Don Stoltz

Molecular diagnosis of Kashmir bee virus infection

SUMMARYMolecular protocols were applied to the diagnosis of Kashmir bee virus (KBV) infection in the honey bee, Apis mellifera. Procedures based on either serology (chemiluminescent Western blotting) or nucleic acid amplification (RT-PCR, reverse transcription-polymerase chain reaction) proved to be very effective, although it was possible in some circumstances to detect viral infection simply by Coomassie blue staining of SDS-PAGE gels. Under laboratory conditions, KBV infections were readily distinguished from those induced by acute paralysis virus, a closely related isolate, by using monospecific antisera raised against the putative picornavirus-like VP4 polypeptide. Since only a very small amount of material was required for either Western blotting or RT-PCR, a single bee could in theory be examined for the presence of a variety of different pathogens. PCR primers based on sequences from the viral RNA polymerase gene amplified a 417 bp amplicon from KBV-infected, but not healthy, pupae. The same prime...

Genomic and Morphological Features of a Banchine Polydnavirus: Comparison with Bracoviruses and Ichnoviruses

ABSTRACT 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 ∼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.

Effect of a virus associated with the reproductive system of the parasitoid wasp, Campoletis sonorensis, on host weight gain☆

Abstract The particulate fraction of the calyx fluid of the endoparasitoid, Campoletis sonorensis, reduces host weight gain when manually injected into healthy Heliothis virescens larvae. Reduced weight gain of the host, H. virescens, is normally associated with parasitism by C. sonorensis. Electron microscopy has confirmed that the particulate fraction of the calyx fluid is composed of virus particles and it appears that this virus, injected with the egg at oviposition, actually reduces host weight gain. The effect of the virus is negated when the calyx fluid is exposed to ultraviolet light prior to injection. Furthermore, the calyx fluid is effective only if injected into hosts; there is no effect on host weight gain when hosts are fed or topically treated with the virus-containing calyx fluid.

Ichnovirus Infection of an Established Gypsy Moth Cell Line

In the present study, a lepidopteran cell line (Ld-652Y, from the gypsy moth, Lymantria dispar) exposed to Hyposoter fugitivus polydnavirus (HfPV) was found to display a variety of cytopathic effects. These included a transient inhibition of cell proliferation, rounding up, aggregation and apoptosis. In addition, unusual paracrystalline structures appeared within the lumen of the rough endoplasmic reticulum; similar structures were observed in the spherulocytes of parasitized Malacosoma disstria. Following Coomassie Blue staining, two new cell-associated polypeptides were detected; one of these, an 8 kDa polypeptide, could also be observed following exposure of LD-652Y cells to media taken from infected cultures or to cell-free haemolymph from parasitized M. disstria. After a period of 2-4 weeks, the L. dispar cell cultures were observed to largely recover from the effects of exposure to virus, and resumed proliferation; transformed cell populations tended to form aggregates, and adhered less tightly to the substrate. Viral DNA was stably maintained in all recovered cell lines, possibly in chromosomally integrated form.

A viral aphrodisiac in the cricket Gryllus texensis

We identified the insect iridovirus IIV-6/CrIV as a pathogen of the cricket Gryllus texensis using electron microscopy (EM) and polymerase chain reaction (PCR) analysis. EM showed that the virus attacks the fat body, an organ important for protein production, immune function and lipid storage. During infection the fat body hypertrophied, but egg production withered, leaving the lateral oviducts empty of eggs; the females were effectively sterile. EM of the testis of infected males suggests that the testis was not invaded by the virus, although sperm taken from the spermatophores of infected males showed little or no motility. Nevertheless, males and females continued to mate when infected. In fact, infected males were quicker to court females than uninfected controls. The virus benefits from the continued sexual behaviour of its host; transmission studies show that the virus can be spread through sexual contact. Sickness behaviour, the adaptive reduction of feeding and sexual behaviour that is induced by an activated immune system, was absent in infected crickets. Total haemolymph protein was reduced, as was phenoloxidase activity, suggesting a reduction in immune protein production by the fat body. The evidence suggests that during IIV-6/CrIV infection, the immune signal(s) that induces sickness behaviour is absent. Curtailment of a hosts sickness behaviour may be necessary for any pathogen that is spread by host sexual behaviour.

