Language：English   Publishing type：Research paper (scientific journal)  

Avian rotaviruses A (RVAs) are occasionally transmitted to animals other than the original hosts across species barriers. Information on RVAs carried by various bird species is important for identifying the origin of such interspecies transmission. In this study, to facilitate an understanding of the ecology of RVAs from wild birds, we characterized all of the genes of an RVA strain, JC-105, that was detected in a fecal sample of a large-billed crow (Corvus macrorhynchos) in Japan. All of the genes of this strain except for the VP4 and VP7 genes, which were classified as novel genotypes (P[56] and G40, respectively), were closely related to those of the avian-like RVA strain detected from a raccoon, indicating the possibility that crows had been involved in the transmission of avian RVAs to raccoons. Our findings highlight the need for further viral investigations in wild birds and mammals to understand the mechanisms of avian-to-mammal RVA transmission.

DOI： 10.1016/j.virol.2024.110114

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Language：English   Publishing type：Research paper (scientific journal)  

Akabane virus (AKAV) is a member of the genus Orthobunyavirus, family Peribunyaviridae. In addition to AKAV strains that cause fetal Akabane disease, which is characterized by abortion in ruminants, some AKAV strains cause postnatal infection characterized by nonsuppurative encephalomyelitis in ruminants. Here, we focused on the NSs protein, a virulence factor for most viruses belonging to the genus Orthobunyavirus, and we hypothesized that this protein would act as a neurovirulence factor in AKAV strains causing postnatal encephalomyelitis. We generated AKAV strains that were unable to produce the NSs protein, derived from two different genogroups, genogroups I and II, and then examined the role of their NSs proteins by inoculating mice intracerebrally with these modified viruses. Our results revealed that the neurovirulence of genogroup II strains is dependent on the NSs protein, whereas that of genogroup I strains is independent of this protein. Notably, infection of primary cultured bovine cells with these viruses suggested that the NSs proteins of both genogroups suppress innate immune-related gene expression with equal efficiency. These results indicate differences in the determinants of virulence of orthobunyaviruses.

DOI： 10.1007/s00705-023-05929-w

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.3390/pathogens12121382

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Lead (Pb), an environmental pollutant, has been widely reported to have contaminated mammals, including humans and birds. This study focuses on the effects of Pb pollution on avian influenza virus (AIV) antibody production. A total of 170 black-headed gulls (Chroicocephalus ridibundus) were captured in Tokyo Bay (TBP) from January 2019 to April 2020 and in Mikawa Bay (MBP) from November 2019 to April 2021. The gulls were weighed, subjected to blood sampling, and released with a ring band on their tarsus. The samples were used to measure blood Pb levels (BLL) and AIV-specific antibodies. The BLL were compared using the Wilcoxon two-sample test between the period when black-headed gulls arrived and the wintering period, defined by the number of gulls counted in each area. A significant increase was found in the TBP. A decrease in BLL significantly increased antibody titer during wintering in TBP and MBP. Pb pollution had a negative effect on the production of AIV antibodies. These findings suggest that wild birds that were contaminated by Pb in the environment may facilitate the spread of zoonotic diseases, further increasing the possibility that environmental pollutants may threaten human health.

DOI： 10.3390/ani13142338

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

During the 2020/21 winter season, 29 and 10 H5N8 high pathogenicity avian influenza viruses (HPAIVs) were isolated from environmental water and wild birds, respectively, in Kagoshima prefecture, Japan. Furthermore, seven subtypes of low pathogenicity avian influenza viruses (LPAIVs) were also isolated; H1N1, H2N9, H3N2, H3N6, H3N8, H4N6, and H6N6 subtypes. While the H5 hemagglutinin (HA) genes of the G1 cluster were isolated throughout the winter season, those of the G2 cluster were also detected in late winter, suggesting that H5 HPAIVs possessing H5 HA genes from the two different clusters were individually introduced into Kagoshima prefecture. Intriguingly, genetic constellations revealed that the H5N8 HPAIVs could be classified into six genotypes, including four previously reported genotypes (E1, E2, E3, and E7), and two new genotypes (tentatively named E8 and E9). The PB1 and PA gene segments of genotypes E8 and E9 shared high similarity with those of LPAIVs, whereas the remaining gene segments were close to those of genotype E1. Furthermore, LPAIVs whose PA gene segment was close to that of genotype E9 were isolated from the environmental water. Overall, we revealed that various HPAIV genotypes circulated in Kagoshima prefecture during the 2020/21 winter season. This study highlights the importance of monitoring both HPAIV and LPAIV to better understand AIV ecology in migratory waterfowl populations.

DOI： 10.1099/jgv.0.001870

DOI： 10.1099/jgv.0.001870

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Newcastle disease caused by highly pathogenic viruses of avian paramyxovirus serotype-1 (APMV-1) is a highly contagious poultry disease. Although a large-scale epidemic of Newcastle disease had occurred in Japan between the 1950s and the 2000s, there have been no outbreaks anywhere since 2010. In addition, there are no reports of epidemiological surveys of APMV-1 in wild birds in Japan in the last 10 years. We conducted the first epidemiological survey of APMV-1 in the Izumi plain, Kagoshima prefecture of southern Japan from the winter of 2018 to 2022. A total of 15 APMV-1 strains were isolated, and isolation rates from roosting water and duck fecal samples were 2.51% and 0.10%, respectively. These results indicate that the isolation method from environmental water may be useful for efficient surveillance of APMV-1 in wild birds. Furthermore, this is the first report on the success of APMV-1 isolation from environmental water samples. Genetic analysis of the Fusion (F) gene showed that all APMV-1 isolates were closely related to virus strains circulating among waterfowl in Far East Asian countries. All isolates have avirulent motifs in their cleavage site of F genes, all of which were presumed to be low pathogenic viruses in poultry. However, pathogenicity test using embryonated chicken eggs demonstrated that some isolates killed all chicken embryos regardless of viral doses inoculated (102 -106 50% egg infectious dose). These results indicated that APMV-1 strains, which are potentially pathogenic to chickens, are continuously brought into the Izumi plain by migrating wild birds.

DOI： 10.1111/1348-0421.13053

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00705-022-05670-w

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Other Link： https://link.springer.com/article/10.1007/s00705-022-05670-w/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Background: The transmissible capacity and toxicity of SARS-CoV-2 variants are continually changing. We report here the follow-up study of hospitalized COVID-19 patients from 2020 to 2022. It is known that the PCR diagnosis for hospitalized patients sometimes causes confusion because of the incompatibility between their diagnosis and symptoms. We applied our sugar chain-immobilized gold-nanoparticles for the extraction and partial purification of RNA from specimens for quantitative RT-PCR assay and evaluated whether the results correlate with patients’ symptoms. Methods and Results: Saliva specimens were taken from hospitalized patients with mild or moderate symptoms every early morning. At the time of RT-PCR diagnosis, two methods for the extraction and partial purification of RNA from the specimen were performed: a commonly used Boom (Qiagen) method and our original sugar chain-immobilized gold nanoparticle (SGNP) method. For symptoms, body temperature and oxygen saturation (SpO2) of patients were monitored every 4 h. Conclusions: It was clear that patients infected with the Delta variant needed more time to recover than those with the Omicron variant, and that the SGNP method showed more realistic correlation with the symptoms of patients compared with the common Qiagen method.

DOI： 10.3390/v14112577

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Society  

A recent study demonstrated the possibility that migratory birds are responsible for the global spread of avian rotavirus A (RVA). However, little is known about what types of RVAs are retained in migratory birds. In this study, to obtain information on RVA strains in migratory birds, we characterised an RVA strain, Ho374, that was detected in a faecal sample from a gull species (Larus sp.). Genetic analysis revealed that all 11 genes of this strain were classified as new genotypes (G28-P[39]-I21-R14-C14-M13-A24-N14-T16-E21-H16). This clearly indicates that the genetic diversity of avian RVAs is greater than previously recognised. Our findings highlight the need for investigations of RVA strains retained in migratory birds, including gulls.

DOI： 10.1099/jgv.0.001792

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

White-tailed sea eagle (Haliaeetus albicilla), a regionally rare species of raptor, is threatened in several countries. To assess the risk of H5 high pathogenicity avian influenza (HPAI) viral infection in rare bird species, we performed experimental infections with a GS/GD96-lineage H5N6 HPAI virus of clade 2.3.4.4e in white-tailed sea eagles. Additionally, during the winter of 2020–2021 in Japan, we accidentally encountered a white-tailed sea eagle that had a fatal outcome due to natural infection with a GS/GD96-lineage H5N8 HPAI virus of clade 2.3.4.4b, allowing us to compare experimental and natural infections in the same rare raptor species. Our experiments demonstrated the susceptibility of white-tailed sea eagles to the GS/GD96-lineage H5 HPAI virus with efficient replication in systemic organs. The potential for the viruses to spread within the white-tailed sea eagle population through indirect transmission was also confirmed. Comprehensive comparisons of both viral distribution and histopathological observations between experimentally and naturally infected white-tailed sea eagles imply that viral replication in the brain is responsible for the disease severity and mortality in this species. These findings provide novel insights into the risk assessment of H5 HPAI viral infection in white-tailed sea eagles, proper diagnostic procedures, potential risks to artificially fed eagle populations and persons handling superficially healthy eagles, potential impact of intragastric infection on eagle outcomes, and possibility of severity of the disease being attributed to viral replication in the brain.

DOI： 10.3389/fmicb.2022.1007350

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Rabies virus (RABV) is a neglected zoonotic pathogen that causes lethal infections in almost all mammalian hosts, including humans. Recently, RABV has been reported to induce intracellular formation of stress granules (SGs), also known as platforms that activate innate immune responses.

DOI： 10.1128/jvi.00810-22

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The Izumi plain in the Kagoshima Prefecture, Japan, is known as an overwintering site for more than 30,000 migratory waterfowl, including endangered crane species. We previously reported that environmental water samples, from artificial wet paddies created as crane roost sites on the Izumi plain, are useful for avian influenza virus (AIV) surveillance. During the 2019/20 winter season, we collected 238 water samples from the crane roost sites and isolated 22 AIVs of six subtypes: one H1N1, one H3N2, seven H3N8, four H4N6, nine H6N6, and one H11N2 subtypes. Genetic analyses revealed that AIVs of the same subtype isolated from the Izumi plain during a single winter season exhibited multiple genetic constellations. Furthermore, phylogenetic analyses suggested that our H3N2 isolate may be a genetic reassortant between close relatives to our H3N8 and H11N2 isolates. Our study highlighted the importance of monitoring AIV circulation to better understand AIV ecology in migratory waterfowl populations.

DOI： 10.3390/pathogens11091013

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Centers for Disease Control and Prevention ({CDC})  

DOI： 10.3201/eid2807.212586

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/tbed.14631

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/tbed.14639

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Recent global outbreaks of highly pathogenic avian influenza viruses (HPAIVs) of the H5N8 subtype in poultry and wild birds have raised concerns about animal and human health, particularly after its first evidence of zoonotic transmission from birds to humans. Here, we report a lethal infection with the H5N8 HPAIV in a mandarin duck that had previously demonstrated resistance to the H5N8 HPAIV infection. In addition, we revealed that the isolated virus was a genetic reassortant between the existing H5N8 HPAIV and LPAIV(s). Although further studies are warranted to assess the impact of the genetic reassortment on virus pathogenicity, the potential role of mandarin ducks in HPAIV dissemination should be re-evaluated.

