Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Medicine　Pathological Science　Department of Microbiology and Immunology

Affiliation (Master)

• Faculty of Medicine　Pathological Science　Department of Microbiology and Immunology

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Profile and Settings

Affiliation

• Hokkaido University, Graduate School of Medicine, Professor

Profile and Settings

• Name (Japanese)

  Fukuhara

• Name (Kana)

  Takasuke

• Name

  201801003859191840

Affiliation

• Hokkaido University, Graduate School of Medicine, Professor

Achievement

Research Areas

• Life sciences / Virology

Published Papers

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

  Naohiro Yasuura, Goki Suda, Masatsugu Ohara, Akimitsu Meno, Takuya Sho, Risako Kohya, Takashi Sasaki, Tomoka Yoda, Sonoe Yoshida, Qingjie Fu, Zijian Yang, Shunichi Hosoda, Osamu Maehara, Shunsuke Ohnishi, Tomoya Saitou, Masaya Sugiyama, Takasuke Fukuhara, Masaru Baba, Takashi Kitagataya, Naoki Kawagishi, Masato Nakai, Mitsuteru Natsuizaka, Koji Ogawa, Akinobu Taketomi, Naoya Sakamoto

  Alimentary pharmacology & therapeutics 2024/09/04 

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

  Md Belal Hossain, Yoshikazu Uchiyama, Samiul Alam Rajib, Akhinur Rahman, Mitsuyoshi Takatori, Benjy Jek Yang Tan, Kenji Sugata, Mami Nagashima, Mamiyo Kawakami, Hitoshi Ito, Ryota Kumagai, Kenji Sadamasu, Yasuhiro Ogi, Tatsuya Kawaguchi, Tomokazu Tamura, Takasuke Fukuhara, Masahiro Ono, Kazuhisa Yoshimura, Yorifumi Satou

  Communications Medicine 4 (1) 2024/08/09
• Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236

  Shigeru Fujita, Arnon Plianchaisuk, Sayaka Deguchi, Hayato Ito, Naganori Nao, Lei Wang, Hesham Nasser, Tomokazu Tamura, Izumi Kimura, Yukie Kashima, Rigel Suzuki, Saori Suzuki, Izumi Kida, Masumi Tsuda, Yoshitaka Oda, Rina Hashimoto, Yukio Watanabe, Keiya Uriu, Daichi Yamasoba, Ziyi Guo, Alfredo A. Hinay, Yusuke Kosugi, Luo Chen, Lin Pan, Yu Kaku, Hin Chu, Flora Donati, Sarah Temmam, Marc Eloit, Yuki Yamamoto, Tetsuharu Nagamoto, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Yutaka Suzuki, Hirofumi Sawa, Keita Mizuma, Jingshu Li, Yume Mimura, Yuma Ohari, Tomoya Tsubo, Zannatul Ferdous, Kenji Shishido, Hiromi Mohri, Miki Iida, Shuhei Tsujino, Naoko Misawa, Kaoru Usui, Wilaiporn Saikruang, Spyridon Lytras, Shusuke Kawakubo, Luca Nishumura, Jarel Elgin Mendoza Tolentino, Wenye Li, Maximilian Stanley Yo, Kio Horinaka, Mai Suganami, Mika Chiba, Ryo Yoshimura, Kyoko Yasuda, Keiko Iida, Adam Patrick Strange, Naomi Ohsumi, Shiho Tanaka, Eiko Ogawa, Kaho Okumura, Tsuki Fukuda, Rina Osujo, Isao Yoshida, So Nakagawa, Akifumi Takaori-Kondo, Kotaro Shirakawa, Kayoko Nagata, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Yoshitaka Nakata, Hiroki Futatsusako, Ayaka Sakamoto, Naoko Yasuhara, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Hisano Yajima, Takashi Irie, Ryoko Kawabata, Kaori Sasaki-Tabata, Ryo Shimizu, M.S.T. Monira Begum, Michael Jonathan, Yuka Mugita, Sharee Leong, Otowa Takahashi, Kimiko Ichihara, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Akatsuki Saito, Anon Kosaka, Miki Kawano, Natsumi Matsubara, Tomoko Nishiuchi, Jiri Zahradnik, Prokopios Andrikopoulos, Miguel Padilla-Blanco, Aditi Konar, Jumpei Ito, Terumasa Ikeda, Shinya Tanaka, Keita Matsuno, Takasuke Fukuhara, Kazuo Takayama, Kei Sato

  eBioMedicine 104 105181 - 105181 2352-3964 2024/06
• Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine.

  Masayuki Aboshi, Kenta Matsuda, Daisuke Kawakami, Kaoru Kono, Yoko Kazami, Takashi Sekida, Mai Komori, Amber L Morey, Shigeru Suga, Jonathan F Smith, Takasuke Fukuhara, Yasumasa Iwatani, Takuya Yamamoto, Nobuaki Sato, Wataru Akahata

  iScience 27 (2) 108964 - 108964 2024/02/16 [Refereed]
   

  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.
• Virological characteristics of the SARS-CoV-2 BA.2.86 variant.

  Tomokazu Tamura, Keita Mizuma, Hesham Nasser, Sayaka Deguchi, Miguel Padilla-Blanco, Yoshitaka Oda, Keiya Uriu, Jarel E M Tolentino, Shuhei Tsujino, Rigel Suzuki, Isshu Kojima, Naganori Nao, Ryo Shimizu, Lei Wang, Masumi Tsuda, Michael Jonathan, Yusuke Kosugi, Ziyi Guo, Alfredo A Hinay Jr, Olivia Putri, Yoonjin Kim, Yuri L Tanaka, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Akatsuki Saito, Jumpei Ito, Takashi Irie, Shinya Tanaka, Jiri Zahradnik, Terumasa Ikeda, Kazuo Takayama, Keita Matsuno, Takasuke Fukuhara, Kei Sato

  Cell host & microbe 32 (2) 170 - 180 2024/02/14 

  In late 2023, several SARS-CoV-2 XBB descendants, notably EG.5.1, were predominant worldwide. However, a distinct SARS-CoV-2 lineage, the BA.2.86 variant, also emerged. BA.2.86 is phylogenetically distinct from other Omicron sublineages, accumulating over 30 amino acid mutations in its spike protein. Here, we examined the virological characteristics of the BA.2.86 variant. Our epidemic dynamics modeling suggested that the relative reproduction number of BA.2.86 is significantly higher than that of EG.5.1. Additionally, four clinically available antivirals were effective against BA.2.86. Although the fusogenicity of BA.2.86 spike is similar to that of the parental BA.2 spike, the intrinsic pathogenicity of BA.2.86 in hamsters was significantly lower than that of BA.2. Since the growth kinetics of BA.2.86 are significantly lower than those of BA.2 both in vitro and in vivo, the attenuated pathogenicity of BA.2.86 is likely due to its decreased replication capacity. These findings uncover the features of BA.2.86, providing insights for control and treatment.
• A rapid and versatile reverse genetics approach for generating recombinant positive-strand RNA viruses that use IRES-mediated translation.

  Tomokazu Tamura, Hirotaka Yamamoto, Saho Ogino, Yuhei Morioka, Shuhei Tsujino, Rigel Suzuki, Takahiro Hiono, Saori Suzuki, Norikazu Isoda, Yoshihiro Sakoda, Takasuke Fukuhara

  Journal of virology e0163823  2024/02/14 

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

  Rigel Suzuki, Akifumi Kamiyama, Hayato Ito, Keita Kawashiro, Takahiro Tomiyama, Tomokazu Tamura, Saori Suzuki, Tomoharu Yoshizumi, Kiyohiko Hotta, Takasuke Fukuhara

  Journal of virological methods 326 114894 - 114894 2024/02/13 

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

  Tomokazu Tamura, Takashi Irie, Sayaka Deguchi, Hisano Yajima, Masumi Tsuda, Hesham Nasser, Keita Mizuma, Arnon Plianchaisuk, Saori Suzuki, Keiya Uriu, Mst Monira Begum, Ryo Shimizu, Michael Jonathan, Rigel Suzuki, Takashi Kondo, Hayato Ito, Akifumi Kamiyama, Kumiko Yoshimatsu, Maya Shofa, Rina Hashimoto, Yuki Anraku, Kanako Terakado Kimura, Shunsuke Kita, Jiei Sasaki, Kaori Sasaki-Tabata, Katsumi Maenaka, Naganori Nao, Lei Wang, Yoshitaka Oda, Terumasa Ikeda, Akatsuki Saito, Keita Matsuno, Jumpei Ito, Shinya Tanaka, Kei Sato, Takao Hashiguchi, Kazuo Takayama, Takasuke Fukuhara

