EPA-600/1-80-020
May 1980
DEVELOPMENT AND STANDARDIZATION OF
IDENTIFICATION AND MONITORING TECHNIQUES
FOR BACULOVIRUS PESTICIDES
by
Dr. Max D. Summers
Department of Entomology
Texas A&M University
College Station, Texas 77843
EPA GRANT 805232
Project Officer
Dr. Clinton Y. Kawanishi
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Environmental Toxicology Division (MD 67)
Toxic Effects Branch
Research Triangle Park, North Carolina 27711
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
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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FOREWARD
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
establishment 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 toxi-
cology, epidemiology, 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 registra-
tion of new pesticides or proposed suspension of those already in use,
conducts research on hazardous 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 testi-
mony and preparation of affidavits as well as expert advice to the Admin-
istrator to assure the adequacy of health care and surveillance of persons
having suffered imminent and substantial endangerment of their health.
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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 microbial. Of these microorganisms, viruses are perhaps the most
unique in structure, biology, and the intimacy of their parasitic rela-
tionship 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
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Abstract
Biological pesticides, in particular the microbial formulations, are
fundamentally different from chemical pesticides in the nature and mode
of action of the active agent. The pesticidal action is dependent on the
activities of living organisms. Identification, detection and monitoring
methods for biological pesticides, because of their nature and characteristics,
are divergent from those classically associated with chemical toxicants.
Therefore, a new class of standardized, specific and sensitive methods
must be developed. Fortunately, much of the sophisticated technology exists
in the area of medical microbiology and can be immediately adapted and
applied to microbial pesticides. Any difficulties encountered in adapting
this technology will be the result of our lack of knowledge about the
biology of the organisms.
One objective of this grant was the development, adaptation and
application of specific sensitive diagnostic and clinical techniques for
identification, detection and monitoring of viral pesticides. A portion
of this research was also commited to study some of the basic biology and
characteristics of baculoviruses so that a more thorough understanding of
the limitations of the developed monitoring technology would be better
understood. This technology can now be applied for the assessment of
health and ecological effects, as well as for regulatory concerns.
A. Baculovirus identification:
1. By analysis of the structural polypeptides of enveloped nucleo-
capsids by analytical SDS-polyacrylamide gel electrophorens
(SDS-PAGE) baculovirus species can be differentiated. This is
one tool for identification which can be used with reliability
and routinely, since different species of baculoviruses contain
several different structural polypeptides. However, MNPVs,
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i.e., those baculoviruses with more than one nucleocapsid per
envelope, reveal qualitative and quantitative differences when
the singly enveloped nucleocapsids are purified from the multiples
and each are analyzed by SDS-PAGE. Such variation might be misin-
terpreted as strain differences if it was not known which biologi-
cal form of the virus was being analyzed.
Furthermore, our biological studies have shown that there
are two infectious forms of a given species of baculovirus which
occur during the infection cycle. Most studies are conducted
on the alkali-liberated from because it is easily obtained in
sufficient quantities for analysis. The non-occluded or extra-
cellular virus is not occluded during the infection cycle, is
200 fold more infectious than the alkali released form and has
different neutralizing antigens. Although this form of virus can
be easily identified by SDS-polyacrylamide gel electrophorens,
care has to be taken when using the appropriate serological iden-
tification technique or it will not be detected.
Polyhedrins and granulins have been purified and identified
by two-dimensional peptide mapping. Although individual baculo-
viruses can be identified by this technique the nature of the
experimental procedure is too complex to allow routine application
for identification.
2. Serological Assays for identification and detection.
Radioimmuneassay (RIA) has been developed for the quantitative
detection of the major structural protein of baculoviruses,
polyhedrin or granulin. By polypeptide mapping (above) and
serological comparisons, it is now known that polyhedrins and
granulins are related and, therefore, group antigens. Standardized
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RIA as developed is able to detect 70-80 picograms of polyhedrin
protein which is equivalent to approximately 40-50 polyhedra.
This assay can significantly detect polyhedrin in as few as 100
infected cells as early as 12 hours psot-infection in susceptible
cells.
3. RIA for polyhedrin can be useful for monitoring the levels of
polyhedra applied in environmental samples. The limitations for
the use of this assay are:
]) polyhedrins are group antigens and not virus specific
as analyzed by RIA;
2) although the RIA is good for a known or standard system
where polyhedra are routinely produced, it is now known
that polyhedrin is not required for infection nor
need be synthesized in cells with replicating virus.
Also, the synthesis of polyhedrin is dependent upon the
cell, tissue or culture conditions of the assay. Therefore, the
assay cannot be reliably used to detect replicating virus,
especially in non-target hosts.
Although RIA has been developed and standardized for poly-
hedrin, there are limitations for the assay which limits its
use for identification and detection. We only discovered
those limitations by these studies. Therefore, emphasis is now
being given to RIA analysis of virus structural proteins to
identify more reliable virus specific proteins for serological
detection.
Immunoperoxidase has been developed and standardized as an
alternative method for detecting the presence of virus antigens
vii
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in cell and tissues. The assay is very sensitive to being able
to detect polyhedrin antigens 2-5 hours prior to visualization
with the phase microscope. Antisera against replicating virus
detected those antigens in cells 2-4 hours before infectious
viruses could be assayed. The technique is also sensitive to the
level of positive detection of 1 in 100 infected cells and can
detect replicating virus in the absence of polyhedrin synthesis.
This technique can be easily adopted to screen a variety of tissues
and cell types in routine monitoring procedures.
4. Restriction endonuclease enzymes are an additional and powerful
tool for identification and for genetics studies. This technology
has been standardized for baculoviruses and every baculovirus
isolate studied to date shows specific genetic markers for iden-
tification. Although serology is useful for clinical applications,
standard monitoring by serological means will not given an accurate
picture of genetic stability or change.
By cloning we have shown that the wild isolates of baculo-
virus of Autqgrapha californica is a mixture of genetic variants.
We do not know what this means concerning mutation or recombina-
tion or what biological significance can be attributed to host
specificity. The genetic stability of these viruses must be under-
stood and such is essential if a realistic assessment is to be
made for potential ability to infect non-target organisms and for
safe use.
viii
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B. Detection and monitoring
The structural proteins of baculoviruses are being identified in
order to develop and apply highly specific and sensitive seriological
detection methodology. RIA and inrmunoperoxidase assays are available for
polyhedrins and granulins. The conditions for extending these assays to
other baculoviruses have been studied so that development of additional
assays could be easily accomplished at the replicating virus level. How-
ever, such assays have limitations due to sensitivity and should be used
along with assays specific for the genetic material of the virus.
The use of restriction endonuclease enzymes for the specific identi-
fication of the structure of baculovirus genomes has been applied in these
studies with good success. This procedure can detect genetic changes in
the virus after exposure to target and nontarget systems. Furthermore,
the methodology as developed with this project is the first step for the
development of molecular hybridization technology. Molecular hybridiza-
tion with nucleic acid probes can be used for detection of less than 0.2
genomes per cellular DNA equivalent in non-target organisms. Therefore,
this can be used for monitoring for persistence and integration in non-
target organisms, both activities of which are essential to evaluate one
potential role of baculovirus genomes to act as carcinogens.
C. Baculovirus Biology
The biological activity of baculoviruses has been shown to exhibit
complex behavior in the susceptible host and in tissue culture. There are
two infectious forms which have different roles and fates during invasion
and infection: the polyhedral form is responsible for persistence and
transmission in the environment, and the extracellular virus which is not
included in the polyhedra and is responsible for secondary infection of
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3
host tissues. Extracellular virus is 2.2 to 2.4 x 10 more infectious
than the alkali released form (from polyhedra), has a different structural
protein composition and different neutralization antigens. These sig-
nificant biological and structural differences reveal why it is necessary
for detection and monitoring that the appropriate diagnostic technique
is used. This reveals the importance of understanding the biology of the
virus and, by knowing this, how to evaluate and assess biological impact.
For example, when evaluating health assessments the results of these
studies suggest that it would be most appropriate to use the most infec-
tious form of the virus.
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CONTENTS
Foreward ii
Abstract ill
Figures viii
Tables x
1. Introduction 1
2. Conclusions 4
3. Recommendations 5
4. Materials and Methods 6
Viruses 6
Isolation of polyhedra 6
Virus purification 6
Purification of capsids 6
Purification of polyhedrin 7
Polyacrylamide gel electrophoresis
and densitometry 7
Molecular weight and protein determination
Cell culture 7
Extracellular Virus 7
Plaque assay of infectious virus 9
Peptide mapping 9
Amino acid analysis 9
Preparation of antigens 9
Polyhedrins 9
Enveloped nucleocapsids from
occlusions (LOVAL) 9
Enveloped nucleocapsids (NOV) 9
Preparation of antisera 10
Preparation of purified antibody (IgG) 10
125I labelling of IgG 10
Microtiter solid-phase immunoradiometric
assay (Micro-SPIRA) 10
Inhibition assay 11
Virus neutralization 11
Absorption of antisera 11
Competition radioimmunoassay (RIA) 11
Immunodiffusion 12
Immunoperoxidase assay 13
Antisera 13
Treatment of fixation of cells 13
Immunoenzymic staining procedure 13
Intracellular and extracellular viral
growth curve 14
Plaque-purification for restriction enzyme
studies 14
Purification of virus and viral DNA for
restriction analysis 15
Restriction endonuclease digestion 15
Agarose electrophoresis 16
XI
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EcoR-1 restriction endonuclease and
molecular weights virus DNAs 16
5. Results and Discussion 17
Granulins and polyhedrins 17
Size 17
Two-dimensional, high voltage
electrophoresis (HVE) 17
Structural polypeptides of enveloped
nucleocapsids and nucleocapsids 22
Virus polypeptides 22
Polypeptide composition of enveloped
singles and enveloped multiples 26
Polypeptides of baculovirus capsules 27
Structural polypeptides of extracellular
virus 28
Competition RIA 30
Immunodiffusion of polyhedrins and
granulins 30
RIA of AcMNPV polyhedrin and TnGV
granulin 31
RIA of AcMNPV polyhedrin synthesized
jln vivo 31
Competitive inhibition studies of
polyhedrins and granulins 31
Discussion 38
Micro-SPIRA: enveloped nucleocapsids 39
Biological properties of infectious virions 40
Physical-infectious particle ratio 40
Immunoperoxidase 45
Production of enveloped nucleocapsids,
polyhedrin and polyhedra 45
Intracellular location of enveloped
nucleocapsid and polyhedrin antigens 45
Infection of Manduca sexta, Mamestra
brassicae, Bombyx mori _5_ and
Carpocapsa pomonella cells 49
Discussion 49
Neutralization 51
Discussion 55
Preliminary studies of phenotypic and geno-
typic variation and/or stability of wild
viral isolates after passage through
alternate host systems 56
Use of restriction enzymes for evaluating
the genome structure of plaque-purified
and wild isolates of baculovirus 59
EcoR-1 endonuclease digests of baculo-
virus DNAs 59
Comparison of wild-type DNAs 60
Restriction analysis of plaque-purified
virus DNA 60
References 65
xii
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FIGURES
Number Page
1 Granulins and polyhedrins 18
2-3 Two-dimensional high-voltage electrophoresis of a tryptic
digest of granulins and polyhedrins 21
4 Polyacrylamide slab gel electrophoresis of enveloped nucleo-
capsid polypeptides 23
5 Baculovirus-enveloped nucleocapsid 24
6 Comparison on all 11% polyacrylamide slab gel of polypeptides
from enveloped single nucleocapsids and multiples 25
7 AcMNPV, RoMNPV, and TnGV were treated with 2% NP-40 in 0.01 M
Tris (pH) 7.8), 0.01 M EDTA for 18 h at 37°C, virus capsids
(density of 1.33 g/ml) were recovered, and 5 yg of each was
prepared for electrophoresis in 11% polyacrylamide slab gels.27
8 A comparison of AcMNPV from the polyhedra of infected TN-368-10
cells with extracellular virus by electrophoresis in 11%
polyacrylamide in the presence of 0.1% SDS. 29
9 Immunodiffusion of polyhedrins and granulins 32
10 Radioimmunoassay of AcMNPV polyhedrin and TnGV granulin 33
11 Detection of AcMNPV polyhedrin in infected cells 35
12 Comparison of polyhedrins and granulins by competitive RIA 36
13 Comparison of polyhedrins and granulins by competitive RIA
utilizing TnGV granulin antiserum 37
14 Accumulation of intra- and extracellular infectious virus of
enveloped nucleocapsid antigens detected by the plaque assay
and immunoperoxidase technique 46
15 Production of TN-368-10 (a) and TN-368-13 (b) Cells of AcMNPV
enveloped nucleocapsid and polyhedrin antigens, as detected
by the immunoperoxidase technique, and of polyhedra 47
16 Micro-SPIRA for enveloped nucleocapsids 41
17 Micro-SPIRA for AcMNPV EV 41
18 Micro-SPIRA for AcMNPV polyhedrin 42
19 Micro-SPIRA for detection of specific antiviral antibodies 42
20 Neutralization of AcMNPV LOVAL (A) and RoMNPV LOVAL (B) with
rabbit antisera generated against AcMNPV LOVAL (a) and RoMNPV
LOVAL (•). 52
xiii
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Number Page
21 Neutralization of AcMNPV LOVAL with heat -inactivated (*) and
untreated (j«) anti-AcMNPV LOVAL serum, and with heat-in-
activated (0) and untreated («) anti-AcMNPV EV serum, 53
22 Virus neutralization with untreated anti-AcMNPV PMB-NOV serum
of AcMNPV PMB-NOV (•) , RoMNPV PMB-NOV (0), and RoMNPV LOVAL
O- 53
23 Neutralization of intracellular nonoccluded virus from both
TN-368-13 cells (f) and the fat body of infected T_. njL larvae
(&•) with heat-inactivated anti-AcMNPV EV serum, 54
24 AcMNPV LOVAL-absorbed anti-AcMNPV PMB-NOV serum versus AcMNPV
EV O and LOVAL (*) . ~ 54
25 SDS-polyacrylamide gel electrophoresis of AcMNPV structural
polypeptides 58
26 Schematic representation of the EcoR-1 restriction fragments for
eight wild-type baculovirus DNAs, 61
27 The DNA from plaque-purified AcMNPVs compared by electrophoresis
of EcoR-1 restriction fragments as described in Fig. 2. 62
xiv
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TABLES
Number Page
1 Basic Peptides -^9
2 Acidic Peptides ^9
3 Neutral Peptides 20
4 Detection of AcMNPV antigens and infectivity by immunoperoxsi-
dase and plaque assay in insect cell lines 4g
5 AcMNPV physical-infectious particle ratios as assayed in vitro 43
6 LD5Q and TCID _ values of LOVAL and EV 45
xv
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SECTION I
INTRODUCTION
The use of insect pathogens, in particular viruses, as a viable
alternative or aid in pest control programs has been comprehensively
documented in the published proceedings of several conferences since
1973 (FAO Report, 1977; WHO Technical Report Series 531, 1973; Summers
et^ al_., 1975; Summers and Kawanishi, 1978). Baculoviruses have been
given favorable attention and consideration for development once a com-
prehensive evaluation of safety is documented and reconfirmed (NAS
Report, 1975: pp. 19-20). Chemicals are the major defense for the
suppression and control of insect pests. However, recent problems
associated with undesirable side effects are now pointing to the urgency
for the development of new or modified methods for arthropod control;
especially highly specific methods for economically important and
dangerous species (FAO Report, 1977: WHO Technical Report Series 585,
1976). It is important that alternative methods do not cause persistent
or cumulative toxic contamination of the environment and, perhaps most
importantly, methods must be utilized against which resistance (bio-
chemical and/or behavioral) is not readily developed. As long as insec-
ticides remain the principle tool for control of agriculturally important
insect pests and vectors of human disease, it is likely that resistance
will continue to be the major threat and perhaps final difficulty (FAO
Report, 1977; WHO Technical Report Series 585).
The number of really useful economic insecticides is limited and new
compounds or compounds with novel modes of action do not appear to be
forthcoming in quantity. The appearance of increasing resistance to new
alternative chemicals makes the situation apparently worse. The poten-
tial advantages of viral pesticides over chemicals are: natural occur-
ence in the environment, specificity, low toxicity, little or no residue,
perhaps the least likelihood of inducing resistance (this has not been
properly tested) and, in some cases, the ability to establish and
maintain persistent infectious forms in the environment,
With the projected problems associated with world food production
and public health the use of microbials for pest control with specific
and novel modes of action may be a new and important approach (Arrata,
1977). EPA has registered two viral pesticides: a formulation of the
nuclear polyhedrosis virus (NPV) of Heliothis zea and NPV of the Douglas
Fur Tussock Moth (Hemerocapsa pseudosugata). The Gypsy Moth NPV (Porthe-
tria dispar) is expected to be registered shortly. Recent reports reveal
that with these registrations the use of microbials on a worldwide basis
is expanding rapidly from an experimental situation to more routine
uses for applied problems (Harrap, 1977, Falcon, 1977).
It seems logical, however, to document the potential problems as
well as benefits for the use of baculoviruses in order to provide the
appropriate forum and background for evaluating possible undesirable
consequences or appropriate risks (Tinsley and Melnick, 1974; Summers
et al., 1975; Summers and Kawanishi, 1978). The testing procedures
and criteria presently used for registration of baculovirus formulation
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have followed those tests utilized for the registration of chemical pesti-
cides. It has been clearly identified in the above documents that those
tests need to be modified for invasive and/or replicating entities such
as viruses. Tests more appropriate for the detection of replication or
persistence in physical and biological systems are badly needed. For
this specific and sensitive detection and monitoring technology needs to
be available.
However, basic to this are other problems which have been fully ad-
dressed (see proceedings of conferences cited above) but not implemented.
Basic to the appropriate detection technology is the use of reliable
identification technology. In fact, identification has been based
primarily upon susceptibility and host range for a given baculovirus.
Identification and cataloguing of baculoviruses by genetic or serological
means has not been established and used. As a result events such as
mutation or recombination cannot be evaluated because of the lack of
appropriate markers. Further, little if anything is known about the
genetics of baculoviruses (Summers and Kawanishi, 1978). It is shown
that baculovirus genomes are large molecules of approximately 108 sugges-
ting that they have a genetic capability comparible for example to the
T-even bacteriophages, Herpes and cytomegaloviruses.
