[Emerging vectorial diseases: West Nile fever, Bluetongue and Schmallenberg]. / Arboviroses émergentes : fièvre West Nile, fièvre catarrhale ovine et virus Schmallenberg.

The increase in international trade over the last thirty years, climate change owing to the industrial revolution, disruption of ecosystems, etc. are some of the factors that may explain the dynamics of disease emergence in regions of the world where they were not present. Thus in 1999, West Nile virus was introduced on the American continent where it spread at high speed. More than 2300 deaths and more than 25,000 neuroinvasive forms were recorded in humans from 1999 to 2019 in the United States of America. In the field of animal diseases, two viruses have made headlines in Europe: bluetongue virus (BTV) and Schmallenberg virus (SBV). The bluetongue virus, previously absent from Europe, was introduced in 1999. Numerous serotypes (1, 2, 4, 6, 8, 9, 25, 27) have since been identified in the European Union. Schmallenberg virus was identified in 2011 in Northern Germany and rapidly spread to other European countries. This virus had never been identified in the world before. These three viruses (WNV, BTV and SBV) are transmitted by arthropod vectors (mosquitoes and Culicoïdes). These emergences are a good illustration of the challenges that our countries will face in the coming years, in public, human and veterinary health.

Usutu virus: A new threat?

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.

Early detection of West Nile virus in France: quantitative assessment of syndromic surveillance system using nervous signs in horses.

West Nile virus (WNV) is a growing public health concern in Europe and there is a need to develop more efficient early detection systems. Nervous signs in horses are considered to be an early indicator of WNV and, using them in a syndromic surveillance system, might be relevant. In our study, we assessed whether or not data collected by the passive French surveillance system for the surveillance of equine diseases can be used routinely for the detection of WNV. We tested several pre-processing methods and detection algorithms based on regression. We evaluated system performances using simulated and authentic data and compared them to those of the surveillance system currently in place. Our results show that the current detection algorithm provided similar performances to those tested using simulated and real data. However, regression models can be easily and better adapted to surveillance objectives. The detection performances obtained were compatible with the early detection of WNV outbreaks in France (i.e. sensitivity 98%, specificity >94%, timeliness 2·5 weeks and around four false alarms per year) but further work is needed to determine the most suitable alarm threshold for WNV surveillance in France using cost-efficiency analysis.

Evidence of exposure of laughing doves (Spilopelia senegalensis) to West Nile and Usutu viruses in southern Tunisian oases.

It has previously been suggested that southern Tunisian oases may be suitable areas for the circulation of flaviviruses. In order to anticipate and prevent possible epidemiological spread of flaviviruses in humans and domestic animals, the ecology of their transmission in the oasis system needs to be better understood. Thus, the aim of this study was to assess the seroprevalence of anti-flavivirus antibodies in the laughing dove (Spilopelia senegalensis), an abundant resident bird in Tunisian oases. Anti-flavivirus antibodies were detected in 17% of sampled doves. Ten per cent of the total tested doves were West Nile virus (WNV) seropositive and 4% were Usutu virus (USUV) seropositive, which provides the first evidence of USUV circulation in Tunisian birds. We also found that the occurrence probability of anti-flavivirus antibodies in dove plasma increased with decreasing distance to coast, suggesting that doves inhabiting coastal oases were more exposed to flaviviruses compared with those inhabiting inland oases. We also found significantly higher antibody occurrence probability in adult doves compared with young doves, which underlines the effect of exposure time. Overall, our results suggest that the laughing dove may be used for WNV and USUV surveillance in southern Tunisia. They also stress the need for investigations combining data on birds and mosquitoes to better understand the ecological factors governing the circulation of flaviviruses in this area.

Seroprevalence of West Nile and Usutu viruses in military working horses and dogs, Morocco, 2012: dog as an alternative WNV sentinel species?

