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BTV and EHDV are closely-related orbiviruses that are transmitted between domestic and wild ruminants via the bites of hematophagous midges. Previous studies have reported seropositivity against BTV antibodies in sheep and goats in two Egyptian governorates (Beni Suef and Menoufia). However, no recent data are available on the BTV serotype(s) circulating in Egypt and the likely presence of EHDV has never been explored. This study investigated the presence of BTV and EHDV among cattle which had been found BTV-seropositive by ELISA method. These cattle living in proximity to sheep and goats previously found BTV-seropositive. These cattle displayed no clinical signs of BT but reproductive problems had been reported in herds. A total of 227 cattle blood samples were therefore collected in 2016 and 2017. Ninety-four of the 227 animals tested by a BTV ELISA were positive for BTV antibodies (41.4%). Of these 94 ELISA-positive cattle, only 83 EDTA-blood samples were available and therefore tested for BTV and EHDV genome detection by RT-PCR and sequencing. Of the cattle sampled in 2016, results revealed that two were RT-PCR-positive for BTV and seven for EHDV. Sequencing showed the presence of EHDV-1 and BTV-3 genome sequences. EHDV-1 S2 shared 99.5% homology with an EHDV-1 S2 from a strain isolated in 2016 in Israel. BTV-3 S2 and S8 sequences shared >99.8% nucleotide similarity with the BTV-3 Zarzis S2 and S8 sequences (Tunisian BTV, also detected in 2016). Of the 66 blood samples tested following their collection in 2017, they were all EHDV-negative by RT-qPCR while five were BTV- positive by RT-qPCR. However, attempts to identify the BTV serotype of these five samples were unsuccessful. Only part of BTV S8 was sequenced and it showed 79% nucleotide similarity with S8 of atypical BTV serotypes (particularly with BTV-26 and another BTV serotype strain isolated from a sheep pox vaccine). Overall, these findings demonstrate that both BTV and EHDV were circulating in Egypt in 2016 and 2017.

International audience; Bluetongue (BT) is an infectious, arthropod-borne viral disease of domestic and wild ruminants. The disease causes animal mortality, production decrease and commercial limits for herds. Despite the active circulation of the disease in the world, few studies have been carried out in Senegal. The objective of this study was to assess the current prevalence of BT in small ruminants and the serotypes circulating in Senegal. A cross-sectional study was conducted in the fourteen regions of Senegal. After the sampling campaign, sera collected in sheep and goats herds were screened for the presence of Bluetongue virus (BTV) specific antibodies using c-Elisa. The whole blood of seropositive animals was further analyzed by RT-qPCR and positive samples were typed to identify BTV serotypes. Analysis of several risk factors such as age, sex and species of animals was performed using logistic regression. The overall seroprevalence of BTV in Senegal was 72.6% (95% CI: 70.3-74.9%) with 75.9% (95% CI: 72.2-79.5%) in goat and 70.6% (95% CI: 67.5-73.6%) in sheep. Female (prevalence=77.1%) and adult (prevalence=80%) animals showed the highest seropositivity to BTV compared respectively to male (55.7%, p=6.133e-09) and young (49.4%, p < 2.2e-16). The RT-qPCR results showed the presence of BT viral genome in 359 small ruminants. The results obtained from serological and genotyping studies showed an active spread of the Bluetongue virus in domestic ruminants and phylogenetic analysis showed that the BTV-2 is one of the circulating serotypes in Senegal. This study allows having baseline information for controlling Bluetongue in Senegal.

International audience; Bluetongue (BT) and epizootic haemorrhagic disease (EHD) are vector-borne viral diseases affecting domestic and wild ruminants. Both are notifiable under OIE rules. BT and EHD viruses (BTV and EHDV) are closely related Orbiviruses with structural, antigenic and molecular similarities. Both viruses can produce analogous clinical signs in susceptible animals. Serological tests are commonly used for BT and EHD diagnosis and surveillance. Competitive ELISA (c-ELISA) is the most widely used serological test for the specific detection of BTV or EHDV viral protein 7 (VP7) antibodies (Abs). The specificity and sensitivity of the BTV c-ELISA kits available on the market are recognized for the detection of BTV Abs. Concerning EHD, a single commercial EHDV c-ELISA kit (ELISA A kit) commonly used for diagnosis in Europe and Africa was available between 2011 and 2018 but is now no longer on the market. In this study, we evaluated a new commercial c-ELISA to detect ruminant EHDV VP7 Abs in 2,199 serum samples from cattle, sheep, goats, wild deer and zoo animals. The results showed that this ELISA kit is specific and can detect the presence of IgG anti-EHDV VP7 with a very good diagnostic specificity and a satisfactory sensitivity in domestic ruminants, zoo animals and wild deer. Therefore, the evaluated c-ELISA can detect the introduction of EHDV into an area where BTV-seropositive domestic animals are present. The performance of this kit is similar to that of the c-ELISA A kit and can thus be used for diagnosis.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) have both been reported in mainland Ecuador, but their occurrence was unknown in the Galapagos Islands, an Ecuadorian province. We aimed to detect BTV or EHDV in cattle from the 3 main cattle-producing Galapagos Islands at a between-herd design prevalence of 20% and a within-herd design prevalence of 15%. Blood samples were collected from 410 cattle in 33 farms and tested for antibodies against BTV and EHDV by competitive ELISAs. All results were negative, suggesting that BTV and EHDV are not present in the Galapagos Islands.

International audience; In November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV-4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV-4 has been introduced to Corsica from Italy (Sardinia) where BTV-4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe.

International audience; Bluetongue virus serotype 8 (BTV-8) caused an epizootic in Europe in 2006/09. Transplacental transmission of BTV-8 was demonstrated leading to abortions, congenital malformations or nervous clinical signs in newborn calves. BTV-8 re-emerged in France in 2015. Although the re-emergent strain is nearly genetically identical to the one that had circulated in 2006/2009, it has caused very few clinical cases. However, from mid-December 2018 to April 2019, cases of calves with congenital malformations or displaying nervous clinical signs occurred in some departments (French administrative unit) in mainland France. Blood samples from these animals were sent to local laboratories, and the positive ones were confirmed at the French Bluetongue reference laboratory (BT-NRL). Out of 580 samples found positive at the local laboratories, 544 were confirmed as RT-PCR BTV-8 positive. The 36 samples found positive in the local laboratories and negative in the BT-NRL were all at the limit of RT-PCR detection. Hundred eighty-eight of the confirmed samples were also tested for the presence of Schmallenberg virus (SBV) and bovine virus diarrhoea virus (BVDV) infection: 4 were found positive for BVDV and none for SBV. The main clinical signs recorded for 244 calves, for which a reporting form was completed by veterinarians, included nervous clinical signs (81%), amaurosis (72%) and decrease/ no suckling reflex (40%). Hydranencephaly and microphthalmia were reported in 19 calves out of 27 in which a necropsy was practiced after death or euthanasia. These results indicate that the re-emergent strain of BTV-8 can cross the transplacental barrier and cause congenital malformations or nervous clinical signs in calves.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an insect-borne virus of ruminants, emerged in livestock in Northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of Euros. Though the outbreak was successfully controlled through vaccination by early 2010, puzzlingly a closely-related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this period without replication, we conclude that the second outbreak was most likely initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Bluetongue virus (BTV) is a Culicoides-borne pathogen infecting both domestic and wild ruminants. In Europe, the Red Deer (Cervus elaphus) (RD) is considered a potential BTV reservoir, but persistent sylvatic cycle has not yet been demonstrated. In this paper, we explored the dynamics of BTV1 and BTV8 serotypes in the RD in France, and the potential role of that species in the re-emergence of BTV8 in livestock by 2015 (i.e., 5 years after the former last domestic cases). We performed 8 years of longitudinal monitoring (2008–2015) among 15 RD populations and 3065 individuals. We compared Culicoides communities and feeding habits within domestic and wild animal environments (51,380 samples). Culicoides diversity (>30 species) varied between them, but bridge-species able to feed on both wild and domestic hosts were abundant in both situations. Despite the presence of competent vectors in natural environments, BTV1 and BTV8 strains never spread in RD along the green corridors out of the domestic outbreak range. Decreasing antibody trends with no PCR results two years after the last domestic outbreak suggests that seropositive young RD were not recently infected but carried maternal antibodies. We conclude that RD did not play a role in spreading or maintaining BTV in France.

