Blowflies are potential vector for avian influenza virus at enzootic area in Japan.

High pathogenicity avian influenza (HPAI) poses a significant threat to both domestic and wild birds globally. The avian influenza virus, known for environmental contamination and subsequent oral infection in birds, necessitates careful consideration of alternative introduction routes during HPAI outbreaks. This study focuses on blowflies (genus Calliphora), in particular Calliphora nigribarbis, attracted to decaying animals and feces, which migrate to lowland areas of Japan from northern or mountainous regions in early winter, coinciding with HPAI season. Our investigation aims to delineate the role of blowflies as HPAI vectors by conducting a virus prevalence survey in a wild bird HPAI-enzootic area. In December 2022, 648 Calliphora nigribarbis were collected. Influenza virus RT-PCR testing identified 14 virus-positive samples (2.2% prevalence), with the highest occurrence observed near the crane colony (14.9%). Subtyping revealed the presence of H5N1 and HxN1 in some samples. Subsequent collections in December 2023 identified one HPAI virus-positive specimen from 608 collected flies in total, underscoring the potential involvement of blowflies in HPAI transmission. Our observations suggest C. nigribarbis may acquire the HPAI virus from deceased wild birds directly or from fecal materials from infected birds, highlighting the need to add blowflies as a target of HPAI vector control.

Identification and epidemiological study of an uncultured flavivirus from ticks using viral metagenomics and pseudoinfectious viral particles.

During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.

Screening and identification of persistent viruses in cell lines derived from medically important arthropods.

Arthropod-derived cell lines serve as crucial tools for studying arthropod-borne viruses (arboviruses). However, it has recently come to light that certain cell lines harbor persistent infections of arthropod-specific viruses, which do not cause any apparent cytopathic effects. Moreover, some of these persistent viral infections either inhibit or promote the growth of arboviruses. Therefore, it is of utmost importance to identify the presence of such persistent viruses and understand their impact on arboviral infections. In this study, we conducted a comprehensive virome analysis of several arthropod-derived cell lines, including mosquito-derived NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, CCL-126 cells, and tick-derived IDE8 cells, along with flesh fly-derived NIH-Sape-4 cells. The aim was to determine if these cells were infected with persistent viruses. The results revealed the presence of 15 persistent viruses in NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, and IDE8 cells. Among these, 11 were already known arthropod-specific viruses, while the remaining 4 were novel viruses belonging to Orthophasmavirus, Rhabdoviridae, Totiviridae, and Bunyavirales. In contrast, CCL-126 and NIH-Sape-4 cells appeared to be free of viral infections. This study provides valuable insights into the diversity and latency of arthropod-specific viruses within arthropod-derived cell lines. Further investigations are required to explore persistent viral infections in other arthropod-derived cell cultures and their effects on arbovirus replication. Understanding these factors will enhance the accuracy and reliability of experimental data obtained using these cell lines.

First Detection of the Jingmen Tick Virus in Amblyomma testudinarium Ticks from the Kanto Region, Japan.

In 2010, Jingmen tick virus (JMTV) was discovered in ticks in China and has been shown to be distributed in several regions worldwide. Recently, cases of JMTV infection in humans have been reported in China and Kosovo, and have attracted much attention as an emerging tick-borne disease. In this study, we detected the JMTV genome in Amblyomma testudinarium ticks collected in Kanagawa Prefecture, Japan, during tick-borne virus surveillance conducted in the Kanto Region. Phylogenetic analysis revealed that the new JMTV strain was closely related to previous strains detected in Japan. This suggests that JMTV may have been maintained during an independent natural transmission cycle in Japan. In addition, unlike other countries and regions, all JMTV strains in Japan were detected only in A. testudinarium ticks, suggesting that this tick species is the primary JMTV vector in Japan. This is the first report of JMTV in the Kanto Region. Further studies are required to elucidate the potential risk of infection with this tick-borne virus in Japan. In particular, the prevalence of JMTV in wild animals should be examined to clarify its geographical distribution, host range, and transmission cycle.

Evaluation of long-term preservation methods for viral RNA in mosquitoes at room temperature.

Mosquitoes are important vectors of various pathogenic viruses. Almost all viruses transmitted by mosquitoes are RNA viruses. Therefore, to detect viral genes, mosquito samples must be kept at low temperatures to prevent RNA degradation. However, prolonged transport from the field to laboratory can pose challenges for temperature control. The aim of this study was to evaluate methods for preserving viral RNA in mosquito bodies at room temperature. Virus-infected mosquito samples were immersed in ethanol, propylene glycol, and a commercially available nucleic acid preservation reagent at room temperature, and viral RNA stability was compared. As a result, for the two RNA viruses (San Gabriel mononegavirus and dengue virus 1) subjected to this experiment, no significant decrease in the viral RNA was observed for at least eight weeks after immersion in the reagents, and the amount of RNA remaining was equivalent to that of samples stored at - 80 °C. These results indicate that immersion storage in these reagents used in this study is effective in preserving viral RNA in mosquitoes under room temperature conditions and is expected to be implemented in epidemiologic surveillance that is not limited by the cold chain from the field to the laboratory.

Comparative analysis of the susceptibility of Aedes aegypti and Japanese Aedes albopictus to all dengue virus serotypes.

BACKGROUND: Dengue fever, caused by the dengue virus (DENV), is the most common viral infection transmitted by Aedes mosquitoes (mainly Ae. aegypti and Ae. albopictus) worldwide. Aedes aegypti is not currently established in Japan, and Ae. albopictus is the primary vector mosquito for DENV in the country, but knowledge of its viral susceptibility is limited. Therefore, we aimed to clarify the status of DENV susceptibility by comparing the infection and dissemination dynamics of Japanese Ae. albopictus to all known DENV serotypes with those of Ae. aegypti. METHODS: After propagation of each DENV serotype in Vero cells, the culture supernatants were mixed with defibrinated rabbit blood and adenosine triphosphate, and the mixture was artificially blood-sucked by two colonies of Ae. albopictus from Japan and one colony of Ae. aegypti from a dengue-endemic country (Vietnam). After 14 days of sucking, the mosquito body was divided into two parts (thorax/abdomen and head/wings/legs) and total RNA was extracted from each sample. DENV RNA was detected in these extracted RNA samples using a quantitative RT-PCR method specific for each DENV serotype, and infection and dissemination rates were analyzed. RESULTS: The Japanese Ae. albopictus colonies were susceptible to all DENV serotypes. Its infection and dissemination rates were significantly lower than those of Ae. aegypti. However, the number of DENV RNA copies in Ae. albopictus was almost not significantly different from that in Ae. aegypti. Furthermore, Japanese Ae. albopictus differed widely in their susceptibility to each DENV serotype. CONCLUSIONS: In Japanese Ae. albopictus, once DENV overcame the midgut infection barrier, the efficiency of subsequent propagation and dissemination of the virus in the mosquito body was comparable to that of Ae. aegypti. Based on the results of this study and previous dengue outbreak trends, Ae. albopictus is predicted to be highly compatible with DENV-1, suggesting that this serotype poses a high risk for future epidemics in Japan.

Blood meal source identification and RNA virome determination in Japanese encephalitis virus vectors collected in Ishikawa Prefecture, Japan, show distinct avian/mammalian host preference.

In Asia, Culex mosquitoes are of particular interest because of their role in maintaining endemic mosquito-borne viral diseases, including the Japanese encephalitis virus (JEV). Nonetheless, host-feeding preferences, along with naturally infecting RNA viruses in certain Culex species, remain understudied. In this study, selected blood-fed mosquitoes were processed for avian and mammalian blood meal source identification. Concurrently, cell culture propagation and high-throughput sequencing (HTS) approaches were used to determine the RNA virome of Culex mosquitoes collected in Ishikawa Prefecture, Japan. The identification of blood meal sources from wild-caught Culex spp. revealed that Culex (Culex) tritaeniorhynchus Giles, 1901, has a robust preference toward wild boar (62%, 26/42), followed by heron (21%, 9/42). The other two species, Culex (Oculeomyia) bitaeniorhynchus Giles, 1901, and Culex (Culex) orientalis Edwards, 1921, showed a distinct preference for avian species, including migratory birds. From the HTS results, 34 virus sequences were detected, four of which were newly identified virus sequences of unclassified Aspiviridae, Qinviridae, Iflaviridae, and Picornaviridae. The absence of observable cytopathic effects in mammalian cells and phylogenetic analysis suggested that all identified virus sequences were insect-specific. Further investigations involving other mosquito populations collected in different areas are warranted to explore previously unknown vertebrate hosts that may be linked to JEV dispersal in nature.

Evaluating the mosquito host range of Getah virus and the vector competence of selected medically important mosquitoes in Getah virus transmission.

BACKGROUND: The Getah virus (GETV) is a mosquito-borne Alphavirus (family Togaviridae) that is of significant importance in veterinary medicine. It has been associated with major polyarthritis outbreaks in animals, but there are insufficient data on its clinical symptoms in humans. Serological evidence of GETV exposure and the risk of zoonotic transmission makes GETV a potentially medically relevant arbovirus. However, minimal emphasis has been placed on investigating GETV vector transmission, which limits current knowledge of the factors facilitating the spread and outbreaks of GETV. METHODS: To examine the range of the mosquito hosts of GETV, we selected medically important mosquitoes, assessed them in vitro and in vivo and determined their relative competence in virus transmission. The susceptibility and growth kinetics of GETVs in various mosquito-derived cell lines were also determined and quantified using plaque assays. Vector competency assays were also conducted, and quantitative reverse transcription-PCR and plaque assays were used to determine the susceptibility and transmission capacity of each mosquito species evaluated in this study. RESULTS: GETV infection in all of the investigated mosquito cell lines resulted in detectable cytopathic effects. GETV reproduced the fastest in Culex tritaeniorhynchus- and Aedes albopictus-derived cell lines, as evidenced by the highest exponential titers we observed. Regarding viral RNA copy numbers, mosquito susceptibility to infection, spread, and transmission varied significantly between species. The highest vector competency indices for infection, dissemination and transmission were obtained for Cx. tritaeniorhynchus. This is the first study to investigate the ability of Ae. albopictus and Anopheles stephensi to transmit GETV, and the results emphasize the role and capacity of other mosquito species to transmit GETV upon exposure to GETV, in addition to the perceived vectors from which GETV has been isolated in nature. CONCLUSIONS: This study highlights the importance of GETV vector competency studies to determine all possible transmission vectors, especially in endemic regions.

Correction: Evaluating the competence of the primary vector, Culex tritaeniorhynchus, and the invasive mosquito species, Aedes japonicus japonicus, in transmitting three Japanese encephalitis virus genotypes.

[This corrects the article DOI: 10.1371/journal.pntd.0008986.].

Growth, Pathogenesis, and Serological Characteristics of the Japanese Encephalitis Virus Genotype IV Recent Strain 19CxBa-83-Cv.

Genotype IV Japanese encephalitis (JE) virus (GIV JEV) is the least common and most neglected genotype in JEV. We evaluated the growth and pathogenic potential of the GIV strain 19CxBa-83-Cv, which was isolated from a mosquito pool in Bali, Indonesia, in 2019, and serological analyses were also conducted. The growth ability of 19CxBa-83-Cv in Vero cells was intermediate between that of the genotype I (GI) strain Mie/41/2002 and the genotype V (GV) strain Muar, whereas 19CxBa-83-Cv and Mie/41/2002 grew faster than Muar in mouse neuroblastoma cells. The neuroinvasiveness of 19CxBa-83-Cv in mice was higher than that of Mie/41/2002 but lower than that of Muar; however, there were no significant differences in neurovirulence in mice among the three strains. The neutralizing titers of sera from 19CxBa-83-Cv- and Mie/41/2002-inoculated mice against 19CxBa-83-Cv and Mie/41/2002 were similar, whereas the titers against Muar were lower than those of the other two viruses. The neutralizing titers of JE vaccine-inoculated mouse pool serum against 19CxBa-83-Cv and Muar were significantly lower than those against Mie/41/2002. The neutralizing titers against the three viruses were similar in three out of the five serum samples from GI-infected JE patients, although the titers against Mie/41/2002 were higher than those against 19CxBa-83-Cv and Muar in the remaining two sera samples. In summary, we identified the basic characteristics of 19CxBa-83-Cv, but further studies are needed to better understand GIV JEV.

Detection of Bartonella quintana (Hyphomicrobiales: Bartonellaceae) Among Day Laborers in Osaka, Japan, 2009-2010.

