Development of Novel Delivery Strategies for Use with Genetically

Chapter 16

Development of Novel Delivery Strategies for Use with Genetically Enhanced Baculovirus Pesticides

Downloaded by EAST CAROLINA UNIV on April 6, 2018 | https://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0595.ch016

H . Alan Wood and Patrick R. Hughes Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, N Y 14853

Phytophagous insect pests account for billions of dollars of losses in agricultural and forest production each year. Synthetic chemical insecticides have limited these losses, but are becoming more costly and more restricted in availability. Because of the increasing development and registration costs, the number of new synthetic pesticides brought to market each year has steadily declined over the past 10 years. At the same time, insect populations have developed resistance to many products, resulting in the need to increase application doses or to abandon pesticides. In response to the documented and potential health/environmental risks associated with synthetic chemical pesticides, in 1988 the U.S. Congress mandated that pesticides registered prior to 1984 must undergo a re-registration process. Because of the costs of satisfying current registration requirements, registration of many pesticides for use in small markets has been abandoned. Subsequently, particularly with medium to small market crops, the need for effective insect control is increasing, but the availability of acceptable chemicals is decreasing. The need to develop "safer pesticides" has become a priority of both the current administration and the Environmental Protection Agency (EPA) (1). The current E P A policy is to facilitate the testing and registration of pesticides which have "reduced risks". In addition the Clinton administration has announced plans to propose legislation which will reduce the use of chemical pesticides, increase the availability of alternatives and promote sustainable agriculture. Use of biological control agents, most notably baculoviruses, as alternatives to chemical pesticides is among the most promising approaches to reaching these goals (2, 3). Hundreds of baculoviruses have been described. They all infect invertebrates and about 90% infect lepidopterous insects, many of which are major agricultural pests (4). In many instances, baculoviruses appear to play an integral role in the natural regulation of insect populations. Extensive health safety and environmental testing with baculoviruses has been conducted over the past 25 years (5). This testing has indicated a total lack of environmental and health concerns with the use of baculovirus pesticides. Based on their safety and potential to replace chemical pesticides, five baculoviruses have been registered as pesticides - Helicoverpa zea nuclear polyhedrosis virus (HzSNPV) in 1975, Orgyia pseudotsugata (Op) M N P V in 1976, Lymantria dispar (Ld) M N P V in 1978, Neodiprion sertifer (Ns) SNPV in 1983, and Spodoptera exigua (Se) M N P V in 1993. However, in the U.S. the only privately produced and commercially available viral pesticide is the SeMNPV (Spod-X ) 0097-6156/95/0595-0221$12.00/0 © 1995 American Chemical Society