  Nature communications 15 (1) 1176 - 1176 2024/02/08 

  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.
• Neutralizing antibody responses and cellular responses against SARS-CoV-2 Omicron subvariant BA.5 after mRNA SARS-CoV-2 vaccination in kidney transplant recipients

  Keita Kawashiro, Rigel Suzuki, Takuto Nogimori, Naoya Iwahara, Takayuki Hirose, Kazufumi Okada, Takuya Yamamoto, Takasuke Fukuhara, Kiyohiko Hotta, Nobuo Shinohara

  2024/01/19 

  Abstract 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 17 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. Twenty recipients and seven healthy controls also received a bivalent omicron-containing booster vaccine, leading to increased IgG and neutralizing antibody titers in both groups. However, the increase was significantly lower in recipients. Recipients did not gain sufficient immunity with a third dose of vaccine, indicating a need to explore methods other than vaccines.
• Rational in silico design identifies two mutations that restore UT28K SARS-CoV-2 monoclonal antibody activity against Omicron BA.1.

  Tatsuhiko Ozawa, Yoshiki Ikeda, Liuan Chen, Rigel Suzuki, Atsushi Hoshino, Akira Noguchi, Shunsuke Kita, Yuki Anraku, Emiko Igarashi, Yumiko Saga, Noriko Inasaki, Shunta Taminishi, Jiei Sasaki, Yuhei Kirita, Hideo Fukuhara, Katsumi Maenaka, Takao Hashiguchi, Takasuke Fukuhara, Kenichi Hirabayashi, Hideki Tani, Hiroyuki Kishi, Hideki Niimi

  Structure (London, England : 1993) 2024/01/05 

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

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

  Microbiology and Immunology 0385-5600 2024 

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

  Kaoru Murakami, Shimpei I Kubota, Kumiko Tanaka, Hiroki Tanaka, Keiichiroh Akabane, Rigel Suzuki, Yuta Shinohara, Hiroyasu Takei, Shigeru Hashimoto, Yuki Tanaka, Shintaro Hojyo, Osamu Sakamoto, Norihiko Naono, Takayui Takaai, Kazuki Sato, Yuichi Kojima, Toshiyuki Harada, Takeshi Hattori, Satoshi Fuke, Isao Yokota, Satoshi Konno, Takashi Washio, Takasuke Fukuhara, Takanori Teshima, Masateru Taniguchi, Masaaki Murakami

  Lab on a chip 23 (22) 4909 - 4918 2023/11/07 

  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.
• Determination of the factors responsible for the tropism of SARS-CoV-2-related bat coronaviruses to Rhinolophus bat ACE2

  Shigeru Fujita, Yusuke Kosugi, Izumi Kimura, Kenzo Tokunaga, Jumpei Ito, Kei Sato, Keita Matsuno, Naganori Nao, Hirofumi Sawa, Shinya Tanaka, Masumi Tsuda, Lei Wang, Yoshikata Oda, Zannatul Ferdous, Kenji Shishido, Takasuke Fukuhara, Tomokazu Tamura, Rigel Suzuki, Saori Suzuki, Hayato Ito, Yu Kaku, Naoko Misawa, Arnon Plianchaisuk, Ziyi Guo, Alfredo A. Hinay, Keiya Uriu, Jarel Elgin M. Tolentino, Luo Chen, Lin Pan, Mai Suganami, Mika Chiba, Ryo Yoshimura, Kyoko Yasuda, Keiko Iida, Naomi Ohsumi, Adam P. Strange, Shiho Tanaka, Kazuhisa Yoshimura, Kenji Sadamasu, Mami Nagashima, Hiroyuki Asakura, Isao Yoshida, So Nakagawa, Akifumi Takaori-Kondo, Kayoko Nagata, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Kazuo Takayama, Rina Hashimoto, Sayaka Deguchi, Yukio Watanabe, Ayaka Sakamoto, Naoko Yasuhara, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Hisano Yajima, Takashi Irie, Ryoko Kawabata, Kaori Tabata, Terumasa Ikeda, Hesham Nasser, Ryo Shimizu, M. S. T. Monira Begum, Michael Jonathan, Yuka Mugita, Otowa Takahashi, Kimiko Ichihara, Chihiro Motozono, Takamasa Ueno, Mako Toyoda, Akatsuki Saito, Maya Shofa, Yuki Shibatani, Tomoko Nishiuchi, Kotaro Shirakawa

  Journal of Virology 97 (10) 0022-538X 2023/10/31 

  ABSTRACT Differences in host angiotensin converting enzyme 2 (ACE2) genes may affect the host range of SARS-CoV-2-related coronaviruses (SC2r-CoVs) and further determine the tropism of host ACE2 for the infection receptor. However, the factor(s) responsible for determining the host tropism of SC2r-CoVs, which may in part be determined by the tropism of host ACE2 usage, remains unclear. Here, we use the pseudoviruses with the spike proteins of two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and the cells expressing ACE2 proteins of eight different Rhinolophus bat species to show that these two spikes have different tropisms for Rhinolophus bat ACE2. Through structural analysis and cell culture experiments, we demonstrate that this tropism is determined by residue 493 of the spike and residues 31 and 35 of ACE2. Our results suggest that SC2r-CoVs exhibit differential ACE2 tropism, which may be driven by adaptation to different Rhinolophus bat ACE2 proteins. IMPORTANCE The efficiency of infection receptor use is the first step in determining the species tropism of viruses. After the coronavirus disease 2019 pandemic, a number of SARS-CoV-2-related coronaviruses (SC2r-CoVs) were identified in Rhinolophus bats, and some of them can use human angiotensin converting enzyme 2 (ACE2) for the infection receptor without acquiring additional mutations. This means that the potential of certain SC2r-CoVs to cause spillover from bats to humans is "off-the-shelf." However, both SC2r-CoVs and Rhinolophus bat species are highly diversified, and the host tropism of SC2r-CoVs remains unclear. Here, we focus on two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and determine how the tropism of SC2r-CoVs to Rhinolophus bat ACE2 is determined at the amino acid resolution level.
• Akaluc bioluminescence offers superior sensitivity to trackin vivodynamics of SARS-CoV-2 infection

  Tomokazu Tamura, Hayato Ito, Shiho Torii, Lei Wang, Rigel Suzuki, Shuhei Tusjino, Akifumi Kamiyama, Yoshitaka Oda, Yuhei Morioka, Saori Suzuki, Kotaro Shirakawa, Kei Sato, Kumiko Yoshimatsu, Yoshiharu Matsuura, Satoshi Iwano, Shinya Tanaka, Takasuke Fukuhara

  2023/10/13 

  Summary Monitoringin vivoviral dynamics can improve our understanding of pathogenicity and tissue tropism. For positive-sense, single-stranded RNA viruses, several studies have attempted to monitor viral kineticsin vivousing reporter genomes. The application of such recombinant viruses can be limited by challenges in accommodating bioluminescent reporter genes in the viral genome. Conventional luminescence also exhibits relatively low tissue permeability and thus less sensitivity for visualizationin vivo. Here we show that unlike NanoLuc bioluminescence, the improved method, termed AkaBLI, allows visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Syrian hamsters. By successfully incorporating a codon-optimized Akaluc luciferase gene into the SARS-CoV-2 genome, we visualizedin vivoinfection, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of neutralizing antibodies and mRNA vaccination by monitoring changes in Akaluc signals. Overall, AkaBLI is an effective technology for monitoring viral dynamics in live animals.
• Prolonged shedding of viable SARS‐CoV‐2 in immunocompromised patients with haematological malignancies: A prospective study

  Takaya Ichikawa, Tomokazu Tamura, Mutsumi Takahata, Takashi Ishio, Makoto Ibata, Ikumi Kasahara, Koichiro Minauchi, Satoshi Yamamoto, Takanori Teshima, Takasuke Fukuhara

  British Journal of Haematology 204 (3) 815 - 820 0007-1048 2023/10/05 

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

  Izumi Kimura, Daichi Yamasoba, Hesham Nasser, Hayato Ito, Jiri Zahradnik, Jiaqi Wu, Shigeru Fujita, Keiya Uriu, Jiei Sasaki, Tomokazu Tamura, Rigel Suzuki, Sayaka Deguchi, Arnon Plianchaisuk, Kumