Polydnavirus Innexins Disrupt Host Cellular Encapsulation and Larval Maturation

Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed that >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock-infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to survive relative to controls and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. Our findings support a role for Vinnexins in CsIV and more broadly Ichnovirus pathology in infected lepidopteran hosts, particularly in disrupting multicellular developmental and immune physiology.

Immunogenicity and Antigenicity of Rabbit Hepatitis E Virus-Like Particles Produced by Recombinant Baculoviruses

Rabbit hepatitis E virus (HEV) is a novel HEV belonging to genotype 3 (HEV-3) in the Orthohepevirus A species of the genus Hepevirus, family Hepeviridae. Rabbit HEV was originally isolated from rabbits and found to cause zoonotic infection. Although rabbit HEV can be successfully grown in culture with several cell lines, including the human carcinoma cell line PLC/PRF/5, it is difficult to obtain the large amounts of viral antigen required for diagnosis and vaccine development. In this study, we expressed N-terminal 13 and 111 aa-truncated rabbit HEV ORF2 proteins using recombinant baculoviruses and obtained two types of virus-like particles (VLPs), RnVLPs and RsVLPs with ~35 and 24 nm diameter, respectively. Anti-rabbit HEV IgG antibodies were induced in high titer by immunizing rabbits with RnVLPs or RsVLPs. The antibody secretion in the serum persisted more than three years. RsVLPs showed stronger antigenic cross-reactivity against HEV-1, HEV-3 and HEV-4 than rat HEV. Moreover, anti-RsVLPs antibodies neutralized not only the cognate virus but also HEV-1, HEV-3 and HEV-4 ex vivo, indicating that rabbit HEV had the same serotype as human HEVs. In contrast, the antibody did not block rat HEV infection, demonstrating that rat HEV belonged to a different serotype. Animal experiments indicated that immunization with either RnVLPs or RsVLPs completely protected the rabbits from challenge by rabbit HEV, suggesting that the VLPs are candidates for rabbit HEV vaccine development.

Simple and rapid plaque assay for recombinant baculoviruses expressing influenza hemagglutinin

Recombinant baculoviruses (rBVs) have been extensively used to generate virus-like particles, and baculoviruses expressing antigenic proteins have become efficient tools for inducing protective immunity. However, current methods for generating baculoviruses are costly and inefficient. Thus, the development of a simple, rapid, and accurate method of baculovirus titration is critically important. We established a method of plaque assay using an immunostaining method by which plaques can be easily visualized in Sf9 cells under a light microscope. Sf9 cells were infected with recombinant baculoviruses expressing influenza hemagglutinin surface proteins from H1N1 (A/California/04/09) or rH5N1 (A/Vietnam/1203/04). The infected cells were incubated with anti-HA antibody and the plaques were visualized using the chromogen 3′3-diaminobenzidine (DAB). Plaques were observed from days 1 to 6 post-infection, and differences in Sf9 cell seeding densities resulted in variations in the final plaque quantification. Sf9 cells seeded at a concentration of 5.5 × 104 cells/well or 7.5 × 104 cells/well showed the higher plaque titers at days 3, 4, and 5 post-infection than those found at days 1, 2, and 6 post-infection. With 5.5 × 104 cells/well or 7.5 × 104 cells/well of cell concentrations, recombinant baculovirus for rBV-HA (H1N1) showed 6 × 107 pfu/ml of titer and rBVs for rBV-HA (rH5N1) showed 5.4 × 107 pfu/ml of titer. Three days of baculovirus incubation with a certain concentration of Sf9 cells seeded are required for a rapid, simple, and accurate plaque assay, which could significantly contribute to all baculovirus-related studies.

Ectopic Expression of Virus Innexins in Heliothis virescens Disrupts Hemocyte-Mediated Encapsulation and Host Viability

1.AbstractPolydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to molt relative to controls, and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. These results, in light of previous findings, support that Polydnavirus Vinnexin gene family members may provide complementary, rather than redundant, effects. This in turn indicates a need to test gene family member functionality across infected hosts for effects to determine member contribution to host range.2.ImportancePolydnaviruses are obligate mutualistic associates of highly speciose wasp taxa that parasitize caterpillars. Expression of Polydnavirus-encoded genes in hosts parasitized by wasps is necessary for successful parasitization, and an unusual genome structure including multiple-membered gene families is hypothesized to contribute to host manipulation. We have tested this hypothesis by in vivo expression of two members of a family of Polydnavirus homologues of Innexins, or insect gap junction proteins. Previous findings demonstrated that the two Vinnexins induce different physiological alterations in heterologous systems. Here, in host caterpillars, we observed differential alteration by the two proteins of host immune cell (hemocyte) bioelectrical physiology and the immune response of encapsulation. Not only do our data suggest a linkage between cellular bioelectricity and immunity in insects, but they support that gene family expansion has functional consequences to both Polydnavirus and host wasp success.

Greater Protection against Virulent Infectious Bronchitis Virus Challenge Conferred by Recombinant Baculovirus Co-expressing S1 and N Proteins

Avian infectious bronchitis virus (IBV) is the causative agent of infectious bronchitis, which causes considerable economic losses to the poultry industry worldwide. It is imperative to develop safe and efficient candidate vaccines to control IBV infection. In the current study, recombinant baculoviruses co-expressing S1 and N proteins, mono-expressing S1 or N proteins alone of IBV were constructed and prepared into subunit vaccines rHBM-S1-N, rHBM-S1 and rHBM-N. The levels of immune protection of these subunit vaccines were evaluated by inoculating specific pathogen-free (SPF) chickens at 14 days of age, boosting with the same dose 14 days later, and following challenge with a virulent GX-YL5 strain of IBV 14 days post-booster (dpb). The commercial vaccine strain H120 was used as a control. The IBV-specific antibody levels as well as the percentages of CD4+ and CD8+ T lymphocytes were detected within 28 days post-vaccination (dpv). The morbidity, mortality, and re-isolation of virus from the tracheas and kidneys of challenged birds were evaluated at 5 days post-challenge (dpc). The results showed that the IBV-specific antibody levels and the percentages of CD4+ and CD8+ T lymphocyte in rHBM-S1-N group were higher than those of rHBM-S1 and rHBM-N groups, especially the cellular immunity response. At 5 dpc, the mortality, morbidity and virus re-isolation rate of rHBM-S1-N were slightly higher than those of H120 group, but were lower than those of rHBM-S1 group and rHBM-N group. The present study demonstrated that the protection of recombinant baculovirus co-expressing S1 and N proteins was better than that of recombinant baculoviruses mono-expressing S1 or N protein alone. Thus, the recombinant baculovirus co-expressing S1 and N proteins could serve as a potential IBV vaccine and this demonstrates that the bivalent subunit vaccine including the S1 and N proteins might be a strategy for the development of an IBV subunit vaccine.