West Nile Virus Lineage 2 Spreads Westwards in Europe and Overwinters in North-Eastern Spain (2017-2020)

Abstract

West Nile virus lineage 2 (WNV-L2) emerged in Europe in 2004; since then, it has spread across the continent, causing outbreaks in humans and animals. During 2017 and 2020, WNV-L2 was detected and isolated from four northern goshawks in two provinces of Catalonia (north-eastern Spain). In order to characterise the first Spanish WNV-L2 isolates and elucidate the potential overwintering of the virus in this Mediterranean region, complete genome sequencing, phylogenetic analyses, and a study of phenotypic characterisation were performed. Our results showed that these Spanish isolates belonged to the central-southern WNV-L2 clade. In more detail, they were related to the Lombardy cluster that emerged in Italy in 2013 and has been able to spread westwards, causing outbreaks in France (2018) and Spain (2017 and 2020). Phenotypic characterisation performed in vitro showed that these isolates presented characteristics corresponding to strains of moderate to 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 very susceptible to WNV infection and may be good indicators of WNV-L2 circulation. Due to the increasing number of human and animal cases in Europe in the last years, this zoonotic flavivirus should be kept under extensive surveillance, following a One-Health approach.

Keywords: Europe; Spain; West Nile virus; avian host; lineage-2; northern goshawk; overwintering; phylogeny.

Figure 1

On the left: Provinces were the study was carried out (dark grey and red). In red: location of the affected provinces of Catalonia: Lleida, and Tarragona, within Spain. On the right: distribution of the RT-PCR-positive and negative wild birds sampled in Catalonia between 2017 and 2020.

Figure 2

Phylogenetic analysis of complete genome nucleotide sequences of WNV-L2. The evolutionary 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. Viral sequences are identified by GenBank accession number, country, and year of isolation. Sequences emphasised in bold and with a black circle (●) were generated during this study.

Figure 3

Phylogenetic analysis of complete genome nucleotide sequences of recent WNV-L2 isolates from western-central Europe. Italian clusters were defined by Barzon et al. 2015 [31]. The evolutionary distances were computed using the optimal TN93+G model and the phylogenetic tree was constructed with the Maximum likelihood method. Bootstrap values of major branches are given for 500 replicates. Viral sequences are identified by GenBank accession number, country, and year of isolation. Sequences emphasised in bold and with a black circle (●) were generated during this study.

Figure 4

Analysis of the plaque size of WNV isolates. Diameter of the plaques is expressed in millimetres. Name of the strains and lineages are indicated. The letters a, b, c and d indicate statistically significant differences at p < 0.05. Columns with no common letters are statistically different (p < 0.05).