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." 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