# Evidence of Exposure to USUV and WNV in Zoo Animals in France

Orianne Constant, Karine Bollore, Marion Clé, Jonathan Barthelemy, Vincent Foulongne, Baptiste Chenet, David Gomis, Laurie Virolle, Serafin Gutierrez, Caroline Desmetz, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Orianne Constant, Karine Bollore, Marion Clé, Jonathan Barthelemy, Vincent Foulongne, et al. Evidence of Exposure to USUV and WNV in Zoo Animals in France. Pathogens, 2020, 9 (12), pp.1005. 10.3390/pathogens9121005. hal-03081918

HAL Id: hal-03081918

https://hal.umontpellier.fr/hal-03081918

Submitted on 21 Dec 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Article

# Evidence of Exposure to USUV and WNV in Zoo Animals in France

Orianne Constant<sup>1,†</sup>, Karine Bollore<sup>1,†</sup>, Marion Clé<sup>1</sup>, Jonathan Barthelemy<sup>1</sup>, Vincent Foulongne<sup>1</sup>, Baptiste Chenet<sup>2 ID</sup>, David Gomis<sup>2</sup>, Laurie Virolle<sup>2 ID</sup>, Serafin Gutierrez<sup>3</sup>, Caroline Desmetz<sup>4</sup>, Rayane Amaral Moares<sup>5</sup>, Cécile Beck<sup>5 ID</sup>, Sylvie Lecollinet<sup>5 ID</sup>, Sara Salinas<sup>1</sup> and Yannick Simonin<sup>1,* ID</sup>

Received: 22 October 2020; Accepted: 29 November 2020; Published: 30 November 2020

<figure><img src="image_3.png" /></figure>

check for
updates

**Abstract:** West Nile virus (WNV) and Usutu virus (USUV) are zoonotic arboviruses. These flaviviruses are mainly maintained in the environment through an enzootic cycle involving mosquitoes and birds. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Nevertheless, there is little data regarding the involvement of other mammals in the epidemiology of these arboviruses. In this study, we performed a serosurvey to assess exposure to these viruses in captive birds and mammals in a zoo situated in the south of France, an area described for the circulation of these two viruses. A total of 411 samples comprising of 70 species were collected over 16 years from 2003 to 2019. The samples were first tested by a competitive enzyme-linked immunosorbent assay. The positive sera were then tested using virus-specific microneutralization tests against USUV and WNV. USUV seroprevalence in birds was 10 times higher than that of WNV (14.59% versus 1.46%, respectively). Among birds, greater rhea (Rhea Americana) and common peafowl (Pavo cristatus) exhibited the highest USUV seroprevalence. Infections occurred mainly between 2016–2018 corresponding to a period of high circulation of these viruses in Europe. In mammalian species, antibodies against WNV were detected in one dama gazelle (Nanger dama) whereas serological evidence of USUV infection was observed in several *Canidae*, especially in African wild dogs (*Lycaon pictus*). Our study helps to better understand the exposure of captive species to WNV and USUV and to identify potential host species to include in surveillance programs in zoos.

**Keywords:** usutu virus; west nile virus; flavivirus; arbovirus; seroprevalence; zoological garden

<footnote n="1">¹ Pathogenesis and Control of Chronic Infections, University of Montpellier, INSERM, EFS, 34000 Montpellier, France; orianne constant@gmail.com (O.C.); karine.bollore@umontpellier.fr (K.B.); marion.cle@umontpellier.fr (M.C.); jonathan.barthelemy@inserm.fr (J.B.); v-foulongne@chu-montpellier.fr (V.F.); sara.salinas@inserm.fr (S.S.)</footnote>

<footnote n="2">² Parc de Lunaret—Zoo de Montpellier, 34090 Montpellier, France; Baptiste.CHENET@ville-montpellier.fr (B.C.); David.GOMIS@ville-montpellier.fr (D.G.); Laurie.VIROLLE@ville-montpellier.fr (L.V.)</footnote>

<footnote n="3">³ ASTRE Research Unit, CIRAD, INRA, 34398 Montpellier, France; serafin.gutierrez@cirad.fr</footnote>

<footnote n="4">⁴ bBioCommunication en CardioMétabolique (BC2M), Montpellier University, 34000 Montpellier, France; caroline.desmetz@umontpellier.fr</footnote>

<footnote n="5">5 UMR 1161 Virology, ANSES, INRAE, ENVA, ANSES Animal Health Laboratory, EURL for Equine Diseases, 94704 Maisons-Alfort, France; rayane.amaralmoraes@anses.fr (R.A.M.); Cecile.BECK@anses.fr (C.B.); Sylvie.LECOLLINET@anses.fr (S.L.)</footnote>

<footnote>* Correspondence: yannick.simonin@umontpellier.fr; Tel.: +33-(0)4-3435-9114</footnote>

<footnote>† Equally contributed.</footnote>
# 1. Introduction

Arboviruses are emerging pathogens on the European continent and, more generally worldwide, although they are already well established in various regions, particularly in Africa and Latin America. Among arboviruses, several flaviviruses (*Flaviviridae* family), e.g West Nile (WNV), Usutu (USUV), tick born encephalitis virus (TBEV), and Meaban (MEAV) viruses are the main flaviviruses that have been known to circulate in France [1–4]. WNV and USUV belong to the Japanese encephalitis (JEV) serocomplex in the *Flavivirus* genus and share many common features [5,6]. Their transmission cycle mainly involves ornithophilic mosquitoes such as *Culex pipiens* and birds. Wild birds play a central role in flavivirus epidemiology as they are the main amplifying hosts in the wilderness. They develop a strong and transitory viremia, are capable of infecting bird-biting mosquitoes, and may contribute to the spread of the virus with their migratory behavior [3]. Moreover, WNV and USUV occasionally infect other vertebrates including humans and horses [7]. TBEV, in the TBEV serocomplex is maintained in an enzootic cycle involving hard ticks (Ixodes ricinus in Western Europe) and rodents as the main reservoir [8]. Birds are suspected to play a role in the spread of the virus [9]. MEAV is a soft tick-borne flavivirus transmitted by *Ornithodoros maritimus* ticks among seabird colonies and has only been identified in Brittany on the Meaban island [1].

WNV was first detected in the West Nile region of Uganda in 1937 from the blood of a febrile woman [10]. Its global distribution has greatly expanded over the last two decades and is currently common in countries of Africa, the Middle East, India, Australia, Central and Southern Europe, and in North, Central, and South America where it can cause disease outbreaks [11–13]. Natural exposure to WNV has also been detected in a wide range of mammals (more than 100 species) [14]. USUV has gained the attention of the scientific community due to its recent incursions in Europe. It was isolated for the first time in South Africa in 1959 [15,16]. Since then, this emerging arbovirus has spread out of Africa, mainly in Europe [3]. Birds and humans infected by USUV can develop neuroinvasive pathologies (including encephalitis and meningoencephalitis). Serologic and molecular evidence of USUV presence in blood donors suggests the silent spread of this virus among asymptomatic humans [3]. Beyond birds and humans, USUV has also been detected in different mammals such as horses, dogs, wild boars, wild ruminants, bats, rodents, and shrews [17–24]. Seroepidemiological studies showed that USUV is probably endemic in several European countries. Whereas WNV is involved in occasional outbreaks or endemic in some of them, mainly in Southern Europe such as in Greece, Italy, or Spain [3,25]. Recently, increased and concomitant transmission of both viruses has been reported in Europe. Beyond a record drought and heat wave, the summer of 2018 saw the largest WNV epidemic reported in terms of countries involved. A total of 16 European countries, including France, were involved and 2083 human cases and 181 deaths were listed [26]. Similarly, the largest epizootic of USUV in Europe was observed in 2018, inducing a massive death of wild birds [3,27,28].

TBEV is the arbovirus with the highest public health impact in Europe. Thousands of TBEV neurological cases are recorded every year in Europe [29]. Human infections can occur through the bite from TBEV-infected ticks and more rarely through the consumption of unpasteurized milk or milk products from cattle, goats, or sheep. TBEV is endemic in many European countries, including the Baltic and Scandinavian countries, Slovenia, and the Czech Republic. In France, the virus has been reported in Eastern France, mainly in Alsace and since 2003 in the Alps (Haute-Savoie) and more recently in Auvergne-Rhône-Alpes (Loire and Haute-Loire) [2,30]. TBEV infections have been detected more largely in Eastern France in a serosurvey on wild boars and roe deer [31].

