Bluetongue and epizootic hemorrhagic disease in La Réunion; a burden on ruminant farming

Since 2002, the livestock of Réunion Island has been subject to reoccurring outbreaks of Bluetongue (BT) and Epizootic Hemorrhage Disease (EHD). To find out a solution to this problem, that can have severe financial implications to ruminant farmers, a study was carried out between March and September of 2011. The aims of this study were: (1) to confirm the circulation of the viral agents in relation to both host reservoirs and insect vectors; (2) to assess the health status of Réunion Island's livestock; and (3) to identify major risk factors pertaining to ruminant farming due to these viruses. A total of 254 cows from 51 farms and 206 sheep and goats were chosen randomly for testing. Multiple blood samples were taken from all sampled animals. Farming procedures and environmental conditions were assessed for each farm via a questionnaire. Any animal exhibiting clinical symptoms of viral infection was also tested. Serology was done using commercially available LSIVET kits, viral detection was performed via RT-PCR and genotyping was performed externally. Logistic regression was used to assess potential risk factors after a fist of univariate analysis (Chi2). Serology studies suggested that ~50% of ruminants were positive for Bluetongue and ~40% for EHD. EHDV-1 virus was detected in ~5% of animals and bluetongue variant 2 was detected in ~1%. Statistical analysis showed that the major risk factors for Bluetongue and EHD viral infection of ruminant livestock include; the category of the reared livestock, the presence of organic waste, the presence of a water source and the proximity of another farm. Those results are the first step to a better understanding of what could happen in the futur in Western Europe. (Texte intégral

Fièvre catarrhale ovine en Europe en 2014 : épizootie dans les Balkans, progression de la circulation en Italie et en Espagne

L'année 2014 a été caractérisée par une situation épidémiologique nouvelle vis-à-vis de la fièvre catarrhale ovine (FCO) en Europe. Suite à la première notification de foyers de sérotype 4 (BTV-4) en Grèce dans la région du Péloponnèse (mai 2014), onze pays de la région des Balkans ont été touchés par l'épizootie de BTV-4 avec un total de 6 485 foyers déclarés. Fin novembre 2014, en Italie, 25 foyers dus au BTV-4 avaient été confirmés. Une diffusion du virus BTV-1 a de plus été observée dans la partie continentale du pays. En Espagne, les premières suspicions impliquant une souche différente de BTV-4 ont été déclarées en septembre 2014. Aucun lien épidémiologique n'existe cependant avec l'épizootie dans les Balkans. Début décembre, 351 foyers avaient été déclarés en Espagne en dehors de la zone de restriction pour le BTV-4. Par ailleurs, sept foyers de BTV-1 ont été déclarés dans le sud du pays. Au vu des stratégies de lutte appliquées par les pays touchés, il ne fait aucun doute que la vaccination de masse reste le seul moyen réellement efficace de lutte contre la FCO. Les traitements insecticides des animaux permettent au mieux de limiter la transmission et ralentir la diffusion, sans la stopper

The 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

Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge.

African horse sickness virus (AHSV) is an orbivirus, a member of the Reoviridae family. Nine different serotypes have been described so far. AHSV is vectored by Culicoides spp. to equids, causing high mortality, particularly in horses, with considerable economic impacts. For development of a safe attenuated vaccine, we previously established an efficient reverse genetics (RG) system to generate Entry Competent Replication-Abortive (ECRA) virus strains, for all nine serotypes and demonstrated the vaccine potential of these strains in type I interferon receptor (IFNAR)-knockout mice. Here, we evaluated the protective efficacies of these ECRA viruses in AHSV natural hosts. One monoserotype (ECRA.A4) vaccine and one multivalent cocktail (ECRA.A1/4/6/8) vaccine were tested in ponies and subsequently challenged with a virulent AHSV4. In contrast to control animals, all vaccinated ponies were protected and did not develop severe clinical symptoms of AHS. Furthermore, the multivalent cocktail vaccinated ponies produced neutralizing antibodies against all serotypes present in the cocktail, and a foal born during the trial was healthy and had no viremia. These results validate the suitability of these ECRA strains as a new generation of vaccines for AHSV

Surveillance et lutte contre l'épizootie 2013 de fièvre catarrhale ovine de sérotype 1 en Corse

