin, R.H., J.F. Vaughn, J.R. Adams, and S.J. Louloudes. Replication
of a Nuclear Polyhedrosis Virus in an Established Insect Cell Line. J.
Invertebr. Pathol., 16:284-288, 1970.
21. Faulkner, P., and J.F. Henderson. Serial Passage of the Nuclear Polyhe
drosis Disease Virus of the Cabbage Looper (Trichoplusia ni) in a Con-
tinuous Tissue Culture Cell Line. Virology, 50:920-924, 1972.
22. Knudson, D.L., and T.W. Tinsley. Replication of a Nuclear Polyhedrosis
Virus in a Continuous Cell Culture of Spodoptera frugiperda; Purifi-
cation, Assay of Infectivity, and Growth Characteristics of the Virus.
J. Virol., 14:934-944, 1974.
23. Knudson, D.L., and K.A. Harrap. Replication of a Nuclear Polyhedrosis
Virus in a Continuous Cell Culture of Spodoptera frugiperda; Micro-
scopy Study of the Sequence of Events of the Virus Infection. J. Virol.,
17:254-268, 1976.
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DISCUSSION
SUMMERS: You are comparing Autographa NPV/TN passage 5 with Spodoptera NPV-S/
SF passage 2. In our hands, Autographa NPV passage 5 drops off significantly
in its ability to produce polyhedra relative to passage 1 or 2, by a rather
large percentage.
KNUDSON: This is in T_. ni cells?
SUMMERS: Yes. I use that as a standard system. We expect comparable pheno-
mena to occur in other cells, although maybe that is a wrong assumption. As
you compare TN-5 with SF-2, would you expect there to be differing levels
in the ability of that passage of the virus to induce polyhedra?
KNUDSON: I am not talking about the number of polyhedra in the cells, but
clearly, that may be possible. I am just listing a group of viruses. For
example, the T_. ni passage 53 which I received from Dr. Faulkner should con-
tain strictly FP virus. In our hands, we find there are many polyhedra using
the Spodoptera cell line and they produce polyhedra very well.
SUMMERS: Still, even with plaques, it would be significantly different from
early passage jT. ni virus.
KNUDSON: I think that is the nature of this meeting. Are they going to be
significantly different, and how may that best be determined? This is a
system, for example, by which you could examine passage effects.
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FIELDS: Have you been able to determine whether this is a surface restric-
tion or if this is intracellular, and do you have any idea of dominance, say,
of fusion? I ask this because it would be a powerful genetic tool in look-
ing at host range differences among this important group.
KNUDSON: I have not had an opportunity to do the types of things that you
asked about, but we got involved in this type of experimentation precisely
for the reason that you mention, that is, the idea of using this for looking
for host range mutants. We are interested in this particular area, and we
wish to set up a system by which we could quantify the differences in host
range and use that as a marker for the genetics.
STOLLAR: Is there any indication that serial high multiplicity passage
leads to something like the generation of defective particles?
KNUDSON: There are a number of accounts that seem to clearly indicate that
serial passage of the virus over a number of periods will produce something
that is referred to as an FP type polyhedra. The degenerative change that
takes place over multiple passage is the formation of polyhedra that are
apparently devoid of virus. For example, this Trichoplusia ni NPV/TN 53 is
an FP phenotype in the T_> ni cell line according to Dr. Faulkner. In the
titration, the classic sort of interference with high multiplicity input
does not appear, but virus replication at high multiplicity does occur. So
at the moment, there is no real evidence of a defective particle that is
interfering. They are defective particles, perhaps, but they do not interfere.
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PART IV
PANEL DISCUSSION
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Viral Pesticides: Implementation and Safety
Moderator: Victor Stollar, M.D.
STOLLAR: Five or six people will begin our discussion, which will be less
formal than the presentations we have had up to now. Incidentally, we would
like initially to limit the discussion to participants; later on other peo-
ple at the meeting may join in.
I would like to make a few remarks before we hear from our first dis-
cussant. The question we are trying to focus on in this meeting is, "What
is the potential harm of using insect viruses as insecticides?" What damage
could they do to man and domestic or wild animals? Thus we must ask, "What
is the capacity for replication of insect virus in vertebrate organisms?
Can there be untoward effects of insect viruses even in the absence of
complete replicative cycles? Could there be abortive infections? Could
there be persistent low-grade infections?"
Perhaps we might be able to get some useful comparative information
from observations made of various arboviruses in mosquito cell lines an
area with which I am familiar.
I think we can rationalize this as follows. The arboviruses and, more
specifically, various of the togaviruses and bunyaviruses are viruses that
specialize in alternate replication and transmission between widely different
organisms. This alternate transmission is required for their maintenance
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in nature. By evolution, they have adapted so that they can replicate well
in very different kinds of hosts or species. Togavirus models could, I
think, be useful because there has been a good deal of information acquired
both at the level of the whole organism (mammalian or arthropod) as well as
in tissue culture. In the case of a virus such as Sindbis, a great deal is
also known about its molecular biology.
Let me very briefly run through some generalizations which can be made
about viruses such as Sindbis, a representative member of the alpha virus
subgroup of the togaviruses. As Dr. Knudson just stated, these viruses
generally have potential for causing disease in vertebrate species and cyto-
pathic effects in vertebrate cells; in contrast, they are generally noncyto-
pathic in the insect host, whether that is the whole mosquito or a tissue
culture. As Knudson pointed out, this is the inverse of what we see with
the insect viruses. Perhaps if we knew why there is this interesting dif-
ference, why togaviruses can kill vertebrate cells, but not insect cells, it
might help us understand not only how insect viruses kill insect cells, but
perhaps also what their potential might be for harmful effects in higher
organisms.
Another generalization is that arboviruses, because of selective pres-
sures exerted during the course of evolution, have developed the capacity,
more so probably than most other viruses, to replicate over a very wide tem-
perature range from 20ฐ to 40ฐ. However, we must point out that in each
system the optimum temperature for virus replication is dictated by the opti-
mum temperature for the host cell. I think when we are considering the
potential of insect viruses, the effects of temperatures on virus replication
in the various cell lines will have to be considered.
Arboviruses are enveloped viruses. They bud off from various cellular
membranes and in the process incorporate carbohydrates and lipids derived
from the host. Thus, a togavirus that is produced in a mosquito cell would
have a very different composition as far as its carbohydrates and lipids from
a virus produced in a mammalian cell. For instance, one major difference
that we have shown is that in contrast to virus grown in mammalian or chick
cells, Sindbis virus grown in mosquito cells contains no sialic acid. But
the variation in carbohydrate and lipid composition, in the case of Sindbis
virus at least, has little, if any, effect on the infectivity of that virus.
In other words, viruses are equally infectious and have the same particle
to PFU ratio, whether they are grown in mosquito cells or mammalian cells.
Similarly, we have shown that the host cell in which the virus is grown had
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no detectable effect on the antigenic properties of the virus. I think this
might bear on some of the questions that have arisen today.
In other words, when we grew virus in chick cells and in mosquito cells,
purified the viruses, produced antisera against each, and reacted each anti-
serum with the virus grown in each cell type we couldn't differentiate
between the virus grown in the chick cells and that grown in mosquito cells.
We said there is no CPE produced by togaviruses in insect cells. That
is a rather sweeping generalization, and there are a number of exceptions.
There is at least one report of some cytopathic effect in the mosquito pro-
duced by Semliki Forest virus. If we turn to the flaviviruses (dengue
Japanese encephalitis, yellow fever viruses), these viruses can produce quite
a massive cytopathic effect in mosquito cell cultures. Furthermore, we have
recently obtained cloned populations of mosquito cells, which in contrast to
all uncloned populations, and to other clones, do show marked cytopathic effect
after infection with Sindbis virus.
Therefore, it appears that some of the earlier generalizations made
and accepted by people working with arboviruses will not hold up; this leads
me to believe that as we study more about the replication of insect viruses
both in insect cells and in cells of higher organisms we may be in for many
surprises.
I also would like to emphasize, from our work dealing with insect cell
cultures, that we have been strongly impressed by the importance of careful
characterization of the cell types and cloning of the cell types.
We have seen in the literature, reports of different clones or strains
of mosquito cells that give equivalent yields of viruses but which differ
very markedly in the response of the cell to the virus. This means that in
looking at insect viruses in mammalian cells, many different cell lines will
have to be tested. That might be a topic for further discussion. I think
it is important that the insect cell lines that people work with be well
characterized, and if possible, cloned. I think it would be useful to have
them karyotyped because there has been at least one instance where people
thought they were working with a mosquito cell line but were in fact using
a moth cell line.
The media used for insect cell lines are usually undefined and rather
complex. In the case of several mosquito cell lines, it has been possible
to adapt them to a simpler medium, namely Eagle's medium supplemented with
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fetal calf sera. This adaptation facilitates many kinds of experiments
involving radioisotopes and might be useful to try in the case of the various
Lepidopteran cell lines.
Lastly, we have to be aware that latent or unsuspected viruses may be
present in our insect cell cultures. There are examples of this already
reported perhaps some examples will be described by the various partici-
pants or, if people are interested, we can describe some of the examples of
unsuspected or latent contamination in some of the mosquito cell lines.
We'll now turn the discussion over to Dr. Shope.
SHOPE: Several of the participants have urged that someone introduce or
reintroduce the concept that one of the needs in the invertebrate pathogenic
virus field is the cataloguing of some of the information that has been pre-
sented this morning, as well as information from investigators throughout
the world. I have had some experience since 1959 with a similar cataloguing
exercise with the arboviruses.
I have with me the International Catalogue of Arboviruses. One might
conceive of an international catalogue of viruses pathogenic to invertebrates
formulated in a similar manner. The Catalogue of Arboviruses evolved from
a group of people sitting down in 1959, very similar to this group, with a
field of viruses, which in its technological development was not as far as
we are today with baculoviruses and the other invertebrate pathogenic viruses.
The decision was made to draw up some format to gather information and then
to collaborate, share information, and have a sort of information exchange,
first on an informal basis. Later on, this evolved into a published book
that is now in its second edition. This contains something over 360
catalogued arbovirus serotypes.
The form that is filled out by the person who catalogues individual
viruses contains information such as: the name and reference type of the
virus, the origin and method of isolation, virus properties, properties of
its DNA or RNA, and reports of virion and nonvirion peptides. The form also
lists the morphology; hemagglutination; antigens and antigenic relationships;
biological characteristics, such as animal host-range; tissue culture sus-
ceptibility; experimental models; the geographic distribution; and a list of
references. Also, in the case of arboviruses, one of the categories reported
is the disease in vertebrates that might or might not be applicable to the
viruses pathogenic to invertebrates. I will pass around the catalogue and
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the latest version of the filled-in catalogue page for people to look at.
Mr. Longworth sat down to see if he could fill in this information with one
or more of the viruses with which he was familiar.
LONGWORTH: With Dr. Shope's help, we looked at the form and condensed it
to get a working information sheet, which investigators throughout the world
can fill in. So if an investigator wishes to compare his isolate with
another, this is one way of getting information. How such a catalogue should
be organized can be left to discussion.
STOLLAR: When a virus is isolated and is submitted, does it have to be
confirmed independently or is only one person involved?
SHOPE: In a working sense, what we initially were interested in was if some-
one had an isolate that he felt was different from anything already in the
catalogue, then he was free to catalogue it, not in a published form, but in
an informal form. We simply encouraged people to submit information. When
it became obvious in certain cases that people were submitting information
on the same agent, they would get together to combine their information.
HARRAP: How was it funded?
SHOPE: Initially, in 1959, there was a small grant from the Rockefeller
Foundation, somewhat under 20 thousand dollars, which paid for the paper
and postage and some secretarial help. The actual professional work was all
volunteer. The catalogue activity was taken over by the National Institutes
of Health, and subsequently, by the Center for Disease Control. It is now
a full-time activity of the Center for Disease Control, which is part of
HEW (U.S. Department of Health, Education, and Welfare).
HEIMPEL: It is all very well to record the differences in viruses and the
various germ plasms that are isolated, but there should be a concerted effort
by everybody to establish some sort of system to preserve these. Now, the
USDA (U.S. Department of Agriculture) is looking into this, rather slowly
but thoroughly, and one of the proposals is to try to commit funds permanently
to get the American Type Culture Collection, which is a private organization,
to store insect cells and insect pathogens and plant pathogens. I've made
my plea all over the world, the last time in Rome to the FAO (Food and Agri-
culture Organization), which now has a scheme for preserving plant germ
plasm, and got a rather positive response from them, that is, they said they
would look into it. But if all those present would support a drive to pre-
serve these organisms in perpetuity, I think it would be of great use to us
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all. Actually, it worries me to see people working with what is called
Autographa californica virus, and they really don't know if that is what
they are working with because the noctuid viruses are so close. If we had
a standard source, we could all start from the same line.
STOLLAR: It seems that the time has arrived for some serious effort at
cataloguing; I'm not quite sure how this will be done, or who will take the
responsibility for it.
SHOPE: I agree, and in the case of arboviruses this can be used as an analogy.
The American Type Culture Collection now contains 116 arboviruses, and the
decision as to which arboviruses would be selected in reference type was
made on the basis of the recommendation of the group that was cataloguing
the viruses, so that the two functions would complement each other.
STOLLAR: Something along this line has been started with various insect cell
lines. Dr. Fred Hink has put together a catalogue of over 100 insect cell
lines, and he's made this catalogue available to everyone.
MCCARTHY: Considering the classification of the viruses, I don't think
enough fundamental knowledge is known about the viruses to classify them as
such, as yet, in terms of the nucleic acids and serological responses.
MILLER: There is an excellent method for identification of baculovirus, and
that is the restriction endonuclease analysis of the viral DNA. It is quite
possible to do this, and a very good catalogue could be made of restriction
enzyme patterns of the various viruses. This will be one way of distinguish-
ing viruses other than the host-range morphology and other types of identifi-
cation that are currently available.
MCCARTHY: I wasn't saying that there is no method available, just that the
data haven't been collected yet.
SUMMERS: I would agree with that, but we have various levels of identifi-
cation with which to deal. One of the routine procedures is a serological
approach and another is restriction endonuclease or physical mapping of viral
genomes; at the moment we are dealing with our serological problems. Later
we will deal more specifically with DNAs and with problems and approaches in
that area. I would like to reinforce what Bill McCarthy said, not so much
in terms of DNAs, but in terms of what we know about baculoviruses, their
complex structure, and so forth. Is the state of our art good enough today
to actually initiate a cataloguing system?
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SHOPE: I think this is the beauty of an informal information exchange as
the initial step, in that you don't have to feel that you must have the
data needed to establish it as a type virus. All you do is put the available
data down on paper and let other people decide whether to confirm it.
IGNOFFO: There is a cataloguing system available from Dr. Martignoni.
SUMMERS: We need to look at that catalogue in terms of how it can be expanded
into the scheme given to us by Dr. Shope. If you look at Martignoni's
catalogue carefully, it cites insects in terms of genus and species, and
virus diseases that have been recorded as associated with the insect.
SHOPE: I am aware of Dr. Martignoni's catalogue. What I had in mind wasn't
meant to supplant what he's done, rather, to continue it and supplement it
with a great deal more information, for instance, on some of the physical
properties.
IGNOFFO: Nor did I mean to exclude that as more and more information is
available, you try to characterize it as much as possible and put it into a
form that everyone can use. All I meant to say is that there is an attempt
to catalogue right now on the basis of what information is known insect
viruses as they are found. It's true in some instances it's very fragmentary
and deals with initial isolation but that's how knowledge is accumulated.
HARRAP: The cataloguing that Martignoni has published is formulated on the
basis of virus host. This is the difference we should emphasize, developing
a catalogue formulated on the basis of the virus. The problem is what pro-
perties of the virus do you use? In the last four years, there have been
national attempts to establish an insect virus classification working group.
It has just been very difficult to get it off the ground. Perhaps the idea
of simply filling out a form for a given virus is the best way of starting.
WOOD: In our rush to characterize and identify these viruses, we have ignored
certain considerations. One is the sources of these viruses. In some cases,
a single virus has been isolated independently by several laboratories, or
from a handful of insects of the same species. How many different viruses
are really even in a single insect? How many variants of the same virus are
present? I think we have a real problem here, particularly when we try to
relate our information to potential health hazards.
Cleanup procedures of the virus populations really only occur through
a filtering process as we pass these viruses through insects and tissue cul-
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tures. I think a lot of attention should be paid to cloning these viruses.
We now have sufficient facilities and techniques for cloning of viruses
which we are particularly interested in. We should work with clones particu-
larly in investigating the physical nature of the particles and immunology.
We also have the opportunity of looking at the biological properties of dif-
ferent clones; possibly we will find clones which have a greater efficacy
in terms of biological control.
We've been looking at these viruses chemically and physically, but not
really biologically. We are worried about health hazards primarily, and not
about characterization. We don't really understand what goes on in an insect
cell, as far as what the viruses are doing. So how are we going to know
what's going on in a nontarget cell? What are the possibilities of a poten-
tial hazard with these viruses? I think this is a very important point, and
I think we have to get into insect cells and look at the viruses to see if
there is a potential for abortive infection or an introduction of some of
the viral genome into the host cells.
McCARTHY: Since these are known as insect viruses, what happens when you
put cloned virus back in the lung? How do you know what you put in is what
you get out?
WOOD: Once you have a clone that has a marker with a particular property,
then if you pick up a latent virus, either in the insect as you have to pass
it through, or in tissue culture, you have a tag to know that something's
gone wrong.
RAPP: There is a tendency for investigators to take a virus from its natural
system and to start passing it in the animal or in vitro. What everyone
looks for, of course, is a virulent virus because they are looking for cyto-
pathic effects, they are trying to get something that grows rapidly to high
titer. This is often far removed from the initial organism, but is the one
that is likely to be used as a pesticide. Some attention ought to be given
to looking for markers of attenuation of some kind. If there is an outbreak
in the field, then one can relate back to whether the outbreak occurred
naturally, or whether it is the field virus, that is, a sprayed virus that
produced it. This is something that turned out to be very valuable, for
example, in the polio vaccination campaign in this country.
Furthermore, it seems to me that there should be an attempt made to
keep isolates of viruses close to fresh isolations and prototype viruses
frozen down, so that they can be compared, whether it's polypeptides or a
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restriction endonuclease analysis of DNA, with what has developed subsequently
down the line. You find, for example, if you look for human or animal iso-
lates in this country, fresh isolates are very hard to find. And if you do
find them, most are less virulent, considerably less cytopathic in cell cul-
tures, than those viruses that have been developed to work with. Yet those
may in fact be more dangerous. So the early isolates have to be maintained,
and this is a good time to start freezing down fresh isolates that have not
been passed and to get multiple isolates to compare.
COLLINS: Since we are dealing with the question of how these viruses may
change with passage, one point that has not been mentioned at all today is,
has anyone looked at the status of endogenous virus information in these
normal insect cell lines that are being used to propagate the virus? Is
there any indication of antigen expression related to these viruses of
nucleic acid?
GRANADOS: I would like to comment on possible adventitious agents, myco-
plasma, or viruses in cell lines currently used by insect virologists.
Recently we came across a problem associated with one of the first insect
cell lines that Hink established, the Heliothis zea. IMC-HZl cell line that
is widely distributed in the U.S., and probably abroad, too. We had diffi-
culty growing it, as we did with a similar cell line obtained from Knudson.
We looked at these with an electron microscope and found low levels of infec-
tion with a nonoccluded baculovirus type particle. We infected TN-368 cells
with the virus. We found a very dramatic infection rate to the extent that
within three days postinoculation, all cells were lysed. I am sure that if
people were to take a good look at what is happening to these cultures before
they get into detailed virus work, more of these adventitious viruses might
be picked up.
KNUDSON: I am very glad that Granados has had a chance to look at the H_. zea
cell line. This is a cell line that I first received many years ago while
I was at Oxford, and it is for the very reason that we had difficulty in hand-
ling that cell line that we did not use it for much of our virological work.
We carry a number of the Lepidoptera cell lines and, in looking for
endogenous agents, we have not by any means examined them all. Certainly
in the S^ frugiperda cell line that I work with, I have never encountered
anything by electron microscope. However, we know that is only one level of
sensitivity. On the other hand, Longworth has detected adventitious agents
in the jS_. frugiperda cell line that I sent him.
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STOLLAR: With the mosquito cell lines, there certainly has been clear evi-
dence of unsuspected contamination, for example, in the report by Buckley
and co-workers. This was first demonstrated in EM sections which showed
that their strain of the Aedes albopictus cell line was full of crystalline
aggregates, subsequently shown serologically to be chikungunya virus.
In Australia, Russell Regnery found an Aedes albopictus cell line con-
taminated with what proved to be Semliki Forest virus. In another instance,
we worked in our laboratory with an Aedes aegypti cell line that looked per-
fectly fine. It grew well and there was no suggestion or evidence of infec-
tion, but medium from that culture, when put on the Aedes albopictus cell
line, caused massive cell fusion within two days. Subsequently, we purified
and characterized to some extent an agent that appears to be a togavirus,
but so far it has not been further identified. It does not seem to be an
alpha or flavivirus .
These studies were done with the help of Dr. Casal at Yale. So here
we have an agent not yet identified of which we would not have become aware
because it does nothing to mammalian or chick cell cultures, even at lower
temperatures. It seems, therefore, that the only indicator cell line we have
for this agent from the Aedes aegypti cell line is the Aedes^ albopictus cell
line.
HARRAP: We have detected vast arrays of small spherical particles in T_. ni
cells from Howard Stockdale at Shell. Because of this, little work has
been done with the T_. ni^ system.
SUMMERS: What was the size of that particle?
HARRAP: It was the size of a parvovirus.
SMITH: Experience with vertebrate viruses has shown that superinfection
brings up a line with a particular virus, which may activate an occult,
latent, or even integrated and completely silent virus. Has this been
observed, and is there any way to test for it?
HARRAP: I can't think of an instance in cell culture, but we have an intrig-
ing instance in insects which it might be useful to describe. The Spodoptera
littoralis insect cultures have been growing on a synthetic diet in our
laboratory for some years now. We did not do any cross-transmission tests
with the other Spodoptera viruses at first because we did not have any cri-
teria for checking what we might get out. As soon as we had some biophysical
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data on the viruses allowing identification, we did this work, and sure
enough, the viruses seemed to be cross-transmissible. But then when we
started to look at the products of the cross-transmission tests, surprising
results were found. When we fed Spodoptera frugiperda NPV to our Spodoptera
littoralis larvae, and we looked at the polyhedra resulting from this cross-
infection, they turned out to be Spodoptera littoralis polyhedra by several
criteria; for example, GC content, structural protein profile, and the anti-
gen-antibody precipitation pattern in immunodiffusion tests. Even the pro-
file on the sucrose gradient after alkali release of virus from polyhedra
was characteristic of J5. littoralis virus in larvae killed as a result of
feeding of J3. frugiperda virus. Initially, we thought the _ฃ>_. frugiperda
inoculum must be a mixture of the two viruses, and that perhaps we had some
sort of selection system occurring.
We got an alternative stock of S_* littoralis larvae from Israel, did
the same tests and found that we could not get infection in these larvae.
However, larvae from the two insect cultures died with the same sort of
kinetics with the homologous virus. So then we came to the conclusion that
in some way we had activated virus latent in our original _S_. littoralis
insect culture. We attempted a variety of temperature shifts and other
stress phenomena on the insects but we could not induce them to die of virus.
We have never seen frank expression of disease in the insects themselves,
and we can't detect the NPV in them in any way, but it seems to be there.
We have now achieved the same result by feeding another NPV, that of
the Heliothis armigera, to this insect stock. The difference here between
the two viruses is even more marked. The Heliothis armigera virus is a
singly enveloped rod, and the Spodoptera littoralis is an enveloped bundle
of nucleocapsids; so all one has to do is to run a gradient to find out which
virus is present. It seems as though we may have a model situation here
for looking at a latent or subclinical NPV infection in an insect population.
MCCARTHY: My laboratory is uniquely connected to a group that is carrying
on a lot of field work. They spray 35-acre plots with gypsy moth NPV, and
they are also treating 2- and 4-acre cabbage plots with Autographa califor-
nica and Pieris rapae granulosis. I think the same considerations are
applied for characterizing baculoviruses for instance, serology and DNA
hybridization. Some of these same considerations can be applied to field
formulations. I am an interested spectator, that's all, on these field
problems. But when I see what goes on with the formulations that are made
up, I think it is a good consideration for safety and toxicity regulations.
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The preparations that we use for characterization, and most everybody
does, bear little relation to the preparations that are being used in field
work. Many of them contain a certain percentage of insect parts in the
case of gypsy moth, they are highly allergenic to some individuals, and
they have certain levels of bacteria. Then comes the addition of stickers,
spreaders and UV protectants, and all these are sprayed out into the environ-
ment. I think that quality control regulations have to be instituted along
these lines, and this eventually leads to the quality control of the virus
that you are using in the first place, which in turn leads to the specific
definition of the type of virus that you are using biologically, chemically,
and genetically. So not only does the strict definition of your baculovirus
become important, but that is carried all the way up the line to the use of
that virus in formulation, and it must be strictly defined also.
STOLLAR: Could you fill us in a little on commercial formulation?
MCCARTHY: I am not really that familiar with commercial formulation.
IGNOFFO: Concerning the nonoccluded virus particles from the HZ-1 cell, I
was wondering if any of you have checked for infectivity in noctuids hosts?
GRANADOS: Sometime today, my research assistants have fed a number of Lepi-
dopterous species with this nonoccluded virus. Heliothis zea will be one of
them.
IGNOFFO: Then you are familiar with the particular lines in question that
have been infected with the DNA of the Heliothis zea NPV?
Viruses have been formulated in many different ways. The earliest and
the easiest method was to grind up the virus-infected insect using water as
the carrier. More recently, dry preparations of technical lots of virus
have been prepared, using the following procedures: freeze-drying, vacuum-
drying, acetone precipitation, air-drying, acetone precipitation with various
adjuvants such as lactose, and spray-drying. Spray-drying (with or without
adjuvants) has been successfully used to prepare viruses. Specific adjuvants
are included to increase shelf life, sunlight persistence, sprayability,
wettability, and to increase the physical properties of the virus, thus
making it easier to apply in the field. Preparations of formulated virus
are generally applied, using water as the carrier. Dusts also have been
successfully used in the past. Current commercial preparations of virus are
wettable powders that are applied using water as the carrier.
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STOLLAR: There is no extensive purification of a specific virus?
IGNOFFO: Not to the level of purification required for biochemical or phy-
sical characterization. In the final analysis, if you can ensure that the
virus in an unpurified technical formulation is safe (not toxic or pathogenic),
then this is probably a better measure of potential risk than if you have
tested a purified preparation which in the process of purification may have
eliminated an entity which was unsafe.
STOLLAR: Would the cost of using more extensively purified virus be prohi-
bitive?
IGNOFFO: I am not in a position to answer that. I can give you an opinion
that it would be more expensive, but there are other people from industry
present here who could give you an estimate of the cost of purification.
HOLOWCZAK: If the viruses are so specific and apparently relatively easy to
come by, in theory at any rate, why haven't they, in fact, just taken over
the field and replaced chemical pesticides completely at this point, when
the chemical people have so many.problems facing them, so to speak, in terms
of carcinogenicity, etc.
IGNOFFO: A big disadvantage to viruses vis-a-vis chemical insecticide, which
is probably true of most entomopathogens, is that they do not kill as quickly
as chemical insecticides. A chemical insecticide, because it is a highly
toxic, broad-spectrum agent, could eliminate pest caterpillars within hours
of application. Biological insecticides cannot do this! Currently, most
persons recommend that biological insecticide be used as the primary control
method and that chemical insecticides only be used as a last resort when
the farmer is facing an imminent loss situation. For example, if the farmer
is in a situation where he would lose his crop in a matter of a few hours
to days (the worms are so large that no disease would knock it down quickly
enough), then a chemical insecticide should be used. We are recommending,
however, that if chemical insecticides must be used, that they be prudently
used, used at lower doses and only when no other recourse is available.
HOLOWCZAK: I assume in almost all cases they would have to be applied each
year the viral pesticide each year.
IGNOFFO: Your assumption is partially correct, depending upon the eco-
system, and the level of controls which is required. In a row-crop system
where plants grow at a rapid rate, and crops are harvested each year, you
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would probably have to apply virus each season and maybe several times during
the season. There are other systems, however (for example, in a forest
environment), in which you could introduce the virus and thus induce an epi-
zootic which could prevent damage by the insect.
FALCON: I would like to make comments regarding quality control. One can
have all the necessary quality control in developing a commercial product,
but yet, when it comes time to apply that product all can be lost. Today
most of the pesticide application work around the world necessitates the
use of water. Water may be used in small quantities, of say 5 gallons or
less per acre, or as much as 800 to 1,200 gallons per acre for application
of a pesticide. The source of the water is important as is its quality.
In some situations water is simply pumped from a local stream or a swamp,
and who is to know what is in the water? So you see, quality control extends
far beyond the product.
With regard to the questions of why the viruses have not taken over
and if they are so specific, why are chemical companies having so many
problems? Unfortunately, in our situation, and with the kinds of games we
play, we are continually asking these same questions of ourselves. Some
factors include the existence of our free enterprise system and the fact
that the chemical industry has a considerable amount of resources because
it is selling products that are producing profits based on royalties. They
engage in heavy advertising, a high degree of merchandising, and personal
customer service. It is a very difficult system to penetrate. The system
has developed over time, and it is a way of life that the farmer and other
users of pesticides are accustomed to. On the other hand, when dealing with
baculoviruses or other biological control agents that have little or no
patent possibilities, there is little money involved, advertising is limited,
and there is little publicity. You seldom see billboards saying, "Use para-
site A or Pathogen S. It is good for you, it will save you money!" There
are a few publications, such as Organic Gardening, that expose this kind of
thing, but generally, it is very, very limited. So in a free enterprise
system where merchandising and patent rights and royalties are the dominant
features, the kinds of things we are trying to do are very difficult.
STOLLAR: Dr. Fields will now discuss certain aspects of the genetics of
viruses, and how these may relate to the problems we are discussing today.
FIELDS: I am a geneticist and I should describe what I heard, and what I
have heard so far is about viruses that have a very specific host range and
a very specific ability to replicate that is safe, and certainly, everything
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that we have heard suggests that is true. The reason I think we are here,
in part, is to ask if this is true both for the viruses that have been
developed, for others that will be developed, and also, will it always be
true, or is it true to a certain finite point, or is there a statistical
or biologic reality that there may be a risk beyond what we are aware of?
Really, what I am saying is not, are these viruses safe and stable, but how
do they change? That is, do they change? How do they change? And what
are the strategies that viruses use to either remain the same or become
different?
I am putting it into a theoretical framework, and I would like to come
back to the practical questions as I see them in terms of some points that
I would like to recommend as a geneticist. First, I would like to briefly
outline how all viruses seem to change and describe their general strategies.
Then, I will outline some of the ways that other animal viruses in the last
few years have been shown to use strategies to change. I think it will be
clear that some viruses change often and easily, and therefore, are very
difficult to eliminate think of influenza and other viruses tend to be
much more stable and have fewer opportunities for change, such as polio
virus, for example. I would then like to focus on some genetic issues that
I have heard of in my very short contact with this type of virus.
The first point is general strategies for change. All viruses and all
living creatures either can mutate or recombine, and mutation can involve
anything from point mutations to frameshift mutations in more complex
creatures with chromosomes, to actual loss of chromosomes, deletions, dupli-
cations, etc. Recombination can either be classic recombination with
breakage and reunion of DNA, or copy choice mechanism, or can occur by
mechanism of reassortment; that is, by exchange of segments rather than a
breaking and reunion. The difference is that mutation is actual creation
of new genes and recombination is shuffling around of old sets of genes.
I will briefly outline some major examples from animal virology that
parallel the viruses we've been hearing about. First, there are herpes and
adenoviruses (which are large DNA viruses), which have been clearly shown
in recent years to undergo recombination, and in many ways these are the
viruses that will have to be most closely modeled. Second, there are
viruses such as influenza, reovirus, and the bunyaviruses (which have seg-
mented genomes), which have been shown to use two primary mechanisms
one is that all of these viruses have been shown to various degrees to
undergo very efficient reassortments. In addition, at least reovirus and
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flu viruses undergo mutation as well. For example, influenza virus,
although not absolutely proven, very likely derives the new segments cross-
species; that is, when a new virus comes in, even though influenza is a
human virus, it is very likely that new genes come from other species.
Even though swine flu did not occur this year, pigs, avian species, and other
nonhuman hosts can act as sources for new genes which I think is going
to become at least something to strongly consider when some of the segmented
insect viruses as genetic systems are developed for them. The term anti-
genie shift refers to insertion of new genes, and antigenic drift refers
to the mutational changes that cause less dramatic changes in the protein.
The third type of virus that is relevant to the small RNA linear vir-
uses, such as polio virus, is somewhat controversial. There is evidence
suggesting that they may undergo recombination, but if so, it occurs at a
very low frequency, and it is quite likely that they undergo mutational
changes as part of their way of changing. Certainly for other linear RNA
viruses, data for true recombination, in the sense of DNA, have been very
difficult to get. This system is obviously going to be applicable to that
other group of picorna-insect viruses.
Now in addition to interactions between two parents of the same type
of virus, a second type of genetic interaction that will have to be consi-
dered in insects is between a virus and an unrelated virus. For example, it
is possible to derive recombinants between adenovirus and SV40 virus, and,
in fact, these viruses were recognized as contaminating the early polio
vaccines. The adeno-SV40 hybrid virus is then a recombinant between two
viruses that are very different.
These types of genetic interactions that I have just described are
genetic in the sense that the genes interact directly. There are two rele-
vant interactions that involve gene products that I think are good examples.
One is the formation of pseudotypes. A pseudotype is a virus that has the
nucleic acid of one virus coated in the protein shell of the outercapsid
of a second type of virus. And there are now numerous examples of this
type of interaction most recently many examples studied involving vesi-
cular stomatitis virus, a rhabdovirus, similar in structure to some of the
viruses we have seen, and leukemia viruses. This is important because it
can alter the host range.
Another type of genetic interaction that seemed to stand out in the
presentations this morning was complementation possibly occurring between
particles that have multiple genomes enclosed within a single coat. That
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is what, in the case of paramyxovirus, we would call polyploid or heterozy-
gous particles that have numerous copies of the same genome. These particles
can interact by having the gene product of one defective member of this
mixture interact with the second and change the biologic properties of
perhaps the first. I would like to point out that the polyploidy is certainly
a very general phenomenon among certain types of viruses, and paramyxoviruses
offer some very interesting parallels, particularly with some studies in-
volving the Newcastle disease virus.
Before briefly outlining impact, 1 would like to point out that host
genetics become very important to discussions of viruses that are affecting
cells and hosts, and the possibility of host-range mutants exists. It is
also possible to take the same virus and get different results in perhaps
different cells in the same animal either cell culture system or insect.
This is a very common finding if one takes one virus and infects a cell
population, and then clones the cells that result. One can see a variety
of biologic effects of such a system, even from a single cloned virus.
There are examples of this in SV40.
In conclusion, the ultimate impact that we are concerned about is host
range and virulence. The questions that we are really asking are: What
is the degree of variability, what are the options of the viruses, and how
stable are these in terms of population dynamics, in terms of host range,
and in terms, ultimately, of virulence? We know that mutations occur in
virtually any large population of viruses and living creatures. We know
that interactions can occur between viruses, and it seems to me that the
genetic systems that are going to have to be developed are going to have to
ask and test concerning the broadness of some of these interactions and the
frequency of mutations by different agents.
Again, I would like to emphasize the fact that one of the reasons the
influenza virus is still with us is because it knows how to vary. It varies
both in the individual genes coating for the two capsid proteins, as well
as by the introduction of new genes. Thus, it is an extraordinarily inter-
esting and variable virus that tells us of the potential for change. We
don't really know this yet about any of the insect viruses; moreover, it will
become very important, particularly when one talks about such creatures as
the cytoplasmic polyhedrosis viruses with segmented genomes, which are not
totally relevant here but have been used, as well as the Nodamura virus
with a segmented RNA genome. Host range and virulence can be altered by a
mutation recombinational mechanism.
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Now the practical versus real summary in this issue is that the use of
chemical insecticides coupled with viruses is a very novel approach which
will raise questions to geneticists. I would like to know whether these
are mutagens because by coupling a chemical insecticide with a virus, one
is, in fact, asking to increase the frequency of mutation and to increase
what may in fact be stable under certain circumstances, to an unstable state.
It is very important to know whether chemical mutagens will have mutagenic
properties in agents that at any point may be co-circulating in the population.
I would like to make the following concluding points. First of all,
from everything that I've heard, genetics work is presently highly feasible
for these viruses. Not only is it feasible, but I personally think it is
quite urgent to develop genetic systems in order to begin to have the right
framework to know what questions to ask. The cloning of the virus is feasi-
ble. Cell culture systems are available along with single cycle experiments
for their study. Host range sounds like it may be available, or certainly
the approach is reasonable, and there are numerous biochemical markers that
serve as superb markers for genetics that have been identified by several
of the presentations. It seems that the genetics of at least selected mem-
bers of these viruses would be highly worthwhile to support.
IGNOFFO: There might have been a misinterpretation when I was talking about
chemical pesticides. What I was trying to relate is that chemical pesticides
normally are not used together with virals in an entire system approach.
What the integrated pest management type system should not use are chemical
insecticides, except as a last resort. We know that some of these chemical
insecticides are mutagens.
FIELDS: In some of the discussions earlier, the issue of having either a
chemical pesticide in the population at the same time or possibly combining
the two in a more potent combination therapy has been raised. I personally
would urge caution in terms of the potential mutagenic capability of that
approach.
FAULKNER: You were asking, do viruses change? We've been quite interested
in this sort of thing, and we worked with plaque-purified strains of nucleo-
polyhedrosis virus of ฅ_ ni. We tried to find out what happened when we
passed them serially in vitro and in vivo. And, as you pointed out, we have
to ask which are to be used. For the in vitro passages, we've tended to use
phenotypic markers, such as what we call many polyhedra and the few poly-
hedra produced per cell. We find the insect tissue culture system that we
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work with tends to fade with the production of the few polyhedra type of
strain of virus, which can be plaque-purified and remains FP, as we call it.
This is fairly well established.
We've also run a series of studies where we've picked plaque-purified
many polyhedra virus; that is, the most virulent virus. These plaques were
picked for the highest virulence that we could obtain and we've serially
passed these in the homologous insect Trichoplusia ni. We initially
started with plaque-purified virus. We harvested the polyhedra from insects
four and seven days post infection and then continued that series to see
what happened to virulence. We continued that for 16 passages and found that
there is no significant change in virulence as far as that particular
marker is concerned, having started off with an MP type of virus. We
examined the hemolymph with each stage of this to see if there were a
phenotypic variation; we did not find it. These viruses have been with us
a very long time, but the virus and the host, in this particular instance,
have brought themselves together pretty well, and we are actually working
with probably the best of the virus.
The other point that I would like to make is that you've said we are
at the stage now where we can look at homologous and heterologous virus
interaction, and so on. I think that a number of people are very interested
in this.
Further, I am quite concerned about the people who produce the viral
insecticides commercially because they tend to produce their inocula from
previously killed populations. Now from what I've told you, there probably
is no major change in virulence, but that seems quite unmicrobiological.
With several of the viruses that we are talking about, we could be producing
the seed inocula in tissue culture and proceed from there.
SUMMERS: I'd like to refocus attention here for just a minute let me
remind you that our present plaque assay is a polyhedral plaque assay.
We've identified several problems associated with this today in terms of
whether or not polyhedrin synthesis or polyhedra is a good and/or reliable
marker. It is within those systems that we have characterized and standard-
ized, and we know how TN368 cells respond to a given multiplicity of infection,
etc. However, I think if we are going to address ourselves to the very
problems that you have summarized, we have to do so with marker systems
specific for and at the level of replicating virus. That is, the enveloped
nucleocapsid, and not some gene product of viral infection such as poly-
hedrin, which we've already seen can be influenced by the cell, by the
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environment, perhaps by the virus, even though it is standardized and
reproducible in a given system. I think this is one of our major problems
in screening monitoring and detecting change. We are not quite there yet,
and I would like to see us reemphasize and focus on the need for those kinds
of studies.
FIELDS: I would agree. Also, I think it is very crucial to distinguish
what is phenotypic (something you are scoring as a phenotype) from a direct
assay for the genetic event (the genotype), and whether it is a biochemical
marker for a segment of nucleic acid, or a restriction cut that shows segre-
gation for a particular marker. The ways to test whether that is a pheno-
type or a genotype would be to use genetics, isolate mutants by traditional
means, and see if there is segregation for polyhedra for a particular region
of the genome.
SUMMERS: I would like to emphasize further that we need to take a compre-
hensive approach to this, which involves the restriction endonuclease mapping,
as well as looking at virus-specific proteins or developing plaque assay
systems for the replicating virus. I don't think the focus here today,
although it has emphasized serology and basic studies on virus structural
polypeptides, is at all excluding direct analysis on the viral DNAs them-
selves .
ZAITLIN: I would like to reinforce what Dr. Fields has said with respect
to strategies for genetic change. And I will give as an example some plant
virus work. Of course, it is appreciated that there are arthropod-transmitted
plant viruses which are, of course, very important in nature. There are
examples of what we would term transcapsidation, rather than the formation
of pseudotypes, which tend to extend the host ranges of virus, or at least
can in theory. For example, the barley yellow dwarf virus is rather specific
in that there are a number of species of aphids and a number of isolates
of the virus. The specificity is very great, but in circumstances involving
a mixedly infected plant, it is possible to get the wrong aphid transmitting
the right virus, which is an example of transcapsidation. This virus, of
course, has the wrong protein coat on it. Then you can visualize the aphid
proceeding to some other species of plant that would not normally host this
virus, but it would then get the virus and the virus could not, theoretically,
replicate in that plant.
IGNOFFO: Is there any way you can relate to us the frequency of drift or
shift in the influenza virus, that they could actually measure and determine
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that there is a shift in comparing passage through cells and through an
in vivo system in man?
FIELDS: I think that is a very important question. I am not talking about
passing this necessarily once through man. First of all, the origin of
that example is not known with certainty, that is, exactly where the new
genes come from. There are hypotheses. But it is very clear that every
10 to 15 years, a new gene is introduced, that is the antigenic shift, and
that correlates with pandemic flu influenza. That is the virus as it infects
people, and it has been shown experimentally that you can, in fact, create
new viruses by co-infecting pigs, for example, with different strains. You
can get recombinants between human and pig strains. The concern is do we
know enough, with any virus, when we are putting a segmented virus into
population, when one has other viruses circulating and one has latent vir-
uses to know that the opportunities under unusual circumstances for intro-
ducing new genes will not exist. In fact, one of the most crucial questions
with flu, reo, and with other segmented viruses is where, in fact, does this
occur? We know it can happen and that it correlates with epidemics. The
question here would be, if you have circulating segmented viruses, can they
interact? I think that is a research question. What is the range of
reaction between cytoplasmic polyhedrosis viruses? And then in getting
interreactions, do you change the host range of these viruses, such that
if you used one as a pesticide, would you really be as specific as you
thought?
(Inaudible comments from the audience.)
This is very high frequency in both systems. That is, the frequency
of reassortment is an experiment that has been done both in cell culture
systems and has been done directly by putting influenza virus into the lungs
of pigs (into the respiratory tract), and it's efficient enough so that a
limited number of animals, when infected with both parents, will come out
with recombinants. So it is an extremely efficient phenomenon, and I cannot
say for sure this is happening with the segmented insect viruses, but it's
unlikely that it's not.
SUMMERS: I think we should go further and say that in the case of baculo-
viruses, or other insect viruses, that this need not be considered a detri-
mental or an undesirable property, because some people immediately extrapo-
late that this is akin to mutation to some other terrible form. This in
fact is natural. What we need with baculoviruses are systems to monitor
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these changes and to begin to develop a data base so that when necessary we
then can discuss with specific examples and data an understanding of mutation,
frequency change, antigenic shift, etc.
FIELDS: I think it is absolutely important to emphasize that. We ask these
questions in order to get the data, not to say that because something could
happen under conditions that are perhaps artificial or unrealistic, one
should not get the information. I think that is a very key point that I
agree with.
HOLOWCZAK: First of all, I would like to say that I've been impressed today
by the presentations of some very elegant data studies, and I think the
systems are very difficult. I also believe there are a lot of good data
resulting from these studies. In general, I want to say that what is being
said here by people who are experienced with their vertebrate models is not
being said because we are endowed with some special knowledge, but it is
because vertebrate virology is a little bit older than insect virology, and
we virologists have made a lot of mistakes. Well, in the sense of having a
good tissue culture system in the study of virus, I think invertebrate viro-
logists have made a lot of mistakes. I think what we are trying to say is,
don't make the same mistakes we did. There are a lot of problems. I recall
particularly the experience with trying to control rabbits in Australia
using a vertebrate virus, which essentially failed.
The other possibility is that of developing resistance to the viruses
that you are using. Perhaps someone will address that issue.
I heard Dr. Tinsley speak recently, and he tells me there are no new
ideas for the development of new insecticides. People are pretty well
exhausted in the area of new chemical development; so therefore, we may be
forced ultimately to go to insect control. And, if when we have insects
that are already resistant to the known chemical insecticides we then
develop resistance to viruses, what is left? I think this is something to
consider; perhaps we should be working on a third control mechanism of
chemicals and viruses, which may be important.
The other thing I think which is intrinsic in what is being said here,
is the fact that it has taken a long time for animal virologists to realize
the different modes of interaction of viruses with cells. The fact that
we cannot only have cell killing, but we can go to the transformation of
cells that live and grow; we don't necessarily have to kill a cell. Most
animal virologists don't understand what causes CPE in animal cells; we've
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been looking at it much longer, perhaps been looking at insect cells, and
we are not really too much ahead in that sense. There are, nevertheless,
situations like slow viruses where you introduce the virus, and it takes many,
many years before you see the effects of that virus. And that is something
that concerns everyone, I think.
The other point is that I have attended four or five seminars now on
insect viruses, and one story that is always told is how much virus one
can get from eating coleslaw. Repeated in many of the conference reprints
is the quote, "You can get X number of milligrams of occluded virus by
eating one good helping of coleslaw." We could say, "So what difference
does it make if we spray with these viruses because it is all there naturally,
and we are going to get a huge dose anyway." That really concerns me
because, as it has been pointed out, the use of these viruses is very limited,
and yet they are already present in nature and we are already eating them.
If we begin to spray extensively, the question is, how much will we be get-
ting at that point? As someone pointed out to me, it is difficult to tell
because of the social changes and organic food people who won't wash their
food, much less cook it, we will probably have a problem with them, because
they will be eating an awful lot of virus. Problems like that arise. So
the question is, what will be the antigenic dose that will be permissible
in our population?
Another thing, people are going to develop all kinds of hypersensiti-
vities, which can be very bad in many ways so to speak. The final thing is
that I am continually struck with the similarity between the entomopox
viruses, which reportedly infect only insects, and those pox viruses that
infect mammalian cells. One looks at the virus, DNA is the same size, and
they contain the same enzymes needed for the replication. They seem to
replicate in the same place in the cell, and the question that emerges is:
How is a mammalian virus different from an insect virus?
I think that's a very interesting question. One thing, I was struck
by the fact that vaccinia virus and the pox viruses in general tend to cause
a delayed sensitivity type of reaction after they are injected into humans
and cause T-cells to activate. I wonder if anyone, for example, has looked
at this phenomenon in a rabbit. Using an entomopox virus that you migh1.
have what would interest me would be to have a nice antigen that doesn't
replicate, hopefully, but would induce T-cell activation because it might be
a very powerful tool to study this phenomenon. But it still bothers me that
these viruses are so similar, and yet they are apparently restricted in
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their host range so very tightly, and one wonders if this won't eventually
break down in some way. There will be insect viruses that will affect mam-
malian cells.
COLLINS: I'd like to follow up on one point that may also be relevant for
tomorrow's discussion about safety. But obviously, one of the things that
we are worrying about is what effect these viruses may have on humans that
come into contact with them, if they are used as pesticides. It seems that
we have a very good captive population right now with people who are working
with these viruses in terms of what sort of an immune response do laboratory
workers who work on these viruses rate. I talked with Dr. Harrap earlier
and I think his experience is rather unusual in terms of the type of pattern
with antibody response that he has seen. But I am curious about the experi-
ences of other labs whether they screen personnel and themselves for
antibodies to these viruses and what sort of antibodies are raised.
Obviously, it is very early in the game to know whether they are group-
specific or type-specific, and this is really just a starting stage, but I
think this would be a very good population to look at in this regard.
HARRAP: We started screening our staff fairly routinely some years ago,
and initially, we could pick up reactions to baculoviruses to the inclusion
body protein as an antigen in a fairly small percentage. It was no more
than 10 percent of the personnel in the lab. We do this roughly every six
months, and in the last two screenings that were done, the staff exhibited
no reactions to baculovirus inclusion body protein.
IGNOFFO: This is routinely done in commercial production, and it has been
done for a while. I am recalling one of the first studies of the screening,
which was done in Japan by Dr. Aizawa on people handling silkworm NPV. There
continues to be regular monitoring for clinical serology and tests for
infectivity using the neonatal system (which employs the youngest larvae
possible). All tests have been negative.
SMITH: I think there are a couple of things we can look at in the context of
history that really should be considered, and perhaps this is an appropriate
time for this. In the case of the myxomatosis viruses that have been used
for control in Australia and so on, it is really quite clear that two orga-
nisms are involved. There is the virus and there is the host, and one of the
things that animal virologists have envied about the insect system is the
genetics that are already available with the host, specifically, Drosophila.
It seems inconceivable to me that this experiment hasn't been done. Perhaps
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it has. You simply take a large population of Drosophila and expose it to a
small or even a large population of one of the virus pathogens that is
pathogenic.
(Tape change.)
What other kinds of biological nonchemical treatments are available?
Specifically, a few years ago there were some attempts to use a bacterial
agent that was infectious for insects.
IGNOFFO: Bacillus thuringiensis has been on the market since 1959. At
present, it probably has sales in the United States of about a million
and a half pounds per year, plus or minus 10%. There are other microbial
agents also. There is Bacillus popilliae (a bacterium of the Japanese
beetle). There are fungi, which have been developed in other countries.
Protozoans are also being developed. So, all typical groups of pathogens
have been looked into as potential "microbial insecticides."
FALCON: There are many examples in the literature especially in the first
part of this century involving the utilization of different forms of biolo-
gical and natural controls. Today this is also true in the types of programs
we are attempting to develop that are termed integrated control or integrated
pest management. In these programs the objective is first to use as much as
possible naturally occurring biological controls, and second, to manipulate
them or to somehow increase and enhance them, etc. For example, there are
privately owned operations in the United States that produce and sell living
insects that are used for biological control. Then there are examples of
pathogens, some of which we have coday. Also more recently, insect hormone
and pheromone research has occurred. There is now a commercial product con-
taining a synthesized insect juvenile hormone that is used to control mos-
quito larvae. Thus, there are many possibilities of interesting and useful
substances that may be combined for pest control purposes. However, there
are many problems associated with their development. There is the problem of
registration, and this immediately creates problems for development because
to put together an insecticide that contains several pathogenic organisms
requires first that each pathogen be registered independently. For each one,
safety, efficacy, and usefulness must be demonstrated. Only after this can one
one begin to talk about a package approach. Simply registering one of these
materials today is very costly. As I pointed out earlier, private industry
is not motivated to do it, and until society, the government, and we are
willing to provide funds for that type of research, the activity will continue
to be very minimal. But certainly your suggestion to develop polyvalent
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types of viruses is a very good possibility. Perhaps within two or three
decades, we will be using polyvalent types of viruses instead of chemical
insecticides.
HOOD: Although I am not a virologist, as a spectator of today's seminar
and an employee of EPA who is worried about registration, I would like to
say that it has been a very good day. One of the things I would like to
hear discussed even a few months from now is how EPA should deal with the
capacity for change possessed by these viruses. If, after a particular
virus is registered with certain qualities, it later invokes a large capa-
city for change and begins to do something it did not do originally, a
myriad of problems could develop. This capacity to change may be innocuous
enough, it may not hurt anything, but on the other hand, if it provides a
threat to a pollinator group, or a threat to mammals or people, then how
do we institutionally cope with this? What should we look for? What are
the signposts in a research program that we should find? What is it going
to cost? In which direction should we go?
There was one other comment about the similarities between insect
viruses I think it's a pox virus and animal viruses. I think this is
the kind of thing that is really kind of frightening when we are going great
guns and all of a sudden it jumps the track and we have released a large
amount of viruses with a great capacity to change. I would like to hear some
discussion on this.
FIELDS: I think it is important to comment on this as I think one of the
attitudes one comes in with is to ask questions, some of which, in fact, are
quite unlikely to be directly relevant to what is going on. For example,
genetically, I think it's fair to say that every living creature has some
intrinsic ability to change, i.e., via mutation; there is a background of
mutation rate, there are opportunities for recombination. Now that does not
say, in fact, that this is directly practical or directly relevant to what
is going on. In fact, questions sometimes can be answered only after many
years and often the answer will be exactly what you thought it was in the
beginning. The real issue in raising the question of genetics is the capa-
bility, the feasibility for asking such questions about mutation rate and
gene structure. Strategies of insect viruses now exist so that you can
really begin to learn which are the right questions. The fact that something
can change doesn't mean that this is practically important or that years of
experience with something that show it to be safe should be curtailed neces-
sarily. For example, just because it is possible that that might change,
and we are all living in an era of heightened awareness of unusual happen-
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ings, it is very easy to create a scenario that would scare us and almost
destroy the ability to do anything.
HOOD: I think what I am looking for is, how do we assess this change? You
said some viruses change very rapidly and some are quite stable. I think
what we really need to have is a set of guidelines or criteria, because,
obviously, we need to sort out the stable from the unstable. It's always
going to change, but I guess sometimes we need help on formulating what do
we do next.
FIELDS: Basically what you have done is ask a question that, until the
focusing on genetics had become feasible, one would not know how to ask.
That is, until you know the gene organization, the gene structure, mutation
frequency, and the general strategies, the question cannot in any way be
asked to provoke a good answer. What we both are saying, I think, is that
we need to know more about the genetics of these viruses; then we can begin
to know the right way to ask proper questions. I think that really is the
crucial lesson: not to make, for example, the parallel that I made for
demonstration purposes of influenza, which is an unusual virus, and not to
translate that into insect viruses as they currently exist, but to translate
that into the need to develop a real information base using a few model
systems, so that we can perhaps answer that question a few years from now.
IGNOFFO: The point that Dr. Fields is making is a very good one, because
the kind of questions that you can ask can't be placed in a theoretical or
a meaningful frame. Do we have the techniques to answer those kinds of
questions? I don't think we have for most of these right now. But they
can be developed along the classical lines that have been developed from
mammalian virology.
ZAITLIN: I think in one case we do have the tools to test this. There is
an example given with respect to the similarity between vaccinia virus and
some pox viruses of insects. I wonder if this is really an example of con-
vergent evolution of viruses that, in fact, aren't really related. Dr.
Fields himself gave the example of SV40 and adenovirus, which are very
similar, although they have differences. If you try to do molecular hybri-
dization between the DNAs of those viruses, they won't hybridize, and I can
give an example from our own studies. We work with several strains of
tobacco mosaic virus, and by all criteria you can judge these to be strains
of the same virus, yet when you do molecular hybridization, they do not
hybridize at all. It turns out that these are viruses originally isolated
from quite diverse species of plants, and I think it is an example of con-
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vergent evolution. I think we could test these sorts of things, at least
with some of these marked similarities between the mammalian and the insect
viruses.
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PARTV
SAFETY: A CRITIQUE
-------
Review of Safety Tests and Methods
of Evaluating Infectivity
Clinton Y. Kawanishi, Ph.D.
Environmental Protection Agency
Research Triangle Park, North Carolina
INTRODUCTION
Viral pesticides can be effective alternatives for problem or ineffi-
cacious chemical pesticides under certain circumstances. To register a pes-
ticide with the EPA, the formulation must be evaluated for hazard according
to certain specified guidelines (see Federal Register 40, pg. 26831, June
1975). The data for a viral pesticide must show that it is not a pathogen
of man, other vertebrates, or nontarget invertebrates. The required tests
are presented in the Guidance for Safety Testing of Baculoviruses. It must
be emphasized that it is the formulated product, not the active ingredient,
that is registered for use with the EPA.
ฎ
Presently, there are two registered baculovirus pesticides; ELCAR for
control of the cotton bollworm, Heliothis zea, and the tobacco budworm,
Heliothis virescens, on cotton; and TM Biocontrol-1 for control of the
Douglas fir tussock moth, Orgyia pseudotsugata, by the U.S. Forest Service
(Federal Register 41, pg. 51067, 1976). Many other baculoviruses are
presently being tested for their safety to man and the floral and faunal
components of the environment.
Much of what I present here has appeared elsewhere (1, 2, 3). Some of
the information was presented at the EPA-USDA working symposium on Baculo-
viruses for Insect Pest Control: Safety Considerations (4).
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IN VIVO SAFETY STUDIES
Extensive in vivo tests have been conducted to assess toxicity-patho-
genicity, allergenicity, teratogenicity, and carcinogenicity of invertebrate
viruses to vertebrate species (1, 2, 5). For a partial list of some of the
viruses tested see Ignoffo (1, Table 8). The largest number of tests were
done with the baculoviruses, especially the H. zea nuclear polyhedrosis
ฎ
virus (NPV), the active ingredient of ELCAR .
The range of doses administered in the safety tests of II. zea NPV, as
an example, are shown by Ignoffo (1, Table 4). The number of polyhedra
7 12
applied ranged between 1 x 10 to 3 x 10 per kg body weight of the test
animal. In addition, virus released by alkali from polyhedra, virus from
the hemolymph of infected insects, polyhedrins, and virus DNA were tested.
The species used for the evaluations are listed in Ignoffo (1, 2, Tables 9
and 3, respectively). Tests were performed on rodents, dogs, birds, fishes,
mule deer, and primates, including man. Cross-infectivity studies among
invertebrates species have also been done (6).
Viral inoculation was done by peroral intubation; diet feeding; dermal
and eye application; intradermal, intramuscular, intracranial, and intra-
venous injections; and by inhalation. In none of the studies were acute
symptoms or other indications of baculovirus infection encountered. The
parameters monitored or evaluated in these studies were most commonly:
weight gains, body temperature, external symptoms of infection, activity
changes, necropsy with examination for gross pathology, and histopathology
of selected tissues. For sensitization studies, wheal formation and size,
as well as other dermal manifestations were checked. Tests for antibodies
to the virus administered, observations for polyhedra in test animal excre-
ment, and bioassay for infectivity of selected tissue homogenates were
performed in some cases.
Aside from the baculoviruses, similar experiments were conducted on
mice, rats, and rabbits with some entomopoxviruses and the parvovirus of
Galleria mellonella, the densonucleosis virus.
A picornavirus, Nodamura virus, which was isolated from apparently
healthy Aedes tritaenorhynchus in Japan, caused illness with hind limb paraly-
sis seven to ten days after it was injected intracranially or subcutaneously
into three-day-old mice (7). Evidence also showed that this virus multiplies
in ticks and Indian meal moths. Bailey and Scott reported on the cultivation
of Nodamura virus in the honey bee and greater wax moth (8).
