United States
                    Environmental Protection
                    Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
                    Research and Development
EPA/600/S1-85/018  Sept. 1985
&EPA         Project  Summary
                    Investigation  on the Potential
                    Environmental  Hazards of
                    Pesticidal Viruses

                    Eng-Shang Huang, Lambert Loh, Yuan-Ming Wu, and Eng-Chun Mar
                                                                                       11
                      Due to the environmental and eco-
                    logical affects of toxic chemical pest-
                    icides, the usage of insect viruses have
                    been considered as one of the alter-
                    natives for the control of agriculture
                    insect pests.  In fact  in the past  3
                    decades, several  baculoviruses have
                    been used as viral  pesticides for pest
                    control.  These viruses have not been
                    demonstrated to be hazardous to non-
                    target organisms  using the classical
                    infectivity and morphological alteration
                    as measuring factors. In this research
                    project, molecular biological approach-
                    es were used to characterize the molec-
                    ular structure of one of the insect vi-
                    ruses in order to investigate and eluci-
                    date the possible  pathogenicity  and
                    oncogenicity of pesticidal viruses to
                    human and other mammals at in vitro
                    level. The study suggests that the pes-
                    ticidal virus Spodoptera Frugiperda (SF)
                    can not productively infect human fibre-
                    blast or  HEP-2 cell lines and cannot
                    induce morphological transformation of
                    human fibroblast.
                      Besides the study on the biopath-
                    ology of a pesticidal virus, Spodoptera
                    fragiperda nuclear polyhedrosis virus
                    (SfNPV), the molecular structure of
                    the genome of this virus was also
                    extensively studied in developing non-
                    hazardous universal pesticidal viruses.
                    The complete set  of virus ON A frag-
                    ments have been  cloned in pBR322
                    plasmid. This set of the recombinant
                    plasmid  is now available  for further
                    gene function study.
                      This work was  carried  out  in  the
                    Cancer Research Center and the Depart-
                    ment of Medicine, University of North
Carolina at Chapel Hill under the sup-
port of U.S. Environmental Protection
Agency. This report is submitted in ful-
fillment of Grant Number R806210 by
the University of North Carolina under
the sponsorship of U.S. Environmental
Protection Agency. The report covers
the period June 10,1978 to September
10,1981.
  This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory, Research Triangle Park, NC, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).

Introduction
  In recent years, there has been great
interest in industry and government in
searching for the possible usage of insect
viruses as an alternative to chemical pes-
ticides to control agricultural insect pests.
The impetus to use viral pesticides  is
based on the environmental and ecologi-
cal effects of toxic" chemical pesticides.
For example, the three most commonly
used chemical insecticides, methylpara-
thion, malathion, andcarbaryl, are highly
toxic and teratogenic to mammals. Oth-
ers such as DDT, are extremely stable in
nature. The accumulation of residual sta-
ble chemicals pose a great problem on
environmental health.
  The nuclear polyhedrosis viruses (NPVs)
are known to be pathogenic to inverte-
brates. This group of viruses causes lethal
disease in their insect hosts. The virus
particles of this group usually consist of
enveloped nucleocapsids which frequent-

-------
ly are included in a large protein lattice or
polyhedron. The nucleocapsids are rod-
shaped with dimensions of about 250 x
50 m/j, and are usually singly enveloped;
but in  some instances, more than one
nucleocapsid can occur within one virus
envelope. Viral genomes  of this group
were found to contain covalently closed
supercoiled double-stranded DNA with a
molecular weight of approximately 75 to
100 x  106 daltons. These viruses com-
prise the best known insect viruses.  All
together, the NPVs  have been found in
more than 200 species of Lepidoptera, in
20 species of Hymenoptera, and in 9 spe-
cies of Diptera. They  are all in the genus
Baculovirus.
  These  bacilliform viruses replicate in
the nuclei of the infected cells. During the
process of infection, a substantial portion
of virions is enveloped and subsequently
occluded in the protein matrix of poly-
hedra. The intact polyhedra are not infec-
tious in in vitro insect cell cultures,  but
they are the key "vector" by which virus
infections  are transmitted in  nature.
When insect larvae ingest the polyhedra,
the infectious virions are  released from
polyhedra through the solubilization of
the  protein matrix of polyhedra in the
alkaline environment and by enzymatic
digestion in larvae gut. The purified NPV
DNA was proved to be infectious in the
insect  cell cultures.

Viral  Pesticides

  Several baculoviruses have been used
as viral pesticides for pest  control during
the last 3 decades;  e.g., the NPV of the
Alfalfa caterpillar (Colias eurytheme); the
NPV of cabbage looper (Trichoplasia  ni);
the NPV of the beet army worm (Spodop-
tera exigua) and NPVs isolated from saw-
flies for forest protection in the USA and
Canada.  T. ni was introduced to  Colum-
bia, South America from California and
has been  used with great success in
recent years.
  Four NPV viral  insecticides have been
registered in the  USA, and are commer-
cially available for field application. The
first, "Elcar", containing the NPV of  the
boll worm (Heliothis zea) is registered by
the pharmaceutical firm Sandoz,  Inc., for
the  control  of  cotton bollworm. The
second available product named "TM
Bioctrol 1" is registered by the US Forest
Service,  and  contains the NPV of  the
Douglas  fir tussock  moth  (Orgyia pseu-
dotsugata). The NPV of the Gypsy Moth
(Porthetria dispar) and NPV of pine sawf ly
(Neodiprion sertifer)  are two other NPVs
which have been registered.
  With  the hazardous environmental
deterioration by chemical pesticides, and
with urgent need for promoting the world
food production in mind, the use of viral
pesticides might be conceptually a practi-
cal and  useful approach. But before any
great revolutionary events happen, a pre-
cise  evaluation of the benefit as well as
the potential environmental health prob-
lem exhibited by this approach should be
made. It is estimated that in the western
hemisphere,  30% of the current  pest
problems in agricultural crop production
can  be treated with viral pesticides. In
California among the pest species group
causing major crop losses, 46% are sus-
ceptible to baculoviruses. Theoretically,
viral pesticides can effectively solve cer-
tain problems such as toxic chemical pol-
lution and inefficiencies of certain chem-
ical pesticides in crop production. As far
as safety and environmental  health is
concerned, relative  amounts of in vivo
and  in vitro tests have been performed.
But most of the tests applied used acute
infectivity, antigenicity, and morphologi-
cal alteration as measuring factors. The
fate  of viral DNA, possibilities of genetic
recombination and viral gene integration,
viral oncogenicity as well as low level of
persistent  infection have  never  been
extensively examined.

Potential Hazards

  There are several important considera-
tions and noteworthy facts to be carefully
examined and evaluated. First, the candi-
date pesticidal virus  may  infect insect
hosts other than the target pest. Second,
insect virus may be  able to induce infec-
tion  in other invertebrate or even verte-
brate via  either permissive or abortive
infection. Third, as  the consequence of
persistent infection or non-fetal infection,
the insects are known to be carriers of a
variety of animal aborviruses. Pesticidal
virus might follow the same pattern, and
introduce itself  into  human  beings or
other vertebrate through its vector host
by an unnatural  route. Fourth,  the so-
called host specificity in virology is neither
a fundamental nor a stable characteristic;
the condition of the host and the nature of
infectious agent (intact virion or naked
DNA) will affect the entire susceptibility
to infection. Although numerous in vitro
and  in vivo experiments have been done
to prove the species specificity and the
safety of pesticide  virus,  the  striking
report of transfection of Fogh-Lund hu-
man amnion cell with the silk worm  NPV-
DNA and the demonstration of viral DNA
and  antigens in vertebrate  cells  have
raised the question of species specific
and real meaning of safety as monitor
solely by  the infectivity and cytopatl
effect.  Furthermore, various cocarcir
gens and  tumor promoting agents, su
as phorbol ester, which probably ex
widely in  nature, might induce an une
pected virus and host interaction whi
might lead to the oncogenic transform
tion of cells infected by pesticidal viruse

Detection and Molecular
Interaction
  In the application of pesticidal viruse
two important issues require immedia
attention.  First of all, it is essential
improve the methodology and sensitiv
in detecting virus and host cell (includii
vertebrate cell and human cell) intera
tion at the molecular level and effects
cocarcinogen on virus and host cell inte
action; the alternative way of virus infe
tion, the fate  of viral DNA, possible vir
gene integration and recombination, vir
oncogenicity  and persistent  infectu
require a molecular biological method
detection  and  observation  other the
infectivity assay. Secondly, the structur
function,  and  genetic  relatedness
baculovirus have to be carefully  studit
and  examined;  a universal  pesticid
virus or a  multifunctional pesticidal viri
may be constructed.
  Other than the  classic  methods
detection  and analysis, there are sever
recent major technical approaches whic
can be applied to insect virus systems ar
will add a great impact to the understani
ing of viral genome  status, gene strui
ture, gene function, and pathogenesi
Such as:

  (a)  Nucleic acid hybridization (inclui
      ing DNA-DNA reassociation kine
      ics  analysis,  in  situ (RNA-DN
      cytohybridization, Southern's bli
      hybridization, etc.

  Detection of viral DNA,  defective c
non-defective, can be achieved by DNX>
DNA reassociation kinetics analysis. Us
ing highly specific radioactive viral DN,
probes,  it has  been possible to  detec
small numbers of copies or portions c
viral genomes in the DNA isolated fror
cells suspected of carrying viral informs
tion. It does not matter whether viral DN;
is replicating or defective, integrated c
plasmid, biologically active or latent. Thi
technique is  able to tell the degree c
homology and relatedness between tw
viruses or two individuals. The degree c
viral gene expression, in regard to trans

-------
criptional mRNA, can also be detected by
this technique.
  As far as localization of viral nucleic
acid and  detection of susceptible cell
types is concerned, the technique of in
s/ft/RNA-DNAcytohybridization will fulfill
the goal.  The great advantage of this
technique is its ability to localize virus-
specific DNA or RNA according to cell
type and intracellular location by  auto-
radiography. In combination with these
nucleic acid hybridization techniques, a
more advanced study of the interaction of
insecticidal  virus with the mammalian
cell, especially  human  cells,  can be
achieved.