Hall and Barry; Biorational Pest Control Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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manufactured by Crop Genetics International. The L d M N P V and OpMNPV are only produced for and used by the U.S. and Canadian forest services. Accordingly, very few viral pesticides are currently available as alternatives to chemical pesticides. A major deterrent to the commercial development of viral pesticides has been the relatively slow pesticidal action of the viruses. After infection, it may take from 5 to 15 days to kill the insects, during which time they can continue to cause significant damage. Based on this, baculoviruses as well as many other microbial pesticides generally have been considered to have limited commercial efficacy. Genetic engineering offers a potential solution to this shortcoming (6, 7). A foreign pesticidal gene can be inserted into the viral genome (8), and expression of the pesticidal gene product during replication will allow the virus to kill insects faster or cause quick cessation of feeding (9). Foreign genes which have been inserted into baculoviruses for this purpose include the Buthus eupeus insect toxin-1 (10), the Manduca sexta diuretic hormone (11), the Bacillus thuringiensis ssp. kurstaki HD-73 delta-endotoxin (12, 13), the Heliothis virescens juvenile hormone esterase (14), the Pyemotes tritici TxP-I toxin (15), Androctonus australis neurotoxin (16, 17), Dol m V gene (18) and T-urf 13 (19) genes. The most efficacious gene inserts have been the neurotoxins and the T-urf 13 gene which is responsible for cytoplasmic male sterility of maize. Several major pesticide companies are currently involved in the commercial development of these and other genetically enhanced viral pesticides. They include American Cyanamid, Dupont and F M C . Potential Problems. Although the insertion and expression of foreign genes in baculoviruses can significantly improve their commercial value, the release of recombinant organisms into the environment is cause for concern and caution. Although naturally occurring baculoviruses appear to have no deleterious environmental/health properties, a careful assessment of the properties of recombinant baculovirus pesticides needs to be performed before they can be used as alternatives to synthetic chemical pesticides. Among the environmental issues raised by the field use of a genetically enhanced baculovirus are the precise determinations of virus host range. One of the major benefits of viral pesticides is their apparent host specificity and inability to infect beneficial insects. Although host range expansion could be commercially attractive, precise host range determinations need to be performed with recombinant viruses to ensure that, if host range expansion has occurred, it has not been expanded to include beneficial or desired host species. From an ecological standpoint, there is a concern that release of any recombinant organism may displace other organisms from the ecological niches which they occupy. Accordingly, displacement of natural virus populations, particularly in non-agricultural settings, could result in unanticipated ecological perturbations over large areas. Although commercially attractive, the release of recombinant viral pesticides with properties which give them a selective advantage over naturally occurring viruses (i.e. increased environmental persistence or improved efficiency of infection) should be viewed with caution and carefully evaluated. Another concern is that recombinant viral pesticides have the potential for reassortment and/or gene transfer. Accordingly, it will be important to consider the frequency and possible consequences of a foreign gene transfer from the released virus into naturally occurring virus populations. It should also be appreciated that, even after the most thorough laboratory evaluation of the biological properties and potential environmental interaction of a


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recombinant organism, there may be unanticipated and unwanted properties associated with the organism. Following release of organisms such as baculoviruses, their removal from the environment would be highly problematic. Baculoviruses occluded within polyhedra or granules can survive in the soil for years (20). Accordingly, the construction of recombinant baculoviruses which have a limited survival capacity in nature is very attractive from an ecological standpoint. The genetic enhancement of baculovirus pesticides has been an extension of the baculovirus expression vector technology developed in the mid 80's (21, 22). This technology has been extremely useful for the expression of foreign proteins of basic research and commercial interest.


Baculovirus Expression Vector Technology. Most genetic engineering of baculoviruses has been the construction of baculovirus expression vectors with the Autographa californica nuclear polyhedrosis virus (AcMNPV). The baculovirus expression vector technology is based on the following biological properties. The naturally occurring virus persists in nature as virions occluded within a crystalline viral protein matrix called a polyhedron. A n A c M N P V polyhedron typically measures 0.8 to 2 |im in diameter and contains approximately 50 virions. When an insect larvae ingests a polyhedron, the alkaline nature of the midgut region results in dissolution of the crystal, releasing the virions for infection of the midgut cells. Early in the replication cycle the progeny virus particles bud through the nuclear membrane into the cytoplasm, lose this membrane and then bud through the plasma membrane. The budded virions are responsible for secondary infections, resulting in eventual systemic infection of the larvae. Late in the virus replication cycle, the virus particles become membrane-bound within the nucleus. At the same time, polyhedrin protein is produced in high concentrations and crystallizes around the membrane-bound virus particles within the nucleus. The baculovirus expression vector system was originally based on the fact that the polyhedrin matrix protein is a late, nonessential protein which is required only for the occluded or late phase of replication. The polyhedrin gene is under the control of one of the strongest eukaryotic transcriptional promoters known. At cell death approximately 50% of the total protein may be polyhedrin protein. It is easy to locate the polyhedrin gene, delete the coding region and insert the coding region for a foreign gene which is then under the transcriptional control of the polyhedrin gene promoter. When the polyhedrin gene is replaced with a foreign gene, the early production of budded virus particles proceeds in a normal fashion. Late in the infection cycle, large amounts of the foreign protein are produced instead of polyhedrin matrix protein. Consequently, the progeny virus particles do not become occluded, and, when the larva dies and its tissues disintegrate, the unprotected virus particles are inactivated by the proteolytic enzymes in the decaying larval tissues. Accordingly, there is a stabilization problem associated with a polyhedrinminus, genetically enhanced baculovirus pesticide. One solution to this problem has been to leave the polyhedrin gene intact and to replace the viral p 10 gene with the foreign gene sequences (16, 23). In this way the foreign gene is expressed under the transcriptional promoter of the nonessential plO gene. The development of occluded, recombinant baculovirus pesticides is rather straightforward and can use previous technologies developed for the production, purification and application of naturally occurring viral pesticides.


