"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.

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.

Turnover Rate of NS3 Proteins Modulates Bluetongue Virus Replication Kinetics in a Host-Specific Manner.

UNLABELLED: 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.

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.

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%.

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.

Analysis by high throughput sequencing of Specific Pathogen Free eggs.

Specific Pathogen Free (SPF) embryonated eggs are used for the production of many veterinary and human vaccines. We have used High Throughput Sequencing to screen allantoic fluids and embryos for the presence of encapsidated viral genomes and viral transcripts, respectively. SPF eggs from two different producers were tested. We evidenced sequences corresponding to known endogenous retroviruses and sequences of Avian Leukosis Virus, but no sequence that might suggest a productive infection of eggs with a virus even distant from known viruses. Our results strongly suggest that SPF eggs such as those used for this study represent a safe substrate for the production of vaccines.

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.

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.

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.

Generation of replication-defective virus-based vaccines that confer full protection in sheep against virulent bluetongue virus challenge.

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.

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.

Colostral antibody induced interference of inactivated bluetongue serotype-8 vaccines in calves.

Since its introduction into northern Europe in 2006, bluetongue has become a major threat to animal health. While the efficacy of commercial vaccines has been clearly demonstrated in livestock, little is known regarding the effect of maternal immunity on vaccinal efficacy. Here, we have investigated the duration and amplitude of colostral antibody-induced immunity in calves born to dams vaccinated against bluetongue virus serotype 8 (BTV-8) and the extent of colostral antibody-induced interference of vaccination in these calves. Twenty-two calf-cow pairs were included in this survey. The median age at which calves became seronegative for BTV was 84 and 112 days as assayed by seroneutralisation test (SNT) and VP7 BTV competitive ELISA (cELISA), respectively. At the mean age of 118 days, 13/22 calves were immunized with inactivated BTV-8 vaccine. In most calves vaccination elicited a weak immune response, with seroconversion in only 3/13 calves. The amplitude of the humoral response to vaccination was inversely proportional to the maternal antibody level prior to vaccination. Thus, the lack of response was attributed to the persistence of virus-specific colostral antibodies that interfered with the induction of the immune response. These data suggest that the recommended age for vaccination of calves born to vaccinated dams needs to be adjusted in order to optimize vaccinal efficacy.

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.