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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.

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes -borne infectious diseases.

Several outbreaks of trench fever caused by Bartonella quintana, occurred in soldiers during World Wars I and II. Although the number of trench fever cases has been decreasing worldwide, the disease has been reported among the homeless population in both developing and developed countries. The current prevalence of B. quintana infection in Japan is unclear. We collected blood and body louse (Pediculus humanus humanus) samples from homeless inpatients who had body lice at the time of emergency hospitalization in Tokyo from January 2013 to March 2015. Patients were tested for B. quintana infections using culture method, polymerase chain reaction, and indirect immunofluorescence assay (IFA). Among the 29 patients tested, the presence of Bartonella spp. was confirmed by genomic sequencing of DNA extracted from the samples from 2 patients (blood culture performed for 13 out of 15 patients), and from body louse samples of 20 patients (69%). Immunoglobulin G against B. quintana was detected in 10 patients (34.5%) at a cut-off titer of 1:256 in IFA. B. quintana infection was detected in samples obtained between 2013 and 2015 in Tokyo and needs to be on the list of differential diagnoses performed for febrile homeless individuals.

BACKGROUND Aedes aegypti is a remarkably effective mosquito vector of epidemiologically important arboviral diseases including dengue fever, yellow fever and Zika. The present spread of resistance against pyrethroids, the primary insecticides used for mosquito control, in global populations of this species is of great concern. The voltage‐gated sodium channel (VGSC) in the nervous system is the known target site of pyrethroids in insects. Past studies have revealed several amino‐acid substitutions in this channel that confer pyrethroid resistance, which are known as knockdown resistance (kdr) mutations. RESULTS This study investigated a laboratory colony of Ae. aegypti, MCNaeg, established from larvae collected in Rio de Janeiro, Brazil in 2016. The MCNaeg colony showed strong resistance against pyrethroids without laboratory selection. Of the two VGSC gene haplotypes present within this colony, one harbored three known kdr mutations, V410L, V1016I, and F1534C, and the other harbored only the known F1534C mutation. In latter haplotype, we also found novel amino‐acid substations including V253F. Previous molecular modeling and electrophysiological studies suggest that this residue serves a pyrethroid‐sensing site in the second receptor, PyR2. Our genetical analysis showed that the haplotype harboring V253F and F1534C is associated with equal or slightly stronger resistance than the other triple kdr haplotype to both Type I and Type II pyrethroids. CONCLUSION The novel substitution V253F is potentially involved in pyrethroid resistance in Ae. aegypti. Further studies are needed to elucidate the role of this substitution in the pyrethroid susceptibility of VGSC. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., We found a novel amino‐acid substitution V253F in the voltage‐gated sodium channel in Aedes aegypti which is potentially involved in pyrethroid resistance of this vector mosquito

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.

Nagano and Yamanashi prefectures of Japan comprise an inland mountain area that extends widely north and south, with elevation varying greatly by location. Mosquitoes transmitting infectious disease have a diversity of habitats in Nagano and Yamanashi, and many species can be expected there. However, there have been few reports on mosquito fauna; in particular, little information is available on mosquitoes such as Aedes albopictus. The mosquito fauna was investigated to clarify their elevational distribution ranging from 317 to 1,534 m, focusing especially on Ae. albopictus, in 24 areas in Nagano and Yamanashi prefectures from July to September in 2012–16. Adult mosquitoes were collected using the Centers for Disease Control and Prevention traps in each area, in addition to larval mosquito collections at several temple and shrine precincts in each area. At sites where elevations were >800 m (14 sites), no adult Ae. albopictus were captured. In addition, larval Ae. albopictus were not collected at elevations >728 m (15 sites). Aedes albopictus was captured at 20 other sites (annual mean air temperature ranged from 9.4 to 15.2°C, July–September mean air temperature ranged from 19.9 to 25.0°C).

Kabuto Mountain virus (KAMV), the new member of the genus Uukuvirus, was isolated from the tick Haemaphysalis flava in 2018 in Japan. To date, there is no information on KAMV infection in human and animals. Therefore, serological surveillance of the infection among humans and wild mammals was conducted by virus-neutralization (VN) test and indirect immunofluorescence assay (IFA). Sera of 24 humans, 59 monkeys, 171 wild boars, 233 Sika deer, 7 bears, and 27 nutria in Yamaguchi Prefecture were analyzed by VN test. The positive ratio of humans, monkeys, wild boars, and Sika deer were 20.8%, 3.4%, 33.9% and 4.7%, respectively. No positive samples were detected in bears and nutria. The correlation coefficients between VN test and IFA in human, monkey, wild boar, and Sika deer sera were 0.5745, 0.7198, 0.9967 and 0.9525, respectively. In addition, KAMV was detected in one pool of Haemaphysalis formosensis ticks in Wakayama Prefecture. These results indicated that KAMV or KAMV-like virus is circulating among many wildlife and ticks, and that this virus incidentally infects humans.

Background In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015. Methods A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a Centers for Disease Control and Prevention (CDC) miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to polymerase chain reaction (PCR)-based sequence analysis of the internal transcribed spacer 2 ( ITS2) region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method with the Kimura 2-parameter model on MEGA X version 10.2.2. Results A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae, was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, namely An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido. Conclusion The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan. Graphical abstract

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 variant can 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 3 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 new members of the Quaranjavirus genus or a related group. The viruses were tentatively named "Ohshima virus" even though only limited sequences of their genomes 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 and other animals and to determine the potential risk of an emerging tick-borne viral disease.

From August 27 to October 15, 2014, a dengue fever outbreak with 158 autochthonous cases occurred after nearly 70 years of no reports of autochthonous cases in Japan. The most competent mosquito vector for dengue virus (DENV) transmission in Japan is Aedes albopictus. Since A. albopictus is widely distributed throughout Japan, we examined the susceptibility of this species to infection by DENV and the relationship of the endosymbiont Wolbachia (wAlbA and wAlbB) with susceptibility to DENV. The A. albopictus YYG strain, collected from the Yoyogi Park in 2014, the epicenter of the dengue fever outbreak, was found to have lower susceptibility to DENV 1 and 3 than that of the indigenous Japanese strains A. albopictus EBN 201808 (F1 from the field) and A. albopictus ISG 201603. Furthermore, the A. albopictus EBN 201808 strain showed the same susceptibility to DENV3 as the A. albopictus ISG 201603tet strain (Wolbachia-free). Susceptibility to DENV3 was not related to Wolbachia strains wAlbA or wAlbB in the A. albopictus ISG 201603 strain.

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: In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015.Methods: A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a CDC miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to PCR-based sequence analysis of the ITS2 region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method. Results: A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae Rueda (2005), was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido.Conclusion: The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.

Several outbreaks of trench fever caused by Bartonella quintana occurred in soldiers during World Wars I and II. Although trench fever cases have been decreasing worldwide, the disease was reported among the homeless population in developing and developed countries. The current prevalence of B. quintana infection in Japan is unclear. Blood and body louse (Pediculus humanus humanus) samples were obtained from homeless inpatients with body lice during emergency hospitalization in Tokyo from January 2013 to March 2015. Patients were tested for B. quintana infections using the culture method, polymerase chain reaction, and indirect immunofluorescence assay (IFA). Among the 29 patients tested, the presence of Bartonella spp. was confirmed by genomic sequencing of DNA extracted from two samples from blood culture performed for 15 out of 29 patients and from body louse samples of 20 patients (69%). Immunoglobulin G against B. quintana was detected in 10 patients (34.5%) at a cut-off titer of 1:256 in IFA. B. quintana infection was detected in samples obtained between 2013 and 2015 in Tokyo and needs to be on the list of differential diagnoses performed for febrile homeless individuals.

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

We conducted a nationwide survey of mosquito distribution in Malawi from November 2011 to April 2012, and from July to September 2012. Using dried specimens of mosquito adults collected during the survey, we analyzed their cytochrome c oxidase subunit I (COI) gene sequences, prepared specimens, and registered the genetic information (658 bp) of 144 individuals belonging to 51 species of 10 genera in GenBank. Using the obtained genetic information, we analyzed the degree of intraspecific variation and investigated the various species from morphological and genetic perspectives. Moreover, we conducted phylogenetic analysis of the medically important species distributed from Africa to Asia and explored their geographical differentiation. Results showed that individuals morphologically classified as Culex univittatus complex included a individual of Cx. perexiguus which, to date, have not been reported in southern Africa. Furthermore, Mansonia uniformis, distributed in Africa and Asia, was revealed to belong to genetically distinct populations, with observed morphological differences of the samples suggesting that they are separate species. The results of genetic analysis further suggested that Cx. ethiopicus is not a synonym of Cx. bitaeniorhynchus, but that it is an independent species; although, in this study, the only definite morphological difference observed was in the shape of the wing scales. Further morphological and genetic investigation of individuals of these species, including larvae, is highly recommended.

Nagano and Yamanashi prefectures of Japan comprise an inland mountain area that extends widely north and south, with elevation varying greatly by location. Mosquitoes transmitting infectious disease have a diversity of habitats in Nagano and Yamanashi, and many species can be expected there. However, there have been few reports on mosquito fauna; in particular, little information is available on mosquitoes such as

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.

The redback spider (Latrodectus hasseltii Thorell) reportedly invaded Japan in September 1995. To date, 84 redback spider bite cases have been reported; 7 of these cases employed the antivenom. Antivenom has been imported from Australia in the past, but because of restrictions on exportation it was evident that nearly all of the antivenom present in Japan would expire during 2014. In 2014, a plan was proposed to experimentally manufacture and stockpile a horse antiserum for ourselves, using redback spiders indigenous to Japan. A total of 11,403 female spiders were captured alive: 1,217 from the vicinity of Nishinomiya City, Hyogo prefecture, and 10,186 from Osaka prefecture. Of these, 10,007 females were dissected, and the venom was extracted from the venom glands of individuals and subjected to crude purification to yield 4 lots, of which the majority was α-latrotoxin. Among them, a large amount of single lots with an estimated protein content of 236 mg is subsequently scheduled to be used for immunizing horses. We also determined lethal toxicity of the venom (LD50: 9.17 μg per mouse), and established the assay for the determination of an anti-lethal titer of antivenom in mice.

Aedes albopictus (Skuse) (Diptera: Culicidae) is distributed widely and is common in much of Japan. In Japan, female adults begin to bite in between April and June, except in the southern subtropics where the mosquito has no dormant period. It is difficult to estimate the first Ae. albopictus biting day because it varies annually depending on the location. Over several years, we surveyed the mosquitoes at different locations that covered a range of warmer to cooler areas of Japan. We found an association between the timing of first biting day by Ae. albopictus and spring temperature. In spring months, the strongest correlation was found with mean April temperatures, followed by March. Based on these data, it may, therefore, be possible to apply a simple formula to predict the timing of the first biting day at various geographical locations in Japan. Forecasting maps were created using a simple prediction formula. We found that the first biting day for Ae. albopictus changed depending on early spring temperatures for each year. There was an approximate 20-d difference in first biting day between years with warmer and cooler springs. This prediction model will provide useful insight for planning and practice of Ae. albopictus control programs, targeting larvae and adults, in temperate regions globally., Article, Journal of Medical Entomology.54(6):1519-1524(2017)