Publications by authors named "Haruhiko Isawa"

59 Publications

Identification and Isolation of Japanese Encephalitis Virus Genotype IV from Culex vishnui Collected in Bali, Indonesia in 2019.

Am J Trop Med Hyg 2021 Jul 19. Epub 2021 Jul 19.

Department of Medical Entomology, National Institute of Infectious Diseases, Shinjuku, Japan.

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.
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http://dx.doi.org/10.4269/ajtmh.20-1554DOI Listing
July 2021

Determining vector competence of Aedes aegypti from Ghana in transmitting dengue virus serotypes 1 and 2.

Parasit Vectors 2021 Apr 29;14(1):228. Epub 2021 Apr 29.

Department of Medical Entomology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

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.
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http://dx.doi.org/10.1186/s13071-021-04728-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082837PMC
April 2021

Discovery of a Novel Flavivirus (Flaviviridae) From the Horse Fly, Tabanus rufidens (Diptera: Tabanidae): The Possible Coevolutionary Relationships Between the Classical Insect-Specific Flaviviruses and Host Dipteran Insects.

J Med Entomol 2021 03;58(2):880-890

Department of Medical Entomology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.

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.
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http://dx.doi.org/10.1093/jme/tjaa193DOI Listing
March 2021

Detection of Bartonella quintana infection among the Homeless Population in Tokyo, Japan, from 2013-2015.

Jpn J Infect Dis 2021 Jan 29. Epub 2021 Jan 29.

Department of Medical Entomology, National Institute of Infectious Diseases, Japan.

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.
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http://dx.doi.org/10.7883/yoken.JJID.2020.505DOI Listing
January 2021

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

PLoS Negl Trop Dis 2020 12 28;14(12):e0008986. Epub 2020 Dec 28.

Department of Medical Entomology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

Japanese encephalitis virus (JEV) is maintained in an enzootic cycle between swine, water birds, and mosquitoes. JEV has circulated indigenously in Asia, with Culex tritaeniorhynchus as the primary vector. In some areas where the primary vector is scarce or absent, sporadic cases of Japanese encephalitis have been reported, with Aedes japonicus japonicus presumed to have the potential as a secondary vector. As one of the world's most invasive culicid species, Ae. j. japonicus carries a considerable health risk for spreading diseases to wider areas, including Europe and North America. Thus, evaluation of its competency as a JEV vector, particularly in a native population, will be essential in preventing potential disease spread. In this study, the two mosquito species' vector competence in transmitting three JEV genotypes (I, III, and V) was assessed, with Cx. tritaeniorhynchus serving as a point of reference. The mosquitoes were virus-fed and the infection rate (IR), dissemination rate (DR), and transmission rate (TR) evaluated individually by either RT-qPCR or focus forming assay. Results showed striking differences between the two species, with IR of 95% (261/274) and 9% (16/177) in Cx. tritaeniorhynchus and Ae. j. japonicus, respectively. Both mosquitoes were susceptible to all three JEV genotypes with significant differences in IR and mean viral titer. Results confirm the primary vector's competence, but the fact that JEV was able to establish in Ae. j. japonicus is of public health significance, and with 2%-16% transmission rate it has the potential to successfully transmit JEV to the next host. This may explain the human cases and infrequent detection in primary vector-free areas. Importantly, Ae. j. japonicus could be a relevant vector spreading the disease into new areas, indicating the need for security measures in areas where the mosquito is distributed or where it may be introduced.
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http://dx.doi.org/10.1371/journal.pntd.0008986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793266PMC
December 2020

A novel nyavirus lacking matrix and glycoprotein genes from Argas japonicus ticks.

Virus Res 2021 01 1;292:198254. Epub 2020 Dec 1.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virusres.2020.198254DOI Listing
January 2021

Establishment and characterization of a cell line from Ghanaian Aedes aegypti (Diptera: Culicidae) focusing on Aedes-borne flavivirus susceptibility.

