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Background: Recent identification of viruses considered responsible for Theiler’s disease (acute hepatitis) in horses include equine parvovirus-hepatitis (EqPV-H) and the most likely of several flaviviruses, equine hepacivirus (EqHV). Both of these viruses have now been detected in North America, Asia, and Europe. Objectives: To determine the prevalence of EqPV-H and EqHV in French and Australian horses and to analyse their respective phylogenetic relationships. Study design: Cross-sectional. Methods: Clinically documented sera from 188 Australian horses and 259 French horses collected between 2016-2019 were analysed. qPCRs validated according to the NF47-600 standard were used to detect EqPV-H and EqHV in sera. Sequencing for phylogenetic analysis was performed on two NS1 fragments (516 nt and 587 nt) for EqPV-H and one NS5B fragment (308 nt) for EqHV. A phylogenic network was built using a Median Joining Network algorithm. Results: 1) EqPV-H: 12/259 (4.6%) and 6/188 (3.2%) samples from French and Australian horses were positive, respectively. Viral loads ranged from 2.3e05 to 6.5e05 copies genome/ml. 2) EqHV: 5/259 (1.9%) and 21/188 (11.2%) samples from French and Australian horses were positive, respectively. Viral loads ranged from 6.1e04 to 1.4e08 copies genome/ml. Both EqPV-H (587 nt fragment) and EqHV phylogenetic analyses showed that Australian strains were clustered, while overall distribution was more heterogenous in French horses. Main limitations: The study population is built by accessing laboratory submissions and potentially biased. The 516 nt NS1 fragment could not be sequenced. Conclusions: This is the first report of EqPV-H detection in both Australian and French horses. The EqPV-H prevalence was similar between the two countries (p-value=0.6) although phylogenetic differences were observed between the two populations. This is also the first reported detection of EqHV in Australian horses, with a higher prevalence when compared with French horses.

International audience; Background: Equine coital exanthema (ECE) is an acute viral infection of horses caused by equid herpesvirus-3 (EHV-3). The presence of the virus and its economic impact is well described in different countries such as Argentina or Japan. However, even though it is included in the HBLB code of practice (UK) and in the list of diseases monitored by the RESPE (FR), only a few studies have been performed in Europe.Objectives: To determine the circulation of EHV-3 in France and measure the efficacy of new antiviral compounds against EHV-3.Study design: Retrospective field study and in vitro experiments.Methods: 71 field samples from suspected ECE cases, and collected between 2010 and 2021, were analysed by qPCR (DNA polymerase gene) and phylogenetic analysis (gG gene) was performed on 10 positive samples. Antiviral compounds (aciclovir, ganciclovir, valganciclovir, decitabine, idoxuridine, pritelivir, cidofovir, aphidicolin, vidarabine) previously described with antiviral properties against EHV-1, were evaluated using Real-Time Cell Analysis (RTCA) on equine dermal cells with the reference strain EHV-3 VR3522, cytopathic effect monitoring (microscopy) and viral load quantification (qPCR).Results: 16/71 (22%) suspected ECE cases were confirmed positive by PCR with 0 to 3 cases per year. French strains were clustered into two of the three observed phylogenic groups, but there was no correlation between the phylogeny and the time of collection or location. Aphidicolin was the most effective compound with an EC50 value of 2.43±0.02 µM (RTCA) and 1.63±0.48 µM (qPCR). Pritelivir and cidofovir were less potent molecules against EHV-3 (EC50 >50 µM).Main limitation: Antiviral compounds were tested against the reference strain only.Conclusions: This study demonstrates for the first time the presence of EHV-3 over a twelve-year period in France. In vitro studies show the antiviral efficacy of aphidicolin.

International audience

National audience; L’herpèsvirus équin 1 (HVE-1) est un pathogène majeur chez les équidés,responsable de 3 formes principales de maladie : la forme respiratoire, la forme abortive et la forme nerveuse. Ce travail présente des avancées significatives dans le traitement antiviral.Une infection expérimentale de poneys Welsh avec la souche HVE-1 FR-56628 (C2254)en présence ou non de Valganciclovir a été réalisée en 2020 sur la Plate-Forme d’Infectiologie Expérimentale (PFIE) de l’INRAe de Nouzilly. Au cours de cette étude, menée par les équipes de LABÉO et la PFIE, l’ensemble des chevaux infectés aprésenté des signes cliniques caractéristiques des infections respiratoires à HVE-1 à des degrés divers (fièvre > 38,5°C; jetage nasal ; toux, inflammation des ganglions, secrétions oculaires, changement de comportement).L’administration du valganciclovir par voie orale au cours de notre étude a permis de maintenir uneconcentration plasmatique à un seuil comparable à celui des concentrations utilisées in vitro. L’ensemble des effets observés en présence duvalganciclovirestactuellement en cours de soumission pour publication dès leur validation par les pairs. Ils serontprésentés de façon exhaustive dans un futur article à venir.

National audience; À ce jour, neuf herpèsvirus (HVE) ont été décrits chez les équidés. L'HVE-1, responsable des formes cliniques les plus graves, est un virus à ADN double brin d'environ 150kb qui contient 80 cadres de lecture ouverts codant 76 gènes uniques. Il se transmet par inhalation ou, après un avortement, par contact direct avec des éléments infectés comme le fœtus ou les annexes fœtales. En l'absence d'anticorps neutralisants dans le mucus, les cellules épithéliales des muqueuses nasale et nasopharyngienne sont infectées. La dissémination de l'HVE-1, via la circulation sanguine, jusqu'au tractus génital de la jument conduit généralement à un avortement. La dissémination du virus au niveau des tissus nerveux peut conduire à la forme nerveuse de la maladie nommée myéloencéphalopathie (EHM) nécessitant trop souvent l'euthanasie de l'animal.Si la vaccination contre l'HVE-1 existe, elle présente aujourd'hui des limitations ce qui conduit les praticiens vétérinaires à se tourner vers l'utilisation de traitements antiviraux pour les formes graves. Bien que son efficacité ne soit pas clairement démontrée, l'aciclovir demeure la molécule la plus utilisée sur le terrain.Notre travail a consisté à cribler in vitro 2891 molécules issues de différentes chimiothèques par des techniques de Real-Time Cell Analysis couplées à des méthodes de qPCR afin d'identifier des hits présentant une efficacité supérieure à celle de l'aciclovir. Ces travaux nous ont conduit à sélectionner 8 molécules parmi lesquelles le valganciclovir (VGCV) qui présentait une activité antivirale in vitro 10 fois supérieure à celle de l'aciclovir (Thieulent et al. 2020). Seul le valganciclovir avait fait l'objet d'études pharmacocinétiques préalables ce qui laissait entrevoir les perspectives d'un essai terrain (Charmichael et al., 2013). Notre étude démontre l'efficacité in vivo du valganciclovir après administration orale chez des poneys infectés par le nouveau variant HVE-1 FR-56628 (C2254) (Sutton et al. ; 2020).Lors de l'étude expérimentale, réalisée au sein de la PFIE (UE 1277 de l'INRAE de Nouzilly ; CEEA VdL, Comité éthique N° 19 ; autorisation APAFiS -22708), l'infection de 8 poneys Welsh mâles âgés de 10 mois a été réalisée par voie intranasale. Quatre poneys ont été traités avec une dose de 6,5 mg/kg de VGCV. Les signes cliniques, l'excrétion virale et la virémie ont été suivis pendant 3 semaines après infection. L'excrétion virale et la virémie ont été mesurées par culture cellulaire et par qPCR.L'ensemble des chevaux infectés ont présenté à des degrés divers des signes cliniques caractéristiques des infections respiratoires à HVE-1 (fièvre > 38,5°C ; jetage nasal ; toux, ganglions et secrétions oculaire). L'excrétion du virus HVE-1 mesurée à partir des écouvillons nasopharyngés est réduite de manière significative dans le groupe traité entre J+1 et J+12. Une diminution significative de la virémie dans le groupe traité a pu être mesurée. Le dosage du VGCV sérique a démontré la présence d'une concentration supérieure à 0.153µg/ml (EC50 déterminée lors de nos expériences in vitro) chez les 4 poneys traités.L'administration orale de valganciclovir réduit les signes cliniques, le jetage nasal et la virémie chez les chevaux infectés par la souche d'HVE1 FR-56628 (C2254).

International audience; Equine herpesvirus 1 isolates from a 2021 outbreak of neurologic disease in Europe have a mutation, A713G, in open reading frame 11 not detected in 249 other sequences from equine herpesvirus 1 isolates. This single-nucleotide polymorphism could help identify horses infected with the virus strain linked to this outbreak.

International audience; Background: Equine herpesvirus type-1 (EHV-1) is an important threat to the equine industry, as illustrated by the ongoing outbreak of neurological disease that was initially reported during a large equestrian event in Valencia, Spain in 2021. Horses returning from this event to their training yards have contributed to the spread of the virus to nine other European countries and to Qatar.Objectives: To design a “tracking” marker in order to specifically follow the dissemination of the Valencia strain, in EHV-1 infected horses with no known epidemiological link with the Valencia outbreak.Study design: Strain isolation and genome sequences’ comparison.Methods: 67 nasopharyngeal swab samples from horses stationed in Valencia were analysed by EHV-1 qPCR and a rapid A/G/C2254 (ORF30) typing test. Positive samples were used for strain isolation in vitro, and Multilocus Sequence Typing (MLST).Results: 19/67 (28%) samples were EHV-1 positive (all 19 typed as A2254). Two strains (FR/Valencia1/2021 and FR/Valencia2/2021) were successfully isolated and characterised by MLST as belonging to clade 10. Analysis of the MLST ORF sequences revealed a mutation at position 713 of the ORF11 (A713G) in FR/Valencia1/2021 and FR/Valencia2/2021, when compared with reference strains Ab4 and V592. This A713G mutation was not present in 104 ORF11 sequences obtained from Genbank (strains from the UK, USA, China, Australia, Belgium, New-Zealand, Japan or India), or 131 and 14 ORF11 sequences from strains isolated in Ireland or France, respectively. This marker allowed subsequent confirmation of suspicious epidemiological links in EHV-1 cases.Main limitation: The limited number of ORF11 sequences available in some countries.Conclusions: Although the existence of this mutation in other field strains cannot be excluded, its absence in the 249 ORF11 sequences analysed, suggests that this SNP constitutes an interesting epidemiological marker to identify horses infected with the EHV-1 strain which was associated with the outbreak in Valencia.

International audience; Data presented in this article are associated with the research article “Identification of antiviral compounds against equid herpesvirus-1 using real-time cell assay screening: efficacy of decitabine and valganciclovir alone and in combination” [1]. These data correspond to the in vitro screening of 2,891 potential antiviral compounds against equid herpesvirus-1 (EHV-1) based on impedance measurements using the xCELLigence® RTCA MP System. This dataset includes compounds from three different libraries: i) 1,199 compounds from the Prestwick® Chemical Library, which contains mostly US Food and Drug Administration approved drugs (Prestwick® Chemical, Illkirch, France); ii) 1,651 compounds from the Centre d'Etudes et de Recherche sur le Médicament de Normandie (CERMN, Caen, France); iii) 41 compounds (called herein in-house antiviral library) selected for their effects against different human viruses. Compounds effective against EHV-1 were selected using the area under normalised curves (AUCn) and the time required for the Cell Index to decrease by 50% after virus infection (CIT50). The full dataset from the screen is made publicly available for further analyses.

National audience; L’herpèsvirus équin1 (HVE-1)est l’un des deux agents responsables de la rhinopneumonie équine. Ce virus peut provoquer des troubles respiratoires, des avortements, une mort néonatale du jeune poulain et des troubles nerveux appelés myéloencéphalopathie équine. Les vaccins existant à l’heure actuelle permettent de réduire les problèmes respiratoires et donc la dissémination du virus. Cependant, ils ne préviennent pas suffisamment les formes graves de la maladie. Par ailleurs, aucune molécule antivirale ne possède d’autorisation de mise sur le marché en santé équine en France. Nous avons développé un système permettant de tester à haut débit un grand nombre de molécules contre l’HVE-1 en conditions de laboratoire. Parmi les 3000 molécules testées, 23 ont été identifiées comme potentiellement efficaces contre ce virus. Le ganciclovir et sa pro-drogue le valganciclovir se sont montrés parmi les molécules les plus efficaces contre l’HVE-1. Ces résultats encourageants, couplés aux données de pharmacocinétique déjà existantes de ces molécules sur le modèle équin nous ont conduits à envisager une étude expérimentale sur des poneys.

Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C2254 (H752) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as UL clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C2254 mutation. When cultured in vitro, the C2254 EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A2254 and G2254 EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C2254 strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C2254 strain and two other A/G2254 field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C2254 and A2254 strains were more sensitive to aciclovir than the G2254 strain, based on viral load measurement.

International audience; Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.

International audience; Respiratory infections are still a major concern in pigs. Amongst the involved viruses, the porcine reproductive and respiratory syndrome virus (PRRSV) and the swine influenza type A virus (swIAV) have a major impact. These viruses frequently encounter and dual infections are reported. We analyzed here the molecular interactions between viruses and porcine tracheal epithelial cells as well as lung tissue. PRRSV-1 species do not infect porcine respiratory epithelial cells. However, PRRSV-1, when inoculated simultaneously or shortly before swIAV, was able to inhibit swIAV H1N2 infection, modulate the interferon response and alter signaling protein phosphorylations (ERK, AKT, AMPK, and JAK2), in our conditions. SwIAV inhibition was also observed, although at a lower level, by inactivated PRRSV-1, whereas acid wash treatment inactivating non-penetrated viruses suppressed the interference effect. PRRSV-1 and swIAV may interact at several stages, before their attachment to the cells, when they attach to their receptors, and later on. In conclusion, we showed for the first time that PRRSV can alter the relation between swIAV and its main target cells, opening the doors to further studies on the interplay between viruses. Consequences of these peculiar interactions on viral infections and vaccinations using modified live vaccines require further investigations.

International audience; Equid alpha-herpesviruses (EHV) are responsible for different diseases in equine population. EHV-1 causes respiratory diseases, abortions and nervous disorders, EHV-4 causes respiratory diseases and sporadic abortion, while EHV-3 is responsible of equine coital exanthema. In view of the lack of efficacy of vaccines against EHV-1 and EHV-4 and in the absence of vaccines against EHV-3, the use of antiviral treatment is of great interest. In this study, we documented the interest of the Real-Time Cell Analysis (RTCA) technology to monitor the cytopathic effects induced by these viruses on equine dermal cells, and established the efficacy of this method to evaluate the antiviral effect of aciclovir (ACV) and ganciclovir (GCV). In addition, the RTCA technology has also been found appropriate for the high-throughput screening of small molecules against EHV, allowing the identification of spironolactone as a novel antiviral against EHV.

National audience

International audience; (1) Background: Equine hepacivirus (EqHV), also referred to as non-primate hepacivirus (NPHV), infects horses—and dogs in some instances—and is closely related to hepatitis C virus (HCV) that has infected up to 3% of the world’s human population, causing an epidemic of liver cirrhosis and cancer. EqHV also chronically infects the liver of horses, but does not appear to cause serious liver damages. Previous studies have been looking to identify route(s) of EqHV transmission to and between horses. (2) Methods: In this retrospective study, we sought to evaluate the prevalence of vertical transmission taking place in utero with measuring by quantitative RT-PCR the amounts of EqHV genome in samples from 394 dead foals or fetuses, paired with the allantochorion whenever available. (3) Results: Detection of EqHV in three foals most likely resulted from a vertical transmission from the mares to the fetuses, consistent with the in utero transmission hypothesis. In support of this observation, the presence of EqHV genome was found for the first time in two of the allantochorions. (4) Conclusions: As seemingly benign viruses could turn deadly (e.g., Zika flavivirus) and EqHV happens to have infected a significant proportion of the world’s horse herds, EqHV infectious cycle should be further clarified.

International audience; Equine hepacivirus (EqHV), also referred to as non-primate hepacivirus (NPHV), infects horses —and dogs in some instances— and is closely related to hepatitis C virus (HCV) that has infected up to 3 % of the world’s human population, causing an epidemic of liver cirrhosis and cancer. EqHV is also chronically infecting the liver of horses, but does not appear to cause serious liver damages. Recent work often associates two other viruses, Theiler Disease Associated Virus (TDAV) and Pegivirus (EqPV) to this research during hepatic syndrome. If the name of the hepatic triad has emerged in recent years, the recent discovery of a new virus, a potentially involved equine parvovirus, raises new questions. Among these, the mode of transmission of these viruses is an essential issue. Previous studies have been looking to identify route(s) of EqHV transmission to and between horses. In a retrospective study, we sought to evaluate the prevalence of vertical transmission taking place in utero with measuring by quantitative RT-PCR the amounts of EqHV genome in samples from 394 dead foals or fetuses, paired with the allantochorion whenever available. Detection of EqHV in three foals most likely resulted from a vertical transmission from the mares to the fetuses, consistent with the in-utero transmission hypothesis. In support of this observation, the presence of EqHV genome was found for the first time in two of the allantochorions. If the first transmission tracks emerge for EqHV, studies are needed for the "triad" or even the "tetrad".

International audience; Few data on cytokine profiles in bronchoalveolar lavage fluid (BALF) are available for racehorses with mild/moderate equine asthma (EA); cytological diagnosis being most frequently made from only one lung. The purpose of the study was to compare cytokine mRNA expressions and protein concentrations in BALF from both lungs. As part of a larger study, 250 ml saline was randomly instilled in one lung and 500 ml in the contralateral lung of 30 clinically healthy Standardbred racehorses. This procedure was repeated 72h later, inversing the volume per lung. Cytological cut-off values for diagnosis of mild EA was neutrophils proportions > 10% when instilling 250 ml. Eleven horses that exhibited unilateral mild inflammatory cytology (i.e. normal cytology [

