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Bruce A Webb

Publications and source records attributed to Bruce A Webb.

16 recordsLinked to original sources
PubMed2006

Divergences in protein activity and cellular localization within the Campoletis sonorensis Ichnovirus Vankyrin family.

Ichnoviruses (IVs) occur in obligate symbiotic associations with endoparasitic ichneumonid wasps. IVs are injected with eggs during parasitization, where viral infection and gene expression alter host physiology to ensure endoparasitoid survival. The seven Campoletis sonorensis IV (CsIV) vankyrin genes encode proteins that possess ankyrin repeat domains resembling the inhibitory domains of NF-kappaB transcription factor inhibitors (IkappaBs). The CsIV vankyrins are divided into two subclasses: those expressed primarily in the host fat body (three genes) and those expressed in host hemocytes (four genes). CsIV vankyrin proteins showed limited antigenic similarity when analyzed by Western blotting. Cellular localization and expression patterns of recombinant vankyrin proteins in High Five and Sf9 insect cells differed within and between the subclasses and in cells exposed to lipopolysaccharide, laminarin, or viral immune challenge. In unstimulated Sf9 cells, five vankyrins were detected in cell nuclei. The remaining two proteins localized predominantly to cytoplasmic granules. Immune stimulation of cells resulted in a nuclear-to-cytoplasmic shift of three vankyrins but did not affect localization of other variants. When expressed from recombinant Autographa californica multiple nucleopolyhedroviruses (AcMNPVs), all vankyrins showed a nuclear localization during early stages of infection with patterns resembling those of immune-challenged cells as the infection progressed. Two fat body vankyrins also produced unique biological effects when expressed from recombinant AcMNPV. Insect cells infected with these viruses exhibited enhanced longevity compared to those infected with viruses expressing other vankyrins. Together, these data suggest that vankyrin proteins in CsIV have divergent physiological functions.

Animals↗
PubMed2006

Effect of Campoletis sonorensis ichnovirus cys-motif proteins on Heliothis virescens larval development.

Polydnaviruses are obligate symbionts of some parasitic hymenopteran wasps responsible for modifying the physiology of their host lepidopteran larvae to benefit the endoparasite. Injection of Campoletis sonorensis ichnovirus (CsIV) into Heliothis virescens larvae alters larval growth, development and immunity but genes responsible for alterations of host physiology are not well described. Recent studies of polydnavirus genomes establish that these genomes encode families of related genes expressed in parasitized larvae. Here we evaluate five members of the CsIV cys-motif gene family for their ability to inhibit growth and development of lepidopteran larvae. To study the function of cys-motif proteins, recombinant proteins were produced from baculovirus expression vectors and injected or fed to H. virescens larvae in diet. rVHv1.1 was identified as the most potent protein tested causing a significant reduction in growth of H. virescens and Spodoptera exigua larvae. H. virescens larvae ingesting this protein also exhibited delayed development, reductions in pupation and increased mortality. Increased mortality was associated with chronic sub-lethal baculovirus infections. Taken together, these data indicate that the cys-motif proteins have pleiotropic effects on lepidopteran physiology affecting both development and immunity.

Amino Acid Motifs↗
PubMed2006

Polydnavirus genes that enhance the baculovirus expression vector system.

The baculovirus expression vector system (BEVS) is a powerful and versatile system for protein expression, which has many advantages. However, a limitation of any lytic viral expression system, including BEVS, is that death and lysis of infected insect cells terminates protein production. This results in interruption of protein production and higher production costs due to the need to set up new infections, maintain uninfected cells, and produce pure viral stocks. Genetic methods to slow or prevent cell death while maintaining high-level, virus-driven protein production could dramatically increase protein yields. Several approaches have been used to improve the BEVS and increase the synthesis of functional proteins. Successful enhancement of the BEVS was obtained when various gene elements were added to the virus, secretion and posttranslational processing were modified, or protein integrity was improved. A gene family from the insect virus Campoletis sonorensis ichnovirus (CsIV) was discovered that delays lysis of baculovirus-infected cells, thereby significantly enhancing recombinant protein production in the BEVS system. By using the CsIV vankyrin gene family, protein production in the vankyrin-enhanced BEVS (VE-BEVS) was increased by a factor of 4- to 15-fold by either coexpressing the vankyrin protein from a dual BEVS or by providing its activity in trans by expressing the vankyrin protein from a stably transformed cell line. In sum, VE-BEVS is an enhancement of the existing BEVS technology that markedly improves protein expression levels while reducing the cost of labor and materials.