In Vitro Cell Dev Biol Anim 2020 Oct 30;56(9):792-798. Epub 2020 Sep 30.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

Mosquitoes are generally considered one of the most important vectors of arboviruses, with Aedes aegypti regarded as the most important in transmission of yellow fever and dengue viruses. To investigate why there are differences in the incidence of dengue fever and Zika in different geographical areas and an absence of outbreaks in Ghana in spite of an abundance of A. aegypti mosquitoes, we established a continuous cell line from embryonic cells of A. aegypti collected in Ghana and assessed its susceptibility to dengue, yellow fever, and Zika viruses. The new cell line (designated AeAe-GH98), having an adhesive spindle-shaped web-like morphology, was serially subcultured in both VP-12 and Schneider's medium supplemented with 10% heat-inactivated fetal bovine serum. AeAe-GH98 cells were found to have a population doubling time of 1.3 d during exponential growth. The mosquito colony used to establish the cell line was confirmed to have originated from Africa using microsatellite assay. In terms of susceptibility to Aedes-borne flaviviruses, AeAe-GH98 cells were found to have different degrees of susceptibility to yellow fever, Zika, and dengue virus infection and propagation. While susceptibility of AeAe-GH98 cells to yellow fever and Zika viruses was comparable with that of C6/36 cells, susceptibility to dengue virus was significantly lower. This cell line will serve as a useful tool for determining molecular factors influencing virus-vector susceptibility in vitro.
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http://dx.doi.org/10.1007/s11626-020-00504-yDOI Listing
October 2020

Identification and characterization of a novel orbivirus, Yonaguni orbivirus, isolated from cattle on the westernmost island of Japan.

Arch Virol 2020 Dec 7;165(12):2903-2908. Epub 2020 Sep 7.

Kyushu Research Station, National Institute of Animal Health, National Agriculture and Food Research Organization, 2702 Chuzan, Kagoshima, 891-0105, Japan.

A novel orbivirus (genus Orbivirus, family Reoviridae), designated Yonaguni orbivirus (YONOV), was isolated from bovine blood collected on a subtropical island of Japan in 2015. The YONOV genome (20,054 nucleotides in total) has a coding arrangement similar to those of mosquito-borne orbiviruses. YONOV has a close genetic relationship to mosquito-borne orbiviruses, especially to Mobuck virus (MBV), which was isolated in North America. However, YONOV and MBV share less than 74% nucleotide sequence identity in the major subcore protein (T2) coding sequence, which satisfies the criterion for species demarcation. It is still uncertain whether YONOV should be assigned to a novel species in the genus Orbivirus.
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http://dx.doi.org/10.1007/s00705-020-04803-3DOI Listing
December 2020

Mosquito-borne viruses, insect-specific flaviviruses (family Flaviviridae, genus Flavivirus), Banna virus (family Reoviridae, genus Seadornavirus), Bogor virus (unassigned member of family Permutotetraviridae), and alphamesoniviruses 2 and 3 (family Mesoniviridae, genus Alphamesonivirus) isolated from Indonesian mosquitoes.

J Vet Med Sci 2020 Jul 25;82(7):1030-1041. Epub 2020 May 25.

Laboratory of Veterinary Microbiology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, 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.
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http://dx.doi.org/10.1292/jvms.20-0261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399325PMC
July 2020

Acquired Functional Capsid Structures in Metazoan Totivirus-like dsRNA Virus.

Structure 2020 08 14;28(8):888-896.e3. Epub 2020 May 14.

The Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Japan; Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan. Electronic address:

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).
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http://dx.doi.org/10.1016/j.str.2020.04.016DOI Listing
August 2020

Deciphering the Virome of Subgroup Mosquitoes, the Major Vectors of Japanese Encephalitis, in Japan.

Viruses 2020 02 28;12(3). Epub 2020 Feb 28.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Japanese encephalitis (JE) remains a public health concern in several countries, and the mosquito plays a central role in its transmission cycle. 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 and . , which are part of the 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 . . A total of 27 viruses, including JEV, were detected from these mosquitoes. Molecular and phylogenetic analyses of the detected viruses classified 15 of the 27 viruses as novel species, notably belonging to the , , , and 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.
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http://dx.doi.org/10.3390/v12030264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150981PMC
February 2020

Entomological Assessment of the Status and Risk of Mosquito-borne Arboviral Transmission in Ghana.

Viruses 2020 01 27;12(2). Epub 2020 Jan 27.

Department of Environmental Parasitology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

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.
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http://dx.doi.org/10.3390/v12020147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077231PMC
January 2020

RNA virome analysis of questing ticks from Hokuriku District, Japan, and the evolutionary dynamics of tick-borne phleboviruses.

Ticks Tick Borne Dis 2020 03 27;11(2):101364. Epub 2019 Dec 27.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Electronic address:

Tick-borne viruses have emerged recently in many parts of the world, and the discoveries of novel tick-borne viruses have been accelerated by the development of high-throughput sequencing technology. In this study, a cost-efficient small benchtop next-generation sequencer, the Illumina MiniSeq, was used for the RNA virome analysis of questing ticks collected from Hokuriku District, Japan, and assessed for their potential utility in a tick-borne virus surveillance system. We detected two phleboviruses [Kabuto Mountain virus (KAMV) and Okutama tick virus (OKTV)], a coltivirus [Tarumizu tick virus (TarTV)], and a novel iflavirus [Hamaphysalis flava iflavirus (HfIFV)] from tick homogenates and/or cell culture supernatants after virus isolation processes. The number of sequence reads from KAMV and TarTV markedly increased when cell culture supernatants were used, indicating a successful isolation of these viruses. In contrast, OKTV and HfIFV were detected only in tick homogenates but not from cell culture supernatants, suggesting a failure to isolate these viruses. Furthermore, we performed genomic and phylogenetic analyzes of these detected viruses. OKTV and some phleboviruses discovered recently by NGS-based methods were probably deficient in the M genome segment, which are herein proposed as M segment-deficient phlebovirus (MdPV). A phylogenetic analysis of phleboviruses, including MdPV, suggested that Uukuniemi and Kaisodi group viruses and kabutoviruses evolved from an ancestral MdPV, which provides insights into the evolutionary dynamics of phleboviruses as emerging pathogens.
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http://dx.doi.org/10.1016/j.ttbdis.2019.101364DOI Listing
March 2020

Persistent viruses in mosquito cultured cell line suppress multiplication of flaviviruses.

Heliyon 2018 Aug 23;4(8):e00736. Epub 2018 Aug 23.

Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo, Japan.

In the growth kinetics analysis of flaviviruses in 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 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.
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http://dx.doi.org/10.1016/j.heliyon.2018.e00736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107885PMC
August 2018

Dengue Virus Infection in during the 2014 Autochthonous Dengue Outbreak in Tokyo Metropolis, Japan.

Am J Trop Med Hyg 2018 05 15;98(5):1460-1468. Epub 2018 Mar 15.

Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

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 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, 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 mosquitoes are distributed.
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http://dx.doi.org/10.4269/ajtmh.17-0954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953391PMC
May 2018

Characterization of a novel thogotovirus isolated from Amblyomma testudinarium ticks in Ehime, Japan: A significant phylogenetic relationship to Bourbon virus.

Virus Res 2018 04 13;249:57-65. Epub 2018 Mar 13.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virusres.2018.03.004DOI Listing
April 2018

Venom and Antivenom of the Redback Spider (Latrodectus hasseltii) in Japan. Part I. Venom Extraction, Preparation, and Laboratory Testing.

Jpn J Infect Dis 2018 Mar 28;71(2):116-121. Epub 2018 Feb 28.

Department of Immunology, National Institute of Infectious Diseases.

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 (LD: 9.17 μg per mouse), and established the assay for the determination of an anti-lethal titer of antivenom in mice.
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http://dx.doi.org/10.7883/yoken.JJID.2017.291DOI Listing
March 2018

Isolation and characterization of Kabuto Mountain virus, a new tick-borne phlebovirus from Haemaphysalis flava ticks in Japan.

Virus Res 2018 01 29;244:252-261. Epub 2017 Nov 29.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.