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its virulence non-structural protein NS3 (BTV-NS3), is able to activate the MAPK/ERK pathway. In response to growth factors, the MAPK/ERK pathway activates cell survival, differentiation, proliferation and protein translation but can also lead to the production of several inflammatory cytokines. By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player of the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease of the MAPK/ERK activation by BTV supporting a model where BTV-NS3 interacts with BRAF to activate this signaling cascade. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members ofOrbivirusgenus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Therefore, the activation of the MAPK/ERK pathway by BTV-NS3 could benefit to BTV replication by promoting its own viral protein synthesis but could also explain the deleterious inflammation associated with tissue damages as already observed in severe cases of BT disease. Altogether, our data provide molecular mechanisms to explain the role of BTV-NS3 as a virulence factor and determinant of pathogenesis.ImportanceBluetongue Virus (BTV) is responsible of the non-contagious arthropod-borne disease Bluetongue (BT) transmitted to ruminants by blood-feeding midges. Despite the fact that BTV has been extensively studied, we still have little understanding of the molecular determinants of BTV virulence. In this report, we found that the virulence protein NS3 interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival, increases protein translation but also contributes to the production of inflammatory cytokines. We showed that BTV-NS3 enhances the MAPK/ERK pathway and this activation is BRAF-dependent. Our results demonstrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to ensure its viral replication. On the other hand, our findings could also explain the deleterious inflammation associated with tissue damages as already observed in severe cases of BT disease.

International audience; A cross-sectional study was conducted to explore the epidemiological situation in Mayotte regarding two orbiviruses: Bluetongue virus (BTV) and Epizootic Haemorrhagic Disease virus (EHDV). In all, 385 individual asymptomatic cattle were blood-sampled (one EDTA and one serum tube per animal) between February and June 2016. Antibody (ELISA) and genome prevalence (PCR) was assessed. Almost all the selected cattle showed antibodies against both BTV and EHDV, at 99.5% (CI95% [98.00, 100]) and 96.9% (CI95% [94.5, 98.3]), respectively. Most of the cattle acquired antibodies in their first years of age. EHDV and BTV genomes were detected in 25.2% (CI95% [21.1, 29.8]) and 18.2% (CI95% [14.6, 22.4]) of samples, respectively. Coinfection with BTV and EHDV was observed in 9.4% of samples (CI95% [6.8, 12.7]). Cattle under three years old were more frequently reported as positive for genome detection by PCR than older cattle. Five serotypes of BTV and one serotype of EHDV were identified from eight samples: BTV-4, BTV-9, BTV-11, BTV-15, BTV-19 and EHDV-6, of which some were reported in neighbouring areas. BTV and EHDV both circulate in Mayotte and in its surrounding territories.

International audience; In November 2017, a 15-day-old calf located in France (Haute-Savoie department) was found positive for bluetongue virus (BTV) RNA by RT-PCR. Laboratory investigations allowed the isolation and identification of the serotype: BTV-4. The analysis of the full viral genome showed that all the 10 genome segments were closely related to BTV-4 strains involved in a large BT outbreak in the Balkan Peninsula, in Italy since 2014 and in Corsica since the end of October 2016. These results together with epidemiological data suggest that BTV-4 has been introduced to mainland France from Corsica or Italy where BTV-4 outbreaks have been reported in summer and autumn 2016. This is the first report of the introduction of BTV-4 in mainland France.

International audience; Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) are closely related Orbiviruses that affect domestic and wild ruminants. In Ecuador previous serological studies reported the presence of BTV; however, no data are available about the presence of EHDV. In this study, 295 cattle without symptoms of infection were sampled from two farms located in Andean and Amazonian regions and from a slaughterhouse in the coastal region. ELISA analyses showed high prevalence of BTV (98.9%) and EHDV (81.3%) antibodies, and RT-qPCRs revealed the presence of EHDV (24.1%) and BTV (10.2%) genomes in cattle blood samples. Viral isolation allowed to identify EHDV serotype 1 (EHDV1) and BTV serotypes 9 (BTV9), 13 and 18. These findings suggest that BTV and EHDV are enzootic diseases in Ecuador.

International audience; Bluetongue virus (BTV) hitherto consisted of 26 recognized serotypes, of which all except BTV-26 are primarily transmitted by certain species of Culicoides biting midges. Three variants of an additional 27th bluetongue virus serotype (BTV-27v01-v03) were recently detected in asymptomatic goats in Corsica, France, 2014-2015. Molecular characterization revealed genetic differences between the three variants. Therefore, invivo characteristics were investigated by experimental infection of a total of 15 goats, 11 sheep and 4 cattle with any one of the three variants in separated animal trials. In goat trials, BTV-naive animals of the same species were kept in a facility where direct contact was unhindered. Of the 15 inoculated goats, 13 and 14 animals were found positive for BTV-RNA and antibodies (Ab), respectively, until the end of the experiments. Surprisingly, BTV-Ab levels as measured with ELISA and neutralization test (SNT) were remarkably low in all seropositive goats. Virus isolation from whole-blood was possible at the peak of viremia until 49dpi. Moreover, detection of BTV-27v02-RNA and Ab in one contact goat indicated thatsimilar to BTV-26at least one of three BTV-27 variants may be transmitted by contact between goats. In the field, BTV-27 RNA can be detected up to 6months in the whole-blood of BTV-27-infected Corsican goats. In contrast, BTV RNA was not detected in the blood of cattle or sheep. In addition, BTV-27 Abs were not detected in cattle and only a transient increase in Ab levels was observed in some sheep. None of the 30 animals showed obvious BT-like clinical signs. In summary, the phenotypes observed for BTV-27v01-v03 phenotypes correspond to a mixture of characteristics known for BTV-25 and 26.

International audience; In French Guiana, the sero- and viro-prevalence of Bluetongue virus (BTV) is high but the circulating serotypes remain unknown. No data are available regarding the prevalence of Epizootic hemorrhagic disease (EHD). This study was conducted to assess the prevalence and to identify the circulating serotypes of these two Orbiviruses in this region (BTV and EHDV). Blood samples were collected in main livestock areas, from 122 young cattle between June and August 2011, to perform virological (PCR and viral isolation) and serological (ELISA) analyses. Moreover, samples from sheep and goat showing BTV-like clinical signs and from newly imported animals were analyzed using the same assays. Results confirmed an important viral circulation, with viro- and seroprevalence of 85% and 84% and 60% and 40% for BTV and EHDV, respectively. Ten Orbivirus serotypes were identified (BTV-1, 2, 6, 10, 12, 13, 17 and 24, EHDV-1 and 6). The circulation of many serotypes in intertropical America and in the Caribbean region underlines the need to establish measures to monitor and control animal movements.

Bluetongue virus serotype 8 (BTV-8) re-emerged in Central France in August 2015. The viral strain identified is nearly identical to the one that circulated during the 2006/2009 massive outbreak throughout Europe. To address the question of an undetected BTV-8 circulation on the French territory, a serological study was conducted on young cattle along a transect of seven departments, three of them located in areas where the virus presence had been confirmed by RT-PCR by winter 2015/2016. Sera from 2,565 animals were collected during the winters preceding and following the re-emergence, with 414 animals being sampled in each of the two consecutive years. All samples were tested by competitive ELISA (IDVet) and, when enough serum was available, ELISA-positive samples were confirmed by seroneutralization tests. In areas with infected holdings, seropositive animals were found before the re-emergence (N = 14 of 511), significantly more on the following year (N = 17 of 257), and eight animals (N = 158) seroconverted over 2015. Seropositive animals were also detected as early as winter 2014/2015 in one department without known infected holdings (N = 12 of 150), and in winter 2015/2016 in three of them (N = 21 of 555), where seven animals (N = 154) seroconverted over 2015. These results suggest that BTV-8 may have spread at low levels before the re-emergence, even in areas considered virus-free. Unfortunately, whole blood from the seropositive animals was not available to definitely confirm the virus presence by RT-PCR.

Emergence and re-emergence of two major diseases in France (bluetongue) and in Mauritius (foot-and-mouth). The emergence of Bluetongue (BT) in continental France (caused by virus of serotype 8 (BTV-8) in 2006 and virus of serotype 1 (BTV-1) in 2007) was a total surprise. End of 2012, six years after the introduction of BT, France was declared free from this disease. However, at the end of August 2015, the BTV-8 made its reappearance in the center of France. In 2016, our laboratory re-isolated this virus. In Corsica, a virus of serotype 4 was identified on 1st December 2016 from sheep samples. On another hand, in 2016, we identified a virus of Foot-and-Mouth disease serotype O in Mauritius. This presentation will describe the conditions of the detection of these viruses as well as the results of phylogenetic analyzes., L’émergence en France continentale de la fièvre catarrhale ovine (FCO) causée en 2006 par le virus de sérotype 8 (BTV-8) puis en 2007, par le virus de sérotype 1 (BTV-1) a constitué une surprise totale. Fin 2012, six ans après l’introduction de la FCO, la France a été déclarée indemne de cette maladie. Pourtant, fin août 2015, le BTV-8 a fait sa réapparition dans le centre de la France. En 2016 notre laboratoire a isolé à nouveau ce virus. En Corse, un virus de sérotype 4 (BTV-4) fut identifié le 1er décembre 2016 à partir de prélèvements de moutons. D’autre part, en 2016, nous avons identifié un virus de la fièvre aphteuse de sérotype O à Maurice. Cette présentation décrira les conditions de détection de ces virus ainsi que les résultats des analyses phylogénétiques., Zientara Stéphan, Sailleau Corinne, Bréard Emmanuel, Vitour Damien, Viarouge Cyril, Belbis Guillaume, Gorna Kamila, Relmy Anthony, Romey Aurore, Blaise-Boisseau Sandra, Cardinale Éric, Bakkali Kassimi Labib. Émergence et ré-émergence de deux dangers sanitaires de catégorie 1 en France (FCO) et à Maurice (FA). In: Bulletin de l'Académie Vétérinaire de France tome 170 n°1, 2017. pp. 66-69.

International audience; Since the unexpected emergence of BTV-8 in Northern Europe and the incursion of BTV-8 and 1 in France in 2006-2007, molecular diagnosis has considerably evolved. Several real-time RT-PCR (rtRT-PCR) methods have been developed and published, and are currently being used in many countries across Europe for BTV detection and typing. In France, the national reference laboratory (NRL) for orbiviruses develops and validates 'ready-to-use' kits with private companies for viral RNA detection. The regional laboratories network that was set up to deal with a heavy demand for analyses has used these available kits. From 2007, ring tests were organized to monitor the performance of the French laboratories. This study presents the results of 63 regional laboratories in the ring trial organized in 2016. Blood samples were sent to the laboratories. Participants were asked to use the rtRT-PCR methods in place in their laboratory, for detection of all BTV serotypes and specifically BTV-8. The French regional laboratories are able to detect and genotype BTV in affected animals. Despite the use of several methods (i.e. RNA extraction and different commercial rtRT-PCRs), the network is homogeneous. The ring trial demonstrated that the French regional veterinary laboratories have reliable and robust BTV diagnostic tools for BTV genome detection.

Summary The Schmallenberg virus (SBV) has recently emerged in Europe, causing losses to the domestic livestock. A retrospective analysis of serodata was conducted in France for estimating seroprevalence of SBV among six wildlife species from 2011–2012 to 2013–2014, that is during the three vector seasons after the emergence of the SBV in France. Our objective was to quantify the exposure of wildlife to SBV and the potential protective effect of elevation such as previously observed for bluetongue. We also compared the spatiotemporal trends between domestic and wild animals at the level of the departments. We tested 2050 sera using competitive ELISA tests. Individual and population risk factors were further tested using general linear models among 1934 individuals. All populations but one exhibited positive results, seroprevalence up to 30% being observed for all species. The average seroprevalence did not differ between species but ranged from 0 to 90% according to the area and period, due to the dynamic pattern of infection. Seroprevalence was on average higher in the lowlands compared to areas located up to 800 m. Nevertheless, seroprevalence above 50% occurred in areas located up to 1500 m. Thus, contrary to what had been observed for bluetongue during the late 2000s in the same areas, SBV could spread to high altitudes and infect all the studied species. The spatial spread of SBV in wildlife did not fully match with SBV outbreaks reported in the domestic livestock. The mismatch was most obvious in mountainous areas where outbreaks in wildlife occurred on average one year after the peak of congenital cases in livestock. These results suggest a much larger spread and vector capacity for SBV than for bluetongue virus in natural areas. Potential consequences for wildlife dynamics are discussed.

International audience; Schmallenberg virus (SBV) is an emerging virus responsible for congenital malformations in the offspring of domestic ruminants. It is speculated that infection of pregnant dams may also lead to a significant number of unrecognized fetal losses during the early period of gestation. To assess the pathogenic effects of SBV infection of goats in early pregnancy, we inoculated dams at day 28 or 42 of gestation and followed the animals until day 55 of gestation. Viremia in the absence of clinical signs was detected in all virus-inoculated goats. Fetal deaths were observed in several goats infected at day 28 or 42 of gestation and were invariably associated with the presence of viral genomic RNA in the affected fetuses. Among the viable fetuses, two displayed lesions in the central nervous system (porencephaly) in the presence of viral genome and antigen. All fetuses from goats infected at day 42 and the majority of fetuses from goats infected at day 28 of gestation contained viral genomic RNA. Viral genome was widely distributed in these fetuses and their respective placentas, and infectious virus could be isolated from several organs and placentomes of the viable fetuses. Our results show that fetuses of pregnant goats are susceptible to vertical SBV infection during early pregnancy spanning at least the period between day 28 and 42 of gestation. The outcomes of experimental SBV infection assessed at day 55 of gestation include fetal mortalities, viable fetuses displaying lesions of the central nervous system, as well as viable fetuses without any detectable lesion.

International audience; At 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.

International audience; Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.

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 in zoo ruminants, France and the Netherlands

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.

In 2006, epizootic haemorrhagic disease (EHD) outbreaks were recorded in the Maghreb (Tunisia, Morocco and Algeria) among cattle, resulting in severe repercussions on herds (oedema of the head, necrotic lesions of the oral mucosa, hyperthermia of the teats, accompanied by anorexia and respiratory distress) and economic losses. The present study gives new information on the molecular characterisation of the EHD virus (EHDV) that had circulated in Tunisia. Genome segments 2, 3, 6, 7 and 10 of EHDV, corresponding to the VP2, VP3, VP5, VP7 and NS3/NS3A proteins, respectively, were amplified from the blood of one animal by RT-PCR and sequenced. Nucleotide sequence comparisons of these five segments with sequences available in the GenBank demonstrated that an EHDV serotype 6 (EHDV-6) had been present in Tunisia in 2006. The possible origin of this strain is discussed.

We announce the complete coding genome sequence of a novel bluetongue virus (BTV) serotype (BTV-n = putative BTV-27) detected in goats in Corsica, France, in 2014. Sequence analysis confirmed the closest relationship between sequences of the novel BTV serotype and BTV-25 and BTV-26, recently discovered in Switzerland and Kuwait, respectively.

