Identification of Three Novel Genes in Phenuiviridae Detected from Aedes Mosquitoes in Hokkaido, Japan.

Mosquitoes are important arthropod vectors of arboviruses. The family Phenuiviridae includes several medically important arboviruses, such as the Rift Valley fever phlebovirus and Toscana phlebovirus. Recent comprehensive genetic analyses have identified many novel mosquito-specific viruses that are phylogenetically related to Phenuiviridae. We collected mosquitoes from Hokkaido in 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 2 genera and 8 species, were collected. Partial RdRp sequences were detected in 97 pools, which allowed us to classify the viruses into 3 clusters provisionally designated as Etutanne virus (ETTV) 1, 2, and 3. The virus most closely related to ETTVs is Narangue virus (family Phenuiviridae, genus Mobuvirus), which was detected in Mansonia mosquitoes; the nucleotide and amino acid sequences of the Narangue virus are 58.4-66.2% and 64.7-86.7% similar, respectively, to those of ETTVs. PCR and RT-PCR using DNA and RNase digestion methods showed that the ETTVs are RNA viruses that do not form non-retroviral integrated RNA virus sequences in the mosquito genome.

Isolation and Characterization of Mosquito-Associated Spiroplasma cantharicola from Aedes japonicus Collected in Hokkaido, Japan.

Species of the genus Spiroplasma are common within arthropods and plants worldwide. Mosquito-associated Spiroplasma spp. have been reported to show pathogenicity toward mosquitoes, which serve as vectors of several infectious diseases that have detrimental effects on public health. Although Spiroplasma spp. are expected to have potential use as biological vector-control tools, characteristics such as their distribution, host species, and cytopathogenic effects (CPEs) are not well understood. In this study, we isolated a Spiroplasma sp. from a female Aedes japonicus collected in Hokkaido, northern Japan. Phylogenetic analysis based on the 16S rRNA gene sequence indicated our isolate was closely related to S. cantharicola. We screened 103 mosquito pools consisting of 3 genera and 9 species, but only detected S. cantharicola in the first isolation. In an in vitro assay, our isolate grew well at 28 °C, but no propagation was observed at 37 °C. Furthermore, the isolate showed strong CPE on a mosquito-derived cultured cell line (C6/36), and its propagation slightly increased when co-cultured with C6/36 cells. To our knowledge, this is the third report of the isolation of S. cantharicola from mosquitoes and the first case in Asia. Our findings provide epidemiological data on S. cantharicola distribution in the region.