Schmallenberg virus infection among red deer, France, 2010-2012.

Schmallenberg virus infection is emerging in European domestic and wild ruminants. We investigated the serologic status of 9 red deer populations to describe virus spread from September 2010 through March 2012 among wildlife in France. Deer in 7 populations exhibited seropositivity, with an average seroprevalence of 20%.

Sensing and control of bluetongue virus infection in epithelial cells via RIG-I and MDA5 helicases.

Bluetongue virus (BTV), an arthropod-borne member of the Reoviridae family, is a double-stranded RNA virus that causes an economically important livestock disease that has spread across Europe in recent decades. Production of type I interferon (alpha/beta interferon [IFN-α/ß]) has been reported in vivo and in vitro upon BTV infection. However, the cellular sensors and signaling pathways involved in this process remain unknown. Here we studied the mechanisms responsible for the production of IFN-ß in response to BTV serotype 8. Upon BTV infection of A549 cells, expression of IFN-ß and other proinflammatory cytokines was strongly induced at both the protein and mRNA levels. This response appeared to be dependent on virus replication, since exposure to UV-inactivated virus failed to induce IFN-ß. We also demonstrated that BTV infection activated the transcription factors IFN regulatory factor 3 and nuclear factor κB. We investigated the role of several pattern recognition receptors in this response and showed that expression of IFN-ß was greatly reduced after small-interfering-RNA-mediated knockdown of the RNA helicase encoded by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5). In contrast, silencing of MyD88, Toll-like receptor 3, or the recently described DexD/H-box helicase DDX1 sensor had no or a weak effect on IFN-ß induction, suggesting that the RIG-I-like receptor pathway is specifically engaged for BTV sensing. Moreover, we also showed that overexpression of either RIG-I or MDA5 impaired BTV expression in infected A549 cells. Overall, this indicates that RIG-I and MDA5 can both contribute to the recognition and control of BTV infection.

Epidemiology, molecular virology and diagnostics of Schmallenberg virus, an emerging orthobunyavirus in Europe.

After the unexpected emergence of Bluetongue virus serotype 8 (BTV-8) in northern Europe in 2006, another arbovirus, Schmallenberg virus (SBV), emerged in Europe in 2011 causing a new economically important disease in ruminants. The virus, belonging to the Orthobunyavirus genus in the Bunyaviridae family, was first detected in Germany, in The Netherlands and in Belgium in 2011 and soon after in the United Kingdom, France, Italy, Luxembourg, Spain, Denmark and Switzerland. This review describes the current knowledge on the emergence, epidemiology, clinical signs, molecular virology and diagnosis of SBV infection.

Colostral antibody induced interference of inactivated bluetongue serotype-8 vaccines in calves.

Since its introduction into northern Europe in 2006, bluetongue has become a major threat to animal health. While the efficacy of commercial vaccines has been clearly demonstrated in livestock, little is known regarding the effect of maternal immunity on vaccinal efficacy. Here, we have investigated the duration and amplitude of colostral antibody-induced immunity in calves born to dams vaccinated against bluetongue virus serotype 8 (BTV-8) and the extent of colostral antibody-induced interference of vaccination in these calves. Twenty-two calf-cow pairs were included in this survey. The median age at which calves became seronegative for BTV was 84 and 112 days as assayed by seroneutralisation test (SNT) and VP7 BTV competitive ELISA (cELISA), respectively. At the mean age of 118 days, 13/22 calves were immunized with inactivated BTV-8 vaccine. In most calves vaccination elicited a weak immune response, with seroconversion in only 3/13 calves. The amplitude of the humoral response to vaccination was inversely proportional to the maternal antibody level prior to vaccination. Thus, the lack of response was attributed to the persistence of virus-specific colostral antibodies that interfered with the induction of the immune response. These data suggest that the recommended age for vaccination of calves born to vaccinated dams needs to be adjusted in order to optimize vaccinal efficacy.

Expression of VP7, a Bluetongue virus group specific antigen by viral vectors: analysis of the induced immune responses and evaluation of protective potential in sheep.

Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.

Bluetongue virus serotype 8 virus-like particles protect sheep against virulent virus infection as a single or multi-serotype cocktail immunogen.

Since 1998, there have been multiple separate outbreaks of Bluetongue disease (BT) in Europe with the largest outbreak ever recorded in Northern Europe caused by Bluetongue virus serotype 8 (BTV-8). Coinciding with the BTV-8 outbreak, a virulent strain of BTV-1 emerged and co-infections of these two serotypes were reported. In response, we generated VLPs for BTV-8 and tested the efficacy of BTV-8 VLPs as a single immunogen and as a component of a multivalent vaccine, with VLPs of BTV-1 and BTV-2, in order to test if there was any interference between serotypes. All pre-Alps sheep vaccinated with BTV-8 VLPs developed a strong neutralising antibody response to BTV-8 and multivalent VLP vaccinated animals also developed neutralising antibodies to BTV-1 and BTV-2. There were no side effects observed due to the vaccination with either the single- or multivalent VLP cocktail. All VLP-vaccinated animals had no clinical manifestation of BT or viraemia after challenge with a virulent BTV-8 isolate. This data indicates that BTV-8 VLPs delivered as a single immunogen or as a component of a multivalent vaccine are highly efficacious. Moreover, there was no interference on the development of a strong protective immune response due to the combination of different phylogenetically unrelated BTV serotypes in the vaccinated animals. This report further highlights that BTV VLPs are safe and efficacious immunogens that are able to afford complete protection against a virulent virus challenge.

Epizootic hemorrhagic disease virus serotype 6 experimentation on adult cattle.

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants closely related to bluetongue virus (BTV). EHDV serotype 6 (EHDV6) has recently caused outbreaks close to Europe in Turkey and Morocco and a recent experimental study performed on calves inoculated with these two EHDV6 strains showed that the young animals have remained clinically unaffected. The aim of this study was to investigate the pathogenicity of an EHDV6 strain from La Reunion Island in adult Holstein (18-month-old heifers). This EHDV6 strain has induced clinical signs in cattle in the field. Samples taken throughout the study were tested with commercially available ELISA and real-time RT-PCR kits. Very mild clinical manifestations were observed in cattle during the experiment although high levels of viral RNA and virus were found in their blood. EHDV was isolated from the blood of infected animals at 8 dpi. Antibodies against EHDV were first detected by 7 dpi and persisted up to the end of the study. Virus was detected in various tissue samples until 35 dpi, but was not infectious. In view of the recent circulation of different arboviruses in Europe, this study demonstrates what the EHD induces a strong viraemia in adult Holstein cattle and shows that a spread of EHD on European livestock cattle is possible.

Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies.

A newly developed Enzym Like Immuno Sorbant Assay (ELISA) based on the recombinant nucleocapsid protein (N) of Schmallenberg virus (SBV) was evaluated and validated for the detection of SBV-specific IgG antibodies in ruminant sera by three European Reference Laboratories. Validation data sets derived from sheep, goat and bovine sera collected in France and Germany (n = 1515) in 2011 and 2012 were categorized according to the results of a virus neutralization test (VNT) or an indirect immuno-fluorescence assay (IFA). The specificity was evaluated with 1364 sera from sheep, goat and bovine collected in France and Belgium before 2009. Overall agreement between VNT and ELISA was 98.9% and 98.3% between VNT and IFA, indicating a very good concordance between the different techniques. Although cross-reactions with other Orthobunyavirus from the Simbu serogroup viruses might occur, it is a highly sensitive, specific and robust ELISA-test validated to detect anti-SBV antibodies. This test can be applied for SBV sero-diagnostics and disease-surveillance studies in ruminant species in Europe.

Co-circulation of bluetongue and epizootic haemorrhagic disease viruses in cattle in Reunion Island.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) in deer have already been isolated in Reunion Island and have caused more or less severe clinical signs in cattle (EHDV) or in sheep (BTV), as observed in 2003. In January 2009, cattle in Reunion Island showed clinical signs suggesting infection by one or the other of these arboviral diseases. A study was set up to determine the etiology of the disease. Analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on blood samples from 116 cattle from different districts of the island detected the presence of the EHDV genome in 106 samples and, in 5 of them, the simultaneous occurrence of BTV and EHDV. One strain of EHDV (7 isolates) and one of BTV were isolated in embryonated eggs and a BHK-21 cell culture. Group and subgroup primer-pairs were designed on the segment 2 sequences available in GenBank to identify and type the EHDV strains. Phylogenetic analysis of the genomic segment 2 (encoding the VP2 serotype-specific protein) of the isolates confirmed the serotypes of these two orbiviruses as BTV-2 and EHDV-6 and allowed them to be compared with previously isolated strains.