A serosurvey of 349 military working horses and 231 military working dogs was conducted in ten sites in Morocco in 2012. This survey revealed a high level of exposure of these animals to flaviviruses: seroprevalence rates of 60% in horses and of 62% in dogs were observed using a competitive West Nile virus (WNV) enzyme-linked immunosorbent assay (cELISA). Seroneutralization test results showed that the majority of cELISA-positive results were due to exposure to WNV. Further assays conducted in vaccinated horses with a DIVA (Differentiating Infected from Vaccinated Animals) test indicated that anti-WNV antibodies had been stimulated through WNV natural infection. Moreover, in both species, seroneutralization tests suggested an exposure to Usutu virus (USUV). Data analysis did not show any significant difference of cELISA seropositivity risk between horses and dogs. Dogs may thus represent an interesting alternative to equines for the serological surveillance of WNV or USUV circulation, especially in areas where equine vaccination precludes passive surveillance (based on the detection of West Nile fever cases) in horses.

Serological evidence for the circulation of flaviviruses in seabird populations of the western Indian Ocean.

Birds play a central role in the epidemiology of several flaviviruses of concern for public and veterinary health. Seabirds represent the most abundant and widespread avifauna in the western Indian Ocean and may play an important role as host reservoirs and spreaders of arthropod-borne pathogens such as flaviviruses. We report the results of a serological investigation based on blood samples collected from nine seabird species from seven islands in the Indian Ocean. Using a commercial competitive enzyme-linked immunosorbent assay directed against the prototypic West Nile flavivirus, antibodies against flaviviruses were detected in the serum of 47 of the 855 seabirds tested. They were detected in bird samples from three islands and from four bird species. Seroneutralization tests on adults and chicks suggested that great frigatebirds (Fregata minor) from Europa were infected by West Nile virus during their non-breeding period, and that Usutu virus probably circulated within bird colonies on Tromelin and on Juan de Nova. Real-time polymerase chain reactions performed on bird blood samples did not yield positive results precluding the genetic characterization of flavivirus using RNA sequencing. Our findings stress the need to further investigate flavivirus infections in arthropod vectors present in seabird colonies.

West Nile virus epizootics in the Camargue (France) in 2015 and reinforcement of surveillance and control networks.

West Nile virus (WNV) infection is a non-contagious disease mainly transmitted by the bites of infected mosquitoes from the genus Culex. The virus is maintained in a mosquito-bird-mosquito cycle, and can accidentally be transmitted to mammalian hosts. Among mammalian hosts, equines and humans are the most sensitive to WNV infection and can develop severe meningoencephalitis. As WNV infections are zoonotic and can be severe in humans and equines, West Nile fever is considered to be a public and animal health concern. After a silent period of almost ten years, WNV re-emerged in France at the periphery of the Camargue area during the summer of 2015, underlining the fact that the Camargue area creates favourable conditions for WNV emergence and amplification in France. The French Network for Epidemiological Surveillance of Equine Diseases (Réseau d'Épidémio-Surveillance en Pathologie Équine [RESPE]) facilitated the early detection of WNV cases in horses. In total, 49 horses were found to be infected; among them, 44 presented clinical signs, 41 with meningoencephalitis and three with hyperthermia only. Six horses among the 41 with nervous symptoms died from the disease or were euthanised (a case fatality rate of 14.6%). The authors describe the characteristics of the 2015 WNV epizootics, the early detection of the first WNV equine cases via the RESPE network and the coordination of WNV surveillance in France.

L'infection par le virus de West Nile est une maladie non contagieuse essentiellement transmise lors de piqûres de moustiques infectés appartenant au genre Culex ; le virus se maintient dans la nature au moyen d'un cycle moustique­ oiseau­moustique ; la transmission à des hôtes mammifères a lieu de manière accidentelle. Parmi les mammifères hôtes, les plus sensibles à l'infection par le virus de West Nile sont les équidés et l'homme, chez qui l'infection peut se manifester sous forme d'une méningo-encéphalite sévère. Les infections par le virus de West Nile étant des zoonoses potentiellement graves chez l'homme et chez les équidés, la fièvre de West Nile doit être considérée comme une priorité de santé publique et animale. Resté silencieux pendant plus d'une décennie, le virus de West Nile est réapparu en France à l'été 2015 en bordure de la Camargue, confirmant que les conditions de cette région sont favorables à l'émergence et à l'amplification du virus. Le réseau français d'épidémiosurveillance en pathologie équine (RESPE) a contribué à la détection précoce du virus de West Nile chez les chevaux. Au total, 49 chevaux étaient infectés, parmi lesquels 44 présentaient des signes cliniques, correspondant à une méningo-encéphalite pour 41 d'entre eux et à une hyperthermie seule pour les trois autres. Six chevaux parmi les 41 qui présentaient des signes neurologiques ont succombé à la maladie ou ont été euthanasiés (taux de létalité de 14,6 %). Les auteurs de cet article décrivent les principales caractéristiques de l'épizootie de 2015 due au virus de West Nile ainsi que la détection précoce des premiers cas équins grâce au réseau RESPE et la coordination des activités de surveillance du virus en France.

