We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.

In this study, we isolated and characterized an insect nidovirus from the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae) in Vietnam, as an additional member of the new family Mesoniviridae in the order Nidovirales. The virus, designated "Dak Nong virus (DKNV)," shared many characteristics with Cavally virus and Nam Dinh virus, which have also been discovered recently in mosquitoes, and these viruses should be considered members of a single virus species, Alphamesonivirus 1. DKNV grew in cultured mosquito cells but could not replicate in the cultured vertebrate cells tested. N-terminal sequencing of the DKNV structural proteins revealed two posttranslational cleavage sites in the spike glycoprotein precursor. DKNV is assumed to be a new member of the species Alphamesonivirus 1, and the current study provides further understanding of viruses belonging to the new family Mesoniviridae.

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.

Two infectious agents were isolated from Culex species mosquitoes in Japan and were identified as distinct strains of a new RNA virus by a method for sequence-independent amplification of viral nucleic acids. The virus designated Omono River virus (OMRV) replicated in mosquito cells in which it produced a severe cytopathic effect. Icosahedral virus particles of approximately 40 nm in diameter were detected in the cytoplasm of infected cells. The OMRV genome was observed to consist of a nonsegmented, 7.6-kb double-stranded RNA (dsRNA) and contain two overlapping open reading frames (ORFs), namely ORF1 and ORF2. ORF1 was found to encode a putative dsRNA-binding protein, a major capsid protein, and other putative proteins, which might be generated by co- and/or post-translational processing of the ORF1 polyprotein precursor, and ORF2 was found to encode a putative RNA-dependent RNA polymerase (RdRp), which could be translated as a fusion with the ORF1 product by a -1 ribosomal frameshift. Phylogenetic analysis based on RdRp revealed that OMRV is closely related to penaeid shrimp infectious myonecrosis virus and Drosophila totivirus, which are tentatively assigned to the family Totiviridae. These results indicated that OMRV is a new member of the family of nonsegmented dsRNA viruses infecting arthropod hosts, but not fungal or protozoan hosts.

The Culex vishnui subgroups, particularly Culex tritaeniorhynchus, are considered the primary vectors of the Japanese encephalitis virus (JEV) in Asia. Recent molecular phylogenetic analyses of JEV isolates from Asian countries have shown that JEVs with diverse genetic variants are present in Asia. Furthermore, some JEV strains have been found to have crossed the East China Sea and been introduced into Japan. In this study, the possibility of overseas migration of the JE vector mosquito, Cx. tritaeniorhynchus was examined from the genetic, physical, and meteorological perspectives. Molecular phylogenetic analysis was performed based on both whole coding sequences and on the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) gene of Cx. vishnui subgroups collected from Asian countries. Culex tritaeniorhymchus was classified into two genetically independent taxa by COI sequences: the Japanese type (Ct-J), which inhabits Japan except for the Amami Islands of southern Japan, and the continental type (Ct-C), which inhabits the Asian region except for Japan. It was confirmed that approximately 10% of Cx. tritaeniorhynchus trapped during the summer in western Kyushu were Ct-C, and that they could fly for up to 38 h continuously. The meteorological analysis also confirmed that the atmospheric flow occurring over the continent coincided with the date of Ct-C capture. This is the first report showing the existence of two taxa in Cx. tritaeniorhynchus. Their physical and physiological characteristics suggest the possibility of long-distance migration from overseas regions to Japan across the East China Sea. Future efforts are expected to provide evidence to support the occurrence of long-distance migration of Cx. tritaeniorhynchus with JEV.

Japanese encephalitis virus (JEV) is maintained in an enzootic cycle between swine, water birds, and mosquitoes. JEV has circulated indigenously in Asia, with Culex tritaeniorhynchus as the primary vector. In some areas where the primary vector is scarce or absent, sporadic cases of Japanese encephalitis have been reported, with Aedes japonicus japonicus presumed to have the potential as a secondary vector. As one of the world's most invasive culicid species, Ae. j. japonicus carries a considerable health risk for spreading diseases to wider areas, including Europe and North America. Thus, evaluation of its competency as a JEV vector, particularly in a native population, will be essential in preventing potential disease spread. In this study, the two mosquito species' vector competence in transmitting three JEV genotypes (I, III, and V) was assessed, with Cx. tritaeniorhynchus serving as a point of reference. The mosquitoes were virus-fed and the infection rate (IR), dissemination rate (DR), and transmission rate (TR) evaluated individually by either RT-qPCR or focus forming assay. Results showed striking differences between the two species, with IR of 95% (261/274) and 9% (16/177) in Cx. tritaeniorhynchus and Ae. j. japonicus, respectively. Both mosquitoes were susceptible to all three JEV genotypes with significant differences in IR and mean viral titer. Results confirm the primary vector's competence, but the fact that JEV was able to establish in Ae. j. japonicus is of public health significance, and with 2%-16% transmission rate it has the potential to successfully transmit JEV to the next host. This may explain the human cases and infrequent detection in primary vector-free areas. Importantly, Ae. j. japonicus could be a relevant vector spreading the disease into new areas, indicating the need for security measures in areas where the mosquito is distributed or where it may be introduced.