Monitoring viral circulation is difficult, especially for viruses with complex transmission cycles. Moreover, serological cross-reactions between flaviviruses can hamper the rapid identification of circulating viruses. Elisa tests suffer from a lack of specificity and need to be confirmed by seroneutralization assays. Zoological gardens represent sites of interest to gain knowledge about the distribution of emerging zoonotic pathogens in different animal species. Therefore, zoos have been proposed as sites adapted to the surveillance of zoonotic viruses, including WNV and USUV [32–36]. These sites generally contain a large diversity of captive species, including birds and mammals,
and are usually close to or within urban areas. Moreover, since captive animals are regularly monitored by veterinarians, samples can be easily available. In addition, WNV and USUV have been frequently detected in birds in zoological gardens and associated with important mortality in some species [23,24,27,28]. Nevertheless, few studies have been published on WNV or USUV exposure in zoo animals [23–26]. Montpellier zoo (south of France) is close to the Camargue area, a wetland that houses a large diversity of wild bird species, including migratory birds and various mosquito populations. This environment is considered to favor the transmission of USUV and WNV [29–32]. In this study, we evaluated in priority the seropositivity against WNV and USUV in captive birds and exotic mammals from the Montpellier zoological garden. TBEV and MEAV circulation has not been evidenced in the south of France. Consequently, when positive samples of competitive enzyme linked immunoabsorbent assay (cELISA) were found negative in WNV and USUV MNTs, TBEV and MEAV virus neutralization assays were carried out to investigate infections by alternative flaviviruses. The objective was to investigate the prevalence of flavivirus infections in a large variety of species and to assess the value of integrating their monitoring within the framework of WNV and USUV surveillance programs. Our results enlarge current knowledge on mammals and birds potentially infected by these viruses and point to species with high seroprevalence as candidates for sentinel surveillance.

## 2. Results

### 2.1. Circulation of USUV/WNV in Zoo Birds

A point to note is that no obvious abnormal bird mortality was observed in Montpellier zoo. Analysis in birds were carried out on 137 specimens of 20 different species (Table 1). Using competitive enzyme linked immunoabsorbent assay (cELISA), we identified antibodies against flaviviruses in 23 out of 137 birds (16.79%, CI95%: 10.53–23.05). Among them, 20 specimens tested positive for USUV-specific antibodies by virus-specific micro neutralization test (MNT) (14.59%, CI95%: 8.686–20.51) and 2 specimens for WNV-specific antibodies (1.45%, CI95%: 0.0–3.46). Overall, our results show a greater proportion of positive specimens for USUV than for WNV.

Moreover, one common ostrich (*Struthio camelus*) presented antibodies against an undetermined flavivirus as we did not detect TBEV or MEAVS-specific antibodies by virus-specific micro neutralization test (Table 1). The two positive birds for WNV were one orange-winged amazon (*Amazona amazonica*) and one marabou stork (*Leptoptilos crumenifer*). The individuals positive to USUV were one Abyssinian ground hornbill (*Bucorvus abyssinicus*), one scarlet ibis (*Eudocimus ruber*), and two emus (*Dromaius novaehollandiae*). We also found antibodies against USUV in 6 common peafowl (*Pavo cristatus*) and 10 greater rhea (*Rhea Americana*) representing respectively 25% and 46.7% seroprevalence in the tested populations. These species, to the best of our knowledge, have never been described as susceptible to flavivirus infection. We analyzed the samples collected from the same animals over several years in order to explore infection temporality. This analysis provided information on viral circulation over time, particularly since USUV and WNV have been only quite recently widely reported in the area. Despite the lack of a continuous sampling over years, we were able to study the temporality of infection for some of the animals. Interestingly longitudinal studies showed that all the sequentially sampled animals found seropositive on the first sampling stayed seropositive throughout the study period as all seropositive birds in 2016 were also positive in 2018 (5/5) (Table A1). Moreover, the majority of specimens were infected between 2016 to 2018 (64.2%, 9/14 for WNV or USUV-positive birds) while the remaining samples show that WNV and USUV were introduced before 2016 in the area.
<table><caption>**Table 1.** Birds species tested by ELISA (enzyme linked immunoabsorbent assay) and for ELISA-positive samples by MNT (microneutralization tests) for West Nile virus (WNV) and Usutu virus (USUV).</caption><thead><tr><th colspan="8">Avian Species</th></tr><tr><th>Species<br>(English Name)</th><th>Species<br>(Scientific Name)</th><th>Collection<br>Date</th><th>Nb of<br>Specimens</th><th>cElisa</th><th colspan="3">MNT Antibodies Titer</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th>USUV</th><th>WNV</th><th>Conclusion</th></tr></thead><tbody><tr><td>Orange-winged amazon</td><td><i>Amazona amazonica</i></td><td>2016–2018</td><td>8</td><td>1</td><td>Neg</td><td>10</td><td>1/8 WNV</td></tr><tr><td>Brazilian teal</td><td><i>Amazonetta brasiliensis</i></td><td>2016–2018</td><td>6</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Buffon's macaw</td><td><i>Ara macao</i></td><td>2016–2018</td><td>2</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Great green macaw</td><td><i>Ara ambiguus</i></td><td>2016–2018</td><td>4</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>White-winged duck</td><td><i>Asarcornis scutulata</i></td><td>2016</td><td>2</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Abyssinian ground hornbill</td><td><i>Bucorvus abyssinicus</i></td><td>2016–2018</td><td>5</td><td>1</td><td>640</td><td>20</td><td>1/5 USUV</td></tr><tr><td>Greater vasa parrot</td><td><i>Coracopsis vasa</i></td><td>2019</td><td>2</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Southern screamer</td><td><i>Chauna torquata</i></td><td>2016–2018</td><td>2</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>White-faced whistling duck</td><td><i>Dendrocygna viduata</i></td><td>2016–2018</td><td>1</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Emu</td><td><i>Dromaius novaehollandiae</i></td><td>2016–2018</td><td>5</td><td>2</td><td>160</td><td>20</td><td>2/5 USUV</td></tr><tr><td>Scarlet ibis</td><td><i>Eudocimus ruber</i></td><td>2016–2018</td><td>38</td><td>1</td><td>640</td><td>10</td><td>1/38 USUV</td></tr><tr><td>Brahma chicken</td><td><i>Gallus domesticus brahma</i></td><td>2016</td><td>4</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Marabou stork</td><td><i>Leptoptilos crumenifer</i></td><td>2016–2018</td><td>3</td><td>1</td><td>10</td><td>40</td><td>1/3 WNV</td></tr><tr><td>Blue-crowned motmot</td><td><i>Momotus momota</i></td><td>2019</td><td>1</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td rowspan="8">Common peafowl</td><td rowspan="8"><i>Pavo cristatus</i></td><td rowspan="8">2016–2018</td><td rowspan="8">24</td><td rowspan="8">6</td><td>10</td><td>Neg</td><td>6/24 USUV</td></tr><tr><td>10</td><td>Neg</td><td></td></tr><tr><td>10</td><td>Neg</td><td></td></tr><tr><td>20</td><td>Neg</td><td></td></tr><tr><td>40</td><td>Neg</td><td></td></tr><tr><td>40</td><td>Neg</td><td></td></tr><tr><td>40</td><td>Neg</td><td></td></tr><tr><td>10</td><td>Neg</td><td></td></tr><tr><td rowspan="10">Grey-winged trumpeter</td><td rowspan="10"><i>Psophia crepitans</i></td><td rowspan="10">2019</td><td rowspan="10">2</td><td rowspan="10">0</td><td>640</td><td>10</td><td>10/21 USUV</td></tr><tr><td>640</td><td>Neg</td><td></td></tr><tr><td>320</td><td>10</td><td></td></tr><tr><td>640</td><td>10</td><td></td></tr><tr><td>320</td><td>Neg</td><td></td></tr><tr><td>320</td><td>Neg</td><td></td></tr><tr><td>640</td><td>10</td><td></td></tr><tr><td>640</td><td>10</td><td></td></tr><tr><td>40</td><td>10</td><td></td></tr><tr><td>20</td><td>Neg</td><td></td></tr><tr><td>African comb duck</td><td><i>Sarkidiornis melanotos</i></td><td>2016–2018</td><td>3</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Common ostrich</td><td><i>Struthio camelus</i></td><td>200–20016</td><td>3</td><td>1</td><td>Neg</td><td>Neg</td><td>Other flavivirus</td></tr><tr><td>Red-crested turaco</td><td><i>Tauraco erythrolophus</i></td><td>2015</td><td>1</td><td>0</td><td>Neg</td><td>Neg</td><td>Neg</td></tr></tbody></table>

Legend: In seropositive birds, titers for USUV and WNV are indicated. Titer was considered specific when MNT titer was at least fourfold higher against one virus compared to the other.