La infección por el virus West Nile es una enfermedad no contagiosa que se transmite básicamente por la picadura de mosquitos infectados del género Culex. El virus, que se instala en un ciclo mosquito­ave­mosquito, también puede transmitirse accidentalmente a mamíferos, de entre los cuales los más sensibles a la infección son los equinos y el ser humano, que pueden contraer graves meningoencefalitis. Puesto que las infecciones por este virus son zoonóticas y pueden revestir gravedad en personas y equinos, se considera que la fiebre West Nile es una enfermedad de importancia sanitaria y zoosanitaria. En Francia, tras un periodo silente de más de diez años, el virus reapareció en verano de 2015 en la periferia de la zona de la Camarga, poniendo así de manifiesto que esta zona genera condiciones propicias al surgimiento y la amplificación del virus en el país. La red francesa de vigilancia epidemiológica de patologías equinas (Réseau d'Épidémio-Surveillance en Pathologie Équine: RESPE]) facilitó la rápida detección de caballos infectados por el virus West Nile. Se detectaron en total 49 animales infectados, entre ellos 44 con signos clínicos, de los que 41 sufrían meningoencefalitis y tres solo presentaban hipertermia. Seis de los 41 caballos que mostraban signos neurológicos murieron a causa de la enfermedad o fueron sacrificados con métodos de eutanasia (lo que supone una tasa de letalidad del 14,6%). Los autores describen las principales características de la epizootia causada por el virus West Nile en 2015, la pronta detección de los primeros casos de caballos infectados gracias a la red RESPE y la coordinación de las labores de vigilancia del virus en Francia.

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model.

West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal Middle-East WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 700 × in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.

Exposure of resident sparrows to West Nile virus evidenced in South Tunisia.

During the last few years, several cases of West Nile virus (WNV) infection in humans have been reported in Tunisia. However, detailed information on WNV infection in wild birds, the primary amplifying host of WNV, are lacking. In this work, we investigated the exposure of wild sparrows (hybrid Passer domesticus × hispaniolensis) living in two oases in southern Tunisia (Gabès and Kébili oases) to WNV, through the detection of WNV-specific antibodies by using ELISA and microneutralization tests. In total, 208 birds were sampled (54 from Kébili, 154 from Gabès). Anti-WNV antibodies were detected in two birds, corresponding to an overall seroprevalence of 1%. There was no significant difference between the two sampled populations [1·85% (1/54) in Kébili, 0·65% (1/154) in Gabès]. These data provide indirect evidence of the exposure of resident sparrows in southern Tunisia to WNV.

High prevalence of West Nile virus in equines from the two provinces of Pakistan.

This study describes the first large-scale serosurvey on West Nile virus (WNV) conducted in the equine population in Pakistan. Sera were collected from 449 equids from two provinces of Pakistan during 2012-2013. Equine serum samples were screened using a commercial ELISA kit detecting antibodies against WNV and related flaviviruses. ELISA-positive samples were further investigated using virus-specific microneutralization tests (MNTs) to identify infections with Japanese encephalitis virus (JEV), WNV and tick-borne encephalitis virus (TBEV). Anti-WNV antibodies were detected in 292 samples by ELISA (seroprevalence 65.0%) and WNV infections were confirmed in 249 animals by MNT. However, there was no animal found infected by JEV or TBEV. The detection of WNV-seropositive equines in Pakistan strongly suggests a widespread circulation of WNV in Pakistan.