DOI： 10.3390/zoonoticdis2010004

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ttbdis.2021.101695

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Language：English   Publishing type：Research paper (scientific journal)  

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.

DOI： 10.1038/s41467-021-22964-w

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease, caused by PRRS virus (PRRSV), that critically affects the swine industry. While the detection of PRRSV genes plays a key role in PRRS control, the PRRSV genome is known to undergo frequent mutation. Nevertheless, primer pairs widely used for the detection of PRRSV genes were designed between 1995 and 2010. The reliability of these primer pairs for the detection of currently circulating PRRSVs is therefore questionable. Here, we investigated the sensitivity of the previously reported primer pairs to detect PRRSV genes that have been recently isolated or detected in Japan. In addition, based on nucleotide sequences from the recent Japanese PRRSVs, we designed four new primer pairs for the detection of PRRSV genes. The sensitivity and specificity of the new primer pairs were evaluated by quantitative reverse transcription PCR using RNA extracted from PRRSV isolates, swine serum, and oral fluid specimens collected from PRRS-affected pigs, and swine sera collected from a PRRSV-free pig farm in Japan. One of novel primer pairs used in our study exhibited greater sensitivity than the previously reported primer pairs, and is thus more reliable for the detection of PRRSV genes.

DOI： 10.1016/j.jviromet.2021.114071

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Monitoring of the epidemic situation is imperative to control the risk of infection with avian influenza H5 and H7 subtype viruses. A microneutralization (MN) assay was employed to detect hemagglutinin (HA) subtype-specific antibodies. However, the conventional MN assay raises biosafety concerns and is labor-intensive and time-consuming. Therefore, a safer and more convenient assay that can be applied in a high-throughput format is warranted. In this study, PB2 knockout (PB2-KO) influenza viruses of H5 and H7 subtypes expressing different colored fluorescent proteins were generated using a reverse genetics system and applied to a novel MN assay for the detection of specific antibodies. The detection sensitivity of our PB2-KO virus-based MN assay was evaluated by observing fluorescent proteins under a fluorescence microscope and measuring fluorescence intensities using a plate reader. In addition, the PB2-KO virus-based MN assay was used for the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Expression of the reporter fluorescent protein from H5 and H7 PB2-KO viruses was restricted toin PB2 protein-expressing cells. The MN titer as determined using fluorescence microscopy and plate reader revealed that the detection sensitivity of our PB2-KO virus-based MN assay was comparable to that of the conventional MN assay. Moreover, H5 and H7 PB2-KO viruses could be usedapplied forto the simultaneous detection of H5 and H7 subtype-specific antibodies in a single assay. Our study demonstrates a safe and convenient assay for the detection of H5 and H7 subtype-specific antibodies.

DOI： 10.1016/j.virusres.2021.198331

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Language：English   Publishing type：Research paper (scientific journal)  

We previously reported that the avirulent fixed rabies virus strain Ni-CE induces a clear cytopathic effect in mouse neuroblastoma cells, whereas its virulent progenitor, the Nishigahara strain, does not. Infection with Nishigahara and Ni-CE mutants containing a single amino acid substitution in the matrix protein (M) demonstrated that the amino acid at position 95 of M (M95) is a cytopathic determinant. The characteristics of cell death induced by Ni-CE infection resemble those of apoptosis (rounded and shrunken cells, DNA fragmentation), but the intracellular signalling pathway for this process has not been fully investigated. In this study, we aimed to elucidate the mechanism by which M95 affects cell death induced by human neuroblastoma cell infection with the Nishigahara, Ni-CE and M95-mutated strains. We demonstrated that the Ni-CE strain induced DNA fragmentation, cell membrane disruption, exposure of phosphatidylserine (PS), activation of caspase-3/7 and anti-poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, an early apoptosis indicator, whereas the Nishigahara strain did not induce DNA fragmentation, caspase-3/7 activation, cell membrane disruption, or PARP-1 cleavage, but did induce PS exposure. We also demonstrated that these characteristics were associated with M95 using M95-mutated strains. However, we found that Ni-CE induced cell death despite the presence of a caspase inhibitor, Z-VAD-FMK. In conclusion, our data suggest that M95 mutation-related cell death is caused by both the caspase-dependent and -independent pathways.

DOI： 10.1099/jgv.0.001594

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Since the influenza pandemic in 2009, the causative agent 'A(H1N1)pdm09 virus', has been circulating in both human and swine populations. Although phylogenetic analyses of the haemagglutinin (HA) gene segment have revealed broader genetic diversity of A(H1N1)pdm09-related swine influenza A viruses (swIAVs) compared with human A(H1N1)pdm09 viruses, it remains unclear whether the genetic diversity reflects the antigenic differences in HA. To assess the impact of the diversity of the HA gene of A(H1N1)pdm09-related swIAVs on HA antigenicity, we characterized 12 swIAVs isolated in Japan from 2013 to 2018. We used a ferret antiserum and a panel of anti-HA mouse monoclonal antibodies (mAbs) raised against an early A(H1N1)pdm09 isolate. The neutralization assay with the ferret antiserum revealed that five of the 12 swIAVs were significantly different in their HA antigenicity from the early A(H1N1)pdm09 isolate. The mAbs also showed differential neutralization patterns depending on the swIAV strains. In addition, the single amino acid substitution at position 190 of HA, which was found in one of the five antigenically different swIAVs but not in human isolates, was shown to be one of the critical determinants for the antigenic difference of swIAV HAs. Two potential N-glycosylation sites at amino acid positions 185 and 276 of the HA molecule were identified in two antigenically different swIAVs. These results indicated that the genetic diversity of HA in the A(H1N1)pdm09-related swIAVs is associated with their HA antigenic variation. Our findings highlighted the need for surveillance to monitor the emergence of swIAV antigenic variants with public health importance.

DOI： 10.1099/jgv.0.001569

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We isolated two highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N8 clade 2.3.4.4b from falcated duck (Anas falcata) feces and environmental water collected at an overwintering site in Japan. Our isolates were almost genetically identical to each other and showed high genetic similarity with H5N8 HPAIVs recently isolated in South Korea, a distant part of Japan, and European countries. These results suggest the potential role of falcated ducks in the dissemination of HPAIVs.

DOI： 10.3390/pathogens10020171

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Language：English   Publishing type：Research paper (scientific journal)  

The West Africa Ebola outbreak was the largest outbreak ever recorded, with over 28,000 reported infections; this devastating epidemic emphasized the need to understand the mechanisms to counteract virus infection. Here, we screen a library of nearly 400 interferon-stimulated genes (ISGs) against a biologically contained Ebola virus and identify several ISGs not previously known to affect Ebola virus infection. Overexpression of the top ten ISGs attenuates virus titers by up to 1000-fold. Mechanistic studies demonstrate that three ISGs interfere with virus entry, six affect viral transcription/replication, and two inhibit virion formation and budding. A comprehensive study of one ISG (CCDC92) that shows anti-Ebola activity in our screen reveals that CCDC92 can inhibit viral transcription and the formation of complete virions via an interaction with the viral protein NP. Our findings provide insights into Ebola virus infection that could be exploited for the development of therapeutics against this virus.

DOI： 10.1038/s41467-020-16768-7

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Language：English   Publishing type：Research paper (scientific journal)  

The sika deer (Cervus nippon) is one of the most common species of wildlife in Japan. This study aimed to reveal the prevalence of tick-borne protozoan parasites in wild sika deer living in western Japan. We used nested polymerase chain reaction (PCR) to detect the 18S rRNA gene of tick-borne apicomplexan parasites (Babesia, Theileria, and Hepatozoon spp.) from 276 blood and liver samples from sika deer captured in the Yamaguchi, Oita, Kagoshima, Okayama, Ehime, Kochi, and Tokushima Prefectures. In total, 259 samples (259/276; 93.8%) tested positive in the nested PCR screening. Gene sequencing revealed that 99.6% (258/259) of positive samples contained Theileria sp. (sika 1), while Theileria sp. (sika 2), another Theileria species, was detected in only 3 samples. We also found that one sample from a sika deer captured in Kagoshima contained the gene of an unidentified Babesia sp. related to Babesia sp. Kh-Hj42, which was previously collected from tick in western Siberia. In conclusion, we found a high prevalence of piroplasms in sika deer from western Japan, and DNA analysis revealed that Theileria sp. (sika 1) had the highest infection rate.

DOI： 10.1016/j.parint.2020.102161

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Language：English   Publishing type：Research paper (scientific journal)  

Akabane virus (AKAV) (genus Orthobunyavirus, family Peribunyaviridae) is an arthropod-borne virus that causes congenital abnormalities in ruminants. Here, we report the complete genome sequences of two AKAV strains causing nonsuppurative encephalomyelitis in cattle by postnatal infection in Japan.

DOI： 10.1128/MRA.00807-20

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1292/jvms.20-0241

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Because broad genetic diversity has recently been detected in Torque teno sus viruses (TTSuV1 and TTSuVk2), the viral genome detection method needs to be improved to understand the prevalence of these viruses. Here, we established single PCR-based detection methods for the TTSuV1 and TTSuVk2a genomes with newly designed primer pairs and applied them to investigate the prevalence of TTSuV1 and TTSuVk2a in Japanese pig populations. The results revealed that 98.2% and 81.7% of the pig farms tested positive for the TTSuV1 and TTSuVk2a genomes, respectively, indicating that both TTSuV1 and TTSuVk2a are widespread in Japan.

DOI： 10.1111/1348-0421.12779

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site for migratory ducks and endangered cranes. We have surveyed avian influenza viruses (AIVs) in this area since 2012 and isolated low-pathogenic AIVs (LPAIVs) of various subtypes every winter season. H3N8 LPAIVs were isolated during the 2012/13 and 2016/17 seasons, and H4N6 LPAIVs were isolated during the 2012/13 and 2013/14 seasons. In the 2017/18 season, one H3N8 and two H4N6 LPAIV strains were isolated from environmental water samples. Genetic and phylogenetic analysis for each gene segment from these H3N8 and H4N6 LPAIVs suggested that our isolates were genetic reassortants generated by intermixing between AIVs circulating not only in Eurasia but also in Africa and/or North America. Comparison of the genetic constellations of our three isolates with their counterparts isolated during previous seasons from the Izumi plain revealed a drastic transition in the genetic constellations of both subtypes. These findings emphasize the importance of continuous surveillance of AIVs on the Izumi plain.

DOI： 10.1007/s00705-019-04519-z

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Language：English   Publishing type：Research paper (scientific journal)  

To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in Madin-Darby canine kidney subclone II cells and detected two mammal-adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased the virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney subclone II, and human lung adenocarcinoma [Calu-3] cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates, in addition to the lungs. Bat influenza viruses have not been tested for their virulence or organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferrets, respectively. A mutant virus possessing both the NA-F144C and NA-T342A mutations was isolated from both the lung and the trachea, suggesting that it has a broader organ tropism than the wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that the propagation of bat influenza H18N11 virus in mammalian cells can result in mammal-adapting mutations that may increase the replicative ability and/or organ tropism of the virus; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets.IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes have been identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammal-adapting mutations that may increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate to high titers in all infected animals.

