West Nile virus in Italy: beyond the bird routes

Context: West Nile virus (WNV) is an arthropod-borne virus considered a One Health challenge because of its increasing impact on human and animal health. It is one the most widely distributed viruses of the encephalitic Flaviviruses. It may cause severe neurological symptoms in humans and animals and is recognized as a serious public health problem also because of its impact on blood transfusion and organ transplantation. First identified in Africa in 1937, it was later introduced and spread in Italy, where in many regions it is now endemic, due to the increasingly favorable climatic and environmental conditions. Aim: The main objectives of this study, based on an interdisciplinary One Health approach, were: (1) to characterize the geographical distribution within specific host and vector populations in Africa; (2) to describe its phylogeographical patterns between Africa and Europe; (3) to define the genetic structure and epidemiology of Italian WNV strains, giving an insight of the viral circulation dynamics in the Italian territory. Methodology: Ecological and epidemiological studies were combined with molecular and phylogenetic analyses, carrying out field sampling activities, cellular culture, viral infection, immunofluorescent assay, multiplexed RT-PCR, sequencing, data analysis, and novel technique design. These activities were carried out both in Italy and in Senegal. Results: Our study evidences: (i) the circulation of several WNV lineages [Lineage 1 (L1), 2 (L2), 7 (L7), and 8 (L8)] in the African Continent; (ii) the presence of diverse competent mosquito vectors in Africa, mainly belonging to the Culex genus; (iii) the lack of vector competence studies for several other mosquito species found naturally infected with WNV in Africa; (iv) the need of more vector competence studies on ticks; (v) the circulation of WNV among humans, animals and vectors in at least 28 African countries; (vi) the lack of knowledge on the epidemiological situation of WNV for 19 African countries, and (vii) the importance of carrying out specific serological surveys in order to avoid possible bias on WNV circulation in Africa (objective 1). Furthermore, a new set of WNV L1 and L2 genome-specific primers for tiled-amplicon sequencing have been designed and a consistent dataset of 64 WNV L2 and 31 WNV L1 Italian genomes and of 3 WNV L2 and 7 WNV L1 Senegalese genome sequences from samples collected in Italy and Senegal between 2006 and 2022 has been produced. Twenty more WNV L1 and L2 Senegalese sequences obtained from samples collected in Senegal between 1985 and 2018 have been shared by the Institut Pasteur Dakar of Senegal and added to the dataset. This allowed the conduction of phylogenetic and phylogeographic analyses, evidencing: (viii) the presence of a strong viral connection between Africa and Europe, with intercontinental circulation supported by birds crossing international boundaries while migrating through the African-Eurasian flyways; (ix) the WNV L1 Western-Mediterranean cluster probable spread from Senegal, where the virus was first reported in 1979, to Italy, where the lineage first appeared in Europe in 1998, and to France in 2000, and the presence of back re-introductory events from Italy, Spain, and France to North and West Africa from the 2010s; and (x) the first African introduction of WNV L2 in Europe in Hungary in 2004, possibly from South African countries (objective 2). Our study also gives an insight of the dynamics of the viral circulation in Italy, demonstrating: (xi) the endemic presence of WNV L1 and L2 in part of Italy supported by resident wild birds and vector competent mosquitoes mainly belonging to the Culex genus; (xii) the current existence of two diverse WNV L1 strains circulating in Italy, one in the North-East, and one circulating intra-regionally in the Campania region; (xiii) suggested characteristic silent periods observed for WNV L1 in the country, with unnoticed circulation lasting sometimes for more than 10 years; (xiv) the 2022 WNV L1 increasing incidence of neurological disease cases in humans; (xv) the presence of genetically stable WNV L2 strains in Italy with continuous circulation throughout the time; (xvi) the presence of overwintering mechanisms supported by bird-to bird, rodent-to bird, or mosquito-to bird transmission routes; (xvii) the existence of WNV L1 and L2 co-infections in birds and mosquitoes; (xviii) the existence of a continuous transmission of the two strains between Western Mediterranean countries, supported by short distance migratory birds; and (xix) the crucial importance of the surveillance system other than the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases in Italy (objective 3). Conclusion: Our work points out the existence of high genetic diversity of WNV strains in Africa, the spread of L1 and L2 strains from Africa to Europe, and the existence of continuous transmission episodes among several Western-Mediterranean countries, with few recently suspected back introductory events from Europe to Africa. The progressive increase of the WNV L2 circulation both temporally and spatially in the Mediterranean countries and the WNV L1 re-appearance in Europe, both associated with a significant impact on humans and animal health, other than the strong WNV incidence in Italy and its endemization in part of its territory, evidence a solid WNV epidemic risk for Italy and a persistent threat for WNV spread into new areas. To predict and control future epidemics, it is crucial to constantly monitor the circulation and evolution of WNV in Europe and Africa, and to implement coordinated surveillance plans in both Continents, even in areas which are not currently affected.