Amino Acid Sequence↗
PubMed2006

Potential uses of Cys-motif and other polydnavirus genes in biotechnology.

Exploiting the ability of insect pathogens, parasites, and predators to control natural and damaging insect populations is a cornerstone of biological control. Here we focus on an unusual group of viruses, the polydnaviruses (PDV), which are obligate symbionts of some hymenopteran insect parasitoids. PDVs have a variety of important pathogenic effects on their parasitized hosts. The genes controlling some of these pathogenic effects, such as inhibition of host development, induction of precocious metamorphosis, slowed or reduced feeding, and immune suppression, may have use for biotechnological applications. In this chapter, we consider the physiological functions of both wasp and viral genes with emphasis on the Cys-motif gene family and their potential use for insect pest control.

Animals↗
PubMed2006

Response of immunocompetent and immunosuppressed Spodoptera littoralis larvae to baculovirus infection.

The Mediterranean lepidopteran pest Spodoptera littoralis is highly resistant to infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via the oral route, but highly sensitive to infection with budded virus (BV) via the intrahaemocoelic route. To study the fate of AcMNPV infection in S. littoralis, vHSGFP, an AcMNPV recombinant that expresses the reporter green fluorescent protein gene under the control of the Drosophila heat-shock promoter, and high-resolution fluorescence microscopy were utilized. S. littoralis fourth-instar larvae infected orally with vHSGFP showed melanization and encapsulation of virus-infected tracheoblast cells serving the midgut columnar cells. At 72 h post-infection, the viral foci were removed during the moult clearing the infection. Thus, oral infection was restricted by immune responses to the midgut and midgut-associated tracheal cells. By contrast, injection of BV into the haemocoel resulted in successful infection of tracheoblasts, followed by spread of the virus through the tracheal epidermis to other tissues. However, in contrast to fully permissive infections where tracheoblasts and haemocytes are equally susceptible to infection, a severe limitation to vHSGFP infection of haemocytes was observed. To investigate the resistance of S. littoralis haemocytes to BV infection with AcMNPV, the larval immune system was suppressed with the Chelonus inanitus polydnavirus or a putatively immunosuppressive polydnavirus gene, P-vank-1. Both treatments increased the susceptibility of S. littoralis larvae to AcMNPV. It is concluded that the resistance of S. littoralis to AcMNPV infection involves both humoral and cellular immune responses that act at the gut and haemocyte levels. The results also support the hypothesis that tracheolar cells mediate establishment of systemic baculovirus infections in lepidopteran larvae. The finding that polydnaviruses and their encoded genes synergize baculovirus infection also provides an approach to dissecting the responses of the lepidopteran immune system to viruses by using specific polydnavirus immunosuppressive genes.

Animals↗
PubMed2005

Polydnavirus genomes reflect their dual roles as mutualists and pathogens.

Symbionts often exhibit significant reductions in genome complexity while pathogens often exhibit increased complexity through acquisition and diversification of virulence determinants. A few organisms have evolved complex life cycles in which they interact as symbionts with one host and pathogens with another. How the predicted and opposing influences of symbiosis and pathogenesis affect genome evolution in such instances, however, is unclear. The Polydnaviridae is a family of double-stranded (ds) DNA viruses associated with parasitoid wasps that parasitize other insects. Polydnaviruses (PDVs) only replicate in wasps but infect and cause severe disease in parasitized hosts. This disease is essential for survival of the parasitoid's offspring. Thus, a true mutualism exists between PDVs and wasps as viral transmission depends on parasitoid survival and parasitoid survival depends on viral infection of the wasp's host. To investigate how life cycle and ancestry affect PDVs, we compared the genomes of Campoletis sonorensis ichnovirus (CsIV) and Microplitis demolitor bracovirus (MdBV). CsIV and MdBV have no direct common ancestor, yet their encapsidated genomes share several features including segmentation, diversification of virulence genes into families, and the absence of genes required for replication. In contrast, CsIV and MdBV share few genes expressed in parasitized hosts. We conclude that the similar organizational features of PDV genomes reflect their shared life cycle but that PDVs associated with ichneumonid and braconid wasps have likely evolved different strategies to cause disease in the wasp's host and promote parasitoid survival.

Animals↗
PubMed2005

Ikappabeta-related vankyrin genes in the Campoletis sonorensis ichnovirus: temporal and tissue-specific patterns of expression in parasitized Heliothis virescens lepidopteran hosts.

Polydnaviruses (PDVs) are unusual insect viruses that occur in obligate symbiotic associations with parasitic ichneumonid (ichnoviruses, or IVs) and braconid (bracoviruses, or BVs) wasps. PDVs are injected with eggs, ovarian proteins, and venom during parasitization. Following infection of cells in host tissues, viral genes are expressed and their products function to alter lepidopteran host physiology, enabling endoparasitoid development. Here we describe the Campoletis sonorensis IV viral ankyrin (vankyrin) gene family and its transcription. The seven members of this gene family possess ankyrin repeat domains that resemble the inhibitory domains of the Drosophila melanogaster NF-kappabeta transcription factor inhibitor (Ikappabeta) cactus. vankyrin gene expression is detected within 2 to 4 h postparasitization (p.p.) in Heliothis virescens hosts and reaches peak levels by 3 days p.p. Our data indicate that vankyrin genes from the C. sonorensis IV genome are differentially expressed in the tissues of parasitized hosts and can be divided into two subclasses: those that target the host fat body and those that target host hemocytes. Polyclonal antibodies raised against a fat-body targeting vankyrin detected a 19-kDa protein in crude extracts prepared from the 3 days p.p. fat body. Vankyrin-specific Abs localized to 3-day p.p. fat-body and hemocyte nuclei, suggesting a role for vankyrin proteins in the nuclei of C. sonorensis IV-infected cells. These data are evidence for divergent tissue specificities and targeting of multigene families in IVs. We hypothesize that PDV vankyrin genes may suppress NF-kappabeta activity during immune responses and developmental cascades in parasitized lepidopteran hosts of C. sonorensis.

Amino Acid Sequence↗
PubMed2004

Evaluation of early fetal loss induced by gavage with eastern tent caterpillars in pregnant mares.

OBJECTIVE: To determine whether gavage of pregnant mares (housed without access to pasture) with starved eastern tent caterpillars (ETCs) or their excreta is associated with early fetal loss (EFL), panophthalmitis, or pericarditis. DESIGN: Randomized clinical trial. ANIMALS: 15 mares. PROCEDURE: 15 mares with fetuses from 40 to 80 days of gestation (dGa) were randomly assigned to 1 of 3 groups and received 2.5 g of ETC excreta, 50 g of starved ETCs, or 500 mL of water, respectively, once daily for 10 days. Mares were housed in box stalls, walked twice daily, and not allowed access to pasture for 12 days before or during the 21-day trial. RESULTS: 4 of 5 mares gavaged with starved ETCs (group 2) aborted on trial days 8 (2 mares), 10, and 13. No control mares or mares that received excreta aborted. Differences between the ETC group and other groups were significant. Abortion occurred on 49, 64, 70, and 96 dGa. Allantoic fluids became hyperechoic the day before or the day of fetal death. Alpha streptococci were recovered from 1 fetus and Serratia marcescens from 3 fetuses. Neither panophthalmitis nor pericarditis was seen. The abortifacient component of the ETCs was not elucidated. CONCLUSIONS AND CLINICAL RELEVANCE: These findings suggest that mares with fetuses from 40 to 120 days of gestation should not be exposed to ETCs because they may induce abortion.

Abortion, Veterinary↗
PubMed2004

Quantitative analysis of hemocyte morphological abnormalities associated with Campoletis sonorensis parasitization.

Endoparasitoids of arthropods evoke host cellular immune responses that result in hemocytic encapsulation of the endoparasitoid, unless these responses are disrupted by the parasite. Our interest has focused on mutualistic viruses found in some hymenopteran endoparasitoids that disrupt hemocyte function and prevent encapsulation. Specifically, the Campoletis sonorensis polydnavirus interacts with wasp factors to suppress immunity via expression of intracellular and secreted viral proteins. To study the roles of specific parasitization-associated factors on immunocyte morphology, fluorescence microscopy was used to visualize the actin cytoskeleton in infected and uninfected cells, or after treatment with C. sonorensis ovarian proteins or plasma from infected larvae. The titer and distribution of F- and G-actin were altered in hemocytes from parasitized insects relative to control cells, with plasma from parasitized larvae having an intermediate effect. This suggests that intracellular and secreted factors contribute to suppression of cellular immune responses in C. sonorensis.