DOI： 10.1128/JVI.01416-19

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Language：English   Publishing type：Research paper (scientific journal)  

We conducted a serological survey to detect antibodies against influenza A virus (IAV) in Japanese wild boars in Kagoshima prefecture, Japan, between 2014 and 2017. Seroprevalence against a pandemic-like swine H1N1 (H1N1pdm) virus was identified in 27.1% of specimens, and 1.7% were positive for both swine H1N2 and H3N2 viruses, indicating that wild boars could play an important role in the dynamics of H1N1pdm viral dispersion in the wild. The high frequency of positive results for sera against the H1N1pdm virus suggests that cross-species IAV transmission between wild boars, livestock, and humans is a threat to veterinary and public health.

DOI： 10.1111/1348-0421.12750

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Language：English   Publishing type：Research paper (scientific journal)  

Viruses are believed to be ubiquitous; however, the diversity of viruses is largely unknown because of the bias of previous research toward pathogenic viruses. Deep sequencing is a promising and unbiased approach to detect viruses from animal-derived materials. Although cranes are known to be infected by several viruses such as influenza A viruses, previous studies targeted limited species of viruses, and thus viruses that infect cranes have not been extensively studied. In this study, we collected crane fecal samples in the Izumi plain in Japan, which is an overwintering site for cranes, and performed metagenomic shotgun sequencing analyses. We detected aviadenovirus-like sequences in the fecal samples and tentatively named the discovered virus crane-associated adenovirus 1 (CrAdV-1). We determined that our sequence accounted for approximately three-fourths of the estimated CrAdV-1 genome size (33,245 bp). The GC content of CrAdV-1 genome is 34.1%, which is considerably lower than that of other aviadenoviruses. Phylogenetic analyses revealed that CrAdV-1 clusters with members of the genus Aviadenovirus, but is distantly related to the previously identified aviadenoviruses. The protein sequence divergence between the DNA polymerase of CrAdV-1 and those of other aviadenoviruses is 45.2-46.8%. Based on these results and the species demarcation for the family Adenoviridae, we propose that CrAdV-1 be classified as a new species in the genus Aviadenovirus. Results of this study contribute to a deeper understanding of the diversity and evolution of viruses and provide additional information on viruses that infect cranes, which might lead to protection of the endangered species of cranes.

DOI： 10.1007/s11262-019-01703-w

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Porcine epidemic diarrhea virus (PEDV) induces an often fatal gastrointestinal disease in piglets. In this study, we performed a PEDV infection experiment with the Microminipig, the smallest of experimental minipigs, as a novel small animal model. We orally inoculated a neonatal Microminipig with an intestinal homogenate of a PEDV-infected pig and housed it in a small cage originally designed for rats in an animal biosafety level 2 facility. The infected Microminipig showed the typical signs of porcine epidemic diarrhea (PED), such as watery diarrhea, loss of appetite and weight loss. We also recognized a high amount of excreted PEDV in its rectal swabs and villus atrophy of the small intestine. These results suggest that the Microminipig is a good small animal model for PED, which may contribute to a better understanding of the pathogenesis of PEDV.

DOI： 10.1177/0300985819839236

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：(公社)日本獣医学会  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

During the 2016-2017 winter season, we isolated 33 highly pathogenic avian influenza viruses (HPAIVs) of H5N6 subtype and three low pathogenic avian influenza viruses (LPAIVs) from debilitated or dead wild birds, duck faeces, and environmental water samples collected in the Izumi plain, an overwintering site for migratory birds in Japan. Genetic analyses of the H5N6 HPAIV isolates revealed previously unreported phylogenetic variations in the PB2, PB1, PA, and NS gene segments and allowed us to propose two novel genotypes for the contemporary H5N6 HPAIVs. In addition, analysis of the four gene segments identified close phylogenetic relationships between our three LPAIV isolates and the contemporary H5N6 HPAIV isolates. Our results implied the co-circulation and co-evolution of HPAIVs and LPAIVs within the same wild bird populations, thereby highlighting the importance of avian influenza surveillance targeting not only for HPAIVs but also for LPAIVs.

DOI： 10.1111/tbed.13087

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

<p>There are many difficult tasks to solve for grasping and transporting operations, specially, one of them has difficult tasks to grasp every object with one type of grasping mechanism. Therefore, robots with gripping mechanism cooperate based on recognition technology to try and solve this problem by grasping and carrying various objects. In this research, we propose a system that shares objects gripped by a robot with a relatively simple gripping mechanism based on recognition technology. Recognition technology based on deep learning recognition using RGB information and recognition technique based on depth information and autonomous mobile robot with different gripping mechanism can handle more objects by sharing grasping transportation for each object. Experimental results have shown the effectiveness of the proposed system.</p>

DOI： 10.1299/jsmermd.2019.2A1-B13

Language：English   Publishing type：Research paper (scientific journal)  

Influenza A viruses cause seasonal epidemics and occasional pandemics. The emergence of viruses resistant to neuraminidase (NA) inhibitors and M2 ion channel inhibitors underlines the need for alternate anti-influenza drugs with novel mechanisms of action. Here, we report the discovery of a host factor as a potential target of anti-influenza drugs. By using cell-based virus replication screening of a chemical library and several additional assays, we identified clonidine as a new anti-influenza agent in vitro. We found that clonidine, which is an agonist of the alpha2-adrenergic receptor (α2-AR), has an inhibitory effect on the replication of various influenza virus strains. α2-AR is a Gi-type G protein-coupled receptor that reduces intracellular cyclic AMP (cAMP) levels. In-depth analysis showed that stimulation of α2-ARs leads to impairment of influenza virus replication and that α2-AR agonists inhibit the virus assembly step, likely via a cAMP-mediated pathway. Although clonidine administration did not reduce lung virus titers or prevent body weight loss, it did suppress lung edema and improve survival in a murine lethal infection model. Clonidine may thus protect against lung damage caused by influenza virus infection. Our results identify α2-AR-mediated signaling as a key pathway to exploit in the development of anti-influenza agents.

DOI： 10.1038/s41598-018-22927-0

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/tbed.12887

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The Izumi plain in Kagoshima Prefecture, Japan, is an overwintering site of endangered cranes (hooded cranes and white-naped cranes) and of many other migratory birds (including wild ducks) that are considered carriers of avian influenza viruses (AIVs). To assess the risks of a highly pathogenic avian influenza outbreak in the crane populations, we tested various environmental samples for AIVs in this area. In the 2014-2015 winter season, we isolated one AIV of the H6N2 subtype from the cranes' roost water and two AIVs of the H11N9 subtype from a crane fecal sample and a cloacal swab of a dead spot-billed duck. Genetic analysis of these AIV isolates indicated that our H6N2 isolate is genetically close to AIVs isolated from wild birds in Southeast Asian countries, except that the PB1 and NS genes belong to the North American virus lineage. All genes of the two H11N9 isolates are related to AIVs belonging to the Eurasian virus lineage. Notably, in our phylogenetic trees, H11 HA and N9 NA genes showing high sequence similarity to the corresponding genes of isolates from wild birds in South Africa and Spain, respectively, did not cluster in the major groups with recent wild-bird isolates from East Asia. These results suggest that AIVs with viral gene segments derived from various locations and bird species have been brought to the Izumi plain. These findings imply a possible association of dynamic movements of wild birds with AIV evolution.

DOI： 10.1007/s00705-017-3698-1

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

An influenza A virus of H4N6 subtype was isolated from the Izumi plain, Japan, in 2013. Genetic analyses revealed that two viral genes (M and NS gene segments) of this isolate were genetically distinct from those of the H4N6 virus isolated from the same place in 2012. Furthermore, three viral genes (PB2, PB1 and M gene segments) of this isolate share high similarity with those of the North American isolates of 2014. These results suggest a high frequency of genetic reassortment of avian influenza viruses in Asian waterfowl and intercontinental movements of avian influenza viruses via migratory waterfowl.

DOI： 10.1111/1348-0421.12545

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Language：English   Publishing type：Research paper (scientific journal)  

Bocaparvoviruses have been studied extensively owing to their ability to cause respiratory illness or gastroenteritis in humans. Some bocaparvoviruses have been detected in non-human primates (gorillas and chimpanzees), but the diversity and evolution of these viruses are not fully understood. In this study, we collected 107 fecal samples from wild western lowland gorillas in Moukalaba-Doudou National Park in Gabon to investigate the presence of bocaparvoviruses. Using a combination of pan-bocaparvovirus PCR and individual identification by microsatellite genotyping, we found that two samples from two apparently healthy infant gorillas were positive for bocaparvovirus. Sequencing and phylogenetic analyses revealed that the two gorilla bocaparvovirus strains are nearly identical and are closely related to viruses in the species Primate bocaparvovirus 2 (with 86.0% nucleotide identity to a human bocavirus 2 isolate). To our knowledge, this is the first report showing the presence of a non-human primate bocaparovirus within Primate bocaparvovirus 2. Our findings provide novel insights into the diversity and evolution of bocaparvoviruses and highlight the importance of surveying these viruses for the safe management of gorilla-based ecotourism.

DOI： 10.1016/j.meegid.2017.05.004

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Language：English   Publishing type：Research paper (scientific journal)  

H5 highly pathogenic avian influenza viruses (HPAIV) have spread in both poultry and wild birds since late 2003. Continued circulation of HPAIV in poultry in several regions of the world has led to antigenic drift. In the present study, we analyzed the antigenic properties of H5 HPAIV isolated in Asia using four neutralizing mAbs recognizing hemagglutinin, which were established using A/chicken/Kumamoto/1-7/2014 (H5N8), belonging to clade 2.3.4.4 and also using polyclonal antibodies. Viruses of clades 1.1, 2.3.2.1, 2.3.4, and 2.3.4.4 had different reactivity patterns to the panel of mAbs, thereby indicating that the antigenicity of the viruses of clade 2.3.4.4 were similar but differed from the other clades. In particular, the antigenicity of the viruses of clade 2.3.4.4 differed from those of the viruses of clades 2.3.4 and 2.3.2.1, which suggests that the recent H5 HPAIV have further evolved antigenically divergent. In addition, reactivity of antiserum suggests that the antigenicity of viruses of clade 2.3.4.4 differed slightly among groups A, B, and C. Vaccines are still used in poultry in endemic countries, so the antigenicity of H5 HPAIV should be monitored continually to facilitate control of avian influenza. The panel of mAbs established in the present study will be useful for detecting antigenic drift in the H5 viruses that emerge from the current strains.

DOI： 10.1111/1348-0421.12478

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Language：English   Publishing type：Research paper (scientific journal)  

Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.

DOI： 10.3201/eid2304.161957

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Language：English   Publishing type：Research paper (scientific journal)  

Adenoviruses are widespread in human population as well as in great apes, although the data about the naturally occurring adenovirus infections remain rare. We conducted the surveillance of adenovirus infection in wild western lowland gorillas in Moukalaba-Doudou National Park (Gabon), in order to investigate naturally occurring adenovirus in target gorillas and tested specifically a possible zoonotic transmission with local people inhabiting the vicinity of the park. Fecal samples were collected from western lowland gorillas and humans, and analyzed by PCR. We detected adenoviral genes in samples from both gorillas and the local people living around the national park, respectively: the overall prevalence rates of adenovirus were 24.1 and 35.0 % in gorillas and humans, respectively. Sequencing revealed that the adenoviruses detected in the gorillas were members of Human mastadenovirus B (HAdV-B), HAdV-C, or HAdV-E, and those in the humans belonged to HAdV-C or HAdV-D. Although HAdV-C members were detected in both gorillas and humans, phylogenetic analysis revealed that the virus detected in gorillas are genetically distinct from those detected in humans. The HAdV-C constitutes a single host lineage which is compatible with the host-pathogen divergence. However, HAdV-B and HAdV-E are constituted by multiple host lineages. Moreover, there is no evidence of zoonotic transmission thus far. Since the gorilla-to-human transmission of adenovirus has been shown before, the current monitoring should be continued in a broader scale for getting more insights in the natural history of naturally occurring adenoviruses and for the safe management of gorillas' populations.

DOI： 10.1007/s11262-016-1360-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Cattle are major reservoirs of the provisionally named influenza D virus, which is potentially involved in the bovine respiratory disease complex. Here, we conducted a serological survey for the influenza D virus in Japan, using archived bovine serum samples collected during 2010-2016 from several herds of apparently healthy cattle in various regions of the country. We found sero-positive cattle across all years and in all the prefectural regions tested, with a total positivity rate of 30.5%, although the positivity rates varied among regions (13.5-50.0%). There was no significant difference in positivity rates for Holstein and Japanese Black cattle. Positivity rates tended to increase with cattle age. The herds were clearly divided into two groups: those with a high positive rate and those with a low (or no) positive rate, indicating that horizontal transmission of the virus occurs readily within a herd. These data demonstrate that bovine influenza D viruses have been in circulation for at least 5 years countrywide, emphasizing its ubiquitous distribution in the cattle population of Japan.

DOI： 10.1371/journal.pone.0163828

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Language：English   Publishing type：Research paper (scientific journal)  

Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED), which is threatening the swine industry all over the world. In Japan, although there were no reported PED cases from 2007 to 2012, a large-scale PED outbreak started in 2013, causing severe economic losses. Although several PEDV studies have been conducted in Japan, more PEDV isolates and sequence information are needed to understand the molecular biology and epidemiology of PEDV. Here, we isolated seven Japanese PEDV strains from intestinal tissue samples collected in 2014 and determined the spike gene sequences of 13 Japanese PEDV strains, including the above seven isolates. Phylogenetic analysis shows that all of the strains are genetically distinct from classical Japanese PEDV strains isolated prior to 2013 and can be classified into two different genotypes: 12 strains belong to the North American clade composed of recent highly pathogenic PEDV strains, and the remaining one strain belongs to the so-called insertion deletion (INDEL) clade. These data suggest multiple PEDV invasions from abroad to Japan. Notably, compared to classical Japanese strains, all of the recent Japanese strains have two amino acid substitutions in a known neutralizing epitope. In addition, one of the strains acquired an additional mutation in another neutralizing epitope that is highly conserved among PEDVs, including the classical and recent isolates. Our isolates and findings will be useful for future investigations aimed at understanding, controlling, and preventing PED.

DOI： 10.1007/s00705-016-2900-1

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Language：English   Publishing type：Research paper (scientific journal)  

Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.

DOI： 10.1038/nmicrobiol.2016.58

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Language：English   Publishing type：Research paper (scientific journal)  

Influenza viruses that express reporter proteins are useful tools, but are often attenuated. Recently, we found that an influenza virus encoding the Venus fluorescent protein acquired two mutations in its PB2 and HA proteins upon mouse adaptation. Here, we demonstrate that the enhanced viral replication and virulence in mice of this Venus-expressing influenza virus are primarily conferred by the PB2-E712D mutation, with only a minor contribution by the HA-T380A mutation.

DOI： 10.1038/srep19933

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Language：English   Publishing type：Research paper (scientific journal)  

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-β 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 ϵ (IKKϵ)-inducible IRF-3-dependent IFN-β promoter activity. Importantly, we revealed that the P proteins from the 1088 and HCM-9 strains, but not from the remaining viruses, interacted with IKKϵ. 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 IKKϵ. 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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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Foot-and-mouth disease virus (FMDV) possess a positive sense, single stranded RNA genome. Internal ribosomal entry site (IRES) element exists within its 5' untranslated region (5'UTR) of the viral RNA. Translation of the viral RNA is initiated by internal entry of the 40S ribosome within the IRES element. This process is facilitated by cellular factors known as IRES trans-acting factors (ITAFs). Foot-and-mouth disease (FMD) is host-restricted disease for cloven-hoofed animals such as cattle and pigs, but the factors determining the host range have not been identified yet. Although, ITAFs are known to promote IRES-mediated translation, these findings were confirmed only in cells derived from FMDV-insusceptible animals so far. We evaluated and compared the IRES-mediated translation activities among cell lines derived from four different animal species using bicistronic luciferase reporter plasmid, which possesses an FMDV-IRES element between Renilla and Firefly luciferase genes. Furthermore, we analyzed the effect of the cellular factors on IRES-mediated translation by silencing the cellular factors using siRNA in both FMDV-susceptible and -insusceptible animal cells. RESULTS: Our data indicated that IRES-mediated translational activity was not linked to FMDV host range. ITAF45 promoted IRES-mediated translation in all cell lines, and the effects of poly-pyrimidine tract binding protein (PTB) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were observed only in FMDV-susceptible cells. Thus, PTB and 4E-BP1 may influence the host range of FMDV. CONCLUSIONS: IRES-mediated translation activity of FMDV was not predictive of its host range. ITAF45 promoted IRES-mediated translation in all cells, and the effects of PTB and 4E-BP1 were observed only in FMDV-susceptible cells.

DOI： 10.1186/S12917-016-0694-8

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Secondary bacterial infections after influenza can be a serious problem, especially in young children and the elderly, yet the efficacy of current vaccines is limited. Earlier work demonstrated that a replication-incompetent PB2-knockout (PB2-KO) influenza virus possessing a foreign gene in the coding region of its PB2 segment can serve as a platform for a bivalent vaccine. METHODS: In the current study, we generated the PB2-KO virus expressing pneumococcal surface protein A (PspA), PB2-KO-PspA virus, the replication of which is restricted to PB2-expressing cells. We then examined the protective efficacy of intranasal immunization with this virus as a bivalent vaccine in a mouse model. RESULTS: High levels of influenza virus-specific and PspA-specific antibodies were induced in the serum and airways of immunized mice. The intranasally immunized mice were protected from lethal doses of influenza virus or Streptococcus pneumoniae. These mice were also completely protected from secondary pneumococcal pneumonia after influenza virus infection. CONCLUSIONS: These findings indicate that our recombinant influenza virus serves as a novel and powerful bivalent vaccine against primary and secondary pneumococcal pneumonia as well as influenza.

DOI： 10.1093/infdis/jiv341

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Language：English   Publishing type：Research paper (scientific journal)  

Non-primate hepacivirus (NPHV) is a recently discovered homolog of the hepatitis C virus in horses. The frequency and distribution of NPHV infections among horses in Japan is unknown. In this study, serum samples from 453 horses across Japan were screened for NPHV RNA using real-time RT-PCR and anti-nonstructural 3 protein (NS3) antibodies using the Gaussia luciferase immunoprecipitation system assay. In order to monitor the course of NPHV infection in horses, we examined 31 stored samples (9 adult horses and 22 young horses) obtained one year ago and compared the results to the recent data. Stored sera from 7 mare-foal pairs were also examined. The NS3 region sequences of 14 NPHV strains from NPHV RNA positive serum samples were determined and analyzed phylogenically. Of the 453 serum samples tested, 33.55% were positive for anti-NS3 antibody and 13.68% were positive for NPHV RNA. We found a higher rate of NPHV RNA detection in serum obtained from young horses (1-2 years of age) than that of adults, in two geographically distinct areas. We observed higher variation in the course of infection over one year in young horses than in adult horses. The foals were infected with NPHV after the weaning period. Phylogenic analysis revealed that while NPHV NS3 genes isolated in Japan clustered with sequences previously classified as NPHV, but the genetic diversity of the Japanese NPHV strains we detected was not correlated with their geographic origin. In conclusion, Japanese horses exhibit a high prevalence of NPHV. Young age appears to be a risk factor for such viral infection in Japan, although the infectious route was not determined.

DOI： 10.1016/j.vetmic.2015.05.028

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The Izumi plain in Kagoshima prefecture, Japan, is an overwintering site of more than 10,000 cranes. The wet paddy areas are artificially created to provide roosting sites for the cranes every winter. Since wild ducks, known to be a natural reservoir of influenza A viruses, also overwinter in this area, the cranes' roost water likely serves as a source of influenza A virus infection. To assess this potential risk, we collected 126 water samples from the cranes' roost in the 2012/2013 winter season for virus isolation. We isolated six influenza viruses of three subtypes (H3N8, H4N6, and H4N8) from the water samples collected in the months of November and December. Genetic analysis of our isolates indicated that these viruses were genetically similar to the low-pathogenic avian influenza viruses circulating among Eurasian waterfowl. These findings suggest the possibility of the cranes becoming infected with the avian influenza viruses that are present in their roost water.

DOI： 10.1007/s00705-015-2610-0

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We determined the complete genome sequences of torque teno sus viruses (TTSuVs) detected in pigs with postweaning multisystemic wasting syndrome (PMWS) and in healthy pigs in Japan. Unexpectedly, we found coinfection of a PMWS-affected pig in Japan with one strain of TTSuV1, five strains of TTSuV2, and one strain of PCV2. Full-genome sequencing of each of these strains, followed by phylogenetic analysis, revealed broad genetic diversity in the TTSuV2 strains infecting the PMWS-affected pig. These results suggest that the geographical bias in the available genetic information about TTSuVs has a limited impact on the evaluation of their genetic diversity.

DOI： 10.1007/s00705-015-2593-x

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Language：English   Publishing type：Research paper (scientific journal)  

Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

DOI： 10.1038/ncomms8491

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We isolated eight highly pathogenic H5N8 avian influenza viruses (H5N8 HPAIVs) in the 2014/15 winter season at an overwintering site of migratory birds in Japan. Genetic analyses revealed that these isolates were divided into three groups, indicating the co-circulation of three genetic groups of H5N8 HPAIV among these migratory birds. These results also imply the possibility of global redistribution of the H5N8 HPAIVs via the migration of these birds next winter.

DOI： 10.2807/1560-7917.ES2015.20.20.21132

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain "undiscovered" in the global porcine populations. We propose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.

DOI： 10.1128/JCM.02941-14

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Language：English   Publishing type：Research paper (scientific journal)  

Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours ('Color-flu' viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.

DOI： 10.1038/ncomms7600

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UNLABELLED: We previously reported that an H5N1 virus carrying the Venus reporter gene, which was inserted into the NS gene segment from the A/Puerto Rico/8/1934(H1N1) virus (Venus-H5N1 virus), became more lethal to mice, and the reporter gene was stably maintained after mouse adaptation compared with the wild-type Venus-H5N1 (WT-Venus-H5N1) virus. However, the basis for this difference in virulence and Venus stability was unclear. Here, we investigated the molecular determinants behind this virulence and reporter stability by comparing WT-Venus-H5N1 virus with a mouse-adapted Venus-H5N1 (MA-Venus-H5N1) virus. To determine the genetic basis for these differences, we used reverse genetics to generate a series of reassortants of these two viruses. We found that reassortants with PB2 from MA-Venus-H5N1 (MA-PB2), MA-PA, or MA-NS expressed Venus more stably than did WT-Venus-H5N1 virus. We also found that a single mutation in PB2 (V25A) or in PA (R443K) increased the virulence of the WT-Venus-H5N1 virus in mice and that the presence of both of these mutations substantially enhanced the pathogenicity of the virus. Our results suggest roles for PB2 and PA in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus. IMPORTANCE: The ability to visualize influenza viruses has far-reaching benefits in influenza virus research. Previously, we reported that an H5N1 virus bearing the Venus reporter gene became more pathogenic to mice and that its reporter gene was more highly expressed and more stably maintained after mouse adaptation. Here, we investigated the molecular determinants behind this enhanced virulence and reporter stability. We found that mutations in PB2 (V25A) and PA (R443K) play crucial roles in the stable maintenance of a foreign protein as an NS1 fusion protein in influenza A virus and in the virulence of influenza virus in mice. Our findings further our knowledge of the pathogenicity of influenza virus in mammals and will help advance influenza virus-related live-imaging studies in vitro and in vivo.

DOI： 10.1128/JVI.01886-15

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The sensitivity of influenza rapid diagnostic tests (IRDTs) currently available in Japan for various influenza virus strains, including human H7N9 and H5N1 isolates, were compared and it was found that all of the IRDTs examined detected these viruses; however, their detection sensitivities differed.

DOI： 10.1111/1348-0421.12185

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Hepatitis B virus (HBV) poses a threat to global public health mainly because of complications of HBV-related chronic liver disease. HBV exhibits a narrow host range, replicating primarily in hepatocytes by a still poorly understood mechanism. For the generation of progeny virions, HBV depends on interactions with specific host factors through its life cycle. Revealing and characterizing these interactions are keys to identifying novel antiviral targets, and to developing specific treatment strategies for HBV patients. In this review, recent insights into the HBV-host interactions, especially on virus entry, intracellular trafficking, genome transcription and replication, budding and release, and even cellular restriction factors were reviewed.

DOI： 10.1002/jmv.23916

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Infections with influenza and respiratory syncytial virus (RSV) rank high among the most common human respiratory diseases worldwide. Previously, we developed a replication-incompetent influenza virus by replacing the coding sequence of the PB2 gene, which encodes one of the viral RNA polymerase subunits, with that of a reporter gene. Here, we generated a PB2-knockout recombinant influenza virus expressing the F protein of RSV (PB2-RSVF virus) and tested its potential as a bivalent vaccine. In mice intranasally immunized with the PB2-RSVF virus, we detected high levels of antibodies against influenza virus, but not RSV. PB2-RSVF virus-immunized mice were protected from a lethal challenge with influenza virus but experienced severe body weight loss when challenged with RSV, indicating that PB2-RSVF vaccination enhanced RSV-associated disease. These results highlight one of the difficulties of developing an effective bivalent vaccine against influenza virus and RSV infections.

DOI： 10.1007/s00705-013-1932-z

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Language：English   Publishing type：Research paper (scientific journal)  

Hepatitis C virus (HCV) infection leads to the development of hepatic diseases, as well as extrahepatic disorders such as B-cell non-Hodgkin's lymphoma (B-NHL). To reveal the molecular signalling pathways responsible for HCV-associated B-NHL development, we utilised transgenic (Tg) mice that express the full-length HCV genome specifically in B cells and develop non-Hodgkin type B-cell lymphomas (BCLs). The gene expression profiles in B cells from BCL-developing HCV-Tg mice, from BCL-non-developing HCV-Tg mice, and from BCL-non-developing HCV-negative mice were analysed by genome-wide microarray. In BCLs from HCV-Tg mice, the expression of various genes was modified, and for some genes, expression was influenced by the gender of the animals. Markedly modified genes such as Fos, C3, LTβR, A20, NF-κB and miR-26b in BCLs were further characterised using specific assays. We propose that activation of both canonical and alternative NF-κB signalling pathways and down-regulation of miR-26b contribute to the development of HCV-associated B-NHL.

DOI： 10.1371/journal.pone.0091373

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Language：English   Publishing type：Research paper (scientific journal)  

Because vaccination is an effective means to protect humans from influenza viruses, extensive efforts have been made to develop not only new vaccines, but also for new adjuvants to enhance the efficacy of existing inactivated vaccines. Here, we examined the adjuvanticity of synthetic hemozoin, a synthetic version of the malarial by-product hemozoin, on the vaccine efficacy of inactivated whole influenza viruses in a mouse model. We found that mice immunized twice with hemozoin-adjuvanted inactivated A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1) virus elicited higher virus-specific antibody responses than did mice immunized with non-adjuvanted counterparts. Furthermore, mice immunized with hemozoin-adjuvanted inactivated viruses were better protected from lethal challenge with influenza viruses than were mice immunized with non-adjuvanted inactivated vaccines. Our results show that hemozoin improves the immunogenicity of inactivated influenza viruses, and is thus a promising adjuvant for inactivated whole virion influenza vaccines.

DOI： 10.1016/j.vaccine.2014.07.079

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Language：English   Publishing type：Research paper (scientific journal)  

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

DOI： 10.1038/nature12392

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Language：English   Publishing type：Research paper (scientific journal)  

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.

DOI： 10.1128/JVI.00076-13

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Language：English   Publishing type：Research paper (scientific journal)  

The influenza A virus NS1 protein affects virulence through several mechanisms, including the host's innate immune response and various signaling pathways. Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype continue to evolve through reassortment and mutations. Our recent phylogenetic analysis identified a group of HPAI H5N1 viruses with two characteristic mutations in NS1: the avian virus-type PDZ domain-binding motif ESEV (which affects virulence) was replaced with ESKV, and NS1-138F (which is highly conserved among all influenza A viruses and may affect the activation of the phosphatidylinositol 3-kinase [PI3K]/Akt signaling pathway) was replaced with NS1-138Y. Here, we show that an HPAI H5N1 virus (A/duck/Hunan/69/2004) encoding NS1-ESKV and NS1-138Y was confined to the respiratory tract of infected mice, whereas a mutant encoding NS1-ESEV and NS1-138F caused systemic infection and killed mice more efficiently. Mutation of either one of these sites had small effects on virulence. In addition, we found that the amino acid at NS1-138 affected not only the induction of the PI3K/Akt pathway but also the interaction of NS1 with cellular PDZ domain proteins. Similarly, the mutation in the PDZ domain-binding motif of NS1 altered its binding to cellular PDZ domain proteins and affected Akt phosphorylation. These findings suggest a functional interplay between the mutations at NS1-138 and NS1-229 that results in a synergistic effect on influenza virulence.

DOI： 10.1128/JVI.02608-12

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Language：English   Publishing type：Research paper (scientific journal)  

It was recently reported by the present team that 3β-hydroxysterol Δ24-reductase (DHCR24) is induced by hepatitis C virus (HCV) infection. In addition, upregulation of DHCR24 impairs p53 activity. In human hepatoma HuH-7 cells, the degree of DHCR24 expression is higher than in normal hepatic cell lines (WRL68) at the transcriptional level. The genomic promoter sequence of DHCR24 was characterized and nucleotide substitutions were observed in HuH-7 cells at nucleotide numbers -1453 (G to A), -1420 (G to T), -488 (A to C) and -200 (G to C). The mutations of these sequences from HuH-7 cell types to WRL68 cell types suppressed DHCR24 gene promoter activity. The sequences were further characterized in hepatocytes from patient tissues. Four tissues from HCV-positive patients with cirrhosis or hepatocellular carcinoma (#1, 2, 3, 5) possessed HuH-7 cell type sequences. Interestingly, one patient with liver cirrhosis (#4) possessed WRL68 cell-type sequences; this patient had been infected with HCV and was HCV negative for 17 years after interferon therapy. Next, the effect of HCV infection on these polymorphisms was examined in humanized chimeric mouse liver and HuH-7 cells. The human hepatocytes possess WRL68 cell type and did not show the nucleotide substitution after HCV infection. The HCV-replicon was removed by interferon treatment and established the cured K4 cells. These cells possess HuH-7 cell type sequences. Thus, this study showed the genomic polymorphism in DHCR24 promoter is not directly influenced by HCV infection.

DOI： 10.1111/1348-0421.12025

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens.

DOI： 10.1038/srep01106

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Although outbreaks of highly pathogenic avian influenza in wild and domestic birds have been posing the threat of a new influenza pandemic for the past decade, the first pandemic of the twenty-first century came from swine viruses. This fact emphasizes the complexity of influenza viral ecology and the difficulty of predicting influenza viral dynamics. Complete control of influenza viruses seems impossible. However, we must minimize the impact of animal and human influenza outbreaks by learning lessons from past experiences and recognizing the current status. Here, we review the most recent influenza virology data in the veterinary field, including aspects of zoonotic agents and recent studies that assess the pandemic potential of H5N1 highly pathogenic avian influenza viruses.

DOI： 10.1146/annurev-animal-031412-103733

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Language：English   Publishing type：Research paper (scientific journal)  

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

DOI： 10.1128/JVI.01925-12

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Language：English   Publishing type：Research paper (scientific journal)  

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. Molecular evolutionary analyses of the 2009 pandemic influenza A H1N1 [A(H1N1)pdm09] virus revealed two major clusters, cluster I and cluster II. Although the pathogenicity of viruses belonging to cluster I, which became extinct by the end of 2009, has been examined in a nonhuman primate model, the pathogenic potential of viruses belonging to cluster II, which has spread more widely in the world, has not been studied in this animal model. Here, we characterized two Norwegian isolates belonging to cluster II, namely, A/Norway/3568/2009 (Norway3568) and A/Norway/3487-2/2009 (Norway3487), which caused distinct clinical symptoms, despite their genetic similarity. We observed more efficient replication in cultured cells and delayed virus clearance from ferret respiratory organs for Norway3487 virus, which was isolated from a severe case, compared with the efficiency of replication and time of clearance of Norway3568 virus, which was isolated from a mild case. Moreover, Norway3487 virus to some extent caused more severe lung damage in nonhuman primates than did Norway3568 virus. Our data suggest that the distinct replicative and pathogenic potentials of these two viruses may result from differences in their biological properties (e.g., the receptor-binding specificity of hemagglutinin and viral polymerase activity).

DOI： 10.1128/JVI.01214-12

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Language：English   Publishing type：Research paper (scientific journal)  

By using two reporter protein-encoding virus-like RNAs derived from identical viral RNA (vRNA) segments, we assessed their incorporation efficiency into single progeny virions. Most plaques formed by the recombinant viruses that were generated in cells positive for both reporter genes expressed only one or the other protein. These results suggest that two virus-like RNAs encoding different reporter proteins compete for incorporation into virions, and individual influenza virions incorporate single, but not multiple, copies of homologous vRNA segments.

DOI： 10.1128/JVI.01204-12

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Language：English   Publishing type：Research paper (scientific journal)  

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans, but currently do not transmit efficiently among humans. The viral haemagglutinin (HA) protein is a known host-range determinant as it mediates virus binding to host-specific cellular receptors. Here we assess the molecular changes in HA that would allow a virus possessing subtype H5 HA to be transmissible among mammals. We identified a reassortant H5 HA/H1N1 virus-comprising H5 HA (from an H5N1 virus) with four mutations and the remaining seven gene segments from a 2009 pandemic H1N1 virus-that was capable of droplet transmission in a ferret model. The transmissible H5 reassortant virus preferentially recognized human-type receptors, replicated efficiently in ferrets, caused lung lesions and weight loss, but was not highly pathogenic and did not cause mortality. These results indicate that H5 HA can convert to an HA that supports efficient viral transmission in mammals; however, we do not know whether the four mutations in the H5 HA identified here would render a wholly avian H5N1 virus transmissible. The genetic origin of the remaining seven viral gene segments may also critically contribute to transmissibility in mammals. Nevertheless, as H5N1 viruses continue to evolve and infect humans, receptor-binding variants of H5N1 viruses with pandemic potential, including avian-human reassortant viruses as tested here, may emerge. Our findings emphasize the need to prepare for potential pandemics caused by influenza viruses possessing H5 HA, and will help individuals conducting surveillance in regions with circulating H5N1 viruses to recognize key residues that predict the pandemic potential of isolates, which will inform the development, production and distribution of effective countermeasures.

DOI： 10.1038/nature10831

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Language：English   Publishing type：Research paper (scientific journal)  

Vaccination is the primary form of protection from influenza virus infection. We recently developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that possesses a reporter gene (the green fluorescent protein gene) in the coding region of the PB2 segment. This virus replicated to high titers in PB2-expressing, but not unmodified, cells, suggesting its potential safety and feasibility as a vaccine. Here, we tested its efficacy in a murine model. The levels of IgG and IgA antibodies against influenza virus in sera, nasal washes, and bronchoalveolar lavage fluids of mice immunized with the PB2-KO virus were higher than those induced by a conventional inactivated vaccine. All PB2-KO virus-immunized mice survived challenges with lethal doses of influenza virus. Moreover, importantly, mice immunized with the PB2-KO virus produced antibodies against the reporter protein, suggesting that the PB2-KO virus has potential as a multivalent vaccine to combat infection with not only influenza virus but also other pathogens.

DOI： 10.1128/JVI.06232-11

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Influenza A virus matrix protein (M1) plays an important role in virus assembly and budding. Besides a well-characterized basic amino acid-rich nuclear localization signal region at positions 101 to 105, M1 contains another basic amino acid stretch at positions 76-78 that is highly conserved among influenza A and B viruses, suggesting the importance of this stretch. To understand the role of these residues in virus replication, we mutated them to either lysine (K), alanine (A), or aspartic acid (D). We could generate viruses possessing either single or combination substitutions with K or single substitution with A at any of these positions, but not those with double substitutions with A or a single substitution with D. Viruses with the single substitution with A exhibited slower growth and had lower nucleoprotein/M1 quantitative ratio in virions compared to the wild-type virus. In cells infected with a virus possessing the single substitution with A at position 77 or 78 (R77A or R78A, respectively), the mutated M1 localized in patches at the cell periphery where nucleoprotein and hemagglutinin colocalized more often than the wild-type did. Transmission electron microscopy showed that virus possessing M1 R77A or R78A, but not the wild-type virus, was present in vesicular structures, indicating a defect in virus assembly and/or budding. The M1 mutations that did not support virus generation exhibited an aberrant M1 intracellular localization and affected protein incorporation into virus-like particles. These results indicate that the basic amino acid stretch of M1 plays a critical role in influenza virus replication.

DOI： 10.1128/JVI.06230-11

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Language：English   Publishing type：Research paper (scientific journal)  

The ability to modify influenza viruses at will has revolutionized influenza research. Reverse genetics has been used to generate mutant or reassortant influenza viruses to assess their replication, virulence, pathogenicity, host range, and transmissibility. Moreover, this technology is now being used to generate approved influenza virus vaccines and develop novel vaccines to combat seasonal and (future) pandemic influenza viruses. Several variations of the original system have been established, all of which are considerably robust and efficient.

DOI： 10.1007/978-1-61779-621-0_12

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Plasmid-based reverse genetics systems allow the artificial generation of viruses with cloned cDNA-derived genomes. Since the establishment of such systems for influenza virus, numerous attempts have been made to tame this pathogenic agent. In particular, several types of viruses expressing foreign genes have been generated and used to further our knowledge of influenza virus replication and pathogenicity and to develop novel influenza vaccines. Here, we review these achievements and discuss future perspectives.

DOI： 10.1016/j.virusres.2011.09.035

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.

DOI： 10.1099/vir.0.037648-0

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The first influenza pandemic of the 21st century was caused by novel H1N1 viruses that emerged in early 2009. An Asp-to-Gly change at position 222 of the receptor-binding protein hemagglutinin (HA) correlates with more-severe infections in humans. The amino acid at position 222 of HA contributes to receptor-binding specificity with Asp (typically found in human influenza viruses) and Gly (typically found in avian and classic H1N1 swine influenza viruses), conferring binding to human- and avian-type receptors, respectively. Here, we asked whether binding to avian-type receptors enhances influenza virus pathogenicity. We tested two 2009 pandemic H1N1 viruses possessing HA-222G (isolated from severe cases) and two viruses that possessed HA-222D. In glycan arrays, viruses possessing HA-222D preferentially bound to human-type receptors, while those encoding HA-222G bound to both avian- and human-type receptors. This difference in receptor binding correlated with efficient infection of viruses possessing HA-222G, compared to those possessing HA-222D, in human lung tissue, including alveolar type II pneumocytes, which express avian-type receptors. In a nonhuman primate model, infection with one of the viruses possessing HA-222G caused lung damage more severe than did infection with a virus encoding HA-222D, although these pathological differences were not observed for the other virus pair with either HA-222G or HA-222D. These data demonstrate that the acquisition of avian-type receptor-binding specificity may result in more-efficient infection of human alveolar type II pneumocytes and thus more-severe lung damage. Collectively, these findings suggest a new mechanism by which influenza viruses may become more pathogenic in mammals, including humans.

DOI： 10.1128/JVI.00859-11

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1111/j.1469-0691.2010.03313.x

Language：English   Publishing type：Research paper (scientific journal)  

In 2009, a swine-origin H1N1 influenza virus caused the first pandemic of the 21st century. To understand the molecular basis of pandemic influenza virus adaptation to new host species, we serially passaged the pandemic (H1N1) 2009 virus strain A/California/04/09 in mouse lungs. After ten passages, the virus became lethal to mice. We found eight amino acid differences between the wild-type and mouse-adapted viruses: one in PB1, three in PA, three in HA, and one in NP. By using reverse genetics to generate mutant viruses, we determined that the amino acid substitutions in PA (at positions 21 and 616), HA (at positions 127 and 222), and NP (at position 375) play independent roles in the increased pathogenicity in mice. Among these five substitutions, an aspartic acid-to-glutamic acid substitution at position 127 in HA contributed to efficient viral replication in mouse lungs. Our results suggest the importance of the viral polymerase complex and of HA in viral adaption to a new host.

DOI： 10.1016/j.virusres.2011.03.022

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Language：English   Publishing type：Research paper (scientific journal)  

Although H5N1 influenza A viruses can cause systemic infection, their neurotropism and long-term effects on the central nervous system (CNS) are not fully understood. We assessed H5N1viral invasion of the CNS and its long-term effects in a ferret model. An H5N1 virus caused nonsuppurative encephalitis, which lasted for 3 months without neurologic signs. Further, another H5N1 virus caused nonsuppurative vasculitis with brain hemorrhage. Three-dimensional analysis of viral distribution in the brain identified the olfactory system as a major route for brain invasion. The efficient growth of virus in the upper respiratory tract may thus facilitate viral brain invasion.

DOI： 10.1128/JVI.00239-11

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.

DOI： 10.1128/JVI.00029-11

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Like the histidine-to-tyrosine substitution at position 274 in neuraminidase (NA H274Y), an asparagine-to-serine mutation at position 294 in this protein (NA N294S) confers oseltamivir resistance to highly pathogenic H5N1 influenza A viruses. However, unlike viruses with the NA H274Y mutation, the properties of viruses possessing NA N294S are not well understood. Here, we assessed the effect of the NA N294S substitution on the replication and pathogenicity of human H5N1 viruses and on the efficacy of the NA inhibitors oseltamivir and zanamivir in mouse and ferret models. Although NA N294S-possessing H5N1 viruses were attenuated in mice and ferrets compared to their oseltamivir-sensitive counterparts, one of the infected ferrets died from systemic infection, demonstrating the potential lethality in ferrets of oseltamivir-resistant H5N1 viruses with the NA N294S substitution. The efficacy of oseltamivir, but not that of zanamivir, against an NA N294S-possessing virus was substantially impaired both in ferrets and in vitro. These results demonstrate the considerable pathogenicity of NA N294S substitution-possessing H5N1 viruses and underscore the importance of monitoring the emergence of the NA N294S mutation in circulating H5N1 viruses.

DOI： 10.1128/JVI.00047-11

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Although influenza virus resistance to the neuraminidase inhibitor zanamivir is reported less frequently than is resistance to the neuraminidase inhibitor oseltamivir in clinical settings, it is unknown whether this difference is due to the limited use of zanamivir or to an inherent property of the drug. We therefore compared the prevalence of drug-resistant viruses and virus shedding in seasonal influenza virus-infected children treated with either oseltamivir or zanamivir. METHODS: Clinical specimens (throat or nasal swab) were collected from a total of 144 pediatric influenza patients during the 2005-2006, 2006-2007, 2007-2008, and 2008-2009 influenza seasons. Neuraminidase inhibitor-resistant mutants were detected among the isolated viruses by sequencing the viral hemagglutinin and neuraminidase genes. Sensitivity of the viruses to neuraminidase inhibitors was tested by neuraminidase inhibition assay. RESULTS: In oseltamivir- or zanamivir-treated influenza patients who were statistically comparable in their age distribution, vaccination history, and type or subtype of virus isolates, the virus-shedding period in zanamivir-treated patients was significantly shorter than that in oseltamivir-treated patients. Furthermore, the frequency of zanamivir-resistant viruses was significantly lower than that of oseltamivir-resistant viruses. CONCLUSION: In comparison with treatment with oseltamivir, treatment of pediatric patients with zanamivir resulted in the emergence of fewer drug-resistant influenza viruses and a shorter virus-shedding period. We conclude that zanamivir shows promise as a better therapy for pediatric influenza patients.

DOI： 10.1093/cid/ciq183

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

A novel influenza (H1N1) virus caused an influenza pandemic in 2009, while highly pathogenic H5N1 avian influenza viruses have continued to infect humans since 1997. Influenza, therefore, remains a serious health threat. Currently, neuraminidase (NA) inhibitors are the mainstay for influenza therapy; however, drug-resistant mutants of seasonal H1N1 and H5N1 viruses have emerged highlighting the need for alternative therapeutic approaches. One such approach is antibody immunotherapy. Here, we show that the monoclonal antibody C179, which recognizes a neutralizing epitope common among H1, H2, H5, and H6 hemagglutinins (HAs), protected mice from a lethal challenge with various H5N1 and pandemic (H1N1) 2009 viruses when administered either intraperitoneally or intranasally. The protective efficacy of intranasally inoculated C179 was comparable to that of intraperitoneal administration. Our results suggest that direct administration of this anti-influenza antibody to viral replication sites is an effective strategy for prophylaxis and therapy.

