"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.

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.

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.

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.

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway.

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 nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). 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 in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. 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 of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection.IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. IMPORTANCE: Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.

Bluetongue virus serotype 27: detection and characterization of two novel variants in Corsica, France.

During the compulsory vaccination programme against bluetongue virus serotype 1 (BTV-1) in Corsica (France) in 2014, a BTV strain belonging to a previously uncharacterized serotype (BTV-27) was isolated from asymptomatic goats. The present study describes the detection and molecular characterization of two additional distinct BTV-27 variants found in goats in Corsica in 2014 and 2015. The full coding genome of these two novel BTV-27 variants show high homology (90-93 % nucleotide/93-95 % amino acid) with the originally described BTV-27 isolate from Corsican goats in 2014. These three variants constitute the novel serotype BTV-27 ('BTV-27/FRA2014/v01 to v03'). Phylogenetic analyses with the 26 other established BTV serotypes revealed the closest relationship to BTV-25 (SWI2008/01) (80 % nucleotide/86 % amino acid) and to BTV-26 (KUW2010/02) (73-74 % nucleotide/80-81 % amino acid). However, highest sequence homologies between individual segments of BTV-27/FRA2014/v01-v03 with BTV-25 and BTV-26 vary. All three variants share the same segment 2 nucleotype with BTV-25. Neutralization assays of anti-BTV27/FRA2014/v01-v03 sera with a reassortant virus containing the outer capsid proteins of BTV-25 (BTV1VP2/VP5 BTV25) further confirmed that BTV-27 represents a distinct BTV serotype. Relationships between the variants and with BTV-25 and BTV-26, hypotheses about their origin, reassortment events and evolution are discussed.

Molecular characterisation of epizootic haemorrhagic disease virus associated with a Tunisian outbreak among cattle in 2006.

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.

Dual modulation of type I interferon response by bluetongue virus.

UNLABELLED: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus that causes an economically important disease in ruminants. BTV infection is a strong inducer of type I interferon (IFN-I) in multiple cell types. It has been shown recently that BTV and, more specifically, the nonstructural protein NS3 of BTV are able to modulate the IFN-I synthesis pathway. However, nothing is known about the ability of BTV to counteract IFN-I signaling. Here, we investigated the effect of BTV on the IFN-I response pathway and, more particularly, the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. We found that BTV infection triggered the expression of IFN-stimulated genes (ISGs) in A549 cells. However, when BTV-infected cells were stimulated with external IFN-I, we showed that activation of the IFN-stimulated response element (ISRE) promoter and expression of ISGs were inhibited. We found that this inhibition involved two different mechanisms that were dependent on the time of infection. After overnight infection, BTV blocked specifically the phosphorylation and nuclear translocation of STAT1. This inhibition correlated with the redistribution of STAT1 in regions adjacent to the nucleus. At a later time point of infection, BTV was found to interfere with the activation of other key components of the JAK/STAT pathway and to induce the downregulation of JAK1 and TYK2 protein expression. Overall, our study indicates for the first time that BTV is able to interfere with the JAK/STAT pathway to modulate the IFN-I response. IMPORTANCE: Bluetongue virus (BTV) causes a severe disease in ruminants and has an important impact on the livestock economy in areas of endemicity such as Africa. The emergence of strains, such as serotype 8 in Europe in 2006, can lead to important economic losses due to commercial restrictions and prophylactic measures. It has been known for many years that BTV is a strong inducer of type I interferon (IFN-I) in vitro and in vivo in multiple cell types. However, the ability of BTV to interact with the IFN-I system remains unclear. Here, we report that BTV is able to modulate the IFN-I response by interfering with the Janus tyrosine kinase (JAK)/signal transducer and activator of transcription protein (STAT) signaling pathway. These findings contribute to knowledge of how BTV infection interferes with the host's innate immune response and becomes pathogenic. This will also be important for the design of efficacious vaccine candidates.

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.

Schmallenberg virus infection among red deer, France, 2010-2012.

Schmallenberg virus infection is emerging in European domestic and wild ruminants. We investigated the serologic status of 9 red deer populations to describe virus spread from September 2010 through March 2012 among wildlife in France. Deer in 7 populations exhibited seropositivity, with an average seroprevalence of 20%.

NS3 of bluetongue virus interferes with the induction of type I interferon.

Upon infection with Bluetongue virus (BTV), an arthropod-borne virus, type I interferon (IFN-I) is produced in vivo and in vitro. IFN-I is essential for the establishment of an antiviral cellular response, and most if not all viruses have elaborated strategies to counteract its action. In this study, we assessed the ability of BTV to interfere with IFN-I synthesis and identified the nonstructural viral protein NS3 as an antagonist of the IFN-I system.

Evidence of excretion of Schmallenberg virus in bull semen.

