The complete sequence of the Adoxophyes orana granulovirus genome

AbstractThe nucleotide sequence of the Adoxophyes orana granulovirus (AdorGV) DNA genome was determined and analysed. The genome contains 99,657 bp and has an A + T content of 65.5%. The analysis predicted 119 ORFs of 150 nucleotides or larger that showed minimal overlap. Of these putative genes, 104 (87%) were homologous to genes identified previously in other baculoviruses. The mean overall amino acid identity of AdorGV ORFs was highest with CpGV ORFs at 48%. Sixty-three ORFs were conserved among all lepidopteran baculoviruses and are considered to be common baculoviral genes. Several genes reported to have major roles in baculovirus biology were not found in the AdorGV genome. These included chitinase and cathepsin, which are involved in the liquefaction of the host, which explains why AdorGV-infected insects do not degrade in a typical manner. The AdorGV genome encoded two inhibitor of apoptosis (iap) genes iap-3 and iap-5. Among all of the granuloviruses genomes there was a very high level of gene collinearity. The genes shared by AdorGV and CpGV had exactly the same order along the genome with the exception of one gene, iap-3. The AdorGV genome did not contain typical homologous region (hr) sequences. However, it contained nine repetitive regions in the genome

Identification and functional analysis of the origins of DNA replication in the Cydia pomonella granulovirus genome

The entire genome of Cydia pomonella granulovirus (CpGV) was systematically screened for origins of DNA replication, using an infection-dependent DNA replication assay in the granulovirus-permissive Cydia pomonella cell line, Cp1 4R. All seven cosmids in an overlapping library that covered the CpGV genome were found to replicate in the assay. A genomic library of 32 overlapping plasmids was subsequently screened. Plasmids that replicated were in turn subcloned into 1-2 kbp overlapping fragments. Eleven subclones replicated, each containing at least one of the 13 single-copy 74-76 bp imperfect palindromes, previously identified in the CpGV genome as possible origins of replication. Genome fragments of 156 bp, each containing one of the 13 palindromes, were cloned to verify replication and provided confirmation that these 13 palindromes are the only origins of replication in the genome. A real-time PCR method was developed for the quantification of DNA replication, which eliminated the need for Southern blotting and hybridization. A set of deletion clones allowed further quantitative characterization of one of the palindromes. The previously proposed non-homologous region origin of replication did not replicate in the assay

The origins of replication of granuloviruses

The genomes of eight granuloviruses (GVs), have been analyzed for the presence of homologous regions (hrs) that may act as origins of replication. Thirteen 74-76-bp palindromes within 11 hrs have previously been identified in the Cydia pomonella GV (CpGV) genome and found to replicate in an infection-dependent DNA replication assay. We report a further palindrome within one of the hrs, which was found to replicate, bringing the total to 14 palindromes. We also report imperfect palindromes, with similar 13-bp end sequences to the CpGV palindromes, within the Adoxophyes orana GV, Cryptophlebia leucotreta GV (CrleGV), Choristoneura occidentalis GV and Phthorimaea operculella GV genomes. No hrs were detected in Agrotis segetum GV, and no additional hrs or palindromes, other than those published, were detected in the Plutella xylostella GV and Xestia c-nigrum GV genomes. Several putative hrs from the GVs were tested for replication in C. pomonella cells using a CpGV-dependent replication assay. Two CrleGV hrs were found to replicate at a low level

