The multiple nucleopolyhedrovirus (AcMNPV) gene encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. In a previous analysis, the gene was found to be under positive selection pressure, suggesting that it may be a determinant of virus host range. To assess the role of ODV-E56 in oral infectivity and host range, we constructed recombinant AcMNPV clones (Ac69GFP-e56lacZ and AcIEGFP-e56lacZ) in which ODV-E56 protein synthesis was eliminated by inserting a -galactosidase () expression cassette into the open reading frame. We also constructed a recombinant virus, Ac69GFP-Roe56, in which the native AcMNPV coding sequence was replaced with that of multiple nucleopolyhedrovirus (RoMNPV), a closely related virus that is significantly more virulent towards some host species than AcMNPV. The recombinant viruses exhibited no alterations in polyhedron production and morphogenesis or in the production of infectious budded virus in cell culture. In bioassays using three lepidopteran host species, the oral infectivities of the mutant viruses Ac69GFP-e56lacZ and AcIEGFP-e56lacZ were profoundly impaired compared with those of wild-type and control recombinant viruses. Oral infectivity was restored fully by marker rescue of the mutant viruses with either the AcMNPV or the RoMNPV gene. In bioassays using two host species that are more susceptible to RoMNPV than to AcMNPV, Ac69GFP-Roe56 killed larvae with LC values similar to those of recombinant viruses expressing AcMNPV ODV-E56. This result indicated that replacement of the AcMNPV gene with the RoMNPV orthologue did not increase virulence against these two species.


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  1. Aguileta, G., Refregier, G., Yockteng, R., Fournier, E. & Giraud, T.(2009). Rapidly evolving genes in pathogens: methods for detecting positive selection and examples among fungi, bacteria, viruses and protists. Infect Genet Evol 9, 656–670.[CrossRef] [Google Scholar]
  2. Banke, S., Lillemark, M. R., Gerstoft, J., Obel, N. & Jorgensen, L. B.(2009). Positive selection pressure introduces secondary mutations at Gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations. J Virol 83, 8916–8924.[CrossRef] [Google Scholar]
  3. Bennett, S. N., Holmes, E. C., Chirivella, M., Rodriguez, D. M., Beltran, M., Vorndam, V., Gubler, D. J. & McMillan, W. O.(2003). Selection-driven evolution of emergent dengue virus. Mol Biol Evol 20, 1650–1658.[CrossRef] [Google Scholar]
  4. Bennett, S. N., Holmes, E. C., Chirivella, M., Rodriguez, D. M., Beltran, M., Vorndam, V., Gubler, D. J. & McMillan, W. O.(2006). Molecular evolution of dengue 2 virus in Puerto Rico: positive selection in the viral envelope accompanies clade reintroduction. J Gen Virol 87, 885–893.[CrossRef] [Google Scholar]
  5. Bonning, B. C.(2005). Baculoviruses: biology, biochemistry, and molecular biology. In Comprehensive Molecular Insect Science, pp. 233–269. Edited by L. Gilbert, K. Iatrou & S. S. Gill. Amsterdam: Elsevier.
