Identification of three novel genes in Phenuiviridae detected from Aedes mosquitoes in Hokkaido, Japan

Mosquitoes are an important arthropod vector of arboviruses. Family Phenuiviridae includes several medically important arboviruses, such as Rift Valley fever phlebovirus and Toscana phlebovirus. Recent comprehensive genetic analyses identified many novel mosquito-specific viruses that are phylogenetically related to Phenuiviridae. We collected mosquitoes in Hokkaido, northern Japan, and conducted reverse transcription polymerase chain reactions (RT-PCRs) targeting the RNA-dependent RNA polymerase (RdRp) gene of Phenuiviridae. A total of 285 pools comprising 3,082 mosquitoes from two genera and eight species were collected. Partial RdRp sequences were detected from 97 pools, which allowed us to classify the viruses into three clusters provisionally designated Etutanne virus (ETTV) 1, 2, and 3. The closest related virus was Narangue virus (family Phenuiviridae, genus Mobuvirus), which was detected from Mansonia mosquitoes, and the similarities of its nucleotide and amino acid sequences with those of the ETTVs were 58.4%-66.2% and 64.7%-86.7%, respectively. PCRs and RT-PCRs using DNA and RNase digestion methods showed that the ETTVs were RNA viruses, but they do not form non-retroviral integrated RNA virus sequences in the mosquito genome.