Molecular characterization of the Neodiprion abietis nucleopolyhedrovirus
Date
2002
Authors
Young, Aaron Michael
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Abstract
Baculoviruses are a large, diverse family of viruses that are pathogenic for Arthropods, particularly members or the Order Insecta. These viruses are comprised of large enveloped rod-shaped capsids that contain supercoiled, double-stranded DNA genomes. Baculoviruses are of particular interest because of their use as gene expression vectors and insect biological control agents. Most of the information currently available on baculoviruses comes from studies of the Autographa californica nucleopolyhedrovirus and closely related species. Very little information is available on baculoviruses from other insect Orders, including Hymenoptera.
The balsam fir sawfly (Neodiprion abietis, Order:Hymenoptera) is a native insect species that is currently causing severe, annual damage to economically valuable balsam fir forests in eastern Canada. Due to environmental regulations, standard chemical pesticides cannot be used to control this pest and as a result, biological control agents have been proposed as a potential alternative.
A nucleopolyhedrovirus (NPV) has recently been isolated from N abietis and has been shown to be able to control balsam fir sawfly population outbreaks in controlled field experiments, however, before the N abietis nucleopolyhedrovirus (NeabNPV) can be registered for use as a biological control agent, research must be conducted to characterize the virus.
I have partially characterized NeabNPV at the nucleotide and protein sequence levels and have concluded that NeabNPV is highly diverged from other members of the Family Baculoviridae. DNA libraries were constructed for NeabNPV using sheared viral genome fragments and HindIII restriction fragments. An EcoRI restriction library was obtained from the Canadian Forestry Centre, Atlantic Division, and was also included in this study. Analysis of the restriction endonuclease profiles obtained for NeabNPV indicate that NeabNPV has a genome approximately 94.7 kilobase pairs in size.
Nucleotide sequence data obtained from these DNA libraries was used to perform comparative sequence analyses and demonstrated that NeabNPV contains many putative baculovirus gene homologues. From the sequence data, four putative complete open reading frames (ORF) were predicted: odv-e56, vlf-1, p74, and polh.
The putative polyhedrin gene homologue was amplified from the NeabNPV genome and sequenced. Analysis of the polyhedrin gene sequence showed that it was 738 nucleotides long and encoded a protein of a predicted molecular weight of 29 .5 kDa. The promoter region of the gene was determined to contain two copies of the baculovirusĀ specific late transcription motif, TAAG. The NeabNPV polyhedrin gene is highly diverged from Lepidopteran NPVs and GVs (Granulovirus) and shares its highest sequence identity with the polyhedrin gene of the Hymenopteran baculovirus Neodiprion sertifer nucleopolyhedrovirus.
Phylogenetic analyses of several putative NeabNPV gene homologues suggested that NeabNPV is highly diverged from the previously described Group I and Group II NPVs and appears in a unique evolutionary clade separate from both the Lepidopteran NPVs and GVs. Phylogenies which included the mosquito (Order: Diptera) baculovirus Culex nigripalpus nucleopolyhedrovirus (CuniNPV) and that used the GV s as a sister outgroup to the NPV s placed N eabNPV and CuniNPV into a unique evolutionary clade
within the NPV group, separate from the Group I and Group II NPV s.
I have also investigated NeabNPV at the protein level and have putatively identified the major nucleocapsid protein, VP39. Structural proteins from budded (BV) and occlusion-derived virions (ODY) were separated on polyacrylamide gels using SDSĀ PAGE and several well-defined proteins were isolated and partially sequenced. The amino acid sequence data was used to perform comparative sequence analyses against our NeabNPV DNA libraries and determined that the genomic DNA library contained several putative overlapping partial vp39 sequences. These sequences were assembled to create an 864 nucleotide long, partial NeabNPV vp39 sequence. Analysis of the inferred amino acid sequence indicated that the NeabNPV VP39 protein shares the most identity to the GVs, but is most similar to the NPVs. The partial VP39 protein demonstrates a predicted secondary structure and hydrophilic profile that is conserved among the Baculoviridae.