In Japan, indigenous tick-borne phleboviruses (TBPVs) and their associated diseases first became evident in 2013 by reported human cases of severe fever with thrombocytopenia syndrome (SFTS). In this study, we report a novel member of the genus Phlebovirus designated as Kabuto Mountain virus (KAMV), which was isolated from the ixodid tick Haemaphysalis flava in Hyogo, Japan. A complete viral genome sequencing and phylogenetic analyses showed that KAMV is a novel member of TBPVs, which is closely related to the Uukuniemi and Kaisodi group viruses. However, unlike the Uukuniemi group viruses, the 165-nt intergenic region (IGR) in the KAMV S segment was highly C-rich in the genomic sense and not predicted to form a secondary structure, which are rather similar to those of the Kaisodi group viruses and most mosquito/sandfly-borne phleboviruses. Furthermore, the NSs protein of KAMV was highly divergent from those of other TBPVs. These results provided further insights into the genetic diversity and evolutionary relationships of TBPVs. KAMV could infect and replicate in some rodent and primate cell lines. We evaluated the infectivity and pathogenicity of KAMV in suckling mice, where we obtained a virulent strain after two passages via intracerebral inoculation. This is the first report showing the existence of a previously unrecognized TBPV in Japan, other than the SFTS virus.
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http://dx.doi.org/10.1016/j.virusres.2017.11.030DOI Listing
January 2018

Isolation and characterization of a new iflavirus from Armigeres spp. mosquitoes in the Philippines.

J Gen Virol 2017 Nov 19;98(11):2876-2881. Epub 2017 Oct 19.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

During an entomological surveillance for arthropod-borne viruses in the Philippines, we isolated a previously unrecognized virus from female Armigeres spp. mosquitoes. Whole-genome sequencing, genetic characterization and phylogenetic analysis revealed that the isolated virus, designated Armigeres iflavirus (ArIFV), is a novel member of the iflaviruses (genus Iflavirus, family Iflaviridae) and phylogenetically related to Moku virus, Hubei odonate virus 4, slow bee paralysis virus and Graminella nigrifrons virus 1. To our knowledge, this is the first successful isolation of iflavirus from a dipteran insect. Spherical ArIFV particles of approximately 30 nm in diameter contained at least three major structural proteins. ArIFV multiplied to high titres (~10 p.f.u. ml) and formed clear plaques in a mosquito cell line, C6/36. Our findings provide new insights into the infection mechanism, genetic diversity and evolution of the Iflaviridae family.
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http://dx.doi.org/10.1099/jgv.0.000929DOI Listing
November 2017

Isolation and characterization of Tarumizu tick virus: A new coltivirus from Haemaphysalis flava ticks in Japan.

Virus Res 2017 10 28;242:131-140. Epub 2017 Sep 28.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address:

During the course of tick-borne virus surveillance in Japan, three independent isolates of probably the same virus were obtained from three geographically distant populations of the hard tick Haemaphysalis flava. Genome analyses of the three isolates demonstrated that they were closely related but distinct strains of a novel virus, designated Tarumizu tick virus (TarTV), which has a genome of 12 double-stranded RNA segments. The development of the virus-induced cytopathic effects on BHK cells significantly varied according to virus strains. Ten out of 12 segments of TarTV appeared to encode putative orthologs or functional equivalents of viral proteins of Colorado tick fever virus (CTFV) and Eyach virus, suggesting that TarTV is the third member of the genus Coltivirus in the family Reoviridae. This was supported by the facts that the 5'- and 3'-terminal consensus sequences of coltivirus genomes were found also in TarTV genome, and segment 9 of TarTV had sequence and structural features that may mediate a stop codon read-through as observed in that of CTFV. However, segment 7 and 10 of TarTV had no significant sequence similarities to any other proteins of known coltiviruses. Electron microscopic analysis demonstrated that TarTV particle had a non-enveloped bilayer icosahedral structure, and viral inclusion bodies were formed in infected cells. TarTV could infect and replicate in several mammalian cell lines tested, but show no clinical symptoms in intracerebrally inoculated mice. Taken together, our findings provide new insights into genetic diversity and evolution of the genus Coltivirus.
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http://dx.doi.org/10.1016/j.virusres.2017.09.017DOI Listing
October 2017

Detection of a novel putative phlebovirus and first isolation of Dugbe virus from ticks in Accra, Ghana.

Ticks Tick Borne Dis 2017 06 23;8(4):640-645. Epub 2017 Apr 23.

Department of Environmental Parasitology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-0034, Japan.

