Reconstructing the recent West Nile virus lineage 2 epidemic in Europe and Italy using discrete and continuous phylogeography

Abstract

West Nile virus lineage 2 (WNV-2) was mainly confined to sub-Saharan Africa until the early 2000s, when it was identified for the first time in Central Europe causing outbreaks of human and animal infection. The aim of this study was to reconstruct the origin and dispersion of WNV-2 in Central Europe and Italy on a phylodynamic and phylogeographical basis. To this aim, discrete and continuous space phylogeographical models were applied to a total of 33 newly characterised full-length viral genomes obtained from mosquitoes, birds and humans in Northern Italy in the years 2013-2015 aligned with 64 complete sequences isolated mainly in Europe. The European isolates segregated into two highly significant clades: a small one including three sequences and a large clade including the majority of isolates obtained in Central Europe since 2004. Discrete phylogeographical analysis showed that the most probable location of the root of the largest European clade was in Hungary a mean 12.78 years ago. The European clade bifurcated into two highly supported subclades: one including most of the Central/East European isolates and the other encompassing all of the isolates obtained in Greece. The continuous space phylogeographical analysis of the Italian clade showed that WNV-2 entered Italy in about 2008, probably by crossing the Adriatic sea and reaching a central area of the Po Valley. The epidemic then spread simultaneously eastward, to reach the region of the Po delta in 2013, and westward to the border area between Lombardy and Piedmont in 2014; later, the western strain changed direction southward, and reached the central area of the Po valley once again in 2015. Over a period of about seven years, the virus spread all over an area of northern Italy by following the Po river and its main tributaries.

Fig 1. Maximum likelihood tree of the 97 West Nile virus complete genome sequences.

The European clades have been highlighted (blue circles). The Italian clades are identified by red triangles. The scale bar indicates 3% of nucleotide divergence.

Fig 2. Phylogeographic analysis of 77 European WNV-2 isolates forming a single monophyletic group.

The branches of the maximum clade credibility (MCC) tree are coloured on the basis of the most probable location of the descendent nodes (AT = Austria; CZ = Czech Republic; GR = Greece; HU = Hungary; IT = Italy; SR = Serbia). The numbers on the internal nodes indicate significant posterior probabilities (pp >0.7), and the scale at the bottom of the tree represents calendar years. The main geographical clades are highlighted.

Fig 3. Population dynamics analysis of the European monophyletic clade of WNV-2. Bayesian skyline plot (BSP).

The effective number of infections is indicated on the Y axis, and time on the X-axis. The coloured area corresponds to the credibility interval based on the 95% highest posterior density interval (HPD).

Fig 4. Spatio-temporal dynamics of the West Nile virus epidemic in Italy.

The inferred spatiotemporal dynamics of WNV-2 in Northern Italy. The figure summarize the most significant migration links in the interested area. More detailed results are reported in S1 Video.