The role of wild animals in the ecoepidemiology of flaviviruses in Northeastern Italy

West Nile virus (WNV) and Usutu virus (USUV), both belonging to the genus Flavivirus, are emerging worldwide as important human and animal pathogens. Migratory birds are involved in the spread of Flaviviruses over long distances, particularly from Africa to Europe. Once introduced, these viruses can be further be dispersed by short-distance migratory and resident bird species. The Region of Trentino-Alto Adige is located on the migratory route of many of the short- and long-distance migratory birds that cross the Alps. Until now, only a silent circulation of WNV and USUV within this territory has been confirmed by serological screening, whilst no cases of infected humans or animals have so far been reported. However, continuous spillover events of both viruses have been reported in neighbouring Regions. The aim of this study was to monitor the circulation of WNV and USUV in Trentino-Alto Adige, in order to detect if active virus shedding occurs in migratory birds captured during their seasonal movements and to evaluate the role that different bird species could play in the spreading of these viruses. All samples tested negative for Flaviviruses. The pattern of flavivirus infection in mosquitoes belonging to the genera Aedes and Culex collected during 2012 in Trentino and Veneto was assessed, using a generic reverse transcription nested-PCR. Virus isolation was attempted in C6/36 insect cell lines.We detected a wide distribution of Aedes flavivirus (AeFV) in Aedes albopictus. In Culex pipiens collected in Veneto, we detected a new sequence of an insect-specific flavivirus and one of Usutu virus. We detected AeFV in Cx. pipiens, for the first time to our knowledge, in both regions. Viral isolation in cell culture was successful for AeFV. AeFV sequences found in Veneto showed a high percentage of similarity to those detected in Trentino and to those previously reported in other areas of northern Italy. The principal vector for WNV and USUV in Europe is Cx. pipiens, with its two forms or biotypes pipiens and molestus, which feeds on a wide variety of vertebrate host species. Host feeding preferences for this species have been little studied in Europe. We estimated the host feeding preference for wild Cx. pipiens using molecular analysis to identify the origin of blood meals, and avian census to control host abundance variations. Additionally, we used host bird odour extracts to test experimentally mosquito preferences in laboratory settings. For the first time, in both regions, we demonstrate a clear feeding preference for the common blackbird (Turdus merula), both for wild collected specimens and in the lab, suggesting a potential important role for this species in the WNV epidemiology in Europe. In Veneto, a seasonal decrease in abundance of blackbirds is associated with increased feeding on Eurasian magpies (Pica pica), and this may be linked to seasonal emergence of WNV in humans. Other species, such as the house sparrow (Passer domesticus) appear to be selected by mosquitoes opportunistically in relation to its abundance. In Trentino, molecular determination of wild-caught mosquitoes in the field were identified as pipiens biotype (85.3%), molestus biotype (7.3%) and hybrids (7.3%) while mosquitoes used in laboratory experiments derived from a population characterized by a large prevalence of molestus biotype (67.5%). Our resuts suggested that the genetic, physiological and behavioral differences between Cx. pipiens biotypes do not affect feeding habits for birds and in particular for blackbird. Arboviruses, as flaviviruses, circulate in enzootic cycles among arthropod vectors and a number of animal species, which act as reservoirs. Each virus requires the contemporary occurrence of competent vertebrate reservoir hosts and mosquito species acting as vectors. Variables such as climate, habitat structure, and the relative abundance and behaviour of vectors and hosts all contribute to the complexity that characterises the dynamics of transmission of vector-borne pathogens The role played by biodiversity in the epidemiology of viral diseases is still debated because it can vary in any single ecoepidemiologic scenario. For these reasons I tried to investigate the influence of avian and mosquito community in Trentino and Veneto on the flavivirus ecoepidemiology by calculating three biodiversity indexes namely, Simpson’s Index, Shannon’s Index and Pielou’s Index, for data collected in 2011 and 2012.