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.

Ichneumonid polydnaviruses, referred to as ichnoviruses (IVs), have so far been observed in only two parasitic wasp subfamilies, the Campopleginae and Banchinae. The IVs carried by banchine wasps have received limited attention, and most of what we know about them is based on the characterization of a single virus, the Glypta fumiferanae ichnovirus (GfIV). The latter differs considerably from the more extensively studied campoplegine IVs, both in terms of virion morphology and features of the packaged genome. These differences have in fact raised the question as to whether campoplegine and banchine IVs have a common ancestor. The present chapter provides a brief review of the current state of knowledge on banchine IVs, including results from recent genomic analyses. It also provides suggestions as to how to address, in future research, the question of whether the campoplegine and banchine IVs have a common origin.

The Neodiprion abietis Gammabaculovirus (Baculoviridae: NeabNPV) is virulent, highly contagious, and infects only midgut epithelial cells of balsam fir sawfly larvae, but infections can carry through to adult sawflies in the midgut. Larval infections are characterized by hypertrophy of midgut epithelial cell nuclei, where virogenic stromata develop to produce nucleocapsids that are singly enveloped before occlusion into occlusion bodies. Infected, occlusion body-laden cells slough from the midgut epithelium as a result of a dissolution of the basal lamina. Infected cells undergo lysis, and viral occlusion bodies exit affected larvae in a watery diarrhea to infect other balsam fir sawfly larvae. A budded virus stage was not observed, but nucleocapsid and occlusion body formation resembled the development of occlusion-derived virions and occlusion bodies in lepidopteran alphabaculoviruses.

Public demand for safer, environmentally-benign alternatives to synthetic chemical pesticides and more stringent barriers put in place by regulatory agencies worldwide has led to increased interest in microbial pest control agents (MPCA) based on viruses, bacteria, fungi, protozoa and nematodes as the active ingredient. The MPCA market has recently experienced an increase of 47% between 2004/2007 with sales worth $396 million in 2007/2008 (CPL Consultants, 2010). Despite this increase, the microbial biopesticide market still only represents about 1% of the sales of chemical pesticides (CPL Consultants, 2010; Marrone, 2007). Factors impeding the establishment of strong MPCA markets are complex (Chandler et al., 2011; Marrone, 2007; Ravensberg, 2011) but include the burdensome costs associated with the registration of commercial products that are aimed at relatively small niche markets (Chandler et al., 2011; Ehlers, 2011). The main priority of regulatory agencies is to protect human health and safety and the environment from potential risks associated with the use of pest control products. A common feature of MPCA registration processes around the world is that they grew out of registration processes designed for chemical pesticides with adjustments allowing for the reduced risks of MCPAs (Chandler et al., 2011; Ehlers, 2011). Even though public attitudes to the use of biological control agents has been favourable (63%) a large proportion of the public (46%) has expressed concerns about the consumption of food treated with microbial pesticides (Cuddleford, 2006).

This chapter provides an overview of the limited amount of work that has been conducted on the genomics of banchine ichnoviruses (IVs), all of which is based on the analysis of a single genome, i.e. that of the Glypta fumiferanae ichnovirus. It also provides more recent analyses and preliminary findings from ongoing studies aimed at characterizing GfIV and elucidating the question of whether campoplegine and banchine IVs have a common origin. Ichneumonid polydnaviruses, referred to as IVs, have so far been observed in only two parasitic wasp subfamilies, the Campopleginae and Banchinae. The IVs carried by banchine wasps have received limited attention, and most of what we know about them is based on the characterization of a single virus, the Glypta fumiferanae ichnovirus (GfIV). The latter differs considerably from the more extensively studied campoplegine IVs, both in terms of virion morphology and features of the packaged genome. These differences have in fact raised the question as to whether campoplegine and banchine IVs have a common ancestor.

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 white-marked 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.

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).

AbstractGypsy 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.

The ichnovirus TrIV, transmitted by the endoparasitic wasp Tranosema rostrale to its lepidopteran host during oviposition, replicates asymptomatically in wasp ovaries and causes physiological dysfunctions in parasitized caterpillars. The need to identify ichnoviral genes responsible for disturbances induced in lepidopteran hosts has provided the impetus for the sequencing and annotation of ichnovirus genomes, including that of TrIV. In the latter, 86 putative genes were identified, including 35 that could be assigned to recognized ichnoviral gene families. With the aim of assessing the relative importance of each TrIV gene, as inferred from its level of expression, and evaluating the accuracy of the gene predictions made during genome annotation, the present study builds on an earlier qPCR quantification of transcript abundance of TrIV rep ORFs, in both lepidopteran and wasp hosts, extending it to other gene families as well as to a sample of unassigned ORFs. We show that the majority (91%) of putative ORFs assigned to known gene families are expressed in infected larvae, while this proportion is lower (67%) for a sample taken among the remaining ORFs. Selected members of the TrV and rep gene families are shown to be transcribed in infected larvae at much higher levels than genes from any other TrIV gene family, pointing to their likely involvement in host subjugation. In wasp ovaries, the transcriptional profile is dominated by a rep gene and a member of a newly described gene family encoding secreted proteins displaying a novel cysteine motif, which we identified among previously unassigned ORFs.

1. Manipulative field studies were carried out to evaluate the foliage age preference-performance relationship for an extreme generalist herbivore, the whitemarked tussock moth (Orygia leucostigma Smith) (Lepidoptera: Lymantriidae), within balsam fir [Abies balsamea (L.) Mill].2. Field surveys indicated that early instar caterpillars fed almost exclusively on young (i.e. current-year) foliage, whereas late instars caterpillars fed on both young and mature (i.e. 1- and 2-year-old) foliage.3. Survival of early instar caterpillars was highest in treatments where current-year and/or 1-year old foliage were available, but decreased significantly on older foliage. In contrast, late instar caterpillars had the highest survival when allowed to feed on all age classes of foliage, whereas potential fecundity was highest for late instars that fed on young foliage.4. Overall, caterpillars had 32-65% higher fitness when able to feed on all rather than just one age class of foliage.5. These results support both the 'complementary diet' hypothesis, which states that dietary mixing of different-aged foliage can increase nutrient uptake and/or dilute harmful secondary plant chemicals, and the 'ontogeny' hypothesis, which attributes changes in diet to changes in the nutritional needs and/or tolerance to plant defences of juvenile insects as they develop.

A novel cypovirus, assigned CoCPV, was isolated from natural populations of the western spruce budworm, Choristoneura occidentalis. The complete nucleotide sequences of genomic segments S2–S5 and S7–S10 were determined. Each segment contained a single open reading frame. Conserved motifs 5′ (AGUUU……UUUGUGC) 3′ were found at the ends of each segment. Analysis of S2, which encoded a putative RNA-dependent RNA polymerase protein, confirmed CoCPV belonged to the genus Cypovirus within the family Reoviridae. Further phylogenetic analysis using S10 (the polyhedrin gene) aligned this virus with species type-16, closely related to a cypovirus isolated from C. fumiferana.

During egg-laying, some endoparasitic wasps transmit a polydnavirus to their caterpillar host, causing physiological disturbances that benefit the wasp larva. Members of the two recognized polydnavirus taxa, ichnovirus (IV) and bracovirus (BV), have large, segmented, dsDNA genomes containing virulence genes expanded into families. A recent comparison of IV and BV genomes revealed taxon-specific features, but the IV database consisted primarily of the genome sequence of a single species, the Campoletis sonorensis IV (CsIV). Here we describe analyses of two additional IV genomes, the Hyposoter fugitivus IV (HflV) and the Tranosema rostrale IV (TrIV), which we compare to the sequence previously reported for CsIV. The three IV genomes share several features including a low coding density, a strong A+T bias, similar estimated aggregate genome sizes (similar to 250 kb) and the presence of nested genome segments. In addition, all three TV genomes contain members of six conserved gene families: repeat element, cysteine motif, viral innexin, viral ankyrin, N-family, and a newly defined putative family, the polar-residue-rich proteins. The three genomes, however, differ in their degree of segmentation, in within-family gene frequency and in the presence, in TrIV, of a unique gene family (TrV). These interspecific variations may reflect differences in parasite/host biology, including virus-induced pathologies in the latter. (C) 2007 Published by Elsevier Inc.

