Alexandra Desprat

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.

Evaluation of humoral response and protective efficacy of two inactivated vaccines against bluetongue virus after vaccination of goats.

Bluetongue serotype 8 has become a major animal health issue in the European Union and the European member States have agreed on a vaccination strategy, which involves only inactivated vaccines. In this study, the efficacy of two inactivated vaccines against bluetongue virus serotype 8 (BTV-8) used in Europe since 2008, BTVPUR ALSAP(®) 8 (MERIAL) and BOVILIS(®) BTV8 (Intervet/SP-AH), was evaluated in goats immunized and challenged with BTV-8 field isolates under experimental conditions. Serological, virological and clinical examinations were conducted before and after challenge. Three groups of 10 goats each (groups A, B and C) were randomly constituted and 2 groups (A and C) were subcutaneously vaccinated twice with one dose of the two commercial vaccines BTVPUR ALSAP 8 (group A) or BOVILIS BTV8 (group C) respectively. Animals of the groups A, C and B (B: controls) were challenged with a virulent inoculum containing BTV-8. During the experiment, it was found out that the BTV-8 challenge inoculum was contaminated with another BTV serotype. However, results demonstrated that vaccination of goats with two injections of BTVPUR ALSAP 8 or BOVILIS BTV8 provided a significant clinical protection against a BTV-8 challenge and completely prevented BTV-8 viraemia in all vaccinated animals. Qualitative data showed no difference in the kinetics and levels of the humoral response induced by these two inactivated vaccines.

Sensing and Control of Bluetongue Virus Infection in Epithelial Cells via RIG-I and MDA5 Helicases

ABSTRACT 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/β]) 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-β in response to BTV serotype 8. Upon BTV infection of A549 cells, expression of IFN-β 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-β. 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-β 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-β 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.

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.

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.

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%.

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.

Evidence of transplacental transmission of bluetongue virus serotype 8 in goats

During the incursion of bluetongue virus (BTV) serotype 8 in Europe, an increase in the number of abortions in ruminants was observed. Transplacental transmission of BTV-8 in cattle and sheep, with subsequent foetal infection, is a feature of this specific bluetongue serotype. In this study, BTV-8 ability to cross the placental barrier at the beginning of the second third of pregnancy and at the end of pregnancy was investigated in goats in two separate experiments. In the first experiment, nine goats were experimentally infected with BTV-8 at 61 days of pregnancy. Foetuses were collected 21 dpi. BTV-8 was evidenced by real time RT-PCR and by viral isolation using blood from the umbilical cord and the spleens of 3 out of the 13 foetuses. All dams were viraemic (viral isolation) at the moment of sampling of the foetuses. Significant macroscopic or histological lesions could not be observed in foetuses or in their infected dams (notably at the placenta level). In the second experiment, 10 goats were infected with BTV-8 at 135 days of pregnancy. Kids were born by caesarean section at the programmed day of birth (15 dpi). BTV-8 could not be detected by rt-RT-PCR in blood or spleen samples from the kids. This study showed for the first time that BTV-8 transplacental transmission can occur in goats that have been infected at 61 days of pregnancy, with infectious virus recovered from the caprine foetuses. The observed transmission rate was quite high (33%) at this stage of pregnancy. However, it was not possible to demonstrate the existence of BTV-8 transplacental transmission when infection occurred at the end of the goat pregnancy.

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.

Acute Schmallenberg virus infections, France, 2012.

To the Editor: After unexpected emergence of bluetongue virus serotype 8 in northern Europe in 2006 (1), another arbovirus, Schmallenberg virus (SBV), which is transmitted by Culicoides spp. biting midges, emerged in Europe in 2011 and caused disease outbreaks among ruminants (2). Nonspecific clinical signs such as fever, decreased milk production, and diarrhea were associated with acute infection in cattle, and late abortions and birth defects in newborns were associated with infection of pregnant cows, ewes, and goats (2,3).