Animals↗
PubMed2004

Polydnavirus genes and genomes: emerging gene families and new insights into polydnavirus replication.

Polydnavirus genome sequencing is providing new insights into viral genome organization and viral gene function. Sequence analyses demonstrate that the genomes of these viral mutualists are largely noncoding but maintain genes and gene families that are unrelated to other viral genes. Interestingly, these organizational patterns in polydnavirus genomes are evident in both the bracovirus and ichnovirus genera, even though these two genera are evolutionarily unrelated. The identity and function of some polydnavirus gene families are considered with some functions experimentally supported and others implied by homology relationships with known insect genes. The evidence relative to polydnavirus origins and evolution is considered but remains an area of speculation. However, sequencing of these viral genomes has been informative and provides opportunities for productive investigation of these unusual mutualistic insect viruses.

Animals↗
PubMed2003

Diversifying selection in a parasitoid's symbiotic virus among genes involved in inhibiting host immunity.

During parasitization of their hosts some insect parasitoids deliver resident viruses which encode genes that must be expressed in the host for successful parasitization. Among these viruses the Campoletis sonorensis Ichnovirus has been well studied and encodes a cys-motif gene family implicated in disruption of host immunity and other physiological systems. Members of this gene family encode one or more intercystine-knot structural motifs in which the non-cysteine residues of the motif are variable. We analyzed patterns of synonymous and non-synonymous substitution within the cys-motif to investigate the evolution of this gene family and the likelihood of virus-host gene coevolution. Maximum likelihood techniques suggest positive selection acts on 8 of 51 codons in the aligned cysteine-rich region. Although the detected positive selection was not strong, it likely contributes to the diversification of this gene family. Comparison of selection pressure relative to tertiary structure of the VHv1.1 cys-motif protein suggests that the hypervariable sites are exposed. Furthermore, invariant residues in the motif exhibit a region-specific pattern of codon bias, suggesting there are unusual mechanisms of effecting selection pressure at work in this system, though the mechanism has yet to be studied. The positive selection and duplication of both the gene family and the cys-motif implies either selection is driving the molecular radiation of immune suppressive genes toward novel hosts, or molecular coevolution with host targets.

Animals↗
PubMed2003

Characterization of Campoletis sonorensis ichnovirus unique segment B and excision locus structure.

Polydnaviruses (PDVs) are segmented, symbiotic, double-stranded DNA viruses that are vertically transmitted as proviruses within the genomes of some parasitoid Hymenoptera. The PDV associated with the ichneumonid wasp Campoletis sonorensis (CsIV) consists of 24 non-redundant DNA segments varying in size from approximately 6 to 20 kbp. CsIV segment B, one of the smallest genome segments, was sequenced and the excision sites of the proviral segment were characterized. The segment B sequence was 83.2% non-coding with only two open reading frames (ORFs). Some non-coding sequences have similarities to database sequences and were likely pseudogenic, but most were unrelated to known nucleic acid or predicted protein sequences. One ORF, BHv0.9, encodes a member of the rep gene family and was expressed only in parasitized insects while transcription of the other ORF could not be detected. Previously, a third region of the segment was shown to hybridize to 0.6 and 1.2 kb poly A+ RNAs from female wasps during virus replication (Theilmann and Summers, 1988) but this region did not have an identifiable ORF in the determined sequence. In contrast to CsIV segment W, segment B had little repetitive sequence. The segment B proviral integration locus contains a 59 bp direct imperfect repeat. Further analyses of this integration locus demonstrated that segment B was excised from wasp genomic DNA with flanking sequences at the integration site rejoined after segment excision. The segment B "excision locus" retained one of the two copies of the 59 bp repeat sequence with the other repeat present in the excised segment. The data indicate that Ichnovirus segments have distinctive characteristics possibly reflecting functional co-evolution between the wasp and individual types of polydnavirus segments.

Animals↗
PubMed2003

Antibiosis-type insect resistance in transgenic plants expressing a teratocyte secretory protein (TSP14) gene from a hymenopteran endoparasite (Microplitis croceipes).

We have isolated a teratocyte secretory protein (TSP14) gene product from a hymenopteran endoparasite that disrupts the growth of lepidopteran insect larvae. To evaluate the insecticidal activity of TSP14 for the protection of crops from insect damage, chimeric gene constructs of TSP14 were expressed in transgenic plants. The coding sequence of the TSP14 gene, with and without its native signal peptide, was placed between the modified peanut chlorotic streak virus (PClSV) full-length transcript (FLt) promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. These chimeric genes, expressed in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) were stably inherited in successive plant generations (R0, R1 and R2 progeny) as shown by molecular analysis. A Western blot analysis of plant extracts showed the presence of a polypeptide of the expected size that cross-reacted with TSP14-specific antibodies. Larvae of the tobacco budworm (Heliothis virescens) and tobacco hornworm (Manduca sexta) which were fed with several independent homozygous transgenic plant lines (R2 progeny) exhibited mortality and reduced growth rates compared to those fed with plants transformed by a vector control. Our results demonstrate the potential for introduction of the TSP14 gene into plants in order to achieve protection against lepidopteran pests.

Journal Article↗
PubMed2002

Characterization of Campoletis sonorensis ichnovirus segment I genes as members of the repeat element gene family.

Campoletis sonorensis ichnovirus (CsIV) is a symbiotic virus associated with the endoparasitic wasp C. sonorensis. The virus is injected into the wasp's host, Heliothis virescens, during oviposition. One CsIV gene has been identified as a repeat element (rep) gene and encodes a ubiquitous imperfectly conserved 540 bp sequence. We report the sequencing and mapping of a rep-containing segment, segment I, that hybridizes to a known rep sequence from segment O(1). Analysis of this 8.6 kbp segment identified three ORFs having high similarity to the 540 bp rep sequence. All three rep sequence ORFs were expressed in parasitized H. virescens as well as in C. sonorensis tissues. Two of these rep genes, I 0.9 and I 1.1, have single copies of the 540 bp repeat sequence, while the third rep gene, I 1.2, has two imperfect copies, which are more similar to each other than to sequences on the segment I single-motif genes. Like the CsIV BHv 0.9 rep gene, the segment I rep genes lack introns and a signal peptide, suggesting that they are not secreted. Based on their similarity in nucleotide sequence, predicted amino acid sequence and gene structure, the three segment I repeat-containing genes, I 0.9, I 1.1 and I 1.2, are new members of the rep gene family.

Amino Acid Sequence↗
PubMedDate not supplied

Eastern tent caterpillars (Malacosoma americanum) cause mare reproductive loss syndrome.

A new equine abortigenic disease, mare reproductive loss syndrome (MRLS), was recognized and significantly impacted the Ohio Valley in the springs of 2001 and 2002. MRLS caused approximately 330 million US dollars in losses in 2001. An epidemiological investigation of MRLS associated occurrence of the disease with exposure to eastern tent caterpillars (M. americanum). This work investigates the epidemiological association between M. americanum and MRLS to determine if this association was correlative or causative. A pilot study and simulated exposure to M. americanum and their excreta on pasture grasses. The pilot study advanced exposure of pregnant mares to M. americanum materials and 18 of the 29 mares in the study aborted with symptoms of MRLS before other cases were reported in the region. In, three of seven mares exposed to M. americanum aborted, while mares in control (n=6) and M. americanum frass (n=7) treatments had no losses. In, mares were fed frozen insect larvae in feed buckets mixed with oats. Abortions occurred in three of five mares receiving frozen M. americanum, while mares that were fed autoclaved M. americanum (n=5) or frozen gypsy moth larvae (n=4) had no abortions due to MRLS. In, M. americanum larvae were dissected and fractionated. Statistically significant numbers of abortions occurred only in the positive control group and in association with the M. americanum exoskeleton. All abortions induced by exposure to M. americanum exhibited changes in echogenicity of fetal fluids and bacteriological findings post abortion that were consistent with MRLS. These studies support the hypothesis that ingestion of M. americanum larvae induces the MRLS-type equine abortions, and provide experimental evidence that this lepidopteran larva can cause an abortigenic disease in a vertebrate host.

Abortion, Veterinary↗