Molecular characterization of flaviviruses from field-collected mosquitoes in northwestern Italy, 2011–2012

BACKGROUND: The genus Flavivirus comprises several mosquito-borne species, including the zoonotic pathogens West Nile and Usutu virus, circulating in animals and humans in Italy since 1998. Due to its ecological and geographical features, Piedmont is considered a risk area for flavivirus transmission. Here we report the results of a flavivirus survey (detection and genetic characterization) of mosquitoes collected in Piedmont in 2012 and the genetic characterization of three strains detected in 2011. METHODS: Pools of 1–203 mosquitoes, upon RNA extraction with TRIzol, were screened by a PCR assay for a 263 bp fragment of the Flavivirus NS5 gene. All positive samples were tested with a specific PCR for the E protein gene of Usutu virus and a generic Flavivirus RT-nested-PCR for a larger tract of the NS5 gene before sequencing. Phylogenetic trees were built with both NS5 fragments of representative Flavivirus species. DNA extracts of part of the positive pools were tested to detect sequences integrated in the host genome. RESULTS: Thirty-four mosquito pools resulted positive for flaviviruses, and twenty-five flavivirus sequences underwent phylogenetic analysis for the short NS5 fragment. Among the 19 sequences correlating with the insect-specific flavivirus group, ten samples, retrieved from Aedes albopictus, clustered within Aedes flavivirus, while the other nine aggregated in a separate clade composed of strains from various mosquito species (mainly Aedes vexans) from Piedmont and the Czech Republic. Six out of these nine also presented a DNA form of the sequence. The remaining sequences belonged to the mosquito-borne group: four, all from Culex pipiens, correlated to Italian Usutu virus strains, whereas two, from Ochlerotatus caspius, were highly similar to Marisma mosquito virus (MMV). CONCLUSIONS: Our findings confirm the circulation of Usutu virus and of the potentially zoonotic Marisma mosquito virus in Piedmont. This is the first detection of Aedes flavivirus in Piedmont. Finally, further evidence for the integration of Flavivirus nucleic acid into the host genome has been shown. These results underline the importance of continuing intense mosquito-based surveillance in Piedmont, supported by a mosquito control program in areas at high risk for human exposure.