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Additional file 1: Supplemental Figure 1. Two USUV EU2 strains induce a specific CPE and persistent viral replication in astrocytes. Supplemental Figure 2. Differential CPE in USUV-infected murine microglia and neurons cells. Supplemental Figure 3. USUV isolates induces CPE in human endothelial cells with an atypical shape for EU2 while there is no CPE in pericytes.

International audience; Arboviruses are viruses transmitted to humans and/or animals by hematophagous arthropods. They have a significant economic and public health impact. Given the number of arboviruses already identified and their great genetic variability, it is essential to have highly flexible tools for their monitoring. Arbovirus circulation within animal populations can be demonstrated by direct and/or indirect screening of a specific virus within vertebrate hosts and/or arthropod vectors. Viruses have great adaptive capacities that enable them to emerge into new geographic areas and/or cross species barriers. Over the decades, arbovirus monitoring has considerably evolved due to innovations in detection technologies. The objectives of this review are to list and assess (i) the current tools for direct or indirect screening for arboviruses, (ii) the new generation tools that best meet expectations in terms of optimal arbovirus monitoring and (iii) the potentials for improved arbovirus monitoring.; Les arbovirus sont des virus transmis à l'Homme et/ou à des animaux par des arthropodes hématophages. Ils sont importants en termes de santé publique mais également d'un point de vue économique. Au vu du nombre d'arbovirus déjà identifiés et de leur grande variabilité génétique, il est nécessaire, voire indispensable, d'avoir des outils ayant une grande flexibilité pour leur surveillance. La circulation des arbovirus au sein d'une population animale peut se démontrer par recherche directe du virus au sein de l'hôte et/ou du vecteur ou par recherche indirecte du virus en mettant en évidence une séroconversion chez l'hôte vertébré. Les virus ont des grandes capacités d'adaptation qui leur permettent d'émerger dans de nouvelles zones géographiques et/ou de franchir les barrières d'espèces. Au fil des décennies, la surveillance des arbovirus a considérablement évolué grâce à toutes les technologies innovantes qui ont vu le jour. Les objectifs de cette revue sont de faire l'état de l'art sur (i) les outils classiques pour la recherche directe ou indirecte des arbovirus, (ii) les outils de nouvelle génération qui répondent au mieux aux attentes en termes de surveillance optimale des arbovirus et (iii) les axes d'amélioration pour la surveillance des arbovirus.

Usutu virus (USUV) is an emerging arbovirus that was first isolated in South Africa in 1959. This Flavivirus is maintained in the environment through a typical enzootic cycle involving mosquitoes and birds. USUV has spread to a large part of the European continent over the two decades mainly leading to substantial avian mortalities with a significant recrudescence of bird infections recorded throughout Europe within the few last years. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. Nonetheless, a few cases of neurological complications such as encephalitis or meningoencephalitis have been reported. USUV and West Nile virus (WNV) share many features, like a close phylogenetic relatedness and a similar ecology, with co-circulation frequently observed in nature. However, USUV has been much less studied and in-depth comparisons of the biology of these viruses are yet rare. In this review, we discuss the main body of knowledge regarding USUV and compare it with the literature on WNV, addressing in particular virological and clinical aspects, and pointing data gaps.

The geographical distribution and impact on animal and human health of both West Nile and Usutu viruses, two flaviviruses of the Japanese encephalitis complex, have been increasing during the past two decades. Both viruses circulate in Europe and Africa within a natural cycle between wild birds and mosquitoes, mainly from the Culex genus. We retrospectively analyzed sera from domestic and wild birds sampled in 2008 in two wetlands, namely the Inner Niger Delta, Mali, and the Lake Alaotra area, Madagascar. Sera were first tested using a commercial ID Screen West Nile Competition Multi-species ELISA kit. Then, positive sera and sera with insufficient volume for testing with ELISA were tested with a Microneutralization Test. In Mali, the observed seroprevalence in domestic birds was 28.5% [24.5; 32.8] 95%CI, 3.1 % [1.8; 5.2] 95%CI, 6.2% [3.4; 10.2] 95%CI and 9.8 % [7.3; 12.8] 95%CI, for West Nile virus (WNV), Usutu virus (USUV), undetermined flavivirus, and WNV/USUV respectively. Regarding domestic birds of Madagascar, the observed seroprevalence was 4.4 % [2.1; 7.9]95%CI for WNV, 0.9% [0.1; 3.1] 95%CI for USUV, 1.3% [0.5; 2.8] 95%CI for undetermined flavivirus, and null for WNV/USUV. Among the 150 wild birds sampled in Madagascar, two fulvous whistling-ducks (Dendrocygna bicolor) were positive for WNV and two for an undetermined flavivirus. One white-faced whistling-duck (Dendrocygna viduata) and one Hottentot teal (Spatula hottentota) were tested positive for USUV. African and European wetlands are linked by wild bird migrations. This first detection of USUV—as well as the confirmed circulation of WNV in domestic birds of two wetlands of Mali and Madagascar—emphasizes the need to improve the surveillance, knowledge of epidemiological patterns, and phylogenetic characteristics of flavivirus in Africa, particularly in areas prone to sustained, intense flavivirus transmission such as wetlands.

National audience

National audience

Usutu virus, an arbovirus discovered in Africa in 1959, has spread over a large part of Europe over the last twenty years causing significant bird mortality as reported in France since 2015. The zoonotic risk, associated with this succession of avian epizootics in Europe, deserves to be taken into account even if human cases remain rare to date. Human infections are most often asymptomatic or present a benign clinical expression. However, neurological complications such as encephalitis or meningoencephalitis have been described. In addition, the recent description of an atypical case of facial paralysis in France suggests that the clinical spectrum of infections caused by Usutu virus is not fully characterized. Finally, the recent history of other arboviral outbreaks invites the scientific community to be extremely vigilant., Le virus Usutu, un arbovirus découvert en 1959 en Afrique, s’est propagé au cours des vingt dernières années sur une grande partie du continent européen provoquant notamment des mortalités aviaires importantes, comme cela est rapporté en France depuis 2015. Le risque zoonotique associé à cette succession d’épizooties aviaires en Europe mérite d’être considéré, même si à ce jour les cas humains restent exceptionnels. L’infection humaine est le plus souvent asymptomatique, ou d’une expression clinique bénigne. Toutefois, des complications neurologiques telles qu’encéphalites ou méningoencéphalites ont été décrites. L’observation récente en France d’un cas atypique de paralysie faciale a frigore1 suggère que le spectre clinique des infections dues au virus Usutu n’est pas complétement connu. L’histoire récente de flambées épidémiques d’autres arboviroses invite ainsi la communauté scientifique à la plus grande vigilance. Les connaissances concernant la physiopathologie de ce virus émergent sont, pour l’heure, très sommaires. Les travaux en cours visent donc à mieux appréhender sa biologie et les mécanismes associés aux atteintes neurologiques.

Objective: West Nile virus (WNV) is considered as the most prevalent arbovirus in Madagascar, yet few studies have focused on WNV circulation in wild birds and horses. The aims of this study are (i) to provide recent estimates of WNV seroprevalence and incidence in horses, (ii) to assess which wild bird species are exposed to WNV and (iii) to identify risk factors associated with exposure in horses and wild birds. Materials and methods: Horses from 4 regions of Madagascar were sampled before and after the 2015-2016 rainy season to estimate prevalence and incidence. Stable and horse characteristics were collected through a questionnaire. A wide range of wild birds species from the same regions were sampled in 2016-2017. Antibodies against WNV in horses and birds were tested using a competition ELISA test; results of a subset of samples were further confirmed using either a specific microsphere immunoassay for horses or a viral neutralization assay for birds. Logistic models were developed to identify risk factors. Results: Overall seroprevalence in horses was 33.5% (n=254) and incidence during the rainy season was 8.8% (n=147). Age, presence of ponds, use of insecticides and combined presence of rice fields and ruminants in the vicinity of stables were identified as risk factors. Overall, 352 birds (belonging to 41 species) were tested and 12.5% (belonging to 19 species) were seropositive. Birds caught outside wetlands, in Alaotra Mangoro region, belonging to the Passeriformes order, and species with an area of distribution in the Indian Ocean islands were significantly more exposed to WNV. Conclusion: This study confirms that WNV is endemic with high levels of circulation in horses, although no clinical cases were recorded. For the first time in Madagascar, 19 species of wild birds (among which 12 of the 21 Passeriformes species) were shown to be exposed to WNV.

Flaviviruses with a major impact in veterinary medicine are widely distributed (e.g. West Nile fever (WNF) has spread across the five continents and Japanese Encephalitis (JE) is reported in South-East Asia) and are mainly responsible for neurological diseases in humans and/or horses.After flavivirus infection, viremia in mammal hosts is generally short and consequently indirect methods are mostly used to diagnose flavivirus infections. However, frequent spatial overlapping in their circulation areas renders the interpretation of serological assays difficult. Indeed, cross-reactions between flaviviruses are observed in rapid serological tests such as in ELISA and immunofluorescence assays (IFA). Serological assay results should thus be confirmed by the tedious comparative virus neutralization test (VNT) using a panel of viruses known to circulate in the area. Moreover, the risk of emergence of new flaviviruses such as reported recently with the Zika virus in Brazil or in North America should be considered when studying flavivirus epidemiology.In the first section, a new strategy aiming at improving the serological diagnosis of flavivirus infections was developed using the multiplexing capacity of microsphere immunoassays (MIA). The flavivirus soluble envelope (sE) glycoprotein ectodomain is composed of three domains (D), e.g. DI, DII and DIII, with EDIII containing virus-specific epitopes. Recombinant EDIIIs of different flaviviruses were synthesized in the Drosophila S2 expression system. The microspheres coupled with rEDIIIs were assayed with equine and ovine sera from natural and experimental flavivirus infections or non-immune samples. Very encouraging results have been obtained and this innovative multiplex immunoassay based on flavivirus rEDIIIs appears to be a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.MIA with WNV nonstructural 1 protein were also implemented to differentiate Infected from Vaccinated Animals (DIVA). Such a DIVA approach was only successful when horses had been immunized with a recombinant canarypox vaccine, while horses receiving inactivated WNV vaccine developed immune responses close to the ones induced after natural infection.Another pitfall in veterinary medicine is the lack of therapeutics for viral diseases and specifically for flaviviruses. The therapeutic arsenal is indeed rather limited and animals are generally administered supportive treatments only. In the second part, the results of the in vitro testing of a broad spectrum antiviral named sr1057 on WNV and JEV replication are presented. This chemical, identified from a unique screening strategy developed by Pasteur Institute, is probably targeting the host cell and was found to inhibit the replication of varied RNA and DNA viruses belonging to different virus families. The sr1057 compound was not as efficient at inhibiting the replication of flaviviruses as for other RNA+ viruses, with a modest antiviral effect demonstrated for WNV and a higher efficacy on JEV. This antiviral presents however potentials for applications in equine veterinary medicine because it efficiently inhibited equine herpes virus-1 and equine arteritis virus in vitro, as clearly shown by other collaborators.; Les flavivirus ayant un impact en médecine vétérinaire sont largement distribués dans le monde (à l’exemple de la fièvre West Nile (WNF) présente sur les cinq continents ou de l’Encéphalite japonaise (EJ) en Asie du Sud-Est) et sont responsables de maladies à dominante neurologique chez l’homme et/ou le cheval.La virémie étant généralement brève lors de ces infections virales, les méthodes de diagnostic utilisées sont essentiellement sérologiques. Or le chevauchement fréquent des aires de répartition des flavivirus complique le diagnostic sérologique. En effet, des réactions sérologiques croisées entre flavivirus sont observées lors de l’utilisation de méthodes de diagnostic usuelles telles que l’ELISA et l’immunofluorescence (IF). Les résultats sérologiques doivent donc être confirmés par la méthode fastidieuse de séroneutralisation (SNT) virale avec les différents flavivirus existants dans la région. De plus, le risque d’émergence sur un territoire donné de nouveaux flavivirus comme le virus Zika au Brésil ou en Amérique du Nord ne peut être exclu.Nous avons donc développé dans la première partie de ce travail une nouvelle stratégie de diagnostic sérologique multiplexe à l’aide de la méthode d’immuno-essai (MIA) sur billes. Sachant que la glycoprotéine d’enveloppe soluble (sE) des flavivirus est divisée en 3 domaines (D) structuraux, DI, DII et DIII et que les épitopes du DIII sont spécifiques de chaque flavivirus, des protéines EDIII recombinantes de différents flavivirus d’intérêt ont été synthétisées en système d’expression Drosophila S2 et leur antigénicité a été testée sur sérums équins et ovins. Nous avons obtenu des résultats très encourageants en démontrant que l’utilisation des EDIII associée avec la capacité de multiplexage de la méthode MIA apparaît comme une réponse adaptée au défi du diagnostic sérologique des infections à flavivirus.Nous avons en outre utilisé la même méthode de multiplexage mais avec des antigènes NS1 du WNV pour mettre en place un test DIVA (Differentiating Infected from Vaccinated Animals) permettant de différencier les chevaux infectés par le WNV des chevaux vaccinés avec un vaccin recombinant WNV.Un autre écueil en médecine vétérinaire est le traitement des affections à flavivirus. En effet l’arsenal thérapeutique est limité et le traitement est avant tout symptomatique. Nous avons dans la seconde partie de ce travail testé in vitro une molécule antivirale à large spectre, le sr1057 sur nos virus d’intérêt (WNV et JEV). Cette molécule qui provient d’une stratégie de criblage développée par l’Institut Pasteur a probablement pour cible la cellule de l’hôte car elle est capable d’inhiber des virus très différents, à génome à ARN de polarité positive ou négative et ADN.Les résultats que nous avons obtenus avec ce composé sont en demi-teinte pour les flavivirus testés avec une efficacité démontrée pour le JEV mais plus modeste pour le WNV. Ils n’excluent cependant pas une possible utilisation de cet antiviral en médecine vétérinaire équine car une activité inhibitrice in vitro sur les virus de l’Herpes équin de type I et de l’artérite virale a été confirmée par d’autres collaborateurs.