Corinne Sailleau

Bluetongue in Belgium, 2006.

Bluetongue has emerged recently in Belgium. A bluetongue virus strain was isolated and characterized as serotype 8. Two new real-time reverse transcription–quantitative PCRs (RT-qPCRs) that amplified 2 different segments of bluetongue virus detected this exotic strain. These 2 RT-qPCRs detected infection earlier than a competitive ELISA for antibody detection.

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.

Bluetongue virus targets conventional dendritic cells in skin lymph.

ABSTRACT Bluetongue virus (BTV) is the etiological agent of bluetongue, a hemorrhagic disease of ruminants (particularly sheep), which causes important economic losses around the world. BTV is transmitted primarily via the bites of infected midges, which inject the virus into the ruminants skin during blood feeding. The virus initially replicates in the draining lymph node and then disseminates to secondary organs where it induces edema, hemorrhages, and necrosis. In this study, we show that ovine conventional dendritic cells (cDCs) are the primary targets of BTV that contribute to the primary dissemination of BTV from the skin to draining lymph nodes. Lymph cDCs support BTV RNA and protein synthesis, as well as the production of infectious virus belonging to several different BTV serotypes, regardless of their level of attenuation. Afferent lymph cell subsets, other than cDCs, showed only marginal levels of BTV protein expression. BTV infection provoked a massive recruitment of cDCs to the sheep skin and afferent lymph, providing cellular targets for infection. Although BTV productively infects cDCs, no negative impact on their physiology was detected. Indeed, BTV infection and protein expression in cDCs enhanced their survival rate. Several serotypes of BTV stimulated the surface expression of the CD80 and CD86 costimulatory molecules on cDCs as well as the mRNA synthesis of cytokines involved in inflammation and immunity, i.e., interleukin-12 (IL-12), IL-1β, and IL-6. BTV-infected cDCs stimulated antigen-specific CD4 and CD8 proliferation as well as gamma interferon production. BTV initially targets cDCs while preserving their functional properties, reflecting the optimal adaptation of the virus to its host cells for its first spread.

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.

Generation of Replication-Defective Virus-Based Vaccines That Confer Full Protection in Sheep against Virulent Bluetongue Virus Challenge

ABSTRACT The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.

Identification of bluetongue virus serotype 2 (Corsican strain) by reverse-transcriptase PCR reaction analysis of segment 2 of the genome.

In October 2000, bluetongue virus was detected on the French island of Corsica. The disease was also reported in Sardinia, Calabria, Sicily and on the Spanish islands of Majorca and Minorca. This paper describes the use of molecular techniques for a rapid identification and serotype determination of serotype 2 of the virus. The nucleotide sequences of segments 2 and 7 of the genome of the Corsican strain were determined and its phylogenetic relationships are described.

Complete Coding Genome Sequence of Putative Novel Bluetongue Virus Serotype 27

ABSTRACT 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.

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.

Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

This paper describes the first RT-PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT-PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT-PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease.

Outbreak of epizootic haemorrhagic disease on the island of Réunion

From January 2003 to April 2003, several cases of disease in cattle which had never been observed on the island before occurred over a large part of the island. The clinical signs were similar to those of infection with bluetongue virus but there was no evidence of clinical disease in sheep on the island. The disease in cattle was characterised by pyrexia (40°C), hyperaemia, oral ulcerations, ptyalism and excessive nasal and ocular secretion. Affected animals presented with torpor and lameness. In some cases, death occurred in eight to 10 days, but the mortality rate was not determined. The morbidity rate in the infected cattle was approximately 7 per cent. During the epizootic, which involved 3607 cattle, 235 clinical cases of EHD were reported. The sheep population of Réunion is very limited (a total of 2000 animals in 18 flocks); the cattle and goat populations are estimated at 30,000 and 40,000 animals, respectively. EDTA-blood samples collected from 24 cattle displaying signs of acute disease were sent to the authors’ laboratory. An aliquot of each EDTA-blood sample was used for virus isolation or RNA extraction. Reverse-transcriptase PCRs (RTPCRs)were carried out, using primers selected to amplify the S7 and S10 regions of bluetongue virus (Zientara and others 2002, Bréard and others 2003). RT-PCRs were also carried out to amplify the S7 and S10 regions of EHDV, using primers that were selected from sequence data on S7 or S10 of EHDV available on GenBank. No amplification products were obtained with the bluetongue-specific primers. In contrast, 1162 base pair (bp) and 809 bp amplification products were obtained with the EHDV S7and S10-specific primers, respectively. All the complete S7 nucleotide sequences were found to be 1162 bp and to have a single open reading frame encoding a predicted protein of 349 residues. The S7 sequences were compared with those available on GenBank (Table 2); 77 per cent and 77·1 per cent homology, respectively, were found at the nucleotide level with EHDV serotypes 1 and 2, which increased to 94·3 per cent and 94·9 per cent, respectively, when the amino acid sequences were compared. The smallest gene (S10) of EHDV is expressed as two proteins in virus-infected cells. The nucleotide sequence of S10 of EHDV contains two in-frame initiation codons, which allow the translation of proteins analogous to NS3 and NS3A of bluetongue viruses (Jensen and others 1994). The nucleotide sequence of S10 of the EHDV strain from Réunion showed 87·3 per cent and 87·8 per cent homology, respectively, when compared with S10 of EHDV serotypes 1 and 2 (Table 2). The predicted amino acid sequence of the NS3 from the Réunion EHDV strain showed 96·9 per cent and 96·5 per cent similarity when compared with EHDV serotypes 1 and 2, respectively. For virus isolation, embryonated chicken eggs were inoculated with aliquots of blood samples which had been stored. The embryonated chicken eggs were incubated for five to seven days at 35°C. They were removed and homogenised in Eagle’s minimum essential medium supplemented with antibiotics. The tissue homogenates were clarified by centrifugation and BHK-21 cells were inoculated with supernatants (Bréard and others 2003). The embryonated eggs were not killed by the blood inoculum five days after the samples had been inoculated, and no lesions were observed in them. However, after two passages through BHK-21 cells, a characteristic cytopathic effect of a viral infection was observed. On the basis of the results of PCR using the EHDV-specific S7 and S10 primers, the virus was identified as EHDV. The virus strain and three sera sampled from acutely affected cattle were sent to the Institute for Animal Health (Pirbright Laboratory), which confirmed the virus as EHDV by ELISA and demonstrated detectable antibody to EHDV in the sera (data not shown). Serotyping of the Réunion EHDV isolate is under investigation. Preliminary results of segment 2 (S2) sequencing have shown that the nucleotide sequence has only 27·6 per cent and Outbreak of epizootic haemorrhagic disease on the island of Réunion