Basically, if baculoviruses are to be evaluated in terms of safe use,
degree of risk by use, or the possibility of there being a risk beyond
of which we are aware, it is necessary to study the structure and stabi-
lity of baculoviruses with the appropriate techniques to see if they do
change as they evolve subject to environmental pressures, If they do
change, what would be the nature and extent of that change?
The ability of a virus to change usually involves mutation or gene-
tic recombination for each of which there may be several potential
mechanisms. It is quite clear by various mechanisms that vertebrate viruses
with large genomes, such as herpes, adeno, and cytomegaloviruses (CMV)
undergo change by recombination. With baculoviruses at present it would
be difficult to document or establish the difference between recombina-
tion and mutation without being able to identify whether a new hybrid
virus had either a rearranged or new set of genes. Also, certain
baculoviruses contain many nucleocapsids common to a viral envelope.
Little is known about whether or not these represent multiple genomes
within a single bundle of particles or whether this is in fact numerous
copies of the same genome. Such combination(s) make it difficult to
predict possible relationships involving defective particles, recombi-
nation, and etc. This may be quite important since it is fairly well
documented that mixed viral infections can occur in nature with insect
viruses. Perhaps of potentially greater importance is possible recom-
bination between a baculovirus and host or unrelated viral DNA. Further-
more, we know nothing about the frequency by which such a genetic change
may occur, since the genomes of our baculoviruses have not been properly
mapped using the appropriate techniques. Once again these kinds of
studies cannot be conducted until the appropriate standards and markers
have been utilized for identification and comparison.
Direct studies identifying the structural properties and anatomy
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of baculovirus DNAs are important. However, it must be recognized
that it is difficult to directly relate ..biological function to
genetic maps. Therefore, studies on virus structural proteins will also
be important in order to make comparisons between proteins
which are specific or contain group associated antigenlc properties.
This is important to develop the basis for routine serological identi-
fication and detection.
Basic to all of this is another rather concrete realization.
Baculoviruses will likely not be used as individual control agents
in pest management applications. The most effective application for
insect viruses is in integrated pest management schemes. This means
that baculoviruses will be applied along, or in conjunction, with
reduced concentration of chemical pesticides, other pathogens such as
bacteria or fungi, or in combination with other organisms such as para-
sites and predators. In particular, little is known about the combined
activities of chemical pesticides or related agents as combinated with
viral replication: little is known about mutagenic potential of chemi-
cal pesticidal agents used in combination with baculovirus.
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SECTION 2
CONCLUSIONS
Because of the structural complexity and biological differences
between extracellular and occluded virions, it is important that identi-
fication and monitoring technology be developed for both forms until
the reasons for these biological differences are better characterized.
It is important to understand better which form is potentially the most
infectious to non-target organisms. The development of serological iden-
tification and monitoring technology for baculovirus granulins and poly-
hedrins has been accomplished and applied. Although the technology is
extremely sensitive, it is presently believed with new data at hand that
RIA for granulins and polyhedrins will not be all that useful from the
standpoint of reliable monitoring and identification. Development of
RIA for virus specific proteins which can directly monitor for virus
specific antigens and, therefore, are more direct indicators of viral
infection should be developed. In addition to radioimmunoassay, immuno-
peroxidase and neutralization techniques have been applied to the inves-
tigation and detection of baculovirus antigens in infected cells.
Preliminary studies have revealed while isolates of baculovirus are
mixtures of genotypic and phenotypic variants the significance and/or
stability of this variation as it exists in nature is not well understood
relative to the genetic stability of baculoviruses in terms of mutation
and/or recombination.
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SECTION 3
RECOMMENDATIONS
It is recommended that for future studies on haculoviruses that
plaque-purified and genetically identified and defined isolates be uti-
lized as the basis for evaluating genetic and genomic stability relative
to mutation, recombination, and/or change with time by passage through
host and alternate host systems. It is necessary to develop identification
and detection technology and both the genomic and phenotypic levels in
order to provide screening technology for evaluating safe and effective
use of baculoviruses in the biological and physical environment. For
serological studies it is important that viral specific structural poly-
peptides be identified for the development and application of radioimmu-
noassay technology. It is suggested that individual structural polypep-
tides be isolated and evaluated in terms of the specificity of their
antigenic reactions and that this should be done for both the extra-
cellular virus and nonoccluded virion forms.
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SECTION IV
METHODS AND MATERIALS
Viruses. Baculoviruses used in this study were: Autographa cali-
fomica (AcMNPV), Rachiplusia ou (RoJylNPV), Anticarsa gemmatalis (AgMNPV) ,
Hello this armigera (HaMNPV) , Trichoplusia ni (TnSNPV) , and Heliothis
zea (HzSNPV), nuclear polyhedrosis viruses; Trichoplusia ni (TnGV) and
Spodoptera frugiperda (SfGV), granulosis viruses. AcMNPV, RoMNPV,
TnSNPV, and TnGV were produced by infecting T_. ni larvae with the appro-
priate polyhedra. SfGV was produced in Spodoptera frugiperda larvae.
HaMNPV and HzSNPV and the original inoculum of RoMNPV were kindly provided
by C. Y. Kawanishi (EPA, Research Triangle Park, N. C.) and J. Hamm
(USDA-ARS, Tifton, Ga.), and AgMNPV, by G. Allen (University of Florida,
Gainesville, Fla.).
Isolation of polyhedra. Polyhedra were isolated essentially as
reported (Summers and Smith, 19761, vith certain modifications: Poly-
hedra purified by banding on sucrose gradients were suspended in 0.01
M Tris (pH 7.8), 0.01 M EDTA, 1.0 M NaCl and were stirred for 30 min at
room temperature. The polyhedra suspension was layered on 40 to 63%
w/w continuous sucrose gradients in 0.01 M Tris buffer and was centri-
fuged at 100,000 g for 30 min. The polyhedra band was removed and
washed free of residual sucrose by differential centrifugation and then
was resuspended in 0.01 M Tris (pH 7.8) 0.01 M EDTA.
Virus purification. Virus particles were removed from purified
polyhedra by incubating the polyhedra for 60 min at 0-4 in 0.1 M Na2C03,
0.01 M EDTA, 0.17 M NaCl, pH 10.9 (Summers and Smith, 1975), The prepar-
ations were layered on sucrose gradients buffered in 0.1 M Tris (pH 7.8),
0.01 M EDTA (ranging in density from 1.17 to 1.25 g/ml) and were
centrifuged for 1 hr at 24,000 rpm (SW-27) at 4°. The bands of virus
were removed from the gradients and the virus particles were washed free
from sucrose by differential centrifugation and were resuspended in water
and stored at -70°. With AcMNPV, AgMNPV, RoMNPV, and HaMNPV which have
one or multiple nucleocapsids per envelope (M = more than one nucleocapsid
per envelope; ^ = enveloped single nucleocapsid per envelope) the top band
containing single-enveloped nucleocapsids was easily separated from the
other bands (referred to as multiples) which contained more than one
microcapsid per envelope particles (Summers and Volkman, 1976) . Compari-
sons of polypeptide composition were made between singles and multiples.
Purification of capsids. Each of the purified virus perparations
was adjusted to a final concentration of 500 pg of protein/ml in 0.01 M
Tris (pH 7.8), 0.01 M EDTA, 2% NP-40 (v/v) and incubated for 18 hr at '
37°. Each preparation was layered on a cesium chloride gradient with
a density ranging from 1.18 to 1.55 g/ml and was centrifuged for 2 hr at
20° at 35,000 rpm (SW-41 rotor). The visible bands were removed, pellet-
ed by differential centrifugation, and later utilized for polyacryla-
mide gel electrophoresis and observation by microscopy. For density
determinations, each gradient was fractionated with an ISCO density
gradient fractionator and gradient scanning profiles were recorded on
an ISCO UA-2 monitor. Densities of the individual samples were deter-
mined using an Abbe refractometer.
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Purification of polyhedrin. Polyhedrin and granulins were purified
using a modification of the procedure reported by Summers and Smith
(1976). Highly purified polyhedra were suspended in 0.01 M Tris (pH
7.8), 0.01 M EDTA (a final concentration of 5.0 mg of protein/ml) and
incubated for 2 hr at 70° to inactivate the associated protease.
Protease-inactivated polyhedra were washed free of buffer by differential
centrifugation. To insure further the inactivation of the protease, the
polyhedra were resuspended in 0.01 M Tris (pH 7,8), 0.01 M HgCl2 (final
concentration of 5 mg of protein/mg) and allowed to equilibrate at room
temperature overnight. The polyhedra were removed from the buffer and
enzyme inhibitor by pelleting, using repeated differential centrifugation
and extensive washing of the pellets with water. Solubilizatifai of the
polyhedra was achieved by adding 0.1 M Na2C03, 0.17 M NaCl, pH '10.8, for
19 min at a concentration of 5 mg of protein ml at 4°, The solution was
centrifuged at 24,000 rpm (SW 27 rotor) for 30 min at 5° to remove any
insoluble material. The solubilized polyhedrins were then utilized for
polyacrylamide gel electrophoresis described previously. Granulins and
polyhedrins were stored at -70° and were shown to be stable relative to
enzyme degradation by polyacrylamide gel electrophoresis.
Polyacrylamide gel electrophoresis and densitometry. Electrophore-
sis in an 11,0% polyacrylamide vertical gel slab in the presence of 0.1%
SDS was conducted as described by Laemmli (1970). The samples were im-
mediately subjected to electrophoresis for 3 hr at a constant power of
7.5 W/gel slab (ISCO Model 493 Constant Power Supply). The gel slab was
14 cm wide, 12 cm high, and 1.5 mm thick. The slabs were fixed in 25%
isopropanol, 10% acetic acid, stained with 0.04% Cootnassie brilliant blue
R-250 for 4 hr, and destained in 10% acetic acid. Stained gels were then
photographed and the transparencies were scanned at 600 nm using a Beckman
DU spectrophotometer equipped with a Gilford 2400 linear transport
accessory.
Molecular weight and protein determination. The method described by
Weber and Osborn (1969) was used to determine the apparent molecular
weights of the viral proteins as compared to the molecular weights of
standard proteins by gel electrophoresis.
The protein determination method of Lowry et al. (1951) was used to
estimate the amount of protein relative to bovine serum albumin as the
standard.
Cell culture. The continuous cell line of Trichoplusia ni, TN-368,
was obtained in the 57th passage from W, F. Hink of Ohio State University
in June 1974. Since then the cells have been passed routinely three times
per week in disposable plastic tissue culture flasks, with the initial
concentration being 1 x 10^ to 2 x 10 cells per ml. Daughter cell lines
were developed from isolated single cells of TN-368 (Volkman and Summers,
1976) and these cloned lines were handled in the same manner as TN-368.
The cultures were grown at 28°C, and the medium used was TNM-FH (Hink,
1970). Antibiotics were not used for routine maintenance of the cells.
Extracellular virus. Initially, AcMNPV was supplied by P. V. Vail
and W. F. Hink in the form of infectious tissue culture medium. TN-368
7
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cells infected with this medium yielded polyhedra that were in turn
used to infect T\ nl larvae for the production of infectious hemolymph.
RoMNPV was obtained in the occluded form from C. Y. Kawanishi. T_, njl
larvae infected with the polyhedra yielded infectious hemolymph, which
was used subsequently to infect TN-368 cells for the production of NOV.
Hemolymph used in the neutralization experiments was collected from
infected T_, ni larvae by removing one or two prolegs and catching the
resultant drops of infectious fluid in a test tube containing TNM-FH
plus antibiotics (200 yg of penicillin and streptomycin per ml and 5 yg
of amphotericin B {Fungizone} per ml).
Preparations of LOVAL used as antigens for generating antisera as
well as in neutralization experiments were made in the following way.
The virions were liberated from gradient-purified polyhedra by incubating
the polyhedra for 10 min at 28°C in 0.1 M Na2C03 + 0.05 M Nad, pH 10.9
(Summers and Smith, 1975). The preparations were then immediately lay-
ered on sucrose gradients made in 0.01 M Tris buffer (pH 7.8)-0.0. M EDTA
(ranging in density from 1.18 to 1.25 g/ml) and centrifuged for 2 hr at
24,000 rpm (SW27 rotor). The distinct bands of one through nany nucleo-
capsids per envelope were collected and pooled (Summers and Volkman,
1976). For some experiments, the band containing one nucleocapsid per
envelope was kept separate from the rest.
Virus from the fat body of AcMNPV-infected larvae was collected in
the following way. The infected fat bodies were removed from five larvae
and homogenized in TNM-FH plus antibiotics with a glass homogenizer. The
mixture was centrifuged at 2,100 x £ for 15 min, and the supernatant
fluid was decanted and filtered through a 0.18- to 14-ym microfiberglass
filter. The resultant infectious preparation was used in neutralization
studies.
Nonoccluded intracellular virus from TN-368-13 was obtained simply
by homogenizing a suspension of AcMNPV-infected cells, which had previ-
ously been pelleted and rinsed twice, in TNM-FH, The disrupted cell
suspension was cleared of heavy and large debris by centrifugation at
4,000 x £ for 15 min. The supernatant fluid was used in the neutrali-
zation experiment.
Extracellular virus or PMB-NOV was obtained from the culture medium
of synchronously infected TN-368-13 (or ^. frugiperda) cells in advance
of cytolysis 36 to 48 hr after infection (EV will refer to TN-368-13
derived virus unless specifically referred to as otherwise). Cells were
pelleted by centrifugation at 300 x j» for 15 min (to minimize cell dis-
ruption) , and the supernatant fluid was further clarified by centrifu-
gation at 4,000 x £ for 15 min. This 4,000 x £ supernatant fluid consti-
tuted the crude virus preparation, which was used in the neutralization
experiments. For some studies (e.g., quantification of physical particles
and the generation of antisera), the preparation was further purified as
follows. The 4,000 x j* supernatant fluid was centrifuged at 12,000 x £
for 45 min to pellet the virus. The pellets were resuspended in 0.01 M
Tris (pH 7.8)-0.01 M EDTA and layered on sucrose gradients in that same
buffer, ranging in density from 1.1 to 1,3 g/ml. The gradients were
centrifuged at 358,000 x £ for 1 hr at 4°C. The
8
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1.17- to 1.18-g/ml region of the gradients containing the virus was
collected and held for experimentation (Summers and Volkman, 1976).
Plaque assay of_ infectious virus, For the plaque assay (Hink and
Vail, 1973), (Volkman and Summers, 1975), log-phase TN-368 cells were
seeded into 30-mm Corning plates at 3,5 x 10^ cells per plate in 2 ml
of medium. Cells were allowed to attach for 1 to 2 hr. The medium was
carefully removed, and the cells innoculated with a 0,1-ml sample (per
plate) containing between 50 and 250 PFU, Plates were rocked every 10
min for 1 hr and then overlaid with Agarose (Wood, 1976) in TNM-FH con-
taining 200 yg each of penicillin and streptomycin per ml and 5yg of
Fungizone per ml. All assays were done in duplicate,
Peptide mapping. Protein samples of 1,5-3.0 mg of purified granulin
or polyhedrin were digested with trypsin at a final substrate:enzyme
ratio of 50:1 in 0.25 M ammonium bicarbonate (pH 8.6) at 38° for 12 hr,
The hydrolysate was applied on Whatman 3MM filter paper and the peptides
resolved in two dimensions by high voltage electrophoresis (Summers and
Smith, 1975) (Brown and Hartly, 1966) as reviewed and outlined by Savant
Instruments, Inc. (1971), "High Voltage Electrophoresis Guide", A
third dimension of the neutral peptides was run using ascending chroma-
tography with butanol:acetic acid:water (4:1:5) for 18 hr at room temp-
erature. Positions of the hydrolysates were detected with 1.0% ninhydrin-
0.1% cadmium acetate.
Amino acid analysis. Samples of 0.3 mg granulin or polyhedrin were
hydrolized in a 6 N HC1 at 105° for 24,48, and 72 hr. The hydrolysates
were analyzed in a Beckman Model 120C automatic amino acid analyzer. The
amounts of serine and threonine were obtained by extrapolating to time
zero. Tryptophan content was based on adsorbance at 288 and 280 nm of
protein dissolved in 6 M guanidine hydrochloride. All data presented are
the mean values obtained from one analysis at 24 and 72 hr and three
analyses at 48 hr.
Preparation of Antigens
Polyhedrins. Highly purified polyhedrin was isolated from the
larval polyhedra of Autographa californica NPV (AcMNPV) by the method of
Summers and Smith (1976) using preparative SDS-polyacrylamide gel electro-
phoresis (SDS-PAGE). The 30,000 molecular weight polyhedrin band was
extracted from the gels and was analyzed for purity and stability by
analytical SDS-PAGE.
Enveloped nucleocapsids from occlusions (LOVAL). Purified enveloped
nucleocapsid preparations described as LOVAL (LOVAL: Larvae-derived
Occluded Viruses which have been Alkali-Liberated, Volkman et^ ail., 1976)
were isolated from purified occluded virus using the method of Summers
and Volkman (1976).
Enveloped nucleocapsids (NOV). AcMNPV EV (Plasma-Membrane Budded
NonOccluded Virus) was purified from infected TN-368-10 (Volkman and
Summers, 1976) culture medium by the method described previously (Summers
and Volkman, 1976). This form of the virus has not been occluded or
exposed to alkali. The physical and chemical properties of all viruses
9
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and viral proteins utilized in this study have been characterized by
SDS-PAGE, and the results of these comprehensive studies are to be
published in a separate report (Summers and Smith, unpubl.). The stabi-
lity and structural integrity of all preparations utilized in this study
were routinely evaluated and documented as stable and reproducible.
Preparation of Antisera
Preimmune - sera were obtained and rabbits were immunized essential-
ly as described by Volkman et al. (1976) with the following purified
antigens: AcMNPV LOVAL, AcMNPV EV, and AcMNPV polyhedrin,
Preparation of Purified Antibody (IgG)
Immunoglobin fractions were separated from antisera using two cycles
of precipitation with 33% saturated ammonium sulfate, The precipitates
were collected by centrifugation at 10.000 rpm for 15 min, and pellets
were dissolved in and dialyzed against 0.0175 M phosphate buffer, pH 6.3.
