Flaviviruses identified in mosquitoes collected in Veneto and Trentino Alto-Adige regions (North-East Italy)

The genus Flavivirus (family Flaviviridae) comprises more than 70 viruses, divided according to their ecological and epidemiological characteristics, and disease associations in three groups: 1) those infecting a range of vertebrate hosts through mosquito or tick bites, called “arthropod-borne viruses”, 2) those spread by an unknown vector, presumed to be limited to infect vertebrates only, and 3) apparently limited to insects alone, called “insect-specific flaviviruses” (“ISFs”). In the first group are present some important or emerging human pathogens, as West Nile virus (WNV) and Usutu virus (USUV). ISFs replicate only in mosquito-derived cells and the first of them to be discovered was Cell Fusing Agent virus, isolated in 1975 from a Aedes aegypti cell line. During the last decades, many others have been isolated and identified, in different geographic regions, from field collected mosquitoes belonging to different species. Other ISFs have been only detected through molecular methods but not isolated. Despite their non-pathogenicity to humans and animals, ISFs have recently gained attention with respect to their ecological and evolutionary relationships with other important flaviviruses. A particular focus of interest is the possible interaction of these viruses within the same vector that can leadto different results as “superinfection exclusion” or ehnanced transmission or replication. In Trentino the only evidence of WNV and USUV have been seroconversion in sentinel chickens in 2005, but ISFs have been detected since 2007. A completely different ecoepidemiological situation is present in Veneto since WNV and USUV have been detected in several studies during the last decades, but there is only one report regarding the detection of ISFs in this region. To gain a better understanding regarding the presence of flaviviruses in these two regions characterized by strong differences in environmenthal, meteoclimatic and ecoepidemiological conditions, we carried out a mosquito screening to detect flaviviruses. Mosquitoes were collected from May to October 2012, using 20 and 10 traps BG-Sentinel (Veneto and Trentino, respectively), located half in a rural and half in an urban environment, with BG-lure attractant and dry ice, checked weekly. Mosquitoes captured were killed at -80°C, identified to species level and pooled according to date, location, species and gender and stored at -80°C until molecular analysis. After RNA extraction, we used a generic RT-nested-PCR targeted on a region of the NS5 gene for the screening of flaviviruses. The phylogenetic analysis were realized on a fragment of 1000bp. For samples positive at the generic NS5 RT-nested-PCR, virus isolation was attempted in C6/36 cell lines (from Aedes albopictus mosquito). Fresh supernatants and cells from cell cultures with evident cytopathic effect, were used for electron microscopy studies. In Veneto we collected a total of 52096 female and 1190 male mosquitoes, belonging to Oc. geniculatus, Oc. caspius, Cx. pipiens, Cx. territans, Cx. modestus, Cs. annulata, An. plumbeus, An. maculipennis, Ae. cinereus/geminus, Ae. albopictus, Ae. vexans and Ae. koreicus species. We detected USUV in Cx. pipiens, and AeFV in Cx. pipiens and in Ae. albopictus. In another pool of Cx. pipiens we also found sequences of an ISF never reported before in literature that could be considered as a new insect-specific flavivirus. We successfully isolated in cell culture AeFV from one pool of Ae. albopictus, with evident cytopathic effect (CPE) (cell aggregation). Transmission electron microscopy performed on C6/36 cells infected by AeFV confirmed the presence of flaviviruses. In Trentino we collected a total of 1622 female and 464 male mosquitoes, belonging to Oc. geniculatus, Cx. pipiens, Cx. hortensis, An. plumbeus, An. maculipennis and Ae. albopictus. We detected only AeFV in pools of Ae. Albopictus, that was also successfully isolated in cell culture. In 1 pool we obtained evident CPE and by electronic microscopy we detected viral particles with the tipical morphological characteristics of Rhabdovirus. Our results confirm the different eco-epidemiological situation present in North-east Italy underlined by previous studies. They strongly support the importance of the influence of the climatic conditions on the transmission viral cycle. Moreover they suggest that a high prevalence of ISFs could damper replication and transmission of other more pathogenic viruses