c Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.T he molecular mechanisms of baculovirus DNA replication have been studied in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of the genus Alphabaculovirus of the family Baculoviridae. Baculoviruses are distinctive in that they have a biphasic replication cycle producing budded virions (BVs) early in infection, and later in infection, single or multiple nucleocapsids are enveloped in the ring zone to form occlusion-derived virus (ODV), which becomes embedded within polyhedra (14). AcMNPV replicates its large (134-kb), circular, double-stranded DNA genome (1) in the nucleus of infected cells following a molecular interaction between virus-encoded transacting factors and cis-acting DNA sequences. To date, the functional roles of only some trans-acting factors involved in DNA replication have been elucidated (24,31).The AcMNPV DNA polymerase (DNApol) plays an essential role in AcMNPV DNA replication (27,48). The 3-kb DNApol open reading frame (ORF) encodes a polypeptide of 984 amino acids (aa) with a predicted molecular mass of 114.31 kDa (46). Previous biochemical studies suggested that baculovirus-encoded DNApol is most closely related to ␣-and ␦-like DNApols of various DNA viruses, such as herpes simplex viruses (HSVs), adenoviruses, and poxviruses (18,30). Moreover, the AcMNPV DNApol is sensitive to aphidicolin, a feature common to ␣-like DNApols (18,37,43). The ␦-like (replicative-form) DNApol requires proliferating cell nuclear antigen (PCNA) (a proc...

The complete genome sequences of Choristoneura occidentalis and C. rosaceana nucleopolyhedroviruses (ChocNPV and ChroNPV, respectively) (Baculoviridae: Alphabaculovirus) were determined and compared with each other and with those of other baculoviruses, including the genome of the closely related C. fumiferana NPV (CfMNPV). The ChocNPV genome was 128,446 bp in length (1147 bp smaller than that of CfMNPV), had a G+C content of 50.1%, and contained 148 open reading frames (ORFs). In comparison, the ChroNPV genome was 129,052 bp in length, had a G+C content of 48.6% and contained 149 ORFs. ChocNPV and ChroNPV shared 144 ORFs in common, and had a 77% sequence identity with each other and 96.5% and 77.8% sequence identity, respectively, with CfMNPV. Five homologous regions (hrs), with sequence similarities to those of CfMNPV, were identified in ChocNPV, whereas the ChroNPV genome contained three hrs featuring up to 14 repeats. Both genomes encoded three inhibitors of apoptosis (IAP-1, IAP-2, and IAP-3), as reported for CfMNPV, and the ChocNPV IAP-3 gene represented the most divergent functional region of this genome relative to CfMNPV. Two ORFs were unique to ChocNPV, and four were unique to ChroNPV. ChroNPV ORF chronpv38 is a eukaryotic initiation factor 5 (eIF-5) homolog that has also been identified in the C. occidentalis granulovirus (ChocGV) and is believed to be the product of horizontal gene transfer from the host. Based on levels of sequence identity and phylogenetic analysis, both ChocNPV and ChroNPV fall within group I alphabaculoviruses, where ChocNPV appears to be more closely related to CfMNPV than does ChroNPV. Our analyses suggest that it may be appropriate to consider ChocNPV and CfMNPV as variants of the same virus species.

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The complete genome of the Orgyia leucostigma nucleopolyhedrovirus (OrleNPV) isolated from the whitemarked tussock moth (Orgyia leucostigma, Lymantridae: Lepidoptera) was sequenced, analyzed, and compared to other baculovirus genomes. The size of the OrleNPV genome was 156,179 base pairs (bp) and had a G+C content of 39%. The genome encoded 135 putative open reading frames (ORFs), which occupied 79% of the entire genome sequence. Three inhibitor of apoptosis (ORFs 16, 43 and 63), and five baculovirus repeated ORFs (bro-a through bro-e) were interspersed in the OrleNPV genome. In addition to six direct repeat (drs), a common feature shared among most baculoviruses, OrleNPV genome contained three homologous regions (hrs) that are located in the latter half of the genome. The presence of an F-protein homologue and the results from phylogenetic analyses placed OrleNPV in the genus Alphabaculovirus, group II. Overall, OrleNPV appears to be most closely related to group II alphabaculoviruses Ectropis obliqua (EcobNPV), Apocheima cinerarium (ApciNPV), Euproctis pseudoconspersa (EupsNPV), and Clanis bilineata (ClbiNPV).