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However, the environmentally persistent nature of occluded baculoviruses is cause for special consideration when dealing with recombinant viruses. Firstly, if an occluded, recombinant viral pesticide is found to have unwanted environmental properties following release, it might be impossible to eliminate such a virus from the environment. Secondly, a genetically enhanced viral pesticide may compete with natural viruses in the environment. Because of the dearth of information concerning the ecology of baculoviruses, it would not be possible to predict the probability of a recombinant virus displacing a natural virus under field conditions. Therefore it is generally considered that several in depth ecological studies will be needed to evaluate the issues related to the commercial application of occluded, recombinant viral pesticides. Co-occlusion Virus Technology. A second solution to inactivation of nonoccluded virions is the co-occlusion process (24, 25). The basic concept of the co-occlusion technology is that if a host cell is infected simultaneously with a wild-type virus particle (containing a polyhedrin gene) and a recombinant virus (lacking a polyhedrin gene), the polyhedrin protein produced by the wild-type virus will occlude both the wild-type and recombinant virus particles. Through co-infection, one achieves co-occlusion. In laboratory studies Hamblin et al. (25) modeled the persistence of a polyhedrin-minus A c M N P V in a virus population as polyhedra containing polyhedrin-plus and polyhedrin-minus virus particles were passaged from insect to insect. The model predicted that persistence of the polyhedrin-minus virus required that insect larvae ingest polyhedra containing equal amounts of the two types of virions at dosages 100 times the dosage required to infect 100% of the larvae. These conditions could not be maintained in nature, and the model predicted that the polyhedrin-minus virus would be eliminated from the progeny virus polyhedra within a few passages. Based on this laboratory data, in 1989 the model was tested in the first field test of a genetically engineered virus in the United States (26). During the first year of the study, A c M N P V polyhedra containing 48% polyhedrin-minus (no foreign gene insert) and 52% wild-type virus particles were applied three times to cabbage plants artificially infested with Trichoplusia ni larvae, a susceptible host. The large progeny polyhedra population produced in the seeded larvae during year one was found to contain 42% recombinant virus particles. During the second and third years, additional progeny polyhedra populations were monitored and found to contain 9% and 6% recombinant virions, respectively. Accordingly, the laboratory model correctly predicted the loss of the recombinant virus during passage. Based on these results, a second field release was conducted in 1993. The test was designed to compare the dynamics of the co-occlusion technology in a forest ecosystem with the row crop study performed in the cabbage field. The recombinant virus was an isolate of the gypsy moth baculovirus, Lymantria dispar (Ld)MNPV (27). The coding region of the L d M N P V polyhedrin gene was replaced with the bacterial lacZ gene which codes for 6-galactosidase. The polyhedrin-minus, 8-galplus virus was occluded within polyhedra following co-infection with the wild-type LdMNPV. The expression of 6-galactosidase by the recombinant virus is being used to track the recombinant virus in time and space. The 1993 release of the cooccluded, recombinant and wild-type L d M N P V established a virus population in a forest plot which will be monitored for the next two years to determine the spread and persistence of the recombinant virus. The co-occlusion technology clearly allows for the release of a recombinant viral pesticide which will not compete with natural virus populations. As it cycles



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from insect to insect, it will be lost from the progeny polyhedra. The 1989 field test did illustrate however a potential problem with the co-occlusion process. A large amount of the progeny virus population produced during year one remained infectious in the soil throughout the study (Wood et al. in press). Over 1,600 biologically active polyhedra per gram dry weight of soil were found in years two and three. With continued applications of co-occluded virus preparations, the concentration of polyhedra containing recombinant virus particles would continue to increase and would persist in the soil with unknown consequences (20, 28, 29). It should also be appreciated that with the co-occlusion technology typically only half of the virus applied in the field would have the genetically enhanced genotype. In addition, although the co-occlusion technology was easily applied to the A c M N P V system, production of co-occluded virus preparations with other hostrvirus systems can be problematic (Wood, unpublished data). Pre-occluded Virus Technology. In appreciation of the stability of occluded, recombinant viruses in the soil, a suicide strategy was developed to eliminate environmental persistence. The suicide strategy is based on the pre-occluded virus technology (30). Following the removal of the polyhedrin gene (with or without insertion of a foreign gene), the early events of budded virus production proceed in a normal fashion. Late in the replication cycle, progeny virus particles, which are normally destined to become occluded, still become membrane bound and accumulate in high concentrations in the nucleus. These virions are called pre-occluded virus particles. Since occluded virions were known to be highly infectious when released and fed to larvae per os (orally) (31), it was reasoned that the pre-occluded virus particles might also be highly infectious per os. Following infections of insect cell cultures with a polyhedrin-minus isolate of A c M N P V , it was shown that infected nuclei contained high concentrations of preoccluded virus particles. Using neonate droplet feeding bioassays (32), it was found that the pre-occluded virions were highly infectious per os. In addition, it was shown that tissue culture samples infected with a polyhedrin-minus virus (producing only pre-occluded virions) always contained higher levels of infectivity than samples infected with wild-type virus (containing polyhedra and pre-occluded virions). It is considered that the reduced metabolic requirements, when large amounts of polyhedrin are not produced, may allow for the production of more virus particles. It was subsequently discovered that infectious pre-occluded virus preparations could be produced in insect larvae. Following death of infected larvae, the occlusion process is required to protect the virions from inactivation. In order to avoid this inactivation process, infected larvae were freeze dried immediately prior to death and release of the proteolytic enzymes. Not only was the infectivity of the preoccluded virions preserved by freeze drying, but the larval tissues also contained higher levels of infectivity per weight of tissue than are produced with wild-type virus infections. Accordingly, the pre-occluded virus technology can be applied to the in vitro and in vivo production of genetically enhanced viral pesticides. In 1993 the pre-occluded virus technology was field tested by AgriVirion Inc. (unpublished data). The test compared the field efficacy of pre-occluded virus preparations with polyhedra samples produced in equivalent numbers of T. ni larvae. The pre-occluded virus samples and polyhedra were sprayed onto cabbage plants infested with T. ni larvae. As expected, the pre-occluded virus samples were highly infectious under field conditions. When compared to comparable samples of polyhedra, the pre-occluded samples infected as many or slightly more larvae than the polyhedra samples. Soil samples were taken one month after death of the larvae



