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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., 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.

© 2020 Pascall et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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 aim of this study was to produce reference sera against the seven serotypes of Epizootic hemorrhagic disease virus (EHDV‑1, EHDV‑2, EHDV‑4, EHDV‑5, EHDV‑6, EHDV‑7, and EHDV‑8). In a high containment unit, seven Prim 'Holstein calves were inoculated at day 0 (D0) with the selected strains (1 EHDV serotype per calf ). Blood samples (EDTA and whole blood) were periodically taken from D0 until the end of the experiment (D31). Sera were tested with two commercially available EHDV competitive ELISAs (c‑ELISA). Viral genome was detected from EDTA blood samples using in‑house real‑time RT‑PCR. Sera taken on D31 post infection (pi) were tested and characterized by serum neutralization test (SNT) and virus neutralization test (VNT) (for calibration of reference sera). Viral RNA was first detected at D2 pi in five calves. All infected animals were RT‑PCR positive at D7 pi. Seroconversion was observed between D10 and D23 pi depending on the EHDV serotype. SNT and VNT have allowed to determine the neutralizing antibody titers of each serum and the potential cross‑reactions between serotypes. The two c‑ELISA used in this study showed similar results. The calibrated sera are now available for the serological identification of an EHDV isolated on tissue culture or to be used as positive control in seroneutralization assay.

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.

Since 1998, Bluetongue virus (BTV) serotypes 1, 2, 4, 6, 8, 9, 11 and 16 have spread throughout Europe. In 2006, BTV serotype 8 (BTV-8) emerged unexpectedly in Northern Europe, in countries such as Belgium, France, Germany, Luxembourg, and the Netherlands, to spread rapidly in the following year throughout the rest of Europe. In 2007, BTV-1 spread in Southern Europe, in Spain and in South of France. In 2008, 2 more BTV serotypes were detected in Northern Europe: BTV-6 in the Netherlands and in Germany, and BTV-11 in Belgium. The European incursion of BTV has caused considerable economic losses, including direct losses from mortality and reduced production, as well as indirect losses generated by ensuing bans on trade of ruminants between infected and non-infected areas. Given the significance of the disease, all affected countries have established control and eradication measures that have evolved together with the availability of detection and prevention tools such as Polymerase Chain Reaction (PCR) tests and vaccines, respectively. This paper describes how the French National Reference Laboratory for BT has managed diagnosis during the fast and massive spread of BTV-1 and 8 in 2007 and 2008.

International audience; Since 1998, Bluetongue virus (BTV) serotypes 1, 2, 4, 6, 8, 9, 11 and 16 have spread throughout Europe. In 2006, BTV serotype 8 (BTV-8) emerged unexpectedly in Northern Europe, in countries such as Belgium, France, Germany, Luxembourg, and the Netherlands, to spread rapidly in the following year throughout the rest of Europe. In 2007, BTV-1 spread in Southern Europe, in Spain and in South of France. In 2008, 2 more BTV serotypes were detected in Northern Europe: BTV-6 in the Netherlands and in Germany, and BTV-11 in Belgium. The European incursion of BTV has caused considerable economic losses, including direct losses from mortality and reduced production, as well as indirect losses generated by ensuing bans on trade of ruminants between infected and non-infected areas. Given the significance of the disease, all affected countries have established control and eradication measures that have evolved together with the availability of detection and prevention tools such as Polymerase Chain Reaction (PCR) tests and vaccines, respectively. This paper describes how the French National Reference Laboratory for BT has managed diagnosis during the fast and massive spread of BTV-1 and 8 in 2007 and 2008.

Objective: Bluetongue (BT) and epizootic haemorrhagic disease (EHD) are arthropod-borne diseases of wild and domestic ruminants caused respectively by viruses belonging to the species Bluetongue virus (BTV) and Epizootic haemorrhagic disease virus (EHDV) within the genus Orbivirus of the Reoviridae. The viruses are transmitted between ruminants by biting midges of the genus Culicoides (Diptera: Ceratopogonidae). BTV went undetected in Reunion Island between its first documented emergence in 1979 and two other serious outbreaks with both BTV-3/ EHDV-6 in 2003, and both BTV-2/EHDV-6 in 2009. In these outbreaks, infected animals developed symptoms including hyperthermia, anorexia, congestion, prostration and nasal discharge. In order to get an overview of the circulation of BT/EHD in Reunion island, an assessment of the prevalence in ruminants native to Reunion Island by a cross-sectional study was undertaken in2011on 67 farms, including a total of 276 cattle, 142 sheep and 71 goats with a total of 489 ruminant samples. Data concerning farm characteristics, type of production, and number of animals were collected through farmer questionnaires for an evaluation of the associated risk factors. In addition, investigation of clinical cases based on the observation of clinical signs was also performed in order to get BTV/EHDV isolates with the aim to track the origins of the circulating strains. Methods: Risk factors analysis Data concerning farm characteristics, type of production, number of animals, closeness to another farm and sugar cane fields, presence of organic and other waste on the farm, exposure to wind, distance to a permanent water point, type of animal housing, presence of ticks on animals, use of treatment against ectoparasites and insects, animal's contacts with other animals or humans, grazing practice, spreading of manure on pastures, presence of Tenrece caudatus, rodent control, number of abortions in the herd in the last 12 months, purchasing behaviour, quarantine of newly purchased animals, other biosecurity factors like hygienic precautions taken by the staff or other people entering the farm (truck driver, vets and other visitors) were taken from a questionnaire which was filled in during an interview with the farmers. This questionnaire was pre-tested on five farms in a preliminary study. The final questionnaire comprised 40 questions of which 75% were closed-ended. Serological assays Specific anti-BTV antibodies were tested in serum samples with a group-specific competitive ELISA based on the VP7 protein using a commercial kit (LSIVetTM Ruminant BT Advanced II- Serum, Life technologies, France). Specific anti-EHDV antibodies were tested using a blocking commercial kit (LSIVetTM Ruminant EHDV-Serum ELISA kit, Life technologies, France). A Sunrise ELISA reader was used for reading at 450 nm (Tecan, France). Optical density values were converted to percentage inhibition (PI). According to the cut-off value of the test, test samples with PI values > 40% for BT and > 60% for EHD were considered as positive. BTV/EHDV genome detection For the BTV group specific real-time RT-PCR, 6 μl of denatured double-stranded RNA prepared with the EZ1 robot and EZ1® Virus Mini Kit v2.0 (Qiagen, France) were reverse transcribed (RT) and amplified using the onestep QuantiTect Probe RT-PCRkit (Qiagen, France) based on segment 1 developed by Toussaint et al. 2007. For the EHDV group specific real-time RT-PCR, 5 μl of denatured double-stranded RNA were reverse transcribed (RT) and amplified using the commercial TaqVetTM EHDV (Life technologies, France).The subgroup-specific EHDV RTPCR based on segment 2 was performed according to Sailleau et al., 2012.Embryonated chicken eggs (ECE) were each inoculated as previously described in Sailleau et al., 2012 Sequence analysis, alignment and phylogenetic analysis To identify the genetic relatedness of the detected virus, phylogenetic analyses were performed with published EHDV sequences. Sixteen full-length VP2 gene sequences were cleaned by hand from the results of several BLAST nucleotide searches as well as direct references from available up-to-date literature and then aligned using the ClustalW translation alignment tool in MEGA (Ver. 5.05). Phylogenetic analysis was performed using the neighbour-joining method using distance measures generated by the p-distance algorithm running 1, 000 iterations with Geneious® Pro. Statistics A Fisher exact test was used to compare differences in prevalence between diseases and species. All statistical procedures were performed using R.3.0.1. A value of P < 0.05 was considered significant. The prevalence rates were estimated as the overall mean and 95% confidence interval (CI). Results: The observed EHD prevalence rate in cattle was 63.77% (95% CI [57.99–69.55]), 5.63% (95% CI [0.03–10.99]) in goats, and 3.70% (95% CI [0.05–6.88]) in sheep, suggesting that EHD occurs more often in cattle than in goats and sheep. These findings were supported by a significant statistical difference in the EHD prevalence rate between species (Fisher exact test, P <

Le 2 septembre 2013, des signes cliniques évocateurs de fièvre catarrhale ovine (FCO) ont été observés dans deux élevages ovins en Corse. Les analyses du laboratoire national de référence en virologie pour la FCO ont confirmé la présence du sérotype 1 du virus de la FCO. L'épizootie s'est ensuite propagée à l'ensemble de l'île. Au 12 novembre 2013, plus de 120 élevages, essentiellement ovins, étaient déclarés infectés. L'impact de la maladie dans ces élevages a été très variable. La localisation des premiers foyers dans le sud de l'île et le séquençage du virus suggèrent que le virus a été introduit depuis la Sardaigne. Pour lutter contre cette épizootie, l'État a mis en place une campagne de vaccination obligatoire de l'ensemble de la population ovine, caprine et bovine de l'île.

La Guadeloupe et la Réunion sont deux départements d'Outre-mer dans lesquels la fièvre catarrhale ovine est enzootique. Depuis 2010, il a été démontré qu'une autre arbovirose transmise par des Culicoides, la maladie épizootique hémorragique des cervidés est aussi présente dans ces îles. Cet article présente les particularités de l'épidémiologie de ces maladies en distinguant les quatre déterminants principaux de l'épidémiologie des maladies vectorielles : animaux/environnement/espèces vectrices de Culicoides/virus. Ces comparaisons montrent la pertinence de la notion de "pathosystème" qui illustre les différences observées pour une même maladie dans des contextes épidémiologiques différents.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropodborne virus of ruminants, emerged in livestock in northern Europe in 2006, preading 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.