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HUMAN TESTS AND MONITORING OF EXPOSED PERSONNEL
Tests were conducted with the H. zea NPV in humans (9). For five days
"CT
volunteers orally ingested 1.2 x 10 polyhedra in gelatin capsules. Subjects
were given complete physical examinations prior to the tests and nine and
then thirty days after the tests began. Ophthalmoscopy, examinations of
the skin and mucous membranes, percussion and auscultation of the chest,
palpations of the abdomen, search for enlarged lymph nodes, and examina-
tion of the extremities were part of the physical. Weight, temperature,
blood pressure, pulse rate, and respiration were also noted. Urinalysis,
hemoglobin level, hematocrit, differential white cell counts, and differen-
tial cell counts, serum glutamic oxaloacetic transaminase, thymol turbidity,
cephalin flocculation, alkaline phosphatase level were determined. As
before, these studies revealed no evidence of viral infection.
Human exposure to the II. zea NPV during manufacture of the viral pesti-
cide was also monitored (10). The general health of the individuals was
checked and urine and blood samples were also tested. Urine samples were
bioassayed against neonate larvae of H_. zea for infectivity. Sera were
tested for the presence of antigens of intact polyhedra by Ouchterlony
immunodiffusion, the presence of antibodies to partially purified NPV
virions, and hemagglutination titer. No allergic response was noted except
for one case of aggravation of a preexisting asthmatic condition. Clinical
symptomatology or immune response indicating subclinical infection was not
detected. Nor was there any indication of viral material in the blood
according to the tests applied.
Serological studies and survey of laboratory workers exposed to insect
viruses were reported by Tinsley and Harrap (11). None of the personnel
exhibited any symptoms of viral infection from material handled in the
laboratory. Sera were tested against an insect parvovirus, an iridovirus,
a granulosis virus, polyhedrin from an NPV, and a small RNA virus. Anti-
bodies of two individuals against the picornavirus were detected.
IN VITRO STUDIES WITH INSECT VIRUSES
Although they are increasing, the number of in vitro studies dealing
with infectivity of insect pathogenic viruses to vertebrate cells are still
relatively few. Some of the cell types previously tested are shown in
Ignoffo (1, Table 11). Unlike the situation with in vivo studies, some
positive tests have been reported. The majority of reported infections
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of vertebrate cells in vitro used viruses that have been grouped in the
past by invertebrate virologists, often for the sake of convenience, into
an artificial category referred to as nonoccluded or noninclusion viruses.
As you are well aware, this encompasses most of the known animal and plant
viruses.
The death of some chick embryos after inoculation with a baculovirus,
the NPV of the silkworm, Bombyx mori, was reported (12). Chorioallantoic
membrane was inoculated with 3000 rpm supernatant of infected silkworm blood.
Virus could be detected by bioassay in silkworm pupae only up to the second
passage. Because no appropriate controls were described, the effects cannot
be definitely attributed to the virus itself. The author also inferred
changes in the morphology of the polyhedra after alternate passage in chick
embryo and insect pupa. Attempts with turkey eggs using alkali released
particles and polyhedra of the nuclear and cytoplasmic polyhedroses viruses
of the silkworm by Cantwell et al. (13) showed no increase in the rate of
parthenogenesis over controls. Polyhedra were not present in histopathological
preparations of parthenogenetic undifferentiated embryonic membranes or in
the embryonic fluid or membranes after injection.
More recently inoculation of WI-38, primary monkey, porcine and bovine
kidney cultures with purified virions, hemolymph from infected hosts, and
DNA of the NPV of Choristoneura fumiferana, the spruce budworm (14), showed
no cytopathic effects (CPE) attributable to the virus. The uninfected
hemolymph of the host, however, proved to cause CPE in the cultures of these
vertebrate cells. Inoculated cultures were carried through three passages
at 28ฐC or 37ฐC before the experiment was terminated. Cells were carried
through only a single passage in the attempted transfection.
Banowetz et al. (15) reported no pathological changes observable with
the light microscope after five passages of Salmonid cell lines, CHSE-214
and STE-137, treated with infectious hemolymph from Douglas fir tussock moth
NPV infected larvae. Electron microscopic examination of cells, 24 hours
post-inoculation, for viral infection did not reveal any indication of virus.
Growth rates did not differ between treated and untreated cell cultures.
Preparations of alkali-released virions of II. zea NPV, when used to
inoculate African green monkey kidney, human primary embryonic kidney, HeLa,
and WI-38 cells produced no CPE after three passages at 37ฐC. No hemadsorp-
tion of guinea pig erythrocytes or interference with ECHO 11 infection was
observed (16).
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Studies by Mclntosh and colleagues indicate association of NPV with
vertebrate cells. Using polyhedra and alkali released particles of E. zea
NPV, they inoculated cultures of primary human amnion, human foreskin,
human embryo, WI-38, and leukocytes (17). No CPE was detected over a 4-
week period, and viability of inoculated and uninoculated cultures were
similar. Treatment with _H. zea NPV did not increase DNA synthesis in leu-
kocyte cultures. No transformed foci in primary human embryo cultures
occurred, and no evidence for leukocyte proliferation in response to viral
inoculation was noted. When inoculated cultures were assayed for infectivity
to cotton bollworm, however, virus was shown to persist in these cultures
up to 4 weeks. However, immunofluorescence studies with conjugated antibodies
to alkali liberated virus failed to detect virus in primary human amnion
cells. NPV inoculation did not enhance or inhibit SV40 infection of PHA
cells.
In a similar study (18) which used immunofluorescence and autoradio-
graphic criteria, the effects of Autographa californica NPV on a poikilo-
thermic vertebrate cell line were assessed. These viper cells, VSW, which
3
were inoculated with [ H]thymidine-labeled virus exhibited grains mainly
distributed over nuclei. Positive immunofluorescence with conjugated anti-
bodies to "infectious hemolymph taken from Trichoplusia ni" was reported.
The fluorescence was prevented by prior treatment with unconjugated antiserum.
The authors interpreted these findings as evidence that _A. californica NPV
became associated with VSW cells. Granados (19) has electron microscopic
evidence of HeLa and FHM cells uptake of _A. californica NPV in vacuoles
and retention of virus in these structures in undegraded form.
The one successful, but unconfirmed, report of transfection of Fogh-
Lund human amnion cell (FL) with the silkworm NPV-DNA (20) utilized the
hypertonic 2.2 M MgSO^ method (21) at pH 7.4. Nuclear inclusions of char-
acteristic polyhedra shape appeared in a small number of cells without CPE
after 13 days. These bodies appeared only in cultures inoculated with
NPV-DNA and NPV-DNA treated with RNase. Comparable FL cells inoculated with
DNase digested NPV-DNA, calf thymus DNA, virions, or Tris-HCl buffer showed
no such structures. The inclusions were dissolved by NaซCO~; were resistant
to trypsin like polyhedra; were not lipid in nature as they were unstained
with Sudan black; and unlike nucleoli and other normal nuclear components
they were only weakly stained by Giemsa. The alkali-dissolved inclusions
gave a positive ring test with antinuclear polyhedra serum and were infec-
tious to silkworm pupae. However, the results should be viewed with caution
because of the small number of animals used in this bioassay. This repre-
sents the only report of baculovirus replication in vertebrate cells.
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Cell culture studies with other insect viruses have been reported. A
member of Reoviridae, the cytoplasmic polyhedrosis virus of Trichoplusia
ni, the cabbage looper, was tested for its infectivity to HeLa, L, and four
mosquito cell lines. Cultures of these cell lines and TN368, a susceptible
cell line derived from the cabbage looper, were inoculated with homogenates
of CPV-infected T_. ni midguts. No evidence of viral infectivity was observed
by phase or electron microscopy, except in the chick TN368 cells (22).
Roberts and Campbell (23) reported that two entomopoxviruses enhanced
exogenous fusion when virions were centrifuged at 1,000 x j> onto L, HeLa,
and BHK-21 cells. At a multiplicity of 100 virions per cell, the extent of
cell fusion was directly related to centrifugation time. The polykaryocytes
increased up to three hours and began to disintegrate after 20 hours.
Remaining uninuclear cells started to divide at 30-36 hours and reached
precentrifugation numbers at 96 hours. Bioassays indicated most virions
were not cell associated. Fat head minnow and 3T3 cultures were not affected
but cells of five mosquito cell lines and TN368 suffered cell death and
lysis without fusion. BTI-EAA cultures, which support the replication of
one of the insect pox viruses, did not fuse or lyse.
The entomopox viruses also interfered with Vaccinia plaque formation.
Multiplicities of 100 and 1,000 virions/cell reduced plaques by 50% and
90-99%, respectively. Interference was increased by trypsinization of
virions, reduced by UV, and eliminated by heating at 56ฐC for 30 minutes.
Experiments indicated that the interference was not due to infectivity but
to host cell impairment and a normal low level of cell fusion.
Chilo iridescent virus (CIV), a member of the Iridoviridae, was shown
to be capable of infecting viper (VSW) cells (24). Evidence indicated an
increase in viral titer, cytoplasmic DNA synthesis after virus inoculation,
positive immunofluorescence for viral antigen at 24 and 48 hours after
inoculation and EM evidence of cytoplasmic viral assays, and particles with
CIV morphology budding from the cells.
Kolomiets and Alekseenko (25) isolated a picornavirus-like 30-70 nm
isometric virus (probably acute bee paralysis virus) from infected hives.
They propagated the virus in 10- to 11-day-old chick embryos by inoculating
filtrates of dead bees into the chorioallantoic membrane. Pathological
changes in the chorioallantoic membrane and embryos with hyperemia in the
region of the head and neck were frequently observed. The authors were able
to reproduce typical clinical symptoms when pathological material was
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injected into healthy bees. This virus hemagglutinated rooster erythrocytes
and a hemagglutination-inhibition test and immunodiffusion test for virus
were developed.
Nodamura virus, a small RNA virus isolated from inapparent infections
of mosquito, multiplies in BHK and mosquito cell lines without CPE (26).
An invertebrate parvovirus, the Galleria mellonella densonucleosis
virus (DNV), has been reported to adapt to mouse L cell line (27). Infected
cells showed fuelgen positive intranuclear inclusions three to four days
after inoculation, positive nuclear fluorescence, and electron microscopic
observation showed particles in L cells. L cells and rat embryo fibroblasts
culture are reported to form foci of transformation by densonucleosis virus.
DISCUSSION
To date the predominant in vivo and in vitro evidence indicates that
baculoviruses are safe for use as pesticides. However, extrapolation and
usefulness of such data can be hampered by our inability to identify baculo-
viruses. For example, published pictures of the so-called nuclear polyhedrosis
virus of Bombyx mori show both S (28) and M (29) NPV. It is fairly common
to encounter both S and M types of NPV infecting a single species (e.g., 30,
31). The occurrence of heteropolyploids, though as yet not reported, would
not be surprising in mixed infections. Where many isolates of a single
morphological type of NPV from one species of insect exist, the problem is
compounded. This points to the importance of developing specific and sen-
sitive identification techniques.
Confirmation of the replication of _A. californica NPV in JB. mori cells
in the absence of CPE and detectable polyhedrin (32) would indicate the
unreliability of these frequently used indicators of infectivity. Because
hemagglutination activity of NPV appears to be associated with polyhedrins
(33), the use of this test as a criterion of infectivity may also be in
doubt.
Qualitative differences in the neutralizing capability of antisera
formed against occluded alkali-liberated and plasma membrane budded parti-
cles (34) suggest caution in interpretation of tests utilizing antioccluded-
virus sera for detection of virus antigen in heterologous systems.
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The evidence for possible prolonged association of some baculovirus
with vertebrate cells in culture (17, 18) calls for careful and detailed
studies. Effective application of newer techniques used in other branches
of virology (e.g., DNA reassociation kinetics, solid phase radioimmunoassay,
etc.) to study inapparent infections should be most fruitful.
Greater caution is advisable in the consideration of insect viruses
belonging to groups with vertebrate or plant pathogens for use as viral
pesticides. With these agents, tests must not only evaluate the direct
effects and the potential for genetic recombination as suggested (35), but
also the possibility of phenotypic mixing, transcapsidation, and genomic
masking with known pathogens. Additionally, the occurrence of many small
viruses in inapparent infections of insects (36) raises the specter of their
presence as adventitious agents in preparations of other more pathogenic
candidate viruses.
I believe that the EPA must take a more comprehensive position regard-
ing biological pesticides. In particular, our registration requirements
should acknowledge the many different properties of biological pesticides.
With viral agents the concern is clearly infectivity and other potential
effects associated with infections, as acknowledged in the Guidance for
Safety Testing of Baculoviruses (4). This is not to say that toxicity
should be ignored. As mentioned previously, it is the formulated product
that is registered and inadvertent introduction of toxicants during manu-
facture of the pesticide is possible. Because viruses are exogenous sources
of genetic information, which may enter cells and possibly produce effects
detrimental to the organism, perhaps detailed information on the viral
nucleic acid (e.g., molecular weight, configuration, density, base composi-
tion, Tm, restriction endonuclease fragments), as well as biochemical and
biophysical characterization of the polyhedrin and virions, should be sub-
mitted. Although analytical methodologies for identifying the virus or its
components are not required for registration, their availability is essential
should a viral pesticide become suspect in health-related problems. In such
circumstances techniques for detecting the virus, its structural and non-
structural proteins, and viral genome or genomic fragments would be especially
invaluable.
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CONCLUSION
Consideration of an insect virus for use as a pesticide should be done
in a rational, prudent manner. Assessment of hazards and benefits associated
with viral pesticides must be done with full cognizance of virus properties,
awareness of virus-nontarget host cell associations that could occur, and
the realization that viruses, like all living things, change with time.
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2. Ignoffo, C.M. Evaluation of ji^ Vivo Specificity of Insect Viruses.
In: Baculoviruses for Insect Pest Control: Safety Considerations,
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5. Valli, V.E., J.C. Cunningham, and B.M. Arif. Tests Demonstrating the
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Pathology, Queen's University Printing Department, Kingston, Ontario,
Canada, 1976. pp. 445-446.
6. Ignoffo, C.M. Specificity of Insect Viruses. Bull. Entomol. Soc.
Amer., 14:265-276, 1968.
7. Scherer, W.F., and H.S. Hurlbut. Nodamura Virus from Japan: A New
and Unusual Arbovirus Resistant to Diethyl Ether and Chloroform. Amer.
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8. Bailey, L., and H.A. Scott. The Pathogenicity of Nodamura Virus for
Insects. Nature, 241:545, 1973.
9. Heimpel, A.M., and L.K. Buchanan. Human Feeding Tests Using a Nuclear-
polyhedrosis Virus of Heliothis zea. J. Invert. Pathol., 9:55-57, 1967.
10. Rogoff, M.H. Exposure of Humans to Nuclear Polyhedrosis Virus During
Industrial Production. In: Baculoviruses for Insect Pest Control:
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Vail, eds. American Society for Microbiology, Washington, D.C., 1975.
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208
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11. Tinsley, T., and K. Harrap. Serological Studies and Surveys of Labora-
tory Workers. In: Baculoviruses for Insect Pest Control: Safety
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12. Aizawa, K. Change in the Shape of Silkworm Polyhedra by Means of
Passage Through Chick Embryo. Entomophaga, 6:197-201, 1961.
13. Cantwell, G.E., R.M. Faust, and H.K. Poole. Attempts to Cultivate
Insect Viruses on Avian Eggs. J. Invert. Pathol., 10:161-162, 1968.
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Registration as a Viral Insecticide for the Control of the Spruce Bud-
worm - Cytopathogenicity in Mammalian Cells. Report to the Insect
Pathology Research Institute, Sault Ste. Marie, Ontario, Canada, 1974.
15. Banowetz, G.M., J.L. Fryer, P.J. Iwai, and M.E. Martignoni. Effects
of the Douglas-fir Tussock Moth Nucleopolyhedrosis Virus (Baculovirus)
on Three Species of Salmonid Fish. USDA Forest Service Research Paper
PNW-214, 1976.
16. Ignoffo, C.M., and R.R. Rafajko. In Vitro Attempts to Infect Primate
Cells With the Nucleopolyhedrosis Virus of Heliothis. J. Invert. Pathol.,
20:321-325, 1972.
17. Mclntosh, A.H., and K. Maramorosch. Retention of Insect Virus Infecti-
vity in Mammalian Cell Cultures. N.Y. Entomol. Soc., 81:175-182, 1973.
18. Mclntosh, A.H., and R. Shamy. Effects of the Nuclear Polyhedrosis Virus
(NPV) of Autographa californica on a Vertebrate Viper Cell Line. Ann.
N.Y. Acad. Sci., 266:327-331, 1975.
19. Granados, R.R. Personal communication, 1977.
20. Himeno, M., F. Sakai, K. Onodera, H. Nakai, T. Fukada, and Y. Kawade.
Formation of Nuclear Polyhedral Bodies and Nuclear Polyhedrosis Virus
of the Silkworm in Mammalian Cells Infected with Viral DNA. Virology,
33:507-512, 1967.
21. Dubes, G.R. Methods for Transfecting Cells with Nucleic Acids of Ani-
mal Viruses: A Review. Birkhauser Verlag, Basel and Stuttgart, 1971.
pp. 18, 41.
22. Granados, R.R. Multiplication of Cytoplasmic Polyhedrosis Virus in
Insect Tissue Cultures. Abstract in Third International Congress on
Virology, Madrid, Spain, 1975. pp. 98.
23. Roberts, D.W., and D.W. Campbell. Effects of Entomopoxviruses on
Vertebrate Cells: Cytotoxicity and Reduction of Vaccinia Plaque Numbers
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on Invertebrate Pathology, Queen's University Printing Department,
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24. Mclntosh, A.M., and M. Kimura. Replication of the Insect Chilo Irides-
cent Virus (CIV) in a Poikilothermic Vertebrate Cell Line. Intervirology,
4:257-267, 1974.
25. Kolomiets, A. Yu., and P.M. Alekseenko. Diagnosis of Viral Paralysis
in Bees. Veterinaria (In Russian), 8:107-108, 1971.
26. Bailey, L., J.F.E. Newman, and J.S. Porterfield. The Multiplication
of Nodamura Virus in Insect and Mammalian Cell Cultures. J. Gen. Virol.,
26:15-20, 1975.
27. Kurstak, E. Small DNA Densonucleosis Virus (DNV). Adv. Virus Res.,
17:207-241, 1972.
28. Himeno, M., S. Yasuda, T. Khosaka, and K. Onodera. The Fine Structure
of a Nuclear-Polyhedrosis Virus of the Silkworm. J. Invert. Pathol.,
11:516-519, 1968.
29. Raghow, R., and T.D.C. Grace. Studies on a Nuclear Polyhedrosis Virus
in Bombyx mori Cells In Vitro. 1. Multiplication Kinetics and Ultra-
structural Studies. J. Ultrastruc. Res., 47:384-399, 1974.
30. Hughes, K.M., and R.B. Addison. Two Nuclear Polyhedrosis Viruses of
the Douglas-Fir Tussock Moth. J. Invert. Pathol., 16:196-204, 1970.
31. Heimpel, A.M. Safety of Insect Pathogens for Man and Vertebrates. In:
Microbial Control of Insects and Mites, H.D. Surges and N.W. Hussey,
eds. 1971. pp. 469-489.
32. Hoops, P., and M.D. Summers. Recent Advances in Baculovirus Serology:
Radioimmunoassay and Immunoperoxidase Assay. 1978. (This proceedings,
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33. Peters, J.E., and R.A. DiCapua- Specific Monosaccharide Inhibition of
Porthetria (Lymantria) dispar NPV Polyhedrin Hemagglutination. Proceed-
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Queen's University Printing Department, Kingston, Ontario, Canada, 1976.
pp. 323-324.
34. Volkraan, L.E., M.D. Summers, and C-H. Hsieh. Occluded and Nonoccluded
Nuclear Polyhedrosis Virus Grown in Trichoplusia ni: Comparative
Neutralization, Comparative Infectivity, and In Vitro Growth Studies.
J. Virol., 19:820-832, 1976.
35. Longworth, J.F., and P.O. Scotti. Properties and Comparative Aspects
of Small Isometric Viruses of Invertebrates. Proceedings of the First
International Colloquium on Invertebrate Pathology, Queen's University
Printing Department, Kingston, Ontario, Canada, 1976. pp. 30-35.
36. Scotti, P.O., A.J. Gibbs, and N.G. Wrigley. Kelp Fly Virus. J. Gen.
Virol. , 30:1-9, 1976.
210
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DISCUSSION
FIELDS: In the human volunteer studies, were any studies done on viral
persistence in the gut, since many of these viruses in insects seem to attack
the gut cells? You mentioned urine and blood; I did not hear stool fil-
trates in terms of a possible additional site that may be, biologically, a
crucial one. Also, you mentioned a few positive serologic tests, but with-
out any denominators. Could you give us some idea of the number of indi-
viduals involved. Two could be out of a thousand or out of ten, and I think
the numbers would be of different significance.
HARRAP: The Ministry of Overseas Development in the United Kingdom wanted
to use two baculoviruses in Africa for pest control, so it had the toxicity
of the viruses tested at the Microbiological Research Establishment, Porton
Down, Salisbury, United Kingdom. For this work, it used the protocol that
is in the WHO Technical Report No. 531. We provided the purified virus to
do the work. We also indicated to the Ministry of Overseas Development that
we thought several of the testing procedures were a waste of time and that
the whole protocol was deficient in one major respect. There are no tests
in the toxicity testing procedures for replication of virus. With the toxi-
city testing that is done and the numbers that are involved, it is difficult
to see how one can usefully set about this task in a reasonable period of
time. To deal with this situation, all we could think of was to see if we
could recover infectious virus from the test animals into a sensitive cell
culture system.
211
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We knew that the two viruses under the test, those of Spodoptera lit-
toralis and Spodoptera exempta, would grow in one of the Spodoptera cell
lines, that of Spodoptera frugiperda. So I transferred one member of my
staff to the Microbiological Research Establishment on a contract for a
year, and we simply used the autopsy organ material, which had been stored
at -70ฐC for a period of 16 months. We took macerated samples both of con-
trol (nonviral-exposed animals) tissues, and tissues of virus-exposed ani-
mals, and applied both filtered and unfiltered tissue homogenates to the
cells to see if we could recover infectious virus. I realize that this is
a rather crude way of approaching the problem, and I am far from satisfied
with it myself. However, it is better than nothing. This work has been
going on for only about four and one-half months and we are only concerned
at the moment with the first set of toxicity tests the acute oral
which are, in fact, the larger group.
9
In this test, a large quantity (I think it is 40 x 10 purified poly-
hedra) is put by tube into the stomach of rats; thereafter, the rats are
sacrificed at intervals and autopsied. Termination sacrifices are done on
the remaining animals at 21 days. We then took the various organs of the
alimentary tract up to the colon and triturated the tissue. To my surprise,
preliminary work seems to indicate that we could recover virus from several
regions of the gut. This is perhaps not surprising, but it looks as if we
can recover virus into cell culture up to ten days after intubation from
several organs along the alimentary tract.
The intriguing thing about this is not that the virus might have multi-
plied in the gut (these data don't obviously show that), but that apparently
the polyhedra must have broken down in the gut lumen to release virus
particles, which are infectious for the cell culture. You cannot normally
infect cell cultures with inclusion body occluded virus, although I am sure
there are instances in which this might occur. So, maybe virus can persist
in the gut for some time in a form that is infectious for insect cells. I
think I can say that at this stage. (Note added in proof: In a second batch
of animals tested since this meeting took place at Myrtle Beach less con-
vincing results were obtained.)
IGNOFFO: There have been tests on humans using orally administered viruses.
If I recall correctly, Art, you did not take stool samples. In the regular
production of a virus, and I am specifically referring to the people hand-
ling the Heliothis virus, there are routine clinical examinations, that
include blood sampling, urine sampling, and testing for both serology based
212
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on gel immunodiffusion with its inherent problems in detection levels, and
a system, which at that time, we still feel has a high level of sensitivity,
that is, the use of the neonatal larvae.
There has been a study done on the fate of Heliothis virus in the
alimentary tract of rats. A known high level based on LC50 units was
administered to rats while they were kept on their regular diets. The
food bolus passed through the animal, and the rats were sacrificed at regular
intervals so that all, or at least all of the activity that we thought we
put in, could be recovered. At the same time those animals were sampled
for blood and various tissues and organs were sampled. So we do have an
idea of the fate of a virus in the alimentary tract of a mammal, though
unfortunately, not in the human. And if I recall, most of the virus, in
the neighborhood of greater than 90 percent, cannot be found after three
days, unless the animals are starved, in which case the passage of food is
not that quick. So the virus can be retained in the animal for a longer
time.
KAWANlSHI: I pointed out that there was one test that took the human gastric
juices and showed inactivation in vitro. I would also like to point out
that during the registration of a pesticide of a virus, there are a lot more
tests sent in with the application than are available in the normal literature.
213
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Methods of Evaluating the Presence
of Viruses and Virus Components
William Meinke, Ph.D.
University of Arizona, Tucson
Tissue culture, though not necessarily representative of the conditions
of an intact animal body, offers a means to test the safety of insect
viruses before they are released in large numbers into the environment.
One advantage of tissue culture, as compared with studies involving animals,
is that growth conditions of the cells and the virus can be carefully con-
trolled and monitored. The investigator can easily assess whether viral
infection leads to virally induced cytopathic effects and/or viral replica-
tion. More subtle changes, such as cell transformation or chromosomal
alteration, can also be determined.
To date, infection of vertebrate cell cultures, including those of
human origin, with intact baculoviruses, has neither produced viral cyto-
pathic effects nor supported virus growth. However, in one study, isolated
DNA of the nuclear polyhedrosis virus of the silk worm caused production of
characteristic nuclear polyhedral bodies in cells originating from human
amnion. These polyhedral bodies appeared to be identical to those found
in infected silk worms and contained infectious viral particles. These
findings suggest that while baculoviruses may not be infectious for verte-
brates for whatever reasons, their genomes may maintain their fidelity in
infected cells. Whether infecting viral DNA is free in the cell or becomes
integrated into chromosomal DNA is not known.
215
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Because of the possibility that baculovirus genomes may retain their
fidelity in vertebrate cells, studies should be implemented not merely to
detect viral CPE or viral replication in infected cell cultures, but more
importantly, to determine the fate of the virus in infected vertebrate cell
cultures. Specifically, experiments designed to determine the fate of the
viral genome in infected cell cultures should be analyzed. The finding
that viral genes are rapidly degraded in infected cell cultures would
definitely support the notion that baculoviruses pose no serious problem
to vertebrates, including man and domestic animals. However, should viral
genes persist in infected cells, the possibility that certain insect viruses
may be potentially hazardous could not be discounted.
Fortunately, the methodology for evaluating the fate of viral genetic
information in infected cell cultures is relatively straightforward. The
presence of viral DNA in infected cell cultures can be detected by extremely
sensitive DNA-DNA hybridization techniques. The sensitivity of DNA-DNA
hybridization is such that as little as one viral genome equivalent per
5-10 cells can be detected. For these types of studies, two parameters must
be met. First, viral DNA labeled to very high radiospecific activities
(greater than 10 counts per minute per pg DNA) is required to serve as a
"probe" for detection of unlabeled viral DNA sequences in cellular DNA.
Secondly, large amounts of total cellular DNA are required in order to
detect the relatively small viral genome within DNA extracted from the
tremendously larger cellular genome.
For the remainder of the discussion, I would like to present some of
the methods we have used to detect viral DNA sequences in infected cells.
While these studies were not done with baculoviruses, the methods and
results that I will present could easily be applied to a study of the fate
of insect viral DNA in infected cell cultures.
For DNA-DNA reassociation studies, viral DNA with specific activities
greater than 10 cpm/Mg is required. In vivo replication of DNA viruses
in the presence of radioactively labeled DNA precursors generally results in
progeny viral DNA molecules with specific activities less than 10 cpm/Mg.
Accordingly, we have developed methods for labeling viral DNA in vitro by
employing "nick-repair" enzymatic reactions (see Table 1). Viral DNA is
first treated with pancreatic deoxyribonuclease I at ratios of 1:500 to
1:1000 in order to introduce a limited number of single-stranded scissions.
The nuclease is then inactivated by heating to 65ฐ for 10 min. Viral DNA
is subsequently labeled by initiating repair synthesis by addition of
216
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TABLE 1. IN VITRO LABELING OF VIRAL DNA
Purified viral DNA in 0.05 M tris-HCl, pH 7.5, 0.05 M KC1, 0.005 M MgCl2
t
Add pancreatic deoxyribonuclease in 0.05 M tris-HCl, pH 7.5, 0.005 M MgCl2
at a ratio of 1:500 (wt/wt) (DNA to DNase). Incubate at 37ฐC for 15-30 min
to introduce "NICKS."
I
Heat to 65ฐC for 19 min.
Dialyze "nicked" DNA against 0.07 M^ potassium phosphate,
pH 7.4, 0.007 M MgCl2
I
Make DNA 0.1 jjmol 2-mercaptoethanol and 0.005 pmol with each necessary
unlabeled deoxynucleotide triphosphate. Add one or two labeled
deoxynucleotide triphosphate(s).
I
Begin "repair" by addition of 20 units DNA polymerase and
incubate at 15-17ฐC for 60 min.
t
Reaction stopped by addition of Sarkosyl to 1%.
f
Pass reaction mixture through Sephadex G-50 equilibrated with 0.02 M tris
HCl, pH 8.0, 0.001 M EDTA, 0.1% Sarkosyl.
t
Pool labeled DNA peak.
t
Extract with phenol.
t
Precipitate with 2 volumes 95% ethanol.
