A new virus of flavivirus：Chaoyang virus isolated in Liaoning province

Objective To identify a new virus of flavivirus by molecular biology,which was isolated from mosquitoes collected in Chaoyang of Liaoning province.Methods Mosquitoes were collected in Chaoyang of Liaoning province and the virus was isolated by cell culture.The isolated virus causing cytopathogenic effect (CPE) on C6/C36 cells was tested by reverse transcription polymerase chain reaction (RT-PCR)and the positive PCR-product was sequenced and analyzed.Coding sequence (CDS)complete genome of the virus was sequenced and compared with the sequences of 36 isolates from GenBank.Phylogenetic tree of CDS complete genome of flavivirus were constructed by neighbor-joining method in Mega 3.1 soft ware.Results Two viral strains were isolated from the mosquitoes and the results of RT-PCR were positive by using universal primers of flavivirus.The length of amplified segment was 986 bp,600-750 bp of which was similar to Dengue virus 2,Iguape virus,Sepik virus and Banzi virus and the nucleotide identities ranged between 66% and 68%.The nucleotide identities between two isolates were 100% and so they actually were one flavivirus.The length of the nucleotide sequence of CDS complete genome of the virus was 10 308 bp and identities of E gene,NS3 gene and NS5 gene were 59.6%,61.7% and 67.0% same,respectively,with the other flavivirus in GenBank.Phylogenetic analysis of CDS complete genome of flavivirus showed the isolate together with Dengue virus group,Japanese encephalitis virus group,Kokobera virus group,Ntaya virus group and Spondweni virus group belonged to one ancestor,but the isolate had its own branch in the tree.Conclusion The isolate from mosquitoes in Chaoyang of Liaoning province belongs to flavivirus,but it is not a member of any other group of flavivirus and it constructs a new group of flavivirus.The new virus is first reported in the world and named Chaoyang virus.The GenBank accession number is FJ883471.