Updated on 2025/05/07

Information

 

写真a

 
FUKUHARA TAKASUKE
 
Organization
Faculty of Medical Sciences Department of Basic Medicine Professor
Title
Professor
Contact information
メールアドレス
External link

Research Areas

  • Life Science / Virology

Degree

  • 医学博士 ( 2010.3 Kyushu University )

Research History

  • Kyushu University 大学院医学研究院 Professor 

    2024.9 - Present

  • Hokkaido University 大学院医学研究院 Professor 

    2020.5 - 2024.8

  • Osaka University 微生物病研究所 Associate Professor 

    2017.12 - 2020.4

  • Osaka University 微生物病研究所 Assistant Professor 

    2011.7 - 2017.11

Education

  • Kyushu University   大学院医学研究院  

    2006.4 - 2010.3

  • Kyushu University   医学部   医学科

    1998.4 - 2004.3

Research Interests・Research Keywords

  • Research theme: Research on viral replication and pathogenicity

    Keyword: 肝炎ウイルス、コロナウイルス、フラビウイルス、RSウイルス

    Research period: 2010.4 - Present

Awards

  • 杉浦奨励賞

    2017.11   日本ウイルス学会  

Papers

  • Rapid Plasmid-Free Generation of Recombinant Positive-Strand RNA Viruses That Use IRES-Mediated Translation Using an Expansion of the Circular Polymerase Extension Reaction (CPER)

    Yamamoto H., Tamura T., Fukuhara T.

    Bio-protocol   15 ( 8 )   e5275   2025.4

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    Reverse genetics systems in virology are technologies used to generate recombinant viruses, enabling the manipulation of viral genes. Recombinant viruses facilitate the investigation of pathogenesis and the development of antivirals. In studies of positive-sense single-stranded RNA (ssRNA) viruses, a reverse genetics approach typically uses infectious viral cDNA clones derived from bacterial artificial chromosomes and plasmids or from the in vitro ligation of viral cDNA fragments. However, these methods are time-consuming, involve complex procedures, and do not always successfully generate recombinant viruses. Possible reasons for unsuccessful outcomes include i) viral sequences exhibiting toxicity in bacterial systems, ii) the duplication of viral genes observed in some strains, complicating the acquisition of correct cDNA clones, and iii) certain cell lines being highly susceptible to infection but difficult to transfect with nucleotides. For these reasons, a simple and rapid reverse genetics system is needed to accelerate research on ssRNA viruses. The circular polymerase extension reaction (CPER) method offers a solution by eliminating the need for molecular cloning in bacteria, enabling the generation of recombinant viruses over a shorter timeframe. This method has been widely adopted for the study of ssRNA viruses, including SARS-CoV-2 and flaviviruses. Recently, we expanded the CPER method for ssRNA viruses using internal ribosome entry site (IRES)-mediated translation. This protocol details the experimental procedures, using bovine viral diarrhea virus as an example—one of the most challenging viruses for generating viral cDNA clones because of the factors listed above.

    DOI: 10.21769/BioProtoc.5275

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  • Application of versatile reverse genetics system for feline coronavirus

    Kida, I; Tamura, T; Kuroda, Y; Fukuhara, T; Maeda, K; Matsuno, K

    MICROBIOLOGY SPECTRUM   13 ( 4 )   e0269224   2025.4   eISSN:2165-0497

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    Language:English   Publisher:Microbiology Spectrum  

    Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV). Although multiple gene mutations in FCoV likely account for FIP pathogenesis, molecular studies for FCoV have been limited due to the lack of a suitable reverse genetics system. In the present study, we established a rapid PCR-based system to generate recombinant FCoV using the circular polymerase extension reaction (CPER) method for both serotype 1 and 2 viruses. Recombinant FCoV was successfully rescued at sufficient titers to propagate the progeny viruses with high sequence accuracy. The growth kinetics of recombinant FCoV were comparable to those of the parental viruses. We successfully generated recombinants harboring the spike gene from a different FCoV strain or a reporter HiBiT tag using the CPER method. The chimeric virus demonstrated similar characteristics with the parental virus of the spike gene. The reporter tag stably expressed after five serial passages in the susceptible cells, and the reporter virus could be applied to evaluate the sensitivity of antiviral inhibitors using the luciferase assay system to detect HiBiT tag. Taken together, our versatile reverse genetics system for FCoV shown herein is a robust tool to characterize viral genes even without virus isolation and to investigate the molecular mechanisms of the proliferation and pathogenicity of FCoV. IMPORTANCE Feline infectious peritonitis is a highly fatal disease in cats caused by feline coronavirus variants that can infect systemically. Due to the lack of a versatile toolbox for manipulating the feline coronavirus genome, an efficient method is urgently needed to study the virus proteins responsible for the severe disease. Herein, we established a rapid reverse genetics system for the virus and demonstrated the capability of the recombinant viruses to be introduced with desired modifications or reporter genes without any negative impacts on virus characteristics in cell culture. Recombinant viruses are also useful to evaluate antiviral efficacy. Overall, our system can be a promising tool to reveal the molecular mechanisms of the viral life cycle of feline coronavirus and disease progression of feline infectious peritonitis.

    DOI: 10.1128/spectrum.02692-24

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  • M49L and other drug resistance mutations emerging in individuals after administration of ensitrelvir in Japanese clinical settings

    Inoue, A; Ichikawa, T; Wada, D; Maruyama, S; Shimazu, H; Kashihara, M; Okuda, K; Saito, F; Fukuhara, T; Nakamori, Y

    ANTIVIRAL RESEARCH   236   106118   2025.4   ISSN:0166-3542 eISSN:1872-9096

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    Recent in-vitro and in-vivo studies and analysis of genomic information registered in GISAID have raised concerns about drug resistance mutations such as M49L after treatment with the 3C-like protease inhibitor ensitrelvir. The aim of this study was to identify resistance gene mutations to 3C-like protease inhibitors, including M49L mutations, in Japanese clinical settings. Genomic analysis of samples from our hospital admissions showed M49L mutations associated with ensitrelvir treatment in three cases and M49L mutation unrelated to ensitrelvir treatment in three cases. In a study of cases with persistent infection or rebound in viral load after 5 days of ensitrelvir treatment, 10 of 16 patients had M49L mutations and 5 had M49I mutations. Resistance gene mutations following treatment with ensitrelvir were shown to emerge even within individual patients who were not immunocompromised. In a study of persistent SARS-CoV-2 infection in severely immunocompromised patients, various drug resistance mutations emerged, with the M49L mutation especially showing a tendency to be a majority mutation. The current status of drug resistance mutations occurring in individuals following administration of ensitrelvir in Japanese clinical settings was clinically investigated for the first time. Considering that the barrier to resistance to ensitrelvir is lower than that to other antiviral drugs and that M49L is a unique mutation that occurs quickly, tends to become a majority mutation, and is maintained thereafter through its ability to replicate, the spread of strains that have acquired ensitrelvir resistance should be closely monitored.

    DOI: 10.1016/j.antiviral.2025.106118

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  • Clinical and molecular landscape of prolonged SARS-CoV-2 infection with resistance to remdesivir in immunocompromised patients

    Iriyama, C; Ichikawa, T; Tamura, T; Takahata, M; Ishio, T; Ibata, M; Kawai, R; Iwata, M; Suzuki, M; Adachi, H; Nao, N; Suzuki, H; Kawai, A; Kamiyama, A; Suzuki, T; Hirata, Y; Iida, S; Katano, H; Ishii, Y; Tsuji, T; Oda, Y; Tanaka, S; Okazaki, N; Katayama, Y; Nakagawa, S; Tsukamoto, T; Doi, Y; Fukuhara, T; Murata, T; Tomita, A

    PNAS NEXUS   4 ( 4 )   pgaf085   2025.4   eISSN:2752-6542

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    Patients with hematologic diseases have experienced coronavirus disease 2019 (COVID-19) with a prolonged, progressive course. Here, we present clinical, pathological, and virological analyses of three cases of prolonged COVID-19 among patients undergoing treatment for B-cell lymphoma. These patients had all been treated with anti-CD20 antibody and bendamustine. Despite various antiviral treatments, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels persisted for >4 weeks, and two of them succumbed to COVID-19. The autopsy showed bronchopneumonia, interstitial pneumonia, alveolar hemorrhage, and fibrosis. Overlapping cytomegalovirus, fungal and/or bacterial infections were also confirmed. Sequencing of SARS-CoV-2 showed accumulation of mutations and changes in variant allele frequencies over time. NSP12 mutations V792I and M794I appeared independently in two cases as COVID-19 progressed. In vitro drug susceptibility analysis and an animal experiment using recombinant SARS-CoV-2 demonstrated that each mutation, V792 and M794I, was independently responsible for remdesivir resistance and attenuated pathogenicity. E340A, E340D, and F342INS mutations in the spike protein were found in one case, which may account for the sotrovimab resistance. Analysis of autopsy specimens indicated heterogeneous distribution of these mutations. In summary, we demonstrated temporal and spatial diversity in SARS-CoV-2 that evolved resistance to various antiviral agents in malignant lymphoma patients under immunodeficient conditions caused by certain types of immunochemotherapies. Strategies may be necessary to prevent the acquisition of drug resistance and improve outcomes, such as the selection of appropriate treatment strategies for lymphoma considering patients' immune status and the institution of early intensive antiviral therapy.

    DOI: 10.1093/pnasnexus/pgaf085

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  • NEDD4-binding protein 1 suppresses hepatitis B virus replication by regulating viral RNAs

    Kobayashi, N; Suzuki, S; Sakamoto, Y; Suzuki, R; Mori, K; Kosugi, Y; Saito, T; Ma, Y; Liang, LH; Izumi, T; Noda, K; Okuzaki, D; Kanegae, Y; Hayashi, S; Tanaka, Y; Yamashita, A; Moriishi, K; Matsuura, Y; Takeuchi, O; Tamura, T; Taketomi, A; Fukuhara, T

    JOURNAL OF GENERAL VIROLOGY   106 ( 3 )   2025   ISSN:0022-1317 eISSN:1465-2099

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    Chronic infection with hepatitis B virus (HBV) (chronic HBV infection) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogues are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug-resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We tested the effect of 132 RBPs on HBV replication by ectopically expressing these proteins along with HBV in hepatocellular carcinoma and evaluated the intracellular capsid-associated HBV DNA level. Our screen identified NEDD4-binding protein 1 (N4BP1) as having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, the overexpression of N4BP1 decreased core-associated HBV DNA levels, while knockdown or knockout of the gene encoding N4BP1 rescued core-associated HBV DNA levels. N4BP1 possesses the KH-like and RNase domains, and both were required for the anti-HBV effect of N4BP1. Additionally, we measured levels of HBV pregenomic RNA (pgRNA) and covalently closed circular DNA in the RBP-transfected cells and confirmed that N4BP1 binds pgRNA directly and regulates both the 3.5 and 2.4/2.1 kb HBV RNA. In summary, N4BP1 is a newly identified host factor able to counteract HBV production by regulating 3.5 and 2.1/2.4 kb HBV RNA.

    DOI: 10.1099/jgv.0.002082

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  • Structural basis for receptor-binding domain mobility of the spike in SARS-CoV-2 BA.2.86 and JN.1

    Yajima H., Anraku Y., Kaku Y., Kimura K.T., Plianchaisuk A., Okumura K., Nakada-Nakura Y., Atarashi Y., Hemmi T., Kuroda D., Takahashi Y., Kita S., Sasaki J., Sumita H., Konar A., Padilla-Blanco M., Andrikopoulos P., Zahradnik J., Nishiuchi T., Matsubara N., Kawano M., Kosaka A., Saito A., Toyoda M., Motozono C., Ueno T., Takahashi O., Leong S., Mugita Y., Jonathan M., Begum M.S.T.M., Shimizu R., Nasser H., Ikeda T., Sasaki-Tabata K., Kawabata R., Irie T., Nakajima Y., Suzuki T., Yasuhara N., Sakamoto A., Futatsusako H., Nakata Y., Watanabe Y., Deguchi S., Hashimoto R., Takayama K., Nakagawa S., Yoshida I., Asakura H., Nagashima M., Sadamas K., Yoshimura K., Osujo R., Fukuda T., Ogawa E., Tanaka S., Ohsumi N., Strange A.P., Iida K., Yasuda K., Chiba M., Suganami M., Horinaka K., Yo M.S., Li W., Pan L., Chen L., Jarel J.E., Fujita S., Kosugi Y., Nishumura L., Kawakubo S., Yoshimura R., Uriu K., Lytras S., Saikruang W., Usui K., Hinay A.A., Guo Z., Misawa N., Ito H., Tsujino S., Suzuki S., Suzuki R., Tamura T., Fukuhara T., Iida M., Mohri H., Shishido K., Ferdous Z., Oda Y., Wang L., Tsuda M., Tanaka S., Ohari Y., Mimura Y., Kida I., Li J., Mizuma K.

    Nature Communications   15 ( 1 )   2024.12

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    Since 2019, SARS-CoV-2 has undergone mutations, resulting in pandemic and epidemic waves. The SARS-CoV-2 spike protein, crucial for cellular entry, binds to the ACE2 receptor exclusively when its receptor-binding domain (RBD) adopts the up-conformation. However, whether ACE2 also interacts with the RBD in the down-conformation to facilitate the conformational shift to RBD-up remains unclear. Herein, we present the structures of the BA.2.86 and the JN.1 spike proteins bound to ACE2. Notably, we successfully observed the ACE2-bound down-RBD, indicating an intermediate structure before the RBD-up conformation. The wider and mobile angle of RBDs in the up-state provides space for ACE2 to interact with the down-RBD, facilitating the transition to the RBD-up state. The K356T, but not N354-linked glycan, contributes to both of infectivity and neutralizing-antibody evasion in BA.2.86. These structural insights the spike-protein dynamics would help understand the mechanisms underlying SARS-CoV-2 infection and its neutralization.

    DOI: 10.1038/s41467-024-52808-2

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  • Positivity of high-sensitivity HBsAg test, not previous HBV infection, indicates poor prognosis in patients with non-HBV-related HCC

    Yasuura, N; Suda, G; Ohara, M; Meno, A; Sho, T; Kohya, R; Sasaki, T; Yoda, T; Yoshida, S; Fu, QJ; Yang, ZJ; Hosoda, S; Maehara, O; Ohnishi, S; Saitou, T; Sugiyama, M; Fukuhara, T; Baba, M; Kitagataya, T; Kawagishi, N; Nakai, M; Natsuizaka, M; Ogawa, K; Taketomi, A; Sakamoto, N

    ALIMENTARY PHARMACOLOGY & THERAPEUTICS   60 ( 10 )   1315 - 1324   2024.11   ISSN:0269-2813 eISSN:1365-2036

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    Language:English   Publisher:Alimentary Pharmacology and Therapeutics  

    Background and Aims: The prognostic impact of previous-HBV-infection (pHBV) in non-HBV-related hepatocellular carcinoma (non-HBV-related-HCC) and the prevalence, characteristics and significance of recently developed high-sensitivity HBs antigen positivity (hHBsAg+) in these patients remain unclear. We aimed to close these gaps. Methods: We retrospectively screened patients with newly diagnosed non-HBV-related-HCC (standard HBsAg-test negative) at Hokkaido University. Patients with complete clinical information and preserved serum for hHBsAg+ were included. We evaluated the prevalence, characteristics and prognostic impact of pHBV and hHBsAg+ in non-HBV-related-HCC. Results: A total of 401 non-HBV-related-HCC patients were included (288 with pHBV/113 without pHBV). In non-HBV-related-HCC, pHBV did not affect overall survival (OS). Among non-HBV-related-HCC patients with pHBV, 11.8% (34/288) were hHBsAg+ and had more advanced stages of HCC, higher AFP levels, higher vascular invasion rates, and significantly shorter OS than others (OS: 19.3 vs. 61.4 months, p = 0.012). Comparison of OS among non-HBV-related-HCC patients without pHBV (group 1), those with pHBV and without hHBsAg+ (group 2), and those with pHBV and hHBsAg+ (group 3) revealed significantly shorter OS in group 3 (19.3, 56.6 and 66.4 months in groups 1, 2 and 3, respectively; p = 0.036). Multivariate Cox regression indicated that compared with group 1, only group 3 was significantly and independently associated with shorter OS (HR: 2.044, p = 0.011). Subgroup analysis revealed that this association was particularly evident in non-HBV-related-HCC patients with non-B-non-C aetiology and advanced HCC. Conclusions: In non-HBV-related-HCC patients, hHBsAg+, not pHBV, is significantly and independently associated with poor prognosis.

    DOI: 10.1111/apt.18229

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  • Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant

    Tsujino, S; Deguchi, S; Nomai, T; Padilla-Blanco, M; Plianchaisuk, A; Wang, L; Begum, MSTM; Uriu, K; Mizuma, K; Nao, N; Kojima, I; Tsubo, T; Li, JS; Matsumura, Y; Nagao, M; Oda, Y; Tsuda, M; Anraku, Y; Kita, S; Yajima, H; Sasaki-Tabata, K; Guo, ZY; Hinay, AA; Yoshimatsu, K; Yamamoto, Y; Nagamoto, T; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Nasser, H; Jonathan, M; Putri, O; Kim, Y; Chen, L; Suzuki, R; Tamura, T; Maenaka, K; Irie, T; Matsuno, K; Tanaka, S; Ito, J; Ikeda, T; Takayama, K; Zahradnik, J; Hashiguchi, T; Fukuhara, T; Sato, K

    MICROBIOLOGY AND IMMUNOLOGY   68 ( 9 )   305 - 330   2024.9   ISSN:0385-5600 eISSN:1348-0421

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    In middle to late 2023, a sublineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. We performed multiscale investigations, including phylogenetic analysis, epidemic dynamics modeling, infection experiments using pseudoviruses, clinical isolates, and recombinant viruses in cell cultures and experimental animals, and the use of human sera and antiviral compounds, to reveal the virological features of the newly emerging EG.5.1 variant. Our phylogenetic analysis and epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T are critical to its increased viral fitness. Experimental investigations on the growth kinetics, sensitivity to clinically available antivirals, fusogenicity, and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 are comparable to those of XBB.1.5. However, cryo-electron microscopy revealed structural differences between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible in our experimental setup. Our multiscale investigations provide knowledge for understanding the evolutionary traits of newly emerging pathogenic viruses, including EG.5.1, in the human population.

    DOI: 10.1111/1348-0421.13165

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  • Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant(タイトル和訳中)

    Tsujino Shuhei, Deguchi Sayaka, Nomai Tomo, Padilla-Blanco Miguel, Plianchaisuk Arnon, Wang Lei, Begum MST Monira, Uriu Keiya, Mizuma Keita, Nao Naganori, Kojima Isshu, Tsubo Tomoya, Li Jingshu, Matsumura Yasufumi, Nagao Miki, Oda Yoshitaka, Tsuda Masumi, Anraku Yuki, Kita Shunsuke, Yajima Hisano, Sasaki-Tabata Kaori, Guo Ziyi, Hinay Alfredo A.Jr., Yoshimatsu Kumiko, Yamamoto Yuki, Nagamoto Tetsuharu, Asakura Hiroyuki, Nagashima Mami, Sadamasu Kenji, Yoshimura Kazuhisa, Nasser Hesham, Jonathan Michael, Putri Olivia, Kim Yoonjin, Chen Luo, Suzuki Rigel, Tamura Tomokazu, Maenaka Katsumi, Irie Takashi, Matsuno Keita, Tanaka Shinya, Ito Jumpei, Ikeda Terumasa, Takayama Kazuo, Zahradnik Jiri, Hashiguchi Takao, Fukuhara Takasuke, Sato Kei

    Microbiology and Immunology   68 ( 9 )   305 - 330   2024.9   ISSN:0385-5600

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  • A micro-disc-based multiplex method for monitoring emerging SARS-CoV-2 variants using the molecular diagnostic tool Intelli-OVI

    Hossain, MB; Uchiyama, Y; Rajib, SA; Rahman, A; Takatori, M; Tan, BJY; Sugata, K; Nagashima, M; Kawakami, M; Ito, H; Kumagai, R; Sadamasu, K; Ogi, Y; Kawaguchi, T; Tamura, T; Fukuhara, T; Ono, M; Yoshimura, K; Satou, Y

    COMMUNICATIONS MEDICINE   4 ( 1 )   161   2024.8   ISSN:2730-664X

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    Language:English   Publisher:Communications Medicine  

    Background: Highly transmissible viruses including SARS-CoV-2 frequently accumulate novel mutations that are detected via high-throughput sequencing. However, there is a need to develop an alternative rapid and non-expensive approach. Here we developed a novel multiplex DNA detection method Intelli-OVI for analysing existing and novel mutations of SARS-CoV-2. Methods: We have developed Intelli-OVI that includes the micro-disc-based method IntelliPlex and computational algorithms of objective variant identification (OVI). More than 250 SARS-CoV-2 positive samples including wastewater ones were analysed to verify the efficiency of the method. Results: IntelliPlex uses micro-discs printed with a unique pictorial pattern as a labelling conjugate for DNA probes, and OVI allows simultaneous identification of several variants using multidimensional data obtained by the IntelliPlex method. Importantly, de novo mutations can be identified by decreased signals, which indicates that there is an emergence of de novo variant virus as well as prompts the need to design additional primers and probes. We have upgraded probe panel according to the emergence of new variants and demonstrated that Intelli-OVI efficiently identified more than 20 different SARS-CoV-2 variants by using 35 different probes simultaneously. Conclusions: Intelli-OVI can be upgraded to keep up with rapidly evolving viruses as we showed in this study using SARS-CoV-2 as an example and may be suitable for other viruses but would need to be validated.

    DOI: 10.1038/s43856-024-00582-z

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  • Generation of recombinant viruses directly from clinical specimens of COVID-19 patients

    Yamamoto, H; Tamura, T; Ichikawa, T; Taguchi, Y; Mori, K; Oguri, S; Suzuki, R; Suzuki, S; Teshima, T; Fukuhara, T

    JOURNAL OF CLINICAL MICROBIOLOGY   62 ( 7 )   e0004224   2024.7   ISSN:0095-1137 eISSN:1098-660X

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    Language:English   Publisher:Journal of Clinical Microbiology  

    Rapid characterization of the causative agent(s) during a disease outbreak can aid in the implementation of effective control measures. However, isolation of the agent(s) from crude clinical samples can be challenging and time-consuming, hindering the establishment of countermeasures. In the present study, we used saliva specimens collected for the diagnosis of SARS-CoV-2—a good example of a practical target—and attempted to characterize the virus within the specimens without virus isolation. Thirty-four saliva samples from coronavirus disease 2019 patients were used to extract RNA and synthesize DNA amplicons by PCR. New primer sets were designed to generate DNA amplicons of the full-length spike (S) gene for subsequent use in a circular polymerase extension reaction (CPER), a simple method for deriving recombinant viral genomes. According to the S sequence, four clinical specimens were classified as BA. 1, BA.2, BA.5, and XBB.1 and were used for the de novo generation of recombinant viruses carrying the entire S gene. Additionally, chimeric viruses carrying the gene encoding GFP were generated to evaluate viral propagation using a plate reader. We successfully used the RNA purified directly from clinical saliva samples to generate chimeric viruses carrying the entire S gene by our updated CPER method. The chimeric viruses exhibited robust replication in cell cultures with similar properties. Using the recombinant GFP viruses, we also successfully characterized the efficacy of the licensed antiviral AZD7442. Our proof-of-concept demonstrates the novel utility of CPER to allow rapid characterization of viruses from clinical specimens.

    DOI: 10.1128/jcm.00042-24

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  • 免疫病原性におけるSARS-CoV-2アクセサリー蛋白質の関与(Involvement of SARS-CoV-2 accessory proteins in immunopathogenesis)

    Ito Hayato, Tamura Tomokazu, Wang Lei, Mori Kento, Tsuda Masumi, Suzuki Rigel, Suzuki Saori, Yoshimatsu Kumiko, Tanaka Shinya, Fukuhara Takasuke

    Microbiology and Immunology   68 ( 7 )   237 - 247   2024.7   ISSN:0385-5600

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    七つのアクセサリー蛋白質(ORF3a、ORF3b、ORF6、ORF7a、ORF8、ORF9b、ORF10)をノックアウトして遺伝子組み換えSARS-CoV-2をde novoで作成し、その特徴についてin vitroとin vivoにて検討した。作成されたウイルス(ORF3-10 KO)は、感染細胞内にてアクセサリー蛋白質を発現せず、ウイルスゲノム内で変異を維持していた。細胞培養でORF3-10 KOウイルスは親株と同等の増殖動態を示した。ORF3-10 KOウイルスに感染したハムスターは軽度の体重減少を示し、口腔および肺組織中でのORF3-10 KOウイルス複製は親株ウイルスと比べて低下していた。ORF3-10 KOウイルス感染は軽度の炎症を誘発したことから、アクセサリー蛋白質を欠如するORF3-10 KOウイルスは自然免疫を回避できないため増殖が低下し、速く体外に排出されると考えられた。

  • Involvement of SARS-CoV-2 accessory proteins in immunopathogenesis

    Ito, H; Tamura, T; Wang, L; Mori, K; Tsuda, M; Suzuki, R; Suzuki, S; Yoshimatsu, K; Tanaka, S; Fukuhara, T

    MICROBIOLOGY AND IMMUNOLOGY   68 ( 7 )   237 - 247   2024.7   ISSN:0385-5600 eISSN:1348-0421

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    Language:English   Publisher:Microbiology and Immunology  

    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the largest single-stranded RNA virus known to date. Its genome contains multiple accessory protein genes that act against host immune responses but are not required for progeny virus production. The functions of the accessory proteins in the viral life cycle have been examined, but their involvement in viral pathogenicity remains unclear. Here, we investigated the roles of the accessory proteins in viral immunopathogenicity. To this end, recombinant SARS-CoV-2 possessing nonsense mutations in the seven accessory protein open reading frames (ORFs) (ORF3a, ORF3b, ORF6, ORF7a, ORF8, ORF9b, and ORF10) was de novo generated using an early pandemic SARS-CoV-2 strain as a backbone. We confirmed that the resultant virus (termed ORF3–10 KO) did not express accessory proteins in infected cells and retained the desired mutations in the viral genome. In cell culture, the ORF3–10 KO virus exhibited similar virus growth kinetics as the parental virus. In hamsters, ORF3–10 KO virus infection resulted in mild weight loss and reduced viral replication in the oral cavity and lung tissue. ORF3–10 KO virus infection led to mild inflammation, indicating that an inability to evade innate immune sensing because of a lack of accessory proteins impairs virus growth in vivo and results in quick elimination from the body. Overall, we showed that SARS-CoV-2 accessory proteins are involved in immunopathogenicity.

    DOI: 10.1111/1348-0421.13157

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  • Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236

    Fujita, S; Plianchaisuk, A; Deguchi, S; Ito, H; Nao, N; Wang, L; Nasser, H; Tamura, T; Kimura, I; Kashima, Y; Suzuki, R; Suzuki, S; Kida, I; Tsuda, M; Oda, Y; Hashimoto, R; Watanabe, Y; Uriu, K; Yamasoba, D; Guo, ZY; Hinay, AA ; Kosugi, Y; Chen, L; Pan, L; Kaku, Y; Chu, H; Donati, F; Temmam, S; Eloit, M; Yamamoto, Y; Nagamoto, T; Asakura, H; Sadamasu, K; Yoshimura, K; Suzuki, Y; Ito, J; Ikeda, T; Nagashima, M; Tanaka, S; Matsuno, K; Fukuhara, T; Takayama, K; Sato, K

    EBIOMEDICINE   104   105181   2024.6   ISSN:2352-3964

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    Background: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. Methods: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. Findings: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. Interpretation: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. Funding: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers “UTOPIA” (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).

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  • Neutralizing antibody responses and cellular responses against SARS-CoV-2 Omicron subvariants after mRNA SARS-CoV-2 vaccination in kidney transplant recipients

    Kawashiro, K; Suzuki, R; Nogimori, T; Tsujino, S; Iwahara, N; Hirose, T; Okada, K; Yamamoto, T; Fukuhara, T; Hotta, K; Shinohara, N

    SCIENTIFIC REPORTS   14 ( 1 )   12176   2024.5   ISSN:2045-2322

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    Although the mRNA SARS-CoV-2 vaccine has improved the mortality rate in the general population, its efficacy against rapidly mutating virus strains, especially in kidney transplant recipients, remains unclear. We examined the anti-SARS-CoV-2 spike protein IgG antibody and neutralizing antibody titers and cellular immunity against B.1.1, BA.1, and BA.5 antigens in 73 uninfected kidney recipients and 16 uninfected healthy controls who received three doses of an mRNA SARS-CoV-2 vaccine. The IgG antibody titers were significantly lower in recipients than in healthy controls. Similarly, neutralizing antibody titers against three viral variants were significantly lower in recipients. When the virus was mutated, the neutralizing antibody titers decreased significantly in both groups. In cellular immunity analysis, the number of spike-specific CD8 + non-naïve T cells against three variants significantly decreased in recipients. Conversely, the frequency of spike-specific Th2 CD4 + T-cells in recipients was higher than that in healthy controls. Nineteen recipients and six healthy controls also received a bivalent omicron-containing booster vaccine, leading to increase IgG and neutralizing antibody titers in both groups. After that, eleven recipients and five healthy controls received XBB.1.5 monovalent vaccines, increasing the neutralizing antibody titers against not only XBB.1.5, but also EG.5.1 and BA.2.86 antigens in kidney recipients. Although kidney recipients did not gain sufficient immunity against Omicron BA.5 with the third dose of vaccine, humoral response against mutant SARS-CoV-2 lineages significantly increased after bivalent Omicron-containing booster vaccine and the XBB.1.5 monovalent vaccine. Therefore, it is important for kidney recipients to continue to administer updated vaccines.

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  • Akaluc bioluminescence offers superior sensitivity to track<i> in</i><i> vivo</i> dynamics of SARS-CoV-2 infection

    Tamura, T; Ito, H; Torii, S; Wang, L; Suzuki, R; Tsujino, S; Kamiyama, A; Oda, Y; Tsuda, M; Morioka, Y; Suzuki, S; Shirakawa, K; Sato, K; Yoshimatsu, K; Matsuura, Y; Iwano, S; Tanaka, S; Fukuhara, T

    ISCIENCE   27 ( 5 )   109647   2024.5   eISSN:2589-0042

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    Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.

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  • The development of a rapid, high-throughput neutralization assay using a SARS-CoV-2 reporter

    Suzuki, R; Kamiyama, A; Ito, H; Kawashiro, K; Tomiyama, T; Tamura, T; Suzuki, S; Yoshizumi, T; Hotta, K; Fukuhara, T

    JOURNAL OF VIROLOGICAL METHODS   326   114894   2024.5   ISSN:0166-0934 eISSN:1879-0984

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    Many methods have been developed to measure the neutralizing capacity of antibodies to SARS-CoV-2. However, these methods are low throughput and can be difficult to quickly modify in response to emerging variants. Therefore, an experimental system for rapid and easy measurement of the neutralizing capacity of antibodies against various variants is needed. In this study, we developed an experimental system that can efficiently measure the neutralizing capacity of sera by using a GFP-carrying recombinant SARS-CoV-2 with spike proteins of multiple variants (B.1.1, BA.5, or XBB.1.5). For all 3 recombinant chimeric genomes generated, neutralizing antibody titers determined by measuring GFP fluorescence intensity correlated significantly with those calculated from viral RNA levels measured by RT-qPCR in the supernatant of infected cells. Furthermore, neutralizing antibody titers determined by visually assessing GFP fluorescence using microscopy were also significantly correlated with those determined by RT-qPCR. By using this high-throughput method, it is now possible to quickly and easily determine the neutralizing capacity of antibodies against SARS-CoV-2 variants.

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  • Research on SARS-CoV-2 and Its Clinical Applications

    Fukuhara T.

    Nihon Kikan Shokudoka Gakkai Kaiho   75 ( 2 )   65 - 65   2024.4   ISSN:00290645 eISSN:18806848

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  • A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation

    Tamura, T; Yamamoto, H; Ogino, S; Morioka, Y; Tsujino, S; Suzuki, R; Hiono, T; Suzuki, S; Isoda, N; Sakoda, Y; Fukuhara, T

    JOURNAL OF VIROLOGY   98 ( 3 )   e0163823   2024.3   ISSN:0022-538X eISSN:1098-5514

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    Reverse genetics systems have played a central role in developing recombinant viruses for a wide spectrum of virus research. The circular polymerase extension reaction (CPER) method has been applied to studying positive-strand RNA viruses, allowing researchers to bypass molecular cloning of viral cDNA clones and thus leading to the rapid generation of recombinant viruses. However, thus far, the CPER protocol has only been established using cap-dependent RNA viruses. Here, we demonstrate that a modified version of the CPER method can be successfully applied to positive-strand RNA viruses that use cap-independent, internal ribosomal entry site (IRES)-mediated translation. As a proof-of-concept, we employed mammalian viruses with different types (classes I, II, and III) of IRES to optimize the CPER method. Using the hepatitis C virus (HCV, class III), we found that inclusion in the CPER assembly of an RNA polymerase I promoter and terminator, instead of those from polymerase II, allowed greater viral production. This approach was also successful in generating recombinant bovine viral diarrhea virus (class III) following transfection of MDBK/293T co-cultures to overcome low transfection efficiency. In addition, we successfully generated the recombinant viruses from clinical specimens. Our modified CPER could be used for producing hepatitis A virus (HAV, type I) as well as de novo generation of encephalomyocarditis virus (type II). Finally, we generated recombinant HCV and HAV reporter viruses that exhibited replication comparable to that of the wild-type parental viruses. The recombinant HAV reporter virus helped evaluate antivirals. Taking the findings together, this study offers methodological advances in virology. IMPORTANCE The lack of versatility of reverse genetics systems remains a bottleneck in viral research. Especially when (re-)emerging viruses reach pandemic levels, rapid characterization and establishment of effective countermeasures using recombinant viruses are beneficial in disease control. Indeed, numerous studies have attempted to establish and improve the methods. The circular polymerase extension reaction (CPER) method has overcome major obstacles in generating recombinant viruses. However, this method has not yet been examined for positive-strand RNA viruses that use cap-independent, internal ribosome entry site-mediated translation. Here, we engineered a suitable gene cassette to expand the CPER method for all positive-strand RNA viruses. Furthermore, we overcame the difficulty of generating recombinant viruses because of low transfection efficiency. Using this modified method, we also successfully generated reporter viruses and recombinant viruses from a field sample without virus isolation. Taking these findings together, our adapted methodology is an innovative technology that could help advance virologic research.

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  • Rational in silico design identifies two mutations that restore UT28K SARS-CoV-2 monoclonal antibody activity against Omicron BA.1

    Ozawa, T; Ikeda, Y; Chen, LA; Suzuki, R; Hoshino, A; Noguchi, A; Kita, S; Anraku, Y; Igarashi, E; Saga, Y; Inasaki, N; Taminishi, S; Sasaki, J; Kirita, Y; Fukuhara, H; Maenaka, K; Hashiguchi, T; Fukuhara, T; Hirabayashi, K; Tani, H; Kishi, H; Niimi, H

    STRUCTURE   32 ( 3 )   263 - 272.e7   2024.3   ISSN:0969-2126 eISSN:1878-4186

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    SARS-CoV-2 rapidly mutates and acquires resistance to neutralizing antibodies. We report an in-silico-designed antibody that restores the neutralizing activity of a neutralizing antibody. Our previously generated antibody, UT28K, exhibited broad neutralizing activity against mutant variants; however, its efficacy against Omicron BA.1 was compromised by the mutation. Using previously determined structural information, we designed a modified-UT28K (VH T28R/N57D), UT28K-RD targeting the mutation site. In vitro and in vivo experiments demonstrated the efficacy of UT28K-RD in neutralizing Omicron BA.1. Although the experimentally determined structure partially differed from the predicted model, our study serves as a successful case of antibody design, wherein the predicted amino acid substitution enhanced the recognition of the previously elusive Omicron BA.1. We anticipate that numerous similar cases will be reported, showcasing the potential of this approach for improving protein-protein interactions. Our findings will contribute to the development of novel therapeutic strategies for highly mutable viruses, such as SARS-CoV-2.

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  • Prolonged shedding of viable SARS-CoV-2 in immunocompromised patients with haematological malignancies: A prospective study

    Ichikawa, T; Tamura, T; Takahata, M; Ishio, T; Ibata, M; Kasahara, I; Minauchi, K; Yamamoto, S; Teshima, T; Fukuhara, T

    BRITISH JOURNAL OF HAEMATOLOGY   204 ( 3 )   815 - 820   2024.3   ISSN:0007-1048 eISSN:1365-2141

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    Prolonged SARS-CoV-2 infection in immunocompromised individuals has been scattered, but the details remain unclear. We conducted a prospective study with 26 COVID-19 patients with haematological malignancies to determine viral shedding kinetics and characteristics. We obtained nasopharyngeal swabs from the patients 21–28 days post-onset for a PCR test and performed virus isolation from the PCR-positive samples. A viable virus was detected in five patients (19.2%), all of whom had malignant lymphoma. Those patients had significantly lower CD4+ T-cell counts than the PCR-negative patients. A comparison of previous chemotherapy showed that anti-CD20 antibodies and bendamustine may be risk factors for prolonged viral shedding.

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  • Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine

    Aboshi, M; Matsuda, K; Kawakami, D; Kono, K; Kazami, Y; Sekida, T; Komori, M; Morey, AL; Suga, S; Smith, JF; Fukuhara, T; Iwatani, Y; Yamamoto, T; Sato, N; Akahata, W

    ISCIENCE   27 ( 2 )   108964   2024.2   eISSN:2589-0042

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    Continuing emergence of variants of concern resulting in reduced SARS-CoV-2 vaccine efficacy necessitates additional prevention strategies. The structure of VLPCOV-01, a lipid nanoparticle-encapsulated, self-amplifying RNA COVID-19 vaccine with a comparable immune response to BNT162b2, was revised by incorporating a modified base, 5-methylcytosine, to reduce reactogenicity, and an updated receptor-binding domain derived from the Brazil (gamma) variant. Interim analyses of a phase 1 dose-escalation booster vaccination study with the resulting construct, VLPCOV-02, in healthy, previously vaccinated Japanese individuals (N = 96) are reported (jRCT2051230005). A dose-related increase in solicited local and systemic adverse events was observed, which were generally rated mild or moderate. The most commonly occurring events were tenderness, pain, fatigue, and myalgia. Serum SARS-CoV-2 immunoglobulin titers increased during the 4 weeks post-immunization. VLPCOV-02 demonstrated a favorable safety profile compared with VLPCOV-01, with reduced adverse events and fewer fever events at an equivalent dose. These findings support further study of VLPCOV-02.

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  • Virological characteristics of the SARS-CoV-2 Omicron XBB.1.5 variant

    Tamura, T; Irie, T; Deguchi, S; Yajima, H; Tsuda, M; Nasser, H; Mizuma, K; Plianchaisuk, A; Suzuki, S; Uriu, K; Begum, MM; Shimizu, R; Jonathan, M; Suzuki, R; Kondo, T; Ito, H; Kamiyama, A; Yoshimatsu, K; Shofa, M; Hashimoto, R; Anraku, Y; Kimura, KT; Kita, S; Sasaki, J; Sasaki-Tabata, K; Maenaka, K; Nao, N; Wang, L; Oda, Y; Sawa, H; Kawabata, R; Watanabe, Y; Sakamoto, A; Yasuhara, N; Suzuki, T; Nakajima, Y; Ferdous, Z; Shishido, K; Mugita, Y; Takahashi, O; Ichihara, K; Kaku, Y; Misawa, N; Guo, ZY; Hinay, A ; Kosugi, Y; Fujita, S; Tolentino, JM; Chen, L; Pan, L; Suganami, M; Chiba, M; Yoshimura, R; Yasuda, K; Iida, K; Ohsumi, N; Strange, AP; Shibatani, Y; Nishiuchi, T; Tanaka, S; Putri, O; Joas, G; Kim, Y; Yamasoba, D; Yoshimura, K; Sadamasu, K; Nagashima, M; Asakura, H; Yoshida, I; Nakagawa, S; Takaori-Kondo, A; Shirakawa, K; Nagata, K; Nomura, R; Horisawa, Y; Tashiro, Y; Kawai, Y; Ueno, T; Motozono, C; Toyoda, M; Ikeda, T; Saito, A; Matsuno, K; Ito, J; Tanaka, S; Sato, K; Hashiguchi, T; Takayama, K; Fukuhara, T

    NATURE COMMUNICATIONS   15 ( 1 )   1176   2024.2   eISSN:2041-1723

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    Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the S486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determine the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. We provide the intrinsic pathogenicity of XBB.1 and XBB.1.5 in hamsters. Importantly, we find that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC suppression. In vivo experiments using recombinant viruses reveal that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, our study identifies the two viral functions defined the difference between XBB.1 and XBB.1.5.

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  • Comparative pathogenicity of SARS-CoV-2 Omicron subvariants including BA.1, BA.2, and BA.5

    Tamura T., Yamasoba D., Oda Y., Ito J., Kamasaki T., Nao N., Hashimoto R., Fujioka Y., Suzuki R., Wang L., Ito H., Kashima Y., Kimura I., Kishimoto M., Tsuda M., Sawa H., Yoshimatsu K., Yamamoto Y., Nagamoto T., Kanamune J., Suzuki Y., Ohba Y., Suzuki S., Kato M., Ferdous Z., Mouri H., Shishido K., Misawa N., Uriu K., Kosugi Y., Fujita S., Suganami M., Chiba M., Yoshimura R., Nakagawa S., Wu J., Takaori-Kondo A., Shirakawa K., Nagata K., Kazuma Y., Nomura R., Horisawa Y., Tashiro Y., Kawai Y., Hashiguchi T., Suzuki T., Kimura K., Sasaki J., Nakajima Y., Sakamoto A., Yasuhara N., Irie T., Kawabata R., Ikeda T., Nasser H., Shimizu R., Begum M., Takahashi O., Ichihara K., Ueno T., Motozono C., Toyoda M., Saito A., Tanaka Y.L., Butlertanaka E.P., Shofa M., Tabata K., Yokota I., Matsuno K., Takayama K., Tanaka S., Sato K., Fukuhara T.

    Communications Biology   6 ( 1 )   2023.12

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    The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.

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  • High-precision rapid testing of omicron SARS-CoV-2 variants in clinical samples using AI-nanopore

    Murakami, K; Kubota, SI; Tanaka, K; Tanaka, H; Akabane, K; Suzuki, R; Shinohara, Y; Takei, H; Hashimoto, S; Tanaka, Y; Hojyo, S; Sakamoto, O; Naono, N; Takaai, T; Sato, K; Kojima, Y; Harada, T; Hattori, T; Fuke, STS; Yokota, I; Konno, S; Washio, T; Fukuhara, T; Teshima, T; Taniguchi, M; Murakami, M

    LAB ON A CHIP   23 ( 22 )   4909 - 4918   2023.11   ISSN:1473-0197 eISSN:1473-0189

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    A digital platform that can rapidly and accurately diagnose pathogenic viral variants, including SARS-CoV-2, will minimize pandemics, public anxiety, and economic losses. We recently reported an artificial intelligence (AI)-nanopore platform that enables testing for Wuhan SARS-CoV-2 with high sensitivity and specificity within five minutes. However, which parts of the virus are recognized by the platform are unknown. Similarly, whether the platform can detect SARS-CoV-2 variants or the presence of the virus in clinical samples needs further study. Here, we demonstrated the platform can distinguish SARS-CoV-2 variants. Further, it identified mutated Wuhan SARS-CoV-2 expressing spike proteins of the delta and omicron variants, indicating it discriminates spike proteins. Finally, we used the platform to identify omicron variants with a sensitivity and specificity of 100% and 94%, respectively, in saliva specimens from COVID-19 patients. Thus, our results demonstrate the AI-nanopore platform is an effective diagnostic tool for SARS-CoV-2 variants.

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  • Impact of Imprinted Immunity Induced by mRNA Vaccination in an Experimental Animal Model

    Fujita, S; Uriu, K; Pan, L; Nao, N; Tabata, K; Kishimoto, M; Itakura, Y; Sawa, H; Kida, I; Tamura, T; Fukuhara, T; Ito, J; Matsuno, K; Sato, K

    JOURNAL OF INFECTIOUS DISEASES   228 ( 8 )   1060 - 1065   2023.10   ISSN:0022-1899 eISSN:1537-6613

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    The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants has led to concerns that ancestral SARS-CoV-2-based vaccines may not be effective against newly emerging Omicron subvariants. The concept of "imprinted immunity"suggests that individuals vaccinated with ancestral virus-based vaccines may not develop effective immunity against newly emerging Omicron subvariants, such as BQ.1.1 and XBB.1. In this study, we investigated this possibility using hamsters. Although natural infection induced effective antiviral immunity, breakthrough infections in hamsters with BQ.1.1 and XBB.1 Omicron subvariants after receiving the 3-dose mRNA-lipid nanoparticle vaccine resulted in only faintly induced humoral immunity, supporting the possibility of imprinted immunity.

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  • Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics

    Kimura, I; Yamasoba, D; Nasser, H; Ito, H; Zahradnik, J; Wu, JQ; Fujita, S; Uriu, K; Sasaki, J; Tamura, T; Suzuki, R; Deguchi, S; Plianchaisuk, A; Yoshimatsu, K; Kazuma, Y; Mitoma, S; Schreiber, G; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Takaori-Kondo, A; Ito, J; Shirakawa, K; Takayama, K; Irie, T; Hashiguchi, T; Nakagawa, S; Fukuhara, T; Saito, A; Ikeda, T; Sato, K

    JOURNAL OF VIROLOGY   97 ( 10 )   e0101123   2023.10   ISSN:0022-538X eISSN:1098-5514

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    Previous studies on the Omicron BA.2 variant suggested that the virological characteristics of BA.2 are determined by the mutations in at least two different regions of the viral genome: in the BA.2 spike gene (enhancing viral fusogenicity and intrinsic pathogenicity) and the non-spike region of the BA.2 genome (leading to intrinsic pathogenicity attenuation). However, the mutations modulating the BA.2 virological properties remain elusive. In this study, we demonstrated that the L371F substitution in the BA.2 spike protein confers greater fusogenicity and intrinsic pathogenicity. Furthermore, we revealed that multiple mutations downstream of the spike gene in the BA.2 genome are responsible for attenuating intrinsic viral pathogenicity and replication capacity. As mutations in the SARS-CoV-2 variant spike proteins could modulate certain virological properties, such as immune evasion and infectivity, most studies have previously focused on spike protein mutations. Our results underpin the importance of non-spike protein-related mutations in SARS-CoV-2 variants. IMPORTANCE Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.

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  • Inhibitors of cannabinoid receptor 1 suppress the cellular entry of Lujo virus

    Kimura, M; Matsuoka, R; Taniguchi, S; Maruyama, J; Paessler, S; Oka, S; Yamashita, A; Fukuhara, T; Matsuura, Y; Tani, H

    VIROLOGY   587   109867   2023.10   ISSN:0042-6822 eISSN:1089-862X

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    Lujo virus (LUJV), which belongs to Mammarenavirus, family Arenaviridae, has emerged as a pathogen causing severe hemorrhagic fever with high mortality. Currently, there are no effective treatments for arenaviruses, including LUJV. Here, we screened chemical compound libraries of Food and Drug Administration (FDA)-approved drugs and G protein-coupled receptor-associated drugs to identify effective antivirals against LUJV targeting cell entry using a vesicular stomatitis virus-based pseudotyped virus bearing the LUJV envelope glycoprotein (GP). Cannabinoid receptor 1 (CB1) antagonists, such as rimonabant, AM251 and AM281, have been identified as robust inhibitors of LUJV entry. The IC50 of rimonabant was 0.26 and 0.53 μM in Vero and Huh7 cells, respectively. Analysis of the cell fusion activity of the LUJV GP in the presence of CB1 inhibitors revealed that these inhibitors suppressed the fusion activity of the LUJV GP. Moreover, rimonabant, AM251 and AM281 reduced the infectivity of authentic LUJV in vitro, suggesting that the antiviral activity of CB1 antagonists against LUJV is mediated, at least in part, by inhibition of the viral entry, especially, membrane fusion. These findings suggest promising candidates for developing new therapies against LUJV infections.

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  • Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study

    Akahata, W; Sekida, T; Nogimori, T; Ode, H; Tamura, T; Kono, K; Kazami, Y; Washizaki, A; Masuta, Y; Suzuki, R; Matsuda, K; Komori, M; Morey, AL; Ishimoto, K; Nakata, M; Hasunuma, T; Fukuhara, T; Iwatani, Y; Yamamoto, T; Smith, JF; Sato, N

    CELL REPORTS MEDICINE   4 ( 8 )   101134   2023.8   ISSN:2666-3791

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    VLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying RNA (saRNA) vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. A phase 1 study of VLPCOV-01 is conducted (jRCT2051210164). Participants who completed two doses of the BNT162b2 mRNA vaccine previously are randomized to receive one intramuscular vaccination of 0.3, 1.0, or 3.0 μg VLPCOV-01, 30 μg BNT162b2, or placebo. No serious adverse events have been reported. VLPCOV-01 induces robust immunoglobulin G (IgG) titers against the RBD protein that are maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5,037 (95% confidence interval [CI] 1,272–19,940) at 0.3 μg to 12,873 (95% CI 937–17,686) at 3 μg compared with 3,166 (95% CI 1,619–6,191) with 30 μg BNT162b2. Neutralizing antibody titers against all variants of SARS-CoV-2 tested are induced. VLPCOV-01 is immunogenic following low-dose administration. These findings support the potential for saRNA as a vaccine platform.

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  • Pembrolizumab plus chemotherapy in Japanese patients with metastatic squamous non-small-cell lung cancer in KEYNOTE-407

    Sugawara, S; Tanaka, K; Imamura, F; Yamamoto, N; Nishio, M; Okishio, K; Hirashima, T; Tanaka, H; Fukuhara, T; Nakahara, Y; Kurata, T; Katakami, N; Okada, M; Horinouchi, H; Udagawa, H; Kasahara, K; Satouchi, M; Saka, H; Tokito, T; Hosomi, Y; Aoe, K; Kishi, K; Ohashi, K; Yokoyama, T; Adachi, N; Noguchi, K; Schwarzenberger, P; Kato, T

    CANCER SCIENCE   114 ( 8 )   3330 - 3341   2023.8   ISSN:1347-9032 eISSN:1349-7006

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    The global phase III KEYNOTE-407 (NCT02775435) trial showed that pembrolizumab plus chemotherapy prolonged overall and progression-free survival (OS/PFS) versus placebo plus chemotherapy in patients with metastatic squamous non-small-cell lung cancer (NSCLC). We present outcomes of patients from Japan enrolled in KEYNOTE-407. Patients were randomized 1:1 to receive pembrolizumab 200 mg or placebo with paclitaxel 200 mg/m2 every 3 weeks (Q3W) or nab-paclitaxel 100 mg/m2 (weekly) plus carboplatin area under the concentration-time curve of 6 mg/mL/min Q3W for four cycles, followed by pembrolizumab or placebo Q3W for a total of 35 cycles. Primary end-points were OS and PFS per RECIST version 1.1 by blinded independent central review. Fifty patients were randomized at Japanese sites (pembrolizumab plus chemotherapy, n = 22; placebo plus chemotherapy, n = 28). Median follow-up time at data cut-off (May 9, 2019) was 15.1 (range, 0.5–24.0) months. Median OS (95% confidence interval [CI]) was 17.3 (12.5–not reached) versus 11.0 (8.6–19.5) months in the pembrolizumab plus chemotherapy versus placebo plus chemotherapy group (hazard ratio [HR] 0.56; 95% CI, 0.27–1.15). Median PFS (95% CI) was 8.3 (6.1–13.0) versus 7.2 (3.9–8.8) months (HR 0.65; 95% CI, 0.35–1.23). Grade 3–5 adverse events (AEs) occurred in 86% and 75% of patients, respectively. There were three fatal AEs, two of which were treatment-related (one from each treatment group, pneumonitis and pulmonary hemorrhage). Efficacy and safety outcomes were consistent with the global study and support the use of pembrolizumab plus chemotherapy in Japanese patients with metastatic squamous NSCLC.

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  • saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern

    Komori, M; Nogimori, T; Morey, AL; Sekida, T; Ishimoto, K; Hassett, MR; Masuta, Y; Ode, H; Tamura, T; Suzuki, R; Alexander, J; Kido, Y; Matsuda, K; Fukuhara, T; Iwatani, Y; Yamamoto, T; Smith, JF; Akahata, W

    NATURE COMMUNICATIONS   14 ( 1 )   2810   2023.5   eISSN:2041-1723

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    Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12 months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains.

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  • A third dose of the BNT162b2 mRNA vaccine sufficiently improves the neutralizing activity against SARS-CoV-2 variants in liver transplant recipients

    Tomiyama, T; Suzuki, R; Harada, N; Tamura, T; Toshida, K; Kosai-Fujimoto, Y; Tomino, T; Yoshiya, S; Nagao, Y; Takeishi, K; Itoh, S; Kobayashi, N; Ito, H; Yoshio, S; Kanto, T; Yoshizumi, T; Fukuhara, T

    FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY   13   1197349   2023.5   ISSN:2235-2988

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    Introduction: We examined the neutralizing antibody production efficiency of the second and third severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine doses (2nd- and 3rd-dose) and neutralizing activity on mutant strains, including, the Ancestral, Beta and Omicron strains using green fluorescent protein-carrying recombinant SARS-CoV-2, in living-donor liver transplantation (LDLT) recipients. Methods: The patients who were administered vaccines other than Pfizer- BioNTechBNT162b2 and who had coronavirus disease 2019 in this study period were excluded. We enrolled 154 LDLT recipients and 50 healthy controls. Result: The median time were 21 days (between 1st and 2nd vaccination) and 244 days (between 2nd and 3rd vaccination). The median neutralizing antibody titer after 2nd-dose was lower in LDLT recipients than in controls (0.46 vs 1.00, P<0.0001). All controls had SARS-CoV-2 neutralizing antibodies, whereas 39 LDLT recipients (25.3%) had no neutralizing antibodies after 2nd-dose; age at vaccination, presence of ascites, multiple immunosuppressive treatments, and mycophenolate mofetil treatment were significant risk factors for nonresponder. The neutralizing activities of recipient sera were approximately 3-fold and 5-fold lower than those of control sera against the Ancestral and Beta strains, respectively. The median antibody titer after 3rd-dose was not significantly different between recipients and controls (1.02 vs 1.22, p=0.0758); only 5% recipients was non-responder. The neutralizing activity after third dose to Omicron strains were enhanced and had no significant difference between two groups. Conclusion: Only the 2nd-dose was not sufficiently effective in recipients; however, 3rd-dose had sufficient neutralizing activity against the mutant strain and was as effective as that in healthy controls.

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  • Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants

    Tamura, T; Ito, J; Uriu, K; Zahradnik, J; Kida, I; Anraku, Y; Nasser, H; Shofa, M; Oda, Y; Lytras, S; Nao, N; Itakura, Y; Deguchi, S; Suzuki, R; Wang, L; Begum, MMST; Kita, S; Yajima, H; Sasaki, J; Sasaki-Tabata, K; Shimizu, R; Tsuda, M; Kosugi, Y; Fujita, S; Pan, L; Sauter, D; Yoshimatsu, K; Suzuki, S; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Yamamoto, Y; Nagamoto, T; Schreiber, G; Maenaka, K; Hashiguchi, T; Ikeda, T; Fukuhara, T; Saito, A; Tanaka, S; Matsuno, K; Takayama, K; Sato, K

    NATURE COMMUNICATIONS   14 ( 1 )   2800   2023.5   eISSN:2041-1723

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    In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.

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  • Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

    Ito, J; Suzuki, R; Uriu, K; Itakura, Y; Zahradnik, J; Kimura, KT; Deguchi, S; Wang, L; Lytras, S; Tamura, T; Kida, I; Nasser, H; Shofa, M; Begum, MM; Tsuda, M; Oda, Y; Suzuki, T; Sasaki, J; Sasaki-Tabata, K; Fujita, S; Yoshimatsu, K; Ito, H; Nao, N; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Yamamoto, Y; Nagamoto, T; Kuramochi, J; Schreiber, G; Saito, A; Matsuno, K; Takayama, K; Hashiguchi, T; Tanaka, S; Fukuhara, T; Ikeda, T; Sato, K; Suzuki, S; Kato, M; Ferdous, Z; Mouri, H; Shishido, K; Misawa, N; Kimura, I; Kosugi, Y; Lin, P; Suganami, M; Chiba, M; Yoshimura, R; Yasuda, K; Iida, K; Ohsumi, N; Strange, AP; Sauter, D; Nakagawa, S; Wu, JQ; Watanabe, Y; Sakamoto, A; Yasuhara, N; Nakajima, Y; Yajima, H; Shirakawa, K; Takaori-Kondo, A; Nagata, K; Kazuma, Y; Nomura, R; Horisawa, Y; Tashiro, Y; Kawa, Y; Irie, T; Kawabata, R; Shimizu, R; Takahashi, O; Ichihara, K; Motozono, C; Toyoda, M; Ueno, T; Shibatani, Y; Nishiuchi, T

    NATURE COMMUNICATIONS   14 ( 1 )   2671   2023.5   eISSN:2041-1723

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    In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022.

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  • Increased flexibility of the SARS-CoV-2 RNA-binding site causes resistance to remdesivir

    Torii, S; Kim, KS; Koseki, J; Suzuki, R; Iwanami, S; Fujita, Y; Jeong, YD; Ito, J; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Sato, K; Matsuura, Y; Shimamura, T; Iwami, S; Fukuhara, T

    PLOS PATHOGENS   19 ( 3 )   e1011231   2023.3   ISSN:1553-7366 eISSN:1553-7374

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    Mutations continue to accumulate within the SARS-CoV-2 genome, and the ongoing epidemic has shown no signs of ending. It is critical to predict problematic mutations that may arise in clinical environments and assess their properties in advance to quickly implement countermeasures against future variant infections. In this study, we identified mutations resistant to remdesivir, which is widely administered to SARS-CoV-2-infected patients, and discuss the cause of resistance. First, we simultaneously constructed eight recombinant viruses carrying the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. We confirmed that all the mutant viruses didn’t gain the virus production efficiency without remdesivir treatment. Time course analyses of cellular virus infections showed significantly higher infectious titers and infection rates in mutant viruses than wild type virus under treatment with remdesivir. Next, we developed a mathematical model in consideration of the changing dynamic of cells infected with mutant viruses with distinct propagation properties and defined that mutations detected in in vitro passages canceled the antiviral activities of remdesivir without raising virus production capacity. Finally, molecular dynamics simulations of the NSP12 protein of SARS-CoV-2 revealed that the molecular vibration around the RNA-binding site was increased by the introduction of mutations on NSP12. Taken together, we identified multiple mutations that affected the flexibility of the RNA binding site and decreased the antiviral activity of remdesivir. Our new insights will contribute to developing further antiviral measures against SARS-CoV-2 infection.

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  • Antiviral Activity of Micafungin and Its Derivatives against SARS-CoV-2 RNA Replication

    Nakajima, S; Ohashi, H; Akazawa, D; Torii, S; Suzuki, R; Fukuhara, T; Watashi, K

    VIRUSES-BASEL   15 ( 2 )   2023.2   eISSN:1999-4915

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    Echinocandin antifungal drugs, including micafungin, anidulafungin, and caspofungin, have been recently reported to exhibit antiviral effects against various viruses such as flavivirus, alphavirus, and coronavirus. In this study, we focused on micafungin and its derivatives and analyzed their antiviral activities against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The micafungin derivatives Mi-2 and Mi-5 showed higher antiviral activity than micafungin, with 50% maximal inhibitory concentration (IC50) of 5.25 and 6.51 µM, respectively (3.8 to 4.7-fold stronger than micafungin) and 50% cytotoxic concentration (CC50) of >64 µM in VeroE6/TMPRSS2 cells. This high anti-SARS-CoV-2 activity was also conserved in human lung epithelial cell-derived Calu-3 cells. Micafungin, Mi-2, and Mi-5 were suggested to inhibit the intracellular virus replication process; additionally, these compounds were active against SARS-CoV-2 variants, including Delta (AY.122, hCoV-19/Japan/TY11-927/2021), Omicron (BA.1.18, hCoV-19/Japan/TY38-873/2021), a variant resistant to remdesivir (R10/E796G C799F), and a variant resistant to casirivimab/imdevimab antibody cocktail (E406W); thus, our results provide basic evidence for the potential use of micafungin derivatives for developing antiviral agents.

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  • Smoking enhances the expression of angiotensin-converting enzyme 2 involved in the efficiency of severe acute respiratory syndrome coronavirus 2 infection

    Suzuki, R; Ono, Y; Noshita, K; Kim, KS; Ito, H; Morioka, Y; Tamura, T; Okuzaki, D; Tagawa, T; Takenaka, T; Yoshizumi, T; Shimamura, T; Iwami, S; Fukuhara, T

    MICROBIOLOGY AND IMMUNOLOGY   67 ( 1 )   22 - 31   2023.1   ISSN:0385-5600 eISSN:1348-0421

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    Smoking is one of the risk factors most closely related to the severity of coronavirus disease 2019 (COVID-19). However, the relationship between smoking history and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is unknown. In this study, we evaluated the ACE2 expression level in the lungs of current smokers, ex-smokers, and nonsmokers. The ACE2 expression level of ex-smokers who smoked cigarettes until recently (cessation period shorter than 6 months) was higher than that of nonsmokers and ex-smokers with a long history of nonsmoking (cessation period longer than 6 months). We also showed that the efficiency of SARS-CoV-2 infection was enhanced in a manner dependent on the angiotensin-converting enzyme 2 (ACE2) expression level. Using RNA-seq analysis on the lungs of smokers, we identified that the expression of inflammatory signaling genes was correlated with ACE2 expression. Notably, with increasing duration of smoking cessation among ex-smokers, not only ACE2 expression level but also the expression levels of inflammatory signaling genes decreased. These results indicated that smoking enhances the expression levels of ACE2 and inflammatory signaling genes. Our data suggest that the efficiency of SARS-CoV-2 infection is enhanced by smoking-mediated upregulation of ACE2 expression level.

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  • 腎移植レシピエントにおけるCOVID-19ワクチン接種後の獲得免疫の検討

    川代 啓太, 鈴木 理滋, 野木森 拓人, 岩原 直也, 広瀬 貴行, 山本 拓也, 福原 崇介, 堀田 記世彦, 篠原 信雄

    Japanese Journal of Transplantation   58 ( Supplement )   s270_1 - s270_1   2023   ISSN:05787947 eISSN:21880034

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    <p>目的 腎移植レシピエントのCOVID-19ワクチン接種の効果について検討。方法 腎移植後にCOVID-19のmRNAワクチン3回接種した未感染レシピエント73名と、同じくワクチン3回接種した健常者17名を対象に、抗体価、中和力価、細胞性免疫を検討した。中和力価は武漢型WT、オミクロンBA.1、BA.5の生ウィルスを用いた中和アッセイにより評価した。細胞性免疫はPBMCをWT、オミクロンBA.1、BA.5のスパイク蛋白抗原を含むペプチドで刺激し、細胞内染色を用いて活性化CD4/CD8メモリーT細胞を評価した。オミクロン対応ワクチンを追加接種したレシピエント20名、健常群7名の抗体価、BA.5に対する中和力価も評価した。結果 レシピエントの年齢中央値は59歳、健常群は35歳であった (p=0.0025)。抗体価中央値はレシピエント91.6AU/ml、健常群369.2AU/mlであった (p<0.0001)。3種類のウィルス株に対する中和力価はいずれもレシピエントで有意に低下していた (p<0.0001)。レシピエントで活性化CD8メモリーT細胞の有意な低下を認めた (p<0.0001)。オミクロン対応ワクチン接種による抗体価、中和力価は両群とも接種前に比べ上昇していたが、その上昇幅はレシピエントで有意に少なかった (抗体価:p=0.0051、中和力価:p=0.0327)。考察 レシピエントはワクチン3回目接種では十分な免疫は得られず、オミクロン対応ワクチン接種による効果も健常群よりも乏しく、ワクチン以外の方法を模索する必要性が示唆された。</p>

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  • Reverse genetics systems for severe acute respiratory syndrome coronavirus 2

    TAMURA Tomokazu, FUKUHARA Takasuke

    Japanese Journal of Thrombosis and Hemostasis   34 ( 4 )   449 - 451   2023   ISSN:09157441 eISSN:18808808

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  • 病原ウイルスの増殖性および病原性発現機構の解明

    福原 崇介

    上原記念生命科学財団研究報告集   36   1 - 5   2022.12

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  • Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant

    Saito Akatsuki, Tamura Tomokazu, Zahradnik Jiri, Deguchi Sayaka, Tabata Koshiro, Anraku Yuki, Kimura Izumi, Ito Jumpei, Yamasoba Daichi, Nasser Hesham, Toyoda Mako, Nagata Kayoko, Uriu Keiya, Kosugi Yusuke, Fujita Shigeru, Shofa Maya, Monira Begum M.S.T., Shimizu Ryo, Oda Yoshitaka, Suzuki Rigel, Ito Hayato, Nao Naganori, Wang Lei, Tsuda Masumi, Yoshimatsu Kumiko, Kuramochi Jin, Kita Shunsuke, Sasaki-Tabata Kaori, Fukuhara Hideo, Maenaka Katsumi, Yamamoto Yuki, Nagamoto Tetsuharu, Asakura Hiroyuki, Nagashima Mami, Sadamasu Kenji, Yoshimura Kazuhisa, Ueno Takamasa, Schreiber Gideon, Takaori-Kondo Akifumi, The Genotype to Phenotype Japan (G2P-Japan) Consortium, Shirakawa Kotaro, Sawa Hirofumi, Irie Takashi, Hashiguchi Takao, Takayama Kazuo, Matsuno Keita, Tanaka Shinya, Ikeda Terumasa, Fukuhara Takasuke, Sato Kei

    Cell Host and Microbe   30 ( 11 )   1540 - 1555   2022.11   ISSN:19313128 eISSN:19346069

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    The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.

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  • SARS-CoV-2 Omicron detection by antigen tests using saliva

    Murakami, K; Iwasaki, S; Oguri, S; Tanaka, K; Suzuki, R; Hayasaka, K; Fujisawa, S; Watanabe, C; Konno, S; Yokota, I; Fukuhara, T; Murakami, M; Teshima, T

    JOURNAL OF CLINICAL VIROLOGY PLUS   2 ( 4 )   100109   2022.11   ISSN:2667-0380

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    The Omicron emerged in November 2021 and became the predominant SARS-CoV-2 variant globally. It spreads more rapidly than ancestral lineages and its rapid detection is critical for the prevention of disease outbreaks. Antigen tests such as immunochromatographic assay (ICA) and chemiluminescent enzyme immunoassay (CLEIA) yield results more quickly than standard polymerase chain reaction (PCR). However, their utility for the detection of the Omicron variant remains unclear. We herein evaluated the performance of ICA and CLEIA in saliva from 51 patients with Omicron and 60 PCR negative individuals. The sensitivity and specificity of CLEIA were 98.0% (95%CI: 89.6–100.0%) and 100.0% (95%CI: 94.0–100.0%), respectively, with fine correlation with cycle threshold (Ct) values. The sensitivity and specificity of ICA were 58.8% (95%CI: 44.2-72.4%) and 100.0% (95%CI: 94.0–100.0%), respectively. The sensitivity of ICA was 100.0% (95%CI: 80.5–100.0%) when PCR Ct was less than 25. The Omicron can be efficiently detected in saliva by CLEIA. ICA also detects high viral load Omicron using saliva.

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  • Virological characteristics of the SARS-CoV-2 Omicron BA.2 subvariants, including BA.4 and BA.5

    Kimura, I; Yamasoba, D; Tamura, T; Nao, N; Suzuki, T; Oda, Y; Mitoma, S; Ito, J; Nasser, H; Zahradnik, J; Uriu, K; Fujita, S; Kosugi, Y; Wang, L; Tsuda, M; Kishimoto, M; Ito, H; Suzuki, R; Shimizu, R; Begum, MM; Yoshimatsu, K; Kimura, KT; Sasaki, J; Sasaki-Tabata, K; Yamamoto, Y; Nagamoto, T; Kanamune, J; Kobiyama, K; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Shirakawa, K; Takaori-Kondo, A; Kuramochi, J; Schreiber, G; Ishii, KJ; Hashiguchi, T; Ikeda, T; Saito, A; Fukuhara, T; Tanaka, S; Matsuno, K; Sato, K

    CELL   185 ( 21 )   3992 - +   2022.10   ISSN:0092-8674 eISSN:1097-4172

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    After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.

    DOI: 10.1016/j.cell.2022.09.018

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  • Impact of JMJD6 on intrahepatic cholangiocarcinoma

    Kosai-Fujimoto, Y; Itoh, S; Yugawa, K; Fukuhara, T; Okuzaki, D; Toshima, T; Harada, N; Oda, Y; Yoshizumi, T; Mori, M

    MOLECULAR AND CLINICAL ONCOLOGY   17 ( 2 )   131   2022.8   ISSN:2049-9450 eISSN:2049-9469

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    The association of Jumonji domain-containing 6 (JMJD6) with the prognosis of various types of cancer has been demonstrated, except in intrahepatic cholangiocarci-noma (ICC). The present study aimed to clarify the impact of JMJD6 on ICC. The liver specimens of 51 patients who underwent surgery for ICC were analyzed for JMJD6 expression using immunohistochemistry staining. The relationship between clinicopathological factors and JMJD6 expression was investigated. The cellular activity was also evaluated in JMJD6 knocked down cells with Transwell migration assay and viability assay. In the immunohistochemistry staining of clinical samples, high expression of JMJD6 was seen in 32 of 51 samples. High expression was also associated with improved overall survival (OS) and recurrence-free survival (RFS) (P=0.0033 and 0.048, respectively). Further analyses revealed that higher JMJD6 expression was one of the improved independent prognostic factors of OS and RFS. Expression of JMJD6 was knocked down in commercial culture cell lines of ICC, and RNA and protein were extracted to analyze the downstream gene expression using RNA-sequencing and western blotting. JMJD6 knockdown was associated with higher programmed death-ligand 1 (PD-L1) expression in RNA-sequencing and western blotting. In addition, PD-L1 expression was higher in JMJD6 low expression clinical samples when measured using immunohistochemistry staining. In conclusion, high expression of JMJD6 was an independent favorable prognostic factor of ICC. JMJD6 may influence the prognosis of ICC through the regulation of PD-L1 expression.

    DOI: 10.3892/mco.2022.2564

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  • SARS-CoV-2 RNA複製をブロックするシクレソニドアセタール誘導体の抗ウイルス活性(Antiviral activity of ciclesonide acetal derivatives blocking SARS-CoV-2 RNA replication)

    Tsuji Genichiro, Nakajima Shogo, Watashi Koichi, Torii Shiho, Suzuki Rigel, Fukuhara Takasuke, Ohoka Nobumichi, Inoue Takao, Demizu Yosuke

    Journal of Pharmacological Sciences   149 ( 3 )   81 - 84   2022.7   ISSN:1347-8613

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    シクレソニド(CIC)のアセタール部位とエステル部位を加水分解して得たCIC-ジオール(16α-ヒドロキシプレドニゾロン)を原料として16種類のCIC-アセタール誘導体をデザイン・合成し、抗重症急性呼吸器症候群コロナウイルス2型(SARS-CoV-2)活性を比較した。その結果、これらのCIC-アセタール誘導体のうち、直鎖アルキル鎖を持つ数種のCIC-アセタール誘導体がCIC-2と比較して強力なウイルスコピー数減少活性を示した。

  • Antiviral activity of ciclesonide acetal derivatives blocking SARS-CoV-2 RNA replication

    Tsuji, G; Nakajima, S; Watashi, K; Torii, S; Suzuki, R; Fukuhara, T; Ohoka, N; Inoue, T; Demizu, Y

    JOURNAL OF PHARMACOLOGICAL SCIENCES   149 ( 3 )   81 - 84   2022.7   ISSN:1347-8613 eISSN:1347-8648

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    Ciclesonide (Cic) is approved as an inhalant for asthma and was clinically tested as a candidate therapy for coronavirus disease 2019 (COVID-19). Its active metabolite Cic2 was recently reported to suppress genomic RNA replication of severe acute respiratory syndrome coronavirus 2. In this study, we designed and synthesized a set of ciclesonide-acetal (Cic-acetal) derivatives. Among designated compounds, some Cic-acetal derivatives with a linear alkyl chain exhibited strong viral copy-number reduction activities compared with Cic2. These compounds might serve as lead compounds for developing novel anti-COVID-19 agents.

    DOI: 10.1016/j.jphs.2022.04.001

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  • Virological characteristics of the SARS-CoV-2 Omicron BA.2 spike

    Yamasoba, D; Kimura, I; Nasser, H; Morioka, Y; Nao, N; Ito, J; Uriu, K; Tsuda, M; Zahradnik, J; Shirakawa, K; Suzuki, R; Kishimoto, M; Kosugi, Y; Kobiyama, K; Hara, T; Toyoda, M; Tanaka, YL; Butlertanaka, EP; Shimizu, R; Ito, H; Wang, L; Oda, Y; Orba, Y; Sasaki, M; Nagata, K; Yoshimatsu, K; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Kuramochi, J; Seki, M; Fujiki, R; Kaneda, A; Shimada, T; Nakada, T; Sakao, S; Suzuki, T; Ueno, T; Takaori-Kondo, A; Ishii, KJ; Schreiber, G; Sawa, H; Saito, A; Irie, T; Tanaka, S; Matsuno, K; Fukuhara, T; Ikeda, T; Sato, K

    CELL   185 ( 12 )   2103 - +   2022.6   ISSN:0092-8674 eISSN:1097-4172

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    Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.

    DOI: 10.1016/j.cell.2022.04.035

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  • Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses

    Tamura, T; Torii, S; Kajiwara, K; Anzai, I; Fujioka, Y; Noda, K; Taguwa, S; Morioka, Y; Suzuki, R; Fauzyah, Y; Ono, C; Ohba, Y; Okada, M; Fukuhara, T; Matsuura, Y

    PLOS PATHOGENS   18 ( 6 )   e1010593   2022.6   ISSN:1553-7366 eISSN:1553-7374

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    Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

    DOI: 10.1371/journal.ppat.1010593

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  • Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant

    Suzuki, R; Yamasoba, D; Kimura, I; Wang, L; Kishimoto, M; Ito, J; Morioka, Y; Nao, N; Nasser, H; Uriu, K; Kosugi, Y; Tsuda, M; Orba, Y; Sasaki, M; Shimizu, R; Kawabata, R; Yoshimatsu, K; Asakura, H; Nagashima, M; Sadamasu, K; Yoshimura, K; Sawa, H; Ikeda, T; Irie, T; Matsuno, K; Tanaka, S; Fukuhara, T; Sato, K

    NATURE   603 ( 7902 )   700 - +   2022.3   ISSN:0028-0836 eISSN:1476-4687

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    The emergence of the Omicron variant of SARS-CoV-2 is an urgent global health concern1. In this study, our statistical modelling suggests that Omicron has spread more rapidly than the Delta variant in several countries including South Africa. Cell culture experiments showed Omicron to be less fusogenic than Delta and than an ancestral strain of SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into two subunits, which facilitates cell–cell fusion2,3, the Omicron S protein was less efficiently cleaved compared to the S proteins of Delta and ancestral SARS-CoV-2. Furthermore, in a hamster model, Omicron showed decreased lung infectivity and was less pathogenic compared to Delta and ancestral SARS-CoV-2. Our multiscale investigations reveal the virological characteristics of Omicron, including rapid growth in the human population, lower fusogenicity and attenuated pathogenicity.