International audience; Since 2000, French Corsica Island has been exposed to the emergence of three different BT virus (BTV) serotypes: serotype 2 in 2000 and 2001, serotype 4 in 2003 and serotype 16 in 2004. Between 2005 and August 2013, no outbreaks have been reported in the French Island. At the beginning of September 2013, sheep located in the south of the island showed clinical signs suggestive of BTV infection. Laboratory analyses identified the virus as BTV serotype 1. Phylogenetic studies showed that the sequences of this strain are closely related to the BTV-1 strain that was circulating in the Mediterranean basin and in Sardinia in 2012.

To the Editor: In 2011, Schmallenberg virus (SBV) emerged in Europe (1); the virus spread into France in January 2012 (2). During January–March 2012, a total of >1,000 cases were reported in France, mainly in stillborn and newborn lambs with congenital malformations. In March 2012, neurologic disorders were detected in five 15-day-old puppies (Belgian shepherd) from a dog breeding kennel in northwestern France (Orne). We report data suggesting that these puppies were infected with SBV. In June 2012, the kennel veterinarian contacted a veterinary school (Unite de Medecine de l’Elevage et du Sport Breeding and Sport Medicine Unit, Maisons-Alfort, France) after neurologic signs of ataxia, exotropia, a head tilt, and stunted growth were observed in a litter of 5 puppies. Four of the puppies had died at 5–6 weeks of age. The veterinarian collected blood samples from the surviving puppy at 3 months of age, and the puppy was euthanized for necropsy. Severe torticollis was observed during the necropsy, but no other macroscopic signs were detected. The brain, including the cerebellum; a part of the spine; and cerebrospinal fluid (CSF) were collected for further investigation. Specific PCR analyses for canine coronavirus, Neospora caninum, Toxoplasma gondii, and canine minute virus were performed on CSF; all test results were negative. The brain tissue was fixed in formalin and processed for histologic examination. Features of degenerative encephalopathy, including neuronal vacuolation, neuropil vacuolation, and minimal gliosis, were observed. Because some clinical signs were evocative of SBV infection and the puppy was born in an area where the virus was circulating actively in cattle and sheep, veterinarians decided to investigate SBV as a possible etiology. Serum samples from the 3-month-old puppy and the dam were tested by virus neutralization test (VNT), according to the protocol used for ruminant serum. The results were negative for the puppy but positive (titer 128) for the mother. Specific competitive SBV ELISA (IDVet, Montpellier, France) against the SBV N protein showed similar results. Real-time reverse transcription PCR (RT-PCR) was performed (3) to detect the presence of the SBV genome in the cerebellum. Because the sample was paraffin-embedded, RNA was extracted from 5-µm sections, as described (4). All of the extracted cerebellum sections had positive test results (cycle threshold range 33−36); the extraction and PCR controls all showed negative results. To confirm these positive results, conventional RT-PCR was used to amplify a 573-nt sequence of the SBV S segment. The amplification product was sequenced, and a BLAST analysis was performed (www.ncbi.nlm.nih.gov/BLAST). An identity of 100% was obtained with the SBV small gene segment from a ruminant (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"KC108860","term_id":"443498566","term_text":"KC108860"}}KC108860). An immunohistochemical assay was also performed; the result was negative. The remaining 7 female dogs in the breeding kennel were tested for SBV in October 2012; 1 showed positive test results by VNT (titer 256), which confirmed that SBV was circulating in the kennel. This positive dam had a litter of puppies in December 2012, but no signs developed, and the puppies were not tested. In March 2013, repeat testing was done on serum samples from the 2 dogs that had shown positive results. Results for both animals were positive by VNT (titers 32 for the dam and 128 for the other dog) and ELISA. Taken together, specific SBV antibodies in the mother and the SBV genome in her puppy suggest that these dogs experienced SBV infection. The absence of detectable SBV antibodies in the puppy in this investigation suggests that transplacental infection occurred before the onset of fetal immune competence. Maternal infection probably occurred in January or February 2012; entomologic monitoring conducted in France showed the presence of Culicoides spp. midges, a vector of SBV, during this period in northwestern France. In addition, because the puppies were born in March 2012 and SBV antibodies were still detectable in the mother in March 2013, the duration of SBV antibodies in dogs appears to be >1 year. In cattle and sheep, the SBV genome persists in an infected fetus and is detectable after birth by real-time RT-PCR, despite gestation length (5,6). Few reports on orthobunyavirus infections in dogs are available. Two serologic studies from the United States (7) and Mexico (8) found antibodies against La Crosse virus, South River virus, and Jamestown Canyon virus in dogs. Two other reports described cases in which La Crosse virus was detected in canine littermates who had clinical encephalitis (9) or neurologic disorders (10). It is unclear if the apparent SBV infection we detected in these dogs was an isolated event or if other cases occurred elsewhere but were not detected because they were not investigated. Further serologic and clinical surveys are needed to estimate SBV prevalence in dogs and the virus’ involvement in the occurrence of neurologic signs in puppies.