IgG was isolated by the method of Levy and Sober (1960),
125 125
I Labeling of IgG. Ig G was labeled with carrier-free Na I
(New England Nuclear) by a modification of the chloramane-T method
described by Greenwood et. al. (1963), [l^IJIgG was separated from
free I25j using a Sephadex G-50 column (0.7 x 20 cm). [I]IgG was
eluted and stored in 0.05 M phosphate buffer, pH 7,5, containing 1%
bovine serum albumin (BSA) and 0.01% sodium azide. Specific activities
of 3-5 x 10" cmp/yg of the protein were routinely obtained. Using 5%
trichloroacetic acid (TCA) precipitation, more than 97% of the radio-
activity was precipitable.
Microtiter Solid-Phase Immunoradiometric Assay (Micro-SPIRA)
Micro-SPIRA was carried out using a modification of the procedure
of Purcell et al. (1973). The polyvinyl microtiter plate wells (Limbro
Chemical Co., Inc.) were coated with 100 yl of antiserum which had been
diluted 1:10 with phosphate-buffered saline, pH 7.3, containing 0.01%
sodium azide (PBS), The microtiter plates were then incubated at 4°C
for 4 hr to allow adsorption to the surfaces of the wells, After as-
pirating the fluid, the wells were washed twice with 200 yl of PBS, Two
hundred microliters of 2% BSA solution (0.05 M phosphate buffer, pH 7.5,
2% BSA, and 0.01% sodium azide) were then added to each well to adsorb
any unreacted sites. After incubation at 4°C overnight, the solution
in each well was aspirated, and 30 yl of the appropriate dilution for
the virus or polyhedrin was added, The microtiter plates were incubated
at 4°C overnight (19-24 hr). The antigen solutions were aspirated, and
each well was washed five times with 200 yl of the 2% BSA solution. Fifty
microliters of [125I]IgG in PBS (30-40 ng, 1 x 105 cpm) was added to each
well and was incubated at 37°C for 4 hr. The wells were aspirated and
washed five times with 200 yl of the 2% BSA solution. Individual wells
were then cut from the microtiter plates, and the bound [^^5I]IgG was
determined in a Searle Model 1195 gamma spectrophotometer,
10
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Inhibition Assay
Microtiter wells were coated with 1:10 dilutions of AcMNPV LOVAL
antiserum and were incubated for 4 hr at 4QC. The wells were then aspi-
rated, and nonreacted sites were coated with the 2% BSA solution over-
night. The BSA solution was aspirated, and 100 yg of AcMNPV LOVAL in
PBS was added to each well and was incubated at 4°C overnight. The
wells were washed three times with PBS. Fifty microliters of the appro-
priate antiserum or IgG was delivered to each well and was incubated at
37°C for 4 hr. After aspiration of the antiserum, each well was rinsed
five times with the 2% BSA solution, and 50 yl of [125I]IgG was added.
Following incubation at 37°C for 4 hr the wells were aspirated and
washed five times with 2% BSA solution. Individual wells were then cut,
and the bound radioactivity was counted as previously described,
Virus neutralization. Dilutions of antisera were made in Grace's
medium, pH 7.5, with 0.5% bovine serum albumin and antibiotics. Virus
samples were diluted in TNM-FH such that 0.1 ml of a 1:'2 dilution would
contain 100 to 200 pfu (Habel, 1969). Equal volumes (0,3 ml) of the
prediluted antisera and the prediluted virus were mixed and allowed to
incubate at either room temperature (28°C) or 37°C for 1 hr. Samples
(0.1 ml) were then plated to determine the remaining pfu by the plaque
assay. For some experiments sera were heated to 56°C for 30 min to
remove any heat-labile nonspecific inhibitors or enhancers or virus
infection.
Adsorption of antisera. Fifty microliters of undiluted anti-AcMNPV
EV was added to 1 ml of 240 yg of AcMNPV LOVAL in 0,01 M Tris (pH 7.8)
-0.01 M EDTA, This mixture was incubated at 37°C for 2 hr with occas-
sional vortexing and then incubated again at 4°C for 72 hr. The suspen-
sion was then centrifuged at 100,000 x £ and 5°C for 1 hr to remove the
LOVAL. The supernatant fluid was carefully removed and used as a 1:20
dilution of adsorbed antiserum. When this serum was tested for residual
LOVAL infectious activity, none was found,
Competition Radioimmunoassay (RIA). For RIA polyhedrins and gran-
ulins were purified by modifications of published procedures (Summers
and Smith, 1976, 1978). The inclusion (polyhedra) associated protease
was inactivitated by HgCl2 and heat treatment (Summers and Smith, 1978)
and polyhedrins and granulins obtained by electrophoresis in preparative
vertical gel slabs in the presence of 0.1% SDS. Following electrophore-
sis the portion of the gel slab containing the polyhedrin or granulin
was removed and extracted as described previously (Summers and Smith,
1975, 1976). Protein solutions were then stored at -90°C, and analyzed
for stability and purity by analytical polyacrylamide vertical slab gel
electrophoresis in the presence of 0,1% SDS (SDS-PAGE) (Laemmli, .1970) .as
modified for baculovirus proteins (Summers and Smith, 1978). For auto-
radiography gel slabs were dried onto filter paper then exposed to Kodak
x-ray film for 48 hours.
AcMNPV polyhedrin and TnGV granulin were labelled with carrier free
(New England Nuclear) by the chloramine - T method (Greenwood et
al., 1963) . -"-"l-protein Was separated from free -*-"lodine using a P-60
11
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(Bio-Rad) column. The labelled protein was stored in 0,01 M Tris buffer,
pH 7.8, containing 2% bovine serum albumin (BSA) and 0.01% sodium azide
(NaN3) at -90°C, and used within two weeks since a progressive loss in
immunoreactivity occurred upon extended storage at -90°C, storage at 4°C,
and labelling of greater than 20 Ci/yg,
Cell extracts of infected or uninfected TN368-10 cell were pre-
pared for RIA in the following manner. Log-phase cells in 25 ml plastic
T-flasks were inoculated in a total volume of 1,0 ml with AcMNPV extra-
cellular virus at a multiplicity of infection (MOI) of 10 to 20. After
1 hour adsorption, 4 ml of TNM-FH medium plus antibiotics were added.
Mock infected cells received 1.0 ml of TNM-FH medium. Following adsorp-
tion, cells were washed free of inoculum by pelleting at 300 g for 15
min, and washed twice with 5.0 ml of TNM-FH media before resuspension
in 5 ml of medium. At designated times post inoculation, cells were
dispersed into a homogenous suspension by gentle shaking and pipeting,
and samples removed for analysis, Cells were counted, adjusted to the
appropriate concentration with medium, and pelleted by centrifugation
at 300 g for 15 minutes. The cells were disrupted by suspension in 0.5
ml of 0.1 M Na2C03 buffer, pH 10.9, containing 0.17 M NaCl, 0.01 M EDTA.
Noindet-P-40 was added to a final concentration of 0,5%; the cellular
suspension was allowed to set for 30 minutes and twice sonicated (30
seconds each at 4°C). The lysate was then centrifuged at 300 g for 15
minutes to remove any insoluble cell debris, and the supernatant carefully
removed for assay of polyhedrin by RIA.
Immunodiffusion. Immunodiffusion tests were conducted by modifi-
cation of the double diffusion method Ouchterlony (1958) .
A modification of the micro-solidphase radioimmunoassay described
by Purcell et al. (1973) was used for competition RIA. Two hundred yl
of 0.05 M Na^C03 - NaHC03 (pH 9.6) containing 5 yg of Protein A (Phar-
macia Fine Chemicals, Inc.) were placed in flexible polyvinyl microtiter
plate wells (Linbro Scientific Co.), incubated at room temperature for
2 hr and then overnight at 4°C. The solution was aspirated, the wells
washed twice with distilled 1^0, and 200 yg of the appropriate dilution
of purified antibody in 0.01 M Tris buffer solution added (pH 7.5; 0.1 M
NaCl: 0.001 M EDTA; 0.01% sodium azide). The microtiter plates were
incubated for 8.0 hr at 4°C. The wells were aspirated, washed twice
with the Tris buffer and 10,000 cpm of ^-^1 polyhedrin or granulin
(150 yl) in Tris-BSA buffer were added. The plate was incubated over-
night at 4°C, twice washed with Tris-BSA buffer solution, and subsequent-
ly washed three times with Tris buffer. The microtiter wells were cut
-IOC
from the plate, and assayed for bound i*-Ji in an automatic gama spectro-
photometer (Searle Model 1195).
Competitive inhibition assays were carried out in the same manner
except the dilution of antibody was used which bound 50% of the homolo-
gous labelled polyhedrin or granulin. Competing proteins were diluted
in the Tris-BSA solution so that each well received 150 yl of the poly-
hedrin or granulin solution. Following 24 hr of incubation at 4°C, 10,000
CMP of -'-^^I-polyhedrin or granulin (0.01 ml) was added and incubated over-
night at 4°C. The wells were then washed, cut from the microtiter plates
and counted.
12
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For RIA detection of polyhedrin infected and non-infected TN368-10
cells, cell lysates were diluted in a 0.01 M Tris-HCl solution (pH 7.5;
containing 0.01 M NaCl; 0.001 M EDTA; 0.05% NP-40; 0.01% sodium azide).
Standard cell lysates with known polyhedrin concentrations were prepared
by the addition of serial dilutions of purified polyhedrin ranging from
10 pg to 10 yg.
Immunoperoxidose Assay
Antisera. Rabbits (from which pre-immune sera were taken) were
injected as previously described with highly purified AcMNPV larvae-
derived occluded virions which had been alkali-liberated (LOVAL; Volk-
man et^ _al.. 1976), or alternatively, with highly purified AcMNPV polyhe-
drin (Summers and Smith, 1976). Antiserum to purified AcMNPV AcMNPV EV
was also tested (Summers and Volkman, 1976; Volkman et^ alU , 1976). All
antisera were heat-inactivated at 56°C for 30 min before use, Peroxi-
dase-conjugated anti-rabbit IgG goat serum which contained 14 mg gamma
globulin conjugated with 2.9 mg peroxidase per ml of solution was pur-
chased from the Research Division, Miles Laboratories, Elkhart, Indiana.
The antisera used in this study were shown to be specific for the homo-
logous antigen(s) by a micro-solid-phase immunoradiometric assay, as they
did not significantly cross-react with heterologous EV, LOVAL, or poly-
hedrin at concentrations less than 200 ng/ml of each antigen (Ohba et
al., 1977)
Treatment of fixation of cells. Log phase cells were infected with
AcMNPV EV at a multiplicity of infection (MOI) of not less than 5 or grea-
ter than 10 (Volkman and Summers, 1976). For time course studies, 10'
cells of each subline, TN-368-10 and TN-368-13, were inoculated with EV
at a MOI of 5. Other insect cell lines were infected at a MOI of 10.
After a 1 hr adsorption period, the cells were pelleted by centrifugation
at 300 g for 5 min and resuspended gently in complete medium. At this
time and at regular intervals thereafter, samples of 10" cells were re-
moved, pelleted by centrifugation at 300 g for 5 min, and rinsed twice
in TNM-FH or TC100 containing 10% fetal calf serum. The cells were
resuspended in a small amount of the appropriate serum-less medium by
pipetting and small drops of each of the samples placed on acid-cleaned
glass slides. The cells were allowed to attach to the slides for 20 min,
then rinsed twice by gentle dipping in 0.01 M-phosphate buffered saline
solution (PBS, pH 7.2). After washing, three different fixing procedures
were compared: (1) the cells on the slide were air-dried for 20 min at
room temperature, fixed in 1.5% paraformaldehyde in 0.067 ^-potassium
phosphate buffer, pH 7.4, for 20 min at room temperature, rinsed three
times with PBS and air-dried again; (2) procedure 1 was used without the
initial air-drying step; (3) the cells were fixed in acetone for 10 min
at room temperature.
The other invertebrate cell lines were processed using the same
routine procedures and reagents employed with TN-368-10 and TN-368-13
cells.
Immunoenzymic staining procedure. The procedure used was basically
that obtained from Dr. Heather Mayor (personal communication; Baylor
13
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Medical Center, Houston, Texas) with a few modifications. After fixation,
the cells were covered with one or two drops of rabbit antiserum diluted
1:100 to 1:200 in PBS, and were incubated at 37°C for 1 hr. The proper
dilution of antiserum was determined experimentally by selecting the
dilution which gave the least nonspecific but the most intense specific
(positive) reaction. Cells were rinsed extensively in five consecutive
changes of PBS before adding one or two drops of a 1:100 dilution of
peroxidase conjugated goat anti-rabbit IgG. After a 1 hr incubation at
37°C, the cells were rinsed extensively in five changes of PBS. The
peroxidase substrate (made up freshly) was 0,4 mg/ml 3-3' diaminoben-
zidine (DAB) in 0.01% hydrogen peroxide (H202) and 0.1 M-Tris, pH 7.6.
The stock bottle of powdered DAB was kept frozen in a desiccator and
the 1^02 working dilution was made fresh each time from a 30% stock
solution. The cells were covered with the substrate solution and were
allowed to incubate for 10 min at room temperature in the dark. Follow-
ing this treatment they were washed extensively in PBS, dehydrated and
mounted with permount for microscopic examination. Dehydration was
accomplished by immersing slides sequentially for 3 min in each of the
following solutions: 25%, 50%, 75%, 90%, and 100% ethanol, and xylene.
A total of 400 cells in each of three replicate samples were scored
to determine the percentage reacting with immunoperoxidase.
Tests for sensitivity and specificity of the assay were performed.
The tests for specificity included the substitution of pre-immune sera
for the enveloped nucleocapsid or polyhedrin antisera, and the use of
non-infected cells. In tests for the appropriate concentrations of
antisera and reagents, dilutions ranging from 1:10 to 1:400 were tested.
Additional controls involved the omission of either virus antiserum,
peroxidase conjugated antiserum, DAB or hydrogen peroxide.
Intracellular and Extracellular viral growth curves. For virus
growth curves TN-368-10 cells were inoculated as described and assayed
in triplicate. At the designated time, 1 x 10" cells were removed,
pelleted by centrifugation at 300 g for 10 min, and the supernatant
saved for extracellular virus tiration. The cells were twice resuspen-
ded in 5 ml of TNM-FH plus 0.1 mg/ml of gentamycin and 5 mg/ml of fungi-
zone, and twice pelleted by centrifugation. After this, they were re-
suspended in 1.1 ml of the medium, pelleted again, and 0.1 ml of final
supernatant tested for residual infectivity by the polyhedral plaque
assay. The pelleted cells were resuspended in the remaining medium,
and disrupted using a Sonifier Cell Disruptor (Heat Systems Ultrasonics,
Inc.) at 4°C for 1 min at setting 3, and then for an additional 0.5 min
at setting 5. The effects of sonication on virus infectivity were
tested by adding known titres of EV to TN-368 cells and disrupting the
cells as above. The procedure routinely destroyed 30% of virus infecti-
vity and all values reported in these studies were corrected accordingly.
The cell debris was pelleted at 2000 g for 10 min and the infectivity
of the supernatant solution was determined by polyhedral plaque assay.
Plaque-purification for restriction enzyme studies. Three forms
of AcMNPV were used to produce the plaques from which each isolate was
purified. These were AcMNPV extracellular (E), AcMNPV single (S), and
AcMNPV multiple (M) (Summers and Smith, 1978). A total of 15 viral
14
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plaque isolates, five each for E, S, and M, were plaque-purified 3 times
and then grown to 20 ml of infectious tissue culture supernatant.
The method used for plaque-purifying wild isolates of AcMNPV was
a modification of the polyhedral plaque assay of Hink and Vail (1973),
as additionally modified by Volkman ejt al,, (1976), Log-phase TN-368
cells were seeded into 35-mm Corning tissue culture dishes at a density
of 7.0 x 10-* cells/plate in 2,0 ml of medium without fetal calf serum.
After the cells attached the medium was removed, and 0,1 ml of virus
dilution was added. The plates were rocked every 15 min for 1 hr,
after which the inoculum was removed and the plates were overlaid with
1.5% agarose (Wood, 1977). In the case of singles (S) and multiples (M),
the overlay contained a 1:20 dilution of AcMNPV antiserum.
Individual plaques were selected from plates showing no more
than five widely separated plaques. Plaques were removed with a 1-cc
disposable tuberculin syringe and 22 gauge needle using a stereoscope
to visualize the plaque. The plaque was diluted and replated on unin-
fected TN-368-10 cells in the same manner described above. Each isolate
was plaque-purified 3 times in this manner before preparing sufficient
quantities of virus for DNA studies.
Purification of virus and viral DNA for restriction analysis. The
polyhedra from infected TN-368-10 cells were purified by sonication (10
sec at a setting of 4 with a model W375 Sonicator equipped with a micro
tip, Ultrasonics, Inc., Plainview, N.Y.). The sonicated suspension was
centrifuged in 40-63% (wt/wt) sucrose gradients, and polyhedra separated
from cellular materials as described by Summers and Smith (1978).
AcMNPV extracellular virus was purified as described by Summers and
Volkman (1976). DNA was purified using a modification of the procedures
of Summers and Anderson (1973) from purified virus (Summers and Smith,
1978). Each virus preparation was prepared at a concentration of 1 mg
protein/ml in 0.1 M Tris, 0.01 M EDTA, pH 7.5, then made 2% with respect
to sodium lauryl sarcosinate and incubated at 37°C for 15 min, Pronase
was added to a final concentration of 2 mg/ml and incubated for 4 hr at
37°C. Each was extracted 3 times with buffer-saturated phenol and the
aqueous layers recovered and layered on CsCl gradients with a mean
density of 1.50 g/ml containing 200 yg/ml ehtidium bromide. Centrifuga-
tion was conducted for 36 hr at 35,000 rpm using an SW-41 rotor. After
centrifugation, the DNA was removed by piercing the bottom of the tubes
and collecting the UV-visible fractions, The two bands which contained
covalently closed DNA (1.58 g/ml) or linear and relaxed circular DNA
(91.54 g/ml) were collected separately, extracted 3 times with isoamyl
alcohol, and dialyzed extensively against 1.0 M Tris, 0,1 M EDTA, pH 7.5.
Restriction endonuclease digestion. Purified viral DNA was di-
gested with EcoR-1 or Hind III restriction enzymes (Boehringer Mannheim,
Indianapolis, Ind.) EcoR-1 digestion mixtures (total of 20 yl) contained
2 yg DNA, 0105 M NaCl, 0.01 M MgCl2, 0.1 M Tris-HCl, pH 7.5, and 10-15
units of enzyme were incubated for 1 hr at 37°. Hind III enzyme digests
(total of 20 yl) contained 2 yg DNA, 0.05 M NaCl, 0,01 M MgCl2, 0.014
M dithiothreitol, 0.01 M Tris-HCl, pH 7.6, and 10-15 units of enzyme and
were incubated for 1 hr at 37°. Enzyme activity was stopped by adding
2.0 yl of 0.1 M EDTA, 50% glycerol, and 0.5% brom-phenol blue. The samples
15
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were heated for 15 min at 60° then quenched on ice before layering on
the gel slab.
Lambda DNA (Miles Laboratories, Inc., Elkart, Ind,) and adenovirus-
2 DNA (gift from Dr. Steve Bachenheimer; University of North Carolina,
Chapel Hill, N.C.) were digested with EcoR-1 as described above.
The restriction endonuclease (Sma-1) isolated from Serratia marces-
cens (Boehringer, Mannheim) was incubated with 2 pg lambda DNA, O.Q15 M
Tris-HCl, pH 8.5, and 10 units of enzyme added for 30 min at 37°. The
sample was prepared for electrophoresis as described above.
Agarose electrophoresis. Horizontal 0.5, 0.75, and 1% agarose
(Bio-Rad Laboratories, Richmond, Calif.) slab gels were prepared on a
LKB model 2117 Multiphore electrophoresis apparatus. Analytical gels
(3 x 150 x 250 mm) were prepared with 4 x 0.75 or 8 x 0.75 mm wells.
Agarose was dissolved in 40 mM Tris, pH 7,8, containing 20 mM sodium
acetate, 1 mM EDTA, and 0,5 yg/ml ethidium bromide, This buffer was
also used as the electrode buffer, Gels were prerun for 30 min before
samples were applied.
The DNA was electrophoresed at a constant voltage of 50 V and at
15° for 18 to 25 hr. DNA fragments in discrete bands were visualized
under long wave UV illumination using a transilluminator (UV Products,
San Gabriel, Calif.). Photographs were taked on Kodak Pan Contrast
Process film using a Wratten No. 15 filter. The agarose gel slabs were
dried under vacuum and the -'^P-labelled fragments detected by exposing
the dried gel to Kodak NS-54T x-ray film for 2 days. Autoradiograms
were scanned with an E-C model scanning densitometer equipped with a
Perkin-Elmer M-2 calculating integrator,
EcoR-1 restriction endonuclease and molecular weights virus DNAs.
Viral DNAs from seven nuclear polyhedrosis viruses and two granulosis
viruses were cleaved with EcoR-1 and electrophoresed in 0.75% agarose.
The molar ratios of fragment bands were determined by plotting the mi-
gration versus the log of the integral area as determined by scanning
autoradiograms or photographic negatives of UV flourescent bands with
a scanning densitometer equipped with a digital integrator. Migration
of EcoR-1 fragments of lambda phage, adenovirus-2 DNA, and the Sma-1
fragments of lambda phage were plotted versus the log molecular weight
to provide a standard curve. Molecular weights of EcoR-1 fragments of
lambda and adenovirus-2 were as reported by Helling et al. (1974 and
Pettersson et^ al. (1973), respectively, and the Sma-1 fragment molecular
weights correspond to values reported by McParland et al. (1976). Mean
baculovirus DNA fragment molecular weights were determined from electro-
phoresis in three agarose gels.
16
-------
SECTION 5
Results and Discussion
A. Granulins and Polyhedrins:
We have reported procedures and techniques which are able to dif-
ferentiate and identify the structural properties of baculovirus poly-
hedrins and granulins (Summers, 1975; Summers and Smith, 1975, 1976,
1978). Two-dimensional high voltage peptide mapping has been a most
useful technique for providing a diagnostic fingerprint for identity
which can be supported by additional data derived from SDS polyacryla-
mide gel electrophoresis, amino acid analysis, and amino-terminal de-
termination (Maruniak and Summers, 1978). These comparisons are
essential as a continuing basis for investigating the structural proper-
ties of polyhedrins relative to the sensitivity and specificity of
serological techniques. Since cross-reactions measured by various
serological techniques have been reported for polyhedrins (Bell and Orlob,
1977; Croizier and Meynadier, 1973, 1975; Guelpa et^ _al, , 1977; Harrap et
al., 1977), a comparison of primary structure peptide mapping of these
proteins will provide information to help predict and explain these
cross-reactions as well as in establishing the identity of individual
granulins and polyhedrins,
1. Molecular Weights. The granulins and polyhedrins of eight
baculoviruses were compared (Figure 1). All polyhedrins and granulins
exhibited a single major component with mobility between chymotryp-
sinogen (25,000) and deoxyribonuclease (31,000). The molecular weights
of each monomer subunit were estimated to the nearest 500 daltons and
were as follows: AcMNPV, 30,000; RoMNPV, 30,000; AgMNPV, 29,000;
HaMNPV, 28,000; TnSNPV, 31,000; HaSNPV, 27,000; TnGV, 25,000; and SfGV,
26,000.
2. Two-dimensional, high-voltage electrophoresis (HVE). Figures
2-3 show the results of HVE of granulins and polyhedrins. Unfortunately,
the neutral peptides migrating on the 0/n line are not highly resolved
using this technique. A third dimension of ascending chromatography
using butanol:acetic acid:water (4:1:5) allows resolution of those
peptides. Visual inspection of each of the maps shows that there are
similarities among all proteins studied.
Tables I-III provide a more detailed analysis of the extent of
similarity and disimilarity. Degrees of similarity may be evaluated by
comparing peptides which are similar by relative migration to the total
number of peptides among those proteins being compared.
Certain peptides of the basic, acidic, and neutral peptides were
found common in all of the granulins and polyhedrins. For example, the
basic peptide 8, of the 1/2 diagonal (Table I) is found in every protein.
Other widely distributed basic peptides are 3 of the 1/3 diagonal, basic
peptides 4, 5, 6, and 9 of the 1/2 diagonal, and basic peptide 4 of the
2/3 diagonal. The most prevalent acidic peptide common to all proteins
17
-------
ABCDEFGH I
_ 160,000
~ 150,000
_ 94,000
~~ 90,000
— 68,000
— 53,000
— 40,000
«* — 31,000
•N - 25,000
'<• — 17,200
t t t t 1 t t !
>O>O >O>O>O >O >O>O
O O CT> O Q.OO.OQ-O Q.O Q-OQ-O
^rO^^^ozOZO Z O ZOZO
OT «)' *~ in W'N ^-r" foo So> So O
CM CM .cvjfroo^
Figure 1. Granulins and polyhedrins were purified as described
in Materials and Methods and disrupted in 2% SDS and 5% mercaptoethanol,
and 1.5 yg of each was electrophoresed in 11% polyacrylamide slab gels
in the presence of 0.1% SDS. SfGV and TnGV granulin and HzjSNPV, TnSNPV,
HaMNPV, AgMNPV, RoMNPV, and AcMNPV polyhedrin are shown in A-H, respec-
tively. The molecular weight of each polyhedrin or granulin is given
below each sample. The standard proteins are shown in I and molecular
weights are indicated on the right.
18
-------
TABLE I: Basic Peptides
Diagonal '4
Peptide No. 12 123456 123456789 10 11 12 12345
AgMNPV ++ + + + + + + + ++ + + + + +
HaMNPV + + + + + + + + + + ++ + +
Hz.VNPV + + + ++ + + + + + +
TnGV +++ + + + + + + + + +
SfGV + ++ + + + + + + + + + +
Tn.SNPV ++ + + + ++ + + + ++ +
RoMNPV ++ + + + + + + + + + + +
AcMNPV ++ 4 + + + + + + + + + +
Composite of tryptic peptides located by two-dimensional
high-voltage electrophoresis and descending chromatography de-
rived from AgMNPV, Anticarsa gemmatalis; HaMNPV, Heliothis
armigera; HzS^NPV, Heliothis zea; TnGV (Summers and Smith, 1976),
Trichoplusia ni; SfGV (Summers and Smith, 1976), Spodoptera
frugiperda; TnS^NPV (Summers and Smith, 1976), Trichoplusia ni;
RoMNPV (Summers and Smith, 1976), Rachiplusia. qu; AcMNPV (Summers
and Smith, 1976), Autographa californica.
TABLE II: Acidic Peptides
Diagonal
Peptide No.
AgMNPV
HaMNPV
HzSNPV
TnGV
SfGV
TnSNPV
RoMNPV
AcMNPV
1
+
+
+
+
+
+
+
2
4-
+
+
+
+
+
\-n/n\>\
345678
+
+ + +
4- +
+-
+ + +
+ +
+ +
-n/n\
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20
-------
are Cl, Dl, E2, and F2. HaMNPV, HzSNPV, and AgMNPV polyhedrins appear
to be most related to previously characterized granulins and polyhedrins
on the 1/2 diagonal (Table I) of the basic peptides, indicating that the
primary sequence of this region of these proteins has been the most sta-
ble and conserved through evolution.
Comparisons of the polyhedrins and granulins by amino-terminal
analysis reveals three different groupings: The amino-terminus for
AcMNPV, RoMNPV, and AgMNPV are proline; for HzSNPV, HaMNPV, and TnSNPV,
methionine; and TnGV and SfGV, glycine. It is interesting to note that
the amino acid sequence of polyhedrin from Bombyx mori NPV (Serebryani,
et. al., 1977) shows the amino-terminal amino acid to be proline which
we have determined as the amino-terminus for AcMNPV, RoMNPV, and AgMNPV.
TnSNPV
c
• •
Asp Glu
S-sib s 2?
< Of-in < 3
Figure 2-3. Two-dimensional high-voltage electrophoresis of a
tryptic digest of granulins and polyhedrins. Electrophoresis was carried
out on Whatman 3 MM filter paper at pH 6.5 (10% puridine - 4% acetic acid)
for the first dimension (3 kV, 35 min) and at pH 1.9 (8% acetic acid - 2%
formic acid) for the second dimension (3 kV, 30 min). Chromatography of
the neutral hydrolysates was run in butanol:acetic acid:water (4:1:5) for
18 h at room temperature, positions of hydrolysates were detected with 1%
ninhydrin - 0.1% cadmium acetate. Dark spots indicate peptides which were
UV-positive; y=yellow peptides; o=orange peptides; AcMNPV=Autographa cali-
fornica NPV; HzSNPV=Heliothis zea nuclear polyhedrosis virus.
21
-------
We have presented data on the primary structure of granulins and
polyhedrins. This information has been useful to identify and differ-
entiate the species specific granulins and polyhedrins. At the same
time, information is provided that shows similarities among the virus
proteins which can account for serological cross-reactions (to be
evaluated later) that have been observed by this and other laboratories.
As summarized in these studies, baculovirus granulins and polyhedrins
demonstrate remarkable similarities in size, composition, and chemical
properties. However, it is obvious from the results reported herein
that for those viruses investigated, each granulin and/or polyhedrin
is a specific protein in association with a given species of occluded
virus. The proteins appear to be of a family or class with degree of
relatedness in terms of similar primary structure. The degree of re-
latedness among the polyhedrins is not nearly as close as between granu-
lins.
Serological studies to date (Mazzone, 1975; Norton and DiCapua,
1975) have shown cross-reactivity in antisera prepared against various
granulins and polyhedrins when tested against heterologous granulins
and polyhedrins, as well as when tested against purified virus prepar-
ations of the DNA baculoviruses. If the chemical relationships hold
true for other viruses as observed in this study, the cross-reactivity
of antigens and antisera is partially understood. However, it is diffi-
cult at this time to draw further conclusions about degrees of similar-
ity among these proteins until more proteins are adequately characterized.
It is now possible, however, to correlate serological studies on these
proteins at the quantitative level with the degree of chemical similari-
ty among polypeptides. The technique of two-dimensional HVE which
arranges peptides along a series of lines, each line ascribed to two
coordinates based upon the net charge on the peptide during HVE at 6.5
and 2.0, is an extremely effective technique for qualitative comparisons
of these proteins. In many cases single amino acid differences can be
detected in major peptides.
It is essential that alkaline protease activity be eliminated for
comparative studies. The very similar structure of the SfGV and TnGV
points to the need for very careful comparisons since it has been prev-
iously shown (Summers and Smith, 1975) that alkaline protease activity
can induce considerable artifact showing significant differences in the
migration of tryptic peptides from enzyme active versus nonac.tive prep-
arations .
B. Structural Polypeptides of Enveloped Nucleocapsids and Nucleocapsids:
1. Virus Polypeptides
Figures 4 and 5 show the relative mobilities of the structural
polypeptides for eight baculoviruses. Only enveloped single nucleocap-
sids were utilized for this initial comparison, and not multiples of
nucleocapsids. The results confirm that baculoviruses are structurally
complex. Although some bands exhibited similar mobility for different
22
-------
viruses, for example, AcMNPV and RoMNPV appear closely related, each
virus was unique when comparing mobility and the total number of bands
resolved.
The molecular weights of enveloped nucleocapsid proteins (VP)
and of polyhedrins and granulins were routinely determined by comparison
with the relative mobilities of standard marker proteins. The molecular
weights of the VPs ranged from 15,000 to 160,000 (Figures 4 and 5).
AcMNPV RoMNPV
A B
AgMNPV HaMNPV
C D
VPI50
VPI25
VPII5 '
VP 90
VP 75
VP73
VP68.64 :
VP58-
VP54
VP46.45 :
VP40-
VP37-
VP36
VP30
VP28.5.28 :
VP23
VP22
VP 19-
VPI8.5-
VPI7-
VPI6'
_VPI60
— VPI40
— VP90
— VP75
— VP73
— VP64
— VP54
— VP46.45
— VP39.5
— VP36
— VP30
— VP28
VPI8
VPI7
VPI6
VP97 —
VP82 —
VP65 —
VP56 —
VP47 —
VP46Z
VP40
VP33-
VP29-
VP25-
VP22.5 —
VP20-
VP 19"
VPI7.5-
VPI7-
^SL
— VP75
= VP73,72
= VP64,62
— VP55
= VP52,5I
= VP48,47
— VP45
— VP39.5
— VP37
— VP32
— VP28
= VP24.5,24
— VP22.5
— VP2I
— VP20
— VPI8.5
— VPI7
— VPI6
Figure 4. Polyacrylamide slab gel electrophoresis of enveloped
nucleocapsid 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 0 to 4° for
60 min. The band containing only one nucleocapsid per envelope (singles)
was removed from the gradient and electrophoresed in 11% polyacrylamide
as described in Materials and Methods, The virus polypeptides (VPs) of
AcMNPV, RoMNPV, AgMNPV, and HaMNPV are shown in A, B, C, and D. The
molecular weights of the virus polypeptides were estimated by comparison
23
-------
of relative mobilities using the method of Weber and Osborn (1969).
Each VP that was resolved is marked with the calculated molecular
o
weight times 10 . 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-glu-
tamic dehydrogenase, 53,000; DNA nuclease, 31,000; chymotrypsinogen,
25,000; and myoglobin, 17,200.
TnSNPV HzSNPV
A B
TnGV SfGV
C D
•is*
VP90-
VP80-
VP64-
VP59-
VP56-
VP5I •
VP45
VP38
VP30
VP28.5
VP24.5
VP24
VP89
VP75
VP72
— VP57
— VP5I
— VP47
— VP38.5
— VP37
— VP3I
— VP28
— VP245
— VP24
VP90 =
VP85 —
VP77 —
VP63,64 =
VP57 —
VP56-
VP52~
VP47~
VP39 —
VP38 —
VP3I —
VP25-
VP24-
. —VPI60
M —VP95
Z — VP89
m —VP7I
«. — VP58
— VP48
— VP42
~VP39
— VP37
— VP3I
— VP29
— VP26
— VP24
VPI9 —
VPI7 —
VPI6_
—VP2I
— VPI9
— VPI7.5
= VPI6,I5
VP2I.5— M»
VP20.5_ A
vWfr
VPI8 —
VPI7 —
VPI6 —
If
-VP2I.5
-VP20.5
VPI8
•VPI7
•VPI6
Figure 5. Baculovirus-enveloped nucleocapsids were prepared
and electrophoresed as described in Fig, 4, The VPs of TnSNPV, HzSNPV,
TnGV, and SfGV are shown in A, B, C, and D, respectively.
24
-------
AcMNPV RoMNPV HaMNPV AgMNPV
ABCDEFGH
160,000 _
150,000 ~~
94,000
90,000 ~
68,000 —
53,000 —
40.000 —
3:,OCX' --
25,000 —
^^ _
r'
VHHHp ^HJHP^
^^^^ ^^^j^ _»,.„„,
OTl^ ^^^^
17,200 —
Figure 6, Comparison on an 11% polyacrylamide slab gel of poly-
peptides from enveloped single nucleocapsids and multiples. Each of the
four MNPVs used in this study was liberated from the polyhedra and gel
electrophoresis was conducted as described in Fig. 4, except that in
addition to collecting the top virus band (singles), the remaining
virus band (multiples) were also collected and analyzed. The VPs of
singles and multiples, respectively, of AcMNPV are observed in A and B;
RoMNPV, C and D; HaMNPV, E and F; and AgMNPV, G and H. The position of
the molecular weight standards are indicated next to the VPs,
25
-------
2. Polypeptide Composition of Enveloped Singles and Enveloped Multiples
of Nucleocapsids
After centrifugation to quasiequilibrium in sucrose gradients,
the least dense virus band (density of 1.20 to 1.21 g/ml) of enveloped
single nucleocapsids from AcMNPV, RoMNPV, AgMNPV, and HaMNPV was separa-
ted from the multiples (usually seven to eight additional bands ranging
in density from 1.22 to 1.25 g/ml) and was compared by polyacrylamide
gel electrophoresis (Figure 6), The results show that both qualitative
and quantitative differences exist between multiples and singles for each
virus. Since comparisons of gel profiles were made by inspection,
qualitative differences in protein composition will be emphasized. It
is not desirable to elaborate upon the more subtle quantitative differen-
ces without a more accurate means of quantitation,
VP30, VT28, VP19, VP18.5, and VP16 of AcMNPV singles are observed
to be in relatively smaller concentration when compared to multiples (Fig-
ure 6A and B). VP58, VP36, and VP23 are apparently absent or diminished
in relative concentration to a level not detectable in the gels. The
polypeptide composition of HaMNPV singles shows that VP64, VP47, VP28,
and VP20 are present in increased amounts relative to the same protein
in multiples (Figure 6E and F), whereas VP89, VP75, VP62, and VP51 are
not observed in multiples. In contrast, there is more of HaMNPV VP45
in multiples as compared to singles. The polypeptides of RoMNPV, singles,
VP90, VP54, VP46, VP45, VP30, VP18, and VP16, exist in greater amounts as
compared to multiples; VP160 is consistently more prominent in multiples
(Figures 6C and D). Quantitative differences are also observed for
VP22.5 andVP17,5 in AgMNPV, however, the quantitative relationship between
singles and multiples is different than those of the other viruses. The
explanation for this is not apparent to us at this time.
3. Polypeptides of Baculovirus Capsids
AcMNPV capsids purified after the above treatment are composed of
two major polypeptides, VP37 and VP18.5, and trace quantities of VP30.5
and VP30 (Figure 7B). RoMNPV capsids have several major polypeptides;
VP61, VP17, VP18, VP30, and VP36, with detectable quantities of VP30.5
(Figure 7D). The presence of two polypeptides between VP30 and VP36
is not presently understood since these two proteins are not detected in
intact virus preparations (Figures 4 and 6). The TnGV capsid is composed
of two major polypeptides, VP31 and VP17 (Figure 71), with trace quanti-
ties of VP29 and VP26. Each capsid preparation consistently contained
several other minor polypeptides (most of which have been discussed) which
could be components of the viral capsid. It is presently assumed that
the differences between capsid and enveloped nucleocapsid protein compo-
sition shown in Figure 7 reveals that the envelope composition of baculo-
viruses is quite complex. For example, AcMNPV contains a total of approx-
imately 18 bands (Figures 6B and 7 A). Since there are only three capsid
proteins, the envelope must contain a majority of the remaining. This
relationship, although different for each virus studied, is consistent
among all comparisons made (Figure 7),
It is easily seen that each of the baculoviruses has a unique
26
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protein composition. However, there is little that can be deduced from
the structural approach presented herein using present information as to
the relationships among baculoviruses in terms of strain, species, etc,
The possible exceptions are AcMNPV and RoMNPV which have many proteins
with similar electrophoretic mobilities, This does not imply that
these proteins are identical, and additional biochemical and serological
characterization of each protein will be necessary to establish the
existence of structural similitaries, A comparison of AcMNPV and RoMNPV
proteins from multiples in Figure 7 shows that two major proteins, VP37
(AcMNPV) and VP36 (RoMNPV), have different electrophoretic mobilities.
Also, AcMNPV has at least three additional major proteins, VP22, VP23,
and VP19, which are not observed in RoMNPV. Peptide mapping analysis
of RoMNPV and AcMNPV polyhedrins has shown that only 40% of the tryptic
peptides of the two proteins have similar mobility (Summers and Smith,
1976), However, Volkman ert_ al. (1976) using comparative neutralization
could not show differences by kinetic neutralization between RoMNPV and
AcMNPV. The results of highly specific and sensitive serological assays
for enveloped nucleocapsids utilizing double gel diffusion, and competi-
tion radioimmunoassay (Hoops and Summers, 1978, manuscript in preparation)
and immunoradiometric assay (Ohba et ajl., 1977) also indicate a great
deal of similarity between AcMNPV and RoMNPV. The apparent close related-
ness of AcMNPV and RoMNPV may provide an interesting example for continued
studies relating structural similarity of baculovirus strains to biologi-
cal activity and specificity.
The quantitative and qualitative differences in virus polypeptides
observed between singles and multiples in MNPVs may, in part, be explained
by the ratio of envelope to nucleocapsid protein. The DNA:protein ratio
is 0.20 for enveloped single nucleocapsids and 0,30 for AcMNPV multiples
(Volkman et^ a\._. , 1976). The significance of the difference in protein
composition between singles and multiples is not clear to us. Whether
the quantitative differences and the presence or absence of certain poly-
peptides may also be related to biological differences is also difficult
to evaluate using present evidence. However, this study has prepared
the basis for comparison with plaque purified viruses in order to reveal
whether or not the polypeptide composition of multiples and singles is
related to strain differences and/or is a genetically stable trait,
4. Structural Polypeptides of Extracellular Virus
The structural polypeptides of AcMNPV purified by alkali extraction
from the polyhedra of infected insects were analyzed by polyacrylamide
electrophoresis (Summers and Smith, 1978), A comparison of the proteins
from polyhedral-derived virus with those of extracellular virus from
cell culture medium (Fig. 8) revealed quantitative and qualitative
differences in polypeptide composition. Extracellular virus has a much
greater quantity of VP64, and several other minor polypeptides, VP70,
VP60, and VP27 were not detected in singles or multiples.
28
-------
A B C D E F
17,200 m
VP-
— IZ5
Figure 8. A comparison of AcMNPV from the polyhedra of infected
TN-368-10 cells with extracellular virus by electrophoresis in 11%
polyacrylamide in the presence of 0.1% SDS. Extracellular virus was
also labeled with L[-"S] methionine and compared to a control as des-
cribed in the Results. Coomassie blue-stained virus polypeptides are
as follows: (B) AcMNPV purified from the polyhedra of infected TN-368-
10 cells, (C) and (D) extracellular virus purified from S-cell protein
+ virus (control) or -"S-virus + cell protein preparations, respectively,
The autoradiogram of L[-"S]methionine-labeled polypeptides in slots C
and D is shown in slots E and F, respectively. The molecular weights
of the standard proteins, electrophoresed in slot A, are: phosphory-
lase a., 90,000; bovine serum albumin, 68,000; L-glutamic dehydrogenase,
53,000; DNA nuclease, 31,000; chymotrypginogen, 25,000; and myoglobin,
17,200. The molecular weight times 10 of extracellular virus polypep-
tides are indicated to the right of the autoradiogram,
29
-------
C. Competition RIA
Because of the complex structure of baculoviruses and problems as-
sociated with obtaining antigens of sufficient purity and quantity
(Harrap ejt al., 1977; Summers and Smith, 1978; Bell and Orlob, 1978),
immunological studies on purified individual structural polypeptides
of the virion have not been reported. Most detailed studies have been
confined to the major paracrystalline protein of the inclusion des-
cribed as granulin for granulosis viruses and polyhedrin for nuclear
polyhedrosis viruses. A single polypeptide of 25,000 to 31,000
molecular weight, polyhedrins and granulins have been shown by two-
dimensional peptide mapping to possess both common and unique tryptic
peptides (Summers and Smith, 1975, 1976; Maruniak and Summers, 1978).
The presence of similar antigenic sites in these group related proteins
have been documented by immunological analysis of several polyhedrins
and granulins (Crozier and Meynadier, 1972a, b, 1973; Norton and DiCapus,
1976; Rohrman e_t al., 1978).
This portion of the study describes the efficacy of preparative
polyacrylamide gel electrophoresis in the presence of sodium dodecyl sul-
fate (SDS) for isolating baculovirus structural polypeptides for use in
immunological studies. Radioimmunoassay (RIA) is employed to compare the
immunoreactivity of SDS purified AcMNPV polyhedrin with the reactivity of
polyhedrin translated in infected cells showing that the detergent
purified protein retains its immunological integrity. Using competitive
inhibition studies as we have also investigated the immunoreactivity of
highly purified granulins and polyhedrins from six baculoviruses to
evaluate the ability of polyhedrin and granulin antisera to discriminate
antigenic differences.
1. Immunodiffusion of polyhedrins and granulins
Preliminary immunological assessment of preparative SDS-PAGE
purified polyhedrins and granulins using immunodiffusion assays resulted
in the precipitin patterns presented in Figures 9 A-F, TnGV granulin
reacted with homologous antiserum to form two major and one minor
precipitin bands (Figure 9 ). SfGV granulin, with primary structure
similar to that of TnGV (Summers and Smith, 1976) showed a reaction of
identity with one major precipitin band. TnGV granulin antiserum also
recognized related antigenic determinants in AcMNPV, RoMNPV, AgMNPV, and
TnSNPV polyhedrins (Figures 9B and C). Under the experimental conditions
utilized HaMNPV and Hz^NPV polyhedrins did not appear to react with TnGV
granulin antiserum (Figures 9B and C), In a similar experiment AcMNPV
polyhedrin antiserum indicated common antigenic determinants between
AcMNPV, RoMNPV, and TnSNPV polyhedrin. However, AcMNPV polyhedrin anti-
serum did not react with HaMNPV and HzSNPV polyhedrins or TnGV and SfGV
granulins,
Because several laboratories have reported that protease activity
does not alter the immunoreactivity of polyhedrins and granulins from
several species of baculovirus (Kalmakoff et^ a_l. , 1978: Norton and DiCap-
ua, 1976), polyhedrins and granulins degraded by enzyme activity were
also reacted with the TnGV granulin and AcMNPV polyhedrin antisera. The
30
-------
immunodiffusion patterns for these proteins and TnGV granulin antiserum
are shown in Figures 9D-F, While immunoreactivity of TnGV granulin
with homologous antiserum is maintained, the reactions are diminished
and patterns with other polyhedrin is noticeably reduced (Figure 9D),
TnSNPV, and AgMNPV polyhedrin reactions are lost (Figure 2F), In
contrast, HaMNPV and HzJJNPV polyhedrins have similar antigenic deter-
minants revealed by proteolytic hydrolysis (Figure 9E-F), Since
alternations also occurred with AcMNPV polyhedrin serum tested against
degraded polyhedrins and granulins (Data not shown), only undegraded
SDS-PAGE purified antigens were used for further immunological testing
with RIA.
2. RIA of AcMNPV polyhedrin and TnGV granulin
Titrations of AcMNPV polyhedrin and TnGV granulin antisera with
homologous iodinated antigen showed that dilutions of 1:2600 (AcMNPV)
and 1:6500 (TnGV) bound 50,% of the respective labelled proteins.
Serial dilutions of unlabelled AcMNPV polyhedrin and TnGV granulin
were then employed to competitively inhibit binding of homologous
labelled antigen at these antiserum dilutions to define assay parameters,
(Figures 10A and B). The standard curve for AcMNPV polyhedrin showed
that 80 pg of AcMNPV polyhedrin would reproductively inhibit binding by
10%, establishing the lower limit or sensitivity of the assay, Five
hundred pg produced 50% inhibition and 3.0 ng were required for 90%
inhibition (Figure 10A). The working range of the TnGV assay was 600
pg to 600 ng (Figure 10B).
3. RIA of AcMNPV polyhedrin synthesized in vivo
A quantitative assay of AcMNPV polyhedrin synthesized in vivo
was made in infected TN-368 cells. The results (Figure 11) show that
a competition curve prepared by use of 10-fold decreasing amounts of
infected TN-368 cell lysates paralleled a standard curve obtained by
adding SDS-purified AcMNPV polyhedrin to uninfected cell lysates. For
each curve the essentially identical slopes produced in the linear regions
of the assay show that SDS purified polyhedrin and polyhedrin synthesized
in infected cells are recognized with equal affinity,
4. Competitive inhibition studies of polyhedrins and granulins
Since studies with AcMNPV polyhedrin j.n vivo indicated no loss in
immunoreactivity of SDS-PAGE purified polyhedrin, a comparison was made
of four polyhedrins and two granulins using competitive inhibition of
-*-^^I-AcMNPV polyhedrin binding to AcMNPV polyhedrin antiserum. The re-
sults (Figures 12A and B) show that all polyhedrins and granulins assayed
contain similar antigenic determinants for AcMNPV polyhedrin recognized
by homologous antiserum. However, the quantity of such determinants
varied among the proteins studied. While 0.5 ng of AcMNPV polyhedrin
inhibited the binding of 125I-AcMNPV polyhedrin by 50%, 5,0, 8.0, and
200 ng of RoMNPV, TnSNPV, and HaMNPV polyhedrins were required to achieve
equivalent inhibition (Figure 12A). Fifty percent inhibition with
HzSNPV polyhedrin and TnGV and SfGV granulins required 50, 55, and 220 ng
31
-------
PoM« nizSN)
RoMN TnSN
TnGV \
YY •'- V
K \ / TnGV TnG» \
Figure 9. Immunodiffusion of polyhedrins and granulins. Trichoplusia ni
granulin antiserum (TnGV As) was reacted with equivalent concentrations
(15 yg/well) of undegraded (protease inactive, A-C) and degraded (protease
active, D-F) polyhedrins and granulins. Plates were incubated for 24 h at
room temperature and photographed directly. The polyhedrins and granulins
used were TnGV, Trichoplusia ni granulin; SfGV, Spodoptera frugiperda,
granulin; AcMN, Autographa californica polyhedrin; RoMN, Rachiplusia cm
polyhedrin; HaMN, Heliothis armigera polyhedrin; HzSN, Heliothis zea poly-
hedrin; TnSN, Trichoplusia ni polyhedrin; AgMN, Anticarsa gemmitalis
polyhedrin.
32
-------
100
~ 90
o
c
S 80
(5
-5 70
£
5 60
s
£ 50
c
^ 40
I 30
Q_
.- 20
N
I 0
-2
AcMNPV polyhtdrln
B
Tn9V gronulin
0 ! 2 3 4 -! 0 123-1
Ncnojrom* Competing Antigen (log)
Figure 10. Radioimmunoassay of AcMNPV polyhedrin and TnGV
granulin. Ten-fold dilutions of AcMNPV polyhedrin (A) and TnGV granulin
(B) in quantities from 10 yg to 10 pg were reacted for 24 h with that
dilution of homologous antiserum which bound 50% of 10,000 CPM of homol-
ogous iodinated antigen. 10,000 CPM of 125I-AcMNPV polyhedrin of IZ5I-
TnGV granulin were added to each well, incubated an additional 24 h, the
wells washed, and the % maximal binding determined as described in
Materials and Methods,
33
-------
respectively (Figure 12B), Also, the slopes of the inhibition curves
for heterologous polyhedrins and granulins indicated that AcMNPV poly-
hedrin antiserum does not bind these proteins with comparable affinity
(Figure 12A and B).
1 9 S
A similar experiment carried out with I-TnGV and TnGV granulin
antiserum resulted in the curves shown in Figure 13A and 13B, Seven
nonograms of the homologous antigen were required to competitively
inhibit by 50% the binding of -^-51-TnGV granulin to granulin antiserum.
For comparison, 9.5 ng of SfGV granulin, 40 ng of AcMNPV polyhedrin, and
400 ng of TnSNPV polyhedrin were necessary to produce 50% inhibition
(Figure 13A). RoMNPV, HzSNPV, and HaMNPV polyhedrins required 45 ng,
330 ng, and 4 pg for the same level of inhibition (Figure 13B). The
possibility of greater degrees of inhibition for TnSNPV, Hzj>NPV, and
HaMNPV polyhedrins was not tested since the upper limit of the assay
was lOy, Of particular note is the failure of SfGV granulin to effect
complete inhibition of I-TNGV granulin at high antigen concentrations
(Figure 13A).
34
-------
Monograms Polyhedrln (log)
-I 0 1234
2 3456
N'jmb»r of Cell* (log)
Figure 11. Detection of AcMNPV polyhedrin in infected cells.
Ten-fold dilutions of TN-368 infected cell lysates (o-o) from 10 cells
were prepared as described in Materials and Methods and reacted with
AcMNPV polyhedrin antiserum at the dilution which bound 50% of the
iodinated AcMNPV polyhedrin indicator. Mock infected cell lysates (•-•)
from 10" cells diluted in a similar manner containing 10 yg to 10 pg of
highly purified AcMNPV polyhedrin served to construct the standard curve.
Following 24 h incubation, 10,000 CPM 125I-AcMNPV polyhedrin were added,
the incubation continued for 24 h, and % maximal binding determined. A
parallel study of the detection of AcMNPV polyhedrin with respect to time
indicated that 1 ng of polyhedrin could be detected at 12 h post infection
in 10 cells, and a ten-fold increase in cell lysate (10^ cells) resulted
in a ten-fold increase in antigen concentration.
35
-------
IOO
- 90
o>
=5 80
£
_ 70
o
g 60
50
I30
J- 20
•i
10
AcMNPV •-•
RoMNPV D-D
TnSNPVe-*
Ho MNPV A-*
- AcMNPV
T«OV O-O
. StOV
HlSNPV
-101234 -101
Monograms Competing Antigen (log)
234
Figure 12. Comparison of polyhedrins and granulins by competitive
RIA. Ten-fold dilutions of purified polyhedrins and granulins ranging
from 10 yg to 10 pg were reacted with the dilution of AcMNPV polyhedrin
antiserum binding 50% of ipdinated homologous antigen. After incubation
for 24 h, 10,000 CPM of I-AcMNPV polyhedrin was added, and the ability
of various polyhedrins and granulins to inhibit binding was determined
and expressed as % maximal binding in the absence of competing antigen.
The competing antigens used were: AcMNPV polyhedrin (•-•), RoMNPV poly-
hedrin (>a); TnSNPV polyhedrin (®-®); HaMNPV polyhedrin (£r£); TnGV
granulin (o-o) ; SfGV granulin (M); and HzSNPV polyhedrin (A-*).
36
-------
100 •
c
o
is
-I 01234
Nonogrami Competing
I 0 1234
Antigen (log )
Figure 13. Comparison of polyhedrins and granulins by competitive
RIA utilizing TnGV granulin antiserum. Ten-fold dilutions of purified
polyhedrins and granulins ranging from 10 yg to 10 pg were tested for
the ability to inhibit the binding of 10,000 CPM of ^25I-TnGV granulin
to homologous antiserum. The results are expressed as % maximal binding
in the absence of competing antigen. The polyhedrins and granulins used
were: TnGV granulin (•-•); SfGV granulin (o-o); AcMNPV polyhedrin
(A"A); TnSNPV polyhedrin (M); Ro MNPV polyhedrin (4-71); HzSNPV poly-
hedrin (o-n); and HaMNPV polyhedrin (®-®),
37
-------
5. Discussion
This study documents the successful utilization of preparative SDS
polyacrylamide gel electrophoresis to isolate and purify sufficient
quantities of a major baculovirus structural protein for immunological
studies. Further, the identical slopes observed in standard (SDS-PAGE
purified) and unknown (cell lysate) competition curves indicates that no
loss of affinity has occurred as a result of SDS-PAGE purification (Fig-
ure 11). Preparative SDS-PAGE also circumvents several basic problems
associated with the serology of baculoviruses and baculovirus proteins.
Antigen purity (Bell and Orlob, 1977) and potential effects of degrada-
tion by an inclusion associated protease (Eppstein and Thoma, 1975;
Kozlov et al., 1975; Summers and Smith, 1975, 1976) represent two areas
of difficulty, particularly for the study of polyhedrins and granulins.
Altered immunoreactivity of protease degraded polyhedrins or granulins
was evidenced by altered immunodiffusion reactions (Figure 9D-F) and
by reduced sensitivity and affinity in RIA (Data not shown),
Two-dimensional high voltage electrophoresis of polyhedrins and
granulins have shown all proteins analyzed contained varying numbers of
similar tryptic peptides (Summers and Smith, 1976; Summers and Maruniak,
1978). Each polyhedrin or granulin, however, also contained peptides
unique in migration. TnGV and SfGV granulins were very similar, which
is confirmed in both immunodiffusion (Figure 9A) and competitive inhibi-
tion studies (Figure 13A). Less similar polyhedrins by tryptic peptide
analysis also demonstrate a large spur formation in immunodiffusion with
TnGV granulin (Figure 9B-C). This result indicates that common antigenic
determinants exist on all polyhedrins and granulins, but that some pro-
teins, in this case TnGV granulin, have other antigenic determinants
which are also available to the immune system. No such distinction
can be seen with AcMNPV polyhedrin antiserum.
Our results from competitive inhibition studies using RIA further
characterized the "group antigen'1 of polyhedrins and granulins. Poly-
hedrins and granulins were shown to be heterologous with respect to the
numbers of antigenic determinants per molecule of protein. For example,
HaMNPV and HzSNPV polyhedrin did not react with TnGV granulin antiserum
in immunodiffusion (Figure 9B-C). When 500 and 1000 fold greater concen-
trations of these proteins were used as competitors in RIA, inhibition
of binding of homologous serum occurred. Thus, the more sensitive irnmu-
nological assay could measure very small numbers of determinants in
HaMNPV and HzSNPV polyhedrins by enzyme hydrolysis which are apparently
not accessible to antibody in the undegraded molecule (Figure 9E and 9F).
Also, aggregation of polyhedrin or granulin to higher molecular weight
complexes may eliminate antigenic determinants formerly accessible to
antibody.
In addition to quantitative differences in polyhedrin and granulin
"group antigens", qualitative differences in the structure of antigenic
determinants were shown to exist. This is suggested by the reduction in
slopes of heterologous competition curves, indicating less affinity for
antibody binding sites compared to the homologous antigen (Figure 12 and
13).
38
-------
Because of the serological similarities identified among apparently
closely and distantly related baculoviruses shown in this study, it will
be difficult to use the present application for identification of baculo-
virus isolates, strains, etc. However, the use of preparative SDS poly-
acrylamide gel electrophoresis for the purification of other baculovirus
structural polypeptides in conjunction with immunological analysis
by competition RIA should provide means to develop sensitive and specific
serological probes for the study of the serology of baculovirus structural
proteins relative to their identity and role in biological specificity.
6. Micro-SPIRA: Enveloped Nucleocapsids
The relative binding of 125I-labelled AcMNPV LOVAL IgG increased
significantly with a concomitant increase in antigen concentration (Fig.
16) within the range of 1-200 ng. At this level there was little if any
significant competition of PMB-NOV and polyhedrin as compared to homolo-
gous binding.
AcMNPV EV was titrated with homologous antiserum and
(Figure 17) . The apparent sensitivity of the homologous binding was
approximately 10 ng. The assay appeared to be specific for AcMNPV EV
at concentrations of 10-200 ng of antigen. The results of the EV binding
assay exhibit similar sensitivity and specificity as did the LOVAL assay.
The binding curve for AcMNPV polyhedrin showed that the sensitivity
of this assay allowed the detection of as little as 200 pg of homologous
antigen. The results of this assay show that the antiserum is specific
for the homologous antigen to at least 200 ng (Figure 18) .
Using LOVAL antisera, the ability of unlabelled antisera to compete
with [I] IgG is shown in Figure 19. Comparisons were made with purified
LOVAL IgG, polyhedrin, and EV antisera. The results of this qualitative
assay indicate the presence of common antigenic determinants among EV,
polyhedrin, and LOVAL.
In the present preliminary study, a micro-SPIRA was successfully
developed at a sensitive level for the detection of AcMNPV enveloped
nucleocapsids , alkali-liberated virus (LOVAL), extracellular virus EV,
and highly purified polyhedrin. The sensitivity of the micro-SPIRA was
5-10 ng for AcMNPV LOVAL and EV. By comparison, the polyhedrin assay
was 10-100 times more sensitive. These assays were specific for the
detection of homologous antigens to the level of 200 ng.
Micro-SPIRA was used to demonstrate the ability of AcMNPV LOVAL,
purified IgG, EV, and polyhedrin antisera to inhibit the specific binding
of AcMNPV LOVAL [^^I]IgG. In a comparison of the degree of inhibition
of the homologous antiserum and IgG, the results likely reflect differen-
ces in antibody titers as a result of purification. The inhibition of
EV and polyhedrin antisera may also involve differences in antibody
titers of specific antibody to determinants in common with AcMNPV LOVAL.
However, the data may also indicate that this inhibition reflects differ-
ences between specific antibodies against related antigenic determinant (s)
39
-------
on AcMNPV LOVAL. Therefore, the results of this study show the ability
to detect specific antiviral antibodies from different sources,
D. Biological Properties of Infectious Virions
One of the significant observations made in early studies is that
virus, as isolated from purified polyhedra, is relatively noninfectious
in cell culture, whereas extracellular, nonoccluded virus recovered from
the hemolymph of infected insects or medium from infected cell cultures
is highly infectious. In one study (Summers and Volkraan, 1976), we
have clarified the nature of the nonoccluded infectious virus by making
biophysical and morphological comparisons of the nonoccluded infectious
material from insect hemolymph and cell culture medium.
Electron microscope examination and buoyant density profiles of
nonoccluded Rachiplusia cm and Autographa californica nuclear polyhedrosis
viruses purified from both infectious insect hemolymph and cell culture
medium revealed that the viruses are enveloped, single nucleocapsids.
The envelopes exhibited variation in the amount and the degree of fit with
regard to the nucleocapsids. This was determined by: (i) electron
microscope observations of virus budding from the surface of infected
cells; (ii) electron microscopic observations of negatively stained prep-
arations of pelleted, highly purified, nonoccluded enveloped particles;
and (iii) the resolution and density distributions of nonoccluded virus
in sucrose gradients after centrifugation to equilibrium; all were com-
pared with virus extracted from polyhedra. Peplomers, observed on the
surface of enveloped nucleocapsids of nonoccluded virus, are not asso-
ciated with polyhedra-derived virus. Density gradient analysis indicated
that virus from insect hemolymph and culture medium exhibited similar
densities of approximately 1.17 to 1.18 g/ml. This is significantly dif-
ferent from the bouyant density of an alkali-liberated, enveloped single
nucleocapsid (1.20 g/ml). Results of this study show that the nonoccluded
forms of two nuclear polyhedrosis viruses from two different sources,
hemolymph and cell culture, are similar with regard to several morpholo-
gical and biophysical characteristics but are quite different from the
alkali-liberated polyhedra-derived form of the virus.
Physical-infectious particle ratio. The physical-infectious particle
ratios for AcMNPV LOVAL and EV are given in Table V. It was determined
that AcMNPV LOVAL had a physical-infectious particle ratio of about 2.2
x 10^:1 for the bundles and 2,4 x 10^:1 for the enveloped single nucleo-
capsids, as assayed by the plaque assay in vitro. In contrast to this,
the AcMNPV EV had a physical-infectious particle ratio of 128:1. If one
can assume that the DNA-protein ratio is the same for the various forms
of RoMNPV as it is for the analogous forms of AcMNPV, then the calculated
physical-infectious particle ratios for these forms are very similar as
well (1.6 x 105:1 for bundles; 2,9 x 105:1 for singles). To test whether
the necessary exposure to alkaline conditions of the LOVAL preparations
could account for their relatively greatly diminished infectivity, a
sample of cell culture-derived extracellular nonoccluded AcMNPV was taken
through the alkaline solubilization procedure and then tested for infec-
tivity. The physical-infectious particle ratio was increased about two-
fold (to 256:1).
40
-------
Q
z
o
u
** c
-03 0 I 2
ANTIGEN CONCENTRATION (log|0 ng)
-oo-i a i 2 3
ANTIGEN CONCENTDATION ( log|0 ng )
Figure 16. Micro-SPIRA for enveloped nucleocapsids . AcMNPV LOVAL
(•) , EV (•) , and polyhedrin (a) , were titrated by using AcMNPV LOVAL
antiserum and AcMNPV LOVAL [125I]IgG (41 ng with a specific activity of
3 fJCi/Mg) . Microtiter wells were coated with a 1:10 serum dilution at
4°C for 4 hr and then were coated with 2% BSA solution at 4°C overnight.
A serial dilution of each antigen was added to the wells and was allowed
to set at 4°C overnight. [-*-2^I]IgG was added to each well. The micro-
titer plates were allowed to set at 37°C for 4 hr, rinsed and counted.
Figure 17. Micro-SPIRA for AcMNPV PMB-NOV. Enveloped nucleocapsid
preparations and polyhedrin were tested in the assay system using AcMNPV
EV antiserum and [125I]IgG (29 ng with a specific activity of 4yCi/pg) .
Microtiter wells were coated with a 1:10 serum dilution using the condi-
tions described in Fig. 1. The antigens used were AcMNPV EV (0), AcMNPV
LOVAL (•) , and AcMNPV polyhedrin (A) .
41
-------
-CO -I 0 1 2
»«TIGEN CONCENTRATION (loglo ng )
Figure 18. Micro-SPIRA for AcMNPV polyhedrin. AcMNPV polyhedrin
and enveloped nucleocapsids were compared using AcMNPV polyhedrin anti-
serum (1:10 dilution) and [-^IJIgG (26 ng with a specific activity of
4yCi/yg). The assay procedure was the same as previously described in
Fig. 1. Antigens tested were: AcMNPV polyhedrin (0), AcMNPV LOVAL
(§), AcMNPV EV (A). Preimmune serum (A) was used as a control and
demonstrated no binding.
Figure 19. Micro-SPIRA for the detection of specific antiviral
antibodies. Microtiter wells were coated with a 1:10 dilution of AcMNPV
LOVAL antiserum at 4°C for 4 hr and then were coated with the 2% BSA
solution at 4°C overnight. One-hundred ng of AcMNPV LOVAL was added to
each well and was incubated at 4°C overnight. Fivefold dilutions of
each antiserum and IgG were added to the wells. After incubation at
37°C for 4 hr, the wells were washed with buffer, and AcMNPV LOVAL
[125x]igG (33 ng with a specific activity of 3 yCi/yg) was added and
incubated at 37°C for 4 hr. AcMNPV LOVAL (0); AcMNPV LOVAL IgG (•). IgG
concentration in the undiluted solution was 1.0 mg/ml; AcMNPV EV (a);
AcMNPV polyhedrin (•); and preimmune serum (a).
100
80
I 60
h-
52
1 40
20
0
o—o
2345
SERUM DILUTION Hog10)
6 00
42
-------
Table V, AcMNPV physical-infectious particle ratios as assayed
in vitro (Summers and Volkman, 1976)
Virus prepn
DNA
^ _ _. . _ Particle/
g/pg of ug of
g/pg or ug or
ig/Ug of protein protein
protein (x 10"7) (x 109)
PFU/yg of
protein
Particles/
PFU
PMB-NOV
LOVAL Ia
LOVAL II-Xb
0.19
0.20
0.30
1.9
2.0
3.0
1,1
1.2
1.8
8,9 x 106
5.0 x 103
8.4 x 103
1.28 x 102
2.40 x 105
2.20 105
Single nucleocapsid per envelope
Many nucleocapsids per envelope
To further characterize the biological differences between NOV and
LOVAL we conducted a study wherein we compared quantitatively the infec-
tivity of AcMNPV LOVAL and EV as assayed both in vitro and in vivo, the
latter as administered by both oral and intercoelomic injection (Volkman
and Summers, 1977).
Table VI gives the LD^Q and the TCID5Q values for the various prep-
arations under the different conditions. The results show that by the
per os route of assay, EV is very low in infectivity: 1 x 10^-fold less
infectious than it is by in vitro assay. When injected into the hemocoel,
however, it is much more highly infectious and approaches the level
obtained in the J.n vitro assay. A further extension of this logic pre-
dicts that LOVAL should be more highly infectious by per os administration
_in vivo than by coelomic administration or by in vitro assay. The results
in Table VI, however, do not support this conclusion. They show that
Table VI. LD50 and TCID50 values of LOVAL and EV
Virus Route of
prepn admins
LOVAL Per os
LOVAL Hemocoelic
PMB-NOV Per os
PMB-NOV Hemocoelic
Per
Purified
viral
protein
(yg)
1.28xlO~5
3.92xlO~5
8.00xlO~3
2.49xlO~7
LD50
Number of
particles
(genome
equiv)a
2.30xl04
7.05xl04
8.80xl06
2.74xl02
Per
Purified
viral
protein
(Ug)
1.16xlO~4
i.iexio"4
7, 84x10" 8
7.84xlO"8
TCID50
Number of
particles
(genome
equiv)a
2.09xl05
2 . 09xl05
8.60X101
8.60X101
Particles/
LD50 +
Particles/
TCID50
1:9
1:3
IxlO5:!
3:1
43
-------
LOVAL is only nine-fold more infectious by the per os route than by assay
in vitro and that the per os route is only three-fold more efficient than
the hemocoelic route. These differences are not comparable to the differ-
ences observed with EV.
In order to investigate whether LOVAL's consistent low efficiency
of infection, even when administered per os, was due to artificial remo-
val of the polyhedrin protein, we assayed highly purified whole polyhedra
by per os infection and found the LDrQ to be about 120. If the efficiency
of infection was the same for the occluded virions as it was for LOVAL,
there would be about 190 genome equivalents per polyhedrin. The results
indicate, then, that occluded virions are more infectious than LOVAL when
administered per os, though only slightly so, about five-fold.
It appears, therefore, that the virions that become occluded, when
assayed either as LOVAL or as occluded virus, are innately less infectious
than EV. There is a 50-fold difference when comparing infectivity under
the best conditions of each.
From this, the results suggest that, whereas the nonoccluded form
of the MNPVs derived from hemolymph and CCM are similar, they are very
different from the alkaline-liberated form of the occluded MNPVs. One
might expect serological differences among viruses from the three sources,
and indeed there are. We have found (Volkman e£_ al_., 1976) that antisera
raised against alkali-liberated virus inhibits in vitro plaque formation
by homologous virus, but does not interfere with plaque formation by the
nonoccluded form. These data suggest the necessity of biochemical, bio-
physical, and related serological comparisons of virus not only from
different sources in the same host, but also from different sources in
alternate hosts, to detect any virus modifications and, once detected, to
determine the nature of virus structure relative to these modifications.
44
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E. Immunoperoxidase
1, Production of enveloped nucleocapsids, polyhedrin and polyhedra
Time course studies were undertaken wherein the onset and the rate
of increase in infected cells of each of the following, as detected by
immunoperoxidase reaction, were compared: synthesis of enveloped nucleo-
capsid antigens, production of infectious intracellular and extracellular
nonoccluded virus, synthesis of polyhedrin, and the formation of polyhedra.
Enveloped nucleocapsid antigens were first detected in infected TN-368-10
cells at 8 h post infection (Figure 14), but not at 6 h. The first evi-
dence of infectious progeny virus (both intra- and extracellular) as
detected by polyhedral plaque assay, however, was at 10 h post infection
(p.i.): a lag of about 2 h between detection of enveloped nucleocapsid
antigens and that of infectious virus,
Polyhedrin was first detected at 12 h p,i. This shows that poly-
hedrin is synthesized late relative to enveloped nucleocapsid proteins,
and its presence did not precede that of infectious virus (Figure 15a),
However, the total amount of infectious nonoccluded virus, both intra-
and extracellular, began to decline at the onset of polyhedrin production
between 12 and 14 h p.i. (Figure 1 and 2).
The crystallization of polyhedrin into polyhedra was initially
observed to occur at 15 h p.i. The rate of production of polyhedra in
infected TN-368-10 cells paralleled the rate of polyhedrin synthesis as
judged by immunoperoxidase reaction with an approximate lag time of 1 to
3 h (Figure 15a and b). In infected TN-368-13 cells, however, the rate
of production of polyhedra was less than that of polyhedrin (Figure 15b).
This difference in the rate of appearance of polyhedra between infected
TN-368-10 cells and TN-368-13 cells has been noted before (Volkman and
Summers, 1976) and presumably is related to the difference in their abi-
lities to serve as indicator cells in the plaque assay (Volkman and Sum-
mers, 1975) and to the difference in their EV yields (Volkman et_ _al_. , 1976)
2. Intracellular location of enveloped nucleocapsid and polyhedrin anti-
gens
With the LOVAL antiserum the first immunoperoxidase staining in
infected cells was observed at 8 h p.i. in distinct regions of the cyto-
plasm indicating the site of enveloped nucleocapsid antigen synthesis
and accumulation. The staining was most intense near the nucleus, but
no stain was detected in the nucleus at that time (Summers js_t al_. , 1978).
At 10 h after infection of TN-368-10 cells, LOVAL antiserum
produced, in addition to the cytoplasmic staining present at 8 h, a very
slight reaction uniformly distributed throughout the nucleus indicating
the presence of some enveloped nucleocapsid antigen(s). This coincided
with the defection of both intracellular and extracellular infectious
virus.
At 12 h p.i., approximately 75% of the TN-368-10 cells treated with
LOVAL antiserum were uniformly stained, although the staining was slightly
45
-------
o.
BO
80
40
c
£
10 20
Time p.i (h)
Figure 14. Accumulation of intra- and extracellular infectious
virus of enveloped nucleocapsid antigens detected by the plaque assay
and immunoperoxidase technique. •*, Percentage of cells in which
enveloped nucleocapsid antigens were detected. Virus titres: •-•,
extracellular; o-o, intracellular virus. Infectivity was determined
by back titration of cell culture medium or disrupted cells in TN-368-
10 cells.
46
-------
100
10 20 30
Time pi (h)
40
80
o 60
40
20
o
o.
10 20 30
Time p.i. (h)
40
Figure 15. Production of TN-368-10 (a) and TN-368-13 (b) cells
of AcMNPV enveloped nucleocapsid and polyhedrin antigens, as detected
by the immunoperoxidase technique, and of polyhedra. The percentage
of cells was determined that reacted with AcMNPV LOVAL (•) or AcMNPV
polyhedrin (•) antisera, or contained polyhedra (0). • ~~
47
-------
more intense in the nucleus than the cytoplasm. At this time, less than
10% of the infected cells became stained with polyhedrin antiserum. The
intensity of staining in the cytoplasm of infected TN-368-10 cells using
LOVAL antiserum began to decrease when the cells first stained with poly-
hedrin, i.e., about 12 h p,i,
At 15 h p.i., 86% of the cells reacted with the LOVAL antiserum,
giving intense staining in the nucleus particularly in the virogenic
stroma. Markedly less staining was observed in the cytoplasm indicating
a possible shutdown in the synthesis of enveloped nucleocapsid antigens.
At 15 h the distribution of polyhedrin was similar to that at 12 h, but
the staining was slightly more intense. Twenty per cent of the cells
reacted with the polyhedrin antiserum, but intact polyhedra were observed
in only 25% (Summers et al., 1978).
At 18 h p.i., 92% of the infected TN-368-10 cells reacted with LOVAL
antiserum giving a somwhat even distribution of stain throughout the
nucleus, with the exception of some localization of stain adjacent to
the inner nuclear membrane. There was greatly diminished staining in
the cytoplasm. When treated with polyhedrin antiserum, 60% of the cells
distinctly stained in the nucleus while reaction in the cytoplasm was
significantly diminished. The staining appeared to be most intense near
the inner nuclear envelope. Polyhedra were observed in 30% of the cells.
At 24 h p.i., staining in TN-368-10 cells treated with LOVAL anti-
serum was similar to that at 18 h except that cells containing numerous
polyhedra in the nuclei reacted less intensely. When polyhedrin antiserum
was used, 70% of the cells stained intensely in the nucleus with some
apparent localization of stain near the inner nuclear membrane; staining
was markedly reduced in the cytoplasm. Polyhedrin were observed in 62%
of the cells.
At 36 h to 42 h p.i., when over 95% of the cells contained polyhedra,
TN-368-10 cells treated with LOVAL antiserum showed only slight staining
in the cytoplasm and a marked reduction of stain in the nucleus. These
results indicated that as enveloped nucleocapsids became occluded, the
reaction with LOVAL antibodies decreased.
TABLE IV: Detection of AcMNPV antigens and infectivity by immunoperoxi-
dase and plaque assay in insect cell lines
Cell lines ___
Bombyx Carpocapsa Manducta Mamestra
mori pomonella sexta brassicae TN-368-10
Time p.i. (h) ... 42
% cells Virus 90%
indicating Polyhedrin <1,0%
antigens
Infection obtained
by back titration
in TN-368-10 cells
Yes
42
0
0
No
48
18
55%
2%
Yes
18
71%
29%
Yes
18
42%
61%
-------
3. Infection of Manduca sexta, Mamestra brassicae^ Bombyx mori 5 and
Carpocapsa pomonella cells
Comparisons of several different invertebrate cell lines were made
in order to test the general utility of the immunoperoxidase technique
as developed with TN-368-10 cells and TN-368-13 cells (Table IV). Al-
though time course studies were not conducted, antisera to enveloped
nucleocapsids and polyhedrin tested against M., brassicae cells at 18 h
p.i. showed reactions typical of those observed in TN-368-10 and TN-
368-13 cells. It is now known from other studies in this laboratory that
the M. brassicae cell line yields titres of EV and polyhedra at least
comparable to those produced by TN-368-10 cells. Carpocapsa pomonella
cells are resistant to infection by AcMNPV and no progeny virus was de-
tected by back titration of cell culture medium in TN-368-10 cells. Also,
neither conjugated antiserum for enveloped nucleocapsids nor for polyhedra
reacted with these cells at time intervals up to 42 h post inoculation.
In ]3. mori cells, different results were obtained. At 42 h p.i.,
90% of the cells reacted with LOVAL antiserum. Staining was more intense
in the cytoplasm adjacent to the outer nuclear membrane with some very
slight staining in the nucleus. The cells did not react, however, with
polyhedrin antiserum. Inspection of 400 cells revealed that only 0.5 to
1.0% contained polyhedra. By 42 h p.i,, cells yielded titres of 10
p.f.u./ml or more of nonoccluded virus particles as confirmed by back
titration of .B. mori cell culture medium in TN-368-10 cells.
4. Discussion
The immunoperoxidase technique can be used to monitor infected
invertebrate cells for the presence of virus antigens during the course
of infection. Some of the advantages that this technique offers over
the popular plaque assay and other polyhedrin-dependent methods are;
(1) it is independent of the production of polyhedra and may be used to
detect enveloped nucleocapsid associated antigens or alternatively,
polyhedrin antigens directly, and (2) it can detect virus antigens in
the absence of infectious virus and polyhedrin. Thus, the method can
be used in the search for: (1) virus mutants which under certain condi-
tions either cannot produce polyhedrin or produce an altered polyhedrin
that cannot crystallize into polyhedra, or (2) virus mutants that do not
produce infectious particles in certain cell types. If used in conjunc-
tion with polyhedral plaque assay, the method could further the under-
standing of the mechanisms involved in the replication and production of
occluded and nonoccluded virus.
It is difficult to evaluate the sensitivity of the assay except,
perhaps, by comparing the sensitivity of antisera when used in this method
with that of the same sera when used in radioimmunoassay. The polyhedrin
antiserum used in this study could detect 75 pg of polyhedrin by binding
studies. Furthermore, both competition radioimmunoassay and the immuno-
peroxidase technique first indicated the appearance of polyhedrin at 12 h
p.i. By radioimmunoassay, polyhedrin was detected by 12 h in as few as
100 cells; at the same time of infection, the immunoperoxidase technique
49
-------
detected polyhedrin in 1 of 100 cells.
The LOVAL antiserum could detect 1 ng .of virus, perhaps less by
competition radioimmunoassay (M, D, Summers, unpublished results). If
it were assumed that similar sensitivity existed for the iiamunoperoxi-
dase method as for the radioimmunoassay, as compared to the polyhedrin
studies, it could be estimated that 1 ng of enveloped nucleocapsid anti-
gens could be detected, which represents, at a maximum, roughly 1,8 x
10 LOVAL particles (Volkman et al. 1976). This is probably an overesti-
mation since the calculation is based upon total virus protein. Although
not yet determined, the antiserum probably recognized only a small pro-
portion of the structural proteins of the enveloped nucleocapsid.
The study relating time, presence and cellular location of anti-
gen-antibody reaction revealed several things about the nature of AcMNPV
infection of TN-368-10 and TN-368-13 cells. The fact that enveloped
nucleocapsid antigens were detected 2 h in advance of infectious virus,
both of which, in turn, were detected 2 to 4 h before polyhedrin, indi-
cates that the synthesis of polyhedrin (in excess of our limits of detec-
tion) is not necessary for the production of infectious nonoccluded virus.
The synthesis of infectious virus began to decline at the onset of poly-
hedrin production as detected by immunoperoxidase staining between 12
and 14 h p.i. This confirms our previous results (Volkman and Summers,
1975). Polyhedrin was synthesized about 2 h before the appearance of
polyhedra. While the rate of appearance of polyhedra paralleled the rate
of appearance of polyhedrin as evaluated by similar slopes (Figure 2) in
TN-368-10 cells, the rate of appearance of polyhedra in TN-368-13 cells
was less when compared to polyhedrin. As the rate of synthesis of poly-
hedrin was approximately the same for TN-368-10 and TN-368-13 cells (see
slopes of reaction curves) (Volkman and Summers, 1975, 1976; Volkman et
al., 1976), perhaps a factor involved in the crystallization of polyhedrin
into polyhedra is related to the two cells lines' abilities to produce
different quantities of polyhedra and at different times. The rate of
appropriate enveloped nucleocapsid protein(s) to serve as a nucleation
site.
Enveloped nucleocapsid antigens initially appeared in the cytoplasm
of infected cells and were transported to the nucleus by 10 h p.i. At
15 h intense staining occurred in the nucleus while staining in the cyto-
plasm was greatly diminished. Intense staining and localization coincided
with the appearance of the virogenic stroma (Volkman et al. 1976) just
prior to polyhedrin formation. The intense staining associated with the
virogenic stroma may be indicative of the insertion of LOVAL specific
proteins into intranuclear virus envelopes. As the number of polyhedra
per nucleus increased between 18 and 42 h, staining of enveloped nucleo-
capsid antigens in the nucleus decreased.
The testing of several invertebrate cell lines indicated that the
immunoperoxidase technique could indeed be useful for host range studies.
In addition, the peroxidase results with infected Bombyx mori 5 cells
revealed that no intense intranuclear staining occurred using LOVAL or
polyhedrin antisera, by contrast with TN-368-10 and TN-368-13 cells. In
fact, the slight staining observed at 10 h p.i. in _B. mori cells was sim-
50
-------
ilar to that at 8 and 10 h p.i, in TN-368-1Q cells, One can speculate
that (1) certain LOVAL specific antigens responsible for the aerological
reaction are not synthesized in JB. mori cells; (2) the transport of
LOVAL specific antigens to the nucleus is inhibited; (3) LOVAL specific
proteins are synthesized and transported to the nucleus, but fail to be
inserted into the LOVAL envelope so that the antigenic structure recog-
nized by the antibodies is not produced; (4) an interfering agent not
detected by our technique may be present, Any of these possibilities
may be directly or indirectly responsible for the lack of nuclear staining,
The observations raise the question of how the Ji, mori cell is able
to block some function related to the synthesis of polyhedrin (Summers and
Smith, 1976), but not replication of enveloped nucleocapsids. The infectious
virus recovered from medium of infected .B. mori 5 and JJ, mori I (from D.
Knudson) infects TN-368-10 cells normally and therefore does not appear
to be defective. In addition, extracellular virus from AcMNPV infected
_B. mori I cells can be 98.5% neutralized by homologous antiserum. More-
over, no plaque forming virus was detected when uninfected !B. mori cells
were sonicated and titrated for virus in TN-368-10 cells,
•*
The finding that infected Bombyx mori cells produced enveloped
nucleocapsid antigens and infectious virus in the absence of detectable
polyhedrin and, consequently, polyhedrin production in nearly all the
cells, illustrates precisely why polyhedrin dependent assays should only
be used as indicators of virus infectivity in well characterized systems.
There is therefore a need for more specific and sensitive techniques for
the direct detection and quantification of enveloped nucleocapsids. The
development of the immunoperoxidase technique as applied herein is one
of the first steps in that direction.
F. Neutralization
Antisera raised against AcMNPV and RoMNPV LOVAL were tested for
neutralization activity against AcMNPV and RoMNPV from various sources,
The results of these experiments were that the antisera neutralized the
homologous and heterologous LOVAL (Figure 20). It did not neutralize
other forms of the virus, however, such as EV, alkali-treated EV, hemo-
lymph-derived NOV, intracellular NOV from culture cells, and intracellu-
lar NOV from the fat body (Volkman ^t jal., 1976),
Figure 21 shows the comparative curves of anti-AcMNPV LOVAL serum
and anti-AcMNPV EV serum versus AcMNPV LOVAL. The curves of the untreated
sera were very nearly identical, indicating that the neutralization titer
in the heterologous serum was equal to that of the homologous serum, but
when the sera were heat inactivated, the 50% neutralization titer of the
homologous antisera (anti-AcMNPV LOVAL) was apparently increased (about
four-fold), whereas the titer of the heterologous antisera appeared to
be relatively unchanged.
Figure 22 depicts the neutralization capability of anti-AcMNPV EV
serum (heat inactivated and untreated) versus AcMNPV EV, and the untreated
serum versus both LOVAL and EV of RoMNPV. It should be noted that the
51
-------
titer of this serum not only was identical with the comparable forms of
AcMNPV and RoMNPV, but was higher against the NOV than against the alka-
li-liberated viruses,
Neutralization of intracellular NOV from both TN-368-10 cells and
the fat body of T_, ni larvae with heat-inactivated anti-AcMNPV EV
(Figure 22) indicated that the intracellular NOV shared a biologically
important antigenic determinant(s) with EV that was not present or
accessible on LOVAL.
Figure 24 shows the results of the neutralization of both AcMNPV
EV and LOVAL with AcMNPV LOVAL-adsorbed anti-AcMNPV EV. As can be~seen,
there was no remaining neutralization activity against LOVAL, whereas
there was no change in the titer against the EV, These results confirm
the conclusion that the LOVAL EV forms are antigenically distinguishable.
Serum concentration
Figure 20. Neutralization of AcMNPV LOVAL (A) and RoMNPV LOVAL
(B) with rabbit antisera generated against AcMNPV LOVAL (0) and RoMNPV
LOVAL (t). Virus samples were incubated for one hr at 37°C with the
indicated concentration of antiserum, and then 0.1 ml was assayed for
biological activity. (The concentration of undiluted antiserum is 1).
The 100% value of biological activity was the average of the control
virus samples incubated with preimmune serum at the various concentra-
tions. All assays were done in duplicate.
52
-------
Figure 21. Neutralization of AcMNPV LOVAL with heat inactivated
and untreated (*) anti-AcMNPV LOVAL serum, and with heat-inactivated
(0) and untreated (•) anti-AcMNPV EV serum. Neutralization was carried
out as described in Fig. 4 and the test. Heat inactivation of antisera
is described in the test.
II ' 10 '
Serum concentration
Figure 22. Virus neutralization with untreated anti-AcHWV EV
serum of AcMNPV PMB-NOV (•), RoMNPV EV (0), and RoMNPV LOVAL 0s , Heat
inactivation of this antiserum did not alter the neutralization curve
of AcMNPV EV (Q).
53
-------
It' II'
S«rum concentration
Figure 23. Neutralization of intracellular nonoccluded virus from
both TN-368-13 cells (•) and the fat body of infected T.. ni larvae
with heat-inactivated anti-AcMNPV EV serum.
f to
c
S 40
I
I »
10 * 10 ' 10 '
Scrum concentration
Figure 24. AcMNPV LOVAL-adsorbed anti-AcMNPV EV serum versus
AcMPV EV O a«d LOVAL (*) . The unadsorbed anti-AcIWPV EV versus
AcMNPV EV curve is shown in comparison (•).
54
-------
Discussion
The results of the neutralization tests showed that anti-HoMNPV
and anti-AcMNPV LOVAL sera neutralize both the homologous and hetero-
logous LOVAL forms of the virus, but neither will neutralize even the
homologous nonoccluded forms of the virus, These results suggest that
antigens important in biological activity are shared in the in vitro
infectious LOVAL of two different viruses, but either are not present
or are not exposed in the infectious forms of the NOV, or are not
important in the expression of their biological activity, In any case,
the two forms of in vitro infectious virus appear to be antigenically
if not biologically different, whereas the same forms of two different
viruses, AcMNPV and RoMNPV, share biologically important antigenic
determinants,
The reciprocal tests, i.e., neutralization of the various forms
with anti-AcMNPV EV, showed that all forms tested of both AcMNPV and
RoMNPV could be neutralized, but that the titer was generally higher
against the nonoccluded forms than the alkaline-liberated forms.
Adsorption of anti-AcMNPV EV with AcMNPV LOVAL completely removed all
neutralization activity against AcMNPV LOVAL, whereas the titer against
AcMNPV EV and other nonoccluded forms was undiminished, These results
confirm the initial findings that those antigens important in the neu-
tralization of LOVAL and EV are different. They also indicate that
whereas LOVAL does not possess an accessible antigen similar to the ones
important for the neutralization of EV, EV does share the antigen that
is active in the neutralization of LOVAL.
The results also indicated that antigens important in the neutral-
ization of NOV other than EV are present and antigenic on EV, That the
same antigen is active in the neutralization of all forms of NOV, however,
has not been determined. The enhancement of the neutralization titer of
untreated AcMNPV EV antisera against alternate nonoccluded forms by its
adsorption with AcMNPV LOVAL indicates that the LOVAL is removing some
component(s) of the serum that interferes with the neutralization of the
alternate nonoccluded forms. This same component(s) does not interfere
with the neutralization of EV, however. That this enhancement is also
achieved by heat inactivation of the serum, and that heat inactivation
and adsorption both does not have an additive effect, indicates that the
component(s) is a nonspecific heat-labile factor.
The neutralization results presented in this report show rather sig-
nificant antigenic differences between LOVAL, EV, and other NOV, and
subtle differences between EV and othe NOV. These antigenic differences
could very possibly be attributable to envelope differences, The results
presented in this paper show that there are significant differences
between the alkali-liberated and nonoccluded forms of the NPVs of R. pu
and A. californica with regard to infectivity and antigenicity, These
results, along with the mounting data indicating significant biochemical,
biophysical, and morphological differences between the alkali-liberated
and nonoccluded virions suggest structural differences that are indica-
tive of differences in specificity, function, and role in the continuance
of the infection cycle.
55
-------
G. Preliminary Studies on Phenotypic and Genotypic Variation and/or
Stability of Wild Viral Isolates After Passage Through. Alternate
Host Systems
In this portion of our studies we compared the stability and re-
producibility of Autographa californica MNPV polyhedrin and virus struc-
tural polypeptides after passage through alternate host systems to the
polypeptides of this virus form several wild isolates (Maruniak et al.,
1978). A biochemical and biophysical comparison of these proteins from
in vivo and in vitro sources will help in determining if changes occur
which could involve host cell modifications, mutation, or recombination.
No differences in mobility of polyhedrin from the different sources
can be detected on SDS polyacrylamide slab gels. When solubilized in
alkali at 37°C for 1 and 2 h polyhedrin from larvae was degraded by the
inclusion associated protease, while polyhedrin which is derived from
infected tissue culture cells remains intact thus indicating lack of or
diminished proteolytic activity.
Tryptic digests of polyhedrin purified from all sources utilized in
this study showed little if any difference from the peptide maps. Keeping
in mind that this technique will discriminate single amino acid differen-
ces in peptides, the polyhedrins were found to be very similar if not
identical.
Using the improved technique of automated Edman degradation, the
N-terminus of purified, undegraded 30,000 M.W, polyhedrin of Autographa
MNPV purified from A. californica and T_. ni larvae was determined as
proline. In contrast, when polyhedrin was dansylated using the dansyl
chloride technique, a weak orange flourescence near dansyl-glx appeared.
