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Molecular and biological studies of fast and slow killing granuloviruses

By Sally Louise Wormleaton

Abstract

The aim of this project was to examine the relationship between granuloviruses (GVs)\ud and factors or genes governing host range, speed of kill and tissue tropism.\ud Hybridisations performed between "fast-killing" (fast) and "slow-killing" (slow) GVs\ud showed that fast GVs appeared to be closely related, as did slow GVs. Fast GVs\ud showed low DNA similarity and only partial collinearity to slow GVs. The exception\ud was Adoxophyes orana GV (AoGV), which is a slow GV but showed relatively high\ud DNA similarity and collinearity to fast GVs. This implied that the differences in\ud speed of kill were not necessarily due to the large variations within the genome\ud between fast and slow GVs. Dose and time mortality studies were performed using\ud AoGV and the genome was physically mapped. This would allow easier access to\ud areas of interest on the genome such as regions that did not hybridise to other GVs,\ud that could be unique to AoGV, and would allow the initiation of a sequencing project\ud of the whole genome. The granulin-containing area of the genome was sequenced for\ud comparisons to other GVs. From these data, it is proposed that the relatedness of GVs\ud is dependent on the family of Lepidoptera they infect rather than the tissue tropism of\ud the virus or its speed of kill.\ud An attempt to expand the host range of Cryptophlebia leucotreta GV (C1GV), to\ud include Cydia pomonella, was undertaken by recombination with Cydia pomonella\ud GV (CpGV). In one experiment C1GV replication was rescued in C. pomonella cells.\ud This was either by complementation of the genome by CpGV or recombination with a\ud gene or factor involving host range and will require further studies to identify the\ud gene/factor responsible.\ud Any recombinant GVs need to be assessed thoroughly for possible expansion of host\ud range to non-target insects. Therefore, transfer vectors were constructed for the\ud production of recombinant GVs containing a reporter gene (lacZ). A recombinant\ud CpGV expressing ß-galactosidase was produced and will need to be cloned before\ud further work is performed. The course of CpGV infection in hosts differing in\ud permissivity to CpGV could also be studied using these recombinant viruses.\ud Another part of this research investigated speed of kill. It involved the study of a 2.45\ud kbp region in CpGV-M1, which was not present in the genotype CpGV-R3. CpGVM1\ud is faster killing than CpGV-R3. A recombinant CpGV-R3 that contained this\ud extra region from CpGV-Ml was produced and was compared to CpGV-R3 and\ud CpGV-M1 by bioassay to assess its speed of kill. The speed of kill was not increased\ud indicating that this region was not responsible. However, the study revealed some\ud putative origins of replication of the CpGV genome

Topics: QR
OAI identifier: oai:wrap.warwick.ac.uk:3844

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  1. 1 Artificial diet for codling moth (C. pomonella), false codling moth (C. leucotreta) and summer fruit tortrix moth (A. orana) larvae.
  2. 1 litre Solution A g KCl 14.35
  3. (1995). 18 baculovirus genes, including lef-11, P35,39k and p47, support late gene expression.
  4. 3.5 Fragments of AoGV DNA that hybridised to CpGV-M 1 cosmids. CpGV cosmid AoGV BamHI fragments hybridising AoGV BgIII fragments hybridising AoGV Pstl fragments hybridising AoGV Sad fragments hybridising M73
  5. 3.7 Fragments of XcGV DNA that hybridised to CpGV-M1 cosmids. CpGV cosmid XcGV BamHI ' fragments hybridising XcGV BglI1 fragments hybridising XcGV EcoRI fragments hybridising M73
  6. 3.9 Fragments of C1GV DNA that hybridised to fragments of TnGV DNA. TnGV CIGV BamHI CIGV EcoRI C1GV KpnI CIGV NdeI fragments fragments fragments fragments fragments hybridising hybridising hybridising hybridising PstI A C,
  7. 5.2 BgIII restriction fragments of AoGV-E DNA and their secondary BamHI fragments
  8. 5.3 BamHI restriction fragments of AoGV-E DNA and their secondary PstI fragments. V Enzyme: single digest BamHI 2° Enzyme: Double digest Pst I Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  9. 5.5 BamHI restriction fragments of AoGV-E DNA and their secondary Sacl fragments.
  10. 5.7 BgIII restriction fragments of AoGV-E DNA and their secondary Pstl fragments.
  11. 5.9 BgIII restriction fragments of AoGV-E DNA and their secondary Sacl fragments
  12. 50 ml vitamin stock solution (100X)
  13. 7.2 Re-calculated sizes in bp of single digest fragments of BamHI, BgIH, PstI and SacI calculated from the summation of smaller double digest fragments. Also estimated sizes in bp of EcoRI fragments.
  14. 9.2 Cumulative number of dead larvae, percentage mortality of larvae infected in assay and percentage mortality of larvae that died at each time point (h p.
  15. 9.3 Cumulative number of dead larvae, percentage mortality of larvae infected in assay and percentage mortality of larvae that died at each time point (h p.
  16. 9.4 Cumulative number of dead larvae, percentage mortality of larvae infected in assay and percentage mortality of larvae that died at each time point (h p.
  17. 9.5 Cumulative number of dead larvae, percentage mortality of larvae infected in assay and percentage mortality of larvae that died at each time point (h p.
  18. A 28,096 b,
  19. A 28096 c, s, h,
  20. A 37,562 h, a,
  21. A 46,000 d, f,
  22. A 46,000 h, g, e, d,
  23. A 46,521 a,
  24. A 46,521 g, j, i, a, h,
  25. a baculovirus gene involved in very late gene-expression.
  26. (1991). A baculovirus homolog of a cu/zn superoxide-dismutase gene. Virology 184,149-161. van
  27. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic acids Research
  28. a, h,
  29. (1999). Activation of baculovirus very late promoters by interaction with very late factor 1.
  30. Add agar after cooling to 70 T.
  31. AGCLDKE FJ B IH A ai hik e0cnf1g MI d , 1Ij Ib CA Sad F Pstl A FED CB b ih ge d' c ýf Sad kbp --1 CA
  32. (1992). Alteration of ecdysteroid metabolism due to baculovirus infection of the fall armyworm Spodopterafrugiperda - host ecdysteroids are conjugated with galactose. Insect Biochemistry and Molecular
  33. (1994). Apple enemy number one.
  34. (1999). Assessment of the application of baculoviruses for control of Lepidoptera. Annual Review of Entomology 44,257-289.
  35. (1996). Baculovirus infection of Spodoptera exigua larvae: lacZ expression driven by promoters of early genes pe38 and me53 in larval tissue.
  36. (1997). Baculovirus Structure.
  37. (1983). Biochemical and biological variation of Cydia pomonella (codling moth) granulosis virus. Virology 124,21-34.
  38. (1995). Biometry: The principles and practice of statistics in biological research
  39. C 15,052 j, i, g,
  40. C 23,000 g, c, h -
  41. (1986). Characterization of 3'-5' exonuclease associated with DNA-polymerase of silkworm nuclear polyhedrosis virus.
  42. (1995). Characterization of v-cath, a cathepsin 1-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis-virus.
  43. Cumulative number of dead larvae, percentage mortality of larvae infected in assay and percentage mortality of larvae that died at each time point (h p.
  44. d, i,
  45. (1985). Death of Cydia pomonella larvae and damage to apple fruit, after field application of codling moth granulosis virus. Entomologia Experimentalis et Applicata
  46. (1996). Development of recombinant baculoviruses for insect control. Annual Review of Entomology
  47. (1992). Differential factor binding at the promoter of early baculovirus gene pe38 during viral infection - GATA motif is recognized by an insect protein.
  48. Dilute with Milli-Q water and adjust pH to 6.4 with IN KOH. Make up to 4.5 litres. Filter sterilize (0.2 µm filter).
  49. Dissolve agar in 1200 ml water. Dissolve in a microwave.
  50. dry ingredients in a blender. Add remaining 800 ml water and formaldehyde.
  51. (2000). Enhancement of baculovirus infection in Spodoptera exigua (Lepidoptera : Noctuidae) larvae with Autographa californica nucleopolyhedrovirus or Nicotiana tabacum engineered with a granulovirus enhancin gene. Applied Entomology and Zoology