Mosquitoes transmit many kinds of arboviruses (arthropod-borne viruses), and numerous arboviral diseases have become serious problems in Indonesia. In this study, we conducted surveillance of mosquito-borne viruses at several sites in Indonesia during 2016-2018 for risk assessment of arbovirus infection and analysis of virus biodiversity in mosquito populations. We collected 10,015 mosquitoes comprising at least 11 species from 4 genera. Major collected mosquito species were Culex quinquefasciatus, Aedes albopictus, Culex tritaeniorhynchus, Aedes aegypti, and Armigeres subalbatus. The collected mosquitoes were divided into 285 pools and used for virus isolation using two mammalian cell lines, Vero and BHK-21, and one mosquito cell line, C6/36. Seventy-two pools showed clear cytopathic effects only in C6/36 cells. Using RT-PCR and next-generation sequencing approaches, these isolates were identified as insect flaviviruses (family Flaviviridae, genus Flavivirus), Banna virus (family Reoviridae, genus Seadornavirus), new permutotetravirus (designed as Bogor virus) (family Permutotetraviridae, genus Alphapermutotetravirus), and alphamesoniviruses 2 and 3 (family Mesoniviridae, genus Alphamesonivirus). We believed that this large surveillance of mosquitoes and mosquito-borne viruses provides basic information for the prevention and control of emerging and re-emerging arboviral diseases.

Entomological surveillance is one of the tools used in monitoring and controlling vector-borne diseases. However, the use of entomological surveillance for arboviral infection vector control is often dependent on finding infected individuals. Although this method may suffice in highly endemic areas, it is not as effective in controlling the spread of diseases in low endemic and non-endemic areas. In this study, we examined the efficiency of using entomological markers to assess the status and risk of arbovirus infection in Ghana, which is considered a non-endemic country, by combining mosquito surveillance with virus isolation and detection. This study reports the presence of cryptic species of mosquitoes in Ghana, demonstrating the need to combine morphological identification and molecular techniques in mosquito surveillance. Furthermore, although no medically important viruses were detected, the importance of insect-specific viruses in understanding virus evolution and arbovirus transmission is discussed. This study reports the first mutualistic relationship between dengue virus and the double-stranded RNA Aedes aegypti totivirus. Finally, this study discusses the complexity of the virome of Aedes and Culex mosquitoes and its implication for arbovirus transmission.

We isolated a new flavivirus from Aedes albopictus mosquito and a related species in Japan. The virus, designated Aedes flavivirus (AEFV), only replicated in a mosquito cell line and produced a mild cytopathic effect. The AEFV genome was positive-sense, single-stranded RNA, 11,064 nucleotides in length and contained a single open reading frame encoding a polyprotein of 3341 amino acids with 5' and 3' untranslated regions (UTRs) of 96 and 945 nucleotides, respectively. Genetic and phylogenetic analyses classified AEFV with the insect flavivirus, but distinct from Cell fusing agent (CFA), Kamiti river virus and Culex flavivirus. Interestingly, a partial sequence of AEFV showed significant similarity to that of Cell silent agent (CSA), the insect flavivirus-related nucleotide sequence integrated in the genome of A. albopictus. These results suggest that AEFV is a new member of the insect flaviviruses, which are intimately associated with Aedes mosquitoes and may share a common origin with CSA.

Omono River virus (OmRV) is a double-stranded RNA virus isolated from Culex mosquitos, and it belongs to a group of unassigned insect viruses that appear to be related to Totiviridae. This paper describes electron cryo-microscopy (cryoEM) structures for the intact OmRV virion to 8.9 Å resolution and the structure of the empty virus-like-particle, that lacks RNA, to 8.3 Å resolution. The icosahedral capsid contains 120-subunits and resembles another closely related arthropod-borne totivirus-like virus, the infectious myonecrosis virus (IMNV) from shrimps. Both viruses have an elevated plateau around their icosahedral 5-fold axes, surrounded by a deep canyon. Sequence and structural analysis suggests that this plateau region is mainly composed of the extended C-terminal region of the capsid proteins. In contrast to IMNV, the infectious form of OmRV lacks extensive fibre complexes at its 5-fold axes as directly confirmed by a contrast-enhancement technique, using Zernike phase-contrast cryo-EM. Instead, these fibre complexes are replaced by a short "plug" structure at the five-fold axes of OmRV. OmRV and IMNV have acquired an extracellular phase, and the structures at the five-fold axes may be significant in adaptation to cell-to-cell transmission in metazoan hosts.

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline-MoNaS (Mosquito Na+ channel mutation Search)-which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.