Clinical signs of deformed wing virus infection are predictive markers for honey bee colony losses.

The ectoparasitic mite Varroa destructor acting as a virus vector constitutes a central mechanism for losses of managed honey bee, Apis mellifera, colonies. This creates demand for an easy, accurate and cheap diagnostic tool to estimate the impact of viruliferous mites in the field. Here we evaluated whether the clinical signs of the ubiquitous and mite-transmitted deformed wing virus (DWV) can be predictive markers of winter losses. In fall and winter 2007/2008, A.m. carnica workers with apparent wing deformities were counted daily in traps installed on 29 queenright colonies. The data show that colonies which later died had a significantly higher proportion of workers with wing deformities than did those which survived. There was a significant positive correlation between V. destructor infestation levels and the number of workers displaying DWV clinical signs, further supporting the mite's impact on virus infections at the colony level. A logistic regression model suggests that colony size, the number of workers with wing deformities and V. destructor infestation levels constitute predictive markers for winter colony losses in this order of importance and ease of evaluation.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

Alphaviral cytotoxicity and its implication in vector development

A great variety of viruses have been engineered to serve as expression vectors. Among them, the alphaviruses Semliki Forest virus and Sindbis virus represent promising tools for heterologous gene expression in a wide variety of host cells. Several applications have already been described in neurobiological studies, in gene therapy, for vaccine development and in cancer therapy. Both viruses trigger stress pathways in the cells they infect, sometimes culminating in the death of the host. This inherent property is either an advantage or a drawback, depending on the type of application. This review covers the development and applications of alphavirus vectors and, as our work has been mainly with Semliki Forest virus, we have focused on this virus with special emphasis on how the understanding of Semliki Forest virus cytotoxicity enables it to be manipulated and used.

Development of a novel marker vaccine platform for protection against Bluetongue Virus (BTV)

Bluetongue virus (BTV) is an economically important member of the genus Orbivirus and closely related to African horse sickness virus (AHSV) and Epizootic hemorrhagic disease virus (EHDV). Currently, 26 different serotypes of BTV are known. The virus is transmitted by blood-feeding Culicoides midges and causes disease (bluetongue [BT]) in ruminants. In 2006/2007, BTV serotype 8 (BTV-8) caused widespread outbreaks of BT amongst livestock in Europe, which were eventually controlled employing a conventionally inactivated BTV vaccine. However, this vaccine did not allow the discrimination of infected from vaccinated animals (DIVA) by the commonly used VP7 cELISA. RNA replicon vectors based on propagation-incompetent recombinant vesicular stomatitis virus (VSV) represent a novel vaccine platform that combines the efficacy of live attenuated vaccines with the safety of inactivated vaccines. Our goal was to generate an RNA replicon vaccine for BTV-8, which is safe, efficacious, adaptable to emerging orbivirus infections , and compliant with the DIVA principle. The VP2, VP5, VP3 and VP7 genes encoding the BTV-8 capsid proteins, as well as the non-structural proteins NS1 and NS3 were inserted into a VSV vector genome lacking the essential VSV glycoprotein (G) gene. Infectious virus replicon particles (VRP) were produced on a transgenic helper cell line providing the VSV G protein in trans. Expression of antigens in vitro was analysed by immunofluorescence using monoclonal and polyclonal antibodies. In a pilot study, sheep were immunized with two different VRP-based vaccine candidates, one comprising the BTV-8 antigens VP2, VP5, VP3, VP7, NS1, and NS3, the other one containing antigens VP3, VP7, NS1, and NS3. Control animals received VRPs containing an irrelevant antigen. Virus neutralizing antibodies and protection after BTV-8 challenge were evaluated and compared to animals immunized with the conventionally inactivated vaccine. Full protection was induced only when the two antigens VP2 and VP5 were included in the vaccine. To further evaluate if VP2 alone, a combination of VP2 and VP5 or VP5 alone were necessary for complete protection, we performed a second animal trial. Interestingly, VP2 as well as the combination of VP2 and VP5 but not VP5 alone conferred full protection in terms of neutralizing antibodies, and protection from clinical signs and viremia after BTV-8 challenge. These results show that the VSV replicon system represents a safe, efficacious and DIVA-compliant vaccine against BTV as well as a possible platform for protection against other Orbiviruses, such as AHSV and EHDV.

Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses

Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma.

UNLABELLED Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth. FROM THE CLINICAL EDITOR Adenovirus dodecahedron, a nanoparticulate proteinaceous biodegradable virus-like particle was used in this study as a vector for the concomitant delivery of cap structure analog and doxorubicine to hepatocellular carcinoma in a rat model, resulting in significant inhibition of tumor growth.

Genome characterization, prevalence and distribution of a Macula-like virus from Apis mellifera and Varroa destructor

Around 14 distinct virus species-complexes have been detected in honeybees, each with one or more strains or sub-species. Here we present the initial characterization of an entirely new virus species-complex discovered in honeybee (Apis mellifera L.) and varroa mite (Varroa destructor) samples from Europe and the USA. The virus has a naturally poly-adenylated RNA genome of about 6500 nucleotides with a genome organization and sequence similar to the Tymoviridae (Tymovirales; Tymoviridae), a predominantly plant-infecting virus family. Literature and laboratory analyses indicated that the virus had not previously been described. The virus is very common in French apiaries, mirroring the results from an extensive Belgian survey, but could not be detected in equally-extensive Swedish and Norwegian bee disease surveys. The virus appears to be closely linked to varroa, with the highest prevalence found in varroa samples and a clear seasonal distribution peaking in autumn, coinciding with the natural varroa population development. Sub-genomic RNA analyses show that bees are definite hosts, while varroa is a possible host and likely vector. The tentative name of Bee Macula-like virus (BeeMLV) is therefore proposed. A second, distantly related Tymoviridae-like virus was also discovered in varroa transcriptomes, tentatively named Varroa Tymo-like virus (VTLV).