    (b)Restriction endonuclease and spe-
       cific DNA fragmentation.

  The  DNA fragmentation by  restriction
endonuclease has become a very power-
ful tool for analyzing not only small viral
genomes but also genomes of increasing
complexity and molecular size. Cleavage
of DNA into specific terminal fragments
and construction of a DNA fragment map
will  provide  elements needed  for the
detailed characterization of viral genome,
and also for the regulation of gene trans-
cription and gene interaction. The restric-
tion  enzyme cleavage  pattern will also
provide a  detailed comparison of strain
variation and strain relatedness.
  In adenovirus system, by DNA frag-
ment transfection and DNA-DNA reasso-
ciation kinetics analysis (using restriction
endonuclease fragments as probe), it was
found that only the extreme left-hand 7%
of the adenovirus type 2 DNA is sufficient
for transformation of rat kidney cell  in
vitro. The EcoR 1 -C fragment, the left 16%
of the viral genome, of adenovirus type 12
DNA has been proved  to carry a trans-
forming gene, and was used as a power-
ful probe for the study of the association
of adenovirus type 2 with various types of
human cancer.
  The  structure and function  of several
viral genomes such as 0X174, SV40,
adenovirus, etc., have been elucidated by
the application of restriction endonucle-
ases. Using DNA fragments generated by
various restriction enzymes and nucleic
acid hybridization techniques, the virus
gene expression and gene regulation in
SV40 and adenovirus-infected permissive
and non-permissive cells have been de-
fined. The utilization of restriction endo-
nuclease and nucleic acid hybridization in
the human cytomegalovirus system has
been very  successfully performed in our
laboratory. We feel that these techniques
can be effectively applied to study gene
interaction and gene expression in pesti-
cidal virus-infected permissive and non-
permissive cells

  (c)  Transfection of viral DNA using
      calcium phosphate and dimethyl-
      sulfoxide(DMSO).

  Viral infection can be initiated in an
alternate route in an in vitro system. By
infection of  cells treated with calcium
phosphate and DMSO, adenovirus DNA
and  herpes simplex DNA have been
proved to be infectious. It is not necessary
to have intact virus particles to initiate the
infection process. Transformation of rat
cells by DNA of  adenovirus type 5  was
also achieved by this method. As men-
tioned above, the specific DNA fragment
carrying the transforming gene has also
been detected  by calcium  phosphate
method. Using this technique to advan-
tage, there  is an urgent need  for the
examination of the biological activity of
pesticidal viral DNA. Mass application of
pesticidal  virus will generate numerous
defective or naked DNA and on some
occasions these particles might become a
potential environmental hazard and dan-
gerous to human health.

  (d)  Gene cloning and  recombinant
      DNA technology.

  Gene cloning  and recombinant DNA
technology has  become  a revolutionary
tool  not only for the study of molecular
biology but also for industrial application.
Numerous genes of biochemical  and
genetic interest have been isolated and
studied due to  the achievements  of
recombinant DNA research. Virus ge-
nomes can be constructed and amplified
in vitro without the natural hosts, and a
wide host range, non-hazardous pesti-
cidal virus might therefore be constructed
with  a  minimum risk  to  health  and
environment.

Summary
  This Summary contains  three  main
elements  which reflect  the work  per-
formed with the support  of a grant from
EPA: the interaction of SfNPV with var-
ious mammalian cells in vitro, the ge-
nomic structure of SfNPV, and cloning of
SfNPV DNA (Hind III fragments) in plas-
mid pBR322. The details are described in
the full report.

-------
     Eng-Shang Huang. Lambert Loh. Yuan-Ming Wu, and Eng-Chun Mar are with
       University of North Carolina, Chapel Hill. NC27514.
     Clinton Kawanishi is the EPA Project Officer (see below).
     The complete report, entitled "Investigation  on the Potential Environmental
       Hazards of Pesticidal Viruses." (Order No. PB 85-242 527/AS; Cost: $11.95,
       subject to change) will be available only from:
             National Technical Information Service
             5285 Port Royal Road
             Springfield, VA 22161
             Telephone: 703-487-4650
     The EPA Project Officer can be contacted at:
             Health Effects Research Laboratory
             U.S. Environmental Protection Agency
             Research Triangle Park, NC27711
United States                        Center for Environmental Research
Environmental Protection               Information
Agency                             Cincinnati OH 45268
Official Business
Penalty for Private Use $300

EPA/600/S1-85/018
                     PS
        CHICAGO

-------