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and bioassayed (Wood et al. in press). The soil from the polyhedra plots contained infectious virus, but, as expected, soil samples from the pre-occluded virus plots were not infectious. Accordingly, the pre-occluded virus technology presents a delivery strategy for recombinant viruses with zero environmental persistence. Following infection and death of a target pest, the pre-occluded virus particles are completely inactivated. Following death of larvae injected with budded virions of a polyhedrin-minus recombinant, Bishop et al. (33) also observed that the virions were quickly inactivated. Besides possessing this highly desirable environmental property, the production cost (in vivo or in vitro) per unit activity with the pre-occluded virus technology is approximately half that of production of polyhedrin-plus (wild-type) viral pesticides. The pre-occluded virus technology has been used with the A c M N P V and L d M N P V systems, and, unlike the co-occlusion technology, should be readily applicable to all occluded baculovirus systems. Conclusions. Although baculoviruses have been proposed as alternatives to synthetic chemical pesticides for over 20 years, these and other biological control agents have not been able to compete with chemical pesticides based on the cost/benefit factors. During the past decade there has been an increase in costs and reduction in benefits associated with the development, commercialization, and use of synthetic pesticides. This has lead to the successful development and marketing of numerous Bt toxin products, the success and public acceptance of which have pointed the way for the development of other biological control agents such as baculoviruses. With the advent of biotechnology, the commercial efficacy of viral pesticides can now be improved significantly. Also, technologies have been developed recently that will allow for significant reductions in production costs (Hughes, unpublished). Concurrent with these advances, public perceptions and government regulatory policies about pesticides clearly are favoring the development of genetically enhanced baculoviruses and other microbial pesticides. Together, it is anticipated that these advances and conditions will foster further investment and development of technologies leading to additional improvements both in formulation and application technologies required for successful commercialization of viral pesticides and other biological control agents. These changes are bringing the widespread application of viruses for insect control closer to realization. With this increased interest and likelihood of success has come increased concern and attention particularly with respect to possible ecological problems associated with recombinant viruses. To address this concern, two delivery strategies, co-occlusion and pre-occluded virus, have been developed to limit or eliminate persistence of the modified viruses in the environment. These approaches reduce the possibility of unwanted interactions and obviate the question of how to remove the viruses from the environment should they have some unforeseen deleterious property. The current academic and commercial interest in viral pesticides will undoubtedly lead to the discovery of several new such technologies for the development and use of viral pesticides. Based on current commercial interest, it is anticipated that in the next five years several new baculovirus pesticide products will be commercially available for forest and agricultural pest management. With changing public attitudes accompanied by more science-based regulatory policies, the commercial development of genetically enhanced viral pesticides will follow in the near future.


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Acknowledgments Preparation of this manuscript was partially supported by National Science Foundation Grant BCS-9208905 and U.S. Forest Service Fed. Agr. No. 23-570. References 1.


2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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25. Hamblin, M.; vanBeek, N.A.M.; Hughes, P.R; Wood, H.A. Applied and Environmental Microbiology, 1990, 56, pp. 3057-3062. 26. Wood, H.A.; Hughes, P.R.; Shelton, A. Journal of Environmental Entomology, in press. 27. Yu, Z.; Podgwaite, J.D.; Wood, H.A. Journal of General Virology, 1992, 73, pp. 1509-1514. 28. Thomas, E.D.; Reichelderfer, C.F.; Heimpel, A.H. Journal of Invertebrate Pathology, 1972, 20, pp. 157-164. 29. Jaques, R. P. Baculoviruses for Insect Pest Control: Safety Considerations; Am. Soc. Microbiol.: Washington, DC. 1975, pp. 90-99. 30. Wood, H.A.; Trotter, K.M.; Davis, T.R.; Hughes, P.R. Journal of Invertebrate Patholology, 1993, 62, pp. 64-67. 31. van Beek, N.A.M.; Wood, H.A.; Hughes, P.R. J. Invertebr. Pathol., 1988, 51, p. 58. 32. Hughes, P.R.; van Beek, N.A.M.; Wood, H.A. Journal of Invertebrate Patholology, 1986, 48, pp. 187-193. 33. Bishop, D.H.L.; Entwistle, P.F.; Cameron, I.R.; Allen, C.J.; Possee, R.D. The Release of Genetically-engineered Micro-organisms; Academic Press: New York, 1988, pp. 143-179. RECEIVED

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Development of Novel Delivery Strategies for Use with Genetically

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