Genomic and Proteomic Analyses Indicate that Banchine and Campoplegine Polydnaviruses Have Similar, if Not Identical, Viral Ancestors

ABSTRACT Polydnaviruses form a group of unconventional double-stranded DNA (dsDNA) viruses transmitted by endoparasitic wasps during egg laying into caterpillar hosts, where viral gene expression is essential to immature wasp survival. A copy of the viral genome is present in wasp chromosomes, thus ensuring vertical transmission. Polydnaviruses comprise two taxa, Bracovirus and Ichnovirus, shown to have distinct viral ancestors whose genomes were “captured” by ancestral wasps. While evidence indicates that bracoviruses derive from a nudivirus ancestor, the identity of the ichnovirus progenitor remains unknown. In addition, ichnoviruses are found in two ichneumonid wasp subfamilies, Campopleginae and Banchinae, where they constitute morphologically and genomically different virus types. To address the question of whether these two ichnovirus subgroups have distinct ancestors, we used genomic, proteomic, and transcriptomic analyses to characterize particle proteins of the banchine Glypta fumiferanae ichnovirus and the genes encoding them. Several proteins were found to be homologous to those identified earlier for campoplegine ichnoviruses while the corresponding genes were located in clusters of the wasp genome similar to those observed previously in a campoplegine wasp. However, for the first time in a polydnavirus system, these clusters also revealed sequences encoding enzymes presumed to form the replicative machinery of the progenitor virus and observed to be overexpressed in the virogenic tissue. Homology searches pointed to nucleocytoplasmic large DNA viruses as the likely source of these genes. These data, along with an analysis of the chromosomal form of five viral genome segments, provide clear evidence for the relatedness of the banchine and campoplegine ichnovirus ancestors. IMPORTANCE Recent work indicates that the two recognized polydnavirus taxa, Bracovirus and Ichnovirus, are derived from distinct viruses whose genomes integrated into the genomes of ancestral wasps. However, the identity of the ichnovirus ancestor is unknown, and questions remain regarding the possibility that the two described ichnovirus subgroups, banchine and campoplegine ichnoviruses, have distinct origins. Our study provides unequivocal evidence that these two ichnovirus types are derived from related viral progenitors. This suggests that morphological and genomic differences observed between the ichnovirus lineages, including features unique to banchine ichnovirus genome segments, result from evolutionary divergence either before or after their endogenization. Strikingly, analysis of selected wasp genomic regions revealed genes presumed to be part of the replicative machinery of the progenitor virus, shedding new light on the likely identity of this virus. Finally, these genes could well play a role in ichnovirus replication as they were overexpressed in the virogenic tissue.

Ultrastructural and genomic characterization of a second banchine polydnavirus confirms the existence of shared features within this ichnovirus lineage.

Polydnaviruses (PDVs) are symbiotic viruses carried by endoparasitic wasps and transmitted to caterpillar hosts during parasitization. Although they share several features, including a segmented dsDNA genome, a unique life cycle where replication is restricted to the wasp host, and immunodepressive/developmental effects on the caterpillar host, PDVs carried by ichneumonid and braconid wasps (referred to as ichnoviruses and bracoviruses, respectively) have different evolutionary origins. In addition, ichnoviruses (IVs) form two distinct lineages, with viral entities found in wasps belonging to the subfamilies Campopleginae and Banchinae displaying strikingly different virion morphologies and genomic features. However, the current description for banchine IVs is based on the characterization of a single species, namely that of the Glypta fumiferanae IV (GfIV). Here we provide an ultrastructural and genomic analysis of a second banchine IV isolated from the wasp Apophua simplicipes, and we show that this virus shares many features with GfIV, including a multi-nucleocapsid virion, an aggregate genome size of ~300 kb, genome segments <5 kb, an impressively high degree of genome segmentation and a very similar gene content (same gene families in both viruses). Altogether, the data presented here confirm the existence of shared characteristics within this banchine IV lineage.

Genomics of Banchine Ichnoviruses: Insights into their Relationship to Bracoviruses and Campoplegine Ichnoviruses

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 Origins and Early History of Polydnavirus Research

Publisher Summary This chapter traces the origins and early history of polydnavirus (PDV) research, linking that to recent progress in the field. It identifies some of the important milestones in PDV research, nonetheless recognizing the inherently subjective nature of any such enterprise. It makes no attempt to provide a comprehensive coverage of events and does not deal with literature describing the effects of PDVs (and/or wasp venoms) on host physiology. PDVs were incapable of replicating in the parasitized host and of course this made sense: a replicative virus might also be a pathogenic virus, with potentially dire consequence for the developing endoparasitoid. The early work from the Summers lab described virus-specific transcriptional activity in various host tissues, it was clear that this was occurring in the apparent absence of viral DNA synthesis; accordingly, the assumption made was that viral replication was necessarily abortive, for the reason already given.

Novel High-throughput Approach for Purification of Infectious Virions

Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700u2009kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible.