Papers - YAMADA Kentaro
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Plasmid-Based Reverse Genetics System Enabling One-Step Generation of Genotype 3 Hepatitis E Virus Reviewed
Kobayashi T., Nishiyama T., Yamada K., Murata K., Okamoto H.
Viruses 17 ( 5 ) 2025.5
Publishing type：Research paper (scientific journal) Publisher：Viruses
Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus that poses a significant public health risk, yet its study is hindered by the complexity of conventional RNA-based reverse genetics systems. These systems require multiple steps, including genome cloning, in vitro transcription, and capping, making them labor-intensive and susceptible to RNA degradation. In this study, we developed a single-step, plasmid-based HEV expression system that enabled direct intracellular transcription of the full-length HEV genome under a cytomegalovirus immediate-early (CMV-IE) promoter. The viral genome was flanked by hammerhead (HH) and hepatitis delta virus (HDV) ribozymes to ensure precise self-cleavage and the generation of authentic 5′ and 3′ termini. This system successfully supported HEV genome replication, viral protein expression, and progeny virion production at levels comparable to those obtained using in vitro-transcribed, capped HEV RNA. Additionally, a genetic marker introduced into the plasmid construct was stably retained in progeny virions, demonstrating the feasibility of targeted genetic modifications. However, plasmid-derived HEV exhibited delayed replication kinetics, likely due to the absence of an immediate 5′ cap. Attempts to enhance capping efficiency through co-expression of the vaccinia virus capping enzyme failed to improve HEV replication, suggesting that alternative strategies, such as optimizing the promoter design for capping, may be required. This plasmid-based HEV reverse genetics system simplifies the study of HEV replication and pathogenesis and provides a versatile platform for the genetic engineering of the HEV genome.
DOI： 10.3390/v17050669
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Mekata H., Yamamoto M., Kaneko Y., Yamada K., Okabayashi T., Saito A.
Pathogens 14 ( 3 ) 2025.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Pathogens
Severe fever with thrombocytopenia syndrome (SFTS), caused by infection with the SFTS virus, is an emerging fatal tick-borne zoonosis endemic to East Asia. Although SFTS is a tick-borne disease, the virus can be transmitted from animals with SFTS without a tick bite. Direct transmission of the SFTS virus from animals to humans has been reported; however, the transmission route is unclear in some cases. Therefore, this study focused on the possibility of SFTS virus transmission through urine and attempted to isolate the infectious virus from the urine of animals with SFTS. Since more efficient cell isolation is needed to determine whether the SFTS virus is present, we first expressed dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), the major receptor for the virus, in Vero cells (Vero-DC-SIGN cells) using a retroviral vector. When inoculated with equal amounts of the SFTS virus strain and SFTS-virus-infected animal serum, Vero-DC-SIGN cells had 42–136% and 20–85% more foci, respectively, than their parent Vero cells. After confirming that Vero-DC-SIGN cells were more suitable for the isolation of the SFTS virus, we investigated whether it could be isolated from the urine of eight cats and two dogs with SFTS. The virus was isolated from 25 μL of urine from two cats with SFTS. Considering that cats excrete 50–100 mL of urine per day, the transmission of the SFTS virus via the urine of cats with SFTS cannot be ruled out. Individuals examining or caring for cats suspected of having SFTS should be aware of the possibility of viral transmission via urine.
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Development of a Multi-Locus Real-Time PCR with a High-Resolution Melting Assay to Differentiate Wild-Type, Asian Recombinant, and Vaccine Strains of Lumpy Skin Disease Virus Reviewed International coauthorship
Bhakha K., Matsui Y., Buakhao N., Wanganurakkul S., Deemagarn T., Oba M., Takemae H., Mizutani T., Misawa N., Chintapitaksakul L., Yamada K., Suwankitwat N.
Veterinary Sciences 12 ( 3 ) 2025.3
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Veterinary Sciences
Lumpy skin disease virus (LSDV) affects cattle and causes significant economic damage. The live vaccine derived from an attenuated strain is effective but is associated with mild disease and skin lesions in some vaccinated cattle. Moreover, recombinant LSDV strains, particularly one with wild-type field and vaccine strains, have recently emerged and spread throughout Asian countries. A cost-effective LSDV typing method is required. We developed a multi-locus real-time PCR with a high-resolution melting (HRM) assay to differentiate between the wild-type, vaccine, and dominant Asian recombinant strains. Based on a multiple alignment analysis, we selected three target genes for the HRM assay, ORF095, ORF126, and ORF145, in which there are insertions/deletions and nucleotide substitutions between wild-type and vaccine strains, and designed primer sets for the assay. Using the synthetic DNA encoding these genes for the two strains, it was shown that the PCR amplicons intercalated with a saturating fluorescent dye could clearly differentiate between wild-type and vaccine strains in the HRM analysis for all three target genes. Further, using clinical samples, our method was able to identify recombinant strains harboring the wild-type ORF095 and ORF145 and the vaccine strain ORF126 genes. Thus, our HRM assay may provide rapid LSDV typing.
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Evaluation of lateral flow devices for rabies diagnosis in decomposed animal brain samples. Reviewed International coauthorship
Todoroki R, Ongtangco JT, Kimitsuki K, Saito N, Mananggit MR, Velasco CR, Mauhay JD, Garcia AM, Demetria CS, Kentaro Y, Nishizono A
Tropical medicine and health 53 ( 1 ) 30 2025.2
Language：English Publishing type：Research paper (scientific journal)
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Mekata H., Yamada K., Umeki K., Yamamoto M., Ochi A., Umekita K., Kobayashi I., Hirai T., Okabayashi T.
BMC Veterinary Research 20 ( 1 ) 190 2024.12
Language：English Publishing type：Research paper (scientific journal) Publisher：BMC Veterinary Research
Severe fever with thrombocytopenia syndrome (SFTS) is a fatal zoonosis caused by ticks in East Asia. As SFTS virus (SFTSV) is maintained between wildlife and ticks, seroepidemiological studies in wildlife are important to understand the behavior of SFTSV in the environment. Miyazaki Prefecture, Japan, is an SFTS-endemic area, and approximately 100 feral horses, called Misaki horses (Equus caballus), inhabit Cape Toi in Miyazaki Prefecture. While these animals are managed in a wild-like manner, their ages are ascertainable due to individual identification. In the present study, we conducted a seroepidemiological survey of SFTSV in Misaki horses between 2015 and 2023. This study aimed to understand SFTSV infection in horses and its transmission to wildlife. A total of 707 samples from 180 feral horses were used to determine the seroprevalence of SFTSV using enzyme-linked immunosorbent assay (ELISA). Neutralization testing was performed on 118 samples. In addition, SFTS viral RNA was detected in ticks from Cape Toi and feral horses. The overall seroprevalence between 2015 and 2023 was 78.5% (555/707). The lowest seroprevalence was 55% (44/80) in 2016 and the highest was 92% (76/83) in 2018. Seroprevalence was significantly affected by age, with 11% (8/71) in those less than one year of age and 96.7% (435/450) in those four years of age and older (p < 0.0001). The concordance between ELISA and neutralization test results was 88.9% (105/118). SFTS viral RNA was not detected in ticks (n = 516) or feral horses. This study demonstrated that horses can be infected with SFTSV and that age is a significant factor in seroprevalence in wildlife. This study provides insights into SFTSV infection not only in horses but also in wildlife in SFTS-endemic areas.
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Involvement of Campylobacter Species in Spotty Liver Disease-like Lesions in Broiler Chickens Detected at Meat Inspections in Miyazaki Prefecture, Japan Reviewed International coauthorship
Jiarpinitnun P., Iwakiri A., Fuke N., Pongsawat P., Miyanishi C., Sasaki S., Taniguchi T., Matsui Y., Luangtongkum T., Yamada K., Misawa N.
Microorganisms 12 ( 12 ) 2024.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Microorganisms
Spotty liver disease (SLD) affects free-range laying hens, leading to mortality and reduced egg production. Campylobacter species, including Campylobacter hepaticus, have been associated with SLD cases worldwide. However, the cause of SLD-like lesions found in broilers in Japan still remains unclear. The present study aimed to investigate the involvement of Campylobacter spp. in broiler SLD by conducting microbiological, molecular biological, serological, histopathological, and immunohistopathological examinations using specimens of liver, bile, cecum, and serum from SLD-like and non-SLD chickens. C. jejuni was predominantly isolated and detected in approximately 40% of both non-SLD livers and SLD-like livers, with no significant difference between them. However, C. hepaticus was neither isolated nor detected in this study. Gross and histopathology revealed multifocal necrotizing hepatitis, suppurative granulomatous hepatitis, and cholangiohepatitis. Hepatitis stages are classified as no hepatitis, subclinical, acute, and chronic hepatitis. C. jejuni was more frequently present in acute-stage SLD-like livers, and high IgG antibody levels were noted in both subclinical and SLD-like-affected chickens, indicating C. jejuni infection. Immunohistochemical examination also supported these findings. The findings suggest that C. hepaticus was not involved in SLD-like in broilers in Japan, but C. jejuni translocation from the intestines to the liver may be a contributing factor.
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Kobayashi T., Takahashi M., Ohta S., Hoshino Y., Yamada K., Jirintai S., Primadharsini P.P., Nagashima S., Murata K., Okamoto H.
Viruses 16 ( 9 ) 2024.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Viruses
The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7–95.3%), as assessed by bioanalyzer, with yields of 13.9–89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development.
DOI： 10.3390/v16091400
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Discovery of a new volcanic soil material, “Akahoya,” as an adsorbent for bacterial and viral pathogens and its application to environmental purification Reviewed International coauthorship
Pongsawat P., Jianpinitnun P., Sasaki S., Miyanishi C., Taniguchi T., Luangtongkum T., Yasui K., Kinoshita H., Kobayashi T., Nagahama H., Yamada K., Misawa N.
Applied and Environmental Microbiology 90 ( 9 ) e0100724 2024.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Applied and Environmental Microbiology
Akahoya is a volcanic soil rich in alumina, primarily deposited in Kyushu, Japan. We have found that Akahoya adsorbs bacteria in the water surrounding cattle grazing areas, suggesting a potential for environmental purification. This study investigated the spectrum of microorganisms adsorbed by Akahoya using a column filled with Akahoya through which a suspension of microorganisms was passed. Shirasu soil, another volcanic soil with a different chemical composition, was used as a control. Akahoya effectively adsorbed a diverse range of microorganisms including Escherichia coli, Campylobacter jejuni, Vibrio parahaemolyticus, Salmonella Enteritidis, Staphylococcus aureus, Clostridium perfringens, spores of Bacillus subtilis and Bacillus anthracis, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), murine norovirus, and avian influenza virus (H3N2), whereas Shirasu soil did not adsorb any of the organisms examined. Moreover, bacteria naturally present in river water, such as aerobic bacteria, total coliforms, and Enterobacteriaceae as indicators of river contamination, as well as E. coli added artificially to sterilized river water, were reduced to below the detection limit (<1 CFU/mL) after being passed through Akahoya. Additionally, the number of viable E. coli continued to decrease after contact with Akahoya for 1 month, suggesting bactericidal effects. Notably, the adsorption of E. coli to Akahoya was influenced by the concentration of phosphate and the pH of the suspension due to the interaction between the surface phosphorylation of organisms and Al2O3, the major chemical component of Akahoya. The present results demonstrate the remarkable ability of Akahoya to remove phosphate and microbes, suggesting that Akahoya could be used for water purification processes. IMPORTANCE Although a safe and sufficient water supply is essential for the maintenance of hygienic conditions, a major challenge is to develop a comprehensive effective, sustainable, and cost-effective technological approach for the treatment and purification of contaminated water. In this study, we demonstrated that a novel volcanic soil, Akahoya, which has unlimited availability, is a highly effective adsorbent for a wide range of bacterial and viral pathogens, suggesting its potential as a sustainable resource for this purpose. It was suggested that the adsorption of microorganisms on Akahoya was mediated by phosphate groups present on the surface structures of microorganisms, which bind to the alumina component of Akahoya according to the phosphate concentration and pH of the liquid phase. The present findings highlight the exceptional ability of Akahoya to eliminate or reduce phosphate and microorganisms effectively in water purification processes, thus contributing to the development of efficient and sustainable solutions for addressing water pollution challenges.
DOI： 10.1128/aem.01007-24
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Complete genome sequence of the avian paramyxovirus serotype 9 strain duck/Miyazaki/128/2021 Reviewed International coauthorship
Mekata H., Yamamoto M., Matsui Y., Niazi A.M., Yamada K., Okabayashi T., Cha S.Y., Jang H.K.
Microbiology Resource Announcements 13 ( 9 ) e0006024 2024.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Microbiology Resource Announcements
Here, we report the complete genome sequence of the avian paramyxovirus serotype 9 strain duck/Miyazaki/128/2021, which was determined using the Illumina MiSeq platform. The position of the hemagglutinin-neuraminidase stop codon differed from that of the only other available completely sequenced prototype strain, duck/New York/22/1977.
DOI： 10.1128/mra.00060-24
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Puppies as the primary causal animal for human rabies cases: three-year prospective study of human rabies in the Philippines Reviewed International coauthorship
Saito N., Inton K.L., Mauhay J.D., Solante R.M., Guzman F.D., Yamada K., Kamiya Y., Saito-Obata M., Quiambao B.P., Yahiro T., Kimitsuki K., Nishizono A.
Frontiers in Microbiology 15 1425766 2024.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers in Microbiology
Introduction: While rabies remains a global concern, detailed studies on human rabies, particularly regarding causal animals and the reasons for not receiving postexposure prophylaxis (PEP), are lacking. Methods: We conducted a 3-year prospective study (October 2019–September 2022) at the Philippines’ largest rabies referral center. We interviewed patients with suspected rabies and their families. We used LN34 qRT-PCR and rapid fluorescent focus inhibition test on saliva samples. We also compared our findings with two retrospective studies at the same hospital. Results: We enrolled 151 patients, including 131 with potential rabies exposure. Similar to retrospective studies, the participants were predominantly males (75.5%), adults (76.8%), low-income individuals (91.4%), and rural dwellers (62.3%). The causal animals were mainly dogs (97.0%), with similar incubation periods, clinical symptoms, and a high proportion not receiving vaccines or immunoglobulins (93.2%). Most causal animals were owned by either the patients’ households or their neighbors (60.2%), with a significant proportion being puppies (58.8%). Most patients had knowledge of rabies; however, reasons for not seeking PEP included misconceptions about minor bites not causing rabies (51.3%), beliefs in traditional healers (33.9%), and economic constraints (22.6%). Despite completing the WHO regimen, two PEP failures were observed. LN34 qRT-PCR detected 98 positive cases (sensitivity, 64.9%; 95% CI 56.7–72.5). These strains belong to the Southeast Asia 4 subclade. Discussion: In conclusion, this study highlights the role of puppies as primary causal animals and the presence of misconceptions that preclude patients from acquiring PEP.
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Shirasaka Y., Yamada K., Etoh T., Noguchi K., Hasegawa T., Ogawa K., Kobayashi T., Nishizono A., Inomata M.
Pharmaceuticals 17 ( 1 ) 79 2024.1
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Pharmaceuticals
The outcomes of unresectable gastric cancer (GC) are unfavorable even with chemotherapy; therefore, a new treatment modality is required. The combination of an oncolytic virus and photodynamic therapy can be one of the promising modalities to overcome this. Mammalian orthoreovirus (MRV) is an oncolytic virus that has been used in clinical trials for several cancers. In this study, we developed and evaluated a recombinant MRV strain type 3 Dearing (T3D) that expresses membrane-targeting KillerRed (KRmem), a phototoxic fluorescent protein that produces cytotoxic reactive oxygen species upon light irradiation. KRmem was fused in-frame to the 3′ end of the σ2 viral gene in the S2 segment using a 2A peptide linker, enabling the expression of multiple proteins from a single transcript. RNA electrophoresis, Western blotting, and immunofluorescence analyses confirmed functional insertion of KRmem into the recombinant virus. The growth activity of the recombinant virus was comparable to that of the wild-type MRV in a cultured cell line. The recombinant virus infected two GC cell lines (MKN45P and MKN7), and a significant cytocidal effect was observed in MKN45P cells infected with the recombinant virus after light irradiation. Thus, recombinant MRV-expressing KRmem has the potential to serve as a novel treatment tool for GC.
DOI： 10.3390/ph17010079
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Development of a real-time RT-PCR system applicable for rapid and pen-side diagnosis of foot-and-mouth disease using a portable device, PicoGene® PCR1100. Reviewed International coauthorship
Matsui Y, Chottikamporn J, Ungvanijban S, Seeyo KB, Vitoonpong R, Suwankitwat N, Songkasupa T, Norimine J, Yamada K, Chintapitaksakul L, Misawa N
Journal of virological methods 319 114753 2023.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Virological Methods
Foot-and-mouth disease (FMD) is a highly contagious viral vesicular disease, causing devastating losses to the livestock industry. A diagnostic method that enables quick decisions is required to control the disease, especially in FMD-free countries. Although conventional real-time reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive method widely used for the diagnosis of FMD, a time lag caused by the transport of samples to a laboratory may allow the spread of FMD. Here, we evaluated a real-time RT-PCR system using a portable PicoGene PCR1100 device for FMD diagnosis. This system could detect the synthetic FMD viral RNA within 20 min with high sensitivity compared to a conventional real-time RT-PCR. Furthermore, the Lysis Buffer S for crude nucleic extraction improved the viral RNA detection of this system in a homogenate of vesicular epithelium samples collected from FMD virus-infected animals. Furthermore, this system could detect the viral RNA in crude extracts prepared using the Lysis Buffer S from the vesicular epithelium samples homogenized using a Finger Masher tube, which allows easy homogenization without any equipment, with a high correlation compared to the standard method. Thus, the PicoGene device system can be utilized for the rapid and pen-side diagnosis of FMD.
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ELGENDY Omnia, KITAHARA Go, YAMADA Kentaro, TANIGUCHI Shin, OSAWA Takeshi
Journal of Reproduction and Development advpub ( 0 ) 261 - 269 2023.8
Language：English Publishing type：Research paper (scientific journal) Publisher：The Society for Reproduction and Development
A high temperature-humidity index during summer has deleterious effects on mitochondrial function, reducing oocyte developmental competence. 5-Aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) are both known to support mitochondrial function and have strong anti-oxidant and anti-apoptotic activities. This study aimed to determine the mechanism of action of 5-ALA/SFC on oocyte quality. Bovine oocytes were collected from medium-sized follicles during summer (July–September, temperature-humidity index:76.6), cultured with 0, 1, 2, 4, and 8 µM 5-ALA with SFC at a molar ratio of 1:0.125, fertilized, and cultured for 10 days. The addition of 8/1 µM 5-ALA/SFC had a deleterious effect on oocyte cleavage rate in comparison with control oocytes, but did not affect the blastocyst rate, while 1/0.125 µM 5-ALA/SFC had a significantly higher increase in blastocyst rate than 8/1 µM 5-ALA/SFC. The addition of 1/0.125 and 2/0.25 µM 5-ALA/SFC improved oocyte quality by increasing the mitochondrial distribution pattern and metaphase-II oocytes, reducing reactive oxygen species and upregulating <i>nuclear factor erythroid-2-related factor 2</i>, <i>heme oxygenase-1</i>, and <i>superoxide dismutase-1</i> in oocytes, and <i>nuclear factor erythroid-2-related factor 2</i> and <i>mitochondrial transcription factor A</i> in cumulus cells. These results indicate that 1/0.125 and 2/0.25 µM 5-ALA/SFC may support oocyte quality and developmental competence and provide anti-oxidant actions in cumulus-oocyte complexes.
DOI： 10.1262/jrd.2023-038
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Kaneko C., Shinohara A., Kikuchi T., Tokuda A., Irie T., Yamada K., Misawa N., Yoshida A.
Mammalian Biology 103 ( 4 ) 363 - 373 2023.8
Language：English Publishing type：Research paper (scientific journal) Publisher：Mammalian Biology
The Japanese badger (Meles anakuma) and the Japanese raccoon dog (Nyctereutes procyonoides viverrinus) are common wild animals in Japan. They are opportunistic omnivores that share similar foods and environments. Previous study has reported a difference in the isolation rates of a specific genus of bacteria from fecal samples of these animals inhabiting the same areas in Japan. This study hypothesized that badgers and raccoon dogs have different gut microbiota, which leads to different metabolisms of nutrients despite their similar ecological niches. This study aimed to verify this hypothesis by comparatively analyzing the gut microbiota of these species. Bacterial 16S rRNA amplicon sequencing analysis was performed using colonic contents collected from 12 badgers and 12 raccoon dogs. As a result, the gut microbiota in badgers and raccoon dogs were completely distinct. Phylum Firmicutes was the most abundant, followed by Proteobacteria, almost without Bacteroidota in badgers. In contrast, Firmicutes and Bacteroidota were abundant in raccoon dogs. Many species-characteristic bacterial taxonomic features were identified in each animal’s gut microbiota. Moreover, raccoon dogs exhibited richer and more diverse species in their gut microbiota than badgers. This study revealed that badgers and raccoon dogs have distinct gut microbiota, which possibly drives different metabolism, though they share similar foods and environments. Considering anatomical feature that badger lacks a cecum which raccoon dog has, the distinct structure of gut microbiota in badger and raccoon dog could be attributed to the differences in the physical structure of the gastrointestinal tract, even though diet and inhabiting environments are quite similar.
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Molecular Analysis of Rabies Virus Using RNA Extracted from Used Lateral Flow Devices Reviewed International coauthorship
Mauhay J.D., Saito N., Kimitsuki K., Mananggit M.R., Cruz J.L., Lagayan M.G., Garcia A.M., Lacanilao P.M., Yamada K., Saito-Obata M., Manalo D.L., Demetria C.S., Quiambao B.P., Nishizono A.
Journal of clinical microbiology 61 ( 3 ) e0154322 2023.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of clinical microbiology
Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas to a central laboratory is challenging owing to biohazard risks and decomposability. We investigated the utility of used lateral flow devices (LFDs) for subsequent molecular analysis and assessed the necessary storage temperatures. Using RNA extracted from used LFD strips, we performed conventional reverse transcription-PCR (RT-PCR) using an LN34 primer set to amplify short fragments (165 bp) for rabies virus detection and the P1-304 primer set to amplify long fragments of the entire N gene amplicon (1,506 bp) for phylogenetic analysis. Among 71 used LFDs stored in a refrigerator and 64 used LFDs stored at room temperature, the LN34 assay showed high sensitivities (96.2% and 100%, respectively) for the diagnosis of rabies, regardless of the storage temperature. A significant reduction in the sensitivity of rabies diagnosis was observed when using the P1-304 primer set for used LFDs stored at room temperature compared to those stored at refrigeration temperature (20.9% versus 100%; P < 0.05). Subsequent sequencing and phylogenetic analysis were successfully performed using the amplicons generated by the P1-304 RT-PCR assays. Used LFDs are thus promising resources for rabies virus RNA detection and sequence analysis. Virus detection via RT-PCR, amplifying a short fragment, was possible regardless of the storage temperature of the used LFDs. However, refrigerated storage is recommended for RT-PCR amplification of long fragments for phylogenetic analysis.
DOI： 10.1128/jcm.01543-22
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Nosocomial Severe Fever with Thrombocytopenia Syndrome in Companion Animals, Japan, 2022 Reviewed
Mekata H., Umeki K., Yamada K., Umekita K., Okabayashi T.
Emerging Infectious Diseases 29 ( 3 ) 614 - 617 2023.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Emerging Infectious Diseases
In Japan, 2 cats that underwent surgery in a room where a sick dog had been euthanized became ill within 9 days of surgery. Severe fever with thrombocytopenia syndrome virus was detected in all 3 animals; nucleotide sequence identity was 100%. Suspected cause was an uncleaned pulse oximeter probe used for all patients.
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Mekata Hirohisa, Kawaguchi Takeshi, Iwao Kosho, Umeki Kazumi, Yamada Kentaro, Umekita Kunihiko, Okabayashi Tamaki
Japanese Journal of Infectious Diseases advpub ( 0 ) 211 - 214 2023.1
Language：English Publishing type：Research paper (scientific journal) Publisher：National Institute of Infectious Diseases
Severe fever with thrombocytopenia syndrome (SFTS) is caused by the severe fever with thrombocytopenia syndrome virus (SFTSV). Although SFTS is a fatal tick-borne zoonosis, it can infect humans without tick bite exposure. Recently, direct transmission of SFTSV from companion pets to humans has become a major problem. We present a case of SFTSV transmission from a dead community cat to a woman who buried the cat in Miyazaki Prefecture, Japan. The community cat died without a diagnosis of SFTS, and the woman buried it without taking any precautions. She developed symptoms of SFTS 9 days later. The woman tested positive for SFTS viral RNA and anti-SFTSV antibodies. The cat’s carcass was exhumed, and tissue samples were collected to confirm the viral infection. Numerous copies of viral RNA were detected. The SFTSV M segment sequences in the cat and the woman were 100% homologous. The woman claimed that she had touched blood that had leaked from the cat’s body while burying it. However, she could have been infected while transporting the cat to the animal hospital. This study highlights the risk of SFTSV infection from contact with sick or dead community cats.
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Fine Particle Adsorption Capacity of Volcanic Soil from Southern Kyushu, Japan. Reviewed International coauthorship
Misawa N, Yasui K, Sakai K, Kobayashi T, Nagahama H, Haraguchi T, Sasaki S, Torrung V, Luangtongkum T, Taniguchi T, Yamada K, Minamimagari M, Usami T, Kinoshita H
Nanomaterials (Basel, Switzerland) 13 ( 3 ) 2023.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Nanomaterials
“Akahoya” is a volcanic soil classified as a special soil deposited in Kyushu, Japan. Many of its properties are not yet clearly understood. We found that Akahoya had the potential to adsorb bacteria in cattle feces, which prompted us to investigate its material properties and perform experiments to comprehensively evaluate its adsorption performance for various fine particles such as acidic and basic dyes, NOx/SOx gas, and phosphoric acid ions, in addition to bacteria. Akahoya had a very high specific surface area owing to the large number of nanometer-sized pores in its structure; it exhibited a high adsorption capacity for both NO2 and SO2. Regarding the zeta potential of Akahoya, the point of zero charge was approximately pH 7.0. The surface potential had a significant effect on the adsorption of acidic and basic dyes. Akahoya had a very high cation exchange capacity when the sample surface was negatively charged and a high anion exchange capacity when the sample surface was positively charged. Akahoya also exhibited a relatively high adsorption capacity for phosphoric acid because of its high level of Al2O3, and the immersion liquid had a very high Al ion concentration. Finally, filtration tests were performed on Escherichia coli suspension using a column filled with Akahoya or another volcanic soil sample. The results confirmed that the Escherichia coli adhered on the Akahoya sample. The results of the Escherichia coli release test, after the filtration test, suggested that this adhesion to Akahoya could be phosphorus-mediated.
DOI： 10.3390/nano13030568
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SODA Kosuke, MEKATA Hirohisa, USUI Tatsufumi, ITO Hiroshi, MATSUI Yuto, YAMADA Kentaro, YAMAGUCHI Tsuyoshi, ITO Toshihiro
Journal of Veterinary Medical Science 85 ( 11 ) 1180 - 1189 2023
Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE SOCIETY OF VETERINARY SCIENCE
In the winter of 2021–2022, multiple subtypes (H5N8 and H5N1) of high pathogenicity avian influenza viruses (HPAIVs) were confirmed to be circulating simultaneously in Japan. Here, we phylogenetically and antigenically analyzed HPAIVs that were isolated from infected wild birds, an epidemiological investigation of affected poultry farms, and our own active surveillance study. H5 subtype hemagglutinin (HA) genes of 32 representative HPAIV isolates were classified into clade 2.3.4.4b lineage and subsequently divided into three groups (G2a, G2b, and G2d). All H5N8 HPAIVs were isolated in early winter and had HA genes belonging to the G2a group. H5N1 HPAIVs belong to the G2b and G2d groups. Although G2b viruses were widespread throughout the season, G2d viruses endemically circulated in Northeast Japan after January 2022. Deep sequence analysis showed that the four HPAIVs isolated at the beginning of winter had both N8 and N1 subtypes of neuraminidase genes. Environmental water-derived G2a HPAIV, A/water/Tottori/NK1201-2/2021 (H5N8), has unique polymerase basic protein 1 and nucleoprotein genes, similar to those of low pathogenicity avian influenza viruses (LPAIVs). These results indicate that multiple H5 HPAIVs and LPAIVs disseminated to Japan via transboundary winter migration of wild birds, and HPAIVs with novel gene constellations could emerge in these populations. Cross-neutralization test revealed that G2a H5N8 HPAIVs were antigenically distinct from a G2b H5N1 HPAIV, suggesting that antibody pressure in wild birds was involved in the transition of the HPAIV groups during the season.
DOI： 10.1292/jvms.23-0121
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Kaneko Chiho, Mekata Hirohisa, Irie Takao, Yamada Kentaro, Misawa Naoaki, Umekita Kunihiko, Okabayashi Tamaki, Umeki Kazumi, Putu Eka Sudaryatma
Ticks and tick-borne diseases 14 ( 2 ) 102115 2022.12
Language：English Publishing type：Research paper (scientific journal)
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Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720. Reviewed International journal
Katsuhiro Ogawa, Kentaro Yamada, Tsuyoshi Etoh, Masahiro Kitagawa, Yoshinori Shirasaka, Kazuko Noguchi, Takeshi Kobayashi, Akira Nishizono, Masafumi Inomata
Journal of virological methods 308 114574 - 114574 2022.10
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
Fluorescence-guided surgery (FGS) is a useful method for removing invasive tumor tissues. For this, near-infrared (NIR) fluorescence probes are suitable for visualizing cancer cells due to their low autofluorescence, and an oncolytic mammalian orthoreovirus (MRV) expressing an NIR fluorescent protein is expected to be a novel tool for FGS. In this study, we identified the optimal insertion site of the NIR fluorescent protein gene iRFP720 (915 nt) in the MRV genome. We constructed genome plasmids for the L1, M1, and S2 segments, where a gene cassette comprising iRFP720 and T2A self-cleaving peptide was inserted in the 5' or 3' region of each segment. Through virus recovery, the recombinant MRV with the gene cassette at the M1 segment's 3' end, T3D-L(M1/3'iRFP720), was capable of replication and passaging with bright NIR fluorescence. However, the replication of T3D-L(M1/3'iRFP720) was approximately 1,000-fold lower than that of the wild-type virus. T3D-L(M1/3'iRFP720) production improved due to the transfection of a fusion-associated small transmembrane protein gene of fusogenic reovirus. Further, fluorescence signals were detected in T3D-L(M1/3'iRFP720)-infected human gastric and pancreatic cancer cells. Thus, the M1 segment's 3' end tolerates the expression of the long iRFP720 gene, which may propel the development of recombinant MRV vectors for FGS.
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Kojima I, Onomoto K, Zuo W, Ozawa M, Okuya K, Naitou K, Izumi F, Okajima M, Fujiwara T, Ito N, Yoneyama M, Yamada K, Nishizono A, Sugiyama M, Fujita T, Masatani T
Journal of virology 96 ( 18 ) e0081022 2022.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Virology
Stress granules (SGs) are dynamic structures that store cytosolic messenger ribonucleoproteins. SGs have recently been shown to serve as a platform for activating antiviral innate immunity; however, several pathogenic viruses suppress SG formation to evade innate immunity. In this study, we investigated the relationship between rabies virus (RABV) virulence and SG formation, using viral strains with different levels of virulence. We found that the virulent Nishigahara strain did not induce SG formation, but its avirulent offshoot, the Ni-CE strain, strongly induced SG formation. Furthermore, we demonstrated that the amino acid at position 95 in the RABV matrix protein (M95), a pathogenic determinant for the Nishigahara strain, plays a key role in inhibiting SG formation, followed by protein kinase R (PKR)-dependent phosphorylation of the a subunit of eukaryotic initiation factor 2a (eIF2a). M95 was also implicated in the accumulation of RIG-I, a viral RNA sensor protein, in SGs and in the subsequent acceleration of interferon induction. Taken together, our findings strongly suggest that M95-related inhibition of SG formation contributes to the pathogenesis of RABV by allowing the virus to evade the innate immune responses of the host.
DOI： 10.1128/jvi.00810-22
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Distribution of Severe Fever with Thrombocytopenia Syndrome Virus and Antiviral Antibodies in Wild and Domestic Animals in Oita Prefecture, Japan. Reviewed International journal
Takehiro Hashimoto, Takaaki Yahiro, Kentaro Yamada, Kazunori Kimitsuki, Minami W Okuyama, Akiko Honda, Miki Kato, Hiroshi Narimatsu, Kazufumi Hiramatsu, Akira Nishizono
The American journal of tropical medicine and hygiene 106 ( 5 ) 1547 - 1551 2022.2
Language：English Publishing type：Research paper (scientific journal)
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The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated. Reviewed International journal
Takashi Nishiyama, Koji Umezawa, Kentaro Yamada, Masaharu Takahashi, Satoshi Kunita, Mulyanto, Isao Kii, Hiroaki Okamoto
Pathogens (Basel, Switzerland) 11 ( 1 ) 2022.1
Language：English Publishing type：Research paper (scientific journal)
The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.
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Comprehensive exploration of chemical space using trisubstituted carboranes. Reviewed International coauthorship International journal
Yasunobu Asawa, Saki Hatsuzawa, Atsushi Yoshimori, Kentaro Yamada, Akira Katoh, Hiroyuki Kouji, Hiroyuki Nakamura
Scientific reports 11 ( 1 ) 24101 - 24101 2021.12
Language：English Publishing type：Research paper (scientific journal)
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Lateral flow devices for samples collected by straw sampling method for postmortem canine rabies diagnosis. Reviewed International journal
Milagros R Mananggit, Daria L Manalo, Nobuo Saito, Kazunori Kimitsuki, Alyssa Marie G Garcia, Patricia Mae T Lacanilao, Joely T Ongtangco, Cornhlo R Velasco, Maria Victoria A Del Rosario, Maria Glofezita O Lagayan, Kentaro Yamada, Chun-Ho Park, Satoshi Inoue, Motoi Suzuki, Mariko Saito-Obata, Yasuhiko Kamiya, Catalino S Demetria, Beatriz P Quiambao, Akira Nishizono
PLoS neglected tropical diseases 15 ( 12 ) e0009891 2021.12
Language：English Publishing type：Research paper (scientific journal)
The direct fluorescent antibody test (dFAT) using brain sample after opening the skull is the standard rabies diagnostic test in animal rabies. However, it is not feasible in many resource-limited settings. Lateral flow devices (LFD) combined with a simple sampling methodology is quicker, simpler, and less hazardous than the standard test and can be a useful tool. We conducted a prospective on-site study to evaluate the diagnostic accuracy of the LFD with the straw sampling method compared with that of the dFAT with the skull opening procedure for post-mortem canine rabies diagnosis. We collected 97 rabies-suspected animals between December 1, 2020 and March 31, 2021. Among the 97 samples, 53 and 50 cases were positive tests for dFAT and LFD, respectively. The sensitivity and specificity of LFD with straw sampling method were 94.3% (95% confidence interval [CI], 84.3-98.8%) and 100% (95% CI, 92.0-100%), respectively. The performance of LFD by the straw sampling method showed relatively high sensitivity and 100% specificity compared with that of dFAT performed on samples collected after opening the skull. This methodology can be beneficial and is a strong tool to overcome limited animal surveillance in remote areas. However, because of our limited sample size, more data using fresh samples on-site and the optimizations are urgently needed for the further implementation in endemic areas.
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Background and descriptive features of rabies-suspected animals in Central Luzon, Philippines. Reviewed International coauthorship
Milagros R Mananggit, Kazunori Kimitsuki, Nobuo Saito, Alyssa Marie G Garcia, Patricia Mae T Lacanilao, Joely T Ongtangco, Cornelio R Velasco, Maria Victoria D Rosario, Maria Glofezita O Lagayan, Kentaro Yamada, Chun-Ho Park, Satoshi Inoue, Motoi Suzuki, Mariko Saito-Obata, Yasuhiko Kamiya, Daria L Manalo, Catalino S Demetria, Beatriz P Quiambao, Akira Nishizono
Tropical medicine and health 49 ( 1 ) 59 - 59 2021.12
Language：English Publishing type：Research paper (scientific journal)
BACKGROUND: The Philippines is one of the major endemic countries for canine rabies in Southeast Asia. However, detailed description and analysis of laboratory-confirmed animal rabies are limited. Highly accurate surveillance requires a thorough understanding of the target area-specific problems and obstacles. Therefore, we aim to describe and analyze the rabies suspect animals in Central Luzon, Philippines, to clarify the characteristics of management and clinical signs by conducting interviews with the owners. METHODS: We prospectively collected information on the rabies suspect animals submitted to the Regional animal laboratory in Central Luzon through passive laboratory-based rabies surveillance between 1st April 2019 and 30th September 2020. We performed active interviews directly or telephonically with the owner. The direct fluorescent antibody test was performed on the hippocampus, brain stem, and cerebellum for laboratory confirmation. Descriptive statistics were used to characterize the number of rabies cases according to management methods and characteristics of suspected animals during the observation period. Clinical symptoms of suspected rabid animals were analyzed by univariate logistic regression analysis. RESULTS: There were 292 sample submissions during the study period. Of these, 160 were positive for dFAT. Samples of pet animals (85.3%) provided by owners or their acquaintances (59.2%) accounted for the majority of laboratory confirmed cases. Case mapping showed that more rabies-suspected cases were sent from areas near the regional laboratory than from those far from the laboratory, despite the incidence of rabies being high in these areas. The management and clinical symptoms of 227 animal cases showed that most owners were managing their animals at home and were allowing them to roam outside (69.6%) and be unvaccinated (78.9%). Rabid animals were more likely to manifest aimless running, restlessness, and agitation. CONCLUSIONS: Our study provided some features of animals with laboratory-confirmed rabies in Central Luzon. However, most of the samples were submitted from areas near the rabies diagnosis laboratory, and the number of samples submitted from remote areas was low. To improve the surveillance capacity, it is necessary to increase sample submissions from remote areas.
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Validation of serum apolipoprotein A1 in rabies virus-infected mice as a biomarker for the preclinical diagnosis of rabies. Reviewed International journal
Kentaro Yamada, Koji Kuribayashi, Naotaka Inomata, Kazuko Noguchi, Kazunori Kimitsuki, Catalino S Demetria, Nobuo Saito, Satoshi Inoue, Chun-Ho Park, Ryo Kaimori, Motoi Suzuki, Mariko Saito-Obata, Yasuhiko Kamiya, Daria L Manalo, Beatriz P Quiambao, Akira Nishizono
Microbiology and immunology 65 ( 10 ) 438 - 448 2021.10
Language：English Publishing type：Research paper (scientific journal)
Rabies is a type of acute fetal encephalitis caused by rabies virus (RABV). While it becomes incurable after symptom onset, it can be prevented by post-exposure prophylaxis (PEP) during the long incubation period. While preclinical diagnosis aids the appropriate PEP administration, it is mostly nonfeasible owing to the absence of viremia or a specific antibody response during the incubation period. Here, an attempt was made to identify a serum biomarker for the preclinical diagnosis of rabies. Using the serum from a mouse inoculated intramuscularly (i.m.) with 5 × 105 focus-forming units (FFU) of recombinant RABV expressing red firefly luciferase (1088/RFLuc) immediately before symptom onset, two-dimensional differential gel electrophoresis was conducted, followed by mass spectrometry, and it was confirmed that apolipoprotein A1 (ApoA1) was up-regulated. ELISA showed that the serum ApoA1 and specific antibody levels increased during the incubation period and on the day of symptom onset. Since a lower infectious dose can be used to induce the unstable and long incubation period generally observed in natural infection, the ApoA1 level in mice inoculated i.m. with 103 FFU of 1088/RFLuc was examined by monitoring viral dynamics using in vivo imaging. The serum ApoA1 and specific antibody levels were up-regulated in 50% and 58.3% of mice exhibiting robust RABV replication, respectively, but not in mice exhibiting weak RABV replication. In addition, it was reported that ApoA1 was found to be a biomarker for neuronal damage. Additional biomarker candidates will be needed for the effective preclinical diagnosis of rabies.
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Elisabet Tangkonda, Meiko Kubo, Satoshi Sekiguchi, Taisuke Shinki, Satomi Sasaki, Kentaro Yamada, Takako Taniguchi, Torrung Vetchapitak, Naoaki Misawa
The Journal of veterinary medical science 83 ( 8 ) 1306 - 1314 2021.8
Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE SOCIETY OF VETERINARY SCIENCE
Workers in poultry abattoirs may be frequently exposed to Campylobacter jejuni, which is a leading cause of bacterial food poisoning in Japan. The present study was conducted to measure the titers of IgG and IgA antibodies against C. jejuni among 104 female workers in a chicken processing plant in Miyazaki prefecture, Japan. Information regarding habitual ingestion of raw chicken meat and potential occupational risk factors was collected using a questionnaire. Acid extracts of four C. jejuni strains representing the genotypes most dominant in Miyazaki were used as antigens. The levels of both immunoglobulins measured by ELISA were not correlated with ingestion of edible raw chicken meat, the amount consumed in one sitting, or its frequency. Although age was correlated with antibody levels, the length of employment was not. Furthermore, the IgG and IgA levels in workers at the evisceration step were significantly higher than those at other locations in the plant. To identify the bacterial proteins recognized by the workers' IgG and IgA antibodies, Western blotting followed by LC/MS was conducted. Flagellin was identified as the common protein recognized in the sera of workers for whom ELISA demonstrated both the highest and lowest antibody levels. We concluded that the titers of IgG and IgA against C. jejuni in workers at the processing plant had been increased by occupational exposure to Campylobacter, regardless of raw chicken meat ingestion.
DOI： 10.1292/jvms.21-0244
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Synthesis of Three-Dimensional (Di)Azatricyclododecene Scaffold and Its Application to Peptidomimetics. Reviewed International journal
Kohei Umedera, Taiki Morita, Atsushi Yoshimori, Kentaro Yamada, Akira Katoh, Hiroyuki Kouji, Hiroyuki Nakamura
Chemistry (Weinheim an der Bergstrasse, Germany) 27 ( 46 ) 11888 - 11894 2021.8
Language：English Publishing type：Research paper (scientific journal)
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Evaluation of the diagnostic accuracy of lateral flow devices as a tool to diagnose rabies in post-mortem animals. Reviewed International coauthorship International journal
Kazunori Kimitsuki, Nobuo Saito, Kentaro Yamada, Chun-Ho Park, Satoshi Inoue, Motoi Suzuki, Mariko Saito-Obata, Yasuhiko Kamiya, Daria L Manalo, Catalino S Demetria, Milagros R Mananggit, Beatriz P Quiambao, Akira Nishizono
PLoS neglected tropical diseases 14 ( 11 ) e0008844 2020.11
Language：English Publishing type：Research paper (scientific journal)
Implementation of lateral flow devices (LFDs) for rabies antigen detection is expected to improve surveillance through the efficient detection of rabid animals in resource-limited settings; however, the use of LFDs for diagnosis remains controversial because some commercially available kits show low sensitivity. Therefore, we compared the diagnostic efficacy of three LFDs (ADTEC, Bionote, and Elabscience kits) paralleled with the direct fluorescent antibody test (dFAT) using fresh samples and investigated the diagnostic accuracies. To do so, we evaluated rabies-suspected samples submitted to the Regional Animal Disease Diagnostic Laboratory III, Philippines. Furthermore, we conducted real-time RT-PCR and sequencing to measure the accuracy of field laboratory diagnosis. The total number of animals submitted during this study period was 184 cases, including negative control samples. Of these, 53.9% (84 cases) were positive in the dFAT. Dogs were the most common rabies-suspected animal (n = 135). The sensitivities of the ADTEC and Bionote kits were 0.88 (74 cases) and 0.95 (80 cases), respectively. The specificity of both kits was 1.00 (100 cases). Furthermore, the sensitivity and specificity of the ADTEC kit after directly homogenizing the samples in assay buffer without dilution in phosphate-buffered saline (ADTEC kit DM) were 0.94 (79 cases) and 1.00 (100 cases), respectively. By contrast, there were no positive results using the Elabscience kit among all dFAT-positive samples. The sensitivity and specificity of LFDs make these tests highly feasible if properly used. Therefore, LFD tests can be used to strengthen the surveillance of rabies-infected animals in endemic and resource-limited settings.
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Genetic and Phenotypic Characterization of a Rabies Virus Strain Isolated from a Dog in Tokyo, Japan in the 1940s. Reviewed International journal
Tatsuki Takahashi, Maho Inukai, Michihito Sasaki, Madlin Potratz, Supasiri Jarusombuti, Yuji Fujii, Shoko Nishiyama, Stefan Finke, Kentaro Yamada, Hiroki Sakai, Hirofumi Sawa, Akira Nishizono, Makoto Sugiyama, Naoto Ito
Viruses 12 ( 9 ) 2020.9
Language：English Publishing type：Research paper (scientific journal)
The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only existent Japanese field strain (street strain). Although this strain potentially provides a useful model to study rabies pathogenesis, little is known about its genetic and phenotypic properties. Notably, this strain underwent serial passages in rodents after isolation, indicating the possibility that it may have lost biological characteristics as a street strain. In this study, to evaluate the utility of the Koma strain for studying rabies pathogenesis, we examined the genetic properties and in vitro and in vivo phenotypes. Genome-wide genetic analyses showed that, consistent with previous findings from partial sequence analyses, the Koma strain is closely related to a Russian street strain within the Arctic-related phylogenetic clade. Phenotypic examinations in vitro revealed that the Koma strain and the representative street strains are less neurotropic than the laboratory strains. Examination by using a mouse model demonstrated that the Koma strain and the street strains are more neuroinvasive than the laboratory strains. These findings indicate that the Koma strain retains phenotypes similar to those of street strains, and is therefore useful for studying rabies pathogenesis.
DOI： 10.3390/v12090914
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狂犬病発症前診断用バイオマーカー候補の探索と評価
西園 晃, 齊藤 信夫, 鈴木 基, 山田 健太郎
感染症学雑誌 94 ( 臨増 ) 329 - 329 2020.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：(一社)日本感染症学会
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フィリピンにおける狂犬病迅速診断キットの有用性評価中間解析(ヒト・動物) Reviewed International coauthorship
齊藤 信夫, 鈴木 基, 西園 晃, 君付 和範, 山田 健太郎, 齊藤 麻理子[小畑], 朴 天鎬, 井上 智, Quiambao Beatriz
感染症学雑誌 94 ( 臨増 ) 322 - 322 2020.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：(一社)日本感染症学会
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狂犬病発症前診断用バイオマーカー候補の探索と評価
西園 晃, 齊藤 信夫, 鈴木 基, 山田 健太郎
感染症学雑誌 94 ( 臨増 ) 329 - 329 2020.3
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：(一社)日本感染症学会
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Reevaluation of the efficacy of favipiravir against rabies virus using in vivo imaging analysis.
Kentaro Yamada, Kazuko Noguchi, Kazunori Kimitsuki, Ryo Kaimori, Nobuo Saito, Takashi Komeno, Nozomi Nakajima, Yousuke Furuta, Akira Nishizono
Antiviral research 172 104641 - 104641 2019.12
Language：Japanese Publishing type：Research paper (scientific journal)
Rabies virus (RABV) is a highly neurotropic virus and the causative agent of rabies, an encephalitis with an almost 100% case-fatality rate that remains incurable after the onset of symptoms. Favipiravir (T-705), a broad-spectrum antiviral drug against RNA viruses, has been shown to be effective against RABV in vitro but ineffective in vivo. We hypothesized that favipiravir is effective in infected mice when RABV replicates in the peripheral tissues/nerves but not after virus neuroinvasion. We attempted to clarify this point in this study using in vivo bioluminescence imaging. We generated a recombinant RABV from the field isolate 1088, which expressed red firefly luciferase (1088/RFLuc). This allowed semiquantitative detection and monitoring of primary replication at the inoculation site and viral spread in the central nervous system (CNS) in the same mice. Bioluminescence imaging revealed that favipiravir (300 mg/kg/day) treatment commencing 1 h after intramuscular inoculation of RABV efficiently suppressed viral replication at the inoculation site and the subsequent replication in the CNS. However, virus replication in the CNS was not inhibited when the treatment began 2 days after inoculation. We also found that higher doses (600 or 900 mg/kg/day) of favipiravir could suppress viral replication in the CNS even when administration started 2 days after inoculation. These results support our hypothesis and suggest that a highly effective drug-delivery system into the CNS and/or the enhancement of favipiravir conversion to its active form are required to improve favipiravir treatment of rabies. Furthermore, the bioluminescence imaging system established in this study will facilitate the development of treatment for symptomatic rabies.
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Near-infrared fluorescent protein iRFP720 is optimal for &ITin vivo&IT fluorescence imaging of rabies virus infection Reviewed
Minori Isomura, Kentaro Yamada, Kazuko Noguchi, Akira Nishizono
JOURNAL OF GENERAL VIROLOGY 98 ( 11 ) 2689 - 2698 2017.11
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：MICROBIOLOGY SOC
In vivo imaging is a noninvasive method that enables real-time monitoring of viral infection dynamics in a small animal, which allows a better understanding of viral pathogenesis. In vivo bioluminescence imaging of virus infection is widely used but, despite its advantage over bioluminescence that no substrate administration is required, fluorescence imaging is not used because of severe autofluorescence. Recently, several far-red and near-infrared (NIR) fluorescent proteins (FPs) have been developed and shown to be useful for whole-body fluorescence imaging. Here, we report comparative testing of far-red and NIR FPs in the imaging of rabies virus (RABV) infection. Using the highly neuroinvasive 1088 strain, we generated recombinant RABV that expressed FPs such as Katushka2S, E2-Crimson, iRFP670 or iRFP720. After intracerebral inoculation to nude mice, the 1088 strain expressing iRFP720, the most red-shifted FP, was detected the earliest with the highest signal-to-noise ratio using a filter set for >700 nm, in which the background signal level was very low. Furthermore, we could also track viral dissemination from the spinal cord to the brain in nude mice after intramuscular inoculation of iRFP720-expressing 1088 into the hind limb. Hence, we conclude that the NIR FP iRFP720 used with a filter set for >700 nm is useful for in vivo fluorescence imaging not only for RABV infection but also for other virus infections. Our findings will also be useful for developing dual-optical imaging of virus-host interaction dynamics using bioluminescence reporter mice for inflammation imaging.
DOI： 10.1099/jgv.0.000950
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A Comparative Study of the RAPINA and the Virus-Neutralizing Test (RFFIT) for the Estimation of Antirabies-Neutralizing Antibody Levels in Dog Samples Reviewed International coauthorship
D. L. Manalo, K. Yamada, I. Watanabe, M. E. G. Miranda, S. M. D. Lapiz, E. Tapdasan, W. Petspophonsakul, S. Inoue, P. Khawplod, A. Nishizono
ZOONOSES AND PUBLIC HEALTH 64 ( 5 ) 355 - 362 2017.8
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：WILEY
The mass vaccination of dogs against rabies is a highly rational strategy for interrupting the natural transmission of urban rabies. According to the World Organization for Animal Health (OIE) and the World Health Organization (WHO), the immunization of at least 70% of the total dog population minimizes the risk of endemic rabies. Knowledge of the virus-neutralizing antibody (VNA) level against the rabies virus (RABV) is required to evaluate protective immunity and vaccine coverage of dogs in the field. The rapid focus fluorescent inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test are recommended by OIE and WHO to determine the VNA levels in serum. However, these tests are cell culture based and require the use of live viruses and specialized equipment. The rapid neutralizing antibody test (RAPINA) is a novel, immunochromatographic test that uses inactivated virus to estimate the VNA level qualitatively. It is a simple, rapid and inexpensive, although indirect, assay for the detection of VNA levels. The RAPINA has shown good positive and negative predictive values and a high concordance with the RFFIT results. In this study, we compared the performance of the two tests for evaluating the vaccination status of dogs in the Philippines, Thailand and Japan. A total of 1135 dog sera were analysed by the RAPINA and compared to the VNA levels determined by the RFFIT. The overall positive and negative predictive values of the RAPINA were 96.2-99.3% and 84.5-94.8%, respectively, with a concordance (kappa) of 0.946-0.97 among the three countries. The RAPINA results were highly homologous and reproducible among different laboratories. These results suggest that this test is appropriate to survey vaccination coverage in countries with limited resources.
DOI： 10.1111/zph.12313
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Pathological lesions in the central nervous system and peripheral tissues of ddY mice with street rabies virus (1088 strain) Reviewed
Kazunori Kimitsuki, Kentaro Yamada, Nozomi Shiwa, Satoshi Inoue, Akira Nishizono, Chun-Ho Park
JOURNAL OF VETERINARY MEDICAL SCIENCE 79 ( 6 ) 970 - 978 2017.6
Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN SOC VET SCI
Most studies on rabies virus pathogenesis in animal models have employed fixed rabies viruses, and the results of those employing street rabies viruses have been inconsistent. Therefore, to clarify the pathogenesis of street rabies virus (1088 strain) in mice, 10(6) focus forming units were inoculated into the right hindlimb of ddY mice (6 weeks, female). At 3 days postinoculation (DPI), mild inflammation was observed in the hindlimb muscle. At 5 DPI, ganglion cells in the right lumbosacral spinal dorsal root ganglia showed chromatolysis. Axonal degeneration and inflammatory cells increased with infection progress in the spinal dorsal horn and dorsal root ganglia. Right hindlimb paralysis was observed from 7 DPI, which progressed to quadriparalysis. However, no pathological changes were observed in the ventral horn and root fibers of the spinal cord. Viral antigen was first detected in the right hindlimb muscle at 3 DPI, followed by the right lumbosacral dorsal root ganglia, dorsal horn of spinal cord, left red nuclei, medulla oblongata and cerebral cortex (M1 area) at 5 DPI. These results suggested that the 1088 virus ascended the lumbosacral spinal cord via mainly afferent fibers at early stage of infection and moved to cerebral cortex (M1 area) using descending spinal tract. Additionally, we concluded that significant pathological changes in mice infected with 1088 strain occur in the sensory tract of the spinal cord; this selective susceptibility results in clinical features of the disease.
DOI： 10.1292/jvms.17-0028
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Increased pathogenicity of rabies virus due to modification of a non-coding region Reviewed International coauthorship
Phatthamon Virojanapirom, Kentaro Yamada, Pakamatz Khawplod, Akira Nishizono, Thiravat Hemachudha
ARCHIVES OF VIROLOGY 161 ( 11 ) 3255 - 3261 2016.11
Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER WIEN
Sub-passaging of QS-05, a street rabies virus (RABV) isolate, in non-neuronal cells resulted in a virus with higher pathogenicity, QS-BHK-P7. Four full-length cDNA plasmids were constructed and the corresponding recombinant viruses were recovered: rQS-05, rQS-BHK-P7 and rQS05-2475G/rQS-BHK-P7-2475A (made by switching of intergenic P-M between these two backbones). rQS-BHK-P7-2475 A virus had eight instead of seven adenosines in its poly(A) sequence. Interestingly, mutant viruses with 6 or 8 adenosines infected more neuroblastoma cells than their parental ones. Mice that were infected intracerebrally and intramuscularly with rQS05-2475G and rQS-BHK-P7 exhibited highest mortality. However, mice infected with rQS-BHK-P7-2475AA had the shortest survival time. This study demonstrates that modifications in the non-coding region may play a role in determining the virulence of RABV.
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Localization of the rabies virus antigen in Merkel cells in the follicle-sinus complexes of muzzle skins of rabid dogs
Taichi Shimatsu, Harumi Shinozaki, Kazunori Kimitsuki, Nozomi Shiwa, Daria L. Manalo, Rodolfo C. Perez, Joselito E. Dilig, Kentaro Yamada, Hassadin Boonsriroj, Satoshi Inoue, Chun-Ho Park
JOURNAL OF VIROLOGICAL METHODS 237 40 - 46 2016.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
The direct fluorescent antibody test (dFAT) on fresh brain tissues is the gold standard for rabies virus antigen detection in dogs. However, this method is laborious and holds a high risk of virus exposure for the experimenter. Skin biopsies are useful for the diagnosis of humans and animals. In mammals, the tactile hair, known as the follicle-sinus complex (FSC), is a specialized touch organ that is abundant in the muzzle skin. Each tactile hair is equipped with more than 2,000 sensory nerve endings. Therefore, this organ is expected to serve as an alternative postmortem diagnostic material. However, the target cells and localization of rabies virus antigen in the FSCs remain to be defined. In the present study, muzzle skins were obtained from 60 rabid dogs diagnosed with rabies by dFAT at the Research Institute of Tropical Medicine in the Philippines. In all dogs, virus antigen was clearly detected in a part of the outer root sheath at the level of the ring sinus of the FSCs, and the majority of cells were positive for the Merkel cell (MC) markers cytokeratin 20 and CAM5.2. Our results suggest that MCs in the FSCs of the muzzle skin are a target for virus replication and could serve as a useful alternative specimen source for diagnosis of rabies. (C) 2016 Elsevier B.V. All rights reserved.
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Efficacy of Favipiravir (T-705) in Rabies Postexposure Prophylaxis Reviewed
Kentaro Yamada, Kazuko Noguchi, Takashi Komeno, Yousuke Furuta, Akira Nishizono
JOURNAL OF INFECTIOUS DISEASES 213 ( 8 ) 1253 - 1261 2016.4
Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS INC
Rabies is a fatal encephalitis caused by rabies virus (RABV), and no antiviral drugs for RABV are currently available. We report for the first time the efficacy of favipiravir (T-705) against RABV in vitro and in vivo. T-705 produced a significant, 3-4 log(10) reduction in the multiplication of street and fixed RABV strains in mouse neuroblastoma Neuro-2a cells, with half-maximal inhibitory concentrations of 32.4 mu M and 44.3 mu M, respectively. T-705 significantly improved morbidity and mortality among RABV-infected mice when orally administered at a dose of 300 mg/kg/day for 7 days, beginning 1 hour after inoculation. T-705 significantly reduced the rate of virus positivity in the brain. Furthermore, the effectiveness of T-705 was comparable to that of equine rabies virus immunoglobulin for postexposure prophylaxis. Collectively, our results suggest that T-705 is active against RABV and may serve as a potential alternative to rabies immunoglobulin in rabies postexposure prophylaxis.
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Contribution of the interaction between the rabies virus P protein and I-kappa B kinase epsilon to the inhibition of type I IFN induction signalling
Tatsunori Masatani, Makoto Ozawa, Kentaro Yamada, Naoto Ito, Masayuki Horie, Aya Matsuu, Kosuke Okuya, Kyoko Tsukiyama-Kohara, Makoto Sugiyama, Akira Nishizono
JOURNAL OF GENERAL VIROLOGY 97 ( 2 ) 316 - 326 2016.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：MICROBIOLOGY SOC
The P protein of rabies virus (RABV) is known to interfere with the phosphorylation of the host IFN regulatory factor 3 (IRF-3) and to consequently inhibit type I IFN induction. Previous studies, however, have only tested P proteins from laboratory-adapted fixed virus strains, and to the best of our knowledge there is no report about the effect of P proteins from street RABV strains or other lyssaviruses on the IRF-3-mediated type I IFN induction system. In this study, we evaluated the inhibitory effect of P proteins from several RABV strains, including fixed and street virus strains and other lyssaviruses (Lagos bat, Mokola and Duvenhage viruses), on IRF-3 signalling. All P proteins tested inhibited retinoic acid-inducible gene-1 (RIG-I)- and TANK binding kinase 1 (TBK1)-mediated IRF-3-dependent IFN-beta promoter activities. On the other hand, the P proteins from the RABV street strains 1088 and HCM-9, but not from fixed strains Nishigahara (Ni) and CVS-11 and other lyssaviruses tested, significantly inhibited I-kappa B kinase epsilon (IKK epsilon)-inducible IRF-3-dependent IFN-beta promoter activity. Importantly, we revealed that the P proteins from the 1088 and HCM-9 strains, but not from the remaining viruses, interacted with IKK epsilon. By using expression plasmids encoding chimeric P proteins from the 1088 strain and Ni strain, we found that the C-terminal region of the P protein is important for the interaction with IKK epsilon. These findings suggest that the P protein of RABV street strains may contribute to efficient evasion of host innate immunity.
DOI： 10.1099/jgv.0.000362
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Evaluation of Rapid Neutralizing Antibody Detection Test against Rabies Virus in Human Sera.
Nguyen KA, Nguyen TT, Nguyen DV, Ngo GC, Nguyen CN, Yamada K, Noguchi K, Ahmed K, Hoang HD, Nishizono A
Tropical medicine and health 43 ( 2 ) 111 - 116 2015.6
Language：Japanese Publishing type：Research paper (scientific journal)
DOI： 10.2149/tmh.2014-35
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Neurogenic Cardiomyopathy in Rabbits With Experimentally Induced Rabies
S. Kesdangsakonwut, Y. Sunden, K. Yamada, A. Nishizono, H. Sawa, T. Umemura
VETERINARY PATHOLOGY 52 ( 3 ) 573 - 575 2015.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SAGE PUBLICATIONS INC
Cardiomyopathies have been rarely described in rabbits. Here we report myocardial necrosis of the ventricular wall in rabbits with experimentally induced rabies. Myocardial lesions were found only in rabbits with brain lesions, and the severity of the cardiac lesions was proportional to that of the brain lesions. Neither the frequency nor the cumulative dose of anesthesia was related to the incidence or the severity of the myocardial lesions. The myocardial lesions were characterized by degeneration and/or necrosis of myocardial cells and were accompanied by contraction band necrosis, interstitial fibrosis, and infiltration of inflammatory cells. The brain lesions due to rabies virus infection were most prominent in the cerebral cortex, thalamus, hypothalamus, brainstem, and medulla. Rabies virus antigen was not found in the hearts of any rabbits. Based on these findings, the myocardial lesions were classified as neurogenic cardiomyopathy.
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Characterization of street rabies virus variants with an additional N-glycan at position 247 in the glycoprotein Reviewed
Kentaro Yamada, Kazuko Noguchi, Akira Nishizono
ARCHIVES OF VIROLOGY 159 ( 2 ) 207 - 216 2014.2
Authorship：Lead author, Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER WIEN
Most street rabies virus glycoproteins (G proteins) possess two N-glycosylation sites, at Asn(37) and Asn(319), whereas an additional N-glycosylation site is present in several fixed (laboratory-adapted) rabies virus strains at Asn(247), which suggests that the N-glycosylation addition may be a marker of fixed viruses. In this study, we successfully cloned two street virus strain 1088 variants, N5B#15 and N5B#10-28, in which the G proteins had an additional N-glycan at position 247, and we examined whether these variants were characterized by cell culture adaptation and attenuation after intramuscular inoculation as fixed viruses. N5B#15 had four mutations, i.e., S148P and D247N in the G protein, and T137A and N2046S in the large (L) protein. N5B#10-28 had an additional mutation in the G protein, R196I. Compared with the parental 1088 virus, both variants exhibited highly efficient replication in mouse neuroblastoma-derived NA cells and reduced pathogenicity in adult mice when inoculated intramuscularly, but not intracerebrally. However, this attenuation was not attributable to the induction of strong immune responses.
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Efficient N-glycosylation at position 37, but not at position 146, in the street rabies virus glycoprotein reduces pathogenicity Reviewed
Kentaro Yamada, Kazuko Noguchi, Akira Nishizono
VIRUS RESEARCH 179 169 - 176 2014.1
Authorship：Lead author, Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
Most street rabies viruses have two N-glycosylation sites in their glycoproteins (G proteins), i.e., at Asn(37) and Asn(319), but Asn(37) is usually not core-glycosylated in an efficient manner. Previously, we reported the possible roles of single additional N-glycosylations at Asn(194) or Asn(247) in the cell adaptation and reduced pathogenicity of a street rabies virus, which suggest that N-glycosylation is closely related to the evolution of rabies viruses. In this study, we characterized two novel N-glycosylation-modified variants, N5C#7 and N5C#8, which were cloned using the limiting dilution method after serial passaging of the street rabies virus strain 1088 in mouse neuroblastoma-derived NA cells. N5C#7 had an L38R mutation in the G protein, which led to efficient core glycosylation at Asn(37). On the other hand, N5C#8 had a D146N mutation in the G protein, which led to an additional N-glycosylation at position 146. Both variants replicated highly efficiently in NA cells compared with the parental strain. Like the parental strain, both variants caused lethal infections in adult mice after intracerebral inoculation. However, N5C#7 exhibited reduced pathogenicity after intramuscular inoculation, whereas N5C#8 displayed the same level of pathogenicity as the parental strain. In summary, the efficient core glycosylation at position 37 was related to cell adaptation and the reduced pathogenicity of the street rabies virus. By contrast, despite of being related to cell adaptation, the additional N-glycosylation at position 146 did not affect the pathogenicity, which is consistent with a report that street rabies virus strains with N-glycosylation sites at positions 37, 146, and 319 have been isolated from rabid animals. Thus, the results of the present study provide additional evidence that supports the relationship between G protein N-glycosylation and rabies virus evolution. (C) 2013 Elsevier B.V. All rights reserved.
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Human Bocavirus in Patients with Encephalitis, Sri Lanka, 2009-2010
Daisuke Mori, Udaya Ranawaka, Kentaro Yamada, Shaman Rajindrajith, Kazushi Miya, Harsha Kumara Kithsiri Perera, Takashi Matsumoto, Malka Dassanayake, Marcelo Takahiro Mitui, Hisashi Mori, Akira Nishizono, Maria Soderlund-Venermo, Kamruddin Ahmed
EMERGING INFECTIOUS DISEASES 19 ( 11 ) 1859 - 1862 2013.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：CENTERS DISEASE CONTROL
We identified human bocavirus (HBoV) DNA by PCR in cerebrospinal fluid from adults and children with encephalitis in Sri Lanka. HBoV types 1, 2, and 3 were identified among these cases. Phylogenetic analysis of HBoV1 strain sequences found no subclustering with strains previously identified among encephalitis cases in Bangladesh.
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Addition of a single N-glycan to street rabies virus glycoprotein enhances virus production Reviewed
Kentaro Yamada, Kazuko Noguchi, Daichi Nonaka, Muneshin Morita, Aiko Yasuda, Hiroaki Kawazato, Akira Nishizono
JOURNAL OF GENERAL VIROLOGY 94 ( Pt 2 ) 270 - 275 2013.2
Authorship：Lead author, Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY
Most street rabies virus G proteins have two N-glycosylation sites, i.e. Asn(37) and Asn(319), whereas additional sites are found in fixed (laboratory adapted) viruses. In this study, we performed a pseudotyped virus assay using G-deficient rabies virus and demonstrated that single-N-glycan additions to the G protein of street rabies virus strain 1088, which are found in adapted strains, enhanced virus production in neural and non-neural cell lines, while additions to Asn(194) or Asn(247) enhanced production greatly. Moreover, we found that N-glycan additions at Asn(194) or Asn(247) facilitated the production of cell-associated virus. In contrast, deletion of the sequon at Asn(37) reduced viral production, while a deletion at Asn(319) resulted in extensive loss of production. Furthermore, G proteins lacking an N-glycan at Asn(319) failed to fold into their correct structure and lost their fusion activity, indicating that Asn(319) N-glycosylation is important for the functional expression of street virus G proteins.
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Relationship between Virus-Neutralizing Antibody Levels and the Number of Rabies Vaccinations: a Prospective Study of Dogs in Japan
Ippei Watanabe, Kentaro Yamada, Akira Aso, Okio Suda, Takashi Matsumoto, Takaaki Yahiro, Kamruddin Ahmed, Akira Nishizono
JAPANESE JOURNAL OF INFECTIOUS DISEASES 66 ( 1 ) 17 - 21 2013.1
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：NATL INST INFECTIOUS DISEASES
A mass rabies vaccination of dogs has been conducted annually in Japan over the last 60 years. To assess both current levels of rabies virus-neutralizing antibody (VNA) in dogs and the rationale for current vaccination procedures, we used a rapid fluorescent focus inhibition test to determine VNA levels in 756 dogs that had visited animal hospitals in Japan. We found that 51.1% of the dogs that had received 1 rabies vaccination had protective VNA levels (>= 0.5 IU/ml) with a geometric mean of 0.61 IU/ml. In contrast, 97.8% of the dogs that had been vaccinated at least twice had protective VNA levels with a geometric mean of 7.86 IU/ml. Furthermore, 97.9-100% of the dogs vaccinated at least twice retained protective VNA levels into the second year after the last vaccination. Although VNA levels in the dogs vaccinated at least twice tended to decline 2 years after the last vaccination, 78.9% retained protective VNA levels. Thus, the current rabies vaccination schedule provides adequate protection, but the registration system and vaccination schedule needs to be improved to ensure that increased numbers of dogs are vaccinated against rabies.
DOI： 10.7883/yoken.66.17
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Passive carriage of rabies virus by dendritic cells
Senba K., Matsumoto T., Yamada K., Shiota S., Iha H., Date Y., Ohtsubo M., Nishizono A.
SpringerPlus 2 ( 1 ) 1 - 12 2013
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SpringerPlus
The rabies virus (RABV) is highly neurotropic and it uses evasive strategies to successfully evade the host immune system. Because rabies is often fatal, understanding the basic processes of the virus-host interactions, particularly in the initial events of infection, is critical for the design of new therapeutic approaches to target RABV. Here, we examined the possible role of dendritic cells (DCs) in the transmission of RABV to neural cells at peripheral site of exposure. Viral replication only occurred at a low level in the DC cell line, JAWS II, after its infection with either pathogenic RABV (CVS strain) or low-pathogenic RABV (ERA strain), and no progeny viruses were produced in the culture supernatants. However, both viral genomic RNAs were retained in the long term after infection and maintained their infectivity. The biggest difference between CVS and ERA was in their ability to induce type I interferons. Although the ERA-infected JAWS II cells exhibited cytopathic effect and were apparently killed by normal spleen cells in vitro, the CVS-infected JAWS II cells showed milder cytopathic effect and less lysis when cocultured with spleen cells. Strongly increased expression of major histocompatibility complex classes I, costimulatory molecules (CD80 and CD86), type I interferons and Toll- like receptor 3, and was observed only in the ERA-inoculated JAWS II cells and not in those inoculated with CVS. During the silencing of the cellular immune response in the DCs, the pathogenic CVS strain cryptically maintained an infectious viral genome and was capable of transmitting infectious RABV to permissive neural cells. These findings demonstrate that DCs may play a role in the passive carriage of RABV during natural rabies infections. © 2013 Senba et al.
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Molecular analysis of the mutational effects of Thai street rabies virus with increased virulence in mice after passages in the BHK cell line
Phatthamon Virojanapirom, Pakamatz Khawplod, Artikaya Sawangvaree, Supaporn Wacharapluesadee, Thiravat Hemachudha, Kentaro Yamada, Kinjiro Morimoto, Akira Nishizono
ARCHIVES OF VIROLOGY 157 ( 11 ) 2201 - 2205 2012.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SPRINGER WIEN
QS-BHK-P7, street rabies virus, after passages in the BHK cell line, had an in vitro phenotype that distinguished it from its parental virus. Both viruses caused lethal infection in mice by central nervous system inoculation; however, only QS-BHK-P7 killed mice by the intramuscular route. We found four mutations, S23R and H424P in ectodomain of the glycoprotein (G), I1711 V in the polymerase genes, and another at the non-coding region between the phosphoprotein and matrix protein genes of QS-BHK-P7. None of the mutations in the G gene occurred in previously reported pathogenic determinants. The roles of mutations in particular non-coding regions remain to be elucidated.
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Evaluation of an improved rapid neutralizing antibody detection test (RAPINA) for qualitative and semiquantitative detection of rabies neutralizing antibody in humans and dogs
Akira Nishizono, Kentaro Yamada, Pakamatz Khawplod, Seiji Shiota, Devika Perera, Takashi Matsumoto, Omala Wimalaratne, Marcelo Takahiro Mitui, Kamruddin Ahmed
VACCINE 30 ( 26 ) 3891 - 3896 2012.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI LTD
Using the principle of immunochromatography, we previously developed a method called RAPINA (Rapid Neutralizing Antibody detection test) that can measure the level of rabies virus -neutralizing antibody (VNA) in serum samples [Shiota S. Mannen K, Matsumoto T, Yamada K, Yasui T, Takayama K. et al. Development and evaluation of a rapid neutralizing antibody test for rabies. J Virol Methods 2009;161:58-62]. RAPINA is faster, simpler, and easier to perform compared with a virus-neutralizing test or enzyme-linked immunosorbent assay (ELISA). The improved version of RAPINA has greater positive and negative predictive values corresponding to a VNA level of 0.5IU/mL, as recommended by the World Health Organization and the World Organization for Animal Health. To verify the efficacy of this improved method, serum samples were collected from humans and dogs before and after immunization against rabies and were tested in Japan, Sri Lanka, and Thailand.
The results were compared between RAPINA and the true VNA levels measured by the Rapid Fluorescent Focus Inhibition Test (RFFIT). The improved RAPINA accurately predicted seropositivity for 182 of 183 seropositive human samples as assessed by RFFIT (99.5%) and for 138 of 140 RFFIT-negative human samples (98.6%). In dog serum samples, the positive and negative predictive values were 99.7% (345/355) and 95.6% (174/182), respectively. RAPINA was also used to estimate VNA levels in a semiquantitative manner by using serial dilution of serum samples.
Our results show that RAPINA is an easy and rapid method for measuring VNA levels before and after immunization with the rabies vaccine and does not need a high skill level or sophisticated equipment. RAPINA can be used to monitor the success of preexposure prophylaxis in at-risk persons, vaccine coverage, and animal control. It can also be used in laboratories with modest facilities and where a large number of samples are screened. (C) 2012 Elsevier Ltd. All rights reserved. -
Detection of Human Bocavirus in the Cerebrospinal Fluid of Children With Encephalitis
Marcelo Takahiro Mitui, S. M. Shahnawaz Bin Tabib, Takashi Matsumoto, Wahida Khanam, Selim Ahmed, Daisuke Mori, Nasima Akhter, Kentaro Yamada, Luthful Kabir, Akira Nishizono, Maria Soderlund-Venermo, Kamruddin Ahmed
CLINICAL INFECTIOUS DISEASES 54 ( 7 ) 964 - 967 2012.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS INC
We report 4 children with encephalitis associated with human bocavirus (HBoV) 1 or 2. All children were severely underweight, and 2 died; 1 of them had a matching HBoV2 nucleotide sequence isolated from serum and bocavirus like particles in the cerebrospinal fluid that were observed with electron microscopy. No further pathogens were detected in the cerebrospinal fluid of these patients.
DOI： 10.1093/cid/cir957
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Serial passage of a street rabies virus in mouse neuroblastoma cells resulted in attenuation: Potential role of the additional N-glycosylation of a viral glycoprotein in the reduced pathogenicity of street rabies virus
Kentaro Yamada, Chun-Ho Park, Kazuko Noguchi, Daisuke Kojima, Tatsuya Kubo, Naoyuki Komiya, Takashi Matsumoto, Marcelo Takahiro Mitui, Kamruddin Ahmed, Kinjiro Morimoto, Satoshi Inoue, Akira Nishizono
VIRUS RESEARCH 165 ( 1 ) 34 - 45 2012.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
Street rabies viruses are field isolates known to be highly neurotropic. However, the viral elements related to their pathogenicity have yet to be identified at the nucleotide or amino acid level. Here, through 30 passages in mouse neuroblastoma NA cells, we have established an attenuated variant of street rabies virus strain 1088, originating from a rabid woodchuck followed by 2 passages in the brains of suckling mice. The variant, 1088-N30, was well adapted to NA cells and highly attenuated in adult mice after intramuscular (i.m.) but not intracerebral (i.c.) inoculations. 1088-N30 had seven nucleotide substitutions, and the R196S mutation of the G protein led to an additional N-glycosylation. Street viruses usually possess one or two N-glycosylation sites on the G protein, 1088 has two, while an additional N-glycosylation site is observed in laboratory-adapted strains. We also established a cloned variant 1088-N4#14 by limiting dilution. Apart from the R196S mutation, 1088-N4#14 possessed only one amino acid substitution in the P protein, which is found in several field isolates. 1088-N4#14 also efficiently replicated in NA cells and was attenuated in adult mice after i.m. inoculations, although it was more pathogenic than 1088-N30. The spread of 1088-N30 in the brain was highly restricted after i.m. inoculations., although the pattern of 1088-N4#14's spread was intermediate between that of the parental 1088 and 1088-N30. Meanwhile, both variants strongly induced humoral immune responses in mice compared to 1088. Our results indicate that the additional N-glycosylation is likely related to the reduced pathogenicity. Taken together, we propose that the number of N-glycosylation sites in the G protein is one of the determinants of the pathogenicity of street rabies viruses. (C) 2012 Elsevier B.V. All rights reserved.
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Nishizono A., Yamada K.
Uirusu 62 ( 2 ) 183 - 196 2012
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Uirusu
The family Rhabdoviridae has a non-segmented single stranded negative-sense RNA and its genome ranges in size from approximately 11 kb to almost 16 kb. It is one of the most ecologically diverse families of RNA viruses with members infecting a wide range of organisms. The five structural protein genes are arranged in the same linear order (3'-N-P-M-G-L-5') and may be interspersed with one more additional accessory gene. For many years, a full of knowledge of the rhabdoviridae has been established on extensive studies of two kinds of prototype viruses; vesicular stomatitis virus (VSV) and rabies virus (RABV). Among them, the genus Lyssavirus includes RABV and rabies-related viruses naturally infect mammals and chiropterans via bite-exposure by rabid animals and finally cause fatal encephalitis. In this review, we describe the sketch of the various virological features of the Rhabdoviridae, especially focusing on VSV and RABV.
DOI： 10.2222/jsv.62.183
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The SI Strain of Measles Virus Derived from a Patient with Subacute Sclerosing Panencephalitis Possesses Typical Genome Alterations and Unique Amino Acid Changes That Modulate Receptor Specificity and Reduce Membrane Fusion Activity Reviewed
Fumio Seki, Kentaro Yamada, Yuichiro Nakatsu, Koji Okamura, Yusuke Yanagi, Tetsuo Nakayama, Katsuhiro Komase, Makoto Takeda
JOURNAL OF VIROLOGY 85 ( 22 ) 11871 - 11882 2011.11
Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY
Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity.