Unlike most viruses, the mature ichnovirus particle possesses two unit membrane envelopes. Following loss of the outer membrane in vivo, nucleocapsids are believed to gain entry into the cytosol via a membrane fusion event involving the inner membrane and the plasma membrane of susceptible host cells; accordingly, experimentally induced damage to the outer membrane might be expected to increase infectivity. Here, in an attempt to develop an in vitro model system for studying ichnovirus infection, we show that digitonin-induced disruption of the virion outer membrane not only increases infectivity, but also uncovers an activity not previously associated with any polydnavirus: fusion from without.

Beginning in the early 1990s, the balsam fir sawfly ( Neodiprion abietis ) became a significant defoliating insect of precommercially thinned balsam fir ( Abies balsamea (L.) Mill.) stands in western Newfoundland, Canada. In 1997, a nucleopolyhedrovirus (NeabNPV) was isolated from the balsam fir sawfly and, as no control measures were then available, NeabNPV was developed for the biological control of balsam fir sawfly. In order to register NeabNPV for operational use under the Canadian Pest Control Products Act, research was carried out in a number of areas including NeabNPV field efficacy, non-target organism toxicology, balsam fir sawfly ecology and impact on balsam fir trees, and NeabNPV genome sequencing and analysis. As part of the field efficacy trials, approximately 22 500 hectares of balsam fir sawfly-infested forest were aerially treated with NeabNPV between 2000 and 2005. NeabNPV was found to be safe, efficacious, and economical for the suppression of balsam fir sawfly outbreak populations. Conditional registration for the NeabNPV-based product, Abietiv™, was received from the Pest Management Regulatory Agency (Health Canada) in April 2006. In July 2006, Abietiv was applied by spray airplanes to 15 000 ha of balsam fir sawfly-infested forest in western Newfoundland in an operational control program.

Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.

Many ichneumonid and braconid endoparasitoids inject a polydnavirus (PDV) into their caterpillar hosts during oviposition. The viral entities carried by wasps of these families are referred to as "ichnoviruses" (IVs) and "bracoviruses" (BVs), respectively. All IV genomes characterized to date are found in wasps of the subfamily Campopleginae; consequently, little is known about PDVs found in wasps of the subfamily Banchinae, the only other ichneumonid taxon thus far shown to carry these viruses. Here we report on the genome sequence and virion morphology of a PDV carried by the banchine parasitoid Glypta fumiferanae. With an aggregate genome size of approximately 290 kb and 105 genome segments, this virus displays a degree of genome segmentation far greater than that reported for BVs or IVs. The size range of its genome segments is also lower than those in the latter two groups. As reported for other PDVs, the predicted open reading frames of this virus cluster into gene families, including the protein tyrosine phosphatase (PTP) and viral ankyrin (ank) families, but phylogenetic analysis indicates that ank genes of the G. fumiferanae virus are not embedded within the IV lineage, while its PTPs and those of BVs form distinct clusters. The banchine PDV genome also encodes a novel family of NTPase-like proteins displaying a pox-D5 domain. The unique genomic features of the first banchine virus examined, along with the morphological singularities of its virions (IV-like nucleocapsids, but enveloped in groups like some of the BVs), suggest that they could have an origin distinct from those of IVs and BVs.

The polydnavirus Toxoneuron nigriceps bracovirus (TnBV) is an obligate symbiont associated with the braconid wasp T. nigriceps, a parasitoid of Heliothis virescens larvae. Previously, to identify polydnavirus genes that allow parasitization by altering the host immune and endocrine systems, expression patterns of TnBV genes from parasitized H. virescens larvae were analysed and cDNAs were obtained. To study the function of the protein from one such cDNA, TnBV1, overexpression of the protein was attempted by using the baculovirus Autographa californica multicapsid nucleopolyhedrovirus. Recovery of stable recombinant virus was unsuccessful, with the exception of recombinants with deletions/mutations within the TnBV1 gene. It was hypothesized that TnBV1 expression was cytotoxic to the Spodoptera frugiperda (Sf21) insect cells that were used to produce the recombinants. Therefore, the Bac-to-Bac system was used to create recombinant baculoviruses maintained in Escherichia coli expressing either TnBV1 (Ac-TnBV1) or an initiator-methionine mutant [Ac-TnBV1(ATG-)]. Microscopy revealed substantial cell death of Sf21 and High Five cells from 48 h post-infection with Ac-TnBV1, but not with the Ac-TnBV1(ATG-) recombinant virus. Ac-TnBV1-infected Sf21 cells, but not those with parental virus infection, showed an increased caspase-3-like protease activity, as well as increased terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) for breaks in host genomic DNA. Although indicative of apoptosis, blebbing and apoptotic bodies were not observed in infected cells. Transiently expressing TnBV1 alone caused TUNEL staining in High Five cells. These data suggest that TnBV1 expression alone can induce apoptosis-like programmed cell death in two insect cell lines. Injection of Ac-TnBV1 budded virus, compared with parental virus, did not result in an alteration of virulence in H. virescens larvae.

The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.

将苜蓿银纹夜蛾多核衣壳核型多角体病毒（Autograph Colifornica nuclear polyhedrosis virus,AcMNPV）的野生型株HR3和温度敏感突变株ts317、ts538、ts8感染草地贪夜蛾（Spodoptera frugiperda）Sf21细胞,并在允许温度（25℃）或非允许温度（33℃）下培养,分别采用过氧化物酶标记的抗P47蛋白、抗P143蛋白、抗多体蛋白和抗病毒结构蛋白的单克隆抗体检测病毒增殖过程各蛋白出现的时间。结果表明:1)P47蛋白是一种晚期（12hpi）表达蛋白,各突变株在允许温度（25℃）能够表达,但在非允许温度（33℃）不能表达。2)P143蛋白是一种早期（8hpi）表达蛋白,在允许温度和非允许温度时都能表达,ts8的表达量较少。3)在非允许温度条件下,蛋白质的合成速度高于允许温度。4)野生型和突变株ts317的病毒结构蛋白（P80、GP64、VP39、P24和PTP）在允许温度增殖下都能检测到,ts538和ts8表达量相对少些。5)除了GP64和P24外,ts538和ts8感染的细胞在非允许温度下不能表达病毒的结构蛋白。6)野生型毒株HR3在允许温度和非允许温度下的蛋白表达无明显差异。

Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV) is a baculovirus pathogenic to spruce budworm, the most damaging insect pest in Canadian forestry. CfMNPV is less virulent to its host insect and its replication cycle is slower than the baculovirus type species Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) but the basis of these characteristics is not known. We have now identified, localized, and determined the sequence of the region of CfMNPV carrying potentially important regulatory genes including p47, lef-12, gta, and pkip. DNA database searches revealed that this region of CfMNPV is most closely related to the homologous OpMNPV genes. Transcription analysis demonstrated that CfMNPV P47 is encoded by a 1.6-kb transcript, LEF-12 is encoded by a 2.6-kb transcript, and GTA is encoded by a 2.1-kb transcript. Transcripts for these genes were detectable at 6 h postinfection but all of them showed a burst in expression levels between 12 and 24 h postinfection corresponding to the time of initiation of CfMNPV DNA replication. A polyclonal antibody, raised against CfMNPV P47, detected a nuclear 43-kDa polypeptide from 12 to 72 h postinfection, demonstrating that the CfMNPV p47 gene product is first expressed at a time corresponding to the burst of transcriptional activity between the early and the late phases. Both AcMNPV and CfMNPV P47 translocate to the nucleus of infected cells.