217
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Escherichia coll DNA polymerase I and the necessary deoxyribonucleotide
triphosphates. Reactions are at 15-17ฐ in order to reduce the possibility
of displacement synthesis. Repair synthesis is terminated by the addition
of Sarkosyl and the DNA subsequently separated from the reaction ingredients
on Sephadex columns. Depending on the type of labeled deoxyribonucleotide
triphosphate employed, specific activities of DNA labeled by these proce-
dures range from about 2-80 x 10 cpm/yg DNA. For example, DNA labeled with
H-TTP generally has specific activities of about 2-6 x 106 cpm/ug while
39 125
utilizing P-TTP or I-dCTP as the labeled precursor yields DNA with
specific activities of 20-80 x 106 cpm/yg.
Our in vitro labeled viral DNA probes generally sediment at about 5.6 S
in alkaline sucrose gradients (Figure 1). This sedimentation velocity value
corresponds to an average single-stranded piece size of about 430 nucleotides,
which also is about the size of an average gene sequence. We have shown
that in vitro labeled viral DNA denatures with the same sharp profile as
observed with viral DNA labeled in vivo, and furthermore, denatured in vitro
labeled viral DNA will reassociate to greater than 90% with normal second
order kinetics.
Thus, in vitro labeled viral DNA should serve as an effective "probe"
to detect unlabeled virus-specific DNA in total cellular DNA derived from
infected cultures. Total cellular DNA can be extracted by a urea-phosphate-
hydroxyapatite column procedure. In brief, cells are suspended in 8 M_ urea,
0.24 M phosphate, pH 6.8, 10~ M EDTA, 1% sodium dodecyl sulfate, 2% isoamyl
alcohol and the cells lysed in a Waring blender (60 seconds, maximum speed).
The solution is applied to a column of hydroxyapatite equilibrated with the
same solution (minus EDTA and isoamyl alcohol). Cellular DNA binds to the
column while cellular proteins and RNA are eluted with several washes.
Urea is then removed by washing the column with 0.14 M phosphate, pH 6.8,
buffer. DNA can then be eluted with 0.48 M phosphate, pH 6.8, buffer
(Figure 2). DNA can be further purified by phenol extraction and then re-
applied to a second hydroxyapatite column equilibrated with 0.14 M phosphate,
pH 6.8, buffer. Following a second elution from the column, the DNA appears
free from protein and RNA contamination. Using these procedures, we usually
obtain about 8 x 10 \ig DNA per mammalian cell (i.e., total cellular DNA).
In order to detect small amounts of viral DNA in cellular DNA, nano-
gram quantities of denatured labeled viral DNA usually are reassociated in
the presence of milligram quantities of cellular DNA. There are several
conditions that will affect the reassociation of the labeled viral probe
to form stable duplex molecules. These include the concentration of DNA,
218
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300
O
E
o 200
o
100
600
E
400 o
o
200
10
20
Fraction no.
30
40
Figure 1. Sedimentation of in vitro labeled viral DNA in alkaline
sucrose gradients. Sedimentation is from right to left in 5 to 20% linear
alkaline sucrose (0.3 N NaOH, l.OMNaCl, 0.001 MEDIA, 0.015% Sarkosyl)
gradients in an SW56 rotor at 15ฐC for 6 hr at 49,000 rpm. , C-labeled
marker DNA 570 nucleotide in average length; oo, in vitro H-labeled viral
DNA.
duration of incubation, salt concentration, temperature, size of the DNA
fragments, solvent viscosity, the guanine + cytosine content of the DNA,
and the nucleotide sequence complexity.
The kinetics of DNA-DNA reassociation follows the equation:
Co/C = 1 + KCot
where Co is the initial concentration of single-stranded viral DNA probe,
C^ is the concentration of the single-stranded viral probe at time J^, and
K. is the reassociation constant. A plot of Co/C versus jt will result in
a straight line if the DNA-DNA reassociation reactions are following second
order kinetics. Should unlabeled viral DNA sequences be present in DNA
extracted from infected cells, the reassociation rate of the probe will be
increased in direct proportion to the amount of unlabeled viral DNA present.
219
-------
O)
700 -
600
160
120
80
40
"" ' ' ' 0.48'M
8M urea * 0.24 M phosphate J 0.14 M phosphate
phosphate"
>, RNA
\t Protein
ป. DNA
40 80 120 160 200 240 280 320 360
Volume (ml)
Figure 2. Hydroxyapatite-urea-phosphate column chromatography isola-
tion of cellular DNA: chemical analysis of fractions. Fractions were
chemically assayed for RNA (FeCl.-orcinol method), DNA (diphenylamine reac-
tion), and protein (Zak and Cohen modification of the Folin phenol-reagent
method).
Thus, from the increased reassociation rates, the total amount of viral
DNA present can be calculated.
The actual methods to follow reassociation of labeled probes are really
straightforward. Labeled viral probes are denatured in the presence of
unlabeled cellular DNA and allowed to reassociate. At various time inter-
vals, samples are removed and applied to columns of hydroxyapatite. Single-
stranded DNA is eluted with 0.14 M phosphate, pH 6.8, buffer while reasso-
ciated duplex molecules are subsequently eluted with 0.48 II phosphate,
pH 6.8, buffer. If the cellular DNA contains viral specific DNA sequences,
the rate of reassociation will be increased over that of control reactions
due to the increase in the concentration of viral DNA sequences.
220
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2.0 ~
20 40 60
HOURS INCUBATION
Figure 3. Reassociation of H-labeled DNA from the human papilloma
virus (HPV) in the presence of unlabeled DNA from HPV infected Wi38 cells.
o
The results are plotted as the ratio (Co/C) of the total H-labeled HPV DNA
o
concentration (Co) to the concentration of single-stranded H-labeled HPV
DNA (c) at various incubation times. Cellular DNA extracted at various
time intervals after HPV infection: , 90 min post-infection; AA, 24
hr post-infection; , 48 hr post-infection; AA, 96 hr post-infection;
nD, 168 hr post-infection;OO> 336 hr post-infection; oo, control
reaction consisting of HPV H-labeled DNA reassociating in the presence of
Wi38 cellular DNA extracted from uninfected cells.
221
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Data derived from a typical DNA-DNA reassociation experiment is pre-
sented in Figure 3. In this case, human cells (WI38) were infected with
the human papilloma virus (wart virus). Like the baculoviruses, the human
papilloma virus (HPV) produced no CPE in infected cells and apparently does
not replicate in cell cultures. The bottom curve shows the reassociation
rate of labeled viral DNA in the presence of Bacillus subtilis DNA (i.e.,
control reaction). The top curve shows the reassociation rate generated
when viral DNA was reassociated in the presence of cellular DNA extracted
immediately after the infection period. From the increased rate of reasso-
ciation, it was calculated that there were at least 27 genome equivalents
per cell 90 minutes after infection. Twenty-four and 48 hours after infec-
tion, there remained 22 and 20 genome equivalents, respectively, per cell.
In fact, viral genetic information could be detected even two weeks after
infection. Thus, while there was no increase in the amount of viral genetic
information in the infected cells, the ability of the viral genes to per-
sist suggests that this virus could remain in a quiescent or latent state
and that some extraordinary event could induce it to either replicate or,
alternatively, to transform cells.
In summary, it may be advisable to utilize DNA-DNA reassociation
experiments to determine the fate of insect viral genetic information in
infected vertebrate cell cultures. These studies could rule out the possi-
bility that certain insect viruses may possess hazardous potentials to
either man or other beneficial living forms.
222
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DISCUSSION
KAWANISHI: Have you tried any of the baculovirus experiments yet?
MEINKE: We are now at the point of in vitro labeling of the baculovirus
DNA. Oddly enough, you'd think that what worked best before would work for
baculovirus DNAs, that a microgram of one should behave like a microgram
of another. They do not, so now we are trying to get a good probe.
IGNOFFO: Can you tell us what your model systems are?
MEINKE: With relation to what?
IGNOFFO: Baculovirus. Which baculovirus and cells are you using?
MEINKE: We are going to use the A. californica baculovirus, and we are
going to use WI38 cells as a representative human cell line.
IGNOFFO: Are you going to use an insect standard cell line also?
MEINKE: Yes, we will use TN368.
223
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Hazard Evaluation for Viral Pesticides:
Test Data Requirements
Reto Engler, Ph.D., and Martin H. Rogoff, Ph.D.
Environmental Protection Agency
Washington, D.C.
Over the last decade, interest in exploiting the pathogenic properties
of insect viruses as pesticides has raised two related issues: one is the
human safety aspect of using insect viruses as pesticides, and the second
is environmental effects on nontarget species. To deal with these issues
one must resolve the problem of which test data requirements would have to
be satisfied in order to register viral products and to promulgate tolerance
regulations for those viruses with a potential for leaving residues in food
and animal feed. The EPA separates these two issues, related to the hazard
assessment responsibilities of the EPA, in order to maintain a proper
perspective and to allow us to assign priorities to the various activities
related to the use of viral pesticides. It is our task at this meeting to
address mainly the second area of concern, the data requirements necessary
for registration and tolerance regulation promulgation purposes.
In order to discuss data requirements from a proper perspective, we
must first clarify EPA's statutory and regulatory mandates as they relate
to registration and tolerance activities. First, a pesticide can be regis-
tered if the EPA can establish a finding (upon examination of supportive
data) that, when used in accordance with its label directions and good
agricultural practices, use of the pesticide is not likely to result in
unreasonable adverse effects on man or on the environment. This concept
does not imply that the EPA guarantees the absolute safety of the pesticide.
225
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However, it does imply that the registration of a pesticide allows its use
under conditions representing a risk to the public which is acceptable to
the administrator. The statute further implies that even pesticides that
pose an unacceptably high risk under general conditions of use may be regis-
tered if they are classified as restricted, which means that they can only
be applied by trained individuals specifically certified as pesticide
applicators.
Similarly, the practice of promulgating tolerance regulations for an
allowable residue or exempting specific pesticides from tolerance regulations
does not imply absolute safety. Establishment of a tolerance implies that
there is a practical certainty that residues of the pesticide below the
tolerance level will not produce adverse effects on the public's health.
Again, the implication is that of an acceptable risk. The concept of incre-
mental risk is also involved in an examination of a pesticide proposed for
registration. Thus, when reviewing a pesticide's supportive data and its
chemical, physical, and biological nature, data reviewers must include evi-
dence that there is a practical certainty that the pesticide will not
produce deleterious effects on the public's health as a result of its use
or due to residues ingested in tolerance situations.
The statute, however, takes one further step, and this step is rein-
forced and formalized in the regulations. The statute holds that in those
instances where risk is demonstrated as unacceptable, such pesticides may
still be registered and tolerances promulgated if benefits from its use
are found to outweigh the risks involved. Such a finding is reached via
the rebuttable presumption procedure.
The preceding indicates that the Agency recognizes a clear distinction
between the universe of data required to support a registration or a toler-
ance and the universe of data that is required to answer the question, "Is
this pesticide safe?" With this background information, we can proceed to
current data requirements for viruses proposed for registration.
Since the early seventies, several attempts have been made to delineate
data requirements necessary to assess the potential hazard posed by the use
of viral pesticides. The publications (1-3) on this subject have the
following two things in common: they are vague, and they address two main
areas of concern the need for precise virus identification methodology,
and the need for an adequate demonstration of lack of infectivity for non-
target species including man.
226
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The following discussion addresses current minimal data requirements
considered adequate for hazard evaluation of viral pesticides. As previously
stated, one should not attempt to relate the utilitarian value of these test
data directly to the value of research studies. Research studies are designed
to answer questions related to determination of the absolute safety of use
of baculoviruses as pesticides. The hazard evaluation consideration is a
dynamic one, and the requirements described will always be subject to change
as the scientific knowledge base expands and alternate and more relevant
approaches are brought forward for consideration.
The first requirement in any application for registration is the identi-
fication of the pesticidal active ingredient (4). It has been stated that
the identification procedures and taxonomic tools applicable to insect vir-
uses need improvement. At present, we cannot state definitely whether
biological, biochemical, or serological methods will be the ultimate identi-
fication tool for regulatory purposes. At this time, the requirement for
identification can be satisfied if the virus is identified by a combination
of valid available methodologies used for virus identification. The use of
a combination of biochemical and biological methods appears to be most
promising (5-7).
In addition to the qualitative identification of the virus, a quantita-
tive description of the virus is required because use rates for the virus
must be established and because of certain regulatory labeling requirements.
In short, an analytical method (in all instances to date, a bioassay) must
be developed. The bioassay is not in actuality an additional data require-
ment, but it is an essential tool required for several regulatory considera-
tions as well as for commercial development of the pesticide. These consi-
derations include quality control and a determination of the stability of
the product, the residual virus levels on food and/or animal feed, and the
fate of the pesticide in the environment (8).
The second requirement in submitting a pesticide for registration is
a proposed label for the product. Although the label addresses many other
aspects of pesticide regulation, such as use directions, rates of application,
and pests controlled, we will only consider those portions of the label
related to hazards and precautions. A complete statement of the ingredients
in the formulation, the active ingredient(s), inerts, and contaminants
must be submitted in order to ascertain whether the inert ingredients used
in the formulation are approved for the intended use. The active ingredient(s)
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are normally listed by weight percent. This poses a problem for biological
pesticides. In the case of biologicals, the bioassay is used as a bridging
tool to relate biological activity to actual weight of the active ingredient.
As in the case of Bacillus thuringiensis, the active ingredient must be
listed in both ways, as weight percent and in terms of biological activity.
Bacillus thuringiensis has an International Unit convention. With JJ.
thuringiensis weight is also under a convention.
The label must also contain the appropriate signal words and precau-
tionary statements derived from the results of the tests which are described
below.
The third submission requirement is related to hazard evaluation. The
testing can be divided into two parts. One deals with the hazard evaluation
on the formulation, the other with the hazard evaluation related to tests
performed on the active ingredient of the pesticide. The hazard evaluation
for the formulation is relatively straightforward. The EPA requires an
acute oral exposure study, an acute dermal exposure study, and primary skin
and eye irritation studies on the formulation. These constitute the full
battery of tests mandated for all products registered. Additional tests
are considered conditional. The acute studies are used to determine the
appropriate label precautionary statements for the product. If the formula-
tion in use can produce a respirable dust, mist, or vapor, an acute inhala-
tion study is also required. If repeated dermal or inhalation exposure is
likely, subacute dermal and inhalation tests and dermal sensitization tests
are required. In short, a viral pesticide as a formulated product has to
undergo the same type of testing as would any other pesticid We empha-
size that these tests do not deal with chronic exposure implications of
actives or inerts but provide the general basis for labeling to protect
users from acute and subacute effects of the formulated product under
expected conditions of handling and use.
The second part of the hazard evaluation testing is carried out on the
virus itself (the active ingredient "technical chemical") and is basically
for chronic effects. The extent of this testing depends on several factors,
including the use pattern proposed for the pesticide and expected environ-
mental effects, if any, upon application of the virus. This testing also
includes determination of natural levels of virus in the environment and
inactivation of the virus in the environment. Last, but of major importance,
evidence must be provided to demonstrate that the virus is innocuous for
nontarget species. We feel that the potential hazard resulting from oral
exposure to a viral pesticide can best be demonstrated by an appropriate
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modified, acute feeding study format. The study should follow a protocol
where relatively high doses of biologically active material are administered
to experimental animals. The protocol must include an investigation of the
stability of the virus in the test animal, rate of inactivation, and the
transfer potential of the virus through the animal or into the tissues of
the animal. In short, even lacking gross infection, invasive and pervasive
properties of the virus should be determined. The study must also determine
whether the test animal experiences any symptoms related to clinical or sub-
clinical infectious diseases in mammals. Classic toxification symptoms are
not expected. Infectious symptoms would include an increase in normal body
temperature and an increase in immunological responses. The selection of
the test species is important because of the well-known refractory proper-
ties of many viral hosts and viral spectrum. Thus, one of the species
selected should be a primate.
In addition, subacute feeding studies should be performed on at least
two species of non-primates using relatively high daily exposures. These
latter studies may not always be necessary, for example, in those cases
where prolonged oral exposure is unlikely, or when other studies indicate
that exposure after viral application is equal to or less than exposure of
the population at risk under natural conditions.
The major hazard of concern with viral agents is infection. Thus, if
infectivity testing provides no overt evidence that the pesticidal virus
interacts with representative vertebrate hosts, the usual studies assessing
chronic effects such as oncogenicity, teratology, and other long-term
effects are not considered as prerequisite test requirements for registration.
Fish and wildlife studies are required depending on the use patterns of
the virus pesticide. These studies include acute and subacute studies in
representative avian species, as well as representative vertebrate and in-
vertebrate aquatic species. The studies must assess acute hazards from
the formulation and the possibility of an infectious process in non-mammalian
species, as opposed to the standard assessment of the virus as a toxicant.
The fourth part of a submission for registration must define biologically
active viral residues in a quantitative manner. The purpose of these studies
is not to establish numerical tolerances for residues, as is the purpose with
chemical pesticides. These studies are intended to provide an understanding
of the fate of the virus in the environment. They should be designed to
reveal what they can about inactivation processes and to quantitate viral
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fate if possible. Submission of evidence that the purposeful application of
virus reduces the total amount of residual virus on food and animal feed
items in the environment is highly significant in hazard assessment in that
the practical certainty that no unreasonable adverse effects can thus be
expected would be considerably strengthened. Environmental residue studies
also serve to fulfill the requirements for environmental chemistry data,
even though the nature of the tests will differ considerably due to the
biological nature of the active ingredient. However, the basic questions
concerning residue quantitation, dissipation rates, and mobility would
apply to a virus as they do to a standard chemical.
The EPA will also consider any other evidence which would contribute
to correctly assessing the hazard posed by viral pesticides. Acceptable
supportive evidence would include recent published or unpublished studies of
the basic virology of the pesticidal virus, or related viruses, or other
investigations relating to the particular virus pesticide. Studies of
interest include attempts to inoculate vertebrate and invertebrate tissue
culture cells using sensitive methods to detect virus-cell interactions,
or studies using unusual routes of administration, such as i.v., i.p., or
even intracerebral inoculations. Basic biochemical studies on viral DNA,
including such identity criteria as annealing or hybridization, may also be
of use in hazard assessment. Although these studies are helpful and are
taken into consideration, they are not considered mandatory regulatory tests.
In accordance with recent advances in molecular biology methodologies,
it is conceivable that insect virus DNA might be hybridized with mammalian
or other DNAs. In a hazard assessment mode, this finding of in vitro
hybridization would not be weighted heavily in terms of risk. On the other
hand, if hybridization with presently available techniques is not possible,
such a finding would be useful in the evaluation.
This is a statement of current general approach. It is neither final
nor static. Hazard evaluation under its present concept is a new art and
will be in a highly dynamic state for the foreseeable future. Therefore,
specific test requirements or test techniques will change as progress is
made and more fundamental understanding of the properties of insect viruses
emerges. Newer techniques and tests must be properly assessed as to their
utilitarian value in the regulatory mode. New techniques will require
standardization with respect to protocol before they can properly become
standard requirements of data to be submitted by a registrant.
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REFERENCES
1. Anon. The Use of Viruses for the Control of Insect Pests and Disease
Vectors. WHO Techn. Rep. Ser, No. 531, 1973.
2. Upholt, W.M., R.A. Engler, and L.E. Terbush. Regulation of Microbial
Pesticides. Ann. N.Y. Acad. Sci., 217:234-237, 1973.
3. Engler, R. Government Regulations of Microbial Insecticides. Devel.
Industr. Microbiol., 15:200-207, 1974.
4. Baculoviruses for Insect Pest Control: Safety Considerations. M.
Summers et al., eds. Am. Soc. Microbiology, Washington, D.C., 1975.
5. Mazzone, H.W., and G.H. Tignor. Insect Viruses: Serological Rela-
tionships. In: Advances of Virus Research, Academic Press, New York,
20:237-270, 1976.
6. Burgerjon, A., G. Biache, and J. Chaufaux. Recherches sur la Specifi-
cite de Trois Virus a Polyedres Nucleaires vis-a-vis de Mamestra
brassicae, Scotia segetum, Trichoplusia ni et Spodoptera exigua.
Entomophaga, 20:153-160, 1975.
7. Harrap, K., C.C. Payne, and J.S. Robertson. Properties of Three Bacu-
loviruses from Closely Related Hosts. Virology (in press).
8. Martignoni, M.E., and P.J. Iwai. Peroral Bioassay of Technical-Grade
Preparations of the Douglas-Fir Tussock Moth Nucleopolyhedrosis Virus.
USDA Forest Service Research Paper PNW-222, 1977.
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DISCUSSION
FIELDS: I have one practical question about primate testing. Do you anti-
cipate any difficulty in obtaining sufficient numbers of primates for test-
ing, considering the difficulties in obtaining such animals?
ENGLER: I don't know the answer to this question, but considering the few
viruses which might be registered, I do not foresee that a vast number of
primates will be needed.
FIELDS: I know, for example, in the vaccine evaluation studies of polio
virus vaccines, and in experimental studies where primates are required,
there really is a current crisis in the availability of such creatures, and
I think it is going to become (since you are talking about subhuman primates
as one of the key experimental hosts that most closely resemble humans) a
very large problem.
RAPP: You are going to have to decide whether you are going to go down
the evolutionary ladder from the chimpanzee, to the baboon, to the rhesus
monkey or the African green monkey, to the marmoset, or wherever you are
heading. As you move further up toward the chimpanzee, things get much
more expensive; and this is in line with your comments that some of the
suggestions have not been specific, and sooner or later someone has to take
this by the tail and decide where and on which animals some of these tests
will be performed. Unfortunately, there probably isn't enough known about
the biology of these agents, especially in noninsect species, to provide
very good clues as to which subhuman primates to use or, for that matter,
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which rodent. Why use the rat, rather than the mouse or the hamster?
Which animal would be the best choice (it might not be the same animal for
a larger number of these different baculoviruses)? We might discuss this
further later on, but this is a very difficult problem. I agree with Dr.
Fields. Many of these animals are on the endangered species list and, for
all practical purposes, are impossible to get. Only a few are being success-
fully bred in this country in limited numbers, like the baboon or the
marmoset, and they are in short supply. If you begin to get into this fairly
extensively, of course, the next question is whether to use newborns, young
adults, or older adults which adds to the problem of cost. You asked a
simple question, but I hope you realize the complexity of it. I don't know
at the moment how one could advise which animal to use for this test.
JOKLIK: I think you gave us a very clear exposition on what the current
regulations are concerning viruses as pesticides as a whole. I don't want
you to get into any scientific discussions but what interests me is what the
mechanism is within the EPA for changing these regulations, for upgrading
them, and so on. Is this done solely within the registration branch of the
EPA, or are outside experts brought in? Is there a mechanism for continuous
upgrading review, evaluation of what is already being done, and so on? How
is this managed administratively?
ENGLER: There is a way to continuously upgrade requirements. We are now
in the process of another reorganization. Subsequently, we will have a
hazard evaluation division and, hopefully, existing in that division will
be a means of seeking outside advice in these areas, not only for viral
pesticides, but for chemical pesticides as well. So I can assure you that
the continuous update of knowledge and research findings will be incorporated
into our evaluation.
SUMMERS: Since the early 70's, I know that you have been actively involved
in writing numerous drafts of guidance testing for biological agents; so I
am more familiar with your historical evolution with this problem than a lot
of people here. It's my understanding that you have reached the sixth
draft, which was published in Baculoviruses for Insect Pest Control; Safety
Considerations, as the guidance for safety testing. And yet, in your safety
testing protocols, I haven't seen any attempt to implement some of the basics
of those recommendations relative to infectivity for viruses. So if you have
a constant mechanism for update, why haven't we seen some reflection of this
in the required safety procedures, at least as they're written into law?
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ENGLER: There is nothing written into law.
SUMMERS: Well, whatever, the Federal Register.
ENGLER: There is nothing in the Federal Register.
IGNOFFO: Could you make a distinction between guidelines and regulations.
ENGLER: In fact, there are no guidelines for safety testing of viruses,
no legally binding guidelines. What is printed in Baculoviruses for Insect
Control is called a "guidance" that was done on purpose.
COLLINS: At least two agents have been registered thus far for use as viral
pesticides. What I would be interested in, and I think might be a lot of
help, would be a review of the testing of these two agents. How the agents
conformed to guidelines at the time they were registered could be examined
to indicate how carefully these guidelines are either being followed or ignored
before registering agents for this kind of use. I think it would be very
useful if we could know what kind of data went into the decision at the time
that these agents were registered.
ENGLER: I would be happy to do that, but I do not think this is an opportune
time.
COLLINS: I just brought it up now so that you would be aware of its useful-
ness .
ENGLER: May I mention two related problems? First of all, the problem which
Dr. Rapp touched on: Is anybody in this room or anywhere else prepared to
say, whether a chimpanzee is a better subject than a marmoset, and, if so,
how much better? These are the questions that we are wrestling with.
The second item is that at the time Heliothis zea NPV, for example, was
registered, we did not know many things we know today about these viruses.
However, a decision was made on the currently available information that the
risk was acceptable. The aspect of changing knowledge has to be considered.
We are still in a developing phase for viral pesticides, much more so than
with chemicals, yet even with chemicals we constantly change some of the
requirements.
COLLINS: I believe the third virus is currently close to registration. It
might also be useful to this discussion if you could tell us what sort of
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data are available on that. This is at a time when these registrations are
under reconsideration.
The other point refers to something you said. If I understand you cor-
rectly, the current guidance situation is that if no signs of infectivity
have been found in nontarget species, and the feeling is that these should
not be looked at for oncogenicity. It seems from what we've heard, in terms
of the biology and some of the biochemical features of these viruses, that
there is that much justification for such an approach. It should be of par-
ticular interest to look at the possible oncogenicity of these agents in non-
target species when you don't find signs of active infection. I think that
a particular guideline, if I heard you correctly, might be worth reconsidering.
IGNOFFO: I would like to make a point about the first questions he brought
up and put it in historical perspective as well. When the attempts were
made to initially register a viral pesticide, EPA did not exist. Registra-
tion was initially done with FDA. Protocols did not exist, so the type of
reasoning that you want to apply has to be placed in the perspective of these
protocols being developed. With a change of reorganization, the kinds of
tests performed were done in consultation with FDA microbiologists and
virologists, and NCI microbiologists and virologists. The ultimate
decision had to be made, of course, on the basis of data that were there.
COLLINS: Has there been any reconsideration or reevaluating of these agents
relative to new guidelines?
IGNOFFO: There has been consideration of reevaluating and redoing critical
tests that were done previously, when and if the sensitive techniques become
available. That has always been a consideration. It has been recommended
from the start that as techniques become more sensitive and more specific,
they should be employed in a monitoring system and also in critical animal
tests; nontarget animal tests should be redone with those new, sensitive
systems.
COLLINS: Should this be done concurrently with the use of these agents, or
should their use cease until these tests are done?
IGNOFFO: You bring up the point that Dr. Fields brought up yesterday. How
certain can you be? We cannot have absolute certainty. So at some point,
we are going to have to make a decision. And that decision can only be
based on the technology that we have and the information we have available
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at that time. We may make a mistake. What certainty do you have that the
vaccines that have been pumped into your children will not develop into
oncogenic viruses in 20 years? Or what reassurance do you have that they
will not develop into any monster?
JOKLIK: This is not really relevant.
IGNOFFO: I believe it is relevant to the point he was trying to make.
JOKLIK: I think we should perhaps discuss that further this afternoon.
Ralph, did you have a comment?
SMITH: It is also something that should invite a more comprehensive discus-
sion. As we heard just now, part of the licensure and registration is an
evaluation of risk. What I think is clear is that we are here to provide
guidance as opposed to guidelines. We might assist both the investigators
and the administrators in some discussion, and perhaps, even a more concrete
formulation of what is an acceptable risk and what is an unacceptable risk.
In other words, one of the things that would assist the administrators, and
even perhaps workers in the field, is that if one incident occurs, this
insecticide should not be used. If that happens, it could be used where
we can perhaps begin to define that if certain things happen there should
be something done.
ENGLER: I think that's a very sensible approach.
HARRAP: Can you clarify a point of information for me? Is it correct that
the only mandatory tests are those required on the formulation, and the tests
required on the active ingredient are conditional and not mandatory?
ENGLER: Yes, in a sense that is correct, the conditional tests are contingent
on the proposed use. If you use your formulation in a little black box,
there are no other than acute tests required. In other words, there is
always a condition for some of the so-called conditional tests.
KAWANISHI: Isn't it correct that the proposed guidelines, published in 1975,
specifically mention formulated product and technical product, at least for
the chemical pesticides?
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ENGLER: That is true. The formulated as well as the active ingredient
has to undergo certain tests. However, the acute tests are and especially
will be only required on the formulation because we need these data for the
purposes of labeling. If the purpose of testing is directed to the overall
safety of the active ingredient, we will have to test the active ingredient.
So again, tests on the active ingredient are conditional, but any time this
pesticide is used outside of a vacuum, some of these conditions will be
invoked. Not all conditional tests will apply to each use; therefore, we
have the apparent ambiguity of conditionality.
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PART VI
PANEL DISCUSSION
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Safety Procedures and Future Recommendations
Moderator: W. K. Joklik, Ph.D.
ENGLER: We consider safety tests a pragmatic approach to the hazard evalua-
tion; furthermore, that the actual tests will not be the only safety con-
siderations. The safety picture of viral pesticides and any other pesticide
is a conglomerate of tests and of considerations, such as use rates, dissi-
pation rates, use location, etc. The actual safety tests are only a part of
the total considerations that are used for making a final recommendation
for registration. After this short preface, I will discuss the pragmatic
approach, which we are presently using.
First, there are four basic acute exposure studies with the formula-
tions. Then we have as many as four conditional acute and subacute tests
on the formulation. These include inhalation, subacute dermal, sensitiza-
tion tests, and the like. Third, we consider feeding tests on the active
ingredient which may be acute and subacute. Depending on the expected expo-
sure, there may also be inhalation tests on the active ingredient at the
acute and subacute levels. What should be emphasized is that especially in
the third category of tests, we are concerned with the invasion and per-
sistence of the virus in the test animal. In the acute tests on the formu-
lation, obviously that is not the most important purpose, but in the tests
on the active ingredient, we are looking for the invasion, the persistence,
and the degradation of the virus as much as technique allows at present.
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JOKLIK: As I understand it, are one and two, in essence, toxicity tests?
ENGLER: That is correct.
JOKLIK: In other words, you feed or administer relatively large amounts of
your formulation to your test animals. I presume these are animals rather
than cultured cells.
ENGLER: These are animals.
JOKLIK: And you observe them over a relatively short period of time for any
adverse effects. Is that correct?
ENGLER: Yes.
JOKLIK: What would the period of time be? Would it be up to a couple of
weeks or a month?
ENGLER: About a couple of weeks. Some of the conditional tests, which may
be subacute dermal exposure tests on the formulation, may continue for as
long as three weeks, with another week for observation. A sensitization test
may be required as well.
JOKLIK: Are serum enzyme elevation tests included, or is that something
different?
ENGLER: You mean antibody?
JOKLIK: No, alkaline phosphatase.
ENGLER: No, that would be included under tests carried out in category three.
JOKLIK: How does three differ from one and two?
ENGLER: For tests under categories one and two, the material is formulated,
and may contain as little as 0.5 percent of the active ingredient. In the
category three tests, the active ingredient or technical material is adminis-
tered to the test animals.
JOKLIK: So in our particular case, it would be highly purified virus?
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ENGLER: It would be what is being proposed to be used in the formulation.
It may be a highly purified virus, it may be virus plus insect fragments,
partially purified insect virus less the fragments, and so on.
(Inaudible.)
ENGLER: Well, maybe the separation into categories is in a sense artificial;
however, the reason I am separating categories one and two is to stress the
point that these tests simply assess the toxicity of the formulation. As
Dr. McCarthy said yesterday that sometimes all kinds of impurities are put
into the formulations, and we need to know what the toxicity of the total
complex of the formulation is the acute toxicity of that complex. If
you have a formulation that has one percent of virus in it, it is somewhat
irrelevant to look for what happens to the active ingredient. One may not
find the needle in the haystack. This is why I am separating the two
approaches, that is, testing on formulation and testing on the active ingre-
dient .
HARRAP: I have in front of me WHO Technical Report No. 531. This is one
of the documents that contains a version of the test protocol. In the acute
oral toxicity test, there used to be quite elaborate procedures for, for
example, organ histopathological examination, a termination report on hema-
tology data, clinical chemistry, gross necropsy findings, microscopic exam-
inations, etc. Is all this still done, or is this detail no longer relevant?
ENGLER: The test you are talking about is identified in category three on
the blackboard, and it is done with the active ingredient.
HARRAP: That is the active ingredient one. It would not be done for the
formulation then?
ENGLER: No. Again, why do it with the formulation in which less than one
percent of the virus may be present?
HARRAP: When you compare the two types of feeding tests, the formulation and
the active ingredient (the virus), you said that the virus need not neces-
sarily be purified. Could the difference between the formulation of the
virus and the active ingredient simply be the presence of the ultraviolet
screening compounds or stickers, and so forth? Or is it a different pre-
paration of viruses taken from the insect?
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ENGLER: The difference between the formulation and the technical material
is that spreaders, stickers, screens, and diluents have been added to make
a formulation, but the technical material of the active ingredient is 100
percent of the technical virus as it is produced.
HARRAP: So, is the requirement that we used to have for that active ingre-
dient being in some purified form now totally dropped because acetone pre-
cipitated slurries of polyhedra were specified originally?
ENGLER: That depends on the particular virus because some virus preparations
in the past the very few that we have registered have been more or
less purified. For example, the gypsy moth at one time had been highly
purified, then they decided to purify it to a lesser degree. In other words,
the tests should be done with that type of material that is eventually used
as a component of the formulation.
HARRAP: 1 can see that when you test the formulation, but I am rather
surprised that you do not do the test on the active ingredient in a more
concentrated or purified virus sample. Any possibility of difficulty with
your formulation would come out in your formulation testing. If you were
going to look for something that is intrinsic to the active ingredient, it
would seem better to just have purer active ingredients to deal with.
ENGLER: It appears that it all boils down to the definition of the active
ingredient and the technical material.
IGNOFFO: We are looking for possible replication and/or development in a
non-homologous host and ways to increase that probability. Dr. Engler is
concerned with registration of products that come off the production line.
From the standpoint of ensuring safety, it is better that the product not
be purified since purification could eliminate extraneous, harmful biotypes.
HARRAP: You just made the point that one of the things we are looking for
in the active ingredient test is some pathological manifestation, and it
would seem to me that you are more likely to get a pathological manifestation
caused by the virus from using the virus alone. That is the only point I
am making. I just cannot see the logic of your distinction between the
formulation and the active ingredient in terms of how you prepare the virus.
COLLINS: Why do these two approaches have to be mutually exclusive? Why
not have three approaches? That seems to be one easy way out, particularly
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concerning the animals used in these tests. Another easy way out would be
to use the active ingredient in its most purified form, then the ingredient
as it goes into the formulation, and thirdly, the formulation. That would
at least give you a distinction between any contaminants in the formulation
that may have toxicity effects, the active ingredient at a concentration
that is somewhat more realistic relative to the formulation, and the intrin-
sic properties of the active ingredient in its purest form.
SHOPE: Regarding tests on the active ingredient, is an immunosuppressed
host or otherwise compromised host used in these tests, in addition to intact
animals with intact immune systems?
ENGLER: We have considered using immunodepressed hosts. However, there
has been no final decision on the true relevancy of such a requirement for
all viruses.
IGNOFFO: If you consider very young mice as immunodepressed hosts, then
there have been tests with viruses. The only other test that I know of with
a biological agent was with a species of nematode which attacks mosquitoes.
This nematode was tested against drug-immunodepressed rats.
SMITH: Who does this testing?
ENGLER: The testing is required from the person or agency that is requesting
registration. This may be private industry, it may be another government
agency, for example, the Forest Service for the tussock moth and gypsy moth.
The actual testing, however, is mostly contracted out by the registrant.
SMITH: Let's say another government agency, which may or may not have the
funds to do the testing, comes to EPA and says, "We would like to use this
as a biological control material." You would not do the testing for them?
They would have to come to you with the data, and if that is the case, who
evaluates their data?
ENGLER: It is true, they have to come in with the data, and we, the EPA, would
then evaluate the data.
JOKLIK: I do understand the difference here. This relates to the testing of
an actual formulation, whereas some of the questions were directed to the
properties of the active ingredient of the virus actually in these formula-
tions, which may require additional testing. To summarize, what total infor-
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mation concerning a formulation is required or has to be on hand before it
is licensed? You said this is the sum total of information that is required.
ENGLER: I have not addressed any auxiliary testing or evidence like environ-
mental persistence studies or fish and wildlife toxicity. Such studies would
be in addition to the ones described so far.
PAGANO: On the acute test, you say there are four. Can you just give us
an idea of what would be four typical acute tests?
ENGLER: Acute oral, acute dermal, dermal irritation, and ocular irritation.
And conditional tests would be inhalation, subacute inhalation, subacute
dermal, and skin sensitization.
JOKLIK: Are there any requirements concerning concentration? How many ani-
mals would be used? And what is the scope of this sort of testing effort?
ENGLER: There are requirements for the number of animals and the number of
test levels. In the particular instance of viral pesticides, probably only
one dose level will be used since the type of formulations we see on viruses
are usually such that the oral toxicity will be more than five grams per
kilogram. If that is the case, one dose level will be sufficient.
JOKLIK: What sort of animals? Does this include both primates, or at least
monkeys, rodents, and does this extend to fish and that type of animal?
ENGLER: No, primates are not included for acute tests on the formulation.
Since you mentioned fish, I should include category four, which includes
"fish and wildlife" toxicity on birds, fish, and aquatic invertebrates.
CURLEY: Could you explain to the group the pathogenic endpoints that result
from each of these tests, regardless of the route of administration, and
whether it is a per os or parenteral route of administration? You mentioned
that these toxicity tests were pathogenic endpoints. I would like you to
tell the group which of these endpoints you evaluate and what you would
consider significant or not significant in your evaluation.
ENGLER: The tests on the formulation are primarily necessary for designing
a label. On the label there will be certain precautionary statements and a
certain signal word, such as "Caution," "Warning," or "Danger." Each toxi-
city category has a certain range within which each formulation must fall,
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based on the acute tests described before. The signal word and thus the
toxicity category are determined by either oral, dermal, eye or skin irrita-
tion; the most severe of these effects determines the required labeling.
HOLOWCZAK: Do all these tests have to be carried out with occluded virus
and free virus, or just with occluded virus? Particularly considering
occluded virus, is there any requirement carried out with tests on free
virus? Or do you just use occluded virus all the time? Since the fates of
these are very different, apparently, in the environment and in the animal,
will the requirement state what will be in the product?
ENGLER: It is what is in the product. If the product contains 70 percent
occluded for 30 percent nonoccluded virus, that is what it is.
KAWANISHI: If, for example, you are studying eye irritation, exactly what
are you looking for? I think what is causing some of the confusion is not
knowing what you are looking for in the tests. Are you expecting the subject
to die?
ENGLER: Well, the acute tests on the formulations are simply designed to
reveal the properties of that particular formulation. If somebody accident-
ally sprays or splashes a chemical on his skin, or gets it in his eyes, we
will have understanding of what might happen to his eyes or to his skin.
That is all the acute tests are designed for. If the rabbit's eyes show
permanent corneal opacity, for example, we should warn the user of this pes-
ticide of the product's potential for causing blindness.
JOKLIK: Have any tests been done on the effect of these agents on plants
rather than on animals or have any tests been done on nontarget insect species?
For example, you may begin to wipe out bees or butterflies.
ENGLER: Although there have been tests done of that sort, I am not that
familiar with them.
JOKLIK: It is not part of the requirement here, is it?
ENGLER: It is not part of the human safety requirement. This is part of the
efficacy and environmental safety. A petition would not get through without
it. Category four includes birds, fish, and aquatic organisms other than
fish. Nontarget insects are included in the efficacy side of the application
because it needs to be shown which insects are susceptible to the virus.
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IGNOFFO: They have been evaluated against a representative species of non-
target beneficial insect parasites and predators in addition to bees, and also
against various species of economic plants.
DIGAPUA: In the acute tests, either on the formulation or on the technical
material, is there any titration done? Or is it a one-dose test?
ENGLER: Titration of how much you give to the animal?
DICAPUA: Yes.
ENGLER: It is a one-dose test, and we have been recommended to use "high
doses," which may be as much as 100- or 10-acre equivalent of the active
ingredient that is used. We felt there was no need for a gradation of expo-
sure if we use as one exposure a rather large quantity. Essentially the
same rationale applies as with the formulation, as I explained a little
earlier. There would be a gradation necessary if a toxic effect is reached.
DICAPUA: So basically, it is a one-level application, and it is based on
what you assume will be put on the field.
ENGLER: Right, on a 100-acre equivalent per man which then is translated
into milligram virus per kilogram body weight.
KAWANISHI: I have had a request for a definition of acute and subacute.
ENGLER: Acute is obvious, that is a one-time exposure. Subacute exposure
is a daily exposure for a number of days, in general, 90 days. There are
some subacute dermal and inhalation tests, which are of a somewhat shorter
duration, about three weeks.
KAWANISHI: I think the Federal Register says something about the subacute,
that it is defined as multiple exposures for less than half the lifetime of
the animal.
ENGLER: That was said at one time and we have wrestled with that definition.
We now define lifetime exposure on one hand and anything less than lifetime
as subacute on the other hand. The lower end of subacute studies is, as I
just mentioned, several weeks to several months.
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IGNOFFO: How do you select a subacute dose when you cannot obtain an acute
dose response? We normally administer the highest dose we can possibly give
an animal. Extremely high doses have been administered over long periods,
sometimes the entire test period, with no observed deleterious effects in
the test animals.
ENGLER: We are dealing with subacute tests. The definition of a subacute
test has nothing to do with the dose. With a toxicant, you necessarily
cannot give an animal an LD50 every day, although there are some toxicants
with which you can do that. The dose, per se, has nothing to do with
the delineation, whether a test is an acute or a subacute test. Again,
we would like to administer as high a dose as possible; practicability and
common sense are, of course, the limitations.
SMITH: In matters like this that are rather important, it seems to me that
everyone would feel better about some of these tests if they were confirmed
by somebody who didn't have a vested interest in producing this pathogen for
us. They make a decision, but whoever is applying for the license brings
the data to them and then sits down and makes a decision; I didn't understand
that they actually confirm the tests themselves.
ENGLER: We make the decision as to whether the test is acceptable; somebody
else, usually a contract laboratory, has performed the test for the regis-
trant .
COLLINS: That's why I hate to be cynical, but if your job or your business
or if your division happens to depend on it if six bugs die one day, that's
going to be a plus-minus test. It certainly would be nice if there were some
kind of neutral group which could at least confirm occasional key tests.
CURLEY: I would like to throw some light on this question since we are the
research and development arm of EPA, the Health Effects Research Lab. It
would be our responsibility, if called upon by the Office of Pesticide Pro-
grams, to test any of these viruses and/or chemical, depending on whether
they have problems identified by the Office of Pesticide Programs. They have
to identify the problem with the chemical or the virus if there is a question
about the data as submitted by industry. They raise the question, and we as
researchers will repeat that study or will commission a study outside the
agency to be done to answer that question. No, we have not tested viruses in
our lab. Dr. Kawanishi is responsible for our work. We are not in the
testing aspect of the problem now.
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DOERFLER: One of your clauses written on the blackboard is to test for inva-
sion and persistence of virus in the test animal. I'm not connected with EPA
at all, but I personally would feel somewhat more satisfied if this were
rephrased to read, "invasion and persistence of virus genes" in test animals.
JOKLIK: We are coming to that. Considering another aspect of this, we now
have a reasonably good idea of which tests and standards are currently
applied to the formulations of viruses that are being considered for licensing.
I wonder if we could focus now more on the actual viruses in these formula-
tions.
Now, viruses, as we know, can cause two types of effects on cells.
First, viruses can cause effects because of the non-nucleic acid constituents
that they contain. We all know that inactive viruses can affect the cells
into which they are taken up. Viruses can affect their host cells without
their nucleic acids expressing themselves at all the toxic effect. And
I think this sort of effect could be tested along these lines, provided that
Dr. Harrap's suggestion is taken up, namely, that the virus be tested in an
optimal diluent or suspension medium, rather than in whatever formulation
is available.
The second effect that viruses have is due to the way their nucleic acid
expresses itself. This was alluded to by Drs. Doerfler and Stollar, and was
not addressed at all in these tests here. I wonder whether we could have some
discussion on what sort of tests might be applied in order to determine how
these viruses express themselves, and the genetic information that they
contain. They probably do not express it completely, otherwise there would
be overt signs of virus multiplication in nontarget hosts, which apparently
have not been detected yet.
But to what extent can these viruses express the genetic information
that they contain with resultant adverse effects on their host cells? One
earlier presentation addressed this type of effect, and that was a descrip-
tion of Meinke's work, in which he develops or probes for the viral nucleic
acid. He can also test whether the viral genome is able to persist and
multiply in cells. Potentially, his test is capable of saying what portion
of the viral genome persists. Additionally he can test, by looking at the
ribonucleic acid, to what extent a DNA-containing virus is transcribed. Can
we arrive at any sort of unanimity, any recommendation that would say that
this type of test should be applied to the viruses, and if so, in what
systems, in what cells, and in what possible animal organs should this sort
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of test be performed? Obviously, one would not be able to do as complete
an analysis, but one would have to select a certain number of cells and the
number of animals.
MEINKE: Primarily, we are contracted to look at mammalian cells. I decided
to look at human cells. Of course, this is only one species and would not
answer the question as to what is happening to other wildlife forms, like
fish or unrelated insects, such as honeybees. You could do these tests ad
infinitum and it could get ridiculous.
I do think it would be a good idea to test certain selected species and
maybe a representative from the major families. In all likelihood, what we
might find is that the genomes are rapidly degraded in these cells. If we
found this, I think it would answer many peoples' doubts and inhibitions about
using these types of vectors. If the reverse were found and sequences per-
sisted, I think we would have to proceed perhaps a little more cautiously.
I do not think you want to make it too rigid because you would have to test
too many species. The obvious benefits for these compounds are very impor-
tant to the economy. I would hate to see an unduly delay, but once again,
one has to consider the safety factors.
HARRAP: Are you going to look for possible integration of pieces of baculo-
virus DNA into the DNA of a target host cell, because we know so little
when discussing anything about replication of these viruses in a sensitive
cell system? I wonder if you are going to start there?
MEINKE: That would be the obvious beginning. I guess it all boils down to
the fact that we know very little about the biology of these viruses. There
is strong evidence that in part of its normal replication, SV40 genes are
inserted into the host chromosomes. It is a normal replication procedure.
We do not know if this occurs in baculoviruses or not.
HARRAP: There is another point, which might be a side issue. In the formu-
lation, you have chemicals, stickers, UV screeners, and so forth. Further-
more, when you put the virus out in the environment, there is the effect of
ultraviolet light (which is one of the inactivators that the agriculturalists
worry about) to consider, which might itself induce mutants. Perhaps we
should think about this.
JOKLIK: Should we concentrate on arriving at a recommendation of a test that
should be carried out? I agree that it is impossible to do everything. But
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it does seem to me that the time is right for trying your type of test in
three or four different types of cells. If the result is positive, one would
then have to go further to see whether the result is adverse. There could
be a bit of baculovirus DNA in human cells without any harm being done. One
would, first of all, like to know what actually happens. We are in a posi-
tion where we can acquire some solid information concerning this aspect, and
I think one has to start somewhere.
COLLINS: One possible approach is based upon some preliminary work that Dr.
Harrap mentioned, which was the question of the persistence of the virus in
the gut of mammals that have been fed test material. I forget whether it
was a formulation or the active agent itself. The data indicated that for
at least ten days or so, virus had persisted in this tissue.
It seems that this would be one approach that would fit in very well
with the safety testing that has already been done, and the animals that are
being used for these tests could be processed in this way. If the virus does
persist in these animals, what sort of immune response occurs? This would
probably be much more relevant to the worries that are obviously in the
backs of our minds. It would also fit in very well with what is being done
already. I do not know if Dr. Harrap or anybody else has any more informa-
tion on this type of approach, but I think that might be one kind of test
that would be very easy to do and that would have a great deal of relevance.
ENGLER: In my presentation, I did not go into details of the way we would
like to see these tests done. In fact, we are requiring this type of interim
sacrifice of animals which were exposed to an acute dose in order to deter-
mine the fate of the virus. This is what I meant with persistence, inactiva-
tion, and degradation of the virus in the animal. Some of these considera-
tions are already included in what is listed here as a category three test,
or acute and subacute testing.
In certain animals, virus has persisted in the gut for some time, al-
though I am not aware that it has persisted for as long as Dr. Harrap has
been reporting. On the other hand, this may reflect a difference in the
detection methodology that was used then, and the one which is used now.
PARTICIPANT: (Inaudible.)
IGNOFFO: Dr. Fields asked a question concerning the fate of virus in verte-
brates and humans and also about the detection level. In a study using rats,
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Heliothis virus was fed via stomach tubes at a rate of about 20 million LD50
units per rat. Animals were sacrificed after various periods of time, and
control tissue was spiked with virus to determine the basal detection level.
The animals were sacrificed at zero minutes, at ten-minute intervals
up to an hour, after two, four, eight hours and then at twenty-four hour
intervals (up to 264 hours). Blood, urine, various tissues, the stomach,
and the alimentary tract were all bioassayed for presence of infective virus.
Using this technique, we could detect about 100 PIBs or about 10 virions.
We feel we can detect about 200 virions using a modified technique and a
large number of test animals. Using the base of 100 percent activity at
zero minutes, about 70-80 percent of the original activity was inactivated
or voided from the intestinal tract after 2 hours. After about 24 hours,
there was less than .005 percent of the original activity. After 48 hours,
it was down to .002 percent, and after 72 hours it was down to less than
.001 percent of the original activity. A value of 0.001 percent was close
to the level of spiked-control samples. All of the tissue homogenates as
well as the urine were negative for the presence of virus.
HARRAP: These were animals that were fed polyhedra?
IGNOFFO: That is correct.
HARRAP: And you were assaying, presumably, for polyhedral inclusion bodies
back into the insect?
IGNOFFO: No. We were able to detect infectious virus, presumably as free
virus particles, infectious viral subunits, or inclusion bodies.
HARRAP: In larvae by feeding?
IGNOFFO: Yes. In fact, we established a temperature-stability curve for
virions by using our sensitive neonatal assay technique.
HARRAP: So when you fed back, how were you certain that you were in fact
assaying free virions? Did you expose the animal to polyhedra and then take
tissue samples?
IGNOFFO: Yes. We were sampling for viral activity per se that would
include polyhedra as well as any other infective unit. The point I wish
to emphasize is that we were testing for the presence of any infective entity.
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HARRAP: Whether the infectivity was from freed virions that might have been
freed in the gut lumen, or whether it was from occluded virus? There is a
slight difference there between what you have done and what I have done,
in that we assume (I think it is generally agreed) that polyhedra will not
infect cell cultures. You need free virions to infect cell culture. Of
course, I was taking tissues from the animals and putting macerates derived
from those tissues back into cell culture and apparently getting infection.
So presumably, the polyhedron dissociated to put free virions in the gut of
the animal. I think I would not, in my system, detect any infectivity due
to polyhedra.
PARTICIPANT: You said you could detect 200 virions?
IGNOFFO: It becomes a numbers game when bioassaying down at a lower level
of detection. I am basing the number of virions we can detect on estimates
per inclusion body. There are about 25 virions per inclusion body. Thus,
we can detect an end point dilution of about ten inclusion bodies (an LC50
detection level is 3 to 5 times higher).
ULVEDAHL: I have a couple of questions for Dr. Meinke. Can the test that
you are developing be performed on a more or less routine basis? How long
does it take to perform one of those probe tests? And what do you estimate
the cost to be?
MEINKE: It is very difficult to estimate costs or time because every virus
that you test probably has different probe characteristics, etc. I have
personally only been involved in this since last November, and we are work-
ing with J_. ni cells 368 and A. californica virus. I have approached this
cautiously; I wanted to work up a quantitation system first, and we now
have a very good plaque assay in the lab.
We are now to the point where we are trying nick-repair of the viral
DNA. To infect cells to get enough cellular DNA, infecting maybe a hundred
cultures or so per virus, extracting the cellular DNA, and doing appropriate
control reactions will be necessary. As soon as we get going we can prob-
ably assay a virus every four or five months. Of course, if someone gives
me purified DNA, I could probably do the whole experiment within four to
five weeks. But now we are starting from scratch, starting our own tissue
culture and preparing our own virus; it is very slow and tedious.
JOKLIK: Dr. Meinke, which DNA is in short supply, the viral DNA, which you
would then label, or the larger, cellular DNA?
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MEINKE: The viral DNA.
JOKLIK: But that should not be such a problem because you could get a bit
of formulation, purify virus from it, and probably make a kilogram without
much trouble. There is plenty of this material available.
MEINKE: Let me point out that I have done all my work in vertebrate virology,
which is really quite simple compared to the invertebrate virology. I point
this out because for a vertebrate virologist working with SV40 to get a virus
9
yield of 10 , or whatever, per milliliter, to go to invertebrate virology, and
get 10 or 10 infectious particles per milliliter is a whole new ball of wax.
For technical reasons, I had decided to do my first experiments with nonoc-
cluded versus occluded virus. In working with nonoccluded virus, low titers
are involved because most of the virus, at least in the T\ ni Autographa
californica NPV system, is, I believe...
JOKLIK: But this is not the point. The reason for the infectivity, why
the titers are so low, is not because the assay system is inefficient.
MEINKE: That is right.
JOKLIK: So you are interested in having the nucleic acid, which is plentiful.
4 5
MEINKE: Well, if you have only 10 and 10 infectious viral particles per
milliliter, there is not much nucleic acid present at all.
JOKLIK: But that is a lot of particles and a lot of DNA, isn't it?
MEINKE: No. It is a very low number, and there is very little DNA.
JOKLIK: I would like to have someone explain that to me.
KNUDSON: May I make a comment and perhaps ask a question of other people who
have worked with these viruses in cell culture? It has been my experience
both with the A. californica and a number of the NPVs not to find titers of
4
10 per milliliter in infected cell cultures. I think a fair figure might be
more in the area of 10 . Would anyone else like to comment on that?
GRANADOS: This is routinely seen in our laboratories with A., californica
virus and I_. ni cells, and in other cell cultures, and the same thing holds
true for entomopoxviruses you get fairly decent yields in the neighborhood
of 107 PFUs per milliliter.
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PAGANO: I mean what everybody's talking about, what Dr. Melnke is getting
at, is that infectious virus is not the point, it is the particle to infec-
tivity ratio that we are trying to get at. What is that?
KNUDSON: As a matter of fact, I have some experience with that using a direct
particle count, and I came up with a figure (this is of a purified virus prep)
in which there is a sonification step involved, so I was not only measuring
my particle to infectious ratio, but the effect of my purification. We came
up with numbers in the neighborhood of 200 to 300 particles per infectious
unit. Dr. Summers has done some direct calculations, and I believe his
figures are a little bit lower, but that has not been taken into account.
SUMMERS: That is just because we do it a little different way. When we
made our calculations they were not significantly different from your results.
So we considered our calculations close enough.
PAGANO: If it is a super-coiled DNA, then you are in a very good position
to purify and recover with very good efficiency (even directly from infected
material). Then you could go on with the labeling.
I would like to help Dr. Meinke clarify his point. You have to divide
this into developmental aspects, but once he has the tests running and he has
the labeled probe, then he can do this test in a matter of days under some
circumstances. We do this with Epstein virus routinely. We accept samples
from all over the world. If you want the complete percentage of the genome
that is present in the tumor tissue or whatever you are analyzing, you may
have to carry on the test for several weeks. But if you simply want to de-
tect presence of viral DNA, then you probably will have an answer in two days.
The tests are strictly controllable; there is no real ambiguity about
them, which is one of the strongest advantages.
There is one other point that keeps coming up about integration. I
think the integration is very interesting as a research problem, but if you
do not find evidence of persistent viral DNA in the noninfectious systems,
which is what we are concerned about, from probe studies, then you know not
to look for integrated sequences because you cannot even identify them. The
more difficult question of deciding about integration does not have to be
approached; it is certainly not the practical level if you do not find
anything persistent.
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MEINKE: An ideal way of doing these experiments is, of course, doing a time
curve in which you take the zero time and find out how many genome equiva-
lents you have put into the cell. Then you just wait and see what happens
to it. Let's say within five hours after infection you no longer find
viral genome sequences, then you have a pretty good indicator that the host
cell is going to degrade it, and there will be no problem with it.
SMITH: I would like to return for just a second to the original discussion.
What kind of guidelines could be applied to where these tests are directed?
I think consideration of these guidelines and their recommendations should
include the biology and actual likelihood of exposure of these agents to
whatever is involved. And, of course, I think that where you have to start
is the insect cell itself. In the homologous system, if the gypsy moth is
being used, what happens to the virus DNA or nucleic acid in the insect or
the gypsy moth cell? The second test system that has to be examined involves
the predators or other natural ecosystems that will be exposed to that virus,
sometimes in a large amount.
Finally, you have to look at either man, vertebrates, or other indicator
systems that indicate whether man is at risk. I think that you are obviously
dealing with limited dollars and limited time, and in some cases, levels of
detection that are relatively sensitive or insensitive. I think these are
the sort of priorities I would recommend.
FIELDS: I thought it might be worth making one point explicitly. The first
issues, as they were outlined, essentially summarized what we have all heard,
underlining the safety tests as they have been done, which have worked quite
reasonably to date. As an editorial aside, I think all of us have been
impressed with the thoroughness with which the work has been done and the
development of this as a system and the enormous amount of data that exist.
Essentially, the issue that has emerged in the last few minutes is trying to
discover how to make feasible more refined probes. Then the distinctions
have to be made about immediate feasibility and immediate applicability to
the issues that are coming up in terms of safety versus the high priority
items for developing the kinds of further, more refined tools for guidelines.
As Meinke presented the data, and as the facts were discussed, it was
clear that there is no absolute timetable. But it is also clear from all
of the comments and concerns that this should be an issue to develop in
terms of immediate priorities, instead of a separate issue from the imme-
diately applicable standards and guidelines. I think it is very important
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to distinguish what is the immediately practical and real first line of
testing, and then we will attempt to refine the issues of safety.
The first issue that certainly involves many of concerns in terms of
persistence, integration, and human effects is the DNA hybridization tech-
niques as they are going to have to be developed over the next year or two.
HOLOWCZAK: I just want to say that if you are going to define a test, then
you are going to have to say something about the kinds of cells that are
going to be used. We have already heard that some of these insect cells
possibly carry latent viruses. We do not want to get into the bag of using
those cells in trying to super-infect it, so to speak, and getting a result
that may be meaningless. Perhaps, since there are people here who are
experienced with these cell lines, they should attempt to define which cell
would be best for use in such tests.
What do you infect with occluded virus, the virus freed from the occlu-
sions, partially degraded virion, perhaps nucleic acid and protein, or
maybe free DNA? All those things should be used to complete an adequate
test. Or should one actually look in the gut of the animal to find out how
the virus is degraded? You might end up with some unusual intermediate
which would gain access to animal cells where something else may not.
I think these are all very important considerations in defining the
tests that could be used. But I do not know if you are going to find any-
thing out from simply throwing occluded virus on cells. From what has been
said, these things do not get into the cells. But apparently, labeled virus
can now be grown; so you could do the straightforward kind of uptake studies
to find out if nucleic acid even gets into the cells, before you begin
hybridization, perhaps, and then go to hybridization studies later. But I
think you have to define which cells you are going to use and how you are
going to apply the virus or subviral particles and make the nucleic acid.
