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-
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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
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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.
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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
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