International audience; In the fields of biology and human and veterinary medicine, much progress has been made over the past 20 years. The new areas of investigation are often linked to technological progress and if big data has not revealed all its secrets, or if 3D bioprinters have not proposed new study models in all expected domains, many innovative technologies have enter in the laboratories of the 21st century. Thus, the cellular culture often presented as a long and tedious technique knows a small revolution with the arrival of real-time technologies. After the Real Time PCR techniques in molecular biology, Real Time Cell Analysis (RTCA) in cell biology opens up new perspectives in many applications. An original platform name Impedancell, labeled in 2019 by the French IBISA (French certification body for technologies applied to research in the living world) was built in Normandy to manage this new paradigm.ImpedanCELL is an innovative and original core facility for studying real-time high-throughput cellular activity using impedance measurement (xCELLigence technology) and real-time cellular imaging. It is localised on two different geographical sites in Caen (Normandy, France): Comprehensive Cancer Center F. Baclesse for all non-microbiology applications and LABÉO (Equine Normandy Equine Valley Biotechnology platform and BIOTARGEN research team) for all applications in virology and in bacteriology (biosafety level 2). The core facility is equipped with cutting-edge technologies (3 xCELLigence MP, 1 xCELLigence DP, 1 iCELLigence, 1 Cellavista, 2 Incucytes…) allowing the study of adhesion, proliferation, cell death, migration and invasion in various domains including oncology, neurosciences, toxicology, immunology, marine biology, virology and bacteriology. In line with one ImpedanCELL’s mission, the core facility is open to any scientists with high-throughput study needs for dynamic tracking of real-time cellular behaviour. ImpedanCELL is open to collaborations and services for both academic and industrial partners and offers theoretical and practical courses. Two different applications performed on each site are presented with the advantage of this new technology for two areas of expertise in each laboratory: equine virology on the one hand and human cancer research on the other.Among the infectious agents found in the equine species, herpesviruses are certainly among those causing the greatest economic losses in the world. Equid alpha-herpesviruses (EHV) are responsible for different diseases in equine population. EHV-1 causes respiratory diseases, abortions and nervous disorders, EHV-4 causes respiratory diseases and sporadic abortion while EHV-3 is responsible of equine coital exanthema. In view of the lack of efficacy of vaccines against EHV-1 and EHV-4 and in the absence of vaccines against EHV-3, the use of antiviral treatment is of great interest. Our team recently documented the interest of the Real-Time Cell Analysis technology to monitor the cytopathic effects (CPE) induced by these viruses on equine dermal cells, and established the efficacy of this method to evaluate the antiviral effect of aciclovir (ACV) and ganciclovir (GCV). In addition, RTCA technology has also been found suitable for high throughput screening of small molecules against EHV , allowing identification of several molecules of interest as a new antiviral against EHV, among them spironolactone is a potential candidate.1In the field of oncology, repositioning is also an interesting approach and, in this respect, the RTCA are technologies of interest. The development of direct Mcl-1 inhibiting molecules constitutes a major challenge for the success of Bcl-xL targeting strategies in ovarian carcinoma. After computational modelling to identify molecules able to target the Mcl 1 hydrophobic pocket, a high throughput functional screening assay based on xCELLigence technology was implemented to evaluate the ability of designed and synthesized molecules called oligopyridines to sensitize ovarian carcinoma cells (in addiction to both Bcl-xL and Mcl-1) to Bcl xL-targeting strategies. After establishing structure-activity relationships and identifying a potent lead (hit) called Pyridoclax, surface plasmon resonance assay demonstrated that pyridoclax directly binds to Mcl-1. Without cytotoxic activity when administered as a single agent, pyridoclax induced apoptosis in combination with Bcl-xL targeting siRNA or with ABT 737 in ovarian cancer cells. These results open up interesting perspectives for the clinical use of Mcl-1 inhibitors to improve the clinical management of ovarian cancer.2 Overall, these two high-throughput studies demonstrated the interest to use impedance based technologies to identify some pertinent targets and molecules for therapeutic intervention in the context of the precision medicine in ovarian cancer an in EHV infectious disease in horses. Our study confirms the efficiency of RTCA to monitor CPE formation induced by EHV-1 on E. Derm cells, and further extends this tool to EHV-4 and EHV-3. In the infectious field, applications in bacteriology and in particular studies on biofilms also use this technology which facilitates the screening of antibiotics. The use in the field of immunology is also booming. While the interest of screening by impedance measurements is well established, real-time monitoring of cell development under the microscope remains essential. Engineers have understood this, which explains the arrival in 2020 of one technology coupling these two approaches.

International audience; Like hepatitis C virus (HCV) in humans, the newly identified equine hepacivirus (NPHV) displays a predominating liver tropism that may evolve into chronic infections. The genomes of the two viruses share several organizational and functional features and are phylogenetically closest amongst the Hepacivirus genus. A limited amount of data is available regarding the spread of hepacivirus infections in horses. In this study, we asked whether in a more representative sample the prevalence and distribution of NPHV infections in France would resemble that reported so far in other countries. A total of 1033 horses sera from stud farms throughout France were analysed by qRT-PCR to determine the prevalence of ongoing NPHV infections and viral loads; in positive samples, partial sequences of NPHV’s genome (50UTR, NS3 and NS5B genes) were determined. Serum concentrations of biliary acids, glutamate dehydrogenase (GLDH) and L-gamma-glutamyl transferase (c-GT) were measured for most horses. We detected NPHV infections in 6.2% of the horses, a prevalence that reached 8.3% in thoroughbreds and was significantly higher than in other breeds. The presence of circulating virus was neither significantly associated with biological disturbances nor with clinical hepatic impairment. Our phylogenetic analysis was based on both neighbour- joining and maximum-likelihood approaches. Its result shows that, like almost everywhere else in the world so far, two major groups of NPHV strains infect French domestic horses. Based on genetic distances, we propose a classification into two separate NPHV subtypes. Viral loads in the serum of horses infected by the main subtype were, in average, four times higher than in those infected by the second subtype. We hypothesize that amino acid substitutions in the palm domain of NS5B between NPHV subtypes could underlie viral phenotypes that explain this result.

Background Avoidance of antigenic stimuli was found to significantly reverse airway obstruction of horses with severe equine asthma (sEA). To date, no published study investigated the influence of steaming hay on lower airway condition of sEA-affected horses. The objectives were to determine the clinical, cytological and cytokine respiratory responses of both sEA and control (CTL) horses experimentally exposed to steamed or dry hay. Results A cohort of 6 sEA horses and 6 CTL horses was involved in this field study. On day 0, both groups were fed with steamed hay for 5 consecutive days, followed by a wash-out period of 26 days prior to be fed with dry hay for 5 consecutive days. Investigations performed 2 days prior to and 5 days after each challenge included clinical score, tracheal mucus accumulation, and bronchoalveolar lavage fluid (BALF) cytology and cytokine mRNA expression. Feeding steamed hay significantly decreased its mould content (P

International audience; An episode of acute equine respiratory infection was reported in western Algeria (Tiaret province) between February and March 2011, affecting a large population of horses. Nasal swabs (n=100) were taken from horses aged between 1 and 27 years, presenting with cough and mucopurulent nasal discharge. The prevalence of equine respiratory virus infections was examined using quantitative polymerase chain reaction (qPCR). One, or more, of four equine respiratory viruses were detected in the nasal swabs of 90 of 100 horses (90%) and the detection rate of equine herpesvirus type 1 (EHV-1), equine herpesvirus type 4 (EHV-4), equine herpesvirus type 2 (EHV-2) and equine herpesvirus type 5 (EHV-5) were 2%, 14%, 90% and 75%, respectively. Equine influenza virus and equine arteritis virus were not detected in any samples. Among the 90 infected horses, 70 were coinfected with EHV-2 and EHV-5 and 14 others were co-infected with EHV-4, EHV-2 and EHV-5. The present study shows a positivity rate of 97.3% for EHV-5 in young horses aged

International audience; Equid gammaherpesviruses-2 and -5 are involved in respiratory problems, with potential clinical man-ifestations such as nasal discharge, pharyngitis and swollen lymph nodes. These viruses are sometimesassociated with a poor-performance syndrome, which may result in a significant and negative economicimpact for the horse industry. The aim of the present study was to develop and validate quantitative PCRmethods for the detection and quantitation of EHV-2 and EHV-5 in equine respiratory fluids. Two dis-tinct tests were characterised: (a) for the qPCR alone and (b) for the whole method (extraction and qPCR)according to the standard model AFNOR XP U47-600-2 (viz., specificity, quantifiable sensibility, linearity,accuracy, range of application, trueness, precision, repeatability and precision of reproducibility). EHV-2and EHV-5 detection were performed on nasal swabs collected from 172 horses, all of which exhibitedclinical signs of respiratory disease. The data revealed a high rate of EHV-2/EHV-5 co-detection that wascorrelated significantly with age. Viral load of EHV-2 was significantly higher in young horses whereasviral load of EHV-5 was not significantly different with age.

International audience

International audience; In virology, different conventional methods are commonly used for virus titration and infectivity studies. Most of them are based on direct examination of viral cytopathic effect, which is time consuming, tedious and requires an endpoint reading. The RTCA xCELLigence system (ACEA Biosciences) has been developed to follow cellular events and their dynamics in real time using a micro-electrical biosensor technology. The aim of this study was to evaluate the feasibility of using this biosensor technology to measure the effect of different equid herpesviruses on equine dermal cell cultures, with or without antiviral acyclovir treatment. Cells were infected with EHV-1 (VR700), EHV-4 (VR2230), EHV-2 (VR701) or EHV-3 (VR352). The viral effects were monitored by 3 different methods: impedance measurements (RTCA), cytopathic effects recording (microscopy) and viral load quantification (qPCR). The RTCA technology showed a dose dependent drop of the cellular index induced by specific impedance variations measured during growth of each virus tested. These results correlated with the cytopathic effect induced by the different herpesvirus species, as well as viral loads measured by qPCR. For example, after 3 days of incubation with EHV-1, RTCA results showed an increase of the cellular index from 0% (infection without antiviral) to 45% and 100% in presence of Acyclovir (10μl/mL and 100 μg/mL, respectively). These results are correlated with the decreased of viral load measured in the presence of Acyclovir: 1010 genome copies/mL with EHV-1 only, 106 copies/mL with EHV-1 + Acyclovir 10 μg/ mL and 105 copies/mL with EHV-1 + Acyclovir 100 μg/ mL. In conclusion, the cellular impedance monitoring system is a new real-time method to monitor cell growth and viral proliferation in cell cultures. This technology is well adapted for high-throughput screening of antiviral molecules. In fact, the xCELLigence system enables continuous impedance measurement and quantification of cell adhesion, proliferation, cell death and detachment during viral infection.

International audience; Hepatitis C virus (HCV) is one of the main causes of end stage liver diseases (cirrhosis and hepatocellular carcinoma) in humans. In vivo studies have been hampered by a lack of access to relevant animal models. Until recently, the genus Hepacivirus was comprised of seven HCV genotypes, plus GBV-B that infects New World monkeys. Within the past few years, new hepaciviruses were identified in dogs and horses (also called canine/equine or non-primate hepaciviruses), in Old World monkeys (Colobus guereza), as well as in rodents (Myodes glareolus and Rhabdomys pumilio) and bats. Yet, the data available regarding newly identified hepacivirus infections is still limited, including in horses. So far, few studies have reported PCR positive horses in different countries (USA, Scotland, Germany, Japan, Brazil and Hungary). The aim of our study was to determine the prevalence of active hepacivirus infections in horses. A RT-qPCR adapted from the test described by Burbello et al. was developed according to the AFNOR norm U47-600-2. We detected 69 positive horses out of 1229 screened with this assay (5,6%). Positive horse samples originated from all parts of France. The number of viral genomes circulating in the blood of hepacivirus-infected horses ranged from 1.15 x 104 to 2.8 x 109 copies/mL, with two third of the results above a threshold of 4.26 x 107 copies/mL. There was no evidence of concomitant hepatic inflammation in the sera, as tested by the levels of gamma-glutamyl transferase and glutamate deshydrogenase. A one-year follow-up study in 7 horses from the same stud farm showed that, from the time of detection, infection persisted up to 18 months. A phylogenetic analysis was performed based on the nucleotide sequences of the 5’-untranslated region of the genome, as well as of the genes encoding the nonstructural proteins 3 and 5B. The result of this analysis suggests that all positive horses identified in France are infected with equine hepacivirus variants distinct from those previously reported. The present study demonstrates for the first time the presence of a significant proportion of ongoing equine hepacivirus infections in the horse herds in France. Determining the consequences of persistent hepacivirus infections in horses warrants further investigation.

At least five equine herpesviruses are known to infect horses : three alpha herpesviruses (equine herpesvirus-1, -3 and -4) and two gamma herpesviruses (equine herpesvirus-2 and -5). Equine herpesvirus-1 is responsible for three clinical presentations : respiratory, abortigenic and neurological. This latter, widely known as EHV-1 associated, myeloencephalopathy has been thoroughly investigated over the past years. Most of the studies focused on the relation between the presence of a mutation in ORF 30 coding for DNA polymerase and the expression of the paralytic (neurological) form. Even if this mutation seems to play a role, other factors related to the host and the environment are certainly involved in the development of the disease. Equine herpesvirus-4 is better known as the rhinopneumonia virus and is responsible for a large part of respiratory disorders in young foals. Equine herpesvirus-3 is responsible for equine coital exanthema, whose transmission through the respiratory tract is now proven. Equine herpesvirus-2 and -5 are associated with subclinical respiratory diseases in equine athletes, even though their pathogenic role has yet to be clarified as they were detected in all continents only relatively recently. The development of molecular genetic tools lead to major progress in the detection of these viruses, but the differentiation between latent and infectious forms is still unresolved. Although studies are currently performed on the typing of strains responsible for different clinical presentations involving equine herpesviruses as well as in other equine viruses (influenza, equine viral arteritis), there is no international consensus on a phylogenic tree for any of the 5 equine herpesviruses. Further studies are definitely required to monitor and characterise the different strains of EHV., Au moins cinq herpèsvirus équins peuvent infecter le cheval : trois alpha-herpèsvirus (les herpèsvirus 1, 3 et 4) et deux gamma-herpèsvirus (les herpèsvirus équins 2 et 5). L’herpèsvirus équin 1 est responsable de trois formes cliniques : une forme respiratoire, une forme abortive et une forme neurologique. Cette dernière, plus connue sous le nom de myéloencéphalopathie à HVE-1, a fait l’objet de nombreuses études ces dernières années. La plupart des travaux ont porté sur la relation entre la présence d’une mutation dans l’ORF 30 codant l’ADN polymérase et l’expression de la forme paralytique. Si cette mutation semble jouer un rôle, d’autres facteurs liés à l’hôte et à l’environnement participent certainement au développement de la maladie. L’herpèsvirus équin 4 est plus connu sous le nom de virus de la rhinopneumonie et est responsable d’une part importante des affections respiratoires chez les jeunes poulains. L’herpèsvirus équin 3 est responsable de l’exanthème coïtal équin et l’on sait aujourd’hui qu’il peut se transmettre par les voies respiratoires. Les herpèsvirus équins 2 et 5 sont associés aux maladies respiratoires subcliniques du cheval athlète même si leur rôle reste à préciser, leur découverte sur l’ensemble des continents étant relativement récente. Le développement des outils de génétique moléculaire a permis de faire des progrès importants dans la détection de ces virus même si un challenge demeure : différencier les formes latentes des formes infectieuses. Si comme pour d’autres virus équins (ex : grippe, artérite virale équine), des travaux sont réalisés pour typer les différentes souches responsables de différentes formes d’expression de la maladie, il n’existe toujours pas de consensus international sur un arbre phylogénique pour aucun de ces cinq herpèsvirus équins. Le suivi et la caractérisation des différentes souches d’HVEs sont très certainement l’autre défi de demain., Pronost Stéphane, Hue Érika, Legrand Loïc, Pitel Pierre-Hugues, Fortier Guillaume. Herpèsviroses des équidés: intérêts et limites des outils de génétique moléculaire. In: Bulletin de l'Académie Vétérinaire de France tome 164 n°4, 2011. pp. 305-316.

International audience; The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

International audience; The aim of the study was to determine whether instillation of either 250 mL or 500 mL of saline for bronchoalveolar lavage (BAL) would influence cytological confirmation of inflammatory airway disease (IAD). Thirty client-owned Standardbred racehorses were sampled via endoscopy with 250 mL of saline in one lung and 500 mL in the contralateral lung. The procedure was repeated 72 h later, reversing the volume per lung. The proportions of BAL fluid (BALF) recovered were significantly higher and neutrophil percentages significantly lower with the larger volume. A poor agreement was found between methodologies in terms of final diagnosis, when based on proportions of neutrophils (>10% from at least one lung). Within the recommended range (250–500 mL), the instilled volume significantly influenced cytological profiles. Establishing specific BALF reference values is warranted.

International audience; Horses are usually infected during the first months of life by several equine herpesviruses, such as EHV-1, EHV-4 or EHV-2. The persistence of these respiratory viruses through establishment of latency, often characterised by an absence of clinical signs, represents a real risk to the horse population. Reactivation of latent EHV could cause disease in both carriers and naive animals in contact. A better understanding of the immune response to EHV infection is necessary to improve our methods of prevention and decrease the risk of transmission. The objectives of this study were 1/ to characterise the cytokine gene expression profile of PBMC after in vitro EHV infection (EHV-1, EHV-4 and EHV-2) and 2/ To determine the efficacy of the inactivated Parapoxvirus ovis (iPPVO), a known immunomodulator in horses , against these same viruses. PBMC were isolated from 3 horses and infected in vitro with EHV-1, EHV-4 or EHV-2, in the presence or absence of iPPVO. In vitro culture of PBMC with EHV-1, EHV-4, EHV-2 and iPPVO induced a significant increase of IFN-α, IFN-β and IFN-γ gene expression. Moreover, EHV-2 induced a significant increase of IL-1β, IL-6 and TNF-α mRNA. The presence of iPPVO induced an earlier and stronger expression of IFN-α, IFN-β and IFN-γ during EHV infection. The presence of iPPVO reduced also the inflammatory response induced by EHV-2. In conclusion, this study suggests that the presence of iPPVO potentiates the development of the immune response during infection EHV in vitro.

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National audience; L'herpèsvirus équin 1 (HVE-1) est le plus redouté des neuf herpès-virus décrits chez les équidés. Le nom usuel pour l'HVE-1 est celui de "virus abortif" et l'expression de "virus de la rhinopneumonie" est dorénavant réservée à l'HVE-4. Ces dernières années, deux types de souches d'HVE-1 ont été décrits. Cependant, toute épizootie à HVE-1 est à considérer avec les mesures sanitaires qui s'imposent quel que soit le type de souche identifié. Des épizooties importantes sévissent toujours en Europe et plusieurs d'entre elles sont bien documentées. Ces données nous renseignent sur l'efficacité des mesures de prévention et des méthodes de diagnostic, les limites actuelles de la vaccination et sur la difficulté à déterminer l'origine de ces crises. Plusieurs travaux ont montré que d'autres espèces que le cheval, en particulier celles présentes dans les zoos, pouvaient être infectées par l'HVE-1 et développer des formes nerveuses de la maladie.

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International audience; The aim of this trial was to investigate the putative involvement of equid herpesvirus 2 (EHV-2) in airway inflammation of adult horses. Six horses received corticosteroid treatment, before either mock infection (n = 2) or EHV-2 strain LK4 inoculation (n = 4). These four horses were also submitted to immunosuppression 84 days post inoculation. EHV-2 was detected by quantitative PCR in respiratory samples up to respectively 21 days and 14 days. Nested PCR, cloning and sequencing allowed the detection of five different ‘field’ strains throughout the trial. Neutrophils proportions were transiently increased in respiratory fluids; neutrophilia being significantly associated with concomitant EHV-2 detection. The laboratory findings reproduced in this trial were compatible with sub-clinical lower airway inflammation and suggest that EHV-2 infection should be suspected when investigating poorly-performing horses.

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The hMSCs are resistant to apoptosis after exposure to different apoptotic inducers, however, these cells do not lack any proteins implicated in apoptosis (Oliver et al. , submitted). Conversely, after an induction of differentiation, these cells acquire a sensitivity to apoptosis. Hence, by analysing the differentiation process of hMSCs, we could gain an insight into the acquisition of a sensibility to apoptosis in these cells. We hope to use this insight into the induction of apoptosis in cancer stem cells that appear to have the same resistance to apoptosis observed in normal stem cells. An analysis of the executioner caspase-3 in undifferentiated MSCs has revealed the presence of the proform (32 kDa) and a 29 kDa cleaved form of caspase-3 and during osteogenesis caspase-3 is cleaved into a 25 kDa form. These cleaved forms of caspase-3, 29 and 25 kDa were previously shown by our group to be the result of a cleavage by calpain (Pelletier et al, 2005). Our data show that caspase-3 is important in the initial stages of osteogenic differentiation but not essential for osteogenic differentiation as a knock-down of caspase-3 delays but does not completely abrogate osteogenic differentiation. An activation of alternative pathways implicating an up-regulation of downstream or upstream partners may be involved., Les cellules souches mésenchymateuses (CSMs) sont résistantes à l'apoptose après traitement avec différents inducteurs apoptotiques alors qu'elles expriment l'ensemble des protéines impliquées dans ce mécanisme (Oliver et coll. , soumis). Cependant, ces cellules acquièrent une sensibilit' à l’apoptose après induction de leur différenciation. Nous étudions les mécanismes de différenciation des CSMs afin de comprendre comment ces cellules acquièrent une sensibilité à l'apoptose au cours de leur différenciation. Ceci nous permettra ensuite d'étudier le défaut responsable de l'échappement des cellules souches cancéreuses à l'apoptose. L'étude protéique des CSMs non différenciées a montré la présence de la procaspase-3 (32 kDa) mais également une forme clivée de 29 kDa. De plus, un fragment de 25 kDa est également formé au cours de la différenciation ostéogénique. Ces formes de 29 et 25 kDa ont été précédemment décrites par notre équipe comme résultant d’un clivage de la procaspase-3 par la Calpaïne (Pelletier et coll. , 2005). En effet, l'inhibition de cette enzyme empêche le clivage de la Caspase-3 et retarde la différenciation. La Caspase3 semble donc jouer un rôle important dans la différenciation des CSMs. Cependant, l'extinction de la Caspase-3 (knock-down) retarde la différenciation mais sans pour autant l'inhiber totalement. Un mécanisme de compensation semble se mettre en place au cours de la différenciation en absence de la Caspase-3.

International audience; Equine rhinopneumonitis, caused by Equid Herpesvirus 4 (EHV-4) and EHV-1, is one of the most frequent respiratory diseases experienced by horse breeders and trainers. The French equine infectious diseases surveillance network (RESPE) reports numerous outbreaks each year, which can significantly disrupt economic activities, as illustrated in France in 2018 when more than 200 equestrian events were cancelled. While the origin of this crisis could date back from the 2016 EHV vaccine shortage, no scientific evidence has been obtained to date.Research teams have often been less interested in the study of EHV-4, probably because EHV-1 is known to induce more severe forms of diseases (i.e. abortions, neonatal deaths and myeloencephalopathy). However, EHV-4 is responsible for approximately 6 times more epizootics (respiratory infection) than EHV-1 (1,2). Several cases of EHV-4 induced abortion have also been observed and reported in recent years (3,4).All of these data attest to the importance of the pathology associated with HVE-4 infection and demonstrate the need to better characterizing EHV-4 common characteristics and differencies with EHV-1.The objectives of this new project are to : 1 / Understand the importance of EHV-4 during the 2018 French crisis in a degraded context of vaccination.2 /Characterise EHV-4 infection in order to improve pathophysiology knowledge, an essential step for future and effective prophylaxis.3 / Characterise EHV-4 strains at the molecular level to identify future vaccine and antiviral targets.

International audience; Among the 9 herpesviruses described in equids, the equine herpesvirus 1 is particularly pathogenic for equine species. It can cause respiratory disorders, abortion, neonatal foal death and neurological disease. As current vaccines do not provide full protection against severe symptoms, EHV-1 represents a major threat for the horse industry, causing serious economic losses every year. Different tools are available or need to be developed in order to improve outbreak management.In this report, we will describe the importance of molecular surveillance of strains circulating after performing a phylogenic analysis of strains isolated from 2012 to 2018, including 2018 major EHV-1 crisis [1]. This study allowed the comparison of three different tools including the typing of the well known A2254G ORF30 mutation, the complete ORF30 sequencing and the Multi Locus Sequence Analysis tool developed by Garvey et al. (2019) [2]. We will also describe the development of new antiviral strategies using the xCELLigence RTCA system, based on cells impedance measurement [3]. Until now, 3.000 compounds have been screened against EHV-1 KyD strain using this technology. Based on calculation of two mathematical models, 26 compounds were identified as effective against EHV-1. Antiviral effects of these compounds were confirmed by dose-response assays. Among these compounds, 6 were selected as they showed an efficacy similar to ganciclovir which is the most potent antiviral compound until now. Antiviral effects of these compounds were then confirmed on different cell models and against two field strains of EHV-1 recently isolated in our laboratory. Three of the retained compounds had never been described before against herpesviruses and some combinations studies showed good synergy between different compounds.References:1. Sutton, G.; Jourdan, M.; Fortier, C.; Maisonnier, V.; Dervin, S.; Marcillaud-Pitel, C.; Legrand, L.; Paillot, R.; Pronost, S. Phylogenic study of Equine Herpesvirus 1 after a major outbreak in France in 2018; 2019;2. Garvey, M.; Lyons, R.; Hector, R.; Walsh, C.; Arkins, S.; Cullinane, A. Molecular Characterisation of Equine Herpesvirus 1 Isolates from Cases of Abortion, Respiratory and Neurological Disease in Ireland between 1990 and 2017. Pathogens 2019, 8, 7.3. Thieulent, C.J.; Hue, E.S.; Fortier, C.I.; Dallemagne, P.; Zientara, S.; Munier-Lehmann, H.; Hans, A.; Fortier, G.D.; Pitel, P.-H.; Vidalain, P.-O.; et al. Screening and evaluation of antiviral compounds against Equid alpha-herpesviruses using an impedance-based cellular assay. Virology 2019, 526, 105–116.

International audience; Purpose of the study: The immune response in asthmatic horses is based on innate immunity with an important role of alveolar macrophages (AM) which are the first line of defense of immune response with phagocytosis and macropinocytosis mechanisms. In fact, AMs are present on the surface of lung cells and are thus directly exposed to inhaled particles. Multiple agents may be implicated in either initiation or maintenance of equine asthma syndrome like dust stables, season, bacteria or beeding. The composition of the administered hay had a greater influence than bedding on breathable dust concentrations and on endotoxin in the inhaled air. The objectives of this study were 1/ characterize alveolar macrophages population from control (CTL) and severe equine asthmatic (sEA) horses; 2/ study the alveolar macrophages phagocytosis and macropinocytosis activities from CTL and sEA horses, in vitro stimulated by different antigens whose hay dust solution; 3/ study the cell behavior and cytokinique response of AMs from CTL and sEA horses, preliminary in vivo hay challenged (Orard et al., 2018) and in vitro stimulated.Methods used: The in vivo hay challenge was previously described in Orard et al. (2018). The AMs were isolated on D-2 (pre-challenge) and D+5 (post-challenge) from the BALF. Hay dust suspensions (HDS) were realized by shaking 1kg of dry or steamed hay with Haygain HG-600, dusts were sieved and suspended at 10 ml of saline per gram of dust. The HDS were characterised for bacteria, mould, endotoxin and β-D-glucan content, inhalable and respirable particles. After 3h of adhesion, the AMs were stimulated with GFP-conjuagted E.coli bioparticles (10 by cells), FITC-conjugated BSA (1 mg/ml), LPS (500 ng/ml), Fumagilline (4 µg/ml), dry HDS (5 µl/ml) or steamed HDS (5 µl/ml). The real-time cellular activities of AMs were analysed by live cell imaging (IncuCyte® S3, Sartorius) and tested for impedance measurement (xCELLigence®, ACEA Biosciences). Immunophenotyping, macropinocytosis and phagocytosis capacities were analysed by flow cytometry (CytoFlex S, Beckman coulter). Griess test was realised at 6h and 24h post-stimulation. Summary of results: Bacteria, mould, endotoxins and beta D glucan decrease in HDS after steaming. Moreover steam treatment decreases significantly and sustainably both inhalable and respirable dust generated by hay shaking. The immunophenotyping of AMs showed that CD14+ cell proportion was more important in sEA horses compared to CTL horse with better phagocytosis and macropinocytosis capacities in CD14+ AMs in sEA horses. No significant difference of phagocytosis and macropinocytosis capacities were observed in CD206+ cells between sEA and CTL horses. When naive AMs (D-2) were only in vitro simulated, we can observe more important cell grouping with LPS and HDS in AMs from CTL horses. We can observe that the size of AMs stimulated with D-HDS is higher compared to the other stimuli. When AMs were preliminary in vivo challenged with steamed hay and in vitro stimulated with HDS, we also observed some grouping cells. This grouping are less important when AMs were preliminary challenged with dry hay. Moreover, the AMs from sEA are smaller size after dry hay challenge and HDS stimulation. Conclusions: The expression of markers of macrophages as CD14 seems to vary according to the status of the horse with better phagocytosis and macropinocytosis capacities in CD14+ AMs frome sEA horses. No difference in phagocytosis and macropinocytosis activity was observed for CD206+ cells regardless of the status of the horse. The behavior of AMs from sEA horses seem to be more affected by a previous in vivo dry hay challenge compared to AMs from CTL horses. In addition, steamed hay challenge in vivo seems appears to have less impact on the behavior of AMs compared dry hay for sEA and CTL horses.

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National audience; This article summarizes the current state of knowledge regarding the recent discovery of three new viruses potentially involved in Equine hepatitis. Identification of an Equine Hepacivirus in 2013 resulted from serendipity; since then, this virus has been identified in horses on all continents. In a recent French study, the prevalence was 6,2 % (64 positive horses out of 1033). Equine Pegiviruses were more recently discovered and remain an enigma. Theiler’s disease associated virus (TDAV) is strongly suspected to be the cause of Theiler’s disease. The three viruses can infect horses via injections of biological products.; Cet article présente l’état des connaissances à propos de la découverte récente de trois virus potentiellement impliqués dans les hépatites chez le cheval. Le premier hépacivirus équin a été découvert fortuitement en 2013 et, depuis, de nombreux travaux rapportent la présence de ce virus sur tous les continents. Une étude française a montré une prévalence de 6,2 % (64 prélèvements positifs/1 033). Les pégivirus, de découverte plus récente, restent une énigme encore actuellement. Le TDAV (Theiler’s disease associated virus) est fortement suspecté d’être responsable de la maladie de Theiler. Ces trois virus présentent la particularité de pouvoir infecter les chevaux par injection sanguine de produits biologiques.

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