D. Vitour

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 (90-93u2009% nucleotide/93-95u2009% 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) (80u2009% nucleotide/86u2009% amino acid) and to BTV-26 (KUW2010/02) (73-74u2009% nucleotide/80-81u2009% 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.

Circulation of bluetongue virus 8 in French cattle, before and after the re-emergence in 2015

Bluetongue virus serotype 8 (BTV-8) re-emerged in Central France in August 2015. The viral strain identified is nearly identical to the one that circulated during the 2006/2009 massive outbreak throughout Europe. To address the question of an undetected BTV-8 circulation on the French territory, a serological study was conducted on young cattle along a transect of seven departments, three of them located in areas where the virus presence had been confirmed by RT-PCR by winter 2015/2016. Sera from 2,565 animals were collected during the winters preceding and following the re-emergence, with 414 animals being sampled in each of the two consecutive years. All samples were tested by competitive ELISA (IDVet) and, when enough serum was available, ELISA-positive samples were confirmed by seroneutralization tests. In areas with infected holdings, seropositive animals were found before the re-emergence (Nxa0=xa014 of 511), significantly more on the following year (Nxa0=xa017 of 257), and eight animals (Nxa0=xa0158) seroconverted over 2015. Seropositive animals were also detected as early as winter 2014/2015 in one department without known infected holdings (Nxa0=xa012 of 150), and in winter 2015/2016 in three of them (Nxa0=xa021 of 555), where seven animals (Nxa0=xa0154) seroconverted over 2015. These results suggest that BTV-8 may have spread at low levels before the re-emergence, even in areas considered virus-free. Unfortunately, whole blood from the seropositive animals was not available to definitely confirm the virus presence by RT-PCR.

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

ABSTRACT 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.

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

ABSTRACT 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.

Complete genome sequence of bluetongue virus serotype 4 that emerged on the French island of Corsica in December 2016

In November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV-4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV-4 has been introduced to Corsica from Italy (Sardinia) where BTV-4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe.

Serological status for BTV-8 in French cattle prior to the 2015 re-emergence

Undetected in Europe since 2010, bluetongue virus serotype 8 (BTV-8) re-emerged in August 2015 in Central France. To gain insight into the re-emergence on the French territory, we estimated the seroprevalence in cattle before the detection of BTV-8 in 2015, in areas differentially affected by the current outbreak. A retrospective survey based on the analysis of stored sera was thus conducted in the winter preceding the re-emergence in seven French departments including the one where the virus was first detected. A total of 10,066 sera were retrieved from animals sampled in 444 different herds in winter 2014/15. Between-herd seroprevalence revealed the presence of seropositive animals in almost all herds sampled (97.4%). The animal-level seroprevalence averaged at 44%, with a strong age pattern reflecting the cumulative exposure to both natural infection and to vaccination. A multivariable analysis allowed separating the respective effects of both exposures. A higher proportion of seropositivity risk was attributed to vaccination (67.4%) than to exposure to natural infection (24.2%). The evolution of seroprevalence induced by the two main risk factors in 74 mainland departments was reconstructed between the vaccination ban (2013) and the re-emergence (2015). We showed a striking decrease in seroprevalence with time after the vaccination ban, due to population renewal, which could have facilitated virus transmission leading to the current outbreak situation.

Bluetongue virus serotype 27: Experimental infection of goats, sheep and cattle with three BTV‐27 variants reveal atypical characteristics and likely direct contact transmission BTV‐27 between goats

Bluetongue virus (BTV) hitherto consisted of 26 recognized serotypes, of which all except BTV-26 are primarily transmitted by certain species of Culicoides biting midges. Three variants of an additional 27th bluetongue virus serotype (BTV-27v01-v03) were recently detected in asymptomatic goats in Corsica, France, 2014-2015. Molecular characterization revealed genetic differences between the three variants. Therefore, inxa0vivo characteristics were investigated by experimental infection of a total of 15 goats, 11 sheep and 4 cattle with any one of the three variants in separated animal trials. In goat trials, BTV-naïve animals of the same species were kept in a facility where direct contact was unhindered. Of the 15 inoculated goats, 13 and 14 animals were found positive for BTV-RNA and antibodies (Ab), respectively, until the end of the experiments. Surprisingly, BTV-Ab levels as measured with ELISA and neutralization test (SNT) were remarkably low in all seropositive goats. Virus isolation from whole-blood was possible at the peak of viremia until 49xa0dpi. Moreover, detection of BTV-27v02-RNA and Ab in one contact goat indicated that-similar to BTV-26-at least one of three BTV-27 variants may be transmitted by contact between goats. In the field, BTV-27 RNA can be detected up to 6xa0months in the whole-blood of BTV-27-infected Corsican goats. In contrast, BTV RNA was not detected in the blood of cattle or sheep. In addition, BTV-27 Abs were not detected in cattle and only a transient increase in Ab levels was observed in some sheep. None of the 30 animals showed obvious BT-like clinical signs. In summary, the phenotypes observed for BTV-27v01-v03 phenotypes correspond to a mixture of characteristics known for BTV-25 and 26.

Fetopathic effects of experimental Schmallenberg virus infection in pregnant goats

Schmallenberg virus (SBV) is an emerging virus responsible for congenital malformations in the offspring of domestic ruminants. It is speculated that infection of pregnant dams may also lead to a significant number of unrecognized fetal losses during the early period of gestation. To assess the pathogenic effects of SBV infection of goats in early pregnancy, we inoculated dams at day 28 or 42 of gestation and followed the animals until day 55 of gestation. Viremia in the absence of clinical signs was detected in all virus-inoculated goats. Fetal deaths were observed in several goats infected at day 28 or 42 of gestation and were invariably associated with the presence of viral genomic RNA in the affected fetuses. Among the viable fetuses, two displayed lesions in the central nervous system (porencephaly) in the presence of viral genome and antigen. All fetuses from goats infected at day 42 and the majority of fetuses from goats infected at day 28 of gestation contained viral genomic RNA. Viral genome was widely distributed in these fetuses and their respective placentas, and infectious virus could be isolated from several organs and placentomes of the viable fetuses. Our results show that fetuses of pregnant goats are susceptible to vertical SBV infection during early pregnancy spanning at least the period between day 28 and 42 of gestation. The outcomes of experimental SBV infection assessed at day 55 of gestation include fetal mortalities, viable fetuses displaying lesions of the central nervous system, as well as viable fetuses without any detectable lesion.

Emergence of bluetongue virus serotype 4 in mainland France in November 2017

In November 2017, a 15-day-old calf located in France (Haute-Savoie department) was found positive for bluetongue virus (BTV) RNA by RT-PCR. Laboratory investigations allowed the isolation and identification of the serotype: BTV-4. The analysis of the full viral genome showed that all the 10 genome segments were closely related to BTV-4 strains involved in a large BT outbreak in the Balkan Peninsula, in Italy since 2014 and in Corsica since the end of October 2016. These results together with epidemiological data suggest that BTV-4 has been introduced to mainland France from Corsica or Italy where BTV-4 outbreaks have been reported in summer and autumn 2016. This is the first report of the introduction of BTV-4 in mainland France.

Development of a Double-Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.