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

Bluetongue and epizootic haemorrhagic disease viruses in Reunion Island

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 <<2.2e-16). The observed BT prevalence rate in cattle was 79.62% (95% CI [74.77– 84.47]), 50.70% in goats (95% CI [39.08–62.33]) and 21.48% in sheep (95% CI [14.55–28.40]) with a significant difference in BT prevalence between species (Fisher exact test, P = 4.367e-10). Additionally, three suspected outbreaks occurred during the 2011 study period, one BTV/EHDV negative, one BTV specific and one combined BTV/EHDV outbreak. In total, 14 EHDV positive cases and 1 BTV/EHDV co - infection case were identified. Two further suspected outbreaks were confirmed to involve EHDV and BTV/EHDV. Isolations of EHDV were successful resulting in the identification of the Reunion -specific EHDV-1 serotype. Phylogenetic analyses of segment 2 showed that the Reunion isolate 6010 _2011 belongs to the group C (hypothesised in Anthony et al. 2009 together with EHDV-1 strains from Australia, 1995, Nigeria, 1967, French Guyana, 2011 and New Jersey, USA, 2011). In January 2014, once more suspected outbreaks occurred on cattle with observed clinical signs such as hyperthermia, congestion and nasal discharge. Virus isolations were successful and led us to identify a new EHDV serotype for Reunion island, the EHDV-7 serotype. Conclusion: Our results confirm that the prevalence of both BT and EHD is high and that both are likely currently circulating. A high risk of BTV and EHDV infections was associated with the introduction of ruminants from neighbouring farms without quarantine, the presence of organic and other waste on the farm, and treatment against ectoparasites and insects. New circulating EHDV serotype 1 and serotype 7 of unknown origin were isolated in 2011 and 2014 respectively. The mechanisms involved in the introduction, maintenance, and perpetuation of both BTV and EHDV orbiviruses in Reunion Island need to be further investigated. How and when the EHDV serotypes were introduced onto the island are unknown, the most likely being the introduction of infected animals from eastern and southern Africa, Madagascar or Australia over a period of many years. The introduction of Malagasy breeds, which could be considered as orbivirus susceptible breeds many decades ago, is one possible hypothesis. Since 1976, importation of domestic ruminants from these countries has stopped. Until 2008, imports were only from mainland France. The maintenance of both viruses in the livestock population could also be due to the presence of reservoirs such as deer as was the case in many places including southern California between 1990 and 2007 (Roug et al., 2012). Pathogens can easily be shared between wildlife and domestic ruminants which has implications for both the animal production industry and wildlife health. Whether animal reservoirs such as Rusa deer Cervus timorensis rusa imported from Mauritius Island and now present in Reunion Island play a role in EHDV epidemiology need to be investigated. The same species of Rusa deer was introduced on the island of Mauritius in 1639 and serological evidence of both EHDV and BTV circulation is documented. Since 1992, in accordance with European Union regulations, importation of live deer from Mauritius to Reunion Island is forbidden. The intermittent detection of certain serotypes and the occasional appearance of new serotypes suggest that, in the past, regular but separate introductions of BTV/EHDV may have also taken place from Madagascar, and from Southeast Asia including Mauritius via windborne Culicoides. Although it exists, the observed herd immunity in Reunion Island is not high enough to prevent the maintenance of an enzootic cycle, which could also be related to the abundance and activity of Culicoides throughout the year. The findings reported here provide additional hypotheses regarding the ecological characteristics of bluetongue and epizootic haemorrhagic disease and other vector -borne livestock diseases. Sentinel surveillance programmes are a useful way of documenting regionalization zones for diseases, which can be of great importance when securing livestock international markets. (Résumé d'auteur

Recombinant capriposviruses expressing proteins of bluetongue virus : evaluation of immune response and protection in small ruminants

Bluetongue is an infectious arthropod-borne viral disease affecting sheep, domestic and wild ruminants caused by Bluetongue Virus (BTV) and transmitted by few species of Culicoides (Diptera: Ceratopogonidae). Since 1998, outbreaks of BT involving 6 distinct serotypes have occurred in the Mediterranean basin. The only BTV vaccines currently available are serotype specific. Objectives: The development of recombinants capripoxvirus for protective immunization of ruminants against bluetongue virus (BTV) infection is described here. Methodology: Sheep (n=11) and goats (n=4) were immunized with bluetongue recombinant capripoxvirus (BTV-Cpox) expressing individually four different genes encoding two capsid proteins (VP2 and VP7) and two non-structural proteins (NS1, NS3) of Bluetongue virus serotype 2 (BTV-2). Results: Seroconversion was observed against NS3, VP7 and VP2 in both groups of animals. A specific BTV antigens lymphoproliferation observed in goats corroborates with a partial protection in sheep challenged with a virulent strain of BTV-2

Évolution de la situation épidémiologique de la fièvre catarrhale ovine en Europe de 2014 à 2017

Depuis 2014, de nombreux foyers de fièvre catarrhale ovine (FCO) sont déclarés en Europe chaque année, principalement de sérotype 4, mais aussi de sérotype 1 (Italie, Croatie, Espagne, Portugal) et de sérotype 8 (France, Chypre, Suisse). On observe une diminution du nombre de foyers de FCO-1 depuis 2014, ainsi qu'une augmentation du nombre de foyers de FCO-4 et, depuis 2015, de FCO-8. L'année 2017 a été marquée par une importante épizootie de sérotype 4 qui a frappé la Sardaigne et la Corse de fin juin à décembre 2017 et l'introduction de ce sérotype 4 en France continentale. Le sérotype 2 a été identifié en Italie en 2014, le sérotype 3 a été détecté pour la première fois en Europe en Sicile en 2017, et le sérotype 16 a été signalé à Chypre en 2014, puis en Grèce et en Turquie à partir de septembre 2017. La situation épidémiologique de la FCO en Europe est donc complexe et le maintien d'un niveau de vigilance élevé est nécessaire car le changement climatique, l'évolution des aires de distribution des insectes vecteurs et les mouvements d'animaux constituent des facteurs de risque d'introduction de nouveaux sérotypes en Europe