  6. Brault, A. C., Huang, C. Y., Langevin, S. A., Kinney, R. M., Bowen, R. A., Ramey, W. N., Panella, N. A., Holmes, E. C., Powers, A. M. & Miller, B. R.(2007). A single positively selected West Nile viral mutation confers increased virogenesis in American crows. Nat Genet 39, 1162–1166.[CrossRef] [Google Scholar]
  7. Braunagel, S. C. & Summers, M. D.(1994).Autographa californica nuclear polyhedrosis virus PDV and ECV viral envelopes and nucleocapsids: structural proteins, antigens, lipid and fatty acid profiles. Virology 202, 315–328.[CrossRef] [Google Scholar]
  8. Braunagel, S. C. & Summers, M. D.(2007). Molecular biology of the baculovirus occlusion-derived virus envelope. Curr Drug Targets 8, 1084–1095.[CrossRef] [Google Scholar]
  9. Braunagel, S. C., Elton, D. M., Ma, H. & Summers, M. D.(1996). Identification and analysis of an Autographa californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus envelope: ODV-E56. Virology 217, 97–110.[CrossRef] [Google Scholar]
  10. Cardenas, F. A., Vail, P. V., Hoffmann, D. F., Tebbets, J. S. & Schreiber, F. E.(1997). Infectivity of celery looper (Lepidoptera: Noctuidae) multiple nucleocapsid polyhedrosis virus to navel orangeworm (Lepidoptera: Pyralidae). Environ Entomol 26, 131–134.[CrossRef] [Google Scholar]
  11. Chen, C. J. & Thiem, S. M.(1997). Differential infectivity of two Autographa californica nucleopolyhedrovirus mutants on three permissive cell lines is the result of lef-7 deletion. Virology 227, 88–95.[CrossRef] [Google Scholar]
  12. Clem, R. J. & Miller, L. K.(1993). Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus. J Virol 67, 3730–3738. [Google Scholar]
  13. Clem, R. J., Fechheimer, M. & Miller, L. K.(1991). Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254, 1388–1390.[CrossRef] [Google Scholar]
  14. Croizier, G., Croizier, L., Argaud, O. & Poudevigne, D.(1994). Extension of Autographa californica nuclear polyhedrosis virus host range by interspecific replacement of a short DNA sequence in the p143 helicase gene. Proc Natl Acad Sci U S A 91, 48–52.[CrossRef] [Google Scholar]
  15. Fang, M., Nie, Y., Harris, S., Erlandson, M. A. & Theilmann, D. A.(2009).Autographa californica multiple nucleopolyhedrovirus core gene ac96 encodes a per os infectivity factor (pif-4). J Virol 83, 12569–12578.[CrossRef] [Google Scholar]
  16. Faulkner, P., Kuzio, J., Williams, G. V. & Wilson, J. A.(1997). Analysis of p74, a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo. J Gen Virol 78, 3091–3100. [Google Scholar]
  17. Harrison, R. L.(2009a). Genomic sequence analysis of the Illinois strain of the Agrotis ipsilon multiple nucleopolyhedrovirus. Virus Genes 38, 155–170.[CrossRef] [Google Scholar]
  18. Harrison, R. L.(2009b). Structural divergence among genomes of closely related baculoviruses and its implications for baculovirus evolution. J Invertebr Pathol 101, 181–186.[CrossRef] [Google Scholar]
  19. Harrison, R. L. & Bonning, B. C.(1999). The nucleopolyhedroviruses of Rachiplusia ou and Anagrapha falcifera are isolates of the same virus. J Gen Virol 80, 2793–2798. [Google Scholar]
  20. Harrison, R. L. & Bonning, B. C.(2000). Use of scorpion neurotoxins to improve the insecticidal activity of Rachiplusia ou multicapsid nucleopolyhedrovirus. Biol Control 17, 191–201.[CrossRef] [Google Scholar]
  21. Harrison, R. L. & Bonning, B. C.(2003). Comparative analysis of the genomes of Rachiplusia ou and Autographa californica multiple nucleopolyhedroviruses. J Gen Virol 84, 1827–1842.[CrossRef] [Google Scholar]
  22. Harrison, R. L. & Bonning, B. C.(2004). Application of maximum-likelihood models to selection pressure analysis of group I nucleopolyhedrovirus genes. J Gen Virol 85, 197–210.[CrossRef] [Google Scholar]
  23. Harrison, R. L. & Summers, M. D.(1995). Mutations in the Autographa californica multinucleocapsid nuclear polyhedrosis virus 25 kDa protein gene result in reduced virion occlusion, altered intranuclear envelopment and enhanced virus production. J Gen Virol 76, 1451–1459.[CrossRef] [Google Scholar]
  24. Holmes, E. C., Woelk, C. H., Kassis, R. & Bourhy, H.(2002). Genetic constraints and the adaptive evolution of rabies virus in nature. Virology 292, 247–257.[CrossRef] [Google Scholar]
  25. Hostetter, D. L. & Puttler, B.(1991). A new broad host spectrum nuclear polyhedrosis virus isolated from the celery looper, Anagrapha falcifera (Kirby), (Lepidoptera: Noctuidae). Environ Entomol 20, 1480–1488.[CrossRef] [Google Scholar]
  26. Hughes, P. R. & Wood, H. A.(1981). A synchronous peroral technique for the bioassay of insect viruses. J Invertebr Pathol 37, 154–159.[CrossRef] [Google Scholar]
  27. Jarvis, D. L., Weinkauf, C. & Guarino, L. A.(1996). Immediate early baculovirus vectors for foreign gene expression in transformed or infected insect cells. Protein Expr Purif 8, 191–203.[CrossRef] [Google Scholar]
  28. Jehle, J. A., Blissard, G. W., Bonning, B. C., Cory, J. S., Herniou, E. A., Rohrmann, G. F., Theilmann, D. A., Thiem, S. M. & Vlak, J. M.(2006). On the classification and nomenclature of baculoviruses: a proposal for revision. Arch Virol 151, 1257–1266.[CrossRef] [Google Scholar]
  29. Kikhno, I., Gutierrez, S., Croizier, L., Croizier, G. & Ferber, M. L.(2002). Characterization of pif, a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus. J Gen Virol 83, 3013–3022. [Google Scholar]
  30. Kitts, P. A. & Possee, R. D.(1993). A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14, 810–817. [Google Scholar]
  31. Kost, T. A., Condreay, J. P. & Jarvis, D. L.(2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567–575.[CrossRef] [Google Scholar]
  32. Kuzio, J., Jaques, R. & Faulkner, P.(1989). Identification of p74, a gene essential for virulence of baculovirus occlusion bodies. Virology 173, 759–763.[CrossRef] [Google Scholar]
  33. Lapointe, R., Popham, H. J., Straschil, U., Goulding, D., O'Reilly, D. R. & Olszewski, J. A.(2004). Characterization of two Autographa californica nucleopolyhedrovirus proteins, Ac145 and Ac150, which affect oral infectivity in a host-dependent manner. J Virol 78, 6439–6448.[CrossRef] [Google Scholar]
  34. Lewis, L. C. & Johnson, T. B.(1982). Efficacy of two nuclear polyhedrosis viruses against Ostrinia nubilalis (Lep.: Pyralidae) in the laboratory and field. Entomophaga 27, 33–38.[CrossRef] [Google Scholar]
  35. Lu, A. & Miller, L. K.(1996). Species-specific effects of the hcf-1 gene on baculovirus virulence. J Virol 70, 5123–5130. [Google Scholar]
  36. Lu, L., Du, Q. & Chejanovsky, N.(2003). Reduced expression of the immediate-early protein IE0 enables efficient replication of Autographa californica multiple nucleopolyhedrovirus in poorly permissive Spodoptera littoralis cells. J Virol 77, 535–545.[CrossRef] [Google Scholar]
  37. Maeda, S., Kamita, S. G. & Kondo, A.(1993). Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV. J Virol 67, 6234–6238. [Google Scholar]
  38. Moscardi, F.(1999). Assessment of the applications of baculoviruses for control of Lepidoptera. Annu Rev Entomol 44, 257–289.[CrossRef] [Google Scholar]
  39. Ohkawa, T., Washburn, J. O., Sitapara, R., Sid, E. & Volkman, L. E.(2005). Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115. J Virol 79, 15258–15264.[CrossRef] [Google Scholar]
  40. O'Reilly, D. R., Miller, L. K. & Luckow, V. A.(1992).Baculovirus Expression Vectors. New York: W. H. Freeman.
  41. Pijlman, G. P., Pruijssers, A. J. & Vlak, J. M.(2003). Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects. J Gen Virol 84, 2041–2049.[CrossRef] [Google Scholar]
  42. Popham, H. J. R., Pellock, B. J., Robson, M., Dierks, P. M. & Miller, L. K.(1998). Characterization of a variant of Autographa californica nuclear polyhedrosis virus with a nonfunctional ORF 603. Biol Control 12, 223–230.[CrossRef] [Google Scholar]
  43. Possee, R. D.(1986). Cell-surface expression of influenza virus haemagglutinin in insect cells using a baculovirus vector. Virus Res 5, 43–59.[CrossRef] [Google Scholar]
  44. Robertson, J. L., Russell, R. M., Preisler, H. K. & Savin, N. E.(2007).Bioassays with Arthropods, 2nd edn. Boca Raton, FL: CRC Press.
  45. Rohrmann, G. F.(2008).Baculovirus Molecular Biology. Bethesda, MD: National Library of Medicine, National Center for Biotechnology Information.