  52. Estimated sizes in bp of restriction fragments of AoGV-E DNA digested with EcoRI and one other mapped restriction enzyme.
  53. (1997). Evidence for rolling circle replication of Autographa californica M nucleopolyhedrovirus genomic DNA.
  54. (1982). Field evaluation of a granulosis virus for the control of the codling moth. Invertebrate Pathology and Microbial control, 3rd International Colloquium on Invertebrate Pathology,
  55. Foetal bovine serum (FBS) is added to 100-200 ml quantities at 10 % to make up complete medium.
  56. (1995). Genetically engineered insect virus pesticides - present and future.
  57. GF DCE Jig chdf C Sad D A ae A B B b BgIII AGCLDKEFJBIHA qr a hol kf ý? 00 b nie cmdg', ia Pstl kbp t A FED C
  58. (1996). Identification of a baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.
  59. (1981). Improved estimation of DNA fragment length from agarose gels.
  60. (1996). Insect protection against viruses.
  61. (1994). Interaction of Trichoplusia ni granulosis virus-encoded enhancin with the midgut epithelium and peritrophic membrane of 4 lepidopteran insects.
  62. IZD04 MEDIUM STOCK SOLUTIONS Soluble amino acid stock solutions (: 25X) (all Sigma, cell culture grade) g g L-arginine x HCl 17.5
  63. K protein missing from baculovirus few polyhedra (FP) mutants. Virology 168,
  64. kbpl ýýýIII d
  65. (1998). Localization of a baculovirus induced chitinase in the insect cell endoplasmic reticulum.
  66. nuclear-RNA polymerases from the host,
  67. (1993). Phosphate cycling on the basic-protein of Plodia interpunctella granulosis virus.
  68. (1996). Physical mapping and identification of interspersed homologous sequences in the Trichoplusia ni granulosis virus genome.
  69. (1988). Preliminary characterization of toxins from the straw itch mite, Pyemotes tritici, which induce paralysis in the larvae of a moth.
  70. (1968). Preliminary evaluation of a granulosis virus for control of codling moth.
  71. (1971). Probit analysis, 3rd edn. Cambridge, England:
  72. Pstl restriction fragments of AoGV-E DNA and their secondary Bgm fragments V Enzyme: single digest Pstl 2° Enzyme: Double digest BgIII Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  73. (1998). Purification and kinetic analysis of a baculovirus ecdysteroid UDP-glucosyltransferase.
  74. (1996). Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodopterafrugiperda cells that accurately initiates late and very late transcription in vitro.
  75. restriction fragments of AoGV-E DNA and their secondary PstI fragments V Enzyme: single digest SacI 2° Enzyme: Double digest Pstl Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  76. Restriction maps of AoGV-E. The upper case letters denote single digest fragments which are above and below the maps and lower case letters denote the double digest fragments of the two enzymes.
  77. s a ý' r ý .. s iý -`
  78. (1995). Sequence analysis and transcriptional mapping of the orf-2 gene of Autographa californica nuclear polyhedrosis virus.
  79. (1999). Sequence and organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome.
  80. single digest BamHI 2° Enzyme: Double digest BgIII Band Fragment Size(kbp) Fragments Summation of fragment sizes (kb p)
  81. single digest BamHI 2° Enzyme: Double digest Sacl Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  82. single digest BgIII 2° Enzyme: Double digest BamHI Band Fragment Size (kbp) Fragments Summation of fragment sizes (kb p)
  83. single digest BgIII 2° Enzyme: Double digest Pstl Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  84. single digest BgIII 2° Enzyme: Double digest Sacl Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  85. single digest Pst! 2° Enzyme: Double digest Sac! Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  86. single digest Pstl 2° Enzyme: Double digest Bam HI Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  87. single digest Sac! 2° Enzyme: Double digest BamHI Band Fragment Size (bp) Fragments Summation of fragment sizes (bp) -
  88. single digest SacI 2° Enzyme: Double digest BgIII Band Fragment Size (bp) Fragments Summation of fragment sizes (bp)
  89. (1986). Specificity and safety of baculoviruses,
  90. (1991). Structural comparison of the Autographa californica nuclear polyhedrosis virus-induced RNA-polymerase and the
  91. (1995). The CAGT motif functions as an initiator element during early transcription of the baculovirus transregulator ie-1.
  92. (1986). The homologous DNA of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) transactivates viral transcription.
  93. (1997). The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome. Virology
  94. (1963). The use of lead citrate at a high pH as an electron opaque stain in electron microscopy.
  95. (1985). Variation in Cydia pomonella granulosis virus isolates and physical maps of the DNA from 3 variants.
  96. Vitamin stock solution (100X) (Sigma, cell culture grade) mg p-aminobenzoic acid 2 Biotin 0.5 D-Ca-Pantothenate 2
  97. ýa ýý,. ý ý` t
  98. (1998). Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P.

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