viruses Article West Nile Virus Lineage 2 Spreads Westwards in Europe and Overwinters in North-Eastern Spain (2017–2020) Pilar Aguilera-Sepúlveda 1 , Sebastián Napp 2,3 , Francisco Llorente 1 , Carlos Solano-Manrique 4 , Rafael Molina-López 5 , Elena Obón 5 , Alba Solé 6 , Miguel Ángel Jiménez-Clavero 1,7 , Jovita Fernández-Pinero 1, * and Núria Busquets 2,3, * 1 Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-CSIC), 28130 Valdeolmos, Spain; aguilera.pilar@inia.es (P.A.-S.); dgracia@inia.es (F.L.); majimenez@inia.es (M.Á.J.-C.) 2 Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Spain; sebastian.napp@irta.cat 3 IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Spain 4 Centre de Fauna de Vallcalent, Àrea de Gestió Ambiental Servei de Fauna i Flora, Forestal Catalana, 25199 Lleida, Spain; carlos.solano@gencat.cat 5 Centre de Fauna de Torreferrussa, Àrea de Gestió Ambiental Servei de Fauna i Flora, Forestal Catalana, 08130 Santa Perpètua de Mogoda, Spain; rafael.molina@gencat.cat (R.M.-L.); elena.obon@gencat.cat (E.O.) 6 Departament d’Acció Climàtica, Alimentació i Agenda Rural, 08007 Barcelona, Spain; asoles@gencat.cat 7 CIBER of Epidemiology and Public Health (CIBERESP), 28029 Madrid, Spain * Correspondence: fpinero@inia.es (J.F.-P.); nuria.busquets@irta.cat (N.B.)   Abstract: West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread Citation: Aguilera-Sepúlveda, P.; across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 Napp, S.; Llorente, F.; Solano- was detected and isolated from four northern goshawks in two provinces of Catalonia (north- Manrique, C.; Molina-López, R.; eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential Obón, E.; Solé, A.; Jiménez-Clavero, M.Á.; Fernández-Pinero, J.; Busquets, overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic N. West Nile Virus Lineage 2 Spreads analyses, and a study of phenotypic characterisation were performed. Our results showed that Westwards in Europe and these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were Overwinters in North-Eastern Spain related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, (2017–2020). Viruses 2022, 14, 569. causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed https://doi.org/10.3390/v14030569 in vitro showed that these isolates presented characteristics corresponding to strains of moderate to Academic Editor: Roy A. Hall high virulence. All these findings evidence that these WNV-L2 strains have been able to circulate and overwinter in the region, and are pathogenic, at least in northern goshawks, which seem to be Received: 21 January 2022 very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the Accepted: 7 March 2022 increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus Published: 9 March 2022 should be kept under extensive surveillance, following a One-Health approach. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: West Nile virus; lineage-2; Spain; phylogeny; avian host; northern goshawk; overwintering; published maps and institutional affil- Europe iations. 1. Introduction Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. West Nile virus (WNV) (genus Flavivirus, family Flaviviridae) is one of the most This article is an open access article widespread zoonotic arboviruses in the world, as it is present in Africa, Europe, the Middle distributed under the terms and East, Asia, and the New World [1]. It circulates in an enzootic cycle where mosquitoes conditions of the Creative Commons (mainly Culex spp.) act as vectors and birds are the reservoir hosts. Many bird species can Attribution (CC BY) license (https:// act as competent hosts [2]. Humans and horses can also be infected and may develop a creativecommons.org/licenses/by/ neurologic disease; however, they are considered dead-end hosts, as the viraemia resulting 4.0/). from the infection is insufficient to infect a new vector. In humans, WNV has also been Viruses 2022, 14, 569. https://doi.org/10.3390/v14030569 https://www.mdpi.com/journal/viruses Viruses 2022, 14, 569 2 of 14 transmitted in a very limited number of cases by transfusions of blood, organ transplants, and during pregnancy, delivery, or breast-feeding. Over 80% of WNV infections in hu- mans are asymptomatic and the most prevalent clinical manifestation is West Nile fever (WNF) [3]. However, severe neuroinvasive illness can occur mainly in elderly and immuno- compromised patients and about 1 out of 10 people who develop severe illness affecting the central nervous system die. There is not specific prophylaxis or treatment for humans, unlike horses, in which the disease can be prevented with some commercial vaccines [4]. In horses, WNV may cause encephalitis or encephalomyelitis that may provoke clinical signs such as ataxia, incoordination, weakness, muscle fasciculation, and cranial nerve deficits. In addition to the consequences on public health, the impact of WNV on animal health may be significant since the equine sector represents a valuable socio-economic resource in rural areas in Europe, and especially in Spain. The WNV genome consists of a single-stranded positive-sense RNA genome of approx- imately 11,000 nt, which encodes and translates a single polyprotein of 3434 amino acids. This polyprotein is further processed by viral and host proteases to yield three structural proteins, –capsid (C), membrane (prM/M), and envelope (E)-, and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), all of them playing an essential role in the viral genome replication [5]. Currently, there are at least eight known lineages of WNV [6], showing its high genetic diversity. Of them, lineages 1 and 2 are the most widely distributed and are responsible for the largest outbreaks in humans and animals. West Nile virus linage 1 (WNV-L1) was first reported in Europe in 1962, concretely in the Camargue [7]. After that, it caused sporadic outbreaks in the following decades in different European countries. Nevertheless, it started to be considered as a pathogen of concern in this continent after the 1990s, and especially since the 2000s, when it re-emerged. Since then, human and animal cases have multiplied across Europe [8]. West Nile virus lineage 2 (WNV-L2) was first detected in Europe in 2004, in a north- ern goshawk (Accipiter gentilis) from Hungary, being the first report of this lineage out- side Africa [9]. This strain (belonging to the defined central-southern European clade) spread rapidly and caused important outbreaks in Hungary and Austria between 2008 and 2009 [10], in Greece in 2010 [11], in Serbia in 2012 [12], and in Italy in 2013 [13]. At the same time, a different WNV-L2, belonging to the named Russian/Romanian clade, affected humans in Russia (2004–2007) [14] and Romania in 2010 [15]. More recently, WNV-L2 has emerged in France [16], Germany [17], the Czech Repub- lic [18], and the Netherlands [19], causing outbreaks in wild birds, and incidental cases in horses and humans. Furthermore, the situation in Italy deserves special attention, as WNV-L1 strains of the western Mediterranean subtype predominated in the region until 2011–2013, when WNV-L2 emerged for the first time in co-circulation with WNV-L1. Since then, lineage 2 has been the most frequently identified, even replacing lineage 1 across the country and originating important outbreaks in humans [13,20,21]. Frequently, after a first outbreak is reported, the disease