  46. Slack, J. & Arif, B. M.(2007). The baculoviruses occlusion-derived virus: virion structure and function. Adv Virus Res 69, 99–165. [Google Scholar]
  47. Smith, G. E. & Summers, M. D.(1980). Restriction map of Rachiplusia ou and Rachiplusia ou–Autographa californica baculovirus recombinants. J Virol 33, 311–319. [Google Scholar]
  48. Song, J., Wang, R., Deng, F., Wang, H. & Hu, Z.(2008). Functional studies of per os infectivity factors of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. J Gen Virol 89, 2331–2338.[CrossRef] [Google Scholar]
  49. Sparks, W., Li, H. & Bonning, B.(2008). Protocols for oral infection of lepidopteran larvae with baculovirus. J Vis Exp 19, 888 [Google Scholar]
  50. Summers, M. D.(2006). Milestones leading to the genetic engineering of baculoviruses as expression vector systems and viral pesticides. Adv Virus Res 68, 3–73. [Google Scholar]
  51. Summers, M. D. & Smith, G. E.(1987).A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures (Texas Agricultural Experiment Station Bulletin no. 1555).
  52. Theilmann, D. A., Chantler, J. K., Stweart, S., Flipsen, H. T., Vlak, J. M. & Crook, N. E.(1996). Characterization of a highly conserved baculovirus structural protein that is specific for occlusion-derived virions. Virology 218, 148–158.[CrossRef] [Google Scholar]
  53. Twiddy, S. S., Woelk, C. H. & Holmes, E. C.(2002). Phylogenetic evidence for adaptive evolution of dengue viruses in nature. J Gen Virol 83, 1679–1689. [Google Scholar]
  54. Vail, P. V., Hoffmann, D. F., Streett, D. A., Manning, J. S. & Tebbets, J. S.(1993). Infectivity of a nuclear polyhedrosis virus isolated from Anagrapha falcifera (Lepidoptera: Noctuidae) against production and postharvest pests and homologous cell lines. Environ Entomol 22, 1140–1145.[CrossRef] [Google Scholar]
  55. van Beek, N. & Davis, D. C.(2007). Baculovirus insecticide production in insect larvae. Methods Mol Biol 388, 367–378. [Google Scholar]
  56. van Oers, M. M. & Vlak, J. M.(2007). Baculovirus genomics. Curr Drug Targets 8, 1051–1068.[CrossRef] [Google Scholar]
  57. Vaughn, J. L., Goodwin, R. H., Thompkins, G. J. & McCawley, P.(1977). The establishment of two insect cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213–217.[CrossRef] [Google Scholar]
  58. Volkman, L. E. & Summers, M. D.(1977).Autographa californica nuclear polyhedrosis virus: comparative infectivity of the occluded, alkali-liberated, and nonoccluded forms. J Invertebr Pathol 30, 102–103.[CrossRef] [Google Scholar]
  59. Volkman, L. E., Summers, M. D. & Hsieh, C. H.(1976). Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia ni: comparative neutralization comparative infectivity, and in vitro growth studies. J Virol 19, 820–832. [Google Scholar]
  60. Weyer, U., Knight, S. & Possee, R. D.(1990). Analysis of very late gene expression by Autographa californica nuclear polyhedrosis virus and the further development of multiple expression vectors. J Gen Virol 71, 1525–1534.[CrossRef] [Google Scholar]
  61. Wickham, T. J., Davis, T., Granados, R. R., Shuler, M. L. & Wood, H. A.(1992). Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system. Biotechnol Prog 8, 391–396.[CrossRef] [Google Scholar]
  62. Woelk, C. H. & Holmes, E. C.(2001). Variable immune-driven natural selection in the attachment (G) glycoprotein of respiratory syncytial virus (RSV). J Mol Evol 52, 182–192. [Google Scholar]
  63. Woelk, C. H., Jin, L., Holmes, E. C. & Brown, D. W.(2001). Immune and artificial selection in the haemagglutinin (H) glycoprotein of measles virus. J Gen Virol 82, 2463–2474. [Google Scholar]
  64. Yang, Z.(2007). Adaptive molecular evolution. In Handbook of Statistical Genetics, 3rd edn, pp. 377–402. Edited by D. Balding, M. Bishop & C. Cannings. New York: Wiley.
  65. Zlateva, K. T., Lemey, P., Vandamme, A. M. & Van Ranst, M.(2004). Molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment G glycoprotein. J Virol 78, 4675–4683.[CrossRef] [Google Scholar]

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