West Nile Virus Lineage 2 Overwintering in Italy

Abstract

In January 2022, West Nile virus (WNV) lineage 2 (L2) was detected in an adult female goshawk rescued near Perugia in the region of Umbria (Italy). The animal showed neurological symptoms and died 15 days after its recovery in a wildlife rescue center. This was the second case of WNV infection recorded in birds in the Umbria region during the cold season, when mosquitoes, the main WNV vectors, are usually not active. According to the National Surveillance Plan, the Umbria region is included amongst the WNV low-risk areas. The necropsy evidenced generalized pallor of the mucous membranes, mild splenomegaly, and cerebral edema. WNV L2 was detected in the brain, heart, kidney, and spleen homogenate using specific RT-PCR. Subsequently, the extracted viral RNA was sequenced. A Bayesian phylogenetic analysis performed through a maximum-likelihood tree showed that the genome sequence clustered with the Italian strains within the European WNV strains among the central-southern European WNV L2 clade. These results, on the one hand, confirmed that the WNV L2 strains circulating in Italy are genetically stable and, on the other hand, evidenced a continuous WNV circulation in Italy throughout the year. In this report case, a bird-to-bird WNV transmission was suggested to support the virus overwintering. The potential transmission through the oral route in a predatory bird may explain the relatively rapid spread of WNV, as well as other flaviviruses characterized by similar transmission patterns. However, rodent-to-bird transmission or mosquito-to-bird transmission cannot be excluded, and further research is needed to better understand WNV transmission routes during the winter season in Italy.

Keywords: West Nile virus; bird-to-bird transmission; birds of prey; flavivirus; hybrid mosquitoes; overwintering; rodent-to-bird transmission; surveillance.

Figure 1

Map of geo-localization site. The northern goshawk was found in the Torgiano municipality (43.0893° N, 12.4410° E) in the Umbria region.

Figure 2

The evolutionary analysis inferred by using the maximum-likelihood method and the general time reversible model is illustrated. The tree with the highest log-likelihood is shown. The percentage of trees in which the associated taxa clustered together is displayed next to the branches. Initial trees for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among the sites (4 categories). The evolutionary distances were computed using the optimal GTR + Γ + I model, with 2000 Γ-rate categories and 5000 bootstrap replications using the Shimodaira–Hasegawa (SH) test. Ten independent MCMC runs with up to 100 million generations were performed to ensure the convergence of the estimates. GenBank accession numbers are indicated for each strain, with country, lineage, and year of isolation. The genome sequence ON032498 WNV L2 (HCov 91%), obtained from the goshawk organ homogenate, is highlighted in red. The WNV L1 MW627239 (Italy 2020) and MT863559 (France 2015), and Koutango virus (KOUTV) EU082200 (Senegal 2013), chosen as outgroups, are highlighted in blue. The new strain (ON032498) showed high genetic similarity with the central-southern European WNV L2 clade, highlighted in pink, with a posterior probability of 100%.