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Japanese encephalitis virus (JEV), a mosquito-borne virus, causes severe clinical symptoms in humans in the Asian-Pacific region, where it circulates in a primary transmission cycle among Culex tritaeniorhynchus mosquitoes, domestic swine (Sus scrofa domesticus), and wading birds. We report here an anomalous result that mosquito-borne JEV was detected in unfed host-questing ticks collected from the field in Japan. JEV genomic RNA was detected in four pools of Haemaphysalis flava nymphs collected in November and December 2019, and March 2020, when Cx. tritaeniorhynchus adults were not presumed to be active. Moreover, JEV antigenomic RNA was detected in some JEV-positive tick samples, suggesting virus replication in ticks. However, taken together with no infectious virus isolated, the possibility that the antigenomic RNA was derived from the undigested bloodmeal source in ticks cannot be ruled out. Thus, the role of the ticks as a natural reservoir for JEV remains to be confirmed.

Viruses belonging to the genus Quaranjavirus in the family Orthomyxoviridae are known as argasid tick-borne viruses. Some viruses in this genus or an unassigned quaranjavirus-like virus have the ability to infect humans although little is known about their pathogenicity. During the surveillance of tick-borne viruses in ixodid ticks in Ehime Prefecture, Japan, novel quaranjavirus-like sequences were detected in three pooled samples of Haemaphysalis histricis nymphs. Phylogenetic analysis revealed that the detected viruses formed a cluster with quaranjaviruses and other related viruses. Specifically, the viruses were closely related to Zambezi tick virus 1 and Uumaja virus, which are quaranjavirus-like viruses recently discovered in ixodid ticks in Africa and Europe, respectively. These findings indicate that the viruses detected in this study were probably a new member of the Quaranjavirus genus or a related group. The viruses were tentatively named Ohshima virus even though only limited sequences of their genome were available. This is the first report on the detection of a quaranjavirus-like virus in the East Asian region. Further investigations are needed to discern its infectivity and pathogenicity against humans or other animals and to determine the potential risk of an emerging tick-borne viral disease.

Ticks are important vector arthropods that transmit various pathogens to humans and other animals. Tick-borne viruses are of particular concern to public health as these are major agents of emerging and re-emerging infectious diseases. The Phenuiviridae family of tick-borne viruses is one of the most diverse groups and includes important human pathogenic viruses such as severe fever with thrombocytopenia syndrome virus. Phenuivirus-like sequences were detected during the surveillance of tick-borne viruses using RNA virome analysis from a pooled sample of Haemaphysalis formosensis ticks collected in Ehime, Japan. RT-PCR amplification and Sanger sequencing revealed the nearly complete viral genome sequence of all three segments. Comparisons of the viral amino acid sequences among phenuiviruses indicated that the detected virus shared 46%-70% sequence identity with known members of the Kaisodi group in the genus Uukuvirus. Furthermore, phylogenetic analysis of the viral proteins showed that the virus formed a cluster with the Kaisodi group viruses, suggesting that this was a novel virus, which was designated "Toyo virus" (TOYOV). Further investigation of TOYOV is needed, and it will contribute to understanding the natural history and the etiological importance of the Kaisodi group viruses.

Tabanid flies (Tabanidae: Diptera) are common hematophagous insects known to transmit some pathogens mechanically or biologically to animals; they are widely distributed throughout the world. However, no tabanid-borne viruses, except mechanically transmitted viruses, have been reported to date. In this study, we conducted RNA virome analysis of several human-biting tabanid species in Japan, to discover and characterize viruses associated with tabanids. A novel flavivirus was encountered during the study in the Japanese horse fly, Tabanus rufidens (Bigot, 1887). The virus was detected only in T. rufidens, but not in other tabanid species, and as such was designated Tabanus rufidens flavivirus (TrFV). TrFV could not be isolated using a mammalian cell line and showed a closer phylogenetic relationship to the classical insect-specific flaviviruses (cISFs) rather than the vertebrate-infecting flaviviruses (VIFs), suggesting that it is a novel member of the cISFs. The first discovery of a cISF from Brachycera provides new insight into the evolutionary history and dynamics of flaviviruses.

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.

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.

