A new cluster of West Nile virus lineage 1 identified in Spain

Abstract

Purpose: West Nile Virus (WNV) is a zoonotic arbovirus worldwide spread. In Spain, its active circulation provokes outbreaks in birds, horses and lately in humans. In September 2017, an outbreak affecting raptors, in the province of Málaga (Andalusia), was investigated. One northern goshawk (Accipiter gentilis) died, while two peregrine falcons (Falco peregrinus) recovered. Here we report the isolation and characterization of a new WNV lineage 1 variant from this outbreak. Methods & Materials: Heart, feathers, brain and kidney samples from the goshawk were collected and firstly analysed by real-time RT-PCR for WNV (Jiménez-Clavero et al., 2006, https://doi.org/doi:10.1177/104063870601800505). Virus isolation was performed in BSR and Vero cells.The whole genome of the isolate was sequenced by Sanger method. Phylogenetic analyses were performed including a wide range of representatives of: 1) all described WNV lineages, and 2) the different clusters of WNV lineage 1. Results: All samples from the goshawk were positive for WNV lineage 1 by RT-PCR. Virus isolation was successful in heart and brain samples. A full genome sequence of 10,919 nt was obtained for the WNV isolated from the brain (named Spain/2017/NG-b). Phylogenetic analyses confirmed that Spain/2017/NG-b belonged to WNV clade 1a. Surprisingly, this isolate was placed neither in the cluster 2 of this clade, which comprises most of the recent European and Mediterranean strains (including the preceding Spanish ones), nor in other closest clusters. When analysing the polyprotein, amino acid substitutions that define WNV clusters 1-4 were not found in Spain/2017/NG-b isolate (May et al., 2011, https://doi.org/10.1128/jvi.01963-10). Moreover, it presented 12 unique residues compared to cluster 2 viruses. Conclusion: These results strongly support that Spain/2017/NG-b represents a new, independent introduction of WNV into the Western Mediterranean since it showed no close relationship to other strains from this region. Therefore, we propose to associate this variant to a new cluster 7 within WNV clade 1a. Finally, a WNV outbreak occurred in equids in December 2017, nearby the raptors´ outbreak above mentioned, suggests the ability of this new variant to infect and cause disease also in horses.