RAPP: There are really two problems. One involves what should be done
now in the light of current information, and the second concerns what should
be done in terms of further developing the research side; obviously, the
two are related. But this discussion has taken the turn that nucleic acid
hybridization may be the cure-all for everything, which it probably is not.
It would seem that one of the problems that has arisen concerns the origin
and genetic make-up of some of these viruses, about which relatively little
is known. That is obviously an area of fruitful further research.
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The second problem is to determine whether these viruses are potentially
dangerous in nontarget tissues. Apparently, this is a very large problem.
And here molecular techniques are valuable, but so are those techniques that
measure RNA, or say, the synthesis of viral protein using the very sensitive
methods that have been developed lately, such as the radioimmunoassay. It
ought to be very easy to develop, as Dr. Summers has already described,
highly sensitive immunologic probes. If you do it Dr. Meinke's way with a
time-curve analysis, it is a very nice intellectual exercise and a nice
research problem. But I don't believe it is really what the EPA needs for
testing viral pesticides.
I think the tests have to be simplified in a rational way, just as the
number of cells and the numbers of animals have to be simplified. Otherwise,
the burden gets so large that the program stops in its tracks, not only
because we do not have the funds, but because there is no available manpower
or time for a great number of tests. One part of the problem that this type
of meeting or any kind of committee meeting like this should address is
minimizing the amount of testing required, while maximizing the information
and the effectiveness of the tests. I think this is one area which should
really be considered by this group.
The next area is research of the future and the kinds of priorities
given to it by various funding agencies. Many of those items are likely to
come up when you find out that a lot of the tests you would like to do and
need to do cannot be done because the basic biological or biochemical
information is not available.
STOLLAR: I would like to make just one technical, but important point. In
looking for the expression of these viruses in nontarget cell lines (work
in vitro), I think it is very important to pay attention to the temperature
at which these experiments are carried out. I think it is unlikely that
these viruses will replicate or express their genome at the normal tempera-
ture for mammalian cells. Perhaps if mammalian cells are used when these
experiments carried out at say 34 to 32 degrees, a great deal of attention
should be directed to using various poikilothermic cell lines at which these
viruses would be more likely to replicate or express their genome.
COLLINS: I think there is one problem with that. If we are using in vitro
tests, particularly in mammalian cells, as valid models for what may be
happening in the field in a natural situation, the viruses are going to
have to adapt and replicate at the body temperature of mammals.
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STOLLAR: But I think to begin with I would look at these temperatures where
it might be more likely, and then probably move up to higher temperatures.
SUMMERS: I would like to get back to what Dr. Rapp said, and what Dr. Meinke
has been trying to get at what cells should be tested, and what would be a
minimum acceptable first attempt?
JOKLIK: It is true that the predator pathway is harmed. Nevertheless, it
seems to me that there is no question that if you spray these viruses from
airplanes, that irrespective of any other way of acquiring them, man will
be exposed to them. What I would recommend is that the minimal test focus
on a human cell. I think the fundamental thing one wants to know is, what
is the fate, the expression, and the effect of viral genomes on human cells?
SUMMERS: I agree with you, but at the same time I disagree. These are in-
vertebrate viruses, and although I consider myself more important than any-
thing else in this world, I think that the possibility for crossing over in
invertebrates is much greater than in man. Now, I agree, we need to go ahead
and test the human cell system, but attention must not be drawn away from
another area that is potentially more realistic in terms of potential infection.
RAPP: The diversity of insects in any given area to be sprayed potentially
causes problems. It would be hard to know where to limit testing procedures.
I suspect it might be a long-range thought, but I am not sure how critical
it is in an attempt to fundamentally protect the human, and perhaps, other
economic aspects of our system. If you tried to protect the whole ecosystem,
the amount of testing that would be involved would be beyond the range of
anything that we could visualize.
SUMMERS: That was not the implication of my statement. If you start with
one human cell line, then you can do the same for one or two other nontarget
invertebrate cell lines.
HARRAP: This is perhaps an unnecessary reminder, but it is worth saying at
this point, that although we may be applying these viruses from airplanes,
large levels of virus can occur naturally in a forest area. Any forest man-
agement authority, and there are some representatives in this room, will
tell us that if there is a large pest situation with a natural epizootic of
these viruses, there are vast quantities of polyhedra in the atmosphere in
the forest anyhow. So it is not as though we are doing something totally
alien. Of course, I realize all the risks and ramifications.
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VOLKMAN: Should port of entry be considered? Do you use lung cells or gut
cells?
JOKLIK: That Is an important point. Could I just come back to Dr. Harrap's
point, which is, of course, a very important one would you extrapolate
from that, and 1 want to play the devil's advocate, seeing that large amounts
of these viruses do occur naturally all the time, and man has been exposed
to them for considerable periods of time with no ill-effects, would you
regard that as some sort of argument that suggests that these viruses really
do not have an effect on man?
HARRAP: It is an argument I consider frequently. Yes, I think one can
base some elements of a case on the fact that there have been a lot of people
involved in veterinary and human virology, and if there had been some clinical
effect of baculoviruses on forest workers, for example, that might well have
been detected by now. I would not like to put undue weight on it, but I do
not think we should lose sight of it.
JOKLIK: I think it is an excellent point. The follow-up question was about
what type of cell to use. Would you like to expand on that?
VOLKMAN: It was just a thought.
GRANADOS: This is somewhat related, and I wanted to come back to this prob-
lem if we are going to understand what is going on in a vertebrate non-
target cell, we obviously are going to need an excellent base line; we have
to know what is going on in the homologous-susceptible system. I wish to
make the point that we still have a long way to go, because except for a small
handful of NPV-type baculoviruses, we do not have good homologous systems
with which to study these viruses. We have no good system for the gypsy moth,
and the same thing can be said about the tussock moth. I am talking about
Autographa californica NPV, _T. ni NPV, and Spodoptera NPVs. No susceptible
insect cell lines have been found for any of the granulosis baculoviruses.
So we definitely are going to run into difficulties when we look at the fate
of granulosis genomes in vertebrate cells. What is going to be our homo-
logous system, what is going to be our baseline for study?
KNUDSON: I would like to re-emphasize that point, which was one of the points
I was trying to make earlier in my presentation on the in vitro specificity,
or host range of these agents. I tried to put forth some sort of methodology,
some sort of mechanism by which we could assess the level of sensitivity in
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the tests, and at the same time, assess the level of susceptibility of a
given cell line, for a given viral agent, in which I have only looked at six
different, or apparently different, NPVs in a handful of cell lines. The
problem really seems that we do not have the baseline data in terms of in
vitro specificity or host range for lepidopterans, let alone the mammalian
or vertebrate systems. First, let's look at things at one level of sensi-
tivity in terms of the detection of the viral-replicated effect or event
in the cell. Then, as new methodologies become available, as they seem
to be coming available, we can refine and improve our sensitivity in our
check, but we do not have the baseline data yet.
IGNOFFO: I want to get back to the original question you posed. What seems
to be coming out of this discussion is that reassociation kinetics is the
best available approach. Do you mammalian virologists agree with that?
MEINKE: No.
IGNOFFO: Well, then, can you tell us what technology can be used to advance
the detection levels beyond those we have dealt with in the past, especially
as it relates to a viral genome in a nonhomologous host?
MEINKE: I do not want to imply that just because I gave a talk on DNA reas-
sociation that it is the very best technique, and I hope it did not come
across in that manner, but it is highly sensitive. You can certainly detect
one viral genome equivalent per cell. Not too many immunological methods can
detect a virus particle per cell. Even though it is a sensitive technique,
I do not want to say it is the only one. We have experts here in other
fields. Let them discuss their areas of expertise.
JOKLIK: Would others like to join you?
FIELDS: Well, since I am not doing the hybridization, I would disagree. I
think you are talking about measurements of DNA and RNA and protein, and
among those there are a variety of systems by which a selected number of
techniques have ultimately become very powerful. Others have more experience
with some of them than I do. But I do think you have to have a focus in
developing DNA hybridization. I think you have to develop a hybridization
analysis for RNA, and then I think when it comes to proteins there are very
sensitive measurements for the gene products in terms of radioimmunoassays.
I think there is enough precedence and a variety of systems that variants
of those three techniques are the ones that should be developed and made
available relatively quickly. There are a lot of techniques that we have
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used and become familiar with, but those are the two in particular, of hy-
bridization and radioimmunoassay for nucleic acid and proteins, that have
turned out to be the most sensitive for answering this particular question
of small amounts of either nucleic acid or protein.
RAPP: The plea I was trying to make was to use them all, because it all
depends on what happens when the nucleic acid gets into the cell. If, in
fact, it is cut and does not do anything, it is going to be very hard to
detect. But suppose it is transcribed and there may be much larger
amounts of RNA there than in the original DNA that is there if that is
translated there may be large amounts of protein compared to either the RNA
or DNA. So an amplification would be possible.
It is impossible to predict, I would think, in advance in these abor-
tive systems, because one could give examples of any one of these circum-
stances as to which technique to use. So the best thing is to simply utilize,
for the three basic compounds one would be looking for, the most sensitive
technology, and for that, nucleic acid reassociation kinetics and the radio-
immunoassay would be the techniques of choice. But I do not think one
would or should exclude any of the other tests.
FIELDS: I agree with that completely.
JOKLIK: Was the technique that Dr. Meinke described, pioneered by Dr. Pagano,
for finding the Epstein-Barr virus genome? That is an extremely powerful
technique. I do not know whether I would subscribe to the fact that cer-
tainly both DNA, RNA, and the radioimmunoassay for protein should be looked
at. Would you subscribe to that, Dr. Pagano?
PAGANO: I was just thinking about the proteins and the RNA. There is a
problem with the proteins. There is one point that has not been made expli-
cit, and that is that we are really concerned about a nonproductive infection.
Let's just underline that, so if you start with the basis of a nonproductive
infection, the DNA, it may- not be expressed in any measurable way. For
example, in the Epstein-Barr virus system, the only product or protein made
is a nuclear antigen. That is an extremely sensitive test in the sense that
if you can find it with a simple immunofluorescence test that anybody can
do, that is fine, but the problem is that you have to have that protein
identified first. And so, whereas you can look for the viral DNA directly
without first having to find which proteins that are novel are made in these
cells, this line of investigation has got to be with the DNA; the RNA would
follow close behind. With luck, there may be a new protein that you can
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identify and also detect. Then you have to select which is the simplest
and most sensitive. But I think it does start with the DNA.
JOKLIK: Now I absolutely subscribe to that. In most transformed cells, cap-
sid antigens do not express themselves. Nevertheless, with these viruses the
only radioimmunoassays that you could devise would be for the capsid proteins
because there is no way of making antibodies for nonvirus proteins. DNA and
its expression, namely, RNA, are the two basic techniques. The radioimmuno-
assay may or may not be useful. I agree that it is most probably amplified.
But at the moment, the only way of doing it would be amplification of a pro-
tein, namely, a capsid protein, which is most likely not to be made.
RAPP: I do not agree.
JOKLIK: Well, in some systems, certainly that would be true, but maybe there
are exceptions. But it would be hard to make the antibody for the right
protein.
FIELDS: It might be worthwhile to point out that the fluorescent antibody
technique which was mentioned is very sensitive and should also be developed.
It also would be a useful other probe for expression in terms of proteins
(both capsid and noncapsid proteins). Could someone comment it is men-
tioned on page 181 of Baculovirusejs for Insect Pest Control: Safety Consid-
erations that "The fluorescent antibody technique, for example, or other
sensitive methods, must be applied to a selected number of exposed tissue
cultures to determine if viral antigens are subunits that would not be
detected by bioassays where produced." The fact that it may be an easier and
more sensitive, in terms of current knowledge, technique to use would be one
point, but the second is to ask if any of this has been followed up or
whether any aspects of this have been developed.
JOKLIK: It is not very sensitive, though. With the RNA, we can certainly
detect one RNA per molecule per cell. But for the fluorescence you need
many protein molecules.
FIELDS: Well, the main point was, historically, if one is looking for evi-
dence of persistence in a nonproducer way, as with the SV40 with adenovirus,
it has been a useful tool. Dr. Rapp, do you want to comment on that in terms
of taking immune sera, and using this as an assay for a protein you may not
have your hands on? The T-antigen is really a good example of one that has
been very difficult to get in terms of the ways we have been talking about,
in the quantity that it is detected.
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RAPP: Of course, the fluorescent antibody tests are not as sensitive, but
you do not need the highly purified probes that you need for the radioimmuno-
assay. You can take a much cruder serum made against a large number of pro-
teins and start a general hunt, if you would like to call it that.
JOKLIK: I think it is undoubtedly a very valuable accessory item.
RAPP: That is right, and the only reason I was disputing the point with you
before was that with all the small viruses the capsid proteins are not made
in transformed cells, but with the larger ones, such as the herpesviruses,
it turns out that in many cell lines they are made.
KNUDSON: I just have one question here I would like discussed, and obviously,
I am not acquainted with the nuances of the significance of certain of these
tests. But what if you do find a genome in the cell, what significance do
you make of that finding? Would you care to discuss that, please?
JOKLIK: Dr. Rapp, would you like to, because you raised the question of
carcinogenicity and teratogenicity?
RAPP: Well, I think that if it is demonstrated that one of the baculoviruses
can exist in a persistent form, whatever that form may be in mammalian cells,
especially if they are human cells, then that would put quite a different
light on safety testing and on whether one is prepared to utilize that parti-
cular reagent in a large-scale testing phase.
Now, it is hard to know what it could do to human cells. For one thing,
not knowing the biology, I would like to know whether these viruses would
have an effect on neural cells. Could they possibly go latent in some cells?
Could they be activated later to cause some unknown disease? I like Dr.
Harrap's point that there are really no good examples of these viruses caus-
ing disease, apparently, in man. But I also have to point out that these
viruses are going to be selected after passage in subculture and that may
change some of their biologic properties. I am convinced, incidentally, I
take it as an article of faith, that the DNA of these viruses will integrate
in certain types of cells. They are, in effect, circular molecules, double-
stranded DNA, seeming for all the world like SV40. There is no reason
whatsoever, given the right conditions and the right cell, that they would
not be able to integrate their genomic material, or at least a portion of it.
If they do that and convert the cells to malignancy in the process, then I
think this should be real cause for much more testing and much thought before
using such an agent in a biological system.
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FIELDS: Well, taking off on Dr. Rapp's last point brings us into the other
category, just briefly, of long-range needs. What Dr. Rapp is essentially
saying is that we still do not know how to formulate the questions
because the biology and genetics of this system remain at a very early
age. Now that does have to be separated from the immediate practical
questions of safety testing and what has to be developed very shortly in
terms of immediate applications for more sensitive probes. But it cannot
be separated from the fact that when you begin to raise these questions
and ask what happens if you get a test that gives a certain result, in order
to really answer the questions that will be raised by anything that turns
out positive, there is no currently available information in terms of the
biology and genetics to use for forming the question.
So that leads to another type of recommendation, a long-term one, that
whatever the political realities, there really needs to be an urging for more
long-term support and attention to the biology and genetics of model systems
in this type of agent.
SMITH: What I thought I might do is discuss some thoughts on these proceed-
ings in three categories. First of all, consider predictions as to how we
might identify some trouble spots. What kinds of things might we envisage
would cause problems? And the top of that list is activation of latent
viruses in invertebrate or vertebrate cells. For example, one might get by
infection of these viruses a new virus which comes out which you do not know
anything about, which then could cause problems. This possibility has ample
precedent in mammalian virology.
The second category is the establishment of persistent infections in
invertebrate or vertebrate cells where you infect the cell with a particular
virus and that virus persists for a long time and causes changes that you
talked about earlier.
The third category is genetic recombination between related viruses
that give rise to new viruses with altered host ranges and other adverse
side effects. Dr. Fields alluded to this. That is, if one considers a poly-
valent material, in which you spray with closely related agents, do those
agents interact with one another to give rise to a new agent that could have
new and unexpected results?
Attenuation is a potential problem. That is, you want the virus to
kill the organism against which it is directed. Well, in the field or in
preparation of the virus you may have the loss of virulence of this material
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and wind up with a very expensive but useless reagent. The other side of that
coin is super-virulence, where you now have, by the propagation or in vivo
passage in the field, a virus which has very unexpected and extremely virulent
reactions.
And lastly, in this category, I think it is very important to pay atten-
tion to those viruses which were termed yesterday, "invertebrate-associated
vertebrate viruses," such as the old category of arboviruses, etc. What is
the exact state of the cell you are dealing with as far as Sindbis and Semliki
Forest virus, and these other viruses which we know grow in insect cells?
The next category is recommendations for future research. At the top
of this list is to characterize the DNA by two methods, restriction enzymes
and hybridization. I think one of the most powerful tools now available in
animal virology is comparing various viruses by their restriction enzyme
patterns. How does virus A differ from related virus B in its pattern of
restriction endonuclease fragments? This can be very, very useful.
Second, to prepare mutants so that you can have markers which you can
follow in a vaccine, or in this case, a deliberately administered virulent
virus. These can be temperature-sensitive or host-range mutants, which
would be useful.
Third, to define the replication of baculoviruses, particularly. I am
encouraged and impressed by the amount of material that is available on
baculovirus structure, but I am also quite concerned that very little is
known about how these viruses replicate and many, many details of the repli-
cation cycle are completely unknown.
Fourth, these viruses, as much as possible, have to be plaque-purified,
biologically purified as much as possible, so that you can work with defined
agents, reproducible from one laboratory to the next, dealing in viruses in
such a way that they are well characterized.
Then, fifth, to continue the development of tissue culture systems
and this will have a variety of ramifications first of all, for virus
production itself. I think most everyone agrees the ultimate production
method for these viruses will be in tissue culture. While, as we understand
now, baculoviruses do not grow that well for field administration in tissue
culture, the nonoccluded virus is not the advantageous virus for spreading in
the field. So you need to develop some type of technique for preparing
occluded viruses.
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Second, tissue culture is very useful for virus purification. When
you want to use a defined agent, you want to get it away from all the other
viruses that may exist in the field, since the squashed bugs that we have
heard about can have other viruses. You get away from crude preparation
by growth in tissue culture, and this requires further development of tissue
culture systems.
And last, tissue culture needs to be continued to develop, to really
understand the growth cycle of baculovirus and other invertebrate viruses.
The last thing in this particular recommendation for future research is that
I am impressed with the production of polyhedrin and granulin as markers of
these particular viruses. They seem to be rather unique and biologically
interesting proteins.
Second in this category is to test in a wide variety of cells. I
have already alluded to the fact that I think invertebrate cells, poikilo-
thermic cells, and mammalian, specifically human, cells, should be used.
And last, most of the tests for replication that I have seen so far
applied have been rather unsophisticated. When one looks for replication in
new cells, one necessarily has to look for production of new virus. That is
the ultimate test for replication and this almost always implies the use of
labels. Could you uniformly apply tritiated thymidine or other nucleic
acid precursors to test for the appearance of new virus?
Most of the tests I have seen applied have used the sole criterion of
new polyhedra formation. You look for the formation of polyhedra in bat
cells or other cells; however, polyhedra formation may be a host-specific
response in that you may find these viruses only form polyhedra in insect
cells. And if you put the same virus in many other species, do these cells,
in fact, form polyhedra? Since we know that, at least it is assumed that
polyhedrin is a virus-specified product. The polyhedron is a relatively
complicated structure and it may be there is not the proper host material
available for its synthesis.
RAPP: I would just like to raise one other issue for future research because
I think it bears very heavily on the practical use of viral pesticides. I
have not heard much about it except for the one study that Dr. Ignoffo men-
tioned, and that is the whole problem of resistance in the field. If, in
fact, insects develop resistance to chemicals, and they obviously do, and
they are like other biological materials, then clearly, they are going to
develop resistance to viruses.
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The question that arises is whether a combination of chemicals would
prevent that phenomenon. I believe the Forest Service has been spraying and
may have some practical field experience, although I suspect it will take
some years for development of resistance, as it took Neisseria some years to
develop resistance against penicillin.
We know from a lot of other experience that it is bound to develop,
and the idea is to try and abort it, if possible.
JOKLIK: Would any of the forestry people like to respond to that?
CUNNINGHAM: In the late 1930's, Diprion hercinia was an important sawfly
pest from Canada. It was accidentally introduced in Europe, and it came
over here without any of its parasites or viruses. In fact, the spruce in
eastern Canada was in great danger. About 1939, a new virus was introduced
along with parasites from Europe, and it spread throughout eastern Canada
over a period of 10 to 12 years. We still have it, and it is an absolutely
perfect example of biological control in that we still have the pest at low
level, it is of no economic importance, and we still have the virus at low
level. There is no indication that there has been any change in the resis-
tance of the insect or the virulence of the virus over a period of approxi-
mately 27 years. From the point of view of resistance we are probably
looking at a short time because the other viruses we have been working with
for only seven or eight years, and there is no indication of change of the
LD50 of the virus we are now working with and there would be two or three
other species.
HARRAP: In our discussions we have worried about the potential of these
viruses for mutation and the frequency of it. Of course, insect populations
may change, but the viruses themselves might also; so that if the insect
developed resistance to the original virus, it may not be resistant to a gene-
tically altered virus. It is rather speculative, of course, but one might
be able to develop this idea and genetically engineer a virus to overcome
any resistance.
SHAPIRO: The gypsy moth was brought to the U.S. in 1869, and in 1906 the
first sign of any type of virus disease was found and it was classically
called "Wilt," described adequately by Glasser and Chapman. This virus
occurs naturally in populations and this virus is found literally every
place the gypsy moth has been.
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When the populations get to be of a high density, at some point in time,
there is a total crash and inevitably, this virus, this nuclear polyhedrosis
virus, is the cause of it and this is 1906 and it is now 1977. The Forest
Service is trying to register this virus for usage, aware that it is there
as a natural part of the ecosystem, but instead of allowing nature to take
its course with these very high population levels existing, to try to bring
the virus into the ascending populations to reduce the ultimate damage.
GRANADOS: At this time, in regard to the spray programs against forest pests,
and in regard to the possible combination of virus insecticide spray programs,
I would like to ask Dr. Rozee from Dalhousie University if he would please
comment on some of the recent findings he has made in the spraying program
in parts of Canada.
ROZEE: I feel a little awkward because I didn't even know when I came to
this conference that you used emulsifiers with virus pesticides. I just
learned that an hour ago, so I now think that perhaps what I have to say
might be pertinent. Could I show a slide?
We became interested in emulsifiers when we looked at a cluster of Reye's
syndrome children and found that the geographic distribution of these child-
ren generally followed the distribution pattern of an aerial spray program.
There were only a dozen of these children but bear in mind, one would expect
from the U.S. experience to only have three or four kids with Reye's syn-
drome in our population. We had fourteen in Canada, which was a very high
incidence.
So we became interested in the components of the aerial spray and
developed a rather difficult animal model, which basically looked at the
effect spray exposure of mice had on the virulence of EMC virus. We have
evolved from this to the certainty, at least in our minds, that the insecti-
cide component itself was not really causing the increased mortality in our
mice. The model we use now is based on tissue culture (Table 1).
First, I will explain the method and then we can look at the results.
It is a very simple experiment. You expose cells (the cells we used were
VERO, human kidney, LLC-MK , L-929, and HeLa) for a period of a few hours
to Toximul MP8, which is one of the more favored emulsifiers for pesticide
aerial sprays, and then you subsequently wash the cells and infect them
with vesicular stomatitis virus. Later I will show results with other
viruses as well. You will see the control number of plaques in the top line,
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TABLE 1. THE ENHANCING EFFECT OF THE EMULSIFIER,
TOXIMUL MP8, ON THE ABILITY OF VARIOUS MAMMALIAN CELL CULTURES
TO SUPPORT VESICULAR STOMATITIS VIRUS (VSV) REPLICATION.
VSV plaques per culture*
Cell
type
L-929
LLC-MK2
VERO
HeLa
HK
Concentration of Toximul
0
50 + 0.6
21 + 1.8
69 + 1.8
68 + 2.8
27 + 2.4
0.25
51 + 4.5
20 + 2.6
72 + 4.3
196 + 6.6
32 + 2.3
1.0
65 + 4.4
23 + 2.4
76 + 3.0
490t + 10
41 + 2.3
2.5
111 + 7.2
35 + 3.2
99 + 3.1
380t + 40
79 + 4.9
ppm
10.0
218 + 10
54 + 3.8
110 + 5.3
Cytotoxic
73 + 2.8
*
The values are the means + standard error.
Estimate; cultures had too many plaques for accurate counting.
and the columns represent parts per million exposure to the emulsifier.
You see that the number of plaques on VERO cells, for example, really does
not change very much, regardless of increasing the amounts of Toximul MP8
to which they are exposed. However, if we consider HeLa cells, we find
they are extremely sensitive to the enhancing effect that Toximul MP8 has.
L-929 cells are also quite sensitive.
These doses of Toximul MP8 are less than the toxic levels of the emul-
sif iers. Many of these concentrations are considerably below the toxic
levels of the emulsifier. This has all sorts of implications when you con-
sider that Toximul MP8 itself is less active as an enhancer than a compound
called Atlox 3409. We are now looking at some 14 others, and we have 80 or
so waiting in our refrigerator. All of these are used widely, not only for
sprays, but also for other things. I think that we should, perhaps, learn
from the chemical pesticide industry, that we should look at the total pack-
age we are going to deliver, rather than just purified virus or a vehicle
alone. I leave that up to you to draw your own conclusions from the data.
The next slide depicts another series of experiments, and it simply
reports in figures what the previous slide showed pictorally. The slide
depicts the cell type running down, concentrations used for the exposure,
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and the number of plaques. You can see that HeLa cells are very sensitive
to the cytotoxic effect of the Toximul MP8.
Now there are two things apparent. First, different cells vary accord-
ing to their sensitivity and second, the plaquing of different viruses is
affected differently. This virus, VSV, happens to be a particularly good
virus to work with, but Reovirus is rather unaffected by the enhancing capa-
city of Toximul MP8. So you have at least three important factors: the
cell, the virus and the various emulsifiers.
COLLINS: Does an emulsifier have the same effect on enhancing the replica-
tion of the insect virus it is being used with? Has this been considered?
ROZEE: No, we have been talking about this during the first part of the
week. We will probably look at it. Dr. Stoltz has volunteered.
PARTICIPANT: (Inaudible).
ROZEE: The commercial spray we were concerned with contained Fenitrothion.
Fenitrothion is immiscible with water, of course, and it was dissolved in
our formulation in Aerotex 3470 as the solvent, and then emulsified with
either Atlox 3409 or Toximul MP8 in water for spraying. It took us awhile
to find out that Toximul MP8, although a nonionic surfactant, is really some
cut distillate, and the company that supplies it does not really know what
it is chemically. We will tell them shortly what it is because we are
examining it by mass spectrophotometry and gas chromatography.
ANGUS: We have never found it necessary to include an emulsifier in our
nuclear polyhedrosis virus preparations.
ENGLER: I would like to make a few comments on Dr. Rozee's presentation.
This is an example which demonstrates that studies done in an in vitro
system may not always lead to results which can be used for a risk assess-
ment without further corroboration. We have seen that a pesticide emulsi-
fier was capable of increasing viral penetration and viral plaque formation
in tissue culture. Although this is interesting, we have a long way to go
to prove and conclude that these emulsifiers are enhancing viral infection
in a living animal, including man. Although this is an important contribu-
tion, jumping to conclusions without further evidence should be avoided. Prior
tests in animals were less than convincing. I think it is extremely impor-
tant to keep this example in mind when we consider and eventually evaluate
in vitro tests with insect viruses for the purpose of safety evaluation.
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KAWANISHI: I would like to point out that there is an article in the jour-
nal, Science, pointing out the same effect with EMC in mice.
ROZEE: We made the original observation in mice and went to tissue cultures
to examine these emulsifiers because it was easier and more accurate. Small
differences between the emulsifiers which affect their enhancing capacity
could not be measured in mice without using a very large number of mice. So
we went to tissue cultures after we found the response in mice. In 1974,
we published the data on mice in Science.
RAPP: I think the point might be made that while it is an interesting
observation, there are a number of different ways of doing the same thing.
For example, light trypsinization of cell cultures will allow certain myxo-
viruses to plaque much more readily. There are many examples of this type
of effect. It is somewhat easy to control in cell culture and show these
effects it is often more difficult with the whole hosts, but it is not an
unusual effect and I would think it did not have any major bearing. I would
be more worried about any dealing with live viruses that such compounds
might, in fact, inactivate certain viruses that would have to be tested
every time we mixed anything together in any live product.
FALCON: I would like to provide some information regarding the approach we
utilize in applying viruses in the field in California. The main objective
of our program is to utilize materials that are environmentally compatible
and to avoid the use of anything we consider to be a foreign agent. We are
working with waterless systems, as I pointed out earlier, because water pro-
vides potential for contamination. So one approach that we utilized is
the suspension of virus in vegetable oil, cotton oil, soybean oil, and corn
oil. And this is applied through very special application equipment, which are
being developed. If we do find the addition of a very small amount of water,
it will give us an invert emulsion, which provides much better coverage,
and of course, you need an emulsifier to accomplish this. We do this using
a medicinal soap, which I think many of you would be familiar with.
JOKLIK: The three questions I see are as follows:
First, do we want to recommend any additions to the procedures to be
applied for testing the safety of agents that are either in use now or that
are about to be licensed? Are there any immediate recommendations that we
want to make? Now, obviously, these would have to be kept simple and
direct, and they would have to be tests that could be carried out in the
foreseeable future.
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Second, could this conference provide a series of high priority topics
for insect virus work, together with recommendations, for increased funding?
Are there some high priority areas where we really need new information?