re-occurs in the same area in the following years, indicating the success of WNV in settling down under favourable conditions. Regarding the situation in Spain, WNV-L1 has been present in the south and central regions of the country during the last two decades [3,22]. Indeed, it has been responsible for the greatest human outbreak in the country during the 2020 transmission season, causing 77 human cases and eight deaths [3]. On the other hand, although WNV-specific antibodies in birds and horses have been detected in Catalonia (north-eastern Spain) during 2010–2019 [23], WNV-L2 was molecularly detected for the first time in 2017 in this area, away from WNV-L1-affected regions of the country. The detection of this lineage in northern goshawks evidenced the capacity of WNV-L2 to spread from central to western regions of Europe [24]. Particularly, Catalonia has implemented a WNV comprehensive surveillance program (including active and passive surveillance in wild birds and horses) and measures were Viruses 2022, 14, 569 3 of 14 intensified due to WNV-L2 detection in 2017 [24]. In 2020, WNV-L2 was again detected in northern goshawks with clinical signs compatible to West Nile disease. However, there have not been reports of human events due to WNV-L2 in the region yet, contrasting with other European countries where this lineage has been responsible for numerous cases [20,25]. The present study analysed the full-genome sequences, their phylogenetic relation- ships with other WNV-L2 strains found in Europe, and the phenotypic characteristics of the first four Spanish isolates of WNV-L2, with the aim of characterising these circulating WNV-L2 strains and elucidating the potential overwintering of the virus in this Mediter- ranean region. These isolates come from the outbreaks that occurred in northern goshawks in Catalonia (north-eastern Spain) during the 2017 and 2020 seasons. 2. Materials and Methods 2.1. Sample Collection Within the surveillance program for WNV implemented in Catalonia, birds found dead or that died at the Wildlife Rehabilitation Centres (WRC) had their heads sent to IRTA-CReSA to be diagnosed for WNV in encephalon samples. Between 2017 and 2020, 373 dead birds from 53 different bird species belonging to 23 families were tested (Table S1). The carcasses of the sampled birds were frozen and, once positive results for WNV were obtained, they were further transferred to IRTA-CReSA. Necropsy was performed at BSL-3 facilities and several organs (kidney, liver, heart and lung) were sampled, collected in 500 µL of DMEM with 6% of Penicillin, Dihydrostreptomycin, and Nystatin (PSN) and stored at −75 ◦ C until further laboratory investigation. 2.2. Initial WNV Molecular Diagnostics Tissue samples were mechanically homogenised using polypropylene pestles and viral RNA was extracted using NucleoSpin® RNA Virus (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. RNA was eluted in a final volume of 50 µL of RNase-free water. WNV RNA was detected by real-time reverse transcription PCR (RT-PCR) using the primers and probe previously described [26] and AgPath-ID™ One- Step RT-PCR reagents (Applied Biosystems, Foster City, CA, USA). The amplification was detected using a Real-Time PCR 7500 Fast System (Applied Biosystems) with the following thermal profile: 48 ◦ C for 10 min; 95 ◦ C for 10 min; and then 45 cycles at 95 ◦ C for 15 s and at 60 ◦ C for 60 s. Further analysis of a partial sequence of the NS5 gene, using the primers and RT-PCR conditions previously described by Scaramozzino et al. [27], were performed in the encephalon samples to determine the WNV lineage. 2.3. Virus Isolation WNV isolation from encephalon samples was performed in Vero cells (ATCC, ref. CCL-81) at IRTA-CReSA facilities. Briefy, cells with 90% of confluence were exposed to the supernatant of homogenised samples for 1 h, and then, post-infection media (DMEM with 2% FBS, 2% PSN and 2% Glutamine) was added to incubate the infected cells for 4 days. Then, the supernatant was collected, centrifuged, aliquoted, and stored at −80 ◦ C. Once WNV isolation was confirmed from one aliquot of obtained isolates by using the same RT-PCR described above and by titration (TCID50 ), WNV isolates were sent to CISA-INIA facilities, where they were propagated similarly in Vero cell cultures (second passage). 2.4. Full Genome Sequencing Total RNA was extracted from 100-µL aliquots of infected cell culture supernatants (2nd passage) using Biosprint 15 workstation (QIAGEN, Valencia, CA, USA), according to the manufacturer’s protocol, with a small modification (carrier RNA was added to a final concentration of 5 µg/mL). RNA was eluted in a final volume of 100-µL RNase-free water, aliquoted, and stored at −80 ◦ C until used. Viruses 2022, 14, 569 4 of 14 The complete genomes of the isolates were obtained by sequencing 23 overlapping PCR amplicons based on L. Barzon’s previous design (personal communication). From this original design, 12 primers remained unchanged and the rest were newly designed by M. Elizalde and J. Fernández-Pinero (personal communication) or were taken from a previously described protocol [28] (Table S2). One-step RT-PCR kit (Qiagen) was used to run conventional RT-PCR assays, following the manufacturer’s instructions. Amplified DNAs were purified using ExoSAP-IT kit (GE Healthcare), and then bidirectionally sequenced by automatic dideoxy cycle sequencing techniques, Big Dye Terminator Cycle Sequencing Kit (v3.1), and an ABI 3730 XL DNA Analyzer, using the same primer sets of the RT-PCR assays. 2.5. Sequence and Phylogenetic Analyses Analysis of the sequences and assembly of the full linear genomes were performed by using SeqMan software (DNASTAR, Madison, WI, USA). The genome sequences obtained in this study were submitted to GenBank database under the accession nos. OM037670, OM037671, OM037672, and OM037673. Multiple alignments were performed with the Clustal W program and phylogenetic trees were created using the Maximum likelihood method (both available in MEGA7 software). Bootstrap analyses were inferred from 500 replicates. 2.6. Characterisation of the Viral Plaques For the in vitro characterisation, the four Spanish WNV-L2 isolates (AC564, AC913, AC923, and AC924) were compared with other WNV known and characterised strains (GenBank accession number of the respective complete genome sequences indicated in parentheses): KUN MP502-66, a putative lineage 6 from Malaysia (GU047874); KUN-KJ359- 11 lineage 1b from Australia (JX014270); B956, an ancient lineage 2 from Uganda (AY532665); NY-99, a lineage 1 from New York (FJ151394); and SPA07, a lineage 1 isolate from Spain (FJ766331). Thus, these nine isolates were inoculated in Vero cells in 6-well culture plates as previously described [29]. After staining with neutral red, plaques were visually examined at 72 h post-infection and photographed. Plaque sizes were then measured using the ImageJ program and statistical analyses were performed by using Student’s t-test. 3. Results Between 2017 and 2020, 5 out of 373 wild birds found dead or that died at the WRC tested positive by RT-PCR for WNV in encephalon samples. Further analysis of a partial sequence of the NS5 gene indicated that all the positive samples belonged to WNV-L2. All these WNV-L2 infected birds were northern goshawks with clinical signs compatible with WNV infection and were transferred to WRC, where they died or were euthanised. In detail, in September 2017, a northern goshawk was found with dehydration, apathy, and low weight near an urban area, a municipality called Alguaire, located 15 km northern of the city of Lleida (Catalonia, Spain), and was transferred to the WRC of Vallcalent. Five days later it developed nervous symptoms (head-shaking, incoordination, and inability to stand upright), and was euthanised. This specimen was further codified as AC568 goshawk (Figure 1). Within days, another sick northern goshawk was admitted to the same WRC and died after 2 days [24]. In September 2020, a sick juvenile northern goshawk was collected in the municipality of Alpicat, located 6 km northwest of Lleida (Catalonia, Spain), and was transferred to the WRC of Vallcalent. It was found with a low weight (508 g). Moreover, it showed blepharitis in the left eye and had a corneal ulcer, possibly due to a contusion. Four days later it was euthanised because it showed apathy, incoordination, and an inability to stand upright. This specimen was further codified as AC913 goshawk. At the same time, in September 2020, two sick juveniles of northern goshawks were found in the Tarragona province (Catalonia, Spain) and were transferred to WRC of Torreferrussa. The specimens presented mild to moderate obtundation and were unable to fly. Muscle tone was normal right. This specimen was further codified as AC913 goshawk. At the same time, in Sep- tember 2020, two sick juveniles of northern goshawks were found in the Tarragona prov- ince (Catalonia, Spain) and were transferred to WRC of Torreferrussa. The specimens pre- sented mild to moderate obtundation and were unable to fly. Muscle tone was normal to mildly increased. They had a right head tilt and intermittent cervical dystonia with the Viruses 2022, 14, 569 head and neck turned to the right. Severe ambulatory tetraparesis and ataxia affecting 5 ofthe 14 wings and the pelvic limbs were observed. Menace response was bilaterally absent. Epi- sodes of quick dilation and constriction of their pupils (eye pinning) were also detected, tomost likely mildly representing increased. Theyahadstrong stress a right or excitement. head Thus, they tilt and intermittent were euthanised. cervical dystonia withThese the specimens head and neckwereturned then codified as AC923 to the right. andambulatory Severe AC924 (Figure 1). tetraparesis and ataxia affecting Virusand the wings isolation from limbs the pelvic encephalon were samples observed. could be carried Menace out in response four was (AC568, AC913, bilaterally absent. AC923, and Episodes AC924) of quick out ofand dilation fiveconstriction WNV-positive northern of their goshawks. pupils No macroscopic (eye pinning) lesions were also detected, werelikely most in therepresenting northern goshawks a strong affected stress orapart from hematoma excitement. Thus, theythatwerewas observed These euthanised. in the brain of AC568, specimens which were then probably codified as was AC923as consequence of a traumatism and AC924 (Figure 1). due to head-shaking. Figure1.1.On Figure Onthe theleft: left:Provinces Provinceswere werethe thestudy study was was carried carried out out (dark (dark grey grey and and red). red). In red: location ofofthe theaffected affectedprovinces provincesofof Catalonia: Catalonia: Lleida, Lleida, and and Tarragona, Tarragona, within within Spain. Spain. OnOnthethe right: right: distribu- distribution oftion theof the RT-PCR-positive RT-PCR-positive and negative and negative wild birds wild birds sampled sampled in Catalonia in Catalonia between between 20172020. 2017 and and 2020. WNVisolation Virus real-timefrom RT-PCR analysissamples encephalon showedcould positive results in be carried allinsamples out examined four (AC568, (Ct AC913, values and AC923, between 14.47 AC924) outand of 35.91) except for thenorthern five WNV-positive liver, heart, and lungNo goshawks. of the AC568 goshawk macroscopic lesions that were were in thenegative northern(Table 1). affected apart from hematoma that was observed in the goshawks brain of AC568, which probably was as consequence of a traumatism due to head-shaking. WNV real-time RT-PCR analysis showed positive results in all samples examined (Ct values between 14.47 and 35.91) except for the liver, heart, and lung of the AC568 goshawk that were negative (Table 1). Table 1. Analysis of the collected samples from the northern goshawks by WNV real-time RT-PCR [26]. Specimens Sample Type/Tissue (Ct) Northern Goshawks Encephalon Kidney Liver Heart Lung (Accipiter gentilis) AC568 15.18 35.91 neg neg neg AC913 14.50 28.73 34.02 31.34 31.26 AC923 14.47 25.99 29.90 30.21 29.02 AC924 18.11 20.85 30.17 27.71 27.88 Viruses 2022, 14, 569 6 of 14 Four full genome linear sequences of 10,972, 10,957, 10,975, and 10,973 nt were obtained from the isolates named AC568, AC913, AC923, and AC924, respectively. Phylogenetic relationships between the new isolates and other WNV-L2 isolates with full genome sequences available in GenBank were established. Firstly, these four isolates were compared with 45 isolates of WNV-L2, including recent European ones from both main defined clades [30] and representative African sequences. Phylogenetic analysis placed the Spanish isolates within the central-southern Euro- pean WNV lineage 2 clade, also defined as the Hungarian clade. Moreover, Spanish isolates were closely related with the Italian (2013) and the French (2018) ones, creating a mono- phyletic group and slightly differing from the Austrian-German group, which comprises representatives from 2014 to 2020 (Figure 2). Then, a further phylogenetic analysis was performed to refine the closest relationships. The Spanish representatives were analysed with 26 selected sequences of the central-southern European clade, incorporating 14 addi- tional sequences, mostly from Italy. The Spanish isolates specifically grouped together with Viruses 2022, 14, 569 the Italian Lombardy cluster, according to Barzon et al.’s 2015 definition [31], which 7 ofalso 15 incorporates the French isolates (Figure 3). 99 OM037672 Spain 2020 Tarragona Goshawk AC923 OM037673 Spain 2020 Tarragona Goshawk AC924 100 OM037670 Spain 2017 Lerida Goshawk AC568 93 OM037671 Spain 2020 Lerida Goshawk AC913 99 KF823806.1 Italy 2013 Mantova/40.1 MT863561.1 France 2018 isolate 7025 100 100 MT863560.1 France 2018 isolate 6125 KF588365.1 Italy 2013 Rovigo/32.1 100 KF647251.1 Italy 2013 Padova/34.1 95 100 KP109691.1 Austria 2014 Vienna blood donor 54 MF984347.1 Austria 2016 Blood donor 2 (BD2/16) MH244510.1 Slovakia 2014 Velky Biel/486.B 99 MW142227.1 Germany 2020 Leipzig isolate LE6 72 100 MH924836.1 Germany 2018 ED-I-33/18-UM 86 MH986056.1 Germany 2018 Rostock 1617 69 KM203860.1 Czech Republic 2013 Cz 13-104 100 MF984345.1 Austria 2015 (Bi2/15) Falcon Central-southern European WNV-L2 clade MH244513.1 Slovakia 2013 Secovce strain 200.B KF179640.1 Austria 2008 100 MH021189.1 Belgium 2017 Antwerpen 65 87 MK947397.1 Slovenia 2018 strain 2950 MN481590.1 Greece 2018 Thessaloniki S14c GrM 85 100 MT341471.1 Greece 2019 Karditsa 13m MT341472.1 Bulgaria 2018 Pleven 10m KC496015.1 Hungary 2010 578/10 100 67 71 KT757323.1 Serbia/Vojvodina 2013 KU206781.1 West Bulgaria 2015/Sofia 73 100 KJ883344.1 Greece 2013 Xanthi 2 KJ883348.1 Greece 2013/Kavala 2 100 98 HQ537483.1 Greece/Nea Santa 2010 61 MH549209.1 Greece 2017 KC496016.1 Serbia/Novi Sad 2010 100 100 KT359349.1 Hungary 2014 DQ116961.1 Hungary 2004 Goshawk 84 EF429197.1 South Africa 1989 SPU116/89 EF429198.1 South Africa 2001 SA93/01 97 100 JN393308.1 South Africa 2008 HS101 08 100 LC318700.1 Zambia 2016 Zmq16m11 95 KY523178.1 Uganda 2009 UG2274 100 DQ318019.1 Senegal ArD76104 100 AY532665.1 B956 99 HM147824.1 Democratic Republic of the Congo 1958 92 FJ425721.1 Russia 2007 Reb VLG 07 H Russian/Romanian WNV-L2 clade 100 100 KJ934710.1 Romania 2013 Hyalomma HM147823.1 Madagascar 1988 AY688948.1 Israel Sarafend unknown GQ903680.1 Cyprus 1968 Q3574-5 MW383507.1 Namibia 2020 M6646-NA-2020 100 HM147822.1 South Africa 1958 0.02 Figure2.2.Phylogenetic Figure Phylogenetic analysis analysis of of complete complete genome genome nucleotide nucleotide sequences sequencesofofWNV-L2. WNV-L2.The Theevolu- evolu- tionary distances were computed using the optimal GTR+I model, and the phylogenetic tree was tionary distances were computed using the optimal GTR+I model, and the phylogenetic tree was constructed with the Maximum likelihood method. Bootstrap values are given for 500 replicates. constructed with the Maximum likelihood method. Bootstrap values are given for 500 replicates. Viral Viral sequences are identified by GenBank accession number, country, and year of isolation. Se- sequences are identified quences emphasised by GenBank in bold and withaccession number, a black circle country, (●) were and year generated of isolation. during this study.Sequences emphasised in bold and with a black circle (•) were generated during this study. Viruses2022, Viruses 2022,14, 14,569 569 87 of of 14 15 OM37670 Spain 2017 Lerida Goshawk AC568 100 OM37671 Spain 2020 Lerida Goshawk AC913 OM37673 Spain 2020 Tarragona Goshawk AC924 57 99 OM37672 Spain 2020 Tarragona Goshawk AC923 MN939560.1 Italy 2016 Vicenza/36.1 KF823806.1 Italy 2013 Mantova/40.1 67 KP789953.1 Italy 2014 Pavia1 KP789958.1 Italy 2014 Pavia5 KP789955.1 Italy 2014 Verona/35.1 Italian Lombardy cluster KP789956.1 Italy 2014 Verona/35.2 65 MN939557.1 Italy 2018 Rovigo/29.1 100 Southwestern Europe 100 69 MN939564.1 Italy 2016 Rovigo/32.1 99 MN939563.1 Italy 2018 Verona/38.1 MN939561.1 Italy 2018 Venice/33.1 98 MN939562.1 Italy 2018 Treviso/35.1 100 KP789957.1 Italy 2014 Cremona4 79 MT863561.1 France 2018 isolate 7025 99 MT863560.1 France 2018 isolate 6125 KF647251.1 Italy 2013 Padova/34.1 KF588365.1 Italy 2013 Rovigo/32.1 Italian Veneto cluster 100 KP789960.1 Italy 2013 Mantova/36.1 KF647249.1 Italy 2013 Rovigo/33.2 100 MF984347.1 Austria 2016 Blood donor 2 (BD2/16) 75 KP109691.1 Vienna 2014 blood donor MH244510.1 Slovakia 2014 Velky Biel/486.B 99 MN794935.1 Germany 2019 BNI-10 Central Europe Central Europe MW142227.1 Germany 2020 Leipzig isolate LE6 100 94 MH986056.1 Germany 2018 Rostock isolate 1617 84 MH924836.1 Germany 2018 isolate ED-I-33/18-UM KJ883344.1 Greece 2013 Xanthi 2 0.0010 Figure3.3.Phylogenetic Figure Phylogeneticanalysis analysisofofcomplete complete genome genome nucleotide nucleotide sequences sequences of recent of recent WNV-L2 WNV-L2 iso- isolates lates western-central from from western-central Europe.Europe. Italian Italian clustersclusters were defined were defined byet by Barzon Barzon et[31]. al. 2015 al. 2015 The[31]. The evo- evolutionary lutionarywere distances distances were using computed computed using the the optimal optimal TN93+G TN93+G model model and the and the phylogenetic phylogenetic tree was tree was constructed constructed with the Maximum likelihood method. Bootstrap values of major branches are given with the Maximum likelihood method. Bootstrap values of major branches are given for 500 replicates. for 500 replicates. Viral sequences are identified by GenBank accession number, country, and year Viral sequences are identified by GenBank accession number, country, and year of isolation. Sequences of isolation. Sequences emphasised in bold and with a black circle (●) were generated during this emphasised study. in bold and with a black circle (•) were generated during this study. The comparison of the Spanish 2017 isolate with the 2020 ones revealed a nucleotide Table 2. Comparison of the amino acid substitutions between the Spanish isolates and representa- divergence from 0.09% tives of the Lombardy and toVeneto 0.27%,clusters being isolates AC923 of WNV-L2. Theand AC924 isolate from(both from2004 Hungary the was Tarragona used as province, 2020) the most closely related (presenting a 99.91% of nucleotide homology), the reference sequence. Black dot indicates the same amino acid as the reference sequence. Amino and the AC913 isolate (from Lleida 2020) the most dissimilar (99.73% acids that are unique in Spanish WNV-L2 polyproteins are highlighted in bold. nucleotide homology). A study of the viral polyproteins was performed comparing Spanish WNV-L2 isolates Initial Refer- Veneto with representatives of the Lombardy (ItalyLombardy 2013 KF823806 Cluster and France 2018 MT863560) ence Cluster and Veneto (Italy 2013 KF647249) clusters; Hungary 2004 isolate (DQ116961) was used as reference. DQ116961 This study KF647249 KF823806 revealed a range of It- MT863560 amino AC568 acidicAC913 Spain divergenceAC923 from 0.03% AC924 (between Amino Spain 2020 Spain 2020 Spain 2020 Viral Pro- HungaryAC913 and Italy AC568) 2013 alyto 20130.41% (between France 2018 the 2017Hungarian Lleida isolate and AC923 and AC924). Acid Posi- Lleida Tarragona Tarragona tein 2004Comparing Rovigo the Spanish Mantova strains, the variation (Common was 0.03–0.12% (Table S3). (Northern tion (Northern (Northern (Northern (Goshawk) Remarkably, (Human) WNV-L2 isolates (Human) Buzzard)detected in Spain were characterised by three unique Goshawk) Goshawk) Goshawk) Goshawk) amino acids changes (i.e., NS1-I123F, NS1-H293R, and NS5-T277I) that were absent in C 34 M · · · · · V · the representatives of the Lombardy and Veneto clusters. Additionally, Spanish isolates 20 T A · · · · · · prM from 2020 presented exclusive amino acids changes: AC913 showed one single change: 29 A · · · · · · V NS1-G41S; AC923 presented two single changes: C-M34V and NS1-M113I, and a change 88 S P P P P P P P E shared with AC924: NS4B-N11S; finally, AC924 showed two unique changes: prM-A29V 159 I T T T T T T T Viruses 2022, 14, 569 8 of 14 and NS5-R599I. Particularly, most of the variation in the amino acid sequences compared in this study was concentrated in the NS1 protein (Table 2). Table 2. Comparison of the amino acid substitutions between the Spanish isolates and representatives of the Lombardy and Veneto clusters of WNV-L2. The isolate from Hungary 2004 was used as the reference sequence. Black dot indicates the same amino acid as the reference sequence. Amino acids that are unique in Spanish WNV-L2 polyproteins are highlighted in bold. Initial Veneto Lombardy Cluster Reference Cluster AC568 AC913 AC923 AC924 DQ116961 KF647249 KF823806 MT863560 Amino Spain 2017 Spain 2020 Spain 2020 Spain 2020 Viral Hungary Italy 2013 Italy 2013 France 2018 Acid Lleida Lleida Tarragona Tarragona Protein 2004 Rovigo Mantova (Common Position (Northern (Northern (Northern (Northern (Goshawk) (Human) (Human) Buzzard) Goshawk) Goshawk) Goshawk) Goshawk) C 34 M · · · · · V · 20 T A · · · · · · prM 29 A · · · · · · V 88 S P P P P P P P E 159 I T T T T T T T 35 Y · H H H H H H 41 G · · · · S · · 69 G E E E E E E E 92 K · · E · · · · NS1 113 M · · · · · I · 123 I · · · F F F F 146 A V · · · · · · 293 H · · · R R R R 1 H Y Y Y Y Y Y Y NS2A 192 S C C C C C C C NS4B 11 N · · · · · S S 26 A T T T T T T T 203 Y H · · · · · · 277 T · · · I I I I 279 K · R · · · · · NS5 299 A T T T T T T T 340 N S · · · · · · 599 R · · · · · · I 886 V A · · · · · · Capacity of cell infection, as a rough approach to virulence estimation of the isolates, was measured by plaque size assays using data from previous studies [6,32,33]. Based on previous observations [6], Malaysian (L6) and Kunjin isolates (L1b) were considered of low virulence, B956 (L2) was of moderate virulence, and NY-99 and SPA07 (both L1) were classified as isolates of high virulence. As expected, a correlation between virulence and plaque size could be observed in previously known isolates examined in this study. Low virulent strains (i.e., KUN MP502-66 and KUN-KJ359-11) formed small plaques in Vero cells cultures (mean: 2.775 mm), high virulent (i.e., NY99 and SP’07) showed big-size plaques (mean: 5.073 mm), and moderate virulent isolates (i.e., B956) formed plaques of medium size (mean: 4.165 mm) (p ≤ 0.05). Considering these categories, Spanish AC568 showed the largest plaque size among all WNV-L2 Spanish isolates examined in this study (mean: 4.89 ± 0.78 mm). This size was statistically larger than the medium-size group (moderate virulence) and most similar to the highly virulent isolates NY-99 and SPA07. On the other hand, AC913 plaques (mean: 4.22 ± 0.83 mm) presented non-significant differences with medium plaque size (B956, moderate virulence), but were statistically distinguishable from the big-size (NY-99 and SPA07) and the small-plaque size (KUN MP502-66 and KUN-KJ359- 11) representatives. Finally, AC923 and AC924 plaques (mean: 4.50 ± 0.94 mm and mean: 4.89 ± 01.17 mm, respectively) showed an intermediate phenotype, between isolates with moderate (medium plaque size) and high virulence (big plaque size) (Figure 4). Viruses 2022, 14, 569 10 of 15 Viruses 2022, 14, 569 9 of 14 Figure 4. Analysis Analysisofofthe theplaque plaquesize sizeofof WNV WNV isolates. Diameter isolates. of the Diameter plaques of the is expressed plaques in mil- is expressed in limetres. Name of the strains and lineages are indicated. The letters a, b, c and d indicate millimetres. Name of the strains and lineages are indicated. The letters a, b, c and d indicate statistically significant differences statistically significant at p < 0.05. Columns differences with at p < 0.05. no common Columns with letters are statistically no common letters aredifferent (p < statistically 0.05). different (p < 0.05). 4. Discussion WNV-L2 WNV-L2was wasfirstly firstlydetected detectedin Europe in Europe in Hungary in Hungary in 2004in [9]; 2004since [9]; then, sinceitthen, has spread it has through the continent, spread through reachingreaching the continent, Greece (south-eastern) in 2010 [11], Greece (south-eastern) Spain in 2010 (south-western) [11], Spain (south- in 2017 [24], western) and Germany in 2017 (northern(northern [24], and Germany Europe) in 2018 [17]. Europe) This[17]. in 2018 apparently fast speedfast This apparently of spread, compared with the European WNV-L1, contrasts speed of spread, compared with the European WNV-L1, contrasts the North Americanthe North American scenario, where scenario,WNV-L1 was able to where WNV-L1 wasreach ablethe west coast to reach the westonly 4 years coast onlyafter its emergence 4 years in New after its emergence York in New in 1999 York[32]. This[32]. in 1999 fact reveals This fact strong revealsdifferences between both strong differences continents between regarding both continents vector-hosts biodiversity, regarding vector-hosts WNV ability biodiversity, WNV to spread, abilitysusceptibility, and immunological to spread, susceptibility, and immuno-status of individuals, logical status ofetc. individuals, etc. The The present present study study focused focused on the first on the first detections detections of of WNV-L2 WNV-L2 in in the the Iberian Iberian Peninsula. Peninsula. The first cases appeared in 2017 in northern goshawks from The first cases appeared in 2017 in northern goshawks from Catalonia (north-eastern Catalonia (north-eastern Spain) Spain) [24]. [24].Seroconversion Seroconversionwas wasobserved observed in in 14 14 bearded beardedvultures (Gypaetus vultures barbatus), (Gypaetus a neara barbatus), threatened-species, at the same time in the same WRC [24]. near threatened-species, at the same time in the same WRC [24]. WNV-L2 was not de- WNV-L2 was not detected again tecteduntil again2020, untilwhen 2020,three whenmorethreenorthern goshawks more northern were affected goshawks in the same were affected (Lleida) in the same and in an adjacent province (Tarragona). WNV-L2 was isolated (Lleida) and in an adjacent province (Tarragona). WNV-L2 was isolated from encephalonfrom encephalon samples, which samples, showed whichthe highest showed theviral loadsviral highest indicating that this tissue loads indicating is suitable that this tissue is forsuitable diagnosis for and viral isolation from northern goshawks infected with WNV-L2. diagnosis and viral isolation from northern goshawks infected with WNV-L2. Moreover, Moreover, neurological clinical signs,clinical neurological which all the affected signs, which allnorthern goshawks the affected northern presented, goshawks correlate perfectly presented, with correlate these high viral loads found in the central nervous system. Even perfectly with these high viral loads found in the central nervous system. Even though though only five RT-PCR positive only fivebirds have positive RT-PCR been detected in Catalonia, birds have recent studies been detected carriedrecent in Catalonia, out in studies the region [23] carried indicated that WNV circulation, not exclusively in birds but also in horses, is much more out in the region [23] indicated that WNV circulation, not exclusively in birds but also in widespread, although the majority of infections are subclinical. horses, is much more widespread, although the majority of infections are subclinical. In 2017, the first isolate of WNV-L2 in Spain was partially sequenced, specifically, In 2017, the first isolate of WNV-L2 in Spain was partially sequenced, specifically, a a segment of 930 nt of the NS5 gene [24]. In the present study, the full-genome of that segment of 930 nt of the NS5 gene [24]. In the present study, the full-genome of that isolate isolate and three more recent ones (2020) have been analysed. More in detail, these four and three more recent ones (2020) have been analysed. More in detail, these four isolates isolates showed a degree of nucleotide homology ranging between 99.73–99.91% and a showed a degree of nucleotide homology ranging between 99.73%–99.91% and a range of range of amino acid homology between 99.88–99.97%, sharing three exclusive amino acid amino acid homology between 99.88%–99.97%, sharing three exclusive amino acid posi- positions. These data evidence that this strain has remained genetically stable since its first tions. These data evidence that this strain has remained genetically stable since its first detection in 2017 in Lleida and has managed to overwinter in the area with some geographic detection in 2017 in Lleida and has managed to overwinter in the area with some geo- spread to another neighbouring province. In 2018 and 2019, no wild birds positive for graphic spread RT-PCR to another were