Bartonella quintana is a gram-negative bacterium causing trench fever, an illness historically acquired by soldiers during World War I. More recently, outbreaks of trench fever have been reported in those experiencing homelessness in the United States, France, Russia, and Tokyo, as well as in children in Nepal and persons in Ethiopia. Reports of B. quintana infection outside of Tokyo are rare in Japan. The aim of this study was to examine body lice and blood obtained from people staying in shelters in Osaka (2009-2010) for B. quintana via polymerase chain reaction and enzyme-linked immunosorbent assays. Day laborers were defined as homeless individuals and shelter residents in this study. We detected genes of B. quintana in body lice by PCR and antibodies against B. quintana. The positive rate of B. quintana genes was 6/10 (60%) in body lice and the seroprevalence (IgG) of B. quintana was 4/10 (40%). This demonstrates that trench fever was endemic in people staying in shelters in Osaka in 2009-2010.

Seroconversion of anti-Getah virus antibody among Japanese native Noma horses around 2012.

Getah virus (GETV), an arthropod-borne virus transmitted by mosquitoes, has been isolated from several animals. GETV infection in horses shows clinical signs such as fever, rash, and edema in the leg. Noma horses are one of the eight Japanese native horses. The present study aimed to clarify the occurrence of GETV infection in Noma horses. Serum samples collected from Noma horses were analyzed using a virus neutralization test and enzyme-linked immunosorbent assay and showed that the anti-GETV antibody titers in the samples collected in 2017 were significantly higher than those collected in 2012. We concluded that a seroconversion of anti-GETV antibodies was occurred in the Noma horse population around 2012, providing evidence of the GETV epidemic in Japan circa 2012.

Detection of Japanese Encephalitis Virus RNA in Host-Questing Ticks in Japan, 2019-2020.

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.

Zoonotic Infection with Oz Virus, a Novel Thogotovirus.

Oz virus is a novel thogotovirus isolated from ticks that causes lethal infection in mice. We conducted serosurveillance of Oz virus infection among humans and wild mammals in Japan using virus-neutralization tests and ELISAs. Results showed that Oz virus may be naturally infecting humans and other mammalian hosts.

Detection of Quaranjavirus-Like Sequences from Haemaphysalis hystricis Ticks Collected in Japan.

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.

Aedes albopictus Strain and Dengue Virus Serotype in the Dengue Fever Outbreaks in Japan: Implications of Wolbachia Infection.

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.

Epidemiological study of Kabuto Mountain virus, a novel uukuvirus, in Japan.

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.

Screening for tick-borne and tick-associated viruses in ticks collected in Ghana.

Ticks are blood-sucking arthropods that transmit many pathogens, including arboviruses. Arboviruses transmitted by ticks are generally referred to as tick-borne viruses (TBVs). TBVs are known to cause diseases in humans, pets, and livestock. There is, however, very limited information on the occurrence and distribution of TBVs in sub-Saharan Africa. This study was designed to determine the presence and distribution of ticks infesting dogs and cattle in Ghana, as well as to identify the tick-borne or tick-associated viruses they harbour. A more diverse population of ticks was found to infest cattle (three genera) relative to those infesting dogs (one genus). Six phleboviruses and an orthonairovirus were detected in tick pools screened by RT-PCR. Subsequent sequence analysis revealed two distinct phleboviruses and the previously reported Odaw virus in ticks collected from dogs and a virus (16GH-T27) most closely related to four unclassified phleboviruses in ticks collected from cattle. The virus 16GH-T27 was considered a strain of Balambala tick virus (BTV) and named BTV strain 16GH-T27. Next-generation sequencing analysis of the BTV-positive tick pool detected only the L and S segments. Phylogenetic analysis revealed that BTV clustered with viruses previously defined as M-segment-deficient phleboviruses. The orthonairovirus detected in ticks collected from cattle was confirmed to be the medically important Dugbe virus. Furthermore, we discuss the importance of understanding the presence and distribution of ticks and TBVs in disease prevention and mitigation and the implications for public health. Our findings contribute to the knowledge pool on TBVs and tick-associated viruses.

Detection of Jingmenviruses in Japan with Evidence of Vertical Transmission in Ticks.

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.

Toyo virus, a novel member of the Kaisodi group in the genus Uukuvirus (family Phenuiviridae) found in Haemaphysalis formosensis ticks in Japan.

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.