Japanese encephalitis virus (JEV) is transmitted between swine, migratory birds, and Culex mosquitoes, and has circulated indigenously in Asia for almost a century. Despite being the country with the highest JEV diversity, surveillance targeting of Indonesia’s vectors is scarce. This study collected mosquitoes from several locations in Tabanan Regency, Bali Island, Indonesia. We captured and classified 3,032 adult Culex mosquitoes into seven species, with Culex vishnui subgroup mosquitoes making up approximately 90% of the total. Japanese encephalitis virus was identified by next-generation sequencing (NGS) analysis of a Cx. vishnui mosquito pool. Genetic and phylogenetic analysis revealed the JEV as genotype (G) IV. The nucleotide identity was 99% with other JEV GIV isolates obtained from swine sera in 2017 on Bali Island and from a human patient in Australia with a travel history to Bali in 2019. This finding indicated that JEV GIV persists in restricted areas and is circulating between swine-mosquito vectors.

Background Dengue virus (DENV) is a mosquito-borne arbovirus transmitted by Aedes mosquitoes, but is not endemic in all areas where this vector is found. For example, the relatively sparse distribution of cases in West Africa is generally attributed to the refractory nature of West African Aedes aegypti (Ae. aegypti) to DENV infection, and particularly the forest-dwelling Ae. aegypti formosus. However, recent studies have shown these mosquitoes to be competent vectors within some West African countries that have suffered outbreaks in the past, such as Senegal. There is however little information on the vector competence of the Ae. aegypti in West African countries such as Ghana with no reported outbreaks. Methods This study examined the vector competence of 4 Ae. aegypti colonies from urban, semi-urban, and two rural locations in Ghana in transmitting DENV serotypes 1 and 2, using a single colony from Vietnam as control. Midgut infection and virus dissemination were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while the presence and concentration of DENV in the saliva of infectious mosquitoes was determined by the focus forming assay. Results There were significant differences in the colonies’ susceptibility to virus infection, dissemination, and transmission. All examined Ghanaian mosquitoes were refractory to infection by DENV serotype 2, while some colonies exhibited potential to transmit DENV serotype 1. None of the tested colonies were as competent as the control group colony. Conclusions These findings give insight into the possible risk of outbreaks, particularly in the urban areas in the south of Ghana, and highlight the need for continuous surveillance to determine the transmission status and outbreak risk. This study also highlights the need to prevent importation of different DENV strains and potential invasion of new highly vector-competent Ae. aegypti strains, particularly around the ports of entry. Graphical Abstract

In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male Aedes albopictus mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2–66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, Ae. albopictus was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no Aedes aegypti mosquitoes are distributed.

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.

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.

Introduction Aedes albopictus (Skuse) is an important vector of arboviral diseases, including dengue, chikungunya and Zika virus disease. Monitoring insecticide resistance and mechanisms by which the mosquito develops resistance is crucial to minimise disease transmission. Aim To determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions. Methods We sampled 33 populations of Ae. albopictus from Asia, Europe and South America, and tested these for susceptibility to permethrin, a pyrethroid insecticide. In resistant populations, the target site for pyrethroids, a voltage-sensitive sodium channel (Vssc) was genotyped. Three resistant sub-strains, each harbouring a resistance allele homozygously, were established and susceptibilities to three different pyrethroids (with and without a cytochrome P450 inhibitor) were assayed. Results Most populations of Ae. albopictus tested were highly susceptible to permethrin but a few from Italy and Vietnam (4/33), exhibited high-level resistance. Genotyping studies detected a knockdown resistance (kdr) allele V1016G in Vssc for the first time in Ae. albopictus. Two previously reported kdr alleles, F1534C and F1534S, were also detected. The bioassays indicated that the strain homozygous for the V1016G allele showed much greater levels of pyrethroid resistance than other strains harbouring F1534C or F1534S. Conclusion The V1016G allele was detected in bothAsian and Italian Ae. albopictus populations, thus a spread of this allele beyond Italy in Europe cannot be ruled out. This study emphasises the necessity to frequently and regularly monitor the V1016G allele in Ae. albopictus, particularly where this mosquito species is the main vector of arboviruses.

There are three main innate immune mechanisms against viruses in mosquitoes. Infection with the flavivirus dengue virus is controlled by RNA interference (RNAi) and the JAK-STAT and Toll signaling pathways. This study showed that another flavivirus, Japanese encephalitis virus (JEV), did not invade the salivary glands of Aedes aegypti and that this may be a result of the innate immune resistance to the virus. Argonaute 2 (Ago2) plays a critical role in the RNAi pathway. To understand the mechanism of JEV resistance, we focused on Ago2 as a possible target of JEV. Here, we show that the expression of MyD88 (a mediator of Toll signaling) and Ago2 mRNAs was induced by JEV in the salivary glands of Ae. aegypti mosquitoes and that Ago2, JAK, and domeless (DOME) mRNAs were induced by JEV in the bodies of Ae. aegypti mosquitoes. Double-stranded (ds) Ago2 RNA enhanced JEV infection, and the virus was detected in salivary glands by immunofluorescence assay. In contrast, MyD88 dsRNA had no effect on JEV infection. These data suggest that Ago2 plays a crucial role in mediating the innate immune response of Ae. aegypti to JEV in a manner similar to that employed by dengue virus.