DOI： 10.1016/j.antiviral.2010.09.007

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Vietnam is one of the countries most affected by highly pathogenic H5N1 influenza A viruses. To evaluate the potential pathogenicity in mammals of H5N1 viruses isolated from humans in Vietnam, we determined the sequences of all eight genes of 22 human isolates collected between 2003 and 2008 and compared their virulence in mice. The isolates were classified into clade 1 and clade 2.3.4 and differed in pathogenicity for mice. Whilst lysine at position 627 of PB2 (PB2-627K) is a critical virulence determinant for clade 2.3.4 viruses, asparagine at position 701 of PB2 and other unknown virulence determinants appear to be involved in the high pathogenicity of clade 1 viruses, warranting further studies to determine the factors responsible for the high virulence of H5N1 viruses in mammals.

DOI： 10.1099/vir.0.021659-0

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Reassortment is an important mechanism for the evolution of influenza viruses. Here, we coinfected cultured cells with the pandemic swine-origin influenza virus (S-OIV) and a contemporary H5N1 virus and found that these two viruses have high genetic compatibility. Studies of human lung cell lines indicated that some reassortants had better growth kinetics than their parental viruses. We conclude that reassortment between these two viruses can occur and could create pandemic H5N1 viruses.

DOI： 10.1128/JVI.01140-10

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.

DOI： 10.1371/journal.ppat.1001079

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Language：English   Publishing type：Research paper (scientific journal)  

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals.

DOI： 10.1371/journal.ppat.1001034

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Language：English   Publishing type：Research paper (scientific journal)  

Simple and rapid diagnosis of influenza is useful for making treatment decisions in the clinical setting. Although many influenza rapid diagnostic tests (IRDTs) are available for the detection of seasonal influenza virus infections, their sensitivity for other viruses, such as H5N1 viruses and the recently emerged swine origin pandemic (H1N1) 2009 virus, remains largely unknown. Here, we examined the sensitivity of 20 IRDTs to various influenza virus strains, including H5N1 and 2009 pandemic H1N1 viruses. Our results indicate that the detection sensitivity to swine origin H1N1 viruses varies widely among IRDTs, with some tests lacking sufficient sensitivity to detect the early stages of infection when the virus load is low.

DOI： 10.1128/JCM.00439-10

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1097/INF.0b013e3181dab225

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 microg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.

DOI： 10.1371/journal.ppat.1000786

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The neuraminidase inhibitors oseltamivir and zanamivi are used to treat H5N1 influenza. However, oseltamivir-resistant H5N1 viruses have been isolated from oseltamivir-treated patients. Moreover, reassortment between H5N1 viruses and oseltamvir-resistant human H1N1 viruses currently circulating could create oseltamivir-resistant H5N1 viruses, rendering the oseltamivir stockpile obsolete. Therefore, there is a need for unique and effective antivirals to combat H5N1 influenza viruses. The investigational drug T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has antiviral activity against seasonal influenza viruses and a mouse-adapted H5N1 influenza virus derived from a benign duck virus. However, its efficacy against highly pathogenic H5N1 viruses, which are substantially more virulent, remains unclear. Here, we demonstrate that T-705 effectively protects mice from lethal infection with oseltamivir-sensitive or -resistant highly pathogenic H5N1 viruses. Furthermore, our biochemical analysis suggests that T-705 ribofuranosyl triphosphate, an active form of T-705, acts like purines or purine nucleosides in human cells and does not inhibit human DNA synthesis. We conclude that T-705 shows promise as a therapeutic agent for the treatment of highly pathogenic H5N1 influenza patients.

DOI： 10.1073/pnas.0909603107

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：{IOS} Press  

DOI： 10.3233/jpi-2010-0256

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Amino acids at positions 627 and 701 in the PB2 protein (PB2-627 and PB2-701, respectively) of avian influenza A viruses affect virus replication in some mammalian cells. Highly pathogenic H5N1 influenza viruses possessing mammalian-type PB2-627 were detected during the Qinghai Lake outbreak in 2005 and spread to Europe and Africa. Via a database search, we found a high rate of viral isolates from Ratitae, including ostrich, possessing mammalian-type PB2-627 or -701. Here, we report that H5N1 avian influenza viruses possessing mammalian-type amino acids in PB2-627 or -701 are selected during replication in ostrich cells in vitro and in vivo.

DOI： 10.1128/JVI.01714-09

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

DOI： 10.1038/nature08260

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The efficient incorporation of influenza virus genome segments into virions is mediated by cis-acting regions at both ends of the viral RNAs. It was shown previously that nt 16-26 at the 3' end of the non-structural (NS) viral RNA of influenza A virus are important for efficient virion incorporation and that nt 27-56 also contribute to this process. To understand further the signalling requirements for genome packaging, this study performed linker-scanning mutagenesis in the latter region and found that nt 27-35 made an appreciable contribution to the efficient incorporation of the NS segment. An NS vRNA library was then generated composed of an RNA population with randomized nucleotides at positions 16-35 such that the virus could select the sequences it required for virion incorporation. The sequences selected differed from the wild-type sequence and no conserved nucleotides were selected. The ability of non-wild-type sequences to function in this manner indicates that the incorporation of influenza A virus genome segments does not absolutely require specific sequences.

DOI： 10.1099/vir.0.010355-0

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Segment 7 of influenza B virus encodes two proteins, M1 and BM2. BM2 is expressed from a stop-start pentanucleotide, in which the BM2 initiation codon overlaps with the M1 stop codon. Here, we demonstrate that 45 nucleotides of the 3' end of the M1 coding region, but not the 5' end of the BM2 coding region, are sufficient for the efficient expression of the downstream protein. Placing these 45 nucleotides and the stop-start pentanucleotide in between the coding sequences induced the expression of at least three noninfluenza proteins, suggesting the utility of this system for expressing multiple proteins from one mRNA.

DOI： 10.1128/JVI.00180-09

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Highly pathogenic H5N1 influenza viruses continue to cause concern, even though currently circulating strains are not efficiently transmitted among humans. For efficient transmission, amino acid changes in viral proteins may be required. Here, we examined the amino acids at positions 627 and 701 of the PB2 protein. A direct analysis of the viral RNAs of H5N1 viruses in patients revealed that these amino acids contribute to efficient virus propagation in the human upper respiratory tract. Viruses grown in culture or eggs did not always reflect those in patients. These results emphasize the importance of the direct analysis of original specimens.

DOI： 10.1128/JVI.00063-09

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.

DOI： 10.1128/JVI.02642-08

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The mechanism by which the influenza A virus genome is packaged into virions is not fully understood. The coding and noncoding regions necessary for packaging of the viral RNA segments, except for the M segment, have been identified. Here, we delineate the M segment regions by incorporating a reporter viral RNA into virions and by generating viruses possessing mutations in the regions. We found that, like the other segments, the M segment coding regions are essential for virion incorporation and that the nucleotide length rather than the nucleotide sequence of the 5' end of the coding region is important.

DOI： 10.1128/JVI.02513-08

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Plasmid-based reverse genetics systems allow the generation of influenza A virus entirely from cloned cDNA. However, since the efficiency of virus generation is dependent on the plasmid transfection efficiency of cells, virus generation is difficult in cells approved for vaccine production that have low transfection efficiencies (e.g., Vero cells). Here we established an alternative reverse genetics system for influenza virus generation by using an adenovirus vector (AdV) which achieves highly efficient gene transfer independent of cell transfection efficiency. This AdV-mediated reverse genetics system will be useful for generating vaccine seed strains and for basic influenza virus studies.

DOI： 10.1128/JVI.01042-07

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The RNA genome of influenza A virus, which forms viral ribonucleoprotein complexes (vRNPs) with viral polymerase subunit proteins (PA, PB1, and PB2) and nucleoprotein (NP), is transcribed and replicated in the nucleus. NP, the major component of vRNPs, has at least two amino acid sequences that serve as nuclear localization signals (NLSs): an unconventional NLS (residues 3 to 13; NLS1) and a bipartite NLS (residues 198 to 216; NLS2). Although both NLSs are known to play a role in nuclear transport, their relative contributions to viral replication are poorly understood. We therefore investigated their contributions to NP subcellular/subnuclear localization, viral RNA (vRNA) transcription, and viral replication. Abolishing the unconventional NLS caused NP to localize predominantly to the cytoplasm and affected its activity in vRNA transcription. However, we were able to create a virus whose NP contained amino acid substitutions in NLS1 known to abolish its nuclear localization function, although this virus was highly attenuated. These results indicate that while the unconventional NLS is not essential for viral replication, it is necessary for efficient viral mRNA synthesis. On the other hand, the bipartite NLS, whose contribution to the nuclear transport of NP is limited, was essential for vRNA transcription and NP's nucleolar accumulation. A virus with nonfunctional NLS2 could not be generated. Thus, the bipartite NLS, but not the unconventional NLS, of NP is essential for influenza A virus replication.

DOI： 10.1128/JVI.01434-06

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Three individual peptide sequences, EVSHPKVG, WVTTSNQW, and SGGSNRSP, which have potentials to bind to Newcastle disease virus (NDV), were identified by the biopanning method using phage display technology. The binding specificities of these peptides presented on phages were confirmed by ELISA competition assay using chicken anti-NDV antiserum. The synthetic peptides designed based on these results partially neutralized the infection of NDV in vitro. The peptide-motives identified here have the potential to lead to the identification of novel molecules that inhibit the NDV infection independent of the immune system.

DOI： 10.1292/jvms.67.1237

PubMed

Total pages：82   Responsible for pages：41-44   Language：Japanese Book type：Scholarly book

J-GLOBAL

J-GLOBAL

Language：Japanese   Publisher：家畜感染症学会誌編集委員会  

あらゆるウイルスは、ヒトや動物などの宿主が存在しない環境中で勝手に自己増殖することはない。畜産現場における感染制御対策の標的は、侵入・増幅・拡散の3つに大別され、特に最も大きな効果が期待できる侵入防止対策は、最も基本的な対策として大きな努力が払われてきた。また近年は「農場内バイオセキュリティ」の重要性が見直され、増幅防止対策の強化が図られているほか、「地域防疫」の概念を支える拡散防止対策にも力が注がれている。養豚農場で発生するウイルス感染症のリスク要因には様々なヒト・モノが挙げられるが、最も警戒すべき要因は、最も頻繁に豚舎へ出入りするヒト、つまり養豚農場の従業員と考えられる。リスクが高いからこそ、うっかり事故の未然防止や健康管理も含め、細心の注意を払った管理体制が求められる。外部からの訪問者も重要なリスク要因で、相手側に立った説明や合理的な仕組み作りが必要となる。外部導入するブタに対しては十分な検疫期間の設定が、モノに関しては品質管理や滅菌消毒、搬入ルールの豚舎レベルでの徹底が重要となる。さらに、豚熱(CSF)の発生要因として大きな注目が集まっている野生イノシシに対する防護柵の敷設のほか、野鳥の侵入防止対策として防鳥ネットの設置も義務付けられることになる。このように、畜産農場が実施すべき防疫対策は多岐に渡るが、重要なことは「できることから着実に進めること」と「持続可能な体制を構築すること」であると考える。(著者抄録)

J-GLOBAL

J-GLOBAL

J-GLOBAL

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

J-GLOBAL

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2017343015

Language：Japanese   Publisher：(公社)日本獣医学会  

J-GLOBAL

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

Language：Japanese   Publisher：(公社)日本獣医学会  

J-GLOBAL

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2016172819

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2016076021

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2015381332

Language：Japanese   Publisher：(公社)日本獣医学会  

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2015175666

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2015060901

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2015014935

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2014144909

Language：Japanese   Publisher：メディカルレビュー社  

CiNii Books

Other Link： http://search.jamas.or.jp/link/ui/2014069624

J-GLOBAL

Language：Japanese  

In most cases, influenza is not fatal, even without treatment. Moreover, vaccination and antivirals have reduced influenza-related mortality in recent years. However, the direct transmission of avian influenza viruses to humans with lethal outcomes in Hong Kong in 1997 was a potent reminder of the devastating potential of the disease. Currently, H5N1 avian influenza viruses are circulating in many Asian countries, and the human death toll continues to rise as the virus spreads to European countries as well. Since the beginning of the outbreak in Asia, more than 120 cases have been confirmed and the mortality rate has been no less than 50%. Current vaccines for H3N2 and H1N1 viruses, of course, have no effect on infection by H5N1 viruses. In addition, H5N1 viruses that are resistant to the antiviral drugs amantadine and oseltamivir have emerged. Fortunately, a virus that is capable of efficient transmission among humans has not emerged. However, it is not a matter of if, but when, such a virus will appear. Here, we review the current situation of avian influenza and pandemic preparedness.

