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.

Experimental infection of cattle, sheep, and goats with the newly emerged epizootic hemorrhagic disease virus serotype 8.

Epizootic hemorrhagic disease virus serotype 8 (EHDV-8) emerged in Europe for the first time in late 2022. In this study, we investigated the kinetics of EHDV-8 infection in cattle, sheep, and goats.  Following experimental infection with EHDV-8, four out of five calves displayed fever, while another calf exhibited ulcerative and crusty lesions of the muzzle. RNAemia peaked at day 7 post infection in all calves and remained relatively stable till the end of the study, at 78 days post infection. Infectious virus was isolated up to 21 days post infection in one calf. As far as small ruminants are concerned, one sheep experienced fever and two out of five had consistent RNAemia that lasted until the end of the study. Remarkably, infectious virus was evidenced at day 7 post infection in one sheep. In goats, no RNA was observed. All infected animals seroconverted, and a neutralizing immune response was observed in all species, with calves exhibiting a more robust response than sheep and goats. Our study provides insights into the kinetics of EHDV-8 infection and the host immune responses. We also highlight that sheep may also play a role in EHDV-8 epidemiology. Altogether, the data gathered in this study could have important implications for disease control and prevention strategies, providing crucial information to policy makers to mitigate the impact of this viral disease on livestock.

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.

Epizootic Hemorrhagic Disease Virus Serotype 8, Italy, 2022.

We describe the detection of epizootic hemorrhagic disease virus (EHDV) serotype 8 in cattle farms in Sardinia and Sicily in October-November 2022. The virus has a direct origin in North Africa; its genome is identical (>99.9% nucleotide sequence identity) to EHDV serotype 8 strains detected in Tunisia in 2021.

Emergence of epizootic hemorrhagic disease in Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

[Emergence of epizootic hemorrhagic disease in Europe]. / Émergence de la maladie hémorragique épizootique en Europe.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-borne disease transmitted by blood-sucking midges of the genus Culicoides. It affects domestic and wild ruminants, mainly white-tailed deer and cattle. At the end of October and in November 2022, outbreaks of EHD were confirmed in several cattle farms in Sardinia and Sicily. This is the first detection of EHD in Europe. The loss of free status and the lack of effective prophylactic measures could have significant economic consequences for infected countries.

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.

African horse sickness: an ancient disease for a current threat / La peste équine : une maladie ancienne pour une menace actuelle

African horse sickness (AHS) is a major arthropod-borne disease that causes significant losses in horses in sub-Saharan Africa. It is caused by the African horse sickness virus (AHSV), which is transmitted during a blood meal by Culicoides biting midges. The distribution of historical African culicoid vectors increases due to global warming. In addition, recent (Thailand, 2020) and earlier (Iberian Peninsula, 1965-66/1987-90) AHS outbreaks outside Africa demonstrate the adaptation of the virus to endogenous species in AHS-free regions, similar to what has been observed for bluetongue disease in recent decades. Therefore, many regions are considered at risk of introduction of AHS which could have important economic consequences for the equine industry. Overall, this prone the European Union to launch research programs to get better diagnostic and prophylactic tools.

La peste équine est une arbovirose majeure qui entraîne des pertes importantes chez les chevaux en Afrique subsaharienne. Elle est provoquée par le virus de la peste équine (African horse sickness virus, AHSV) dont la transmission s'effectue au cours d'un repas sanguin par des petits moucherons hématophages appartenant au genre Culicoides. En outre, les espèces vectrices historiques de culicoïdes présentes en Afrique voient leur aire de répartition s'étendre en lien avec le réchauffement climatique à l'échelle mondiale. Par ailleurs, des épisodes épizootiques récents (Thaïlande, 2020) ou un peu plus anciens (péninsule ibérique, 1965-66/1987-90) en dehors du continent africain soulignent la capacité d'adaptation du virus à des espèces vectrices autochtones, à l'instar de ce qui a été observé pour la fièvre catarrhale ovine ces dernières décennies. Ces facteurs laissent craindre à tout moment une introduction de la peste équine dans des régions indemnes. L'urgence est donc donnée actuellement par l'Union européenne pour se doter de meilleurs outils diagnostiques et prophylactiques afin de prévenir des conséquences économiques brutales pour l'industrie équine.

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.

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.

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.

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.

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.

The Genome Segments of Bluetongue Virus Differ in Copy Number in a Host-Specific Manner.

Genome segmentation is mainly thought to facilitate reassortment. Here, we show that segmentation can also allow differences in segment abundance in populations of bluetongue virus (BTV). BTV has a genome consisting in 10 segments, and its cycle primarily involves periodic alternation between ruminants and Culicoides biting midges. We have developed a reverse transcription-quantitative PCR (RT-qPCR) approach to quantify each segment in wild BTV populations sampled in both ruminants and midges during an epizootic. Segment frequencies deviated from equimolarity in all hosts. Interestingly, segment frequencies were reproducible and distinct between ruminants and biting midges. Beyond a putative regulatory role in virus expression, this phenomenon could lead to different evolution rates between segments.IMPORTANCE The variation in viral gene frequencies remains a largely unexplored aspect of within-host genetics. This phenomenon is often considered to be specific to multipartite viruses. Multipartite viruses have segmented genomes, but in contrast to segmented viruses, their segments are each encapsidated alone in a virion. A main hypothesis explaining the evolution of multipartism is that, compared to segmented viruses, it facilitates the regulation of segment abundancy, and the genes the segments carry, within a host. These differences in gene frequencies could allow for expression regulation. Here, we show that wild populations of a segmented virus, bluetongue virus (BTV), also present unequal segment frequencies. BTV cycles between ruminants and Culicoides biting midges. As expected from a role in expression regulation, segment frequencies tended to show specific values that differed between ruminants and midges. Our results expand previous knowledge on gene frequency variation and call for studies on its role and conservation beyond multipartite viruses.

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-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. Although 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 long period without replication, we hypothesise that the second outbreak could have been 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.

Evaluation of a commercial ELISA for detection of epizootic haemorrhagic disease antibodies in domestic and wild ruminant sera.

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.

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.

Red deer (Cervus elaphus) Did Not Play the Role of Maintenance Host for Bluetongue Virus in France: The Burden of Proof by Long-Term Wildlife Monitoring and Culicoides Snapshots.

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.