detected neighbouring even though 120 province. and 56 In 2018 were and 2019, tested, no wild birds respectively. positive Absence of for RT-PCR were detected even though 120 and 56 were tested, RT-PCR positivity does not necessarily mean lack of WNV circulation. In fact, in 2018, respectively. Absence of RT-PCR positivity does not necessarily mean lack of WNV circulation. positivity for WNV by SNT was detected in an age-category 3 (i.e., born in 2018) Eurasian In fact, in 2018, Viruses 2022, 14, 569 10 of 14 magpies (Pica pica) sampled in the same area where WNV had been reported in 2017 [34], which suggested that the virus might have overwintered in the area and recirculated in the following year. Furthermore, in 2018, a horse with symptoms compatible with WNV and IgM positive was detected in the municipality of Vilanova i la Geltrú, in central-eastern Catalonia [34]. In 2018, Europe experienced the largest WNV epidemic to date with 2083 human cases [35], which contrasts with the limited circulation observed in Catalonia in the same year. High WNV transmission in Europe in 2018, mainly in south-eastern areas, was associated with favourable climatic conditions (e.g., increased precipitation in March or increased temperatures in May) [17,21], which did not occur in Catalonia [36]. Phylogenetic analyses showed that the Spanish isolates from 2017 and 2020 belonged to the central-southern European WNV-L2 clade [30]. This clade also includes recent strains from Germany, France, the Czech Republic, Greece, and Italy, where important outbreaks in humans and animals have occurred [13,16,18,20,37–39]. Our results corroborate the spread of this cluster to southern and western areas of the European continent, and represent the westernmost point in Europe this WNV lineage has reached and established to date. Furthermore, these Spanish isolates were closely related with the Italian (2013) and the French (2018) ones, constituting a monophyletic group diverging from the Austrian-German group, with representatives from 2014 to 2020 [37]. A subsequent phylogenetic analysis revealed that the Spanish isolates specifically grouped to an Italian cluster, namely the Lombardy cluster [31]. This cluster, also known as clade B, has shown more ability to spread than the Veneto cluster (also known as clade A) [20]. Indeed, the Veneto cluster apparently became extinct in 2013–2014, as no new sequences from this clade (neither from Italy nor from other countries) have been detected since then and, remarkably, none were collected in Italy in 2018 during the largest epidemic of WNV infection [16,20]. Even more, the Lombardy cluster, which has been detected in the Veneto region since 2014 (in Verona, Vicenza, Rovigo, Venice, and Treviso regions), has even replaced the Veneto cluster, becoming the dominant one in the area [21]. Besides, the Lombardy cluster, which initially caused outbreaks in consecutive years in northern Italy (2013–2014) [31], was able to spread from northern Italian regions to north-eastern Spain in 2017 and south-eastern France in 2017 or 2018 (considering that the 2017 human cases and the WNV-L2 confirmed outbreaks in birds in 2018 reported in the same French area likely shared the same origin) [16]. This replacement of strains in Italy, followed by a successful spread towards Spain and France, suggests that this variant from the Lombardy cluster might have acquired improved fitness [21]. Moreover, this variant has demonstrated to have the ability to cause outbreaks at least in wild birds [16] and humans [21], and probably in horses [40]. Accordingly, the phylogenetic analyses suggest one single introduction of WNV-L2 from northern Italy into France and Spain, probably via infected birds, as has been usually hypothesised [16]. Our phylogenetic analyses together with the seropositivity found before and after the first detection of WNV-L2 [23,41] suggest the presence and overwintering of WNV in the region. Furthermore, it is unknown whether the WNV seropositivity in the region was due to WNV-L1 or WNV-L2. While only the latter has been identified in this territory, silent WNV-L1 circulation should not be discarded. Therefore, the molecular analysis of different samples is essential in order to characterise isolates and study the spread and evolution of WNV over time. In the same way, it is important to point out the close phylogenetic relationship between certain Italian and Spanish isolates, as has been highlighted in this study of WNV-L2 and in previous ones focused on WNV-L1 [42]. This fact could reveal a particular dissemination route from Italy to Spain, although this hypothesis could be biased due to the absence of sequences from other western Mediterranean countries. Likewise, while the isolate AC568 (Lleida, 2017) displayed phenotypic characteristics of isolates of high virulence in mouse models, according to previously described classifi- cation in relation to plaque size in cell culture [6,32,33], the isolate AC913 (Lleida, 2020) showed characteristics of moderate virulence (similar to B956 strain) [6]. On the other hand, Viruses 2022, 14, 569 11 of 14 isolates AC923 and AC924 (Tarragona, 2020) presented an intermediate phenotype between moderate and high virulence. This presumable virulence estimated in vitro might correlate with the virulence observed in the affected birds, although it is uncertain if it holds a link to the horse outbreaks occurred in the region during 2020 and 2021 seasons [40,43]. In any case, these plaque size assays must be considered only rough estimates to approach virulence of WNV strains, and further in vivo studies of pathogenicity in vertebrate hosts will be necessary in order to accurately evaluate the virulence of these new strains. Wild raptors, especially northern goshawks, seem to be particularly susceptible to WNV neuro-invasive infections since they have been repeatedly found affected due to WNV infection in different countries in Europe [9,10,12,16,22,38,44–46]. In fact, from more than 350 birds sampled over 2017–2020, only five individuals were WNV positive by PCR, all of them northern goshawks that finally died or were euthanised due to the infection. This fact could be explained either because of their predation patterns, as they mainly feed on Passeriformes [46,47], which could be infected, and this may result in a subsequent infection of the predator [2], or due to mosquito feeding preferences [48]. Samples tested in this study were obtained from individuals of two WRC from Catalo- nia, highlighting the key role that these kind of centres can play in the surveillance and detection of emerging pathogens, as has been previously observed in other instances in Spain [41,49]. Finally, the detection of WNV-L2 in these northern goshawks in combination with the detection of specific antibodies against WNV in birds and horses from Catalonia [23] indicates an increased risk of WNV spillover into the human population in this area. Since WNV-L2 has been responsible for the largest outbreak of WNV in Europe in 2018 [35], it is necessary to reinforce WNV surveillance in Spain in order to better anticipate future outbreaks in humans. 5. Conclusions This study confirmed the presence and overwintering of WNV-L2 in north-eastern Spain between 2017 and 2020. Northern goshawk was the affected species, further confirm- ing the susceptibility of these raptors to WNV infection. In addition, this work highlighted the spread of WNV-L2 from central regions of Europe (such as Italy) to the west, causing outbreaks in France and Spain. Taking into account the results of this study and the situ- ation observed in neighbouring countries (such as Italy, Germany, or Greece), the risk of a further spillover to humans is high. Thus, it is essential to reinforce surveillance in the country, under a One-Health approach, in order to provide an early warning and to take the appropriate prevention and control measures. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/v14030569/s1, Table S1: Summary of the wild bird species analysed during the passive surveillance program carried out between 2017 and 2020; Table S2: Primers’ sequences used in this study for the whole genome sequencing of the first Spanish WNV- L2 isolates; Table S3: Percent identity matrix of the polyprotein between the Spanish isolates and representatives of the Lombardy and Veneto clusters of WNV-L2. The isolate from Hungary 2004 was used as the reference sequence. Author Contributions: Conceptualization, M.Á.J.-C., J.F.-P. and N.B.; investigation: C.S.-M., E.O., R.M.-L., A.S., P.A.-S. and N.B.; formal analysis, P.A.-S., F.L., J.F.-P. and S.N.; resources: C.S.-M., E.O., R.M.-L. and A.S.; writing—original draft preparation, P.A.-S., F.L., J.F.-P. and M.Á.J.-C.; writing— review and editing, P.A.-S., S.N., F.L., C.S.-M., E.O., R.M.-L., A.S., J.F.-P., M.Á.J.-C. and N.B.; supervi- sion: J.F.-P., F.L., M.Á.J.-C., S.N. and N.B.; funding acquisition, M.Á.J.-C., J.F.-P. and N.B. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by National Institute for Agricultural and Food Research and Technology (E-RTA2015-00002-CO2-01 project) and Research State Agency (PID2020-116768RR- C21/C22 project) funding programs. Institutional Review Board Statement: Not applicable. Viruses 2022, 14, 569 12 of 14 Informed Consent Statement: Not applicable. Data Availability Statement: The sequences presented in this study are openly available in GenBank database. Acknowledgments: We thank Amalia Villalba, Nuria Pujol and Raquel Rivas for their technical support, Encarnación Madueño for sequencing the samples, the neurologist Juanjo Minguez for the technical support in the description of clinical signs at WRC of Torreferrussa, as well as Maia Elizalde and Luisa Barzon for kindly providing the primer sets sequences for whole genome sequencing. Conflicts of Interest: The authors declare no conflict of interest. References 1. Mencattelli, G.; Ndione, M.H.D.; Rosà, R.; Marini, G.; Diagne, C.T.; Diagne, M.M.; Fall, G.; Faye, O.; Diallo, M.; Faye, O.; et al. Epidemiology of West Nile Virus in Africa: An Underestimated Threat. PLoS Negl. Trop. Dis. 2022, 16, e0010075. [CrossRef] [PubMed] 2. Komar, N.; Langevin, S.; Hinten, S.; Nemeth, N.; Edwards, E.; Hettler, D.; Davis, B.; Bowen, R.; Bunning, M. Experimental Infection of North American Birds with the New York 1999 Strain of West Nile Virus. Emerg. Infect. Dis. 2003, 9, 311–322. [CrossRef] [PubMed] 3. García San Miguel Rodríguez-Alarcón, L.; Fernández-Martínez, B.; Sierra Moros, M.J.; Vázquez, A.; Julián Pachés, P.; García Villacieros, E.; Gómez Martín, M.B.; Figuerola Borras, J.; Lorusso, N.; Ramos Aceitero, J.M.; et al. Unprecedented Increase of West Nile Virus Neuroinvasive Disease, Spain, Summer 2020. Eurosurveillance 2021, 26, 2002010. [CrossRef] [PubMed] 4. Rizzoli, A.; Jimenez-Clavero, M.A.; Barzon, L.; Cordioli, P.; Figuerola, J.; Koraka, P.; Martina, B.; Moreno, A.; Nowotny, N.; Pardigon, N.; et al. The Challenge of West Nile Virus in Europe : Knowledge Gaps and Research Priorities. Eurosurveillance 2015, 20, 21135. [CrossRef] [PubMed] 5. Habarugira, G.; Suen, W.W.; Hobson-Peters, J.; Hall, R.A.; Bielefeldt-Ohmann, H. West Nile Virus: An Update on Pathobiology, Epidemiology, Diagnostics, Control and “One Health” Implications. Pathogens 2020, 9, 589. [CrossRef] [PubMed] 6. Pérez-Ramírez, E.; Llorente, F.; Del Amo, J.; Fall, G.; Sall, A.A.; Lubisi, A.; Lecollinet, S.; Vázquez, A.; Jiménez-Clavero, M.Á. Pathogenicity Evaluation of Twelve West Nile Virus Strains Belonging to Four Lineages from Five Continents in a Mouse Model: Discrimination between Three Pathogenicity Categories. J. Gen. Virol. 2017, 98, 662–670. [CrossRef] [PubMed] 7. Murgue, B.; Murri, S.; Triki, H.; Deubel, V.; Zeller, H.G. West Nile in the Mediterranean Basin: 1950–2000. Ann. N. Y. Acad. Sci. 2006, 951, 117–126. [CrossRef] [PubMed] 8. Barrett, A.D.T. West Nile in Europe: An Increasing Public Health Problem. J. Travel Med. 2018, 25, tay096. [CrossRef] 9. 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] 10. 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] 11. Papa, A.; Bakonyi, T.; Xanthopoulou, K.; Vázquez, A.; Tenorio, A.; Nowotny, N. Genetic Characterization of West Nile Virus Lineage 2, Greece, 2010. Emerg. Infect. Dis. 2011, 17, 920–922. [CrossRef] [PubMed] 12. Petrović, T.; Blázquez, A.B.; Lupulović, D.; Lazić, G.; Escribano-Romero, E.; Fabijan, D.; Kapetanov, M.; Lazić, S.; Saiz, J.C. Monitoring West Nile Virus (WNV) Infection in Wild Birds in Serbia during 2012: First Isolation and Characterisation of WNV Strains from Serbia. Eurosurveillance 2013, 18, 20622. [CrossRef] [PubMed] 13. Barzon, L.; Pacenti, M.; Franchin, E.; Lavezzo, E.; Masi, G.; Squarzon, L.; Pagni, S.; Toppo, S.; Russo, F.; Cattai, M.; et al. Whole Genome Sequencing and Phylogenetic Analysis of West Nile Virus Lineage 1 and Lineage 2 from Human Cases of Infection, Italy, August 2013. Eurosurveillance 2013, 18, 20591. [CrossRef] [PubMed] 14. Platonov, A.E.; Karan, L.S.; Shopenskaia, T.A.; Fedorova, M.V.; Koliasnikova, N.M.; Rusakova, N.M.; Shishkina, L.V.; Arshba, T.E.; Zhuravlev, V.I.; Govorukhina, M.V.; et al. Genotyping of West Nile Fever Virus Strains Circulating in Southern Russia as an Epidemiological Investigation Method: Principles and Results. Zhurnal Mikrobiol. Epidemiol. Immunobiol. 2011, 2, 29–37. 15. Sirbu, 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] [PubMed] 16. 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] 17. Ziegler, U.; Lühken, R.; Keller, M.; Cadar, D.; Van Der Grinten, E.; Michel, F.; Albrecht, K.; Eiden, M.; Rinder, M.; Lachmann, L.; et al. West Nile Virus Epizootic in Germany, 2018. Antivir. Res. 2019, 162, 39–43. [CrossRef] [PubMed] 18. Zelená, H.; Kleinerová, J.; Šikutová, S.; Straková, P.; Kocourková, H.; Stebel, R.; Husa, P.; Husa, P.; Tesařová, E.; Lejdarová, H.; et al. First Autochthonous West Nile Lineage 2 and Usutu Virus Infections in Humans, July to October 2018, Czech Republic. Pathogens 2021, 10, 651. [CrossRef] Viruses 2022, 14, 569 13 of 14 19. Vlaskamp, D.R.M.; Thijsen, S.F.T.; Reimerink, J.; Hilkens, P.; Bouvy, W.H.; Bantjes, S.E.; Vlaminckx, B.J.M.; Zaaijer, H.; van den Kerkhof, H.H.T.C.; Raven, S.F.H.; et al. First Autochthonous Human West Nile Virus Infections in the Netherlands, July to August 2020. Eurosurveillance 2020, 25, 2001904. [CrossRef] 20. Veo, C.; Della Ventura, C.; Moreno, A.; Rovida, F.; Percivalle, E.; Canziani, S.; Torri, D.; Calzolari, M.; Baldanti, F.; Galli, M.; et al. Evolutionary Dynamics of the Lineage 2 West Nile Virus That Caused the Largest European Epidemic: Italy 2011–2018. Viruses 2019, 11, 814. [CrossRef] 21. Pacenti, M.; Sinigaglia, A.; Franchin, E.; Pagni, S.; Lavezzo, E.; Montarsi, F.; Capelli, G.; Barzon, L. Human West Nile Virus Lineage 2 Infection: Epidemiological, Clinical, and Virological Findings. Viruses 2020, 12, 458. [CrossRef] [PubMed] 22. Aguilera-Sepúlveda, P.; Gómez-Martín, B.; Agüero, M.; Jiménez-Clavero, M.