## 2.2. Circulation of USUV/WNV in Zoo Mammals

The analysis of seroprevalence in mammals was performed on 274 specimens of 50 different species (Table 2). A total of 11 mammals were positive with the pan-flavivirus ELISA kit (4.01%, CI95%:
1.69–6.33). Similarly to our results with birds, most of the positives mammals had antibodies against USUV, including one Asian lion (*Panthera leo persica*), one maned wolf (*Chrysocyon brachyurus*), one Iberian wolf (*Canis lupus signatus*), and two African wild dogs (*Lycaon pictus*) (Table 2). In contrast, we detected antibodies against WNV in one dama gazelle (*Nanger dama*) (Table 2). Three animals presented antibodies against non-identified flaviviruses (*Acinonyx jubatus*, *Camelus dromedarius*, and *Panthera leo leo*) (the corresponding sera turned out to be negative in TBEV and MEAV neutralization assays). Thus, according to the available data, only a small proportion of exotic mammals' species was exposed to USUV or WNV infections. African wild dog has never been identified as a susceptible species to infection by USUV. To confirm the susceptibility of this *Canidae* to USUV infection, we analyzed the sera of 8 African wild dogs from another zoological garden (Réserve Africaine de Sigean) located 100 km from Montpellier zoo, also in the south of France. We identified two more animals with antibodies against USUV confirming a relatively high prevalence for USUV in this species (33.3%, 4/12).

Among all mammalians and more particularly in the order *Carnivora*, *Canidae* presented the highest USUV seroprevalence as we detected 25% (CI95%: 7.67–42.32) of positive specimens (Table A2).

To conclude, in this study statistically significant associations between seropositivity and sex or age were not observed. Among the species for which a relatively large sampling was available, Greater rhea (10/21) and to a lesser extent African wild dogs (4/12) had the highest infection rates. Blood samples from species showing high seroprevalence were additionally analyzed by RT-PCR for the presence of USUV or WNV RNA but we did not identify acute viral infection in these available samples.

<table><caption>**Table 2.** Mammals species tested by ELISA and by MNT for ELISA-positive samples.</caption><thead><tr><th colspan="7">Mammal Species</th></tr><tr><th>Species<br>(English Name)</th><th>Species<br>(Scientific Name)</th><th>Collection<br>Date</th><th>Nb of<br>Specimens</th><th colspan="3">MNT Antibodies Titer</th><th>Conclusion</th></tr><tr><th></th><th></th><th></th><th></th><th>cELISA</th><th>USUV</th><th>WNV</th><th></th></tr></thead><tbody><tr><td>Cheetah</td><td>Acinonyx jubatus</td><td>2016–2018</td><td>6</td><td>1</td><td>Neg</td><td>Neg</td><td>Other<br>flavivirus</td></tr><tr><td>Addax</td><td>Addax nasomaculatus</td><td>2015–2019</td><td>12</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Black howler</td><td>Alouatta caraya</td><td>2015–2018</td><td>3</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Barbary sheep</td><td>Ammotragus lervia</td><td>2014–2019</td><td>10</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Hog deer</td><td>Axis porinus</td><td>2016–2018</td><td>5</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Sichuan takin</td><td>Budorcas taxicolor<br>tibetana</td><td>2015</td><td>1</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Common marmoset</td><td>Callithrix jacchus</td><td>2017</td><td>1</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Dromedary</td><td>Camelus dromedarius</td><td>2016–2018</td><td>2</td><td>1</td><td>Neg</td><td>Neg</td><td>Other<br>flavivirus</td></tr><tr><td>Iberian wolf</td><td>Canis lupus signatus</td><td>2013–2018</td><td>10</td><td>1</td><td>40</td><td>Neg</td><td>1/10 USUV</td></tr><tr><td>White rhinoceros</td><td>Ceratotherium simum</td><td>2004–2019</td><td>5</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Sika deer</td><td>Cervus nippon</td><td>2016</td><td>8</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Linnaeus's two-toed<br>sloth</td><td>Choloepus didactylus</td><td>2016</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Maned wolf</td><td>Chrysocyon brachyurus</td><td>2015–2018</td><td>2</td><td>1</td><td>40</td><td>Neg</td><td>1/2 USUV</td></tr><tr><td>Prehensile-tailed<br>porcupine</td><td>Coendou prehensilis</td><td>2016</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Lowland paca</td><td>Cuniculus paca</td><td>2017</td><td>3</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Blesbok</td><td>Damaliscus pygargus<br>phillipsi</td><td>2017</td><td>1</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Somali wild ass</td><td>Equus africanus</td><td>2004</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th colspan="8">Mammal Species</th></tr><tr><th>Species<br>(English Name)</th><th>Species<br>(Scientific Name)</th><th>Collection<br>Date</th><th>Nb of<br>Specimens</th><th colspan="3">MNT Antibodies Titer</th><th>Conclusion</th></tr><tr><th></th><th></th><th></th><th></th><th>cELISA</th><th>USUV</th><th>WNV</th><th></th></tr></thead><tbody><tr><td></td><td>somaliensis</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Grévy's zebra</td><td>Equus grevyi</td><td>2017</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Persian onager</td><td>Equus hemionus onager</td><td>2004–2016</td><td>5</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Hartmann's<br>mountain zebra</td><td>Equus zebra hartmannae</td><td>2017–2019</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Crowned lemur</td><td>Eulemur coronatus</td><td>2017–2018</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Mongoose lemur</td><td>Eulemur mongoz</td><td>2016–2017</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Rothschild's giraffe</td><td>Giraffa camelopardalis<br>rothschildi</td><td>2015</td><td>1</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Alaotran gentle<br>lemur</td><td>Hapalemur alaotrensis</td><td>2016–2018</td><td>7</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Sable antelope</td><td>Hippotragus niger</td><td>2017</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Nile lechwe</td><td>Kobus megaceros</td><td>2018–2019</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Ring-tailed lemur</td><td>Lemur catta</td><td>2017</td><td>5</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Northern tiger cat</td><td>Leopardus tigrinus</td><td>2018</td><td>1</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>African wild dog</td><td>Lycaon pictus</td><td>2016–2019</td><td>4<sup>a</sup> + 8<sup>b</sup></td><td>2<sup>a</sup> + 2<sup>b</sup></td><td>40</td><td>Neg</td><td>4/12 USUV</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>160</td><td>Neg</td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>40<sup>a</sup></td><td>Neg<sup>a</sup></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td>40<sup>a</sup></td><td>Neg<sup>a</sup></td><td></td></tr><tr><td>Bennett's wallaby</td><td>Macropus rufogriseus</td><td>2016–2018</td><td>12</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Kirk's dik-dik</td><td>Madoqua kirkii</td><td>2016–2019</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Reeves's muntjac</td><td>Muntiacus reevesi</td><td>2017</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Giant anteater</td><td>Myrmecophaga<br>tridactyla</td><td>2018</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Dama gazelle</td><td>Nanger dama</td><td>2017–2019</td><td>14</td><td>1</td><td>Neg</td><td>10</td><td>1/14 WNV</td></tr><tr><td>Beisa oryx</td><td>Oryx beisa</td><td>2017</td><td>6</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Scimitar-horned<br>oryx</td><td>Oryx dammah</td><td>2016–2018</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Arabian oryx</td><td>Oryx leucoryx</td><td>2016–2019</td><td>6</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Caussenarde Sheep</td><td>Ovis aries</td><td>2016–2019</td><td>34</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Cameroon Sheep</td><td>Ovis aries cameroon</td><td>2018–2019</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Atlas lion</td><td>Panthera leo leo</td><td>2003–2019</td><td>4</td><td>1</td><td>Neg</td><td>Neg</td><td>Other<br>flavivirus</td></tr><tr><td>Asian lion</td><td>Panthera leo persica</td><td>2017</td><td>1</td><td>1</td><td>40</td><td>Neg</td><td>1/1 USUV</td></tr><tr><td>White-faced saki</td><td>Pithecia pithecia</td><td>2018</td><td>1</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Bharal</td><td>Pseudois nayaur</td><td>2015–2019</td><td>26</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Emperor tamarin</td><td>Saguinus imperator</td><td>2019</td><td>3</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>South American<br>tapir</td><td>Tapirus terrestris</td><td>2017</td><td>1</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Nyala</td><td>Tragelaphus angasii</td><td>2016–2019</td><td>4</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Eastern bongo</td><td>Tragelaphus eurycerus<br>isaaci</td><td>2017–2019</td><td>3</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Common eland</td><td>Tragelaphus oryx</td><td>2016–2019</td><td>5</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Lesser kudu</td><td>Tragelaphus imberbis</td><td>2016</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Syrian brown bear</td><td>Ursus arctos syriacus</td><td>2015–2017</td><td>3</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Red ruffed lemur</td><td>Varecia rubra</td><td>2018–2019</td><td>2</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr><tr><td>Vicuna</td><td>Vicugna vicugna</td><td>2017–2019</td><td>5</td><td>Neg</td><td>Neg</td><td>Neg</td><td>Neg</td></tr></tbody></table>

Legend: <sup>a</sup>—Montpellier zoo; <sup>b</sup>—Sigean zoo.
3. Discussion

Among arboviruses, WNV and USUV are neurotropic viruses characterized by an overlapping
geographic distribution, host and vector ranges, as well as similar clinical manifestations [7].
The epidemiology of both WNV and USUV in Europe has changed dramatically over the last
two decades. Studies performed in Italy provided evidence that USUV circulation in humans seems to
be higher than WNV infection in some endemic regions where both viruses circulate [3,12,37,38]. At the
end of the 90 s, there have been several reports of WNV outbreaks in Europe. Subsequently, this virus has
spread widely in Central and Southern European countries, causing more important epidemics [39–46].
In 2018, WNV infections in Europe increased considerably compared to previous transmission seasons
causing the biggest epidemic ever listed on the European continent [26]. This epidemic has affected
more people in one year than during the previous 10 years across Europe and was concomitantly
associated with a major USUV epidemic affecting several European countries and causing massive
mortality in several bird species [3,28,47,48]. In accordance with human epidemiological data, our
results suggest that USUV, and to a lesser extend WNV, may have circulated in zoo animals between
2016 and 2018 given that the two-thirds of seropositive animals analyzed were infected between
2016 and 2018. This higher seroprevalence during this period supports observations of an intense
transmission in the surveyed area in the 2016–2018 period compared to previous transmission seasons.
This exceptional level of viral circulation in 2018 for both USUV and WNV is probably related to
several environmental and ecological factors influencing the spreading of these viruses such as elevated
temperatures and precipitation anomalies, and other environmental factors that can favor mosquito
breading [49,50].

Wild birds play a major role as reservoir hosts and as carriers for zoonotic arboviruses and their
arthropod hosts to Europe. Every year, millions of birds migrate between Europe and Africa and play
an important role in the spread of viruses to new areas. Both WNV and USUV can commonly infect at
least 36 species of birds. Studying the seroprevalence of WNV and USUV in the avian population from
zoos is therefore a good way to monitor the circulation of these viruses notably linked to the greater
awareness of avian mortality in zoos compared to wild avifauna. Moreover, zoos are places where
humans, mosquitoes, and birds are gathered in a relatively small space. Birds from zoological gardens
have been described as victims of USUV infection. The first major outbreaks of USUV was identified in
2001 with the death of five great grey owls (Strix nebulosa) in a Vienna zoo [34]. In 2006 massive death
were also reported in wild and captive avian species members of the orders Passeriformes (blackbirds
and sparrows) and Strigiformes (owls) at a Zurich zoo [34] and deaths were reported in captive owls
in Italy the same year [51]. USUV seroprevalence was studied in the zoos of Vienna, Zurich, and
Basel with 5.3% to 8.75% of birds showing USUV-specific antibodies by PRNT-90, whereas no USUV
antibodies were detected in the birds of the Budapest zoo [32]. Moreover, one of the first recorded WNV
outbreaks in America occurred at the Bronx zoo in 1999 [52]. We identified several new avian species
that appear to be susceptible to infection by WNV and USUV. Moreover, the seroprevalence appeared
to be more important for USUV than for WNV in the Montpellier zoo. Of note USUV infections in the
same area had previously been reported in mosquitoes [53], birds [54], and human [55] whereas WNV
was reported in birds [54], horses [56–58], and humans [57,59,60]. One of the surprising outcomes of
this study was that among USUV positive samples we found a very high seroprevalence for common
peafowls (25.0%), emus (40.0%), and greater rheas (47.6%). To the best of our knowledge, these species
have never been described as susceptible to flavivirus infection. Interestingly we identified four new
orders susceptible to USUV infection (Casuariiformes, Pelecaniformes, Psittaciformes, and Rheiformes) and
in particular we detected a very high seroprevalence for Casuariiformes and Rheiformes orders (40.0%
and 47.6% respectively). At this point, the higher prevalence observed in Casuariiformes and Rheiformes
could be due to either a higher susceptibility to flavivirus infection or to a higher rate of vector feeding
(for example, due to their large size). Montpellier zoo has abundant populations of Culex pipiens, the
main mosquito vector of USUV and WNV in the area (S. Gutierrez, personal communication). Studies
on the blood-meal preference in these populations could help to define if those vertebrate species are
preferred by mosquito vectors over other vertebrates in the zoo. In our study the detection of stable
antibodies titers is probably associated with either the persistence of antibodies for a long time as
previously described for USUV and WNV or to periodic circulation of these viruses in the area [29,30].

Interestingly we found no flavivirus antibodies in non-human primates in the 27 individuals
from the nine species that we studied (Alouatta caraya, Callithix jacchus tibetana, Eulemur coronatus,
Eulemur mongoz, Hapalemur alaotrensis, Lemur catta, Pithecia pithecia, Saguinus imperator, and
Varecia rubra). Although these observations must be interpreted with caution due to the limited
number of animals it seems that primates are not particularly sensitive to infections by USUV and WNV
may be due to the feeding preferences of *Culex pipiens* for avian species. Another study previously
described a low prevalence in primates from several zoological gardens [32]. On the other hand, unlike
what has been found previously in another zoo, we did not detect flavivirus antibodies in the order
Perissodactyla and especially in the Equidae family in the 18 animals studied although horses have
been shown to be animals particularly susceptible to USUV and WNV [22,32,61–63]. Interestingly we
found a high seropositivity for USUV in Carnivora, especially in Canidae, as previously described
mainly in studies on domestic dogs [23,24,64]. Our results show that other Canidae than dogs could
be potentially used as sentinel animals for virus surveillance especially since a longer exposure to
arboviruses can be inferred from the fact that they spend most of the time outside. Free-living mammals
are naturally exposed to flavivirus infections and, thus additional studies on their host competence
would help in determining if they could be involved in the virus transmission cycle.

To conclude, our study shows a relatively high exposure of some species of exotic birds and
mammals to USUV and to a lesser extent to WNV in a particularly exposed area of France. The overall
seroprevalence was 0.73% for WNV and 6.57% for USUV. Our results correlate with a circulation
of these two viruses reported in particular in 2018. This study highlights the value of the interest
developing monitoring programs to better prevent, detect, and alert arboviruses circulation in areas
gathering a large numbers of animals, humans, and vectors such as zoological gardens.

**4. Materials and Methods**

4.1. *Animal Samples*

Montpellier zoo (south of France) covers an area of 80 hectares and is surrounded by an urban
area and host some 106 animal species including birds and mammals. The sera were collected in
this zoo between 2003 and 2019. In total, the sera of 411 animals belonging to 70 species and to
18 different orders were analyzed (for birds: *Anseriformes*, *Casuariiformes*, *Ciconiiforme*, *Coraciiformes*,
*Galliformes*, *Gruiformes*, *Musophagiformes*, *Pelecaniformes*, *Psittaciformes*, *Rheiformes*, and *Struthioniformes*,
for mammals: *Artiodactyla*, *Carnivora*, *Diprotodontia*, *Perissodactyla*, *Pilosa*, *Primates*, and *Rodentia*).
Among them, samples from 137 zoo-birds, belonging to 20 different species and from 274 mammals
from 50 different species were investigated. Samples originate from serum banks and animals
undergoing medical check-ups. For all samples, 0.3 to 3 mL of blood was drawn from the cutaneous
ulnar vein, the jugular vein, or a femoral vein. The blood was centrifuged at 2000× g for 10 min,
the serum was then separated from the clot, and stored at -20 °C until analysis. Experiments were
performed according to national regulations. The collection of samples was performed by veterinarians
of the zoo. Samples were collected from serum banks or from animals subjected to sanitary check-ups,
health programs, or surgical interventions. No animal was sampled for the only purpose of this study.

4.2. cELISA

Sera were screened using a cELISA test (ID Screen West Nile Competition Multi species ELISA kit, (IDVet, France). The technique is based on plates pre-coated with the envelope (E) protein of WNV and measure the competition between antibodies present in the animal serum tested and a monoclonal anti-WNV.E antibody conjugated to horseradish peroxidase (HRP). This test produces cross-reactions notably with other flaviruses and can therefore detect a large spectrum of flaviviruses including WNV
and USUV and other related flaviviruses [65,66]. Assays were performed and interpreted according manufacturer instructions as previously described [66].

### 4.3. Seroneutralization Assays

The MNT was carried out in flat-bottomed 96 well microplates after serum heat-inactivation at 56 °C for 30 min. First 50 µL of Dulbecco's modified Eagle's (DMEM) medium (Thermoscientific) supplemented by 2% heat-inactivated fetal bovine serum (Sigma) was added in each well. Serum was subsequently added to a well in the first column to obtain a starting dilution of 1:5. From this well, two-fold dilutions were made by pipetting 50 µL of each well in the next, until the last column. The sera were mixed with 50 µL of virus suspension (at 100 tissue culture infectious dose 50 (TCID₅₀) of USUV (France2018, MT863562) or WNV (lineage 2, MT863560) or TBEV (strain Hypr, Genbank ID U39292.1) or MEAV (Brest ART707, Genbank ID AF013386.1, DQ235144)), incubated at 37 °C for 90 min to allow neutralization of the virus. Wells were then supplemented with 100 µL of Vero E6 cell suspension (ATCC) (DMEM with 2% fetal bovine serum) and incubated 3 to 5 days at 37 °C and under 5% CO₂ following the virus. After 3 days (WNV, USUV) and 5 days (TBEV, MEAV) microplates were read under the microscope to determine the presence or absence of cytopathogenic effect in samples and in positive and negative sera controls. In regards to MEAV, PRNT90 assays with sera diluted 1:20 was performed on SW13 cells as previously described [67]. Each sample was assigned a titer that was the reciprocal of the dilution in that well. Sera that showed neutralization at dilutions ≥1:10 (WNV, USUV) and ≥1:20 (TBEV, MEAV) were considered positive. When neutralization was observed against WNV and USUV, the neutralizing immune response was considered specific if the VNT titer for any given virus was at least fourfold higher than titers obtained against the other viruses. Duplicates were performed to confirm positive results.

### 4.4. RT-qPCR

Viral RNAs were extracted from 50 µL of serum with the EZ1 apparatus running the EZ1 DSP virus kit (Qiagen). Viral RNA levels were measured by a one-step quantitative reverse transcriptase PCR assay (RT-qPCR) on the Light Cycler 480 (Roche) with primers, probe, and cycling conditions previously described for USUV [68] and WNV [69].

### 4.5. Statistical Analysis

Seroprevalence for USUV and WNV was estimated by dividing the number of positive animals by total animals tested, using two-sided exact binomial 95% confidence intervals (95%CI). Correlation between the seroprevalence and other independent variables (sex, age, etc.) were analyzed by means of a Pearson's chi-square test or by the Fisher's exact test (less than 6 samples per group).

**Author Contributions:** Conceptualization, Y.S.; methodology, Y.S., K.B., C.B., S.L. and V.F.; investigation, O.C., M.C., J.B., C.D., K.B., R.A.M.; resources, B.C., D.G., L.V., C.B., S.L.; writing—original draft preparation, Y.S.; writing—review and editing, Y.S., B.C., D.G., L.V., S.G., S.S., C.B., S.L., C.D.; funding acquisition, Y.S. and S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by REACTing (reference: YY/FC/2018-032) and the Montpellier University of Excellence (MUSE) through ANR (the French National Research Agency) under the "Investissements d'avenir" program with the reference ANR-16-IDEX-0006.

**Acknowledgments:** We thank the zoological garden of Montpellier and the "Réserve Africaine de Sigean", especially Marielle Beltrame, for providing samples for analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
## Appendix A

<table><caption>**Table A1.** WNV and USUV seroprevalence in longitudinally sampled birds (2016 and 2018).</caption><thead><tr><th rowspan="2">Species</th><th colspan="2">2016</th><th colspan="2">2018</th><th rowspan="2">Period of Infection</th></tr><tr><th>USUV</th><th>WNV</th><th>USUV</th><th>WNV</th></tr></thead><tbody><tr><td>Amazona amazonica</td><td>Neg</td><td>Neg</td><td>Neg</td><td>10</td><td>between 2016 and 2018</td></tr><tr><td>Dromaius novaehollandiae</td><td>80</td><td>10</td><td>160</td><td>10</td><td>before 2016</td></tr><tr><td>Eudocimus ruber</td><td>Neg</td><td>Neg</td><td>640</td><td>10</td><td>between 2016 and 2018</td></tr><tr><td>Leptoptilos crumenifer</td><td>10</td><td>20</td><td>10</td><td>40</td><td>before 2016</td></tr><tr><td>Pavo cristatus</td><td>Neg</td><td>Neg</td><td>40</td><td>10</td><td>between 2016 and 2018</td></tr><tr><td rowspan="10">Rhea americana</td><td>10</td><td>Neg</td><td>10</td><td>Neg</td><td>before 2016</td></tr><tr><td>640</td><td>20</td><td>640</td><td>20</td><td>before 2016</td></tr><tr><td>Neg</td><td>Neg</td><td>640</td><td>Neg</td><td>between 2016 and 2018</td></tr><tr><td>20</td><td>Neg</td><td>320</td><td>10</td><td>before 2016</td></tr><tr><td>Neg</td><td>Neg</td><td>640</td><td>10</td><td>between 2016 and 2018</td></tr><tr><td>Neg</td><td>Neg</td><td>320</td><td>Neg</td><td>between 2016 and 2018</td></tr><tr><td>Neg</td><td>Neg</td><td>320</td><td>Neg</td><td>between 2016 and 2018</td></tr><tr><td>Neg</td><td>Neg</td><td>640</td><td>10</td><td>between 2016 and 2018</td></tr><tr><td>Neg</td><td>Neg</td><td>640</td><td>10</td><td>between 2016 and 2018</td></tr></tbody></table>

<table><caption>**Table A2.** Prevalence of WNV or USUV antibodies in birds and mammalian species sampled classified by order and family.</caption><thead><tr><th>Classification</th><th>Number</th><th>Positives</th><th>%WNV</th><th>%USUV</th></tr></thead><tbody><tr><td><strong>Class Aves Order Anseriformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Anatidae</td><td>12</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Anhimidae</td><td>2</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Casuariiformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Casuariidae</td><td>5</td><td>2 USUV</td><td>0.0</td><td>40.0</td></tr><tr><td><strong>Order Ciconiiforme</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Ciconiidae</td><td>3</td><td>1 WNV</td><td>33.0</td><td>0.0</td></tr><tr><td><strong>Order Coraciiformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Momotidae</td><td>1</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Galliformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Phasianidae</td><td>28</td><td>6 USUV</td><td>0.0</td><td>17.2</td></tr><tr><td><strong>Order Gruiformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Psophiidae</td><td>2</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Musophagiformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Musophagidae</td><td>1</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Pelecaniformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Threskiornithidae</td><td>38</td><td>1 USUV</td><td>0.0</td><td>2.6</td></tr><tr><td><strong>Order Psittaciformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Psittacidae</td><td>16</td><td>1 WNV</td><td>6.2</td><td>0.0</td></tr><tr><td>Family Psittaculidae</td><td>5</td><td>1 USUV</td><td>0.0</td><td>20.0</td></tr><tr><td><strong>Order Rheiformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Rheidae</td><td>21</td><td>10 USUV</td><td>0.0</td><td>47.6</td></tr><tr><td><strong>Order Struthioniformes</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Struthionidae</td><td>3</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>TOTAL Birds</strong></td><td>137</td><td>22</td><td>1.45</td><td>14.59</td></tr></tbody></table>
<table><caption>Table A2. *Cont.*</caption><thead><tr><th>Classification</th><th>Number</th><th>Positives</th><th>%WNV</th><th>%USUV</th></tr></thead><tbody><tr><td><strong>Class Mammalia Order</strong></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Artiodactyla</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Bovidae</td><td>142</td><td>1 WNV</td><td>0.7</td><td>0.0</td></tr><tr><td>Family Cervidae</td><td>17</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Camelidae</td><td>7</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Giraffidae</td><td>1</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Carnivora</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Canidae</td><td>24</td><td>6 USUV</td><td>0.0</td><td>25.0</td></tr><tr><td>Family Felidae</td><td>12</td><td>1 USUV</td><td>0.0</td><td>8.3</td></tr><tr><td>Family Ursidae</td><td>3</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Diprotodontia</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Macropodidae</td><td>12</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Perissodactyla</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Equidae</td><td>13</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Rhinocerotidae</td><td>5</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Tapiridae</td><td>1</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Pilosa</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Choloepodidae</td><td>2</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Myrmecophagidae</td><td>2</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Primates</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Atelidae</td><td>3</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Callitrichidae</td><td>4</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Lemuridae</td><td>20</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Pitheciidae</td><td>1</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>Order Rodentia</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>Family Cuniculidae</td><td>3</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td>Family Erethizontidae</td><td>2</td><td>0</td><td>0.0</td><td>0.0</td></tr><tr><td><strong>TOTAL Mammalian</strong></td><td><strong>274</strong></td><td><strong>8</strong></td><td><strong>0.36</strong></td><td><strong>2.55</strong></td></tr><tr><td><strong>TOTAL Animals</strong></td><td><strong>411</strong></td><td><strong>30</strong></td><td><strong>0.73</strong></td><td><strong>6.57</strong></td></tr></tbody></table>

## References

1. Chastel, C.; Main, A.J.; Guiguen, C.; le Lay, G.; Quillien, M.C.; Monnat, J.Y.; Beaucournu, J.C. The isolation of Meaban virus, a new Flavivirus from the seabird tick Ornithodoros (Alectorobius) maritimus in France. *Arch. Virol.* **1985**, *83*, 129–140. [CrossRef]

2. Velay, A.; Solis, M.; Kack-Kack, W.; Gantner, P.; Maquart, M.; Martinot, M.; Augereau, O.; De Briel, D.; Kieffer, P.; Lohmann, C.; et al. A new hot spot for tick-borne encephalitis (TBE): A marked increase of TBE cases in France in 2016. *Ticks Tick Borne Dis.* **2018**, *9*, 120–125. [CrossRef]

3. Clé, M.; Beck, C.; Salinas, S.; Lecollinet, S.; Gutierrez, S.; Van de Perre, P.; Baldet, T.; Foulongne, V.; Simonin, Y. Usutu virus: A new threat? *Epidemiol. Infect.* **2019**, *147*, e232. [CrossRef]

4. Beck, C.; Goffart, I.L.; Franke, F.; Gonzalez, G.; Dumarest, M.; Lowenski, S.; Blanchard, Y.; Lucas, P.; de Lamballerie, X.; Grard, G.; et al. Contrasted epidemiological patterns of west nile virus lineages 1 and 2 infections in France from 2015 to 2019. *Pathogens* **2020**, *9*, 908. [CrossRef] [PubMed]

5. Calisher, C.H.; Gould, E.A. Taxonomy of the virus family Flaviviridae. *Adv. Virus Res.* **2003**, *59*, 1–19. [CrossRef] [PubMed]

6. Beck, C.; Jimenez-Clavero, M.A.; Leblond, A.; Durand, B.; Nowotny, N.; Leparc-Goffart, I.; Zientara, S.; Jourdain, E.; Lecollinet, S. Flaviviruses in Europe: Complex circulation patterns and their consequences for the diagnosis and control of West Nile disease. *Int. J. Environ. Res. Public Health* **2013**, *10*, 6049–6083. [CrossRef] [PubMed]

7. Zannoli, S.; Sambri, V. West nile virus and usutu virus co-circulation in europe: Epidemiology and implications. *Microorganisms* **2019**, *7*, 184. [CrossRef] [PubMed]
8. Michelitsch, A.; Wernike, K.; Klaus, C.; Dobler, G.; Beer, M. Exploring the reservoir hosts of tick-borne encephalitis virus. *Viruses* **2019**, *11*, 669. [CrossRef] [PubMed]

9. Waldenström, J.; Lundkvist, Å.; Falk, K.I.; Garpmo, U.; Bergström, S.; Lindegren, G.; Sjöstedt, A.; Mejlon, H.; Fransson, T.; Haemig, P.D.; et al. Migrating birds and tickborne encephalitis virus. *Emerg. Infect. Dis.* **2007**, *13*, 1215–1218. [CrossRef]

10. Smithburn, K.C.; Hughes, T.P.; Burke, A.W.P.J. A neurotropic virus isolated from the blood of a native of Uganda. *Am. Soc. Trop. Med. Hyg.* **1940**, *s1–s20*, 471–492. [CrossRef]

11. Petersen, L.R.; Brault, A.C.; Nasci, R.S. West Nile virus: Review of the literature. *JAMA J. Am. Med. Assoc.* **2013**, *310*, 308–315. [CrossRef]

12. Lustig, Y.; Sofer, D.; Bucris, E.D.; Mendelson, E. Surveillance and diagnosis of west nile virus in the face of flavivirus cross-reactivity. *Front. Microbiol.* **2018**, *9*, 2421. [CrossRef] [PubMed]

13. Lecollinet, S.; Pronost, S.; Coulpier, M.; Beck, C.; Gonzalez, G.; Leblond, A.; Tritz, P. Viral equine encephalitis, a growing threat to the horse population in Europe? *Viruses* **2019**, *12*, 23. [CrossRef] [PubMed]

14. Root, J.J.; Bosco-Lauth, A.M. West Nile Virus Associations in Wild Mammals: An Update. *Viruses* **2019**, *11*, 459. [CrossRef] [PubMed]

15. McIntosh, B.M. Usutu (SAAr 1776); nouvel arbovirus du groupe B. *Int. Cat. Arboviruses* **1985**, *3*, 1059–1060.

16. Woodall, J. The viruses isolated from arthropods at the East African Virus Research Institute in the 26 years ending December 1963. *Proc. E. Afr. Acad.* **1964**, *2*, 141–146.

17. Escribano-Romero, E.; Lupulović, D.; Merino-Ramos, T.; Blázquez, A.-B.; Lazić, G.; Lazić, S.; Saiz, J.-C.; Petrović, T. West Nile virus serosurveillance in pigs, wild boars, and roe deer in Serbia. *Vet. Microbiol.* **2015**, *176*, 365–369. [CrossRef]

18. Cadar, D.; Becker, N.; de Mendonca Campos, R.; Börstler, J.; Jöst, H.; Schmidt-Chanasit, J. Usutu virus in bats, Germany, 2013. *Emerg. Infect. Dis.* **2014**, *20*, 1771–1773. [CrossRef]

19. Diagne, M.; Ndione, M.; Di Paola, N.; Fall, G.; Bedekelabou, A.; Sembène, P.; Faye, O.; Zanotto, P.; Sall, A. Usutu Virus Isolated from Rodents in Senegal. *Viruses* **2019**, *11*, 181. [CrossRef]

20. Nikolay, B.; Diallo, M.; Boye, C.S.B.; Sall, A.A. Usutu Virus in Africa. *Vector-Borne Zoonotic Dis.* **2011**, *11*, 1417–1423. [CrossRef]

21. Bažanów, B.; Jansen van Vuren, P.; Szymański, P.; Stygar, D.; Frącka, A.; Twardoń, J.; Kozdrowski, R.; Pawęska, J. A Survey on West Nile and Usutu Viruses in Horses and Birds in Poland. *Viruses* **2018**, *10*, 87. [CrossRef]

22. Ben Hassine, T.; De Massis, F.; Calistri, P.; Savini, G.; BelHaj Mohamed, B.; Ranen, A.; Di Gennaro, A.; Sghaier, S.; Hammami, S. First Detection of Co-circulation of West Nile and Usutu Viruses in Equids in the South-west of Tunisia. *Transbound. Emerg. Dis.* **2014**, *61*, 385–389. [CrossRef]

23. Montagnaro, S.; Piantedosi, D.; Ciarcia, R.; Loponte, R.; Veneziano, V.; Fusco, G.; Amoroso, M.G.; Ferrara, G.; Damiano, S.; Iovane, G.; et al. Serological Evidence of Mosquito-Borne Flaviviruses Circulation in Hunting Dogs in Campania Region, Italy. *Vector-Borne Zoonotic Dis.* **2019**, *19*, 142–147. [CrossRef]

24. Durand, B.; Haskouri, H.; Lowenski, S.; Vachieri, N.; Beck, C.; Lecollinet, S. Seroprevalence of West Nile and Usutu viruses in military working horses and dogs, Morocco, 2012: Dog as an alternative WNV sentinel species? *Epidemiol. Infect.* **2016**, *144*, 1857–1864. [CrossRef]

25. Vilibic-Cavlek, T.; Savic, V.; Petrovic, T.; Toplak, I.; Barbic, L.; Petric, D.; Tabain, I.; Hrnjakovic-Cvjetkovic, I.; Bogdanic, M.; Klobucar, A.; et al. *Emerging Trends in the Epidemiology of West. Nile and Usutu Virus Infections in Southern Europe*; Frontiers Media S.A.: Lausanne, Switzerland, 2019; Volume 6, p. 437.

26. European Centre for Disease Prevention and Control (ECDC). *West Nile Fever in Europe in 2018*; ECDC: Solna, Sweden, 2018.

27. Čabanová, V.; Šikutová, S.; Straková, P.; Šebesta, O.O.; Vichová, B.; Zubríková, D.; Miterpáková, M.; Mendel, J.; Hurníková, Z.; Hubálek, Z.; et al. Co-Circulation of West Nile and Usutu Flaviviruses in Mosquitoes in Slovakia, 2018. *Viruses* **2019**, *11*, 639. [CrossRef]

28. Nagy, A.; Mezei, E.; Nagy, O.; Bakonyi, T.; Csonka, N.; Kaposi, M.; Koroknai, A.; Szomor, K.; Rigó, Z.; Molnár, Z.; et al. Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary, 2018. *Eurosurveillance* **2019**, *24*, 1900038. [CrossRef]

29. Süss, J. Tick-borne encephalitis 2010: Epidemiology, risk areas, and virus strains in Europe and Asia-An overview. *Ticks Tick Borne Dis.* **2011**, *2*, 2–15. [CrossRef]
30. Botelho-Nevers, E.; Gagneux-Brunon, A.; Velay, A.; Guerbois-Galla, M.; Grard, G.; Bretagne, C.; Mailles, A.; Verhoeven, P.O.; Pozzetto, B.; Gonzalo, S.; et al. Tick-borne encephalitis in auvergne-rhône-alpes region, France, 2017-2018. *Emerg. Infect. Dis.* **2019**, *25*, 1944–1948. [CrossRef]

31. Bournez, L.; Umhang, G.; Faure, E.; Boucher, J.-M.; Boué, F.; Jourdain, E.; Sarasa, M.; Llorente, F.; Jiménez-Clavero, M.A.; Moutailler, S.; et al. Exposure of Wild Ungulates to the Usutu and Tick-Borne Encephalitis Viruses in France in 2009-2014: Evidence of Undetected Flavivirus Circulation a Decade Ago. *Viruses* **2020** **19**, 12, 10. [CrossRef] [PubMed]

32. Buchebner, N.; Zenker, W.; Wenker, C.; Steinmetz, H.W.; Sós, E.; Lussy, H.; Nowotny, N. Low Usutu virus seroprevalence in four zoological gardens in central Europe. *BMC Vet. Res.* **2013**, *9*, 153. [CrossRef]

33. Caballero-Gómez, J.; Cano-Terriza, D.; Lecollinet, S.; Carbonell, M.D.; Martínez-Valverde, R.; Martínez-Nevado, E.; García-Párraga, D.; Lowenski, S.; García-Bocanegra, I. Evidence of exposure to zoonotic flaviruses in zoo mammals in Spain and their potential role as sentinel species. *Vet. Microbiol.* **2020**, *247*, 108763. [CrossRef]

34. Steinmetz, H.W.; Bakonyi, T.; Weissenböck, H.; Hatt, J.M.; Eulenberger, U.; Robert, N.; Hoop, R.; Nowotny, N. Emergence and establishment of Usutu virus infection in wild and captive avian species in and around Zurich, Switzerland-Genomic and pathologic comparison to other central European outbreaks. *Vet. Microbiol.* **2011**, *148*, 207–212. [CrossRef] [PubMed]

35. Hidalgo-Martínez, A.; Puerto, F.I.; Farfán-Ale, J.A.; García-Rejón, J.E.; Rosado-Paredes, E.D.P.; Méndez-Galván, J.; Figueroa-Ocampo, R.; Takashima, I.; Ramos, C. Prevalencia de infección por el virus del Nilo occidental en dos zoológicos del estado de Tabasco. *Salud Pública de México* **2008**, *50*, 76–85. [CrossRef] [PubMed]

36. Ziegler, U.; Fast, C.; Eiden, M.; Bock, S.; Schulze, C.; Hoeper, D.; Ochs, A.; Schlieben, P.; Keller, M.; Zielke, D.E.; et al. Evidence for an independent third Usutu virus introduction into Germany. *Vet. Microbiol.* **2016**, *192*, 60–66. [CrossRef] [PubMed]

37. Percivalle, E.; Sassera, D.; Rovida, F.; Isernia, P.; Fabbi, M.; Baldanti, F.; Marone, P. Usutu Virus Antibodies in Blood Donors and Healthy Forestry Workers in the Lombardy Region, Northern Italy. *Vector-Borne Zoonotic Dis.* **2017**, *17*, 658–661. [CrossRef]

38. Grottola, A.; Marcacci, M.; Tagliazucchi, S.; Gennari, W.; Di Gennaro, A.; Orsini, M.; Monaco, F.; Marchegiano, P.; Marini, V.; Meacci, M.; et al. Usutu virus infections in humans: A retrospective analysis in the municipality of Modena, Italy. *Clin. Microbiol. Infect.* **2017**, *23*, 33–37. [CrossRef]

39. Tsai, T.F.; Popovici, F.; Cernescu, C.; Campbell, G.L.; Nedelcu, N.I. West Nile encephalitis epidemic in southeastern Romania. *Lancet* **1998**, *352*, 767–771. [CrossRef]

40. Bakonyi, T.; Ivanics, É.; Erdélyi, K.; Ursu, K.; Ferenczi, E.; Weissenböck, H.; Nowotny, N. Lineage 1 and 2 strains of encephalitic West Nile virus, Central Europe. *Emerg. Infect. Dis.* **2006**, *12*, 618–623. [CrossRef]

41. Kutasi, O.; Bakonyi, T.; Lecollinet, S.; Biksi, I.; Ferenczi, E.; Bahuon, C.; Sardi, S.; Zientara, S.; Szenci, O. Equine encephalomyelitis outbreak caused by a genetic lineage 2 west nile virus in Hungary. *J. Vet. Intern. Med.* **2011**, *25*, 586–591. [CrossRef]

42. Popović, N.; Milošević, B.; Urošević, A.; Poluga, J.; Lavadinović, L.; Nedeljković, J.; Jevtović, D.; Dulović, O. Outbreak of west Nile virus infection among humans in Serbia, August to October 2012. *Eurosurveillance* **2013**, *18*, 20613. [CrossRef]

43. Bakonyi, T.; Ferenczi, E.; Erdélyi, K.; Kutasi, O.; Csörgo, T.; Seidel, B.; Weissenböck, H.; Brugger, K.; Bán, E.; Nowotny, N. Explosive spread of a neuroinvasive lineage 2 West Nile virus in Central Europe, 2008/2009. *Vet. Microbiol.* **2013**, *165*, 61–70. [CrossRef]

44. Savini, G.; Capelli, G.; Monaco, F.; Polci, A.; Russo, F.; Di Gennaro, A.; Marini, V.; Teodori, L.; Montarsi, F.; Pinoni, C.; et al. Evidence of West Nile virus lineage 2 circulation in Northern Italy. *Vet. Microbiol.* **2012**, *158*, 267–273. [CrossRef]

45. Barzon, L.; Pacenti, M.; Franchin, E.; Pagni, S.; Lavezzo, E.; Squarzon, L.; Martello, T.; Russo, F.; Nicoletti, L.; Rezza, G.; et al. Large human outbreak of West Nile virus infection in north-eastern Italy in 2012. *Viruses* **2013**, *5*, 2825–2839. [CrossRef]

46. Sribu, A.; Ceianu, C.S.; Panculescu-Gatej, R.I.; Vázquez, A.; Tenorio, A.; Rebreanu, R.; Niedrig, M.; Nicolescu, G.; Pistol, A. Outbreak of West Nile virus infection in humans, Romania, July to October 2010. *Eurosurveillance* **2011**, *16*, 19762. [CrossRef]
47. Papa, A. Emerging arboviruses of medical importance in the Mediterranean region. *J. Clin. Virol.* **2019**, *115*, 5–10. [CrossRef]

48. Aberle, S.W.; Kolodziejek, J.; Jungbauer, C.; Stiasny, K.; Aberle, J.H.; Zoufaly, A.; Hourfar, M.K.; Weidner, L.; Nowotny, N. Increase in human West Nile and Usutu virus infections, Austria, 2018. *Eurosurveillance* **2018**, *23*, 1800545. [CrossRef]

49. Semenza, J.C.; Tran, A.; Espinosa, L.; Sudre, B.; Domanovic, D.; Paz, S. Climate change projections of West Nile virus infections in Europe: Implications for blood safety practices. *Environ. Health A Glob. Access Sci. Source* **2016**, *15*, 125–136. [CrossRef]

50. Haussig, J.M.; Young, J.J.; Gossner, C.M.; Mezei, E.; Bella, A.; Sirbu, A.; Pervanidou, D.; Drakulovic, M.B.; Sudre, B. Early start of the west nile fever transmission season 2018 in Europe. *Eurosurveillance* **2018**, *23*, 1800428. [CrossRef]

51. Manarolla, G.; Bakonyi, T.; Gallazzi, D.; Crosta, L.; Weissenböck, H.; Dorrestein, G.M.M.; Nowotny, N. Usutu virus in wild birds in northern Italy. *Vet. Microbiol.* **2010**, *141*, 159–163. [CrossRef]

52. Lanciotti, R.S.; Roehrig, J.T.; Deubel, V.; Smith, J.; Parker, M.; Steele, K.; Crise, B.; Volpe, K.E.; Crabtree, M.B.; Scherret, J.H.; et al. Origin of the West Nile virus responsible for an outbreak of encephalitis in the Northeastern United States. *Science* **1999**, *286*, 2333–2337. [CrossRef]

53. Eiden, M.; Gil, P.; Ziegler, U.; Rakotoarivony, I.; Marie, A.; Frances, B.; L'Ambert, G.; Simonin, Y.; Foulongne, V.; Groschup, M.H.; et al. Emergence of two Usutu virus lineages in Culex pipiens mosquitoes in the Camargue, France, 2015. *Infect. Genet. Evol.* **2018**, *61*, 151–154. [CrossRef] [PubMed]

54. Vittecoq, M.; Lecollinet, S.; Jourdain, E.; Dé, F.; Thomas, R.; Blanchon, T.; Arnal, A.; Lowenski, S.; Gauthier-Clerc, M. Recent Circulation of West Nile Virus and Potentially Other Closely Related Flaviviruses in Southern France. *Vector-Borne Zoonotic Dis.* **2013**, *13*, 610–613. [CrossRef] [PubMed]

55. Simonin, Y.; Sillam, O.; Carles, M.J.; Gutierrez, S.; Gil, P.; Constant, O.; Martin, M.F.; Girard, G.; Van de Perre, P.; Salinas, S.; et al. Human Usutu Virus Infection with Atypical Neurologic Presentation, Montpellier, France, 2016. *Emerg. Infect. Dis.* **2018**, *24*, 875. [CrossRef]

56. Durand, B.; Chevalier, V.; Pouillot, R.; Labie, J.; Marendat, I.; Murgue, B.; Zeller, H.; Zientara, S. West Nile virus outbreak in horses, Southern France, 2000: Results of a serosurvey. *Emerg. Infect. Dis.* **2002**, *8*, 777–782. [CrossRef]

57. Bahuon, C.; Marcillaud-Pitel, C.; Bournez, L.; Leblond, A.; Beck, C.; Hars, J.; Leparc-Goffart, I.; L'ambert, G.; Paty, M.-C.C.; Cavalerie, L.; et al. West Nile virus epizootics in the Camargue (France) in 2015 and reinforcement of surveillance and control networks. *Rev. Sci. Tech. L'OIE* **2016**, *35*, 811–824. [CrossRef]

58. Pradier, S.; Sandoz, A.; Paul, M.C.; Lefebvre, G.; Tran, A.; Maingault, J.; Lecollinet, S.; Leblond, A. Importance of wetlands management for west nile virus circulation risk, camargue, Southern France. *Int. J. Environ. Res. Public Health* **2014**, *11*, 7740–7754. [CrossRef]

59. Calistri, P.; Giovannini, A.; Hubalek, Z.; Ionescu, A.; Monaco, F.; Savini, G.; Lelli, R. Epidemiology of West Nile in Europe and in the Mediterranean Basin–!2009-11-17–!2009-12-11–!2010-04-22–! *Open Virol. J.* **2010**, *4*, 29–37. [CrossRef]

60. Young, J.J.; Coulombier, D.; Domanović, D.; Zeller, H.; Gossner, C.M.; Aberle, S.; Schmoll, F.; Christova, I.; Chakarova, S.; Novosel, I.P.; et al. One health approach for West Nile virus surveillance in the European Union: Relevance of equine data for blood safety. *Eurosurveillance* **2019**, *24*, 1800349. [CrossRef] [PubMed]

61. Barbic, L.; Vilibic-Cavlek, T.; Listes, E.; Stevanovic, V.; Gjenero-Margan, I.; Ljubin-Sternak, S.; Pem-Novosel, I.; Listes, I.; Mlinaric-Galinovic, G.; Di Gennaro, A.; et al. Demonstration of Usutu Virus Antibodies in Horses, Croatia. *Vector-Borne Zoonotic Dis.* **2013**, *13*, 772–774. [CrossRef]

62. Csank, T.; Drzewnioková, P.; Korytár, L.; Major, P.; Gyuranecz, M.; Pistl, J.; Bakonyi, T. A Serosurvey of Flavivirus Infection in Horses and Birds in Slovakia. *Vector-Borne Zoonotic Dis.* **2018**, *18*, 206–213. [CrossRef]

63. Guerrero-Carvajal, F.; Bravo-Barriga, D.; Martín-Cuervo, M.; Aguilera-Sepúlveda, P.; Ferraguti, M.; Jiménez-Clavero, M.Á.; Llorente, F.; Alonso, J.M.; Frontera, E. Serological evidence of co-circulation of West Nile and Usutu viruses in equids from western Spain. *Transbound. Emerg. Dis.* **2020**. [CrossRef]

64. Maquart, M.; Dahmani, M.; Marié, J.L.; Gravier, P.; Leparc-Goffart, I.; Davoust, B. First Serological Evidence of West Nile Virus in Horses and Dogs from Corsica Island, France. *Vector-Borne Zoonotic Dis.* **2017**, *17*, 275–277. [CrossRef]
65. Chevalier, V.; Marsot, M.; Molia, S.; Rasamoelina, H.; Rakotondravao, R.; Pedrono, M.; Lowenski, S.; Durand, B.; Lecollinet, S.; Beck, C. Serological evidence of west nile and usutu viruses circulation in domestic and wild birds in wetlands of mali and madagascar in 2008. *Int. J. Environ. Res. Public Health* **2020**, *17*, 1998. [CrossRef]

66. Beck, C.; Leparc-Goffart, I.; Desoutter, D.; Debergé, E.; Bichet, H.; Lowenski, S.; Dumarest, M.; Gonzalez, G.; Migné, C.; Vanhomwegen, J.; et al. Serological evidence of infection with dengue and zika viruses in horses on French pacific Islands. *PLoS Negl. Trop. Dis.* **2019**, *13*, e0007162. [CrossRef]

67. Arnal, A.; Gómez-Díaz, E.; Cerdà-Cuéllar, M.; Lecollinet, S.; Pearce-Duvet, J.; Busquets, N.; García-Bocanegra, I.; Pagès, N.; Vittecoq, M.; Hammouda, A.; et al. Circulation of a Meaban-like virus in yellow-legged gulls and seabird ticks in the Western Mediterranean Basin. *PLoS ONE* **2014**, *9*, e89601. [CrossRef] [PubMed]

68. Nikolay, B.; Weidmann, M.; Dupressoir, A.; Faye, O.; Boye, C.S.; Diallo, M.; Sall, A.A. Development of a Usutu virus specific real-time reverse transcription PCR assay based on sequenced strains from Africa and Europe. *J. Virol. Methods* **2014**, *197*, 51–54. [CrossRef]

69. Garcia, M.; Alout, H.; Diop, F.; Damour, A.; Bengue, M.; Weill, M.; Missé, D.; Lévêque, N.; Bodet, C. Innate Immune Response of Primary Human Keratinocytes to West Nile Virus Infection and Its Modulation by Mosquito Saliva. *Front. Cell. Infect. Microbiol.* **2018**, *8*, 387. [CrossRef]

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_4.png" /></figure>

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).