The pattern of flavivirus infection in mosquitoes belonging to the genera Aedes and Culex collected in two regions of north-eastern Italy (Trentino and Veneto) was assessed. Mosquitoes were collected during 2012 and screened for flaviviruses using a generic reverse transcriptionnested-PCR targeted on a region of the non-structural NS5 gene. The phylogenetic analysis was performed on a fragment of~1000 bp. Virus isolation was attempted in C6/36 insect cell lines and the infected cell cultures were studied by electron microscopy. We detected a wide distribution of Aedes flavivirus (AeFV) in Aedes albopictus, with higher infection prevalence in Trentino than in Veneto. In Culex pipiens collected in Veneto, we detected a new sequence of an insect-specific flavivirus and one of Usutu virus. Interestingly, we detected AeFV in C. 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. Co-infections with different flaviviruses were not detected.
INTRODUCTIONThe genus Flavivirus (family Flaviviridae) comprises .70 viruses that, according to their mechanism of transmission, are included in one of the following three groups: (1) those infecting a range of vertebrate hosts through mosquito or tick bites, called 'arthropod-borne viruses', (2) those spread without a known vector, presumed to be limited to infecting vertebrates only, and (3) those apparently limited to insects alone, called 'insect-specific flaviviruses' (ISFs) (Ishikawa & Konishi, 2011;Huhtamo et al., 2012 and references therein). The flavivirus genome contains genes coding for three structural proteins (capsid, premembrane and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Lindenbach et al., 2006). Regions encoding envelope, NS3 and NS5 are the most frequently used for phylogenetic analysis of flaviviruses. The inclusion of ISFs in the genus Flavivirus is supported by similarities with other flaviviruses in terms of genomic organization, polyprotein hydropathy profiles and cleavage sites, but they are not able to replicate in mammalian cells and they have been isolated only in mosquito-derived cells (Kuno, 2007;Hoshino et al., 2009;Bolling et al., 2011;Cook et al., 2012;Haddow et al., 2013).The 'arthropod-borne viruses' group includes some important emerging human and animal pathogens, such 3These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession numbers for representative sequences obtained in this work are KM871198-KM871202. Lelli et al., 2008;Manarolla et al., 2010;Tamba et al., 2011; Vázquez et al., 2011;Cerutti et al., 2012;Ravanini et al., 2012;Buchebner et al., 2013;Calzolari et al., 2013a; Höfle et al., 2013). In Europe, USUV has been recognized as a candidate human pathogen in Austria (Weissenböck et al., 2007), Italy (Cavrini et al., 2009;Pecorari et al., ...