Genetic characterization of a new insect flavivirus isolated from Culex pipiens mosquito in Japan.
ABSTRACT We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.
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ABSTRACT: Flavivirus is a highly heterogeneous viral genus that includes important human pathogens and several viral strains with unknown zoonotic potential. Mosquito-borne flaviviruses have been isolated and characterized in Northern Italy: West Nile virus and Usutu virus were detected in mosquitoes and in different host species and recent studies provided evidence about the circulation of "insect Flavivirus" strains. In order to clarify the diffusion and the distribution of the mosquito-transmitted flaviviruses in Italy, we analyzed Culex and Ochlerotatus mosquitoes collected in 2009 and 2010 in an area divided evenly between hills and plains and where the landscape is dominated by mixed agricultural patches, rice fields, deciduous tree forests, and urban environments. Each mosquito pool was tested for the presence of Flavivirus strains and we characterized positive samples by genetic sequencing. Positive mosquito pools revealed low infection prevalence, but suggested a continuous circulation of both Usutu virus and insect Flavivirus. Interestingly, phylogenetic analyses based on NS5 gene partial sequences showed a closer relationship among new Usutu virus strains from Piedmont and the reference sequences from the Eastern Europe, with respect to Italian samples characterized so far. Moreover, NS5 gene phylogeny suggested that mosquito flaviviruses found in Italy could belong to different lineages. Our results contribute to a wider point of view on the heterogeneity of viruses infecting mosquitoes suggesting a taxonomical revision of the Mosquito-borne Flavivirus group.Parasites & Vectors 05/2012; 5:99. · 2.94 Impact Factor
Article: Culex Flavivirus and West Nile Virus in Culex quinquefasciatus Populations in the Southeastern United States[show abstract] [hide abstract]
ABSTRACT: Little is known of the interactions between insect-only flaviviruses and other arboviruses in their mosquito hosts, or the potential public health significance of these associations. The specific aims of this study were to describe the geographic distribution, prevalence, and seasonal infection rates of Culex flavivirus (CxFV) and West Nile virus (WNV) in Culex quinquefasciatus Say in the Southeastern United States, investigate the potential association between CxFV and WNV prevalence in Cx. quinquefasciatus and describe the phylogenetic relationship among CxFV and WNV isolates from the Southeastern United States and around the world. Using ArboNET records, 11 locations were selected across Georgia, Mississippi, and Louisiana that represented a range of WNV human case incidence levels. Cx. quinquefasciatus were trapped weekly throughout the summer of 2009 and pools were screened for flavivirus RNA by reverse transcriptase polymerase chain reaction. Cx. quinquefasciatus from Georgia had significantly higher CxFV infection rates than either Mississippi or Louisiana. CxFV was not detected in Mississippi after July, and no CxFV was detected in Cx. quinquefasciatus in Louisiana. In Georgia, CxFV infection rates were variable between and within counties and over time. WNV infection rates were not significantly different across states or months, and WNV sequences from all three states were identical to each other in the envelope and NS5 gene regions. Phylogenetically, NS5 and E gene sequences from Georgia CxFV isolates clustered with CxFV from Japan, Iowa, and Texas. Multiple CxFV genetic variants were found circulating simultaneously in Georgia. No evidence was found supporting an association between WNV and CxFV infection prevalence in Cx. quinquefasciatus.Journal of Medical Entomology 01/2012; · 1.76 Impact Factor
Article: Entomological Surveillance for Flaviviruses at Migratory Bird Stopover Sites in Hokkaido, Japan, and a New Insect Flavivirus Detected in Aedes galloisi (Diptera: Culicidae)[show abstract] [hide abstract]
ABSTRACT: To investigate the possible spread of West Nile virus (WNV) into Japan, we carried out entomological surveillance for flaviviruses at migratory bird stopover sites in Hokkaido, Japan, during 2003–2006. A total of 3,826 mosquitoes, identified as 15 species in five genera, were collected and 2,465 of these were grouped into 123 pools that were assayed for cytopathic effects on mosquito and mammalian cell cultures and for flavivirus RNA by reverse transcription-polymerase chain reaction using flavivirus universal primer sets for fragments of the NS3 and NS5 genes. Neither WNV nor other mosquito-vertebrate transmitted flaviviruses were detected in mosquitoes collected at any of the sites in Hokkaido, but five Culex flaviviruses and one novel Aedes galloisi flavivirus were identified from Culex pipiens L. s. l. and Aedes galloisi Yamada, respectively. Genetic and phylogenetic analyses based on the partial NS5 nucleotide sequences classified Aedes galloisi flavivirus with the insect flavivirus, but distant from Cell fusing agent, Kamiti river virus, and Culex flaviviruses, showing <74% sequence identities. Polymerase chain reaction-based bloodmeal analysis of 79 females showed that all of the Aedes and Ochlerotatus mosquitoes fed on mammals (deer and humans), whereas, Cx. pipiens s. 1. mosquitoes fed on both of avian (ducks and sparrows, 85.7%) and mammalian hosts (dog, 14.3%). We suggest that to date WNV has not become established in Japan.Journal of Medical Entomology 01/2012; · 1.76 Impact Factor