Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus.
ABSTRACT We have described in the accompanying paper by Sang, et al., (, Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5' and 3' non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.
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ABSTRACT: 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.Parasites & Vectors 08/2014; 7(August 2014):395. · 3.25 Impact Factor
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ABSTRACT: Mosquitoes are highly effective vectors for transmission of human and animal pathogens. Understanding the relationship between pathogen and vector is vital in developing strategies to predict and prevent transmission. Cell lines derived from appropriate mosquito hosts provide a relatively simple tool for investigating the interaction between the host and viruses transmitted by mosquitoes. This review provides a brief overview of the development of mosquito cell lines, methods of isolation, their availability and application for investigating insect-virus interactions.Parasites & Vectors 08/2014; 7(1):382. · 3.25 Impact Factor
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ABSTRACT: Most known flaviviruses, including West Nile virus (WNV), are maintained in natural transmission cycles between hematophagous arthropods and vertebrate hosts. Other flaviviruses such as Modoc virus (MODV) and Culex flavivirus (CxFV) have host ranges restricted to vertebrates and insects, respectively. The genetic elements that modulate the differential host ranges and transmission cycles of these viruses have not been identified.Virology journal. 08/2014; 11(1):150.