African horse sickness.

African horse sickness (AHS) is a devastating disease of equids caused by an arthropod-borne virus belonging to the Reoviridae family, genus Orbivirus. It is considered a major health threat for horses in endemic areas in sub-Saharan Africa. African horse sickness virus (AHSV) repeatedly caused large epizootics in the Mediterranean region (North Africa and southern Europe in particular) as a result of trade in infected equids. The unexpected emergence of a closely related virus, the bluetongue virus, in northern Europe in 2006 has raised fears about AHSV introduction into Europe, and more specifically into AHSV-free regions that have reported the presence of AHSV vectors, e.g. Culicoides midges. North African and European countries should be prepared to face AHSV incursions in the future, especially since two AHSV serotypes (serotypes 2 and 7) have recently spread northwards to western (e.g. Senegal, Nigeria, Gambia) and eastern Africa (Ethiopia), where historically only serotype 9 had been isolated. The authors review key elements of AHS epidemiology, surveillance and prophylaxis.

West Nile virus epidemiology and factors triggering change in its distribution in Europe.

West Nile virus (WNV) has historically been considered among the least virulent members of the Japanese serogroup complex (family Flaviviridae, genus Flavivirus). The WNV natural cycle involves birds as the main amplifying hosts and several species of mosquito as vectors. Many outbreaks occurred during the past decade, causing severe human encephalitis in the Old World, and the virus has become established in many European countries. Emergence of WNV is difficult to predict and even more difficult to prevent. In this review, the latest information on the epidemiology, transmission dynamics and clinical aspects of WNV is presented, with particular focus on specific factors likely to trigger changes in the distribution of the disease in Europe, such as climate changes and their consequences on the potential vectors of WNV or bird migration routes. The control of some anthropogenic and environmental factors could help prevent extension and re-emergence of WNV epidemics.

Spread of West Nile virus in Iran: a cross-sectional serosurvey in equines, 2008-2009.

We report the first large-scale serosurvey for West Nile virus (WNV) conducted in the equine population in Iran. Blood samples were obtained in 2008-2009 from 1054 equines collected from 260 districts located in 27 provinces. The overall seroprevalence rate for WNV neutralizing antibodies was 23·7%. Marked geographical variations were observed as province-specific seroprevalence rates ranged from 1% to 88%, the highest values being observed in the southern and western parts of the country. The presence of IgM-positive animals (n=9) indicated a recent circulation of WNV in several provinces. Logistic modelling confirmed this result with a significant effect of age on seropositivity. This study revealed extensive circulation of WNV in Iran particularly in southwestern provinces where the virus probably circulates every year.

Evidence of exposure to zoonotic flaviviruses in zoo mammals in Spain and their potential role as sentinel species.

A serosurvey was carried out to assess emerging flavivirus exposure in zoo mammals in Spain and to determine the dynamics of seropositivity in species that were longitudinally sampled during the study period. Sera from 570 zoo animals belonging to 120 mammal species were collected at ten zoos (A-J) in Spain between 2002 and 2019. Twenty-one of these animals, belonging to ten different species, were sampled longitudinally at four of the zoos during the study period. Antigenically-related flavivirus antibodies were detected in 19 (3.3 %; 95 %CI: 2.0-5.2) of the 570 animals analyzed using bELISA. Seropositivity was observed in ten (8.3 %) of the 120 species tested. Five (23.8 %) of the 21 animals sampled more than once presented seropositivity in all samplings whereas seroconversion was only observed in one white rhinoceros (Ceratotherium simum). Flavivirus antibodies were found at six of the ten sampled zoos and in consecutive years between 2008 and 2018. Virus neutralization tests confirmed West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) infection in ten (1.8 %; 95 %CI: 0.7-2.8), five (0.9 %; 95 %CI: 0.1-1.6) and one (0.2 %; 95 %CI: 0.0-0.5) animal, respectively. Antibodies against Meaban virus (0 %; 95 %CI: 0.0-0.7 %) were not found in the tested sera. The results demonstrate WNV, USUV and TBEV exposure in zoo mammals, which may be of public health and conservation concern. Seropositivity to WNV and USUV was detected in regions where these viruses have not been reported previously. Anti-WNV antibodies found in zoo animals sampled in 2009 point to WNV circulation at least one year before the first outbreaks were reported in horses and humans in Spain. Our results indicate that zoo mammals could be useful sentinel species for monitoring emerging flavivirus activity in urban areas.