Viruses are highly diverse and are the sole agents that can infect organisms in all domains of life. Viruses are defined as capsid-encoding organisms as opposed to ribosome-encoding cellular organisms. However, recent advances in virology indicate the existence of unique viruses that do not meet this basic definition, such as capsidless viruses. During virome analysis of the soft tick Argas japonicus, we identified virus-like sequences closely related to the members of genus Nyavirus (family Nyamiviridae). Further analysis revealed sequences derived from a novel nyavirus that lacks two structural protein genes, matrix (M) and glycoprotein (G). This unique nyavirus is tentatively named Sekira virus (SEKRV). To our knowledge, this is the first study to report a nyavirus deficient in M and G genes in nature. The mechanism of infection, replication, and persistence of SEKRV remain unknown, yet this finding provides new insight into virus evolution and the diverse way of viral life in nature.

Non-enveloped icosahedral double-stranded RNA (dsRNA) viruses possess multifunctional capsids required for their proliferation. Whereas protozoan/fungal dsRNA viruses have a relatively simple capsid structure, which suffices for the intracellular phase in their life cycle, metazoan dsRNA viruses have acquired additional structural features as an adaptation for extracellular cell-to-cell transmission in multicellular hosts. Here, we present the first atomic model of a metazoan dsRNA totivirus-like virus and the structure reveals three unique structural traits: a C-terminal interlocking arm, surface projecting loops, and an obstruction at the pore on the 5-fold symmetry axis. These traits are keys to understanding the capsid functions of metazoan dsRNA viruses, such as particle stability and formation, cell entry, and endogenous intraparticle transcription of mRNA. On the basis of molecular dynamics simulations of the obstructed pore, we propose a possible mechanism of intraparticle transcription in totivirus-like viruses, which dynamically switches between open and closed states of the pore(s).

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&ndash, 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&rsquo, knowledge, this is the first study to characterize the viromes of the major JE vectors in Japan.

In the growth kinetics analysis of flaviviruses in Aedes albopictus C6/36 cell lines obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank and the European Collection of Authenticated Cell Culture (ECACC), these two cells line showed different viral susceptibility for Zika virus (ZIKV), Dengue virus (DENV), and Japanese encephalitis virus (JEV). Next-generation sequencing (NGS) analysis revealed that the C6/36 JCRB strain was persistently infected with two viruses without showing any cytopathic effects. The complete sequence analysis demonstrated that the one virus was Menghai rhabdovirus (MERV), which has been found from Aedes albopictus mosquito. The other virus was a novel virus, designated as Shinobi tetravirus (SHTV). Interestingly, the viral susceptibility of these two strains was almost even for Sindbis virus and Getah virus. We cloned SHTV and MERV from JCRB C6/36 cell line and then re-infected them into another C6/36 cell line, resulting in the reproduction of persistent infection with each virus. ZIKV growth was suppressed in SHTV and/or MERV re-infected C6/36 cells also. To our knowledge, this is the first demonstration that persistent infection with rhabdovirus and/or permutotetravirus suppressed flavivirus replication in mosquito cells.

The genus Thogotovirus, as represented by Thogoto virus and Dhori virus, comprises a group of arthropod-borne viruses, most members of which are transmitted by ticks. Here we report the genetic and biological characterization of a new thogotovirus, designated Oz virus (OZV), isolated from the hard tick Amblyomma testudinarium in Ehime, Japan. OZV efficiently replicated and induced a cytopathic effect in Vero cells, from which enveloped pleomorphic virus particles were formed by budding. OZV could also replicate in BHK-21 and DH82 cells and caused high mortality in suckling mice after intracerebral inoculation. Phylogenetic analyses of six viral proteins indicated that OZV is clustered with Dhori and related viruses, and is most closely related in glycoprotein (GP) and matrix protein (M) sequences to Bourbon virus, a human-pathogenic thogotovirus discovered recently in the United States. Our findings emphasize the need for understanding the geographic distribution and ecology of OZV and related viruses and for reevaluation of the medical and public health importance of thogotoviruses.

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1741-4) contains supplementary material, which is available to authorized users.

Amino acid position 123 in the E protein of Japanese encephalitis virus (JEV) determines viral growth properties and pathogenicity. The majority of JEV strains have a serine residue at this position (E123S); however, JEV with an asparagine residue (E123N) has also been isolated. To compare the growth properties and pathogenicity of E123S and E123N JEV, we produced recombinant JEV with a serine-to-asparagine substitution at position 123 (rJEV-Mie41-ES123N) in the E123S-type strain Mie/41/2002 background. The growth rate of rJEV-Mie41-ES123N was similar to that of Mie/41/2002 in mammalian and mosquito cell lines. Mouse challenge experiments showed that there was only a slight difference in neuroinvasiveness between the parent strain (Mie/41/2002) and rJEV-Mie41-ES123N. Thus, our results indicate that the Ser-to-Asn substitution in the JEV E protein has weak impact on viral growth properties in vitro or on pathogenicity in vivo.

Dengue virus (DENV) isolation from mosquitoes is necessary for providing definitive evidence of virus circulation, and is critical for further virological characterization and determination of epidemiological characteristics. By using Aedes albopictus mosquitoes captured during an outbreak in Tokyo in 2014, we compared the DENV isolation rates of a conventional virus isolation method that uses C6/36 mosquito cells as assay cells with those of a virus isolation method that relies on an antibody-dependent enhancement (ADE) mechanism by using FcγR-expressing baby hamster kidney (BHK) cells and an antibody with ADE activity. The number of DENV genome copies and infectious virus titers in cell culture supernatant fluids of FcγR-expressing BHK cells were significantly higher than those of the C6/36 cells. In addition, DENV was isolated from a mosquito pool by using FcγR-expressing BHK cells only in the presence of infection-enhancing antibody. Infectious virus was detected in six mosquito pools only by using FcγR-expressing BHK cells. The results suggest that the method that relies on ADE mechanism by using the FcγR-expressing BHK cells and an antibody with ADE activity is useful for DENV isolation from mosquitoes caught in the field.

We isolated two distinct viruses from mosquitoes collected in Bustos, Bulacan province, Philippines, in 2009. These viruses show rapid replication and strong cytopathic effects in mosquito C6/36 cells. Whole-genome analysis of these viruses demonstrated that both viruses belong to the negevirus group. One of the viruses, from Culex vishunui mosquitoes, is a new strain of Negev virus. The other virus, from a Mansonia sp. mosquito, is a new negevirus designated Bustos virus. Gene expression analysis of the Bustos virus revealed that infected cells contain viral subgenomic RNAs that probably include open reading frame (ORF) 2 or ORF3. Purified Bustos virus particles contained at least three proteins, and the major component (a probable major capsid protein) is encoded by ORF3. Bustos virus did not show infectivity in mammalian BHK-21 cells, suggesting that it is an insect-specific virus, like other known negeviruses.

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.

Culex flavivirus (CxFV) is an insect-specific flavivirus that has recently been detected in various Culex spp. mosquitoes worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of a Tokyo strain, CxFV-NIID21. The full-length CxFV-NIID21 cDNA was cloned into the low-copy-number plasmid pMW119, which was stably amplified in Escherichia coli. Transfection of a mosquito cell line with in vitro-transcribed RNA from the cDNA clone resulted in the production of recombinant progeny virus with growth properties, cytopathogenicity, and virion morphology similar to the parental virus.

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 .

Summary A German traveler developed dengue fever in late August 2013, following a direct flight from Germany. Autochthonous dengue virus (DENV) infection has not been reported in Japan. To evaluate the risk of autochthonous DENV transmission in Japan, the authors performed a retrospective search of the five areas visited by the German patient to determine the population density of dengue vector mosquito, Aedes albopictus . The annual mean temperature of each area was higher than 12°C, which is considered suitable for the establishment of A. albopictus populations. Our retrospective search revealed the population density of A. albopictus to be high in the urban areas of Japan.

To evaluate the vectorial capacity of mosquitoes for viruses in Japan, the host-feeding habits of the mosquitoes were analyzed by sequencing polymerase chain reaction-amplified fragments of the cytochrome b and 16S ribosomal RNA regions of the mitochondrial DNA of 516 mosquitoes of 15 species from seven genera that were collected from residential areas during 2003-2006. Culex pipiens L. and Aedes albopictus Skuse were the most commonly collected species in urban and suburban residential areas. Anautogenous Culex pipiens pallens Coquillett was distinguished from the autogenous Cx. pipiens form molestus Forskal using a polymerase chain reaction-based identification method. Both Cx. p. pallens and Cx. p. form molestus exhibited similar host-feeding habits, broadly preferring avian (50.0 and 42.5% of avian, respectively) and mammalian (38.6 and 45.0% of avian, respectively) hosts, such as tree sparrows, ducks, and humans. Conversely, Ae. albopictus exhibited a highly mammalophilic and anthropophilic feeding pattern, with 84.2% feeding on mammalian hosts and 68.5% of these on humans. We concluded that in Japan, Cx. pipiens might play a significant role in the avian-to-mammal transmission of viruses, such as West Nile virus, whereas Ae. albopictus might play a role in the human-human transmission of dengue and Chikungunya viruses.

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.

In a previous study, the highly pathogenic avian influenza (HPAI) H5N1 viruses were isolated from blow flies collected at the Tamba Town of Kyoto prefecture during the outbreak period in March 2004. In this study, we carried out virus exposure experiments to investigate whether the H5N1 virus would survive in a blow fly, Calliphora nigribarbis. The virus exposure experiments showed that the H5N1 influenza virus was isolated from the crop and intestine of C. nigribarbis for at least 24 h, and the viruses remained viable with titers ranging from 0.5 to 4.63 TCID50. This result suggests that C. nigribarbis could possibly transport the H5N1 virus over a distance of 2 km, which is the distance they can migrate within 24 h.

Among the tick-borne orbiviruses (genus Orbivirus, family Reoviridae), 36 serotypes are currently classified within a single virus species, Great Island virus. In this study, we report the first characterization of a tick-borne orbivirus isolated from the tick Ixodes turdus in Japan, which we identified as a new member of the species Great Island virus. The virus isolate, designated Muko virus (MUV), replicated and induced cytopathic effects in BHK-21, Vero E6, and CCL-141 cells and caused high mortality in suckling mice after intracerebral inoculation. Full genome sequence analysis showed that MUV shared the greatest phylogenetic similarity with Tribec virus in terms of the amino acid sequences of all viral proteins except for outer capsid protein 1 (OC1; VP4 of MUV). Analysis of genome segment 9 in MUV detected an uninterrupted open reading frame that overlaps with VP6 (Hel), which putatively encodes a molecular and functional equivalent of NS4 from Great Island virus. Our study provides new insights into the geographic distribution, genetic diversity, and evolutionary history of the members of the species Great Island virus.

A total of 160 autochthonous dengue cases transmitted by Aedes albopictus were reported between August and October of 2014 in Tokyo Metropolis, Japan. Ae. albopictus is a medically important vector of dengue virus, which has expanded its geographic distribution in temperate regions. Understanding the distribution and biting density of Ae. albopictus during the 2014 dengue outbreak in Tokyo is important to evaluate the epidemic risks of dengue fever in other highly populated cities in Europe and Asia. Of the 160 patients, 134 visited the same park (Yoyogi Park) located in central Tokyo. Mosquitoes infected with dengue virus were collected from this park, suggesting that it was the exclusive location for the transmission of dengue. This study aimed to collect referential data to estimate the transmission threshold of dengue virus in terms of biting density of Ae. albopictus and determined high transmission risk areas of dengue virus in Yoyogi Park and its vicinity. The overall mean density of biting Ae. albopictus (7.13/man/8 min) was sufficiently high for successful transmission of dengue virus, and areas with biting densities higher than the overall mean density were classified as high risk areas for the transmission of dengue virus in Yoyogi Park.

Japanese encephalitis virus (JEV) infection in mosquitoes was monitored in Vietnam from 2006 to 2008. A total of 15,225 mosquitoes, identified as 26 species in five genera were collected and 12,621 were grouped into 447 pools for examination of JEV infection by assays for cytopathic effects in C6/36 cells and by RT-PCR to detect flavivirus RNA. Three JEV strains were isolated from Culex tritaeniorhynchus Giles collected in northern and southern Vietnam and two JEV strains were isolated from Culex vishnui Theobald collected in the highlands of Vietnam. Genetic and phylogenetic analyses, based on complete E gene nucleotide sequences, revealed that the five JEV strains were classified into the genotype I group and six amino acid differences were found in these five strains. These results indicated that multiple JEV genotype I populations are circulating countrywide in Vietnam, transmitted by bites of their Cx. tritaeniorhynchus and Cx. vishnui.

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.

SUMMARYSerum specimens were collected from 25 wild boars in Hiroshima prefecture located in the western region of Japan from November 2004 to February 2005. The sera were tested for antibodies to Japanese encephalitis virus (JEV) by IgM capture and IgG enzyme-linked immunosorbent assays (ELISA), and plaque reduction neutralization test. Seventeen samples (68%) were positive for neutralizing antibody to JEV. All the neutralizing antibody-positive samples were positive for IgG-ELISA. One was also positive for IgM. The results indicate that approximately 70% of the wild boars were positive for anti-JEV antibody, and raises the possibility that wild boars may play a role in the infectious cycle of JEV in this region.

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.