The nonstructural protein NSs of Schmallenberg virus is targeted to the nucleolus and induces nucleolar disorganization

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death

Activation of Inflammasome during Bluetongue Virus Infection

Bluetongue virus (BTV), a double-stranded RNA virus belonging to the Sedoreoviridae family, provokes an economically important disease in ruminants. In this study, we show that the production of activated caspase-1 and interleukin 1 beta (IL-1β) is induced in BTV-infected cells. This response seems to require virus replication since a UV-inactivated virus is unable to activate this pathway. In NLRP3-/- cells, BTV could not trigger further IL-1β synthesis, indicating that it occurs through NLRP3 inflammasome activation. Interestingly, we observed differential activation levels in bovine endothelial cells depending on the tissue origin. In particular, inflammasome activation was stronger in umbilical cord cells, suggesting that these cells are more prone to induce the inflammasome upon BTV infection. Finally, the strength of the inflammasome activation also depends on the BTV strain, which points to the importance of viral origin in inflammasome modulation. This work reports the crucial role of BTV in the activation of the NLRP3 inflammasome and further shows that this activation relies on BTV replication, strains, and cell types, thus providing new insights into BTV pathogenesis

Re-Emergence of Bluetongue Virus Serotype 8 in France, 2015

International audienceAt the end of August 2015, a ram located in central France (department of Allier) showed clinical signs suggestive of BTV (Bluetongue virus) infection. However, none of the other animals located in the herd showed any signs of the Bluetongue disease. Laboratory analyses identified the virus as BTV serotype 8. The viro and sero prevalence intraherd were 2.4% and 8.6% in sheep and 18.3% and 42.9% in cattle, respectively. Phylogenetic studies showed that the sequences of this strain are closely related to another BTV-8 strain that has circulated in France in 2006-2008. The origin of the outbreak is unclear but it may be assumed that the BTV-8 has probably circulated at very low prevalence (possibly in livestock or wildlife) since its first emergence in 2007-2008

Novel Bluetongue Virus in Goats, Corsica, France, 2014

During 2000–2013, 4 genotypes of bluetongue virus (BTV) were detected in Corsica, France. At the end of 2013, a compulsory BTV-1 vaccination campaign was initiated among domestic ruminants; biological samples from goats were tested as part of a corresponding monitoring program. A BTV strain with nucleotide sequences suggestive of a novel serotype was detected

Comparative Virus-Host Protein Interactions of the Bluetongue Virus NS4 Virulence Factor

This study was funded by the Blue-Med project in the framework of the PRIMA program, an Art.185 initiative supported and funded under European Union Horizon 2020International audienceBluetongue virus (BTV) is the etiologic agent of a non-contagious arthropod-borne disease transmitted to wild and domestic ruminants. BTV induces a large panel of clinical manifestations ranging from asymptomatic infection to lethal hemorrhagic fever. Despite the fact that BTV has been studied extensively, we still have little understanding of the molecular determinants of BTV virulence. In our report, we have performed a comparative yeast two-hybrid (Y2H) screening approach to search direct cellular targets of the NS4 virulence factor encoded by two different serotypes of BTV: BTV8 and BTV27. This led to identifying Wilms’ tumor 1-associated protein (WTAP) as a new interactor of the BTV-NS4. In contrast to BTV8, 1, 4 and 25, NS4 proteins from BTV27 and BTV30 are unable to interact with WTAP. This interaction with WTAP is carried by a peptide of 34 amino acids (NS422−55) within its putative coil-coiled structure. Most importantly, we showed that binding to WTAP is restored with a chimeric protein where BTV27-NS4 is substituted by BTV8-NS4 in the region encompassing residue 22 to 55. We also demonstrated that WTAP silencing reduces viral titers and the expression of viral proteins, suggesting that BTV-NS4 targets a cellular function of WTAP to increase its viral replication

The VP3 Protein of Bluetongue Virus Associates with the MAVS Complex and Interferes with the RIG-I-Signaling Pathway

International audienceBluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges, is a major concern of wild and domestic ruminants. While BTV induces type I interferon (alpha/beta interferon [IFN-α/β]) production in infected cells, several reports have described evasion strategies elaborated by this virus to dampen this intrinsic, innate response. In the present study, we suggest that BTV VP3 is a new viral antagonist of the IFN-β synthesis. Indeed, using split luciferase and coprecipitation assays, we report an interaction between VP3 and both the mitochondrial adapter protein MAVS and the IRF3-kinase IKKε. Overall, this study describes a putative role for the BTV structural protein VP3 in the control of the antiviral response

Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections

International audienceMicrovascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine

Complete genome sequence of bluetongue virus serotype 8, which reemerged in France in August 2015

We announce here the complete genome sequence (coding and noncoding) of the bluetongue virus (BTV) serotype 8, isolated from a ram in Allier department, France, 2015. Sequence analysis confirms the reemergence of the BTV-8 strain that previously circulated in France until 2009 and other European countries until 2010

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization