Lineage 2 West Nile virus (WNV) caused a vast epidemic in Europe in 2018, with the highest incidence being recorded in Italy. To reconstruct the evolutionary dynamics and epidemiological history of the virus in Italy, 53 envelope gene and 26 complete genome sequences obtained from human and animal samples were characterised by means of next-generation sequencing. Phylogenetic analysis revealed two Italian strains originating between 2010 and 2012: clade A, which apparently became extinct in 2013−2014, and clade B, which was responsible for the 2018 epidemic. The mean genetic distances in clade B increased over time and with the distance between sampling locations. Bayesian birth-death and coalescent skyline plots of the clade B showed that the effective number of infections and the effective reproduction number (Re) increased between 2015 and 2018. Our data suggest that WNV-2 entered Italy in 2011 as a result of one or a few penetration events. Clade B differentiated mainly as a result of genetic drift and purifying selection, leading to the appearance of multiple locally circulating sub-clades for different times. Phylodynamic analysis showed a current expansion of the infection among reservoir birds and/or vectors.