Although polyhedrin primary structure was not significantly differ-
ent as determined by peptide mapping and amino acid analysis, minor
quantitative differences in AcMNPV virus structural polypeptides were
found when comparing it from one in vitro and four in vivo sources by
SDS-PAGE (Figure 25). As accurately as possible to 30 yg of each was
loaded on gels, yet obvious quantitative differences appeared especially
with VP68 (Virus Protein), VP33, VP22, VP18, and VP16. There appeared
to be more of VP68 in AcMNPV isolate D (Falcon) while there was signifi-
cantly less in isolate B (Ruud). A difference in VP32 was noted by more
of this protein in isolate C obtained from Vail. More of VP22 was found
in that from isolate D (Falcon), while there was much less of the protein
if any in AcMNPV isolate B (Ruud). The lower molecular weight protein,
VP18, was reduced in amount in our AcMNPV isolate E. Finally, a quanti-
tative difference appeared in VP16 being diminished in AcMNPV isolate D
(Falcon) and also diminished in our preparation of enveloped nucleocapsids,
F, isolated from tissue culture derived polyhedra. Some qualitative
differences noted were VP22 being apparently absent or diminished compared
to the amount loaded on the gel in the AcMNPV preparation from L. Ruud (B)
and perhaps the presence of the diffuse VP18.5 from the AcMNPV prepared
in this laboratory (E), It should be noted that the polypeptides of
enveloped nucleocapsids derived from tissue culture polyhedra closely
paralleled those for the same isolate derived from larvae.
56
-------
It was shown in one of our studies (Maruniak and Summers, 1978)
that TT. ni_ granulosis virus, T_. ni nuclear polyhedrosis virus, Autographa
californica (AcMNPV), and Rachiplusia cm RoMNPV polyhedrin and granulin
primary sequences were specific for each virus. Using the same biochemical
techniques, we could not detect any alteration in primary sequence of
AcMNPV polyhedrin after passage through alternate host systems. It should
be noted that AcMNPV from this laboratory has been propagated 20 to 30
times in T\ ni larvae and likewise, AcMNPV extracellular virus which was
used to infect tissue culture cells has been cultured many times through
the cells since 1975. This data supports the hypothesis that polyhedrin
is a virus specific protein, and by this method of evaluation appears to
remain stable upon continuous passage through alternate biological systems.
R. J. Cibulsky, et al., (1977) also found peptide maps of polyhedrin
from AcMNPV from A. californica and T_. ni larvae to be indistinguishable.
However, we cannot directly compare our results due to: (1) the use of a
different system for electrophoresis; (2) the virus associated protease
was not inactivated in that study; and, (3) peptide mapping was carried
out on a protein preparation with bands of several molecular weights.
It has been discussed previously in this report that an active virus
protease and a heterogenous protein can cause artifacts in peptide
mapping, and therefore, it is essential to inactivate the protease and
provide evidence of purity of the polypeptide preparation. However, as
compared to polyhedra from larvae, there appears to be little if any
protease activity associated with tissue culture derived polyhedra
when alkaline solubilized up to 2 hours at 37°C and, hence little or no
degradation of polyhedrin. We therefore recommend that investigators
consider tissue culture derived polyhedra as an additional source for
undegraded polyhedrin.
Further consideration is being given to enveloped nucleocapsids
showing AcMNPV derived from various sources, both in vivo and in vitro,
exhibits very similar polypeptide profiles on SDS polyacrylamide slab
gels. SDS-PAGE has been demonstrated to be a valid technique for showing
differences in virus strains by Montcastle and Choppin (1977), among four
measles virus strains and be Gupta et al., (1977) for several strains of
human cytomegalovirus where some qualitative and quantitative differences
are seen. Furthermore, we do note some quantitative and qualitative
differences in virus structural proteins, for which the molecular basis
of these differences and their functional significance remain to be
elucidated, the major polypeptides were conserved in the viruses from the
three laboratories as well as those collected from the field. We have
also determined in studies we are continuing that plaque purified isolates
show distinct phenotypic and genotypic differences existing within a wild
isolate of AcMNPV.
57
-------
A B C D E F
VP
_I25
94,000 — -- -- - 90
_ ..... ^ ______ . ___™^.» _. ._ "7 c 7 °3
68,000 •* ~ --- =68,64
.. _____ _ . ~r
53 OOO MI " ....... JZTI^L. ^"^L ^^^ ^^^ _ 'jo. I
46
45,40
37
TO
3I'°°° " """"^ — , =28.5,28
25,000 •»
-23
- . - 22
- 19
~~ 18.5
17,200 »
Figure 25. SDS-polyacrylamide gel electrophoresis of AcMNPV
structural polypeptides. The SDS-gel composition was 10.8% acrylatnide
(Acrylamide/Bisacrylamide = 37,5/1), (A) Molecular weight standards
are listed in Fig. 1, (B) AcMNPV from infected field insects from L. Ruud,
(C) AcMNPV in Autographa californica obtained from P. Vail. (D) AcMNPV
in T_. ni obtained from L, Falcon, (E) AcMNPV in T_, ni from this labora-
tory. (F) AcMNPV derived from TN-368-10 insect tissue culture cells in
this laboratory.
58
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H. Use of Restriction Enzymes for Evaluating the Genome Structure of
Plaque-Purified and Wild Isolates of Baculovirus
The original source(s) of baculoviruses used in basic and field
applied research or for commercial development has been from field-
collected, infected insects subsequently propagated in the laboratory,
For the characterization of wild isolate viruses, structural, immunolo-
gical and biological criteria can be employed. A knowledge of the pro-
tein compositon and viral DNA structure would provide identifiable
phenotypic and genotypic markers.
In order to identify baculoviruses, it is necessary to take into
account the complex replication behavior they exhibit both in vivo and in
cell culture, producing infectious forms with different biological and
structural properties that have not yet been fully characterized. The
nuclear polyhedrosis virus (NPV) of Autographa californica is known to
produce at least three infectious forms: a) viruses occluded in the
nucleus with a single nucleocapsid per envelope (singles); b) with multi-
ple nucleocapsids per envelope (multiples); and c) extracellular virus
that has budded from the plasma membrane (Summers and Volkman, 1976;
Volkman ej^ al., 1976). The structural polypeptide composition of AcMNPV
singles and multiples, has been shown (Summers and Smith, 1978) to be
both quantitatively and qualitatively different. Herein, we show that
the structural polypeptides of extracellular AcMNPV exhibit different
electrophoretic patterns in polyacrylamide when compared to singles or
multiples. Polypeptide differences among these three infectious forms
suggests the possibility of genetic variation. Therefore, we isolated
the three forms from wild-type, AcMNPV to compare their viral DNAs using
restriction endonuclease digestion and fragment analysis.
The uncertainty of the genetic purity and the possibility of genetic
change (or contamination by other baculoviruses) during passage in labor-
atory insect colonies and in cell cultures emphasizes the need to deter-
mine the genomic heterogeneity and the genetic stability of wild baculo-
virus isolates. The NPV of A. californica was purified in cell culture
by multiple plaque purifications and the viral DNAs compared using agarose
electrophoresis of EcoR-1 and HindIII restriction endonuclease fragments.
By comparing the restriction fragment patterns of each of the plaque-puri-
fied viral DNAs to the wild-type viral DNAs, we detected and quantified
genetic variants existing in wild-type AcMNPV,
EcoR-1 Endonuclease Digests of Baculoviruses DNAs
The EcoR-1 restriction patterns of nine DNAs from A, californica
MNPV (Ac) , Rachiplusia ou MNPV (Ro), Trichoplusia nil SNPV (TnS), Porthe-
_trla dispar MNPV (Pd), Spodoptera exigua MNPV (Se), Heliothis zea SNPV
(Hz), and Heliothis armigera MNPV (Ha), and from T_, ni (TnG) and Spod-
optera frugiperda (SfG) granulosis virus are shown in Figure 26 (Smith
and Summers, 1978).
Each DNA restriction pattern was further analyzed by scanning den-
sitometry and molarity of each fragment determined as described in Materi-
als and Methods, Submolar restriction fragments could be detected in
four wild-type baculovirus DNAs, AcMNPV, TnGV, SfGV, and HaMNPV.
59
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Comparison of Wild-Type DNAs
Wild-type AcMNPV was obtained from three sources: 1) our laboratory
where the virus was originally received from Dr. Pat Vail and subsequently
grown for several generations over a period of 4 years in T_. ni larvae;
2) field-collected virus obtained from Dr. Lou Falcon, University of
California, Berkeley; and 3) an original isolate of AcMNPV from Dr. Pat
Vail grown in A. californica larvae. Restriction fragment analysis using
EcoR-1 (not shown) and HindiII endonucleases with viral DNA from these
three isolates were identical.
A comparison of the DNA restriction patterns using EcoR-1 and
Hindlll endonucleases of three wild-type AcMNPV forms, extracellular
virus (E), singles (S), and multiples (M), showed no detectable differen-
ces (data not shown). The EcoR-1 and Hindlll DNA restriction patterns
from wild-type AcMNPV grown in larvae (L) or cell culture (C) were also
identical.
Restriction Analysis of Plaque-Purified Virus DNA
DNA was purified from 11 plaque-purified AcMNPV isolates. Of the
11 plaque-purified viruses, three were extracellular virus (El, 2, 3)
and four each from singles (SI, 2, 4, 5) and multiples (Ml, 2, 3, 4).
An analysis of each by EcoR-1 digestion gave a total of three restriction
patterns designated as Types I, II, and III. Each type of AcMNPV had
molecular weights of 79.0, 79.1, 81.0 megadaltons, respectively. The
plaque-purified virus isolates El, 2, and 3, S4, and 5, and Ml, 2, and
4 were of Type I; S2 and M3 were of Type II: and only SI was of the Type
III EcoR-1 restriction pattern (Figure 27).
A feature common to uncloned AcMNPV DNAs, when cleaved with EcoR-1
and electrophoresed in 0.75 and 1.0% agarose is the presence of 23
restriction fragments. All but one of the fragments are major and in
molar concentrations. A submolar fragment with a molecular weight of
2.5 x 10 , which is missing. Type II DNA restriction pattern only in
the absence of a fragment with a molecular weight of 2.4 x 10 and
the presence of a 2.5 x 10 band as a major molar fragment. The EcoR-1
Type II DNA was identified in two of the eleven clones. This shows that
uncloned AcMNPV DNA isolates contain detectable EcoR-1 Type I and II
genomes. The EcoR-1 Type III restriction pattern is like the Type II
except for a 7.0 x 106 fragment. This EcoR-1 Type III pattern
was not detected in uncloned DNA. Inasmuch as only one of the 11 virus
clones tested exhibited this pattern, it is likely a genomic variant in
the wild isolate that is present in only a small percentage of the viral
DNA population.
The restriction patterns of the 11 plaque-purified AcMNPVs produced
by the Hindlll enzyme showed that 8 of 11 viral DNAs were different.
Digestion with Hindlll gave more resolution and revealed differences in
plaque-purified virus DNAs not detected with EcoR-1 digests. Consistent
with the results of the EcoR-1 restriction enzyme analysis, Hindlll pat-
60
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AcM RoM TnS TnG SfG SeM Hz& HoM
=* —" —3
—IOS.IOS
9090
70
6 3 ——
-40 —40.40 ~1* 40 "•3T .,., . 40
• 2 6 =:J, ft — * *
-2 15
2 65
2 S. -* »» ___ ,47 __ 2 4C
i~j£ —235.235 ?4" "S
-I 10
• II
1 §5
' I •» ^^^ I •>
— ' '»•' '» ^M H , „ 1 75
_ | 65 ' 'O -' '0 ' "
=!8 =i» _140 ^,45 -'»
—I 15,135
. I 30 ~~~l 32
. 1 10
. lOt.101 10» 101
=8'.? - °« —0.0
- o 12 - o 12
- 0 76
0 70 — ^— 0 70 ^— o 70
0 61 - 0 6« - 0 6> _ o ^
_
0 60 — — 0 60
- 0 55
- 0 53
Figure 26. Schematic representation of the EcpR-1 restriction
fragments for eight wild-type baculovirus DNAs. Molecular weights in
megadaltons are given for each fragment and are the mean values from
three separate agarose electrophoresis gels. The restriction pattern
of PdMNPV DNA is not included as the co-migration of numerous high
molecular weight EcoR-1 fragments made analysis difficult. Minor or
submolar fragments have been indicated (*).
61
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EcoR I
C I II ill
2.5-
Figure 27. The DNA from plaque-purified AcMNPVs compared by
electrophoresis of EcoR-1 restriction fragments as described in Fig.
2. The DNA restriction pattern from virus clones, El, E2, E3, S4,
S5, Ml, M2, and M4 were Type I; S2 and M3 were Type II; and SI was
Type III. EcoR-1 restriction fragment of wild-type AcMNPV DNAs from
infected insect larvae (L) and infected all culture (C) are shown.
A minor fragment present wild-type DNA with a molecular weight of 2,5
x 10" daltons has been indicated.
62
-------
terns of uncloned AcMNPV DNAs also contained several minor fragments.
These minor fragments have mobilities that are the same as major; molar
fragments found in several of the plaques-purified viral DNAs.
A comparison of the EcpR~l restriction patterns of the DNAs from
nine wild isolates of baculoviruses shows that this method is specific
for virus genome identification (Figure 26), Only two of the baculo-
virus DNAs that were analyzed, AcMNPV and RoMNPV, showed similarity in
restriction fragments, of which about 40% of the EcpR-1 and HindiII
fragments co-migrated. The EcpR-1 restriction patterns of DNA from
Orgy a pseudotsugat £>NPV and MNPV (Rohrman et al. , 1978) also appear to
be unique in comparison with the DNAs analyzed in this report,
Each infectious form of AcMNPV whether derived from in vitro, extra-
cellular virus, or in vivo, singles or multiples from polyhedra (Summers
and Smith, 1978), have certain structural polypeptides which differ quan-
titatively and qualitatively. These infectious forms are apparently
phenotypes inasmuch as 1) DNA purified from wild-type AcMNPV extracellular
virus or singles and multiples from polyhedra has similar restriction
patterns, and 2) restriction enzyme digests of plaque-purified isolates
for each infectious type revealed the presence of genetic variants; how-
ever, it was not possible to correlate a given restriction pattern with
any of the three infectious forms. It is possible that minor genomic var-
iants, not detected with EcoR-1 or Hindlll restriction enzymes, could be
responsible for the observed differences, but we presently feel this is
unlikely. Furthermore, extracellular virus is 2000-fold more infectious
than singles or multiples derived from polyhedra, yet no difference
was detected in plaque-purified isolates from the three infectious forms
which would account for a more efficient variant (Volkman e_t al., 1977).
The use of other restriction enzymes and molecular hybridization will be
needed to additionally confirm that these distinctly different viral
forms do have the same genome.
The DNA restriction patterns of wild-type AcMNPV, TnGV, SfGV, and
HaMNPV consistently showed submolar fragments. These fragments could not
be attributed to incomplete digestion products since concentration did
not change the pattern. The presence of submolar fragments suggests that
intermolecular heterogeneity is present in these wild-type virus DNAs.
Variation in the viral DNA molecules could be of two types: 1) molecules
with the same overall base and gene composition but with a rearrangement
of certain genome segments, for example, inversion of specific regions
detected in herpes virus DNA (Hayward et al., 1975); or 2) molecules with
different gene composition as would result from recombination, site-spec-
ific mutations, or contamination with host or other virus DNAs.
The nature of the genomic variation found in wild-type baculovirus
DNAs, as evidenced by the presence of submolar fragments, is not known.
However, the restriction enzyme patterns of DNA from plaque-purified
AcMNPV suggests that heterogeneity is not due to contamination of other
viral DNA(s) but to a mixture of AcMNPV genetic variants. This is because
the submolar restriction fragments found in wild-type AcMNPV DNA had the
same mobility as molar fragments in the restriction patterns of certain
cloned AcMNPV DNAs.
63
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Of 11 plaque-purified AcMNPVs compared in this study; eight had
different _EcaR-l and/or Hindlll restriction patterns, With certain of
the plaque-purified viruses, no change was detected in viral DNA res-
triction endonuclease fragment patterns upon either additional plaque
purification or undiluted serial passage 16 times through cell culture,
This provides additional evidence that AcMNPV variants are a natural
occurrence and are not induced by laboratory manipulation,
The DNAs of wild-type AcMNPV from two other sources when analyzed
with EcoR-1 and HindiII and compared to DNA of AcMNPV grown in this lab-
oratory, had identical restriction patterns with respect to both major
and minor fragments. This suggests that these sources of AcMNPV are
also genetically heterogenous and may contain some of the same variants.
Plaque purification of a wild-type baculovirus, should it prove to be
concomitant with biological or phenotypic changes, will have significant
implications as to the specificity and any potential for altered host
range.
-------
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68
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO.
EPA-600/1-80-020
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Development and Standardization of Identification and
Monitoring Techniques for Baculovirus Pesticides.
5. REPORT DATE
May 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOFUS)
Max D. Summers
8. PERFORMING ORGANIZATION REPORT NO.
9. "ErlFGfiMING ORGANIZATION NAME AND ADDRESS
Department of Entomology
Texas A&M University
College Station, Texas 77843
10. PROGRAM ELEMENT NO.
1EA615
11. CONTRACT/GRANT NO.
Grant #805232
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Lab - Toxic Effects Branch
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
600/11
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Biological pesticides, in particular the microbial formulations, are fundamentally
different from chemical pesticides in the nature and mode of action of the active
agent. The pesticidal action is dependent on the activities of living organisms.
Identification, detection and monitoring methods for biological pesticides, because
of their nature and characteristics, are divergent from those classically associated
with chemical toxicants. Therefore, a new class of standardized, specific and
sensitive methods must be developed.
One objective of this grant was the development, adapttation and application of
specific sensitive diagnostic and clinical techniques for identification, detection
and monitoring of viral pesticides. A portion of this research was also commited
to study some of the basic biology and characteristics of baculoviruses so that a
more thorough understanding of the limitations of the developed monitoring technology
would be better understood. This technology can now be applied for the assessment
of health and ecological effects, as well as for regulatory concerns.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEIW ENDED TERMS c. COSATI Field/Group
Biological pesticides
Chemical pesticides
Baculoviruses
06F,T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NC OF PAGES
84
20. SECURITY CLASS {This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-I (Rev. 4—77) PREVIOUS EDITION is OBSOLETE
69
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