Ticks are ectoparasites that transmit various types of human and animal pathogens. In particular, emerging and re-emerging diseases caused by tick-borne viruses are public health concerns around the world. However, in many countries of the sub-Saharan African region, epidemiological information on tick-borne viral infections is limited, and their prevalence and distribution remain largely unknown. In this study, we conducted surveillance on ticks to detect medically important tick-borne bunyaviruses in three study sites in and near to Accra, the capital city of Ghana, in 2015. Domestic dogs and cattle were surveyed and were found to be infested with various tick species belonging to the genera Rhipicephalus, Amblyomma and Haemaphysalis. Importantly, we detected a novel putative phlebovirus in Rhipicephalus ticks, and successfully isolated a new strain of Dugbe virus from Am. variegatum ticks. To our knowledge, this is the first report of tick-associated viruses in Ghana other than Crimean-Congo hemorrhagic fever virus.
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http://dx.doi.org/10.1016/j.ttbdis.2017.04.010DOI Listing
June 2017

Dengue Virus Isolation in Mosquito Aedes albopictus Captured During an Outbreak in Tokyo, 2014, by a Method Relying on Antibody-Dependent Enhancement Mechanism Using FcγR-Expressing BHK Cells.

Vector Borne Zoonotic Dis 2016 12 24;16(12):810-812. Epub 2016 Oct 24.

2 Department of Virology 1, National Institute of Infectious Diseases , Tokyo, Japan .

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.
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http://dx.doi.org/10.1089/vbz.2016.1982DOI Listing
December 2016

Complete Genome Sequencing and Phylogenetic Analysis of a Getah Virus Strain (Genus Alphavirus, Family Togaviridae) Isolated from Culex tritaeniorhynchus Mosquitoes in Nagasaki, Japan in 2012.

Vector Borne Zoonotic Dis 2016 12 9;16(12):769-776. Epub 2016 Nov 9.

2 Department of Medical Entomology, National Institute of Infectious Diseases , Tokyo, Japan .

Getah virus (GETV; genus Alphavirus, family Togaviridae) is a mosquito-borne virus known to cause disease in horses and pigs. In 2014, for the first time in ∼30 years, a sudden GETV outbreak occurred among racehorses in Ibaraki, Japan. Two years before this outbreak, we obtained multiple GETV isolates from Culex tritaeniorhynchus mosquitoes collected in Nagasaki, Japan and determined the whole genome sequence of GETV isolate 12IH26. Our phylogenetic analysis of GETV strains revealed that the isolate 12IH26 forms a robust clade with the epidemic strains 14-I-605-C1 and 14-I-605-C2 isolated from horses in the 2014 outbreak in Ibaraki. Furthermore, the complete genomic sequence of the isolate 12IH26 was 99.9% identical to those of the 2014 epidemic strains in Ibaraki. Phylogenetic analysis also showed that the recent Japanese GETV strains, including the isolate 12IH26, are closely related to the Chinese and South Korean strains rather than the previous Japanese strains, suggesting that GETV strains may be transported from overseas into Japan through long-distance migration of the infected mosquitoes or migratory birds.
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http://dx.doi.org/10.1089/vbz.2016.2017DOI Listing
December 2016

Bustos virus, a new member of the negevirus group isolated from a Mansonia mosquito in the Philippines.

Arch Virol 2017 Jan 26;162(1):79-88. Epub 2016 Sep 26.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

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.
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http://dx.doi.org/10.1007/s00705-016-3068-4DOI Listing
January 2017

The infectious particle of insect-borne totivirus-like Omono River virus has raised ridges and lacks fibre complexes.

Sci Rep 2016 09 12;6:33170. Epub 2016 Sep 12.

The Laboratory of Molecular Biophysics, Department of Cell and Molecular biology, Uppsala University, Sweden.

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.
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http://dx.doi.org/10.1038/srep33170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018817PMC
September 2016

Argonaute 2 Suppresses Japanese Encephalitis Virus Infection in Aedes aegypti.

Jpn J Infect Dis 2017 Jan 9;70(1):38-44. Epub 2016 May 9.

Department of Medical Entomology, National Institute of Infectious Disease.

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.
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http://dx.doi.org/10.7883/yoken.JJID.2015.671DOI Listing
January 2017

Characterization of a novel negevirus isolated from Aedes larvae collected in a subarctic region of Japan.

Arch Virol 2016 Apr 21;161(4):801-9. Epub 2015 Dec 21.

Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.

We isolated and characterized a novel positive-sense, single-stranded RNA virus from Aedes larvae collected on Okushiri Island, Hokkaido, Japan. This virus, designated Okushiri virus (OKV), replicated in the Aedes albopictus cell line C6/36 with severe cytopathic effects and produced a large number of spherical viral particles that were 50-70 nm in diameter and released into the cell culture medium. The OKV genome consisted of 9,704 nucleotides, excluding the poly(A) tail at the 3'-terminus, and contained three major open reading frames (ORF1, ORF2, and ORF3). ORF1 encoded a putative protein of approximately 268 kDa that included a methyltransferase domain, FtsJ-like methyltransferase domain, helicase domain, and RNA-dependent RNA polymerase domain. The genome organization and results of a phylogenetic analysis based on the amino acid sequence predicted from the nucleotide sequence indicated that OKV is a member of a new insect virus group of negeviruses with a possible evolutionary relationship to some plant viruses. ORF2 and ORF3 were suggested to encode hypothetical membrane-associated proteins of approximately 45 kDa and 22 kDa, respectively. This is the first study on a novel negevirus isolated from mosquito larvae in Japan.
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http://dx.doi.org/10.1007/s00705-015-2711-9DOI Listing
April 2016

Genetic and biological characterization of Muko virus, a new distinct member of the species Great Island virus (genus Orbivirus, family Reoviridae), isolated from ixodid ticks in Japan.

Arch Virol 2015 Dec 8;160(12):2965-77. Epub 2015 Sep 8.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

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 Tribeč 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.
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http://dx.doi.org/10.1007/s00705-015-2588-7DOI Listing
December 2015

Analysis of Mosquito-Borne Flavivirus Superinfection in Culex tritaeniorhynchus (Diptera: Culicidae) Cells Persistently Infected with Culex Flavivirus (Flaviviridae).

J Med Entomol 2015 Mar 5;52(2):222-9. Epub 2015 Feb 5.

Department of Medical Entomology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.

Superinfection exclusion is generally defined as a phenomenon in which a pre-existing viral infection prevents a secondary viral infection; this has also been observed in infections with mosquito-borne viruses. In this study, we examined the superinfection exclusion of the vertebrate-infecting flaviviruses, Japanese encephalitis virus (JEV) and dengue virus (DENV), by stable and persistent infection with an insect-specific flavivirus, Culex flavivirus (CxFV), in a Culex tritaeniorhynchus Giles cell line (CTR cells). Our experimental system was designed based on the premise that wild Cx. tritaeniorhynchus mosquitoes naturally infected with CxFV are superinfected with JEV by feeding on JEV-infected animals. As a result, we found no evidence of the superinfection exclusion of both JEV and DENV by pre-existing CxFV infection at the cellular level. However, JEV superinfection induced severe cytopathic effects on persistently CxFV-infected CTR cells. These observations imply the possibility that JEV superinfection in CxFV-infected Cx. tritaeniorhynchus mosquitoes has an adverse effect on their fitness.
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http://dx.doi.org/10.1093/jme/tju059DOI Listing
March 2015

Persistent natural infection of a Culex tritaeniorhynchus cell line with a novel Culex tritaeniorhynchus rhabdovirus strain.

Microbiol Immunol 2015 Sep;59(9):562-6

Transboundary Animal Diseases Research Center, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065.

Culex tritaeniorhynchus rhabdovirus (CTRV) is a mosquito virus that establishes persistent infection without any obvious cell death. Therefore, occult infection by CTRV can be present in mosquito cell lines. In this study, it is shown that NIID-CTR cells, which were derived from Cx. tritaeniorhynchus, are persistently infected with a novel strain of CTRV. Complete genome sequencing of the infecting strain revealed that it is genetically similar but distinct from the previously isolated CTRV strain, excluding the possibility of contamination. These findings raise the importance of further CTRV studies, such as screening of CTRV in other mosquito cell lines.
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http://dx.doi.org/10.1111/1348-0421.12279DOI Listing
September 2015
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