Japanese encephalitis (JE) remains a public health concern in several countries, and the Culex mosquito plays a central role in its transmission cycle. Culex mosquitoes harbor a wide range of viruses, including insect-specific viruses (ISVs), and can transmit a variety of arthropod-borne viruses (arboviruses) that cause human and animal diseases. The current trend of studies displays enhanced efforts to characterize the mosquito virome through bulk RNA sequencing due to possible arbovirus–ISV interactions; however, the extent of viral diversity in the mosquito taxon is still poorly understood, particularly in some disease vectors. In this study, arboviral screening and RNA virome analysis of Culex tritaeniorhynchus and C. pseudovishnui, which are part of the Culex vishnui subgroup mosquitoes, were performed. Results from these two mosquito species, known as the major vectors of JE virus (JEV) in Asia, collected in three prefectures in Japan were also compared with the sympatric species C. inatomii. A total of 27 viruses, including JEV, were detected from these Culex mosquitoes. Molecular and phylogenetic analyses of the detected viruses classified 15 of the 27 viruses as novel species, notably belonging to the Flaviviridae, Rhabdoviridae, Totiviridae, and Iflaviridae families. The successful isolation of JEV genotype I confirmed its continuous presence in Japan, suggesting the need for periodic surveillance. Aside from JEV, this study has also reported the diversity of the RNA virome of disease vectors and broadened the knowledge on mosquito virome profiles containing both arbovirus and ISV. Mosquito taxon seemed to contribute largely to the virome structure (e.g., virome composition, diversity, and abundance) as opposed to the geographical location of the mosquito species. This study therefore offers notable insights into the ecology and evolution of each identified virus and viral family. To the authors’ knowledge, this is the first study to characterize the viromes of the major JE vectors in Japan.

Superinfection exclusion is generally defined as a phenomenon in which a pre-existing viral infection prevents a secondary viral infection; this has also been observed in infections with mosquito-borne viruses. In this study, we examined the superinfection exclusion of the vertebrate-infecting flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV), by stable and persistent infection with an insect-specific flavivirus, Culex flavivirus (CxFV), in a Culex tritaeniorhynchus Giles cell line (CTR cells). Our experimental system was designed based on the premise that wild Cx. tritaeniorhynchus mosquitoes naturally infected with CxFV are superinfected with JEV by feeding on JEV-infected animals. As a result, we found no evidence of the superinfection exclusion of both JEV and DENV by pre-existing CxFV infection at the cellular level. However, JEV superinfection induced severe cytopathic effects on persistently CxFV-infected CTR cells. These observations imply the possibility that JEV superinfection in CxFV-infected Cx. tritaeniorhynchus mosquitoes has an adverse effect on their fitness.

Among members of the order Mononegavirales , RNA splicing events have been found only in the family Bornaviridae . Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae , but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae .

Jingmen tick virus (JMTV) and the related jingmenvirus-termed Alongshan virus are recognized as globally emerging human pathogenic tick-borne viruses. These viruses have been detected in various mammals and invertebrates, although their natural transmission cycles remain unknown. JMTV and a novel jingmenvirus, tentatively named Takachi virus (TAKV), have now been identified during a surveillance of tick-borne viruses in Japan. JMTV was shown to be distributed across extensive areas of Japan and has been detected repeatedly at the same collection sites over several years, suggesting viral circulation in natural transmission cycles in these areas. Interestingly, these jingmenviruses may exist in a host tick species-specific manner. Vertical transmission of the virus in host ticks in nature was also indicated by the presence of JMTV in unfed host-questing Amblyomma testudinarium larvae. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan.