Detection, Characterization and Sequencing of BTV Serotypes Circulating in Cuba in 2022.

In Cuba, despite a high sero-prevalence of bluetongue virus (BTV), circulating serotypes remain unknown. The aim of this study was to identify circulating BTV serotypes in farms throughout the western region of Cuba. Blood samples were collected from 200 young cattle and sheep between May and July 2022 for virological analyses (PCR, viral isolation and virus neutralization) and genome sequencing. The results confirmed viral circulation, with viro-prevalence of 25% for BTV. The virus was isolated from 18 blood samples and twelve BTV serotypes were identified by sequencing RT-PCR products targeting the segment 2 of the BTV genome (BTV-1, 2, 3, 6, 10, 12, 13, 17, 18, 19, 22 and 24). Finally, the full genome sequences of 17 Cuban BTV isolates were recovered using a Sequence Independent Single Primer Amplification (SISPA) approach combined to MinION Oxford Nanopore sequencing technology. All together, these results highlight the co-circulation of a wide diversity of BTV serotypes in a quite restricted area and emphasize the need for entomological and livestock surveillance, particularly in light of recent changes in the global distribution and nature of BTV infections.

Circulation of Bluetongue Virus Serotypes 1, 4, 8, 10 and 16 and Epizootic Hemorrhagic Disease Virus in the Sultanate of Oman in 2020-2021.

The circulation of Bluetongue (BT) and Epizootic Hemorrhagic Disease (EHD) in the Middle East has already been reported following serological analyses carried out since the 1980s, mostly on wild ruminants. Thus, an EHD virus (EHDV) strain was isolated in Bahrain in 1983 (serotype 6), and more recently, BT virus (BTV) serotypes 1, 4, 8 and 16 have been isolated in Oman. To our knowledge, no genomic sequence of these different BTV strains have been published. These same BTV or EHDV serotypes have circulated and, for some of them, are still circulating in the Mediterranean basin and/or in Europe. In this study, we used samples from domestic ruminant herds collected in Oman in 2020 and 2021 for suspected foot-and-mouth disease (FMD) to investigate the presence of BTV and EHDV in these herds. Sera and whole blood from goats, sheep and cattle were tested for the presence of viral genomes (by PCR) and antibodies (by ELISA). We were able to confirm the presence of 5 BTV serotypes (1, 4, 8, 10 and 16) and the circulation of EHDV in this territory in 2020 and 2021. The isolation of a BTV-8 strain allowed us to sequence its entire genome and to compare it with another BTV-8 strain isolated in Mayotte and with homologous BTV sequences available on GenBank.

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

Bluetongue 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.

Serological Responses in Cattle following Booster Vaccination against Serotypes 4 and 8 Bluetongue Virus with Two Bivalent Commercial Inactivated Vaccines.

Since the outbreak of bluetongue in Northern Europe in 2006, numerous outbreaks involving several serotypes have been observed. Since 2008, compulsory or voluntary vaccination campaigns with inactivated vaccines have been carried out to eradicate these serotypes. In France, serotypes 8 and 4 have been enzootic since 2017, and currently, the majority of vaccinations take place in the context of animal movements, to comply with the regulations of the importing countries. Several vaccine manufacturers have developed inactivated vaccines against serotypes 4 and 8 (mono or bivalent). In this study, we investigated and compared the serological responses to a booster vaccination with two different bivalent inactivated vaccines (BTVPUR suspension injectable® 4 + 8, Boehringer Ingelheim or SYVAZUL ® BTV 4 + 8, Biové) following a primary vaccination with BTVPUR® 4 + 8 in the previous year. The results show that using an alternative vaccine for booster vaccination is at least as effective as using the homologous vaccine. Indeed, the antibody response against BTV-8 is higher in the case of a heterologous vaccination and identical for BTV-4. This information could allow more flexibility in the choice of vaccines used for booster vaccination, particularly in cases where homologous vaccines are in short supply or unavailable.

Insight on Bluetongue virus transmission in small ruminants in Senegal.

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.

Epizootic Haemorrhagic Disease Virus Serotype 8 in Tunisia, 2021.

Epizootic haemorrhagic disease (EHD) is a Culicoides-borne viral disease caused by the epizootic haemorrhagic disease virus (EHDV) associated with clinical manifestations in domestic and wild ruminants, primarily white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus). In late September 2021, EHDV was reported in cattle farms in central/western Tunisia. It rapidly spread throughout the country with more than 200 confirmed outbreaks. We applied a combination of classical and molecular techniques to characterize the causative virus as a member of the serotype EHDV-8. This is the first evidence of EHDV- 8 circulation since 1982 when the prototype EHDV-8 strain was isolated in Australia. This work highlights the urgent need for vaccines for a range of EHDV serotypes.

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

Bluetongue 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/ß]) 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-ß 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.

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.

Clinical cases of Bluetongue serotype 8 in calves in France in the 2018-2019 winter.

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.

Bluetongue Virus in France: An Illustration of the European and Mediterranean Context since the 2000s.

Bluetongue (BT) is a non-contagious animal disease transmitted by midges of the Culicoides genus. The etiological agent is the BT virus (BTV) that induces a variety of clinical signs in wild or domestic ruminants. BT is included in the notifiable diseases list of the World Organization for Animal Health (OIE) due to its health impact on domestic ruminants. A total of 27 BTV serotypes have been described and additional serotypes have recently been identified. Since the 2000s, the distribution of BTV has changed in Europe and in the Mediterranean Basin, with continuous BTV incursions involving various BTV serotypes and strains. These BTV strains, depending on their origin, have emerged and spread through various routes in the Mediterranean Basin and/or in Europe. Consequently, control measures have been put in place in France to eradicate the virus or circumscribe its spread. These measures mainly consist of assessing virus movements and the vaccination of domestic ruminants. Many vaccination campaigns were first carried out in Europe using attenuated vaccines and, in a second period, using exclusively inactivated vaccines. This review focuses on the history of the various BTV strain incursions in France since the 2000s, describing strain characteristics, their origins, and the different routes of spread in Europe and/or in the Mediterranean Basin. The control measures implemented to address this disease are also discussed. Finally, we explain the circumstances leading to the change in the BTV status of France from BTV-free in 2000 to an enzootic status since 2018.