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Green water is a technique commonly used in aquaculture, that consists of adding live algae in the water culture and its benefits have been shown for several species. Several hypotheses exist to explain the benefits of green water: increase in nutritional value; action as a probiotic; increase in contrast to reveal preys for larvae; or increase in predator behaviour of larvae. Green water produced with a mix of strains Isochrysis galbana and Chaetoceros muelleri (50:50, cells:cells) applied in four different ways was tested. The survival and the growth of American lobster (Homarus americanus) between stage I and stage IV post-larvae were not affected by the addition of live algae. The lipid classes were not affected by the addition of algae and limited variation was observed in the fatty acids and bacterial profiles. Furthermore, the green water techniques had a limited effect on the behaviour of post-larvae stage IV lobster at releasing. Behaviour was mostly affected by the age of post-larvae. The bacteria Lewinella sp., Leucothrix sp. and Thiothrix sp. appeared to represent a common and core component of Stage IV lobster post-larvae microflora. The results show that the algae do not increase either nutritional value or feed intake of the lobster larvae. Probiotic effect may be more important when larvae are raised in a close system where potential bacterial pathogens could have more chances to colonize the culture. Also, the dark green colour of the larval tank used in this study may have mimicked the effect of green water in the control group. Biochemical results suggest that dietary supplementation with phospholipids and DHA is needed in a lobster hatchery using frozen Artemia and our open formula Dry mix.

Food quality can influence the performance of immature insects and their interactions with pathogens, such as viruses. In manipulative field studies, virus-free caterpillars of the whitemarked tussock moth (WMTM) (Orgyia leucostigma (Smith)) had higher survival, more female-biased sex ratios, and were larger when feeding on white birch (Betula papyrifera Marshall) versus balsam fir (Abies balsamea (Linnaeus) Miller) or red spruce (Picea rubens Sargent). Subsequent laboratory studies with two nucleopolyhedroviruses, derived from WMTMs and Douglas-fir tussock moths, indicated that caterpillars fed high quality food (i.e., artificial diet) prior to infection had less mortality associated with virus infection than those feeding on lower quality foliage (i.e., birch). In field studies, caterpillars fed birch following infection had significantly lower mortality than those feeding on relatively lower quality foliage (i.e., balsam fir). We postulate that higher nutritional quality in artificial diet relative to birch (previrus-ingestion nutrition) and in birch relative to balsam fir foliage (postvirus-ingestion nutrition) has a positive effect on the ability of tussock moth caterpillars to resist or recover from viral infections, although the specific mechanisms responsible for observed resistance remain unclear.

Gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), multicapsid nucleopolyhedrovirus (LdMNPV) has been registered as a microbial pest-control product in the United States (Gypchek®) and Canada (Disparvirus®). Similarly, Douglas-fir tussock moth, Orgyia pseudotsugata (McDunnough) (Lepidoptera: Lymantriidae), multicapsid nucleopolyhedrovirus (OpMNPV) is registered in the United States and Canada as TM BioControl-1® and a product derived from TM BioControl-1 (Virtuss®) is also registered in Canada. To determine changes that may have occurred in these products over time, we compared DNA from Gypchek with Disparvirus and DNA from TM BioControl-1 with Virtuss using restriction fragment length polymorphism (RFLP) analysis. Gypchek and Disparvirus showed the same RFLP banding patterns when viral genomic DNA was digested with BamH I, EcoR V, and Hind III and only a single band difference at approximately 1.6 kilobase (kb) when digested with Bgl II. TM BioControl-1 and Virtuss showed no differences in genomic DNA when digested with Bgl II, Sam I or Hind III. Twelve viral open reading frames (ORFs) were amplified from Gypchek and Disparvirus and nine from TM BioControl-1 and Virtuss by polymerase chain reactions (PCR). The amplified ORFs ranged from highly conserved (polyhedrin) to least conserved (vp91 capsid associated protein). The products were sequenced and the deduced protein products compared. Amino acid sequences deduced from the sequenced PCR products indicated that 8 of the 12 proteins were identical in the two LdMNPV products. The four proteins showing minor sequence variations were DNA polymerase, LEF-8, P74 envelope protein, and VP 91 capsid associated protein. No differences were detected in the protein products deduced from the nine sequenced ORFs from TM BioControl-1 and Virtuss. Comparative RFLP and protein phylogenetic analyses of Gypchek with Disparvirus and TM BioControl-1 with Virtuss revealed little difference between the respective LdMNPV and OpMNPV populations that make up these product pairs.