Exposure to West Nile virus and tick-borne encephalitis virus in dogs in Spain.

In the past decade, the spread of emerging zoonotic flaviviruses (genus Flavivirus, family Flaviviridae) has been reported in many regions worldwide, representing a threat to both human and animal health. A serosurvey was carried out to assess exposure and risk factors associated with antigenically related flaviviruses, particularly West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV), in dogs in Spain. Flavivirus antibodies were detected in 39 of 815 dogs (4.8%; 95% CI: 3.3-6.3) by bELISA. Significantly higher seropositivity was observed in hunting dogs compared to pet dogs. Virus neutralization tests confirmed WNV-specific and TBEV-specific antibodies in 11 and 14 bELISA-positive dogs, respectively. This is the first serosurvey of WNV and TBEV in dogs in Spain and the first report of TBEV circulation in this country. The seropositivity obtained indicates widespread, but not homogeneous, distribution of WNV and TBEV in dogs in Spain. In 2013 and 2015, WNV-seropositive dogs were detected in those areas of Andalusia where the highest number of WNV outbreaks were reported in both horses and humans. Antibodies against TBEV have been found in dogs sampled in two different periods and regions in Spain. Serosurveillance in dogs could be a complementary way of monitoring the activity of emerging flaviviruses in Spain.

Japanese encephalitis circulation pattern in swine of northern Vietnam and consequences for swine's vaccination recommendations.

Japanese encephalitis (JE) is the largest worldwide cause of infectious encephalitis in humans and is caused by a mosquito-borne flavivirus. JE transmission cycle involves Culex mosquitoes, pigs and aquatic birds as principal vertebrate amplifying hosts. JE infection is responsible for reproductive disorder in pigs when occurring after sexual maturity. In tropical areas, JE is endemic and the majority of pigs get infected before the age of 6 months. However, in subtropical areas, pigs may be infected after sexual maturity and thus experience clinical signs, inducing economic loss. The study aimed at better characterizing the influence of seasonal temperature variations (through estimates of degree days, DD) on JE circulation in pigs in subtropical area and inferring on the potential impact on JE symptomatic infection in reproductive pigs. Six hundred and forty-one pig's sera sampled in northern Vietnam were analysed for Japanese encephalitis virus (JEV) by pan-flavivirus ELISA. A subset of 108 ELISA-positive samples, representative of each sampling occasion, were confirmed by JEV neutralization test compared with West Nile virus neutralization test. We modelled the seroprevalence of pigs according to a DD variable using a generalized additive model. We then predicted the age of infection in pigs according to their month of birth, using averaged temperature data over 10 years. The model predicts that only 80 percentage of pigs born between July and September will be protected against JEV when reaching sexual maturity contrary to the rest of the year when almost all pigs will seroconvert before sexual maturity. In subtropical area such as northern Vietnam, pigs could thus show symptomatic infection due to JE, and consequently reproductive disorders. Vaccination of future breeder pigs in epidemic areas could avoid the occurrence of JE-associated reproductive disorders.

Are tree squirrels involved in the circulation of flaviviruses in Italy?

West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) are emerging zoonotic flaviviruses (family Flaviviridae), which have circulated in Europe in the past decade. A cross-sectional study was conducted to assess exposure to these antigenically related flaviviruses in eastern grey squirrels (Sciurus carolinensis) in Italy. Seventeen out of 158 (10.8%; CI95% : 5.9-15.6) squirrels' sera tested through bELISA had antibodies against flaviviruses. Specific neutralizing antibodies to WNV, USUV and TBEV were detected by virus neutralization tests. Our results indicate that tree squirrels are exposed to Culex and tick-borne zoonotic flaviviruses in Italy. Moreover, this study shows for the first time USUV and TBEV exposure in grey squirrels, broadening the host range reported for these viruses. Even though further studies are needed to define the real role of tree squirrels in the epidemiology of flaviviruses in Europe, this study highlights that serology could be an effective approach for future investigations aimed at broadening our knowledge about the species exposed to these zoonotic infections.

Comparative Risk Analysis of Two Culicoides-Borne Diseases in Horses: Equine Encephalosis More Likely to Enter France than African Horse Sickness.

African horse sickness (AHS) and equine encephalosis (EE) are Culicoides-borne viral diseases that could have the potential to spread across Europe if introduced, thus being potential threats for the European equine industry. Both share similar epidemiology, transmission patterns and geographical distribution. Using stochastic spatiotemporal models of virus entry, we assessed and compared the probabilities of both viruses entering France via two pathways: importation of live-infected animals or importation of infected vectors. Analyses were performed for three consecutive years (2010-2012). Seasonal and regional differences in virus entry probabilities were the same for both diseases. However, the probability of EE entry was much higher than the probability of AHS entry. Interestingly, the most likely entry route differed between AHS and EE: AHS has a higher probability to enter through an infected vector and EE has a higher probability to enter through an infectious host. Consequently, different effective protective measures were identified by 'what-if' scenarios for the two diseases. The implementation of vector protection on all animals (equine and bovine) coming from low-risk regions before their importation was the most effective in reducing the probability of AHS entry. On the other hand, the most significant reduction in the probability of EE entry was obtained by the implementation of quarantine before import for horses coming from both EU and non-EU countries. The developed models can be useful to implement risk-based surveillance.

West Nile virus infection in horses, Indian ocean.

The circulation of West Nile virus (WNV) in horses was investigated in the Southwest Indian ocean. In 2010, blood samples were collected from a total of 303 horses originating from Madagascar, Mauritius, Reunion and the Seychelles and tested for WNV-specific antibodies. An overall seroprevalence of 27.39% was detected in the Indian Ocean with the highest WNV antibody prevalence of 46.22% (95% CI: [37.4-55.2%]) in Madagascar. The age and origin of the horses were found to be associated with the WNV infection risk. This paper presents the first seroprevalence study investigating WN fever in horses in the Southwest Indian Ocean area and indicates a potential risk of infection for humans and animals. In order to gain a better understanding of WN transmission cycles, WNV surveillance needs to be implemented in each of the countries.

A Serological Protein Microarray for Detection of Multiple Cross-Reactive Flavivirus Infections in Horses for Veterinary and Public Health Surveillance.

The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection with WNV and JEV can lead to severe neurological disease and death, while USUV, SLEV and TBEV infections are mainly asymptomatic, however, and induce antibody responses. Horses often serve as sentinels to monitor active virus circulation in serological surveillance programmes specifically for WNV, USUV and JEV. Here, we developed and validated a NS1-antigen protein microarray for the serological differential diagnosis of flavivirus infections in horses using sera of experimentally and naturally infected symptomatic as well as asymptomatic horses. Using samples from experimentally infected horses, an IgG and IgM specificity of 100% and a sensitivity of 95% for WNV and 100% for JEV was achieved with a cut-off titre of 1 : 20 based on ROC calculation. In field settings, the microarray identified 93-100% of IgG-positive horses with recent WNV infections and 87% of TBEV IgG-positive horses. WNV IgM sensitivity was 80%. Differentiation between closely related flaviviruses by the NS1-antigen protein microarray is possible, even though we identified some instances of cross-reactivity among antibodies. However, the assay is not able to differentiate between naturally infected horses and animals vaccinated with an inactivated WNV whole-virus vaccine. We showed that the NS1-microarray can potentially be used for diagnosing and distinguishing flavivirus infections in horses and for public health purposes within a surveillance setting. This allows for fast, cheap, syndrome-based laboratory testing for multiple viruses simultaneously for veterinary and public health purposes.