Schmallenberg virus (SBV) is a novel orthobunyavirus, discovered in Germany in late 2011. It mainly infects cattle, sheep and goats and could lead to congenital infection, causing abortion and fetal abnormalities. SBV is transmitted by biting midges from the Culicoides genus and there is no evidence that natural infection occurs directly between ruminants. Here, we could detect SBV RNA in infected bull semen using qRT-PCR (three bulls out of seven tested positive; 29 positive semen batches out of 136). We also found that highly positive semen batches from SBV infected bulls can provoke an acute infection in IFNAR-/- mice, suggesting the potential presence of infectious virus in the semen of SBV infected bulls.

Spread and impact of the Schmallenberg virus epidemic in France in 2012-2013.

BACKGROUND: The Schmallenberg virus (SBV) emerged in Europe in 2011 and caused a widespread epidemic in ruminants.In France, SBV emergence was monitored through a national multi-stakeholder surveillance and investigation system. Based on the monitoring data collected from January 2012 to August 2013, we describe the spread of SBV in France during two seasons of dissemination (vector seasons 2011 and 2012) and we provide a large-scale assessment of the impact of this new disease in ruminants. RESULTS: SBV impact in infected herds was primarily due to the birth of stillborns or deformed foetuses and neonates. Congenital SBV morbidity level was on average moderate, although higher in sheep than in other ruminant species. On average, 8% of lambs, 3% of calves and 2% of kids born in SBV-infected herds showed typical congenital SBV deformities. In addition, in infected herds, farmers reported retrospectively a lower prolificacy during the vector season, suggesting a potential impact of acute SBV infection during mating and early stages of gestation. CONCLUSIONS: Due to the lack of available control and prevention measures, SBV spread quickly in the naive ruminant population. France continues to monitor for SBV, and updated information is made available online on a regular basis [http://www.plateforme-esa.fr/]. Outbreaks of congenital SBV are expected to occur sporadically from now on, but further epidemics may also occur if immunity at population level declines.

Evidence of Schmallenberg virus circulation in ruminants in Greece.

During March 2013, we investigated the presence and the levels of Schmallenberg virus (SBV) circulation in three dairy cow herds and three sheep flocks in Central Macedonia, Greece. In two cow herds, a high number of abortions had been observed during the winter. Six bulk-tank milk samples and 147 individual sera were screened for SBV-specific antibodies by ELISA. Positive reactions were obtained from 5 out of 6 bulk-tank milk samples, 58 out of 90 sera from the 3 cow herds, and 2 sera from 2 of the 3 sheep flocks. Twenty-two ELISA-positive sera were tested by serum neutralization test (SNT). SNT confirmed the presence of neutralizing antibodies against SBV in all samples tested, with titers ranging between 1:32 and ≥1:256. No neutralizing antibodies against Akabane virus (AKAV) or Shamonda virus (SHAV) were detected, indicating that neutralizing antibodies against SBV do not cross react with AKAV or SHAV in SNT. ELISA testing of bulk-tank milk samples proved to be convenient and reliable. None of the tested sera was found positive for SBV by real-time RT-PCR, indicating that the sampling was conducted past the viremia stage. This is the first report of SBV circulation in Greece.

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.

Sensing and control of bluetongue virus infection in epithelial cells via RIG-I and MDA5 helicases.

Bluetongue virus (BTV), an arthropod-borne member of the Reoviridae family, is a double-stranded RNA virus that causes an economically important livestock disease that has spread across Europe in recent decades. Production of type I interferon (alpha/beta interferon [IFN-α/ß]) has been reported in vivo and in vitro upon BTV infection. However, the cellular sensors and signaling pathways involved in this process remain unknown. Here we studied the mechanisms responsible for the production of IFN-ß in response to BTV serotype 8. Upon BTV infection of A549 cells, expression of IFN-ß and other proinflammatory cytokines was strongly induced at both the protein and mRNA levels. This response appeared to be dependent on virus replication, since exposure to UV-inactivated virus failed to induce IFN-ß. We also demonstrated that BTV infection activated the transcription factors IFN regulatory factor 3 and nuclear factor κB. We investigated the role of several pattern recognition receptors in this response and showed that expression of IFN-ß was greatly reduced after small-interfering-RNA-mediated knockdown of the RNA helicase encoded by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated gene 5 (MDA5). In contrast, silencing of MyD88, Toll-like receptor 3, or the recently described DexD/H-box helicase DDX1 sensor had no or a weak effect on IFN-ß induction, suggesting that the RIG-I-like receptor pathway is specifically engaged for BTV sensing. Moreover, we also showed that overexpression of either RIG-I or MDA5 impaired BTV expression in infected A549 cells. Overall, this indicates that RIG-I and MDA5 can both contribute to the recognition and control of BTV infection.