DOI： 10.11150/kansenshogakuzasshi1970.80.1

PubMed

J-GLOBAL

Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2023.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2023.7

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2022.12

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2022.11

Language：English   Presentation type：Oral presentation (general)  

Venue：長崎  

Event date： 2022.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2022.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2022.1 - 2022.2

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2021.11

Language：English   Presentation type：Oral presentation (general)  

Event date： 2021.11

Language：English   Presentation type：Oral presentation (general)  

Event date： 2021.11

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2021.9

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2021.5

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2020.12

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2020.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2020.7

Language：English   Presentation type：Oral presentation (general)  

Event date： 2020.1

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2020.1

Language：English   Presentation type：Oral presentation (general)  

Event date： 2019.10

Language：English   Presentation type：Poster presentation  

Event date： 2019.9

Language：English   Presentation type：Poster presentation  

Event date： 2019.9

Language：English   Presentation type：Poster presentation  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2019.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2019.6

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2018.10

Language：English   Presentation type：Poster presentation  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2018.9

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Event date： 2018.5

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Event date： 2018.4

Language：English   Presentation type：Oral presentation (invited, special)  

Event date： 2017.10

Language：English   Presentation type：Oral presentation (general)  

Event date： 2017.10

Language：Japanese   Presentation type：Poster presentation  

Event date： 2017.9

Language：English   Presentation type：Oral presentation (general)  

Event date： 2017.5

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Event date： 2016.12

Language：Japanese   Presentation type：Poster presentation  

Event date： 2016.10

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2016.10

Language：English   Presentation type：Oral presentation (general)  

Event date： 2016.10

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2016.9

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2016.2

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2015.11

Language：English   Presentation type：Oral presentation (general)  

Event date： 2015.11

Language：Japanese   Presentation type：Poster presentation  

Event date： 2015.11

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.9

Language：English   Presentation type：Oral presentation (general)  

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2014.11

Language：Japanese  

Venue：横浜  

Event date： 2014.11

Language：Japanese  

Venue：横浜  

Event date： 2014.11

Language：Japanese  

Venue：横浜  

Event date： 2014.11

Language：Japanese  

Venue：横浜  

Event date： 2014.11

Language：Japanese  

Venue：横浜  

Event date： 2014.10

Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Event date： 2014.9

Language：Japanese  

Venue：札幌  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：札幌  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：札幌  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：札幌  

国内学会

Event date： 2014.7

Language：English  

Venue：カナダ  

国際学会

Event date： 2013.11

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2013.10

Language：English  

Venue：韓国  

国際学会

Event date： 2013.9

Language：Japanese  

Venue：岐阜  

国内学会

Event date： 2013.9

Language：Japanese  

Venue：岐阜  

国内学会

Event date： 2013.9

Language：Japanese  

Venue：岐阜  

国内学会

Event date： 2013.9

Language：English  

Venue：南アフリカ  

国際学会

Event date： 2013.9

Language：Japanese  

Venue：岐阜  

国内学会

Event date： 2013.6

Language：English  

Venue：スペイン  

国際学会

Event date： 2013.2

Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：東京  

国際学会

Event date： 2012.11

Language：Japanese  

Venue：大阪  

国内学会

Event date： 2012.9

Language：Japanese  

Venue：岩手  

国内学会

Event date： 2011.9

Language：English  

Venue：北海道  

国際学会

Event date： 2011.5

Language：English  

Venue：香港  

国際学会

Event date： 2011.4

Language：English  

Venue：アメリカ合衆国  

国際学会

Event date： 2010.6

Language：English  

Venue：ベルギー  

国際学会

Event date： 2010.6

Language：English  

Venue：ベルギー  

国際学会

Event date： 2007.5

Language：English  

Venue：カナダ  

国際学会

Event date： 2006.9

Language：English  

Venue：兵庫  

国際学会

Audience： General,　Company,　Governmental agency

Type：Other

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Other

高病原性鳥インフルエンザの現状と対策

Audience： General,　Company,　Governmental agency

Type：Other

Audience： General,　Company,　Governmental agency

Type：Other

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Other

高病原性鳥インフルエンザの現状と対策

Audience： General,　Company,　Governmental agency

Type：Other

Audience： General,　Company,　Governmental agency

Type：Other

Audience： General,　Company,　Governmental agency

Type：Other

Audience： General,　Company,　Governmental agency

Type：Other

Audience： General,　Company,　Governmental agency

Type：Other

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Other

高病原性鳥インフルエンザの現状と対策

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Other

高病原性鳥インフルエンザの現状と対策

Audience： Company

Type：Lecture

Audience： Company

Type：Lecture

Audience： General,　Company,　Governmental agency

Type：Other

Audience： General,　Company,　Governmental agency

Type：Other

Audience： High school students,　College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Seminar, workshop

Audience： General,　Company,　Governmental agency

Type：Other

Audience： Company

Type：Lecture

Audience： Company

Type：Visiting lecture

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Seminar, workshop

養豚の基礎知識

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Other

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

Audience： Company

Type：Visiting lecture

Audience： Company

Type：Lecture

Audience： General,　Company,　Governmental agency

Type：Other

Audience： Company

Type：Visiting lecture

Audience： Company

Type：Lecture

Audience： Company

Type：Lecture

Audience： Company

Type：Lecture

Audience： General,　Company,　Governmental agency

Type：Other

動物に感染するウイルス

Audience： Governmental agency

Type：Lecture

鳥インフルエンザについて

Audience： Researchesrs,　General,　Company,　Governmental agency

Type：Visiting lecture

豚インフルエンザ・概論

Audience： General,　Company,　Governmental agency

Type：Lecture

動物に感染するウイルス

Audience： Company

Type：Visiting lecture

PRRSウイルスについて

Audience： Governmental agency

Type：Lecture

Audience： Governmental agency

Type：Lecture

鳥インフルエンザについて

Audience： General,　Company,　Governmental agency

Type：Lecture

豚コレラ再発の背景と今後の展望

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

高病原性鳥インフルエンザの現状と対策

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Seminar, workshop

養豚の基礎知識

Audience： High school students,　College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Seminar, workshop

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Seminar, workshop

ロボット技術ならびにＩＣＴ技術を活用した肉用鶏飼養衛生管理システム

Audience： General,　Company,　Governmental agency

Type：Lecture

豚コレラとアフリカ豚コレラ

Audience： Company

Type：Visiting lecture

養豚農場におけるウイルス感染症対策

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Seminar, workshop

ウイルス感染症に対する分子生物学的研究

Audience： Company

Type：Lecture

Audience： Company

Type：Visiting lecture

「鳥インフルエンザのウイルス伝播様式および出水平野における調査について」
「AIを活用したブロイラー生産の取組み」

Audience： Teachers,　General

Type：Visiting lecture

動物に感染するウイルス

Audience： General,　Company,　Governmental agency

Type：Lecture

口蹄疫をはじめとする家畜のウイルス感染症とその制御

Audience： Researchesrs,　General,　Company,　Governmental agency

Type：Lecture

人工知能ロボットを活用したブロイラー養鶏飼養衛生管理システム

Audience： Teachers,　General,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

野鳥における鳥インフルエンザの流行状況

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　Governmental agency

Type：Lecture

鹿児島県のツル渡来地における鳥インフルエンザの流行状況調査

Audience： Teachers,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

今シーズンに向けた高病原性鳥インフルエンザの防疫対策

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

野鳥における鳥インフルエンザの流行状況と家きんへの感染リスク

Audience： General,　Company,　Governmental agency

Type：Lecture

養豚の飼養衛生管理について

Audience： Researchesrs,　General,　Company

Type：Lecture

動物インフルエザの現状とヒトへ感染リスク

Audience： Governmental agency,　Media

Type：Other

鳥インフルエンザ対策の現状について

Audience： Company

Type：Lecture

鳥インフルエンザ・豚インフルエンザの現状と ウイルスに対する洗浄・消毒の効果

Audience： General,　Company,　Governmental agency

Type：Lecture

豚インフルエンザの特性と現状

Audience： Company

Type：Lecture

国内養豚における 豚インフルエンザの流行状況

Audience： Researchesrs,　Company,　Governmental agency

Type：Lecture

養豚で問題となるRNAウイルス感染症 ～SI・PED・PRRS～

Audience： Company

Type：Other

国内における豚インフルエンザの現状 ～最新知見に基づくウイルス学的考察～

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Governmental agency

Type：Lecture

野鳥における高病原性鳥インフルエンザ

Audience： General,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

国内における豚インフルエンザの現状 ～最新知見に基づくウイルス学的考察～

Audience： General,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

国内における豚インフルエンザの現状 ～最新知見に基づくウイルス学的考察～

Audience： General,　Company,　Governmental agency

Type：Lecture

豚インフルエンザの現状 ～最新知見に基づくウイルス学的考察～

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

TADセンターの高病原性鳥インフルエンザ検査体制

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

鳥インフルエンザの検査方法および人への感染リスクについて

Audience： General,　Company,　Civic organization,　Governmental agency,　Media

Type：Lecture

豚流行性下痢(PED)の制圧へ向けて

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Company,　Governmental agency

Type：Lecture

インフルエンザA(H7N9)ウイルスの正体は？

Audience： General,　Company,　Governmental agency

Type：Lecture

豚インフルエンザの現状

Audience： College students,　Graduate students,　Teachers,　Researchesrs,　General,　Scientific,　Company,　Governmental agency

Type：Lecture

TADセンターにおける動物インフルエンザ研究

Audience： College students,　Graduate students,　Teachers

研究者の海外留学 -メリット・デメリットに関する超主観的考察-

Audience： General,　Company,　Governmental agency

Type：Lecture

豚におけるインフルエンザについて