    DOI: 10.1038/s41586-022-04462-1

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  • 特集 ウイルスを創る,ウイルスを視る 新型コロナウイルスを創る

    福原 崇介

    医学のあゆみ   280 ( 9 )   901 - 904   2022.2   ISSN:00392359

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    Publisher:医歯薬出版  

    DOI: 10.32118/ayu28009901

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  • 最先端医療の今 フラビウイルスの複製における非構造タンパク質NS1の役割

    田村 友和, 福原 崇介, 松浦 善治

    Medical Science Digest   51 ( 1 )   40 - 43   2025.1   ISSN:1347-4340

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    フラビウイルス感染症は,節足動物が媒介するジカ・デング・ダニ媒介性脳炎・日本脳炎ウイルスなどを原因とする人獣共通感染症である。本邦でも地球温暖化とヒトの移動範囲・経済活動の拡大で,ジカウイルス感染症およびデング熱の感染例が近年報告されている。また,北海道でダニ媒介性脳炎が断続的に発生している。しかし,有効な治療法がないため,その制御に向けた基礎研究を推進する必要がある。本研究では,これまで全く不明であったフラビウイルスの粒子産生の分子メカニズムの解明を目的に,多角的な手法(ウイルス学,生化学,バイオインフォマティクス)でウイルスタンパク質NS1を解析した。その結果,NS1タンパク質の感染細胞内での多量体形成がウイルスゲノムの複製と感染性粒子の産生に関与することが初めて明らかになった。本成果により,NS1タンパク質を標的とした新規抗フラビウイルス薬の開発が期待される。(著者抄録)

  • 【次のパンデミックに備えた新しい感染症研究 みんなで連携し、迅速に研究し、成果を社会に還元する】次のパンデミックに向けて教訓を得て,いま動き出す 公衆衛生・臨床・基礎研究の最前線で感じた課題とこれからの取り組み

    佐藤 佳, 福原 崇介, 福永 興壱, 押谷 仁, 忽那 賢志

    実験医学   42 ( 8 )   1238 - 1247   2024.5   ISSN:0288-5514

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    Language:Japanese   Publisher:(株)羊土社  

  • 【次のパンデミックに備えた新しい感染症研究 みんなで連携し、迅速に研究し、成果を社会に還元する】超高速なウイルス人工合成法の確立と応用研究

    福原 崇介

    実験医学   42 ( 8 )   1217 - 1221   2024.5   ISSN:0288-5514

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    2020年5月,コロナウイルスを人工合成するリバースジェネティクス法はすでに開発されていたが,複雑かつ高度な遺伝子操作技術と数ヵ月もの期間が必要であった.それを受けて,われわれはCircular Polymerase Extension Reaction(CPER)法というPCRを活用した簡便な手法で,感染性組換えSARS-CoV-2を作製する技術を開発した.より多くの研究者が迅速・簡便に新型コロナウイルスを合成できるようになることで,病原性解析やワクチン・抗ウイルス薬の開発,また,次々と現れる変異に対するこれまで以上に素早い解析が可能となる.筆者はこのCPER法を駆使することで,新たに出現した変異株の解析を含むさまざまなSARS-CoV-2の解析を行ってきた.(著者抄録)

  • 新型コロナウイルスにおけるリバース・ジェネティクス法

    田村 友和, 福原 崇介

    日本血栓止血学会誌   34 ( 4 )   449 - 451   2023.8   ISSN:0915-7441

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  • CPER法による高速リバースジェネティクスを駆使したSARS-CoV-2の応用研究

    福原 崇介

    日本臨床細胞学会九州連合会雑誌   54   1 - 5   2023.7   ISSN:0912-6600

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    Language:Japanese   Publisher:日本臨床細胞学会-九州連合会  

    コロナウイルスのリバースジェネティクス法はすでに開発されていたが,複雑かつ高度な遺伝子操作技術と数ヵ月もの期間が必要であった.しかし,次々に現れる変異ウイルスに対応し,かつ病原性の解明や治療法・予防法の開発を行うためには,迅速かつ簡便な感染性ウイルスを作出する技術の開発が必要である.今回,我々はCircular Polymerase Extension Reaction(CPER)法というPCRを活用した簡便な手法で,感染性組み換えSARS-CoV-2を作製する技術を開発した。CPERを用いたリバースジェネティクスではウイルスゲノムを改変した組換えウイルスを作製することが極めて容易である.これまでに,全てのVOCまたはVOIのスパイクを持つ組換えウイルスの作製に成功し,その性状解析を行ってきた。また,GFPやルシフェラーゼといったレポーターを搭載した組換えSARS-CoV-2の作製にも成功した.本稿では,SARS-CoV-2の変異株であるデルタ株,オミクロン株(BA.1株),ステルスオミクロン株(BA.2株)に関する成果を示す(Cell Host Microbe 2021,Nature 2021,Nature 2022,Ce22 2022).より多くの研究者が迅速・簡便に新型コロナウイルスを合成できるようになることで,人工的に遺伝子改変したウイルスを用いた病原性解析やワクチン・抗ウイルス薬の開発,また,次々と現れる変異に対するこれまで以上に素早い解析が可能となり,新型コロナウイルス感染症克服に向けた研究が飛躍的に進むことが期待される.また,今回構築したウイルス感染症に対する解析プラットフォームは今後訪れる新興感染症対策に間違いなく有益である.(著者抄録)

  • 【ウイルス性肝炎学2023-最新の病態・診断・治療情報-】総論 C型慢性肝疾患治療のeliminationに向けて

    鈴木 紗織, 福原 崇介

    日本臨床   81 ( 増刊7 ウイルス性肝炎学2023 )   33 - 39   2023.7   ISSN:0047-1852

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  • 【ウイルス感染症の克服に向けた新展開】ウイルス感染症に対する創薬やワクチン開発に関わる技術開発

    福原 崇介

    細胞   54 ( 5 )   249 - 252   2022.5   ISSN:1346-7557

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    新型コロナウイルスの世界的感染拡大は、多数の感染者および死亡者を引き起こし、世界に大混乱を起こした。その中で、世界の研究者が多くの技術を開発し、ウイルスの増殖機構の解明や治療法の確立に大きく貢献した。今後の新興再興ウイルス感染症の制御において、SARS-CoV-2に関する技術開発の経験をプラットフォーム化することが重要と考えられる。SARS-CoV-2の研究モデルとしては、(1)in vitro感染モデルの確立、(2)in vivo感染モデルの確立、(3)ウイルス感染や複製の疑似モデルの確立および(4)リバースジェネティクス系の確立は、それぞれが大きく研究推進に寄与した技術である。特に、コロナウイルスゲノムがRNAウイルスの中では極めて大きいことを考慮すると、レプリコンやリバースジェネティクスが短期間で樹立できたことは大きな進歩である。本稿では、SARS-CoV-2感染症に対する創薬やワクチン開発に関わる技術開発を概説し、今後の新興再興ウイルス感染症の技術開発に関しても考察を加える。(著者抄録)

  • 【ウイルスを創る、ウイルスを視る】新型コロナウイルスを創る

    福原 崇介

    医学のあゆみ   280 ( 9 )   901 - 904   2022.2   ISSN:0039-2359

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    Language:Japanese   Publisher:医歯薬出版(株)  

    コロナウイルスのリバースジェネティクス法はすでに開発されていたが、複雑かつ高度な遺伝子操作技術と数ヵ月もの期間が必要であった。しかし、次々に現れる変異ウイルスに対応し、かつ病原性の解明や治療法・予防法の開発を行うためには、迅速かつ簡便な感染性ウイルスを作出する技術の開発が必要である。今回、筆者らはcircular polymerase extension reaction(CPER)法というPCRを活用した簡便な手法で、感染性組換え新型コロナウイルス(SARS-CoV-2)を作製する技術を開発した。より多くの研究者が迅速・簡便にSARS-CoV-2を合成できるようになることで、人工的に遺伝子改変したウイルスを用いた病原性解析やワクチン・抗ウイルス薬の開発、また、次々と現れる変異に対するこれまで以上にすばやい解析が可能となり、新型コロナウイルス感染症(COVID-19)克服に向けた研究が飛躍的に進むことが期待される。(著者抄録)

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Professional Memberships

  • 日本ウイルス学会

    2009.4 - Present

  • 分子生物学会

    2009.4 - Present

Committee Memberships

  • 日本ウイルス学会   評議員   Domestic

    2024.10 - Present   

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    Committee type:Academic society

Research Projects

  • Promotion of comprehensive interdisciplinary virology for the post-COVID era

    Grant number:23K20041  2023.11 - 2030.3

    Grants-in-Aid for Scientific Research  Fund for the Promotion of Joint International Research (International Leading Research )

    佐藤 佳, 福原 崇介, 松野 啓太, 齊藤 暁, 木村 香菜子

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    Grant type:Scientific research funding

    ウイルスのふるまいについて、マクロスケールな流行動態を規定する因子を捕捉し、研究室内におけるメゾスケールな挙動として理解し、ミクロケールな構造生物学の見地から解明する。多階層で学際的なウイルス研究を有機的に展開する。新たなパンデミックリスクに備えた、学際的、国際的、持続的なウイルス学研究を展開するための、将来性と世界的なインパクトに満ちた研究課題である。医学・獣医学ウイルス学領域における若手育成を目指し、「ウイルス学若手ネットワーク」と連携することで、本課題が推進する学際的なウイルス研究に参画する若手ウイルス学者の啓蒙を進め、世界で活躍できる若手研究者の育成を図る。

    CiNii Research

  • Development of novel anti-viral therapeuticing an advanced screening technologys us

    Grant number:22KK0112  2022.10 - 2025.3

    Grants-in-Aid for Scientific Research  Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))

    小林 弘一, 田村 友和, 福原 崇介, 應田 涼太

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    Grant type:Scientific research funding

    ヒトHLA分子特に、MHCクラスIと言われる分子群は、ウイルス感染時にウイルス抗原をリンパ球に提示し、感染状態を知らせる為に必須であり、感染細胞およびウイルスの排除に必要な仕組みである。このため、多くのウイルスがMHCクラスIのメカニズムを抑えて、免疫から逃避し、感染を維持しようとする。COVID19を引き起こすSARS-CoV-2ウイルスはそのうちの一つである。MHCクラスIを増強することはウイルス治療に直結するにも関わらず、現在まで治療薬としての開発は進んでいない。Baylor医科大学の創薬部門とTexasA&M 大学の微生物免疫部門との共同研究により治療薬の開発に取り組む。

    CiNii Research

  • Promotion of emerging and re-emerging virus research using high-speed reverse genetics

    Grant number:21H02736  2021.4 - 2024.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Fukuhara Takasuke

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    Grant type:Scientific research funding

    Using the rapid reverse genetics approach of SARS-CoV-2, we generated various recombinant viruses and conducted the following investigations and development of novel experimental systems:1.Characterization of mutant strains, 2.Elucidation of mechanisms for acquiring resistance to antiviral drugs, 3.Clarification of the efficacy of vaccines in post-transplant patients, 4.Development of a direct reverse genetics method through direct amplification of viral genomes from saliva samples, 5.Construction of an efficient neutralization assay system using GFP-tagged recombinant viruses, 6.Development of a mouse model of SARS-CoV-2 with assessable pathogenicity, 7.Construction of an in vivo imaging system using recombinant SARS-CoV-2 incorporating Akaluc.

    CiNii Research

Educational Activities

  • ウイルス学に関する教育を学部生、修士学生、博士学生に対して行なっている。

Class subject

  • ウイルス学

    2024.9 - Present   Second semester

Year of medical license acquisition

  • 2004