DOI： 10.1128/JVI.05067-11
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Whole-genome analysis of a human rabies virus from Sri Lanka
Takashi Matsumoto, Kamruddin Ahmed, Omala Wimalaratne, Kentaro Yamada, Susilakanthi Nanayakkara, Devika Perera, Dushantha Karunanayake, Akira Nishizono
ARCHIVES OF VIROLOGY 156 ( 4 ) 659 - 669 2011.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SPRINGER WIEN
The complete genome sequence of a human rabies virus, strain H-08-1320, from Sri Lanka was determined and compared with other rabies viruses. The size of the genome was 11,926 nt, and it was composed of a 58-nucleotide 3' leader, five protein genes - N (1353 nt), P (894 nt), M (609 nt), G (1575 nt), and L (6387 nt) - and a 70-nt 5' trailer. The intergenic region G-L contained 515 nt. The sizes of the nucleoprotein, phosphoprotein, matrix-protein, glycoprotein and large-protein was 450, 296, 202, 524 and 2,128 residues, respectively. The phosphoprotein and large protein were one amino acid shorter and longer, respectively, than those of most rabies viruses. The glycoprotein of H-08-1320 had a unique amino acid substitution at antigenic site I. Whole-genome phylogenetic analysis showed that strain H-08-1320 formed an independent lineage and did not cluster with rabies viruses from other countries.
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A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells Reviewed
Shigeo Nagashima, Masaharu Takahashi, Jirintai, Toshinori Tanaka, Kentaro Yamada, Tsutomu Nishizawa, Hiroaki Okamoto
JOURNAL OF GENERAL VIROLOGY 92 ( Pt 2 ) 269 - 278 2011.2
Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY
We have previously demonstrated that the release of hepatitis E virus (HEV) from infected cells depended on ORF3 protein, which harbours one or two PSAP motifs. To elucidate the PSAP motif(s) in the ORF3 protein during virion egress, five PSAP mutants derived from an infectious genotype 3 cDNA clone of pJE03-1760F/wt that can grow efficiently in PLC/PRF/5 cells were analysed. Four mutants, including mutLSAP, mutPSAL, mutLSAL (the substituted amino acids in the authentic PSAP motif are underlined) and mutPLAP/PSAP (the changed amino acid in the additional PSAP motif is underlined) generated progenies as efficiently as the wild-type virus. Conversely, the HEV RNA level in the culture supernatant of mutPLAP/LSAL RNA-transfected cells was significantly lower than in cells transfected with the wild-type RNA, similar to an ORF3-null mutant. Consistent with the ORF3-deficient mutant, the mutPLAP/LSAL mutant with no intact PSAP motifs banded at 1.26-1.27 g ml(-1) in sucrose, and was captured by anti-ORF2, but not by anti-ORF3, with or without prior treatment with detergent (0.1% sodium deoxycholate). The absence of the ORF3 protein on the mutant particles in the culture supernatant was confirmed by Western blotting, despite the expression of ORF3 protein in the RNA-transfected cells, as detected by immunofluorescence and Western blotting. Therefore, at least one of the two intact PSAP motifs in the ORF3 protein is required for the formation of membrane-associated HEV particles possessing ORF3 proteins on their surface, thus suggesting that the PSAP motif plays a role as a functional domain for HEV budding.
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Five-year (January 2004-December 2008) surveillance on animal bite and rabies vaccine utilization in the Infectious Disease Hospital, Dhaka, Bangladesh Reviewed
Moazzem Hossain, Tania Bulbul, Kamruddin Ahmed, Ziauddin Ahmed, Mohammad Salimuzzaman, Mohammad Shahidul Haque, Ajmat Ali, Shohrab Hossain, Kentaro Yamada, Kazuhiko Moji, Akira Nishizono
VACCINE 29 ( 5 ) 1036 - 1040 2011.1
Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI LTD
The magnitude of animal bite and utilization of rabies vaccine was determined at the Infectious Disease Hospital, Dhaka, Bangladesh. From January 2004 to December 2008, 150,068 patients with animal bite visited the hospital, 86.2% and 13.8% of them received nerve tissue and tissue culture vaccine (TCV), respectively. Dog bite was most frequent, found in 90.7% cases. In 794 rabies cases only 24.4% had a history of post-exposure vaccination. Only a negligible number of patients received rabies immunoglobulin (RIG). To prevent further human deaths and economic losses intra-dermal TCV regime and equine RIG should be immediately introduced in Bangladesh. (C) 2010 Elsevier Ltd. All rights reserved.
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Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus
Takashi Matsumoto, Kentaro Yamada, Kazuko Noguchi, Kantou Nakajima, Kenzo Takada, Pakamatz Khawplod, Akira Nishizono
MICROBIOLOGY AND IMMUNOLOGY 54 ( 11 ) 673 - 683 2010.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL
Rabies is a fatal viral encephalitis which is transmitted by exposure to the bite of rabid animals. Human and equine rabies immunoglobulins are indispensable pharmacological agents for severe bite exposure, as is vaccine. However, several disadvantages, including limited supply, adverse reactions, and high cost, hamper their wide application in developing countries. In the present study, two novel huMabs which neutralize rabies virus were established from vaccinated hyperimmune volunteers using the Epstein-Barr virus transformation method. One MAb (No. 254), which was subclass IgG3, effectively neutralized fixed rabies viruses of CVS, ERA, HEP-Flury, and Nishigahara strains and recognized a well-conserved epitope located in antigenic site II of the rabies virus glycoprotein. No. 254 possessed 68 ng/ml of FRNT(50) activity against CVS, 3.7 x 10-7 M of the Kd value, and the enhancing effect of complement-dependent virolysis. In addition, No. 254 showed effective neutralization potency in vivo in the mouse challenge test. The other MAb, 4D4, was subclass IgM and showed neutralizing activity against CVS and Nishigahara strains. 4D4 recognized a novel antigenic site which is associated with the neurovirulence of rabies, a glycoprotein located between antigenic site I and VI. Both human MAbs against rabies are expected to be utilized as a tool for future post-exposure prophylaxis.
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Determination of the 5'-terminal sequence of subgenomic RNA of hepatitis E virus strains in cultured cells
Koji Ichiyama, Kentaro Yamada, Toshinori Tanaka, Shigeo Nagashima, Jirintai, Masaharu Takahashi, Hiroaki Okamoto
ARCHIVES OF VIROLOGY 154 ( 12 ) 1945 - 1951 2009.12
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：SPRINGER WIEN
Using RNA preparations extracted from PLC/PRF/5 cells transfected with infectious genotype 3 hepatitis E virus (HEV) cDNA clones or inoculated with a fecal suspension containing a genotype 4 HEV, the 5'-terminal sequence of a 2.2-kb subgenomic RNA of genotype 3 and 4 HEVs was determined. Despite an insertion of T after nucleotide 5116 or an ORF3-null mutation in genotype 4 HEV and/or one of the genotype 3 variants, it was found that the subgenomic RNA of genotype 3 and 4 HEVs initiates exclusively at nucleotide 5122 with the common sequence of 5'-GC, which is identical to that of the prototype genotype 1 HEV.
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Evaluation of a New Tumor Necrosis Factor-alpha-Inducing Membrane Protein of Helicobacter pylori as a Prophylactic Vaccine Antigen Reviewed
Kunimitsu Inoue, Seiji Shiota, Kentaro Yamada, Kazuyo Gotoh, Masami Suganuma, Toshio Fujioka, Kamruddin Ahmed, Hidekatsu Iha, Akira Nishizono
HELICOBACTER 14 ( 5 ) 135 - 143 2009.10
Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY
Background:
Tumor necrosis factor (TNF)-alpha-inducing protein (Tip alpha) is a newly identified carcinogenic factor present in Helicobacter pylori. Tip alpha has the unique function of inducing TNF-alpha production by gastric cells in vitro and is assumed to be related with the development of gastritis and gastric cancer. We investigated the effects of vaccination with Tip alpha against H. pylori infection and analyzed the immune responses.
Methods:
C57BL/6 mice were immunized via the intranasal route with CpG, recombinant Tip alpha + CpG, and recombinant del-Tip alpha (a mutant of Tip alpha) + CpG. Eight weeks after the mice were infected with H. pylori (5 x 10<SU7</SU CFU), the number of colonizing bacteria in the stomach was calculated, and the histological severity of gastritis was evaluated. Levels of Tip alpha-specific IgG and IgA antibodies in mouse serum were measured by an enzyme-linked immunosorbent assay (ELISA). Local production of cytokines including Interleukin (IL)-10, TNF-alpha and Interferon (IFN)-gamma in gastric mucosa was also measured by real time-PCR.
Results:
Levels of Tip alpha-specific antibodies were significantly higher in Tip alpha-immunized and del-Tip alpha-immunized mice than in the infection control group. The numbers of colonizing bacteria were significantly reduced in Tip alpha-immunized mice (4.29 x 10<SU5</SU CFU/g) and del-Tip alpha immunized mice (2.5 x 10<SU5 </SUCFU/g) compared with infection control mice (5.7 x 10<SU6</SU CFU/g). The levels of IFN-gamma and IL-10 were significantly higher in del-Tip alpha-immunized mice than the infection control group.
Conclusion:
Vaccinations with Tip alpha and del-Tip alpha were effective against H. pylori infection. The inhibition of H. pylori colonization is associated mainly with Th1 cell-mediated immunity. -
Development and evaluation of a rapid neutralizing antibody test for rabies
Seiji Shiota, Kazuaki Mannen, Takashi Matsumoto, Kentaro Yamada, Takehito Yasui, Katsuyoshi Takayama, Yukuharu Kobayashi, Pakamatz Khawplod, Kazuyo Gotoh, Kamruddin Ahmed, Hidekatsu Iha, Akira Nishizono
JOURNAL OF VIROLOGICAL METHODS 161 ( 1 ) 58 - 62 2009.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
The level of virus-neutralizing antibody, which plays a crucial role in the prevention of rabies, is determined by rabiesviruis (RABV) neutralizing test, which are time- and cost-consuming. In order to determine the level of neutralizing antibody in vaccinees, an easy and reliable method is needed. Based on the principle of immunochromatography, we developed a RAPINA (RAPId Neutralizing Antibody) test to determine the presence of neutralizing antibody in serum. In the RAPINA test, if neutralizing antibody equivalent to 0.5 IU/ml of serum sample are mixed with an optimal amount of inactivated RABV (iRABV) and are completely absorbed by the virus, none of the iRABV can bind with monoclonal antibody that recognizes the iRABV glycoprotein (G) on the test strip. A total of 115 human sera samples were tested. The sensitivity, specificity and accuracy of the RAPINA test compared with rapid fluorescent focus inhibition test (RFFIT) as a standard test, were 88.7, 91.9 and 90.4%, respectively. The RAPINA test is a simple, safe and rapid method, which can be a substitute for neutralizing tests that use live viruses, cultured cells and fluorescence microscopy. This test might be useful for screening a large number of sera. (C) 2009 Elsevier B.V. All rights reserved.
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ORF3 protein of hepatitis E virus is essential for virion release from infected cells Reviewed
Kentaro Yamada, Masaharu Takahashi, Yu Hoshino, Hideyuki Takahashi, Koji Ichiyama, Shigeo Nagashima, Toshinori Tanaka, Hiroaki Okamoto
JOURNAL OF GENERAL VIROLOGY 90 ( Pt 8 ) 1880 - 1891 2009.8
Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY
The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein remains unclear. To elucidate the role of the ORF3 protein in the virus life cycle, an infectious cDNA clone (pJE03-1760F/wt) that can replicate efficiently in PLC/PRF/5 and A549 cells and release progeny into the culture medium was used to generate a derivative ORF3-deficient (Delta ORF3) mutant whose third in-frame AUG codon of ORF3 was mutated to GCA. The Delta ORF3 mutant in the culture medium of mutant RNA-transfected PLC/PRF/5 cells was able to infect and replicate within PLC/PRF/5 and A549 cells as efficiently as the wild-type pJE03-1760F/wt virus. However, less than 1/100 of the number of progeny was detectable in the culture medium of Delta ORF3 mutant-infected PLC/PRF/5 cells compared with wild-type-infected PLC/PRF/5 cells, and the HEV RNA level in the culture medium of Delta ORF3 mutant-infected A549 cells was below or near the limit of detection. An immunocapture PCR assay revealed that the ORF3 protein is present on the surface of cell-culture-generated wild-type HEV but not on the Delta ORF3 mutant. Wild-type HEV in the culture supernatant peaked at a sucrose density of 1.15-1.16 g ml(-1), in contrast with the Delta ORF3 mutant in culture supernatant, which banded at 1.27-1.28 g ml(-1), similar to HEV in cell lysate and faecal HEV. These results suggest that the ORF3 protein is responsible for virion egress from infected cells and is present on the surface of released HEV particles, which may be associated with lipids.
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Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells
Kentaro Yamada, Masaharu Takahashi, Yu Hoshino, Hideyuki Takahashi, Koji Ichiyama, Toshinori Tanaka, Hiroaki Okamoto
JOURNAL OF GENERAL VIROLOGY 90 ( Pt 2 ) 457 - 462 2009.2
Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY
A full-length infectious cDNA clone (pJE03-1760F/wt) of a genotype 3 hepatitis E virus (HEV) (strain JE03-1760F) obtained from a faecal specimen was constructed ill this study. Upon transfection of the capped in vitro transcripts of pJE03-1760F/wt into PLC/PRF/5 cells, the viral RNA levels in the culture supernatant started to increase on day 6 post-transfection (p.t.) and reached 10(7) copies ml(-1) on day 28 p.t. Detection of increasing numbers of cells with ORF2 protein expression by immunofluorescence assay at 5, 7, 11 and 15 days p.t. indicated the spread of HEV infection in cell culture. When the cDNA-derived virus in culture supernatant was inoculated into PLC/PRF/5 or A549 cells, it grew as efficiently as the faeces-derived virus in both cells, reaching 10(6) copies ml(-1) at 30 days post-inoculation. Our reverse genetics system for HEV that is usable in a robust cell-culture system will be useful for elucidation of the mechanism of HEV replication and functional roles of HEV proteins.
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Amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus
Tetsuo Mita, Kenta Shimizu, Naoto Ito, Kentaro Yamada, Yuki Ito, Makoto Sugiyama, Nobuyuki Minamoto
VIRUS RESEARCH 137 ( 1 ) 33 - 39 2008.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
The molecular mechanism involved in cytopathogenicity of rabies virus has not been fully elucidated yet. A fixed rabies virus Nishigahara strain does not induce clear cytopathic effect (CPE) in mouse neuroblastoma (NA) cells, whereas Ni-CE strain, which was established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, induces CIPE that is characterized by rounding, shrinkage and detachment of the cells. In this study, to identify which viral gene is associated with the CPE of Ni-CE Strain, we analyzed chimeric viruses between Nishigahara and Ni-CE strains generated by reverse genetics systems of both strains. We showed that the matrix gene of Ni-CE strain is responsible for the CPE in NA cells. It was also demonstrated by infection of Nishigahara and Ni-CE mutants with a single amino acid substitution in the matrix protein (M) that an amino acid at position 95 of M is a cytopathic determinant of the virus. We also demonstrated that the CPF is, at least partly, due to apoptosis. This is the first report of identification of an amino acid residue in a rabies virus protein that is important for the cytopathogenicity of the virus. (C) 2008 Elsevier B.V. All rights reserved.
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Mutational events during the primary propagation and consecutive passages of hepatitis E virus strain JE03-1760F in cell culture
Felipe R. Lorenzo, Toshinori Tanaka, Hideyuki Takahashi, Koji Ichiyama, Yu Hoshino, Kentaro Yamada, Jun Inoue, Masaharu Takahashi, Hiroaki Okamoto
VIRUS RESEARCH 137 ( 1 ) 86 - 96 2008.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 cells, using a genotype 3 HEV (JE03-1760F strain). Thirteen generations of consecutive passages of culture supernatant were successfully carried out in PLC/PRF/5 cells, with the highest HEV load reaching 10(8) copies/ml in the culture medium. Based on continuous release of progenies into culture medium, 50% tissue culture infectivity doses were estimated to be 2.0 x 10(3) Copies for wild-type JE03-1760F and 1.4 x 10(2) copies for p 13 (progeny in the thirteenth passage). Earlier appearance and greater increase in the yield of progenies in the culture supernatant were evident in p13 compared with wild-type. The cell culture-produced variants in primary propagation (p0)and consecutive passages (p5 [fifth passage], p10 [tenth], and p13)differed from the wildtype virus by 1. 9, 18, and 19 nucleotides (nt), respectively, over the entire genome of 7226 nt, excluding the poly(A) tail. Three of five non-synonymous Mutations in p13 were shared by a variant (fifth passage) in another series of passages of JE03-1760F. These results suggest that adaptation of HEV variants to growth in vitro is associated with a limited number of mutations similar to hepatitis A virus. (C) 2008 Elsevier B.V. All Fights reserved.
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Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces Reviewed
Masaharu Takahashi, Kentaro Yamada, Yu Hoshino, Hideyuki Takahashi, Koji Ichiyama, Toshinori Tanaka, Hiroaki Okamoto
ARCHIVES OF VIROLOGY 153 ( 9 ) 1703 - 1713 2008.9
Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER WIEN
Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of > 10(6) copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm(3) in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.
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Adult measles Invited
Yamada K., Okada H.
Nippon rinsho. Japanese journal of clinical medicine 65 Suppl 3 364 - 367 2007.3
Authorship：Lead author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Nippon rinsho. Japanese journal of clinical medicine
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Involvement of nucleoprotein, phosphoprotein, and matrix protein genes of rabies virus in virulence for adult mice
Kenta Shimizu, Naoto Ito, Tetsuo Mita, Kentaro Yamada, Junji Hosokawa-Muto, Makoto Sugiyama, Nobuyuki Minamoto
VIRUS RESEARCH 123 ( 2 ) 154 - 160 2007.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
Rabies virus Ni-CE strain causes nonlethal infection in adult mice after intracerebral inoculation, whereas the parental Nishigahara strain kills mice. In this study, to identify viral gene(s) related to the difference in pathogenicity between Ni-CE and Nishigahara strains, we generated chimeric viruses with respective genes of the virulent Nishigahara strain in the background of the avirudent Ni-CE genome. Since chimeric viruses, which had the N, P, or M genes of the Nishigahara strain, respectively, killed adult mice after intracerebral inoculation, it became evident that the N, P, and M genes are related to the difference in pathogenicity between Ni-CE and Nishigahara strains. Previously, we showed that the G gene is a major contributor to the difference in pathogenicity between another avirulent strain, RC-HL, and the parental Nishigahara strain. These results imply that the attenuation mechanism of the Ni-CE strain is different from that of the RC-HL strain, thus suggesting that rabies virus can be attenuated by diverse mechanisms. This is the first report of changes in viral genes other than the G gene of rabies virus causing the reversion of pathogenicity of an avirulent strain. (c) 2006 Elsevier B.V. All rights reserved.
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Multiple amino acids in the glycoprotein of rabies virus are responsible for pathogenicity in adult mice
M Takayama-Ito, N Ito, K Yamada, M Sugiyama, N Minamoto
VIRUS RESEARCH 115 ( 2 ) 169 - 175 2006.2
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV
We have reported that the region between amino acids 164 and 303 in the glycoprotein of rabies Nishigahara strain is important for lethality in adult mice. The region contains nine amino acid substitutions between the virulent Nishigahara and the avirulent RC-HL strains. In order to determine key residues for the pathogenicity, two chimeric strains and seven mutants were generated and examined for pathogenicities. The R(G 242/255/268) strain, in which amino acids at positions 242, 255, and 268 were replaced by those from the Nishigahara strain in the genomic backbone of the RC-HL strain, showed the same lethality as that of the Nishigahara strain in mice. Mutants in which one or two of these three amino acids were replaced by those from the Nishigahara strain did not revert to the lethality of the R(G 242/255/268) strain. These results demonstrate that at least these three amino acids are related to enhancement of pathogenicity. It is thought that multiple amino acids of the G protein are related to the pathogenicity of rabies viruses. (c) 2005 Elsevier B.V. All rights reserved.
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Characterization of recombinant rabies virus carrying double glycoprotein genes Reviewed
J Hosokawa-Muto, N Ito, K Yamada, K Shimizu, M Sugiyama, N Minamoto
MICROBIOLOGY AND IMMUNOLOGY 50 ( 3 ) 187 - 196 2006
Language：English Publishing type：Research paper (scientific journal) Publisher：CENTER ACADEMIC PUBL JAPAN
A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UV-inactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.
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Multigenic relation to the attenuation of rabies virus Reviewed
K Yamada, N Ito, M Takayama-Ito, M Sugiyama, N Minamoto
MICROBIOLOGY AND IMMUNOLOGY 50 ( 1 ) 25 - 32 2006
Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：CENTER ACADEMIC PUBL JAPAN
Rabies virus Nishigahara strain causes lethal infection in adult mice after intracerebral inoculation. On the other hand, the RC-HL strain, derived from the Nishigahara strain, does not cause lethal infection in adult mice. We previously demonstrated that a chimeric virus, R(G), with the open reading frame of the G gene (G-ORF) from the Nishigahara strain in the background of the RC-HL genome, is virulent. Reversely, in order to demonstrate that the G gene of the RC-HL strain is related to the attenuated phenotype, we established a reverse genetics system of the Nishigahara strain and generated a chimeric virus, Ni(G), with the G-ORF from RC-HL in the background of the Nishigahara genome. Contrary to our prediction, Ni(G) killed adult mice after intracerebral inoculation with neuropathic symptoms like those of Nishigahara strain infection. Therefore, the G-ORF of the RC-HL strain is not the sole determinant of the attenuated phenotype. In additional investigation, we examined other genes, including N, P, M and L genes, and generated chimeric viruses exhaustively. We found that chimeric viruses with a single gene from the RC-HL were not attenuated and that chimeric viruses with the G-ORF and at least one other ORF from the RC-HL were attenuated. In conclusion, attenuation from the Nishigahara to RC-HL strain is multigenic.
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Ito N., Sugiyama M., Yamada K., Shimizu K., Takayama-Ito M., Hosokawa J., Minamoto N.
Microbiology and Immunology 49 ( 11 ) 971 - 979 2005
Language：English Publishing type：Research paper (scientific journal) Publisher：Microbiology and Immunology
Matrix (M) protein of rabies virus is known to play an important role in assembly and budding of the progeny virus. We generated an M gene-deficient rabies virus, RC-HLΔM, using a reverse genetics system of rabies virus RC-HL strain to develop a novel type of vaccine. RC-HLΔM infection was confined within a single cell in mouse neuroblastoma cells. This deficient virus failed to generate the progeny virus in the cells. In contrast, RC-HLΔM propagated in BHK cells inductively expressing M protein. Suckling and adult mice inoculated intracerebrally with the parental RC-HL strain showed lethal infection and transient body weight loss, respectively, whereas both suckling and adult mice inoculated with RC-HLΔM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 10 focus-forming units of RC-HLΔM but not UV-inactivated RC-HL. Intranasal immunization with RC-HLΔM resulted in almost the same antibody titer to rabies virus as that in the case of immunization with live RC-HL strain. These findings indicate that RC-HLΔM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines. 5
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Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain. Reviewed
Ito N, Sugiyama M, Yamada K, Shimizu K, Takayama-Ito M, Hosokawa J, Minamoto N
Microbiology and immunology 49 ( 11 ) 971 - 979 2005
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Region at amino acids 164 to 303 of the rabies virus glycoprotein plays an important role in pathogenicity for adult mice
M Takayama-Ito, N Ito, K Yamada, N Minamoto, M Sugiyama
JOURNAL OF NEUROVIROLOGY 10 ( 2 ) 131 - 135 2004.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS INC
The authors have previously reported that the glycoprotein of the pathogenic Nishigahara strain of rabies virus is required to lethality for adult mice. A cluster region of amino acid substitutions exists at the positions 164 to 303 on the glycoprotein between avirulent and virulent strains. In this study, the authors generated a chimeric strain having the region at the positions 164 to 303 of the glycoprotein derived from the pathogenic Nishigahara strain in the genetic background of the avirulent RC-HL strain. The chimeric R(G 164-303) strain restores the lethality for adult mice. This result clearly shows that the region at the position 164 to 303 of glycoprotein plays an important role in the lethality for adult mice. Moreover, the authors observed that the lethality for adult mice correlated well with the viral growth in a brain but not with the pH-dependent fusion activity in vitro.
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Ito N., Takayama-Ito M., Yamada K., Hosokawa J., Sugiyama M., Minamoto N.
Microbiology and Immunology 47 ( 8 ) 613 - 617 2003
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Microbiology and Immunology
To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7-9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC-HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase-transcripts from the plasmid cap-independent for translation. After co-transfection of these helper plasmids and the previously constructed full-length genome plasmid of the RC-HL strain to BHK/T7-9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.
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Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system Reviewed
N Ito, M Takayama-Ito, K Yamada, J Hosokawa, M Sugiyama, N Minamoto
MICROBIOLOGY AND IMMUNOLOGY 47 ( 8 ) 613 - 617 2003
Language：English Publishing type：Research paper (scientific journal) Publisher：CENTER ACADEMIC PUBL JAPAN
To improve efficiency of recovery of rabies virus from cloned cDNA, we established a BHK cell clone that stably expresses T7 RNA polymerase, which we named BHK/T7-9. We also constructed new helper plasmids for expression of nucleoprotein and RNA polymerase of the RC-HL strain using the pTM1 plasmid vector, which makes the T7 RNA polymerase-transcripts from the plasmid cap-independent for translation. After co-transfection of these helper plasmids and the previously constructed full-length genome plasmid of the RC-HL strain to BHK/T7-9 cells, recombinant rabies virus was efficiently recovered from the cloned cDNA.
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Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: Glycoprotein gene is associated with virulence for adult mice Reviewed
N Ito, M Takayama, K Yamada, M Sugiyama, N Minamoto
JOURNAL OF VIROLOGY 75 ( 19 ) 9121 - 9128 2001.10
Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY
In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.