International audience; So far, no published data was available concerning the circulation of Bluetongue virus (BTV) in Madagascar. During a survey on Rift Valley Fever, we were able to detect a virus belonging to BTV. Therefore, we conducted a study aiming at characterizing molecularly the BTV isolated and assess the importance of circulation of BTV in Madagascar. A total of 4393 sera from ruminants selected randomly by stratification and sampled in 30 districts of Madagascar were tested for BTV. Moreover, 175 cattle were followed during 11 months.Phylogenetic analyses were performed from virus isolated from unfed pools of mosquitoes.Overall, the estimated mean seroprevalence of infection at the national level was 95.9% (95% CI: [95.2–96.5]) in cattle and 83.7% (95% CI: [81.4–85.9]) in small ruminants. Estimation of incidence rate was 54 per 100 cattle-years assuming that the incidence rate is constant allyear along. Phylogenetic analyses revealed that BTV detected belong to serotype 2.In conclusion, our results showed that BTV is endemic in Madagascar and highly prevalent among cattle. In our study we did not work on the vector involved in transmission of BTV in cattle. Thus, research should be conducted to better describe epidemiology of BTV in Madagascar including vectors and assess economic impact of the disease associated to BTV infections.

International audience; Eradication of bluetongue virus is possible, as has been shown in several European countries. New serotypes have emerged, however, for which there are no specific commercial vaccines. This study addressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheep were randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercial vaccines against BTV strains 2, 4, and 9. A fifth positive control group was given a vaccine against BTV-8. The other 2 groups were unvaccinated controls. Sheep were then challenged by subcutaneous injection of either BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groups developed detectable antibodies against the vaccinated viruses. Furthermore, sheep that received heterologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when compared to unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinated sheep were not as common after challenge with BTV-8. This study shows that heterologous protection can occur, but that it is difficult to predict if partial or complete protection will be achieved following inactivated-BTV vaccination.

International audience; Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter-and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive.

Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate. IMPORTANCE BTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection.

International audience; Recently, a contamination incident was described in which the challenge inoculum used in a bluetongue virus serotype 8 (BTV-8) vaccination trial was contaminated with a BTV-11 virus that was closely related to the Belgian BTV-11 virus from 2008. This study reports the first complete genome sequences of four BTV-11 viruses: the BTV-11 contaminant, BTV-11 reference strain, BTV-11 vaccine strain and a recently isolated BTV-11 field strain from Martinique. Full-genome analysis showed that these viruses belong to serotype 11/nucleotype A and cluster together with other western topotype bluetongue viruses. Detailed comparisons of the genomes further indicated that the contaminant was derived from the BTV-11 reference strain, as they were distinguished by a single synonymous nucleotide substitution. The previously reported partial sequence of genome segment 2 of the Belgian BTV-11 was found to be identical to that of the BTV-11 vaccine strain, indicating that it most likely was the BTV-11 vaccine strain. These findings also suggest that the BTV-11 contaminant and the Belgian BTV-11 are not the same viruses. Finally, comparison of the reference and vaccine strain did not allow determining the amino acid substitutions that contribute to the attenuated phenotype.