Third, what mechanism could be suggested to have at least the first of
these listened to by the EPA? How could we have some impact on the laws
and regulations as they are written? Will it be sufficient for this con-
ference to come out with a report? Should this report be published, perhaps
in a journal with national visibility, or in the form of a report or a
letter? And how could we have some impact on the decision-making process?
MEINKE: It seems very apparent from yesterday and today that present testing
methods for these viruses, which are probably not implicated in any disease
entity in man, are probably adequate. I think what has come out in these
meetings is that we should stress in whatever final report we give that if
we recommend some kind of funding from EPA, it should be for basic biology
related to the baculoviruses. And we should not be so concerned with the
doubtful possibility that these viruses are going to be infectious to man.
It seems like you just cannot answer any questions until you learn the basic
biology of what you are working with, and it seems to me that should be
stressed. This is an area that should be funded. The people who are work-
ing in the field and have developed it over the years have far to go.
JOKLIK: To a large extent, I agree with you. But on the other hand, we
are living in an age where the public is becoming very conscious of safety.
We have seen this in a variety of areas. I fear that if there is publicity
without definitive information that large amounts of virus are being
sprayed without any effect, that this could very well kill the program. I
do not expect these viruses to have any drastic effects on the majority of
the population, but on the other hand, 1 feel we need to take precautions
to a certain extent.
MEINKE: Well, to some extent we have already taken some precautions. Dr.
Ignoffo's studies and others have shown that there is probably little like-
lihood that the viruses do have an effect.
JOKLIK: And it is not going to be simple toxicity because the DNA recom-
binant research has shown that.
MEINKE: Well, I was going to bring that up secondarily. When the public
becomes involved, it is sometimes not good for science; using recombinant
DNA as an obvious example of an incident the public exaggerated.
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IGNOFFO: I think the insect virologists, insect pathologists, or whatever
you want to call us, should be proud of what we have done and how we have
judiciously and prudently tried to do those tests that would demonstrate
possible hazards. We will never have absolute assurance of safeness or
lack of hazard. We must continue to evaluate possible hazards, using the
best currently available techniques. That is why I asked a question
earlier...what are the best kinds of tests to demonstrate that the viral
genome is in the host and to indicate what it is doing in the host? If
specific techniques are necessary and not available, then let's develop
these and apply them to the most critical vertebrate system available and
preferably use a system which has previously demonstrated lack of suscep-
tibility to an insect virus.
COLLINS: In terms of steps, I think that could be put into action in a
relatively short period of time. I would like to suggest two things, one of
which I think could be done rather easily, very quickly, and with relatively
little trouble compared to what is being done already. The other one is a
bit more complicated and I imagine would be more controversial here.
The first one would be in terms of the toxicity testing that is now
being done on the formulation and the active agent that goes into the formu-
lation. I think there should be testing, and I think the state of the art
in this field is now such that the most biologically pure form of the
infectious agent is now testable and does not require doing any other types
of tests than are already being done. It would just be another set of
experiments along these lines, using that as the material being tested, and
that is the first thing.
The second thing is this came up before, and I do not think it got
the amount of attention it is due I think there are real questions here
about the fact that, as it is presently set up, EPA accepts the data from
testing organizations who obviously have a strong vested interest in the
approval of these data. I think if one considers what animal virus vaccines
have gone through in terms of independent testing, then it is very appropriate
to recommend that if EPA, or whoever is going to be the regulatory agency,
be adequately funded and adequately staffed. I do not really think, given
the kinds of testing being done so far, that this would require a great deal
of additional staff. In any event, that they be given the funding and staff
necessary to confirm these results and repeat these tests is very important,
particularly if any problems arise with this program relative to the public.
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So far, only two insect viruses have been registered, although perhaps one
more is close and more are being tested. So it is a relatively small number
of agents we are talking about, and the experience that so far has been
relatively positive and trouble-free may be very misleading. I think it is
very important when talking about this as a long-range program, especially
one to replace chemical pesticides, that a program be set up where these
sorts of data can be independently confirmed and confirmed by the organiza-
tion that is involved in regulating their use.
ENGLER: I would like to add something to the three points the chairman
made. He suggested recommending topics of high priority maybe we should
look at these topics and separate out those types of tests that are near
completion, and are near the point where they can be easily used to assess
the safety in a different fashion than we do at present. Alternatively we
should separate out those other types of tests which are concerned with the
basic advancement of knowledge on these viruses. In other words, we should
red flag those types of areas that would give us an immediate benefit.
DOERFLER: With regard to the types of tests that have been discussed, it
might be of interest if we commented on the type of program that has started
in our country, in Germany.
As most of you are probably aware, the Farbwerke-Hoechst Company, with
the support of the Federal Ministry of Research and Technology, is trying
to develop these viruses, and there have been several people at universities,
acting as consultants, who voiced their opinions about the most appropriate
safety tests to use. I do not want to go through the details of these dis-
cussions, but I would like to express my personal opinion here. Earlier,
I said something about what I thought would be applicable from the field
of adenovirology, with which I am familiar and believed could be applied
to this type of problem. Further, I very much support Dr. Meinke.
We have been involved a little bit in tooling up to the same type of
experiment Dr. Meinke is using, and I believe it would be interesting to
see what the implication of reassociation kinetics would have in this regard.
I personally feel that this is the most powerful tool and the most immediate
thing to be done because, as much as I agree that one should look for expres-
sion, if you do not find one genome or fragments of it, there is little
likelihood that you will find its expression. I think those things even-
tually need to be done, but maybe the first one is to look for DNA.
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ANGUS: When we decided to test a virus against spruce budworm under field
conditions, we thought it appropriate to have some safety testing done. We
considered doing it ourselves, and then the question came up of who would
believe us since we have a vested interest in proving that what we are
going to use is safe. We made the decision to take it out of the house
and took it to the Ontario Veterinary College, where the work was done on
a research contract. This decision was made to use this institution
because of its standing and experience with the equipment.
When I discussed this with our own Canadian authorities, they were
reluctant to set up a scientific group of their own. They muttered things
like, "It is an old established practice that the judge and the jury shall
not be the same person." Now, I don't know if that was said because our
own regulatory people did not want to take on the responsibility of doing
the technical work. But there is this to be considered: There will not be
that many of these products coming on-line that we could go ahead and set
up a research organization with an appropriate expertise and appropriate
equipment for what may be a sporadic enterprise. But that is my only comment.
We made the decision that we would take it to a group whose scientific
credentials were impeccable, and we did not tell them that we wanted them
to prove this virus is safe. We put the question in the honest way, "Would
you have a look at this and tell us what it might do in a variety of animals?"
COLLINS: I think this is a possible alternative. All I would say is that
industry, which is not known to be terribly altruistic, clearly feels that
it is worth their while to keep a testing program like this available, even
if it is only for occasional use. And I would say that if they feel it is
worthwhile, given their cost-balance ratios, it might be worthwhile, whatever
the inefficiency would be, for a regulatory agency to have a similar kind
of program. But I think an alternative possibility would be, if they are
adequately equipped, to have outside agencies do this. All I am saying is,
and this is not to impugn the honesty and integrity of industrial research
labs, the fact is that there exists the considerable possibility of conflict
of interest, and from the safety standpoint, this is what we are all worried
about. It seems that we ought to bend over backwards for the sake of care,
particularly if we are worried about what the public is going to say relative
to any possible problems that will arise in the future.
HEIMPEL: How long does this go on? There is no evidence that these viruses
are harmful. They do not affect other animals. How long do we continue
attempts to prove that they do?
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COLLINS: I am not asking for absolute guarantees. All I am saying is it
seems worthwhile to have confirmed the data that are available and I think
that the point Dr. Ignoffo has brought up should address itself more to the
second point that Dr. Joklik mentioned, namely what additional tests, now
that technology is advanced further, should be used. I agree, I think addi-
tional tests are needed.
HEIMPEL: I agree too.
SUMMERS: I think what you are really asking is, can we reaffirm some of
the critical safety experiments using more sensitive and appropriate
techniques?
COLLINS: Yes, I think a lot of these techniques have the advantage of
representing a form of basic research that is going to occur in a lot of
laboratories, given adequate funding, not merely as a safety program, but
as a part of basic research. I think from that standpoint, they are parti-
cularly valuable. They will probably be confirmed in a variety of labs
and by a variety of workers using different cell systems and different vir-
uses. All I am saying is whatever the criteria are going to be that are
used in safety testing with new agents coming on, the results should be con-
sidered, and criteria confirmed.
SUMMERS: Then do we go back to Meinke's lead-off statement that our data
appear adequate, but we need more research in the area of the biology of
these viruses? What we need to do is identify that research in terms of
immediate and longer-term goals.
FIELDS: I think we have answered the first part, in terms of immediate high
priority items, in terms of reassociation kinetics and radioimmunoassays,
and have discussed those back and forth. It might be reasonable to start
making a list in terms of explicit recommendations for the next level of
priority work. We are essentially saying that what has been done to date
has been well done and is solid, and we are now just trying to set the next
two levels of priorities. The immediate methodology that you have just
discussed falls into the three groups of the hybridization methodology for
DNA, RNA, and radioimmunoassays or other immunologic tests for proteins.
Should we now identify a list of topics for more long-term development?
JOKLIK: I think it would be more profitable to appoint some subcommittees.
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HOLOWCZAK: It is not clear to me what and how good immunological tests have
been used and how good antisera have been for identifying these viruses.
IGNOFFO: This has been done but the tests have not been done with sensitive,
specific antisera.
HOLOWCZAK: I consider it a very high priority to have agent-specific anti-
sera available to identify these viruses and to monitor their occurrence
in nature. This should be a part of any new safety testing procedures.
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PART VII
PANEL DISCUSSION
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Discussion of Preliminary Draft
of Panel Recommendations
Presenter: Robert E. Shope, M.D.
Baculoviruses have been shown to provide a promising alterna-
tive approach to pest control. Current evaluation methods suggest
viruses are effective; furthermore, available data have not revealed
any deleterious effects for other components of the ecosystem (i.e.,
other invertebrates, plants, and vertebrates, including man). Safety
testing criteria should continually respond to improved technology.
We draw attention to EPA guidelines for safety testing of baculo-
viruses, Section B-l, b-2. "It is important that the most sensitive
methods for detecting virus replication are incorporated into the
EPA guidelines for safety testing of baculoviruses." Recent devel-
opments in molecular biology have provided more sensitive and refined
tools and offer the potential for testing at improved levels of
specificity. By their implementation, the opportunity can be taken
to improve further the safety tests for baculovirus pesticides.
We draw attention to the identification criteria listed on
page 179 of Baculoviruses for Insect Pest Control. The recent
development of restriction fragment analysis of a variety of DNA-
containing viruses has provided a powerful new tool which should be
included among the identification techniques. A fundamental requi-
site for safety is to demonstrate the lack of persistence or expres-
sion of the viral genome or parts of the viral genome in nontarget
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systems. Such tests should include two human cell types, as well
as rodent, avian, and fish cells.
The development of such tests should embrace a variety of tech-
niques such as molecular hybridization and radioimmunoassay. We
strongly advise that EPA move with all speed to implement the above
recommendations for safety involving identification, baculovirus
genome tests, and sensitive methods for virus replication, at least
by January 1979.
Research Program
To provide a continuing basis for improvement and an evaluation
of new products, it is imperative that certain areas bearing on the
safety of insect viruses should be more fully understood. A better
understanding is needed of the replication and pathogenesis of bacu-
loviruses in their natural hosts and cell culture systems. Also,
the genetics of insect viruses and the development of specific genetic
markers are vital for precise ecological study. For example, the
possible range of interaction between the viral pesticide and other
viruses in the biologic environment needs to be explored.
These general recommendations of the invited panel are intended to focus
attention on areas urgently in need of research. They represent certain
elements only of major issues raised in the panel discussion and are put
forward as the basis for the formulation of a specific program at an early
date. We feel that this could best be achieved by a permanent and indepen-
dent advisory panel.
DISCUSSION
COLLINS: The two things I would like to open for discussion would be about
what changes in safety testing procedures ought to be made. The only things
being tested right now are the formulation and the active ingredient that
goes into formulation.
The third possibility was that the most biologically pure preparation of
the active ingredient also could be run through the same types of toxicity
tests that are being done. That is one point that I think would be worth
mentioning specifically. Second, I think it is very important to establish
confirmatory testing of data.
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HARRAP: I think as far as this is concerned we looked at the recommendations
from the previous EPA meeting in Bethesda and found that several of the points
you raised were covered in the recommendations there. That is why we draw
attention to "Guidance for Safety Testing," Baculoviruses for Insect Pest
Control, and cite certain paragraphs because we were not taken notice of the
last time, and we hope that by repeating this, some note will be taken this
time.
COLLINS: Except if you are drawing attention to a whole body of information,
it is obviously unclear which aspects you think are important and which ones
you don't. You might want to say that we are drawing attention to certain
specific things, but if you are drawing attention to a whole body of infor-
mation, the ones you want to stress are going to be lost.
IGNOFFO: Could we review each specific point in the recommendations?
SHOPE: It is stated in the first paragraph that, "Baculoviruses have been
shown to provide a promising alternative approach to pest control. Current
evaluation methods suggest viruses to be effective; furthermore, available
data have not revealed any deleterious effects for other components of the
ecosystem (i.e., other invertebrates, plants, and vertebrates, including man).
Safety testing criteria should continually respond to improved technology.
We draw attention to EPA guidelines for safety testing of baculoviruses,
Section B-l, b-2."
IGNOFFO: Would you please read the section about that particular recommen-
dation?
SHOPE: Section B-l, b-2: It's under "Data To Be Collected from Experimental
Animals." "Attempts to isolate and detect insect virus inclusion bodies
or virions in gastrointestinal tract, blood, urine, and selected body tissues
which would normally be affected by a viral infection (e.g., lymph nodes,
kidneys, cerebrospinal system, spleen, and liver) use the most specific and
sensitive assay for virus detection." Attention should be focused on, "It is
important that the most sensitive methods for detecting virus replication
are incorporated into EPA guidelines for safety testing of baculoviruses."
PARTICIPANT: I don't really understand who is going to be responsible for
the updating of these tests. It is certainly not going to be the licensee.
I do not understand what organization or group is going to be responsible.
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SHOPE: First of all, these are recommendations considerations that have
been derived from our discussions for the past two days, which reflect what
we feel the need for applying new and approved detection technology to
evaluate safety on a continuing basis. These recommendations do not bring
with them the immediate application of this technology. We have recommended
that it now be superimposed upon future testing procedures or schemes.
COLLINS: If I understand it, the section you are drawing attention to really
deals more with detection procedures than with what materials are going to be
administered in these tests. Is that right?
SHOPE: That is correct.
COLLINS: This might be a possible place to bring in the testing of the...
IGNOFFO: That section, as he read it, specified the most active material.
COLLINS: No, not as I heard it. The most sensitive techniques were stressed.
IGNOFFO: I thought it also stressed that you utilize the most active sub-
stance the most biologically active fraction.
SHOPE: No, I do not believe that is right. Let me read it again from the
"Safety Testing Procedure," concerning the data to be collected from the
experimental animals. "The attempts to detect and isolate insect virus
inclusion bodies or virions in gastrointestinal tract, blood, urine, and
selected body tissues which would normally be affected by viral infection,
use the most specific and sensitive assay for virus detection."
SMITH: If I might just draw attention to that Section A is where the
material that is administered is described, and there might be the place to
make a change. The very first sentence in Section A is titled, "Schedule
of Feeding and Sacrifice," and it follows: "The insect virus is adminis-
tered in a single dose to young adult laboratory animals, in both sexes."
One could simply amend that by saying the most active insect virus, or the
most active preparation, or the cleanest preparation, or however one might
propose to do it, would be done in that fashion.
IGNOFFO: The licensee would also have to test the formulation to meet EPA
requirements. If a person is trying to establish whether these agents are
safe, he would not only want to meet the formulation requirements of EPA,
but also to test the most infectious available entity.
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COLLINS: I think one of the things we are trying to establish is what the
intrinsic biological properties of these agents are, and although, clearly,
testing the formulation is very good, as far as it goes, one of the things
we are stressing here is that the biological systems are still relatively
undefined, and I think this is very important information.
ENGLER: I would like to comment briefly on the issue of purity criteria of
the inoculum which is used for testing. I think basically you should not
be too concerned about this aspect and give the regulatory agency some lati-
tude. In extreme cases, where the active ingredient which comes off the
production line is 30 percent virus or even less, EPA has the foresight to
ask for additional tests on the purified material. On the other hand, if
that material that is produced is 95 percent virus and 5 percent insect
fragments, almost anyone would be satisfied that that is as close as we can
come to purified material. In reality, the test protocols take such
variations into consideration. Making purity requirements too stringent
may in fact box us in, and we may lose the ability of good scientific judg-
ment for determining on what type of virus preparation the tests should be
run.
COLLINS: Let me stress that I do not think we are looking for 100 percent
purity, the important word in what I have been saying is "available," the
most highly purified preparation available, recognizing all the problems
inherent in finding this. The only thing I can say from what your comments
have been is that, based on what I've heard in the last two days, EPA does
not appear to have demonstrated the foresight in the past to really say that
a virus preparation which is going into the formulation is not pure enough
and that they are asking for something more. I see no evidence of that kind
of control.
SHOPE: Can I ask Dr. Collins for a point of clarification? It seems to me,
from what we know about what happens in the pathogenesis in the insect, that
when a preparation is sprayed, it is consumed by the insect, and then at
some point actual free virlons appear in the hemolymph of the insect, or in
tissues of the insect. Therefore, in order to test this properly in a
tissue culture system, one would have to have free virions. The material
that is being safety tested has, under the current procedures, a very small
percentage of free rods and, therefore, if one does a test in vitro for inte-
gration of DNA into the genome, the basic starting material would have been
polyhedra and thus inappropriate to initiate in vitro infection. Does this
answer your question?
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COLLINS: No. I am specifically referring to the animal toxicity testing
that is being done already. If anyone wants to modify the tests that are
being used, we can talk about that. That is not what I am talking about at
the moment. All I am saying is that whatever criteria are being used right
now for toxicity testing in animals, the four acute, the subacute, or what-
ever, the same series of tests should be run parallel with the best purified
preparation available at that time. That's all I am saying, I am saying
nothing about in vitro testing on tissue culture or anything like that.
IGNOFFO: Do you really want to formalize this into a regulation? Why not
permit EPA the flexibility of deciding the best alternatives?
COLLINS: I have not seen where it has been done.
IGNOFFO: Oh, yes, it has been.
COLLINS: The preparations that have been spoken about so far in these last
two days came across as being very...
IGNOFFO: Tests have been done with purified inclusion bodies, liberated
virus particles, insect hemolymphs, etc. Not every test that was done in-
cluded all these substances.
COLLINS: So if it has been done, I do not think there is any problem in
including it in the guidelines that already exist. Let's formalize it
then.
IGNOFFO: From a technical standpoint, purity is only a secondary factor to
infectivity. I can give you a preparation that is 99.9% pure but is inactive.
COLLINS: The other word that was in the phrase that I have used is "biolo-
gically active."
ENGLER: Let me illustrate the problem with an actual case, the forthcoming
registration of the gypsy moth NPV. The safety tests were done with a highly
purified material. The registrant later decided that a less purified material
will eventually be used. This has led to the condition where the safety tests
were actually performed with a more purified material than that which is go-
ing to be used. The question then came up, "Must all the tests be repeated
with the less purified material?" EPA decided that some tests had to be
repeated and some did not; it became a matter of judgment. This is all I am
asking, that a margin of judgment be maintained in conducting these tests.
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COLLINS: I am not trying to suggest hard and fast rules. I think what I am
suggesting fits in very well with what is being done and gives you as much
flexibility as you want within the guidelines that you already have. I would
ask and imagine that the preparation that you are talking about at the time
it was submitted to you for testing was presumably the preparation that was
going to be used in the formulation, and that is why if it was pure, it was
because they were going to use a pure preparation in the formulation. If
they had not, you never would have thought to look at that kind of prepara-
tion.
IGNOFFO: I would like to comment on the type of the inoculum which is
used. I believe that virologists or registrants interested in evaluating
risk factors do more than is required by regulatory agencies and will, in
fact, test the most infectious agent as well as formulations. These mate-
rials, for reasons of time and cost, will not be tested in every animal
system. Investigators will probably test only in the most critical system(s)
in order to increase the probability of infection in a nonhomologous host.
COLLINS: There is no problem with that because the current guidelines would
dictate that they would have to test the material that is going into the
formulation. So, if they test 100 percent purified material, and later
decide to use something that is 30 percent purified, then, as I understand the
present guidelines, they have to go back and test that 30 percent because
that is what is going into the formulation. I am suggesting that the test-
ing of the most highly purified biologically active material available should
be included in the toxicity testing along with the formulation and the
material that is going into the formulation.
IGNOFFO: The recommendation is acceptable to me from a technical standpoint.
I am not going to argue in EPA's behalf. In the final analysis it is their
decision. But from a technical standpoint, I think your suggestion is
desirable and has been used in the past.
SMITH: I think in terms of the administrator, we can recommend whatever we
want. However, I think that it is important that we recognize that what I
would consider a law is that whatever is put on the field is what has to be
tested and that has to be given the most attention.
On the other hand, I think if there is any dangerous procedure, it
would be to test the biologically purified material and then spray unpurified
matter on the field. Parallel testing is extremely acceptable as far as I
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can see, but I think the real requirement must be that whatever is put on the
field is what is tested for sure. And this precedent of having tested a
purified product and then spraying an impure product is unacceptable.
SUMMERS: Shall this be included then as a recommendation?
RAPP: First of all, the group putting this together was conscious of the
fact that EPA's main function concerns safety. And I think we should make
the point very strongly that whatever we are writing here is strictly advi-
sory. The third area that I would like to emphasize is that we were very
careful not to get too specific. If you are going to start making highly
specific, direct recommendations, you need much more information than I
believe we have. You need the impact on the products, on costs, and on test-
ing facilities. It does not simply involve putting in some more guidelines
to run more tests. I think this is a very complex thing, and that is one
of the reasons why we recommended a more permanent advisory panel to look
into this to see if these guidelines should be written in great detail.
To reamend all the previous documents that have been put together by various
groups would, although we could have done that, have been an extraordinary
mission.
I for one am very reluctant to put my name on highly specific recom-
mendations. Moปt of these are general and trying to upgrade, in a general
way, the level of safety testing to take into account newer technology and
to urge new research programs that would enable better safety testing in
the future as new products become available. I sympathize with Dr. Collins
if we really start writing very specific recommendations into this, it would
change the whole character of the document, the whole thing would have to
be reworked and reexamined.
COLLINS: I view this as a fairly general recommendation. I am not saying
anything about how the purified material should be prepared, what percentage
of purity is necessary. I am putting it forward as a fairly general recom-
mendation and I think, as I read the old recommendations that were written,
the material that is being inoculated is being presented in a fairly general
way. And I think it is in with that kind of terminology.
So, while I agree that this is not the appropriate forum for recommend-
ing very specific measures, I am really looking at this as a fairly general
modification of what has already been done, without any specificity. This
is where the flexibility comes in as to what is the most highly purified
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biologically active material available occluded virus and nonoccluded
virus, or whatever. That is something for whoever is going to get involved
with this to work out, and that is where flexibility comes in. So I say,
if people feel that this is over-specific, they should vote against it. I
am presenting it as a general modification and one that I think is important
and has a reasonable amount of flexibility in it.
SUMMERS: Dr. Engler, do you visualize this as a general statement?
ENGLER: The more I hear Dr. Collins talk, the more I feel it is a general
suggestion. The only reason why I was outspoken against restrictive and too
specific purity criteria is that such criteria have a tendency to go beyond
the scientific intent and will become legalistic. Once this has happened,
the intended scientific flexibility and judgment are lost.
To summarize what we have done in the past and what will be done in the
future: not necessarily all tests are performed with all different grades
of purities. We are using bridging type tests, that is, some tests are done
with more dirty material, some are done with more pure material, and some
with both, giving us a basis for comparison. If there are significant dif-
ferences, we will ask for more tests on the two components. If there are no
differences, we can draw the conclusion that the state of purity does not
affect the overall safety evaluation of the virus product. This is essen-
tially what is being done and I hope that this is satisfactory.
COLLINS: The other point is that we should look at this meeting as some-
thing of a continuum in a series of meetings. I would like to see us moving
in the right direction as far as safety testing goes. If a year or two from
now, people refer to this meeting and the recommendations that were made,
I hope they will be moving in the direction of more sensible testing. I
think this discussion may be a small step in that direction, not a very
great one, but a small one.
IGNOFFO: I think a lot of people, including me, would disagree with your
comments on the validity and technical worth of the protocols of previous
evaluations.
COLLINS: That is a separate question. That is not what I am dealing with.
If you want to challenge the toxicity testing on the basis of whether it
is scientifically valid or not, that is fine.
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IGNOFFO: I was not challenging the tests or the protocols, but your intro-
ductory statements concerning the validity of these tests.
COLLINS: 1 am saying the material that is being used in the test should be
expanded.
SUMMERS: You are right, Dr. Collins, this is a continuation of many things
and I am sure it is going to continue. For example, the National Research
Council of The National Academy of Sciences is putting together a study team
to look at the same things that we are concerned with here, and perhaps in
a more comprehensive way with more time available than we have had. I would
have a tendency to support Dr. Rapp's comments. We spend a lot of time
wanting to present a general statement that really focuses upon the very
thing you are talking about, but doing it in a way that is compatible with
the sequence of things the time that is available and yet, what we
consider to be the urgent applications needed for improving safety or a more
comprehensive evaluation of safety, not only now, but in the future. I
myself would prefer to stick with the general statement, however, if this
needs to be voted upon should we do it now?
COLLINS: I move that currently used animal toxicity tests be carried out
with the most highly purified biologically active material available in
addition to the tests that are already being done with the formulated mate-
rial and the active ingredient used in the formulation.
CUNNINGHAM: Could I have some clarification on defining the term formulation,
because I think everybody involved in the applied side of things knows that
there is no ideal formulation yet available. We are all fiddling around
with different things, and, in all probability, the formulations that are
used this year will be altered by next year. Most of the things that are
used in formulations at the moment are pretty innocuous materials, such as
molasses, for example. Do I understand that a virus should be safety tested
in the formulated state, and if in another two years there is a good change
in formulation (for instance, one is using carbon again, another innocuous
substance) that the whole routine should be run through again with the latest
formulation? Or can the materials that are used in the formulation be safety
tested independently from the virus? And then we can make up various permu-
tations and combinations? How do we stand on that line?
ENGLER: That depends very much on what components are being used or substi-
tuted in a formulation. Certainly, if the formulation's "inerts" are as
innocuous as molasses or milk sugar, a conclusion can be reached by the EPA
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as to how safe or how irritating these formulations are, whether or not
additional tests should be run. If we cannot reach that conclusion, some or
all tests pertaining to the formulation may have to be repeated. So it is
an open question and an open-ended issue.
HUANG: What is meant by the "most purified" or "biologically active mate-
rial"? Now as you all know, you can make up 2-3 micrograms of material that
is highly purified and that is available. If you want about 30 grams per-
haps, which is needed to run a toxicity test, that is a task of a different
order of magnitude. So what kind of availability are you talking about?
COLLINS: I think this is the flexibility that we talked about. Whoever
is going to be regulating this testing is going to have to decide that. I
am trying to keep this as a general recommendation with flexibility for EPA,
if that is who it is going to be, to decide how much they need and how pure
it has to be. This may differ from virus to virus, and I would not even
try to give you a number.
HUANG: So in other words, you are leaving it to the discretion and judgment
of. ..
COLLINS: Exactly the same people whose judgment and discretion are now
controlling it.
PULL1AM: You all ought to fill out your 171 Forms, send them to EPA, and
come to work with us because I think you are begging a legalistic issue
that is probably not appropriate for this particular meeting. You are, as
many of you pointed out, making recommendations to EPA for safety testing
and improvement on present methods that are being used to evaluate the safety
of products that have been submitted for registration. I think that should
be kept in focus. We are not making laws here and we are not making regula-
tions we are not formulating guidelines that are going to be published
in the Federal Register in the next couple of months. I think EPA welcomes
any recommendations that you folks have to make, and I think they should
be taken in that spirit. I do not think we need to argue the fine points
of whether these things are available and how much they cost at this time.
I think as you pointed out earlier, that is something that has to be done
with more available time. I think the recommendations that you make are
important, that they should be made, and that they should be transmitted so
that they can be considered in perhaps a more practical light if necessary.
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HARRAP: I think this whole conversation illustrates and, indeed, emphasizes
very well what some of us were afraid of if we tried to get too specific. If
you start to use a word like "purity" and the virologist in me has great
attraction to that word purity as soon as you use it you will have to
start defining it. You have to say what you mean, and then things start to
change. So we have to have some guidance proposals to respond to that. For
example, we have to decide whether, in toxicity testing, purity is important,
and for virus replication tests whether it is important or not. So I really
think this is what we are seeing, a good illustration of the value of trying
to keep specific items out of these recommendations at this stage.
SUMMERS: All those in favor of Dr. Collins1 proposed modification? Three.
Those opposed? Thirteen.
SHOPE:
"Recent developments in molecular biology have provided
more sensitive and refined tools and offer the potential for test-
ing at improved levels of specificity. By their implementation,
the opportunity can be taken to improve further the safety tests
for baculovirus pesticides. We draw attention to the identification
criteria listed on page 179 of Baculovirus for Insect Pest Control.
The recent development in restriction fragment analysis of a variety
of DNA-containing viruses has provided a powerful tool that should
be included among the identification techniques."
SHOPE: This is on page 179 under the heading of "Guidance for Safety Testing
of Baculoviruses." I will read the paragraph: "The virus must be an
insect pathogen, which is identified taxonomically according to host spec-
trum and serological and biochemical and biophysical tests." We want to
call your attention to this new test which we feel should be included.
SHOPE: Next section:
"A fundamental requisite for safety is to assure the lack
of persistence or expression of the viral genome or parts of
viral genome in nontarget systems. Such tests should include
two human cell types, and rodent, avian, and fish cells. The
development of such tests should embrace a variety of techniques,
such a molecular hybridization and radioimmunoassay."
KNUDSON: I would just like a clarification of the first part of that sen-
tence, for example, "A fundamental requisite of safety is to assure a lack
of persistence." Does that imply that if there is persistence or an
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expression of a viral genome or any part thereof, then the agent in any cell
line that you are using, nontarget systems, is not safe? Is that what
that means?
SMITH: I think that if at this point we can identity a single criterion
for potential problems, persistence has to be it. I do not know that that
indeed will be the case, that if there is persistence in the form of an
integrated genome and so on, it is unsafe. On the other hand, if there is a
single criterion that I think can be identified at this meeting as a poten-
tial problem for further safety testing, I would say that persistence
probably is as good a criterion as we can identify at this time. I think
that that is what this recommendation is really saying.
KNUDSON: You redefined the word persistence in that discussion. If you
want to define what you are talking about when you say persistence or
expression, fine. I do not think it is very clear.
MEINKE: I think the statement is much too strong. You might have persis-
tence of these DNAs, but for how long? Say it lasts for a week you can
call it persistence, but it might clear. It could last 24 hours, whereas it
could last for five. How long is persistence? Taking a ridiculous thing,
suppose we grind up some cabbage leaves and extract the DNA. And then sup-
pose I put that DNA into a million cells and it persists that would mean
we should not eat cabbage anymore. It is much too strong a statement. It
could stop the whole program, I think.
IGNOFFO: If you really want to make a distinction between the justification
for the tests and actually doing the tests, I think we all agree that what
we want is to do the tests. Thus, I suggest deleting the philosophical
and justification phrases and stressing what the evaluation should include.
SMITH: No, again may I use the previous arguments all over again. We are
trying to write in this communication recommendations to be taken to EPA
for consideration. And I think the way it is written, the concerns of the
advisory panel are underlined by this kind of phrasing. This is not going
to be law, this is not going to be in the guidelines, as I understand it,
at least that is what was said just a moment ago. And this is the way we
express our concerns about this kind of biochemical or biological event,
and nothing more than that.
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IGNOFFO: Dr. Smith, what you have to ask yourself is, can you visualize a
situation, any situation, where persistence would occur that would in fact
completely prevent the development of this particular virus?
SMITH: Oh, yes indeed.
IGNOFFO: And, can you visualize a situation where persistence could occur,
and yet not be a safety hazard?
SMITH: Yes, indeed.
IGNOFFO: If you can visualize these situations, then what we must not do
is formulate a statement that would be all-inclusive and could in fact stop
a program when persistence was demonstrated; yet this persistence did not
present a safety hazard.
SMITH: That is right, but it is not the job of this panel to conduct those
tests. What we are saying is, if persistence is demonstrated it is of con-
cern, period.
KNUDSON: I think the point that I would like to make, the one word in the
sentence that I think throws the whole context off is the word "lack." If
you could change that maybe to something like "to examine the extent of any
possible" or "what is the significance thereof." The word "lack" is the
problem.
IGNOFFO: I think it makes it a hard and fast rule that does not leave the
possibility of backing out because we do have persistence and yet there is
no safety hazard.
HARRAP: May I explain the reason for this statement? Dr. Joklik was vir-
tually adamant that a specific recommendation like this was made. We had
some discussion about its implications. He felt maybe it would not totally
rule out a specific virus in a certain instance, but he wanted this recommen-
dation in so that if there was any persistence or expression of baculovirus
genome in nontarget cells, a more thorough look would be taken at the situ-
ation by having the recommendation there.
IGNOFFO: That is exactly what that will do! If we are primarily concerned
with evaluating the existence of persistence, then O.K. If tests are recom-
mended that will evaluate persistence, then at least the next step (if
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persistence does exist) is to decide whether to proceed. But if it is in-
cluded as recommended, I think there is only one recourse after an evaluation
has been made to stop everything.
HARRAP: No, that was not his feeling. What he wanted to do was to stop
things while people took a more thorough look at them.
MEINKE: I think it is too strong. Can't we just say something to the effect
that "a fundamental concern of"?
COLLINS: Something like, "The fundamental concerns of safety require the
investigation of the question of persistence..."
SHOPE: Full quote:
"A fundamental concern for safety requires the investigation
of the question of persistence or expression of the viral genome,
or parts of the viral genome in nontarget systems. Such tests
should include two human cell types, and rodent, avian, and fish
cells. The development of such tests should include or embrace a
variety of techniques such as molecular hybridization and radio-
immunoassay."
MEINKE: Now I think the second part of that is also too strong and too
specific. I do not think we should spell out two of these kinds of cells,
one of this and that. I think we should just use nontarget systems and leave
it up to EPA to decide what are the nontarget systems. Can't we just stop
it at nontarget systems?
FIELDS: Well, as a minimum you want a fibroblast and a epithelial cell and
this is not an absolute number, but certainly I believe that all of us felt
that this was a minimum screen that certainly could and probably should be
supplemented. But this was a relatively specific and strong feeling that
most of us had, that this really was in many ways the crux of the problem
of altered host range and if there was to be any major, however remote,
issue of safety, this would be the most crucial but specific recommendation
to come out.
IGNOFFO: You are concerned with cell type?
FIELDS: Yes, well...and I think it was really the feeling that if one were
to choose there would be absolutely no way of having a single cell type that
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could cover it. Two is really a minimum, but at least one could cover fibro-
blast and epithelial. But it would be advisable in developing this to
broaden it and add further cells. But I think to leave this too general
would be to dilute what really is the remote but focal issue that we are
coming to of tests that can be added, and really further refine safety.
HARRAP: In fact, in my notes I have the words "at least." I do not know
whether it was noted, but we did say it.
MEINKE: I would like to point out that in almost every mammalian system I
can think of, you can use a monkey cell line to mimic any kind of occurrences
in other cells. I am not saying that there are not exceptions, but whatever
happens in human cells in cultures, usually happens in monkey cells in cultures,
So, I do not know why you would really have to lock in on human cells for
one thing. I am trying to think of a system where one virus does not repli-
cate on the monkey cell line and does replicate on a human cell line. Maybe
I should say primate cell lines. I hate to see anything just locked in like
that where you just have to do those experiments like that. It seems, it is
such a firm statement, that's all.
RAPP: I will give him a very famous example. Herpes simplex virus probably
grows in more types of cells than any other mammalian virus. It does not
grow in rhesus monkey cells, which was a favorite cell for most investiga-
tions until about fifteen years ago. So, examples exist, and I could give
you others, but that is the most obvious one.
MEINKE: Yes, but there are a lot of examples in reverse where these viruses
do grow better in other primates besides human cells.
RAPP: In the final analysis, the species we want to protect most is the
human species and that is the reason for using human cells.
IGNOFFO: If that is the ultimate concern, why not concentrate on primate
lines? Forget about avian, mammalian, and fish lines. Let's assume that
we do not get viral replication or cytopathic effect in primate cell lines
but do get effects in other vertebrate lines. Then what?
SMITH: Well, again, getting back to the spirit of what we are trying to
prepare here, let's propose now for a moment this particular virus integrates
and persists in fish cells, but does not in chicken or mammalian or human
cells. Then you put it to the EPA and they make the judgment that it is not
important in this case. On the other hand, if you present data that says
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the genome integrates into mammalian, into human cells, as well as fish and
insect cells, then it is a different ball of wax. And what we are saying
in this communication is that this information should be available for safety
testing.
RAPP: Well, what we had hoped, without listing 500 cell types, was that the
EPA would use its latitude. If, for example, you were spraying an area in
which dairy cattle grazed regularly, one might use bovine cells. We were
looking from the point of view of various other economically important and
ecosystem important species. This was just a suggestion that is a minimum
and the reason is that these types of species are found in areas that would
generally be sprayed. I mean, there would be water in most of them, and so
on. It is rather arbitrary to say fish because we knew that there were
fish cell lines available. But if I were spraying in an area where I knew
there were a lot of cattle, I would try to use some bovine cells.
SHOPE:
"We strongly advise that EPA move with all speed to implement
the above recommendations for safety involving identification,
baculovirus genome tests, and sensitive methods for virus replica-
tion, at least by January 1979."
COLLINS: I hesitate to bring this up, but I really think we should address
the question of confirmatory testing, whether it be by EPA or by anybody
else. I think this is extremely important and to emphasize what was said
before, we are making recommendations and not using the force of law. The
point is that if we do not make these recommendations, presumably they will
not even be discussed by EPA since they are not even going to come before
them. I think this is a very, very important concern, and I think it is one
that should be brought to EPA's attention. I recognize that implicit in
this would be increased funding, increased staffing, but again we are here
to make recommendations on what would be the most rational approaches to take
from this point on, and I think that this is an extremely important one.
ULVEDAL: I have been pretty quiet in this discussion because I am on the
research side of the house and not the regulatory side of the house to which
most of these questions have been addressed. However, from the research
side, I would like to hear as many specific recommendations and strong
recommendations as possible. That gives us the option to examine, evaluate,
and then sift out what we can do and what we cannot do. So I would like to
hear as many specific recommendations as possible.
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SUMMERS: Would it be possible for you to modify that statement to include
something like "most critical" tests?
COLLINS: I would say before an agent is registered for use as a viral pesti-
cide that data relevant to the most critical safety tests should be confirmed
either by a regulatory agency or outside independent investigators. EPA
does not have to feel that certain of the data are inconclusive. I mean,
they can take data that look perfectly reasonable on the surface. The fact
is that they have to decide which of those tests are most critical.
SUMMERS: "Before a biological agent is registered as a viral pesticide,
the data most relevant to the safety test should be reconfirmed by EPA."
Is that a motion to be added to this section?
COLLINS: Yes, I move that the motion be added.
IGNOFFO: Dr. Engler, what are your comments on this? You have been very
quiet. I would like to hear what you have to say.
ENGLER: I made my comments before and as long as it has been said around
this table that these are recommendations which may or may not be imple-
mented, I have no comments on that.
IGNOFFO: Just out of curiosity, would you recommend that the recommendation
on reconfirmation of tests' results be implemented?
ENGLER: No.
SUMMERS (restatement of the motion): "Before a biological agent is regis-
tered as a viral pesticide, the data most relevant to the safety tests
should be reconfirmed by EPA."
ANGUS: Mr. Chairman, could you entertain some advice from the floor?
SUMMERS: Yes.
ANGUS: Although I know it is not directly pertinent to your consideration
today, there are some international implications in the wording of this
because in some jurisdictions, and I am not aware of what is the rule here,
it is the premise that the applicant is responsible for whatever costs arise
from trying to find the registration for something. Now as I hear this
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wording, it means that EPA will have to go out and do the tests itself, and
this implies that EPA is now going to set up its own testing facility, that
they will have protocols and results and data submitted to them, and that
they will decide to do some part of it or all of it all over again. Is
that what is implied?
COLLINS: No, I think the discussion we just had made it very clear that
that could either be done in-house or they could send that out.
SHOPE: I think I need some clarification here also. Let us assume there
is a virus which is proposed for registration by the Forest Service and that
presumably the cost for the development of that registration would be borne
by public funds. You are then suggesting that we use more public funds to
confirm what a government agency has already found to be true. I personally
feel that that would be an unwarranted use of public funds.
COLLINS: I would consider that an appropriate-use of public funds. I think
that situation is not going to be the norm, or at least it will be a mixture
of that and where the initial testing has been done with private money.
SUMMERS: Once again I think the recommendation has inherent in it that
flexibility because it states that the data most relevant to the safety tests
be reconfirmed. So I think that is left to EPA's, or whomever's, discretion.
All in favor of this motion? Twelve. Those opposed? Two.
SHOPE: There was a question about January 1979.
"We strongly advise that EPA move with all speed to implement
the above recommendations for safety involving identification,
baculovirus genome tests, and sensitive methods for virus replica-
tion, at least by January 1979."
RAPP: The fact is that there were many people on the panel who felt that we
ought to put in January 1979 because the technology is so advanced. No one
is asking for results by January 1979, just that things be under way, so to
speak, and that some of these tests be put into operation. That is a very,
very loose kind of recommendation which is simply intended to call attention
to the fact that EPA ought to be heading in that general direction. That
is all it really implies.
IGNOFFO: If I had a new virus, how long would it take me to develop the
radioimmunoassay technique?
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RAPP: Well, to some extent it would depend on how well it would grow in
your test systems, how fast you could purify the proteins, but if you already
have the technology, and apparently the technology that purifies the various
proteins associated with baculoviruses is available, I do not think it would
take long.
HARRAP: Developing the radioimmunoassay...four months.
SUMMERS: Four to six, because you would want time for standardization.
FALCON: I would like to know why it is necessary to specifically tell EPA
to implement these things. Isn't it assumed that the conference, the fact
that EPA gave sanction to this conference, aren't you begging the question?
I see no need at all for that particular sentence.
SHOPE: At least a partial response, the document, which is an appendix to
the book, Baculovirus Safety Testing, has not been implemented...at least
parts of it. And I think that this is an effort at least to have specific
areas implemented.
FALCON: The fact that it has not been implemented does not mean that EPA
has not reviewed that information and considered it and taken from it what
they feel they can utilize. I think in this case you are making recommenda-
tions and then you are moving one step further where you say, "You better
implement these recommendations." Now that is going beyond what a recom-
mendation is designed to do, I feel.
HARRAP: No, I do not agree with you. The historical fact is that we have
made recommendations before. All that I have seen at this meeting, and what
has happened since, is that EPA has taken no notice of it. There seems to
be no rational attempt to identify the viruses, as far as I can see. So we
are drawing attention to this again. This time we are pointing out certain
techniques by which this can be done, which we think are practicable. If
they choose to disregard it again, that is their affair, but I think it is
our duty to recommend. We are recommending again what we recommended before.
ENGLER: Since the Bethesda meeting, EPA has actually registered only one
virus, the tussock moth virus, so, implying that the guidance or recommenda-
tions of the Bethesda meeting have not been implemented is not quite fair;
how can EPA implement recommendations if there are no viruses registered?
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HARRAP: In that instance did you have certain criteria for identity? Did
you have a battery of data on the virus properties and, for example, anti-
sera against components of the virus before you agreed to register it?
ENGLER: One thing that the people assembled in this room have to realize
is that we need almost a "cookbook" type recommendation for identification,
as well as for any other tests such as those that are suggested by Dr. Meinke
and colleagues. Before we standardize this approach, we cannot proceed, we
cannot tell the registrant, whoever he may be, "Do something along the line
of DNA identity." This is one thing that I have to stress here, and that
all the people in research have to keep in mind, the tests which will be used
eventually must be standardized and tested for their predicted value.
HARRAP: But can't you contract this work out to competent labs? The Minis-
try of Overseas Development in England contracts out characterization work
to us and pays us for it, and then they contract out the toxicity work to
MRE, in Porton Down, and pay them for it. They do not have all the expertise
themselves.
SMITH: One of the things we are recommending for implementation by this date
is a set-up of a sort of permanent advisory panel. I would envisage one of
the functions of this permanent advisory panel is to make specific "cookbook,"
if you wish, recommendations to the EPA. What we are saying by setting this
date is that this is a reasonable time by which this panel should be well
under way. Other things that we think are more pressing, in terms of safety
testing, etc., are also included in this timetable. So I think that within
the confines of this short time that we have here, and this is a brief docu-
ment, I do believe this is a reasonable timetable with which this permanent
panel can be set up. You can ask that panel to do whatever you wish, but one
of these things is what you have suggested.
SUMMERS: Is there a motion to remove that date or to modify that statement?
Is there a specific motion before us on that statement at all? Is there
additional discussion?
SHOPE:
"Research Program. To provide a continuing basis for improve-
ment and an evaluation of new products, it is imperative that cer-
tain areas bearing on the safety of insect viruses be more fully
understood: 1. A better understanding is needed of the replica-
tion and pathogenesis of baculoviruses in their natural hosts
and cell culture systems...."
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GRANADOS: I suggest that we drop back to baculoviruses and insert insect
viruses to make that more general.
SHOPE:
"To provide a continuing basis for improvement and an evalua-
tion of new products, it is imperative that certain areas bearing
on the safety of insect viruses should be more fully understood.
A better understanding is needed of the replication and patho-
genesis of baculoviruses in their natural hosts and cell culture
systems," and the suggested modification would be "A better under-
standing is needed of the replication and pathogenesis of insect
viruses in their natural hosts and cell culture systems."
IGNOFFO: The primary concern at this stage is for those viruses we believe
can be developed feasibly. Thus, I believe we should continue to stress the
baculoviruses. I am fully in favor of a study of all insect viruses, their
modes of replication, pathogenicity, the whole pattern of replication, and
specificity. But I think this conference directly relates to the baculoviruses,
SMITH: Now, I would like to disagree with that. I think it is relevant to
safety to know what insect viruses do, and one of the things that can happen
is that you can spray a forest or a family with an uncharacterized virus
that you really should know something about. How do you prevent its repli-
cation? Do you have antiserum?
IGNOFFO: How do you know something about an unknown virus?
SMITH: I did not say "unknown," I said "uncharacterized." Let's say, for
example, we had the cricket paralysis virus unwittingly sprayed on the people.
Are antisera available, how does it replicate, what does it replicate in?
That kind of information is necessary. What you are trying to say here, I
hope, is that the panel advises further research along lines that may be
relevant to safety.
COLLINS: What about a compromise, something like, since the word "insect
viruses" is used in either the previous sentence or the previous phrase in
that sentence..."of these agents with particular emphasis on baculoviruses"?
SHOPE: I can read it if you like.
"To provide a continuing basis for improvement and an
evaluation of new products, it is imperative that certain areas
bearing on the safety of insect viruses, with particular emphasis
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on baculoviruses, should be more fully understood. A better
understanding is needed of the replication and pathogenesis of
viruses in their natural hosts and cell culture systems. The
genetics of viruses and the development of specific genetic
markers are vital for precise ecological study. For example,
the possible range of interaction between the viral pesticide
and other viruses in the biologic environment needs to
be explored."
SUMMERS: All in favor? Unanimous. Proceed.
SHOPE:
"These general recommendations of the invited panel are
intended to focus attention on areas urgently in need of research.
They represent certain elements only of major issues raised in
the panel discussion and are put forward as the basis for the
formulation of a specific program at an early date. We feel that
this could be best achieved by a permanent and independent advi-
sory panel."
SMITH: I move that we unanimously adopt it.
SUMMERS: Is there a second to that? All in favor? It is unanimous.
I just want to say thanks to the Environmental Protection Agency for
providing the opportunity for an entirely new forum or atmosphere in which
to think intelligently and discuss the use of viruses as biological alter-
natives for chemical pesticides. I want to thank my colleagues who took
part in the formal presentation and participations. We have been working
together for some time. For those of you not on the panel who do not know
us well, I want you to know that they really did take this task seriously,
and they did a superb job.
I want to thank the members, many of whom are not here now, of the
Virology Advisory Panel. As I said earlier, only two out of the original 12
declined. From your comments, my interpretation is that from the standpoint
of or evaluation of basic research, you have been very complimentary, and
I think you recognize what the important problems are in our programs. You
really revealed through an honest response and evaluation the promise of
this area of virology, and I think you perhaps recognize the promise and the
problems of this area better than some of us do, perhaps even the government.
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I am personally gratified for this exchange. It has been a very intel-
lectual one, and I hope we have the opportunity to do it again. I hope we
now can begin to consider you as colleagues, as I hope you will consider us
colleagues and virologists.
To the EPA, I think it is clear that all has been said that can be said.
The serious problem that we have is really implementation. We keep saying
this at every program, every meeting, over and over again. And my final
comment to the Environmental Protection Agency is that I would hope that
you would somehow identify your responsibility in this area and provide a
mechanism to deal with it.
CURLEY: Thank you, Max. I, too, would like to, on behalf of the Office of
Research and Development and the Health Effects Lab at Research Triangle
Park, North Carolina, thank you and your colleagues for putting this sympo-
sium together, and for the recommendations that will be forthcoming from
you. I would like to point out here, and I register only one concern that
I would like to share with you.
We are in the Office of Research and Development. Our primary respon-
sibility is research, and Reto Engler's office is the office that is a pro-
gram office, the Office of Pesticide Programs. We respond to two different
administrators. We have not attempted to provide a forum for looking at
basically the research in this area. You have not only done that, you have
also looked at some of the regulatory aspects of the viral pesticides pro-
gram. So we got two things, we got recommendations on the regulatory process,
and we got recommendations on research. Through my administrator, we will
provide the recommendations that you have made to us on the regulatory process
to Reto's administrator, and hopefully, they will consider, or give due con-
sideration to all of these recommendations that have come out of this symposium.
Again, I thank you and your colleagues and we hope that we will again
be able to provide further symposia, further research through the Office of
Research and Development, and hopefully, through OPP, through Reto Engler's
group. Both his group and my group can respond to various aspects of these
recommendations put to us. We will look forward to working with you again
and I hope that Reto Engler and his group will also work with you.
ENGLER: From all the conferences on safety aspects of insect viruses held
in the past, I feel that this has been the most rewarding one, primarily for
the reasons which Dr. Curley mentioned. It has become clear to the scien-
306
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tists and to the virologists that there are two different needs related to
the viral pesticides.
The recommendations clearly state that we should categorize the basic
research and the testing for regulatory purposes that need to be done. The
testing presently done was found largely acceptable, some additional testing
was highly recommended to be included in the near future, and, in addition,
basic research is recommended as a long-term goal. I also would like to re-
emphasize that any safety test should be standardized and tried out as com-
pletely as possible. This applies to identification as well as to the
sophisticated virus-cell interaction tests.
Along with that, I would also make a statement regarding our plight in
the registration part of the pesticide effort of EPA. One must consider
that we are working very close to the legal side of pesticide regulation.
If in the future this panel of experts recommends a test and EPA is going to
adopt the test, it will be difficult to deviate from the requirement unless
flexibility is built into it. That goes back to how many tissue cultures
we are going to use and the other topics that were discussed around the table
this morning. So please keep that in mind, that when a test becomes a
requirement it is not only a scientific requirement, it is also a legal
requirement. Along with these thoughts, maybe some of the terms need to be
redefined, namely, guidelines. Guidelines have in the past become a legal
document, whereas guidance is not considered a legal document.
As a final statement I would strongly support what is also contained in
the recommendation, that this or a very similar panel of experts will con-
tinue as an advisory group to finalize those standard tests and to finalize
the future research needs.
SUMMERS: I am assuming that this session is finished.
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Conference Attendees
DR. GEORGE ALLEN
Professor
Department of Entomology and
Nematology
University of Florida
3103 McCarty Hall
Gainesville, Florida 32611
DR. MARIO ANDRES
University of Toronto
540 College Street
Toronto, Ontario
CANADA
DR. THOMAS A. ANGUS
Insect Pathology Research Institute
Post Office Box 490
Sault Ste. Marie, Ontario, P6A 5M7
CANADA
DAVID J. BROWN, M.SC.
Plant Products Division
Canada Department of Agriculture
Ottawa, Ontario K1A OC5
CANADA
DR. JOHN A. COUCH
Research Pathologist
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32503
DR. JOHN C. CUNNINGHAM
Insect Pathology Research Institute
Post Office Box 490
Sault Ste. Marie, Ontario, P6A 5M7
CANADA
DR. S.C. CHOI
University of Toronto
540 College Street
Toronto, Ontario
CANADA
AUGUST CURLEY
Chief, Toxic Effects Branch
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Environmental Toxicology Division
(MD66)
Research Triangle Park, North
Carolina 27711
DR. WALTER DOERFLER
Professor of Genetics
University of Koln
Weyertal 121
5000 Koln 41
WEST GERMANY
DR. PETER FAULKNER
Professor
Department of Microbiology
Queen's University
Kingston, Ontario, K7L 3N6
CANADA
309
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DR. JOHN J. HAMM
Research Entomologist
U.S. Department of Agriculture, ARS
Southern Grain Insects Research
Laboratory
Tifton, Georgia 31794
DR. A.M. HEIMPEL
Chief, Insect Pathology Laboratory
Plant Protection Institute
U.S. Department of Agriculture, ARS
BARC West
Beltsville, Maryland 20705
DR. KENNETH J. HOOD
Office of Research and Development
U.S. Environmental Protection Agency
(RD-683)
401 M Street, S.W.
Washington, D.C. 20460
DR. H.T. HUANG
Program Manager
National Science Foundation
1800 G Street, N.W.
Washington, D.C. 20550
DR. ROBERT L. KAISER
Director
Bureau of Tropical Diseases
Center for Disease Control
1600 Clifton Road, N.E.
Atlanta, Georgia 30333
DR. WILLIAM H.R. LANGRIDGE
Virologist
Boyce Thompson Institute for Plant
Research
1086 North Broadway
Yonkers, New York 10701
DR. HORACE MAZZONE
Forest Insect and Disease Laboratory
U.S. Department of Agriculture
151 Sanford Street
Hamden, Connecticut 06514
DR. A.H. MCINTOSH
Waksman Institute of Microbiology
Rutgers University
New Brunswick, New Jersey 08903
DR. LOIS K. MILLER
Assistant Professor
Department of Biochemistry
The University of Idaho
Moscow, Idaho 83843
DR. H.G. MILTENBURGER
Professor
Institute of Zoology
Technical University
D-6100 Darmstadt
WEST GERMANY
DR. JOHN D. PASCHKE
Professor
Department of Entomology
Purdue University
West Lafayette, Indiana 47907
DR. ROGER L. PIERPONT
Lead Registration Support Section
Ecological Effects Branch
Criteria and Evaluation Division
U.S. Environmental Protection Agency
Crystal Mall, Building #2, Room 824
Arlington, Virginia 20460
DR. JEAN E. PULL I AM
Chief, Pesticide Research
Coordination Staff
Office of Pesticide Programs
U.S. Environmental Protection Agency
East Tower 501 (WH-566)
401 M Street, S.W.
Washington, D.C. 20460
ANN L. RALSTON
Microbiologist
U.S. Environmental Protection Agency
(MD67)
Research Triangle Park, North
Carolina 27711
RUSSELL REGNERY
Department of Microbiology
Rutgers Medical School
College of Medicine and Dentistry
of New Jersey, Box 101
Piscataway, New Jersey 08854
310
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DR. DONALD W. ROBERTS
Insect Pathologist
Boyce Thompson Institute for Plant
Research
1086 North Broadway
Yonkers, New York 10701
DR. K.R. ROZEE
Professor and Head Microbiology/
Medicine
Dalhousie University
Sir Charles Tupper Building
Halifax, Nova Scotia, B3H 4H7
CANADA
DR. PATRICIA L. RUSSELL
Staff Biologist
Special Science Programs
American Institute of Biological
Sciences
1401 Wilson Boulevard
Arlington, Virginia 22209
DR. ERNST-FRIEDRICH SCHULZE
Hoechst Aktiengesellschaft
Pflanzenschutzforschung Biologie
Postfach 800320
Frankfurt/Main 6230
WEST GERMANY
DR. MARTIN SHAPIRO
Research Entomologist
U.S. Department of Agriculture, ARS
Building 1398
Otis Air Force Base, Massachusetts
02542
DR. T.R. SHIEH
Manager
Biological Research and Development
Sandoz Incorporated
15995 SW 78th Place
Miami, Florida 33157
GALE E. SMITH
Department of Entomology
Texas A&M University
College Station, Texas 77843
DR. GORDON R. STAIRS
Professor of Entomology
Ohio State University
1735 Neil Avenue
Columbus, Ohio 43210
DR. D.B. STOLTZ
Assistant Professor of Microbiology
Dalhousie University
Sir Charles Tupper Building
Halifax, Nova Scotia, B3H 4H7
CANADA
DR. FRODE ULVEDAL
Acting Director
Health Effects Division
U.S. Environmental Protection
Agency (RD 683)
401 M Street, S.W.
Washington, D.C. 20460
DR. STAN WEBB
Department of Microbiology
Medical College of Virginia
Box 487
Richmond, Virginia 23298
DR. H. ALAN WOOD
Associate Virologist
Biological Control Program
Boyce Thompson Institute for
Plant Research
1086 North Broadway
Yonkers, New York 10701
311
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/9-78-026
2.
3. RECIPIENT'S ACCESSION NO.
TITLE AND SUBTITLE
Viral Pesticides: Present Knowledge and Potential
Effects on Public and Environmental Health
5. REPORT DATE
September 1978
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Toxicology Division
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park. N.C. 27711
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental Protection Agency
Office of Research and Development
Health Effects Research Laboratory
Research Triangle Park. N.C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
RTP,NC
14. SPONSORING AGENCY CODE
EPA 600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Baculoviruses appear to be effective alternatives to chemical pest control. To
date deleterious effects on other components of the ecosystem have not been demonstra
ted. However, safety testing recommended for registration utilize protocols develope
for chemical pesticides. Safety testing should respond to improving technology. The
concensus of the symposium participants was that (1) safety testing protocols be
modified such that they are appropriate for biological agents, (2) the sensitive and
refined tools- of molecular biology such as restriction fragment analysis, nucleic aci
hybridization techniques, radioimmunoassay, etc., offer improved levels of specificit
for virus identification and/or detection of virus replication, (3) the question of
persistence or expression of viral genome or parts of viral genome in non-target
systems should be of primary concern, (4) EPA confirm the data most relevant to safet
tests before a biologic agent is registered, and (5) detailed studies on replication,
pathogenesis and genetics of these agents in their natural hosts and cell culture
systems should be carried out. These general recommendations emphasize only certain
major topics urgently in need of research to provide basic information necessary for
more precise and rational assessment of possible health effects of biologic agents
used as pesticides.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
biological agents
pest control
chemical tests
virology
pesticides
baculoviruses
06 F, M
15 B
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS {ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
332
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
312
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