A bacmid approach to the genetic manipulation of granuloviruses

A Cydia pomonella granulovirus (CpGV) bacmid has been constructed, which allows rapid and efficient production of recombinant baculoviruses in Escherichia coli. An 8.6 kbp bacterial DNA cassette derived from the AcMNPV Bac-to-Bac (R) system was ligated into a unique Pacl restriction site within an intergenic region flanking the DNA ligase gene of the CpGV genome. The CpGV bacmids produced in E. coli were transfected into a CpGV-permissive C. pomonella cell line and the transfected cells fed to larvae to amplify the virus. The enhanced green fluorescent protein (EGFP) gene under the constitutive Drosophila heat-shock promoter was transposed into the mini-attTn7 transposition site, using a modified pFASTBAC (TM) donor plasmid, to generate a recombinant CpGV bacmid which caused infected larvae to glow under UV light. Targeted homologous recombination was also achieved in a recombinant proficient E. coli strain (BJ5183). A chloramphenicol acetyl transferase (CAT) gene replaced the cathepsin (v-cath) gene in the bacmid to produce a v-cath-deletion mutant. This is the first published report of a granulovirus bacmid, which will allow easy manipulation of the CpGV genome, enabling future studies on granulovirus genes and biology. (C) 2008 Elsevier B.V. All rights reserved

The development of endemic baculoviruses of Plutella xylostella (diamondback moth, DBM) for control of DBM in East Africa

Abstract A project to develop non-chemical methods of DBM control on Brassica crops in Kenya has been exploring the use of endemic pathogens as potential control agents. Initial surveys for endemic pathogens identified P. xylostella granulovirus (PlxyGV) on farms in Kenya. Subsequently 14 genetically distinguishable isolates were identified from field collected material. These were purified and ranging bioassays showed these isolates were pathogenic to Kenyan strains of DBM with LC 50s varying from 2.36 x 10 6 to 3.95 x 10 7 occlusion bodies (OB) per ml for II instar DBM. One isolate (Nya-01) was selected and subsequently used for field trials in Kenya. The trials showed that unformulated PlxyGV applied at weekly intervals at a rate of 3.0 x 10 13 OB/ha could control DBM on kale more effectively than available chemical insecticides. After application, infection rates in DBM can reach 90%. Further field trials are currently underway to determine the lowest effective dose rate for this virus when applied as a formulation. Initial virus production studies using in vivo propagation in II instar DBM reared on cabbage showed an initial productivity of 4.0 ± 0.44 x 10 10 OB/larva

Genetic and biological comparisons of four nucleopolyhedrovirus isolates that are infectious to Adoxophyes honmai (Lepidoptera : Tortricidae)

The smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae), is one of the most important pests of tea plants in Japan. Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) isolates from Tsukuba (AdhoNPV-Ts) and Tokyo (AdhoNPV-To), Japan, and Adoxophyes orana nucleopolyhedrovirus (AdorNPV) isolates from England (AdorNPV-En) and the Netherlands (AdorNPV-Ne) were subjected to genetic and biological comparisons to select a candidate NPV isolate to control A. honmai. Restriction endonuclease (REN) analysis demonstrated that AdhoNPV-Ts and AdhoNPV-To had similar REN patterns, whereas AdorNPV-En and AdorNPV-Ne exhibited different REN patterns from each other as well as those of AdhoNPV-Ts and AdhoNPV-To. Bioassays with fourth-instar A. honmai larvae showed that AdorNPV-En was most pathogenic, with the lowest LD50 of 37 occlusion bodies (OBs) per larva. When A. honmai neonates were inoculated with each isolate, most larvae infected with AdhoNPV-Ts and AdhoNPV-To were killed in the final (fifth)-instar, whereas larvae infected with AdorNPV-Ne were killed at every instar and larvae infected with AdorNPV-En were killed at the first- to third-instar. AdorNPV-En or AdhoNPV-Ts fed to neonates had the shortest or longest killing times, respectively, with ST50 values of 6 and 19 days. AdhoNPV-To and AdorNPV-Ne had intermediate killing times. The OB yield per larva of AdhoNPV-Ts and AdhoNPV-To was significantly higher than that of AdorNPV-En and AdorNPV-Ne. Our results suggest that AdorNPV-En is suitable as an inundative agent because it is a quick-killing, highly virulent NPV, and AdhoNPV-Ts and AdhoNPV-To are more appropriately used as inoculative agents because of their high OB production. (C) 2008 Elsevier Inc. All rights reserved