Á.; Fernández-Pinero, J. A New Cluster of West Nile Virus Lineage 1 Isolated from a Northern Goshawk in Spain. Transbound. Emerg. Dis. 2021, 1–7. [CrossRef] [PubMed] 23. Napp, S.; Llorente, F.; Beck, C.; Jose-Cunilleras, E.; Soler, M.; Pailler-García, L.; Amaral, R.; Aguilera-Sepúlveda, P.; Pifarér, M.; Molina-López, R.; et al. Widespread Circulation of Flaviviruses in Horses and Birds in Northeastern Spain (Catalonia) between 2010 and 2019. Viruses 2021, 13, 2404. [CrossRef] [PubMed] 24. Busquets, N.; Laranjo-González, M.; Soler, M.; Nicolás, O.; Rivas, R.; Talavera, S.; Villalba, R.; San Miguel, E.; Torner, N.; Aranda, C.; et al. Detection of West Nile Virus Lineage 2 in North-Eastern Spain (Catalonia). Transbound. Emerg. Dis. 2019, 66, 1–5. [CrossRef] [PubMed] 25. Petrovic, T.; Šekler, M.; Petric, D.; Vidanovic, D.; Debeljak, Z.; Lazic, G.; Lupulovic, D.; Kavran, M.; Samojlovic, M.; Ignjatovic Cupina, A.; et al. Intensive West Nile Virus Circulation in Serbia in 2018 Results of Integrated Surveillance Program. Pathogens 2021, 10, 1294. [CrossRef] [PubMed] 26. Linke, S.; Ellerbrok, H.; Niedrig, M.; Nitsche, A.; Pauli, G. Detection of West Nile Virus Lineages 1 and 2 by Real-Time PCR. J. Virol. Methods 2007, 146, 355–358. [CrossRef] [PubMed] 27. Scaramozzino, N.; Crance, J.; Jouan, A.; De Briel, D.A.; Stoll, F.; Garin, D. Comparison of Flavivirus Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5 Gene Sequences. J. Clin. Microbiol. 2001, 39, 1922–1927. [CrossRef] 28. Jourdain, E. Oiseaux Sauvages et Virus West Nile: Étude Éco-Épidémiologique en Camargue. Ph.D. Thesis, Université Joseph Fourier–Grenoble 1, Grenoble, France, 2006; p. 215. 29. Payne, A.F.; Binduga-Gajewska, I.; Kauffman, E.B.; Kramer, L.D. Quantitation of Flaviviruses by Fluorescent Focus Assay. J. Virol. Methods 2006, 134, 183–189. [CrossRef] [PubMed] 30. Ravagnan, S.; Montarsi, F.; Cazzin, S.; Porcellato, E.; Russo, F.; Palei, M.; Monne, I.; Savini, G.; Marangon, S.; Barzon, L.; et al. First Report Outside Eastern Europe of West Nile Virus Lineage 2 Related to the Volgograd 2007 Strain, Northeastern Italy, 2014. Parasites Vectors 2015, 8, 418. [CrossRef] [PubMed] 31. Barzon, L.; Papa, A.; Lavezzo, E.; Franchin, E.; Pacenti, M.; Sinigaglia, A.; Masi, G.; Trevisan, M.; Squarzon, L.; Toppo, S.; et al. Phylogenetic Characterization of Central/Southern European Lineage 2 West Nile Virus: Analysis of Human Outbreaks in Italy and Greece, 2013–2014. Clin. Microbiol. Infect. 2015, 21, 1122.e1–1122.e10. [CrossRef] 32. Davis, C.T.; Beasley, D.W.C.; Guzman, H.; Siirin, M.; Parsons, R.E.; Tesh, R.B.; Barrett, A.D.T. Emergence of Attenuated West Nile Virus Variants in Texas, 2003. Virology 2004, 330, 342–350. [CrossRef] [PubMed] 33. Jia, Y.; Moudy, R.M.; Dupuis, A.P.; Ngo, K.A.; Maffei, J.G.; Jerzak, G.V.S.; Franke, M.A.; Kauffman, E.B.; Kramer, L.D. Charac- terization of a Small Plaque Variant of West Nile Virus Isolated in New York in 2000. Virology 2007, 367, 339–347. [CrossRef] [PubMed] 34. Napp, S.; Montalvo, T.; Piñol-Baena, C.; Gómez-Martín, M.B.; Nicolás-Francisco, O.; Soler, M.; Busquets, N. Usefulness of Eurasian Magpies (Pica pica) for West Nile Virus Surveillance in Non-Endemic and Endemic Situations. Viruses 2019, 11, 716. [CrossRef] [PubMed] 35. European Centre for Disease Prevention and Control. Epidemiological Update: West Nile Virus Transmission Season in Europe. 2018. Available online: https://www.ecdc.europa.eu/en/news-events/epidemiological-update-west-nile-virus-transmission- season-europe-2018 (accessed on 9 January 2022). 36. Copernicus Climate Change Service. Monthly Summaries of Precipitation, Relative Humidity and Soil Moisture 2018. Available online: https://climate.copernicus.eu/ (accessed on 9 January 2022). 37. Ziegler, U.; Santos, P.D.; Groschup, M.H.; Hattendorf, C.; Eiden, M.; Höper, D.; Eisermann, P.; Keller, M.; Michel, F.; Klopfleisch, R.; et al. West Nile Virus Epidemic in Germany Triggered by Epizootic Emergence, 2019. Viruses 2020, 12, 448. [CrossRef] [PubMed] 38. Hubálek, Z.; Tomešek, M.; Kosina, M.; Šikutová, S.; Straková, P.; Rudolf, I. West Nile Virus Outbreak in Captive and Wild Raptors, Czech Republic, 2018. Zoonoses Public Health 2019, 66, 978–981. [CrossRef] [PubMed] 39. Papa, A.; Papadopoulou, E.; Chatzixanthouliou, C.; Glouftsios, P.; Pappa, S.; Pervanidou, D.; Georgiou, L. Emergence of West Nile Virus Lineage 2 Belonging to the Eastern European Subclade, Greece. Arch. Virol. 2019, 164, 1673–1675. [CrossRef] [PubMed] 40. Ministerio de Agricultura, Pesca y Alimentación. Consulta de Notificación de Enfermedades de los Animales de Declaración Obligatoria. Available online: https://servicio.mapa.gob.es/rasve/Publico/Publico/BuscadorFocos.aspx (accessed on 9 January 2022). 41. Alba, A.; Allepuz, A.; Napp, S.; Soler, M.; Selga, I.; Aranda, C.; Casal, J.; Pages, N.; Hayes, E.B.; Busquets, N. Ecological Surveillance for West Nile in Catalonia (Spain), Learning from a Five-Year Period of Follow-Up. Zoonoses Public Health 2014, 61, 181–191. [CrossRef] [PubMed] Viruses 2022, 14, 569 14 of 14 42. Sotelo, E.; Fernández-Pinero, J.; Llorente, F.; Vázquez, A.; Moreno, A.; Agüero, M.; Cordioli, P.; Tenorio, A.; Jiménez-Clavero, M.Á. Phylogenetic Relationships of Western Mediterranean West Nile Virus Strains (1996–2010) Using Full-Length Genome Sequences: Single or Multiple Introductions? J. Gen. Virol. 2011, 92, 2512–2522. [CrossRef] 43. European Centre for Disease Prevention and Control (ECDC). Weekly Updates: 2021 West Nile Virus Transmission Season. Available online: https://www.ecdc.europa.eu/en/west-nile-fever/surveillance-and-disease-data/disease-data-ecdc (accessed on 9 January 2022). 44. Feyer, S.; Bartenschlager, F.; Bertram, C.A.; Ziegler, U.; Fast, C.; Klopfleisch, R.; Müller, K. Clinical, Pathological and Virological Aspects of Fatal West Nile Virus Infections in Ten Free-Ranging Goshawks (Accipiter gentilis) in Germany. Transbound. Emerg. Dis. 2021, 68, 907–919. [CrossRef] 45. Hubálek, Z.; Kosina, M.; Rudolf, I.; Mendel, J.; Straková, P.; Tomešek, M. Mortality of Goshawks (Accipiter gentilis) Due to West Nile Virus Lineage 2. Vector-Borne Zoonotic Dis. 2018, 18, 624–627. [CrossRef] 46. Vidaña, B.; Busquets, N.; Napp, S.; Perez-Ramírez, E.; Jiménez-Clavero, M.Á.; Johnson, N. The Role of Birds of Prey in West Nile Virus Epidemiology. Vaccines 2020, 8, 550. [CrossRef] [PubMed] 47. García-Salgado, G.; Rebollo, S.; Pérez-Camacho, L.; Martínez-Hesterkamp, S.; Navarro, A.; Fernández-Pereira, J.M. Evaluation of Trail-Cameras for Analyzing the Diet of Nesting Raptors Using the Northern Goshawk as a Model. PLoS ONE 2015, 10, e0127585. [CrossRef] [PubMed] 48. Victoriano Llopis, I.; Tomassone, L.; Grego, E.; Serrano, E.; Mosca, A.; Vaschetti, G.; Andrade, D.; Rossi, L. Evaluating the Feeding Preferences of West Nile Virus Mosquito Vectors Using Bird-Baited Traps. Parasites Vectors 2016, 9, 479. [CrossRef] [PubMed] 49. Bravo-Barriga, D.; Aguilera-Sepúlveda, P.; Guerrero-Carvajal, F.; Llorente, F.; Reina, D.; Pérez-Martín, J.E.; Jiménez-Clavero, M.Á.; Frontera, E. West Nile and Usutu Virus Infections in Wild Birds Admitted to Rehabilitation Centres in Extremadura, Western Spain, 2017–2019. Vet. Microbiol. 2021, 255, 109020. [CrossRef] [PubMed]