Detection of West Nile virus lineage 2 in North‐Eastern Spain (Catalonia)

Abstract In September 2017, West Nile virus (WNV) lineage 2 was detected in Catalonia (Northern Spain) in northern goshawks by passive surveillance. The phylogenetic analyses showed that it was related to the Central/Southern European strains, evidencing WNV lineage 2 spread to Western Europe. WNV local transmission was later detected in bearded vultures housed at the Wildlife Recovery center where the goshawk was transferred to. Further studies, before the following period of high mosquito activity, indicated that WNV had circulated intensively in poultry and horses but only surrounding of the area where the virus was detected. In other areas of Catalonia, circulation of flaviviruses different to WNV was identified. Public Health investigations failed to detect WNV infection in humans.

In Spain, cases of WNV in horses and humans were reported in 2010 in the South of the country (Andalusia region), which were caused by a WNV lineage 1 strain (García-Bocanegra et al., 2011).
Since then, WNV became endemic in Southern Spain, re-emerging every year and expanding northwards, causing outbreaks in horses, with further confirmed human cases in 2016 (López-Ruiz et al., 2018).

| RESULTS AND DISCUSSION
In September 2017, a northern goshawk (Accipiter gentilis) was found with dehydration, apathy and low weight near an urban area of Lleida province (Catalonia, Spain), and was transferred to the Wildlife Recovery Center (WRC) of Vallcalent. Five days later it developed nervous symptoms (head-shaking, incoordination, and inability to stand upright), and was euthanized. Samples of nervous tissue were positive to WNV infection by RT-qPCR (Linke, F I G U R E 1 Molecular phylogenetic analysis by maximum likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura & Nei, 1993). The tree with the highest log likelihood (−2,521.85) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 34 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 933 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar, Stecher, & Tamura, 2016). The Spanish WNV isolate is marked using a dot. The sequence obtained in this study was submitted to GenBank Nucleotide Sequence Database under the following accession number: MH327930. The Spanish WNV isolate is marked using a green dot. The sequence obtained in this study [Colour figure can be viewed at wileyonlinelibrary.com] Ellerbrok, Niedrig, Nitsche, & Pauli, 2007). WNV-positivity was confirmed by the Central Veterinary Laboratory (CVL) in Algete by RT-qPCR (Jiménez-Clavero, Agüero, Rojo, & Gómez-Tejedor, 2006).
The analysis of a partial sequence of the NS5 gene using the primers described by Scaramozzino et al., 2001; indicated that it belonged to lineage 2. After confirmation, the case was reported to the World Organization for Animal Health (OIE). Within days, WNV lineage 2 was also detected in another sick northern goshawk that was admitted to Vallcalent-WRC and had died after 2 days. Three days later, a third northern goshawk found with an old fracture tested positive by cELISA (IDvet-ID Screen ® West Nile Competition) and was confirmed by SNT at the CVL (titer of 1/40).
West Nile virus infection was detected in three northern goshawks, including two symptomatic animals, even though, according to the ornithologists consulted, this species is uncommon in the affected area, and probably just arrived (autumn migration) from Northern-Eastern Europe. Interestingly, in Europe, WNV lineage 2 has been repeatedly isolated from northern goshawks (Bakonyi et al., 2013). This may be explained by a higher susceptibility of this F I G U R E 2 (a) Locations where the WNV-positive goshawk was detected (triangle), and of Vallcalent-WRC (empty square). Also results of the serological cross-sectional survey in poultry (round symbol) and horses (square symbol) carried out in the surveillance area (10 km radius). (b) Results of the cross-sectional survey in poultry (round symbol) and horses (square symbol) carried out in the surveillance area (10 km radius). (c) Location of Catalonia, and the affected area, within Spain [Colour figure can be viewed at wileyonlinelibrary.com] T A B L E 1 Results of the cross-sectional survey in chickens and horses within the surveillance area. Results of cELISA and SNT for both individual animals and holdings  That seems to suggest that there was no widespread circulation of WNV in humans in the area.
The fact that WNV was detected in Catalonia was probably due to the implementation of a comprehensive surveillance program (including active and passive surveillance in wild birds and horses) for more than 12 years. In fact, since 2010, WNV positivity by SNT has been detected in wild birds (mainly raptors) in this same area, which was not included among the high-risk areas for WNV in Catalonia (Alba et al., 2014). However, local WNV circulation could never be demonstrated as positive animals were always potentially migratory.
Given the ability of WNV to overwinter (Rudolf et al., 2017), the re-emergence of WNV lineage 2 in Catalonia would be possible.
After the detection of WNV circulation in 2017, surveillance measures implemented in the affected area were intensified.
Furthermore, WNV lineage 2 recurrence combined with the northward expansion of WNV lineage 1, the emergence of novel WNV genotypes and the circulation of other flaviviruses, e.g., USUV (Busquets et al., 2008) results in a complex scenario for the future, with several co-circulating flaviviruses. The consequences of flaviviruses co-infections in both the hosts and the vectors are unpredictable (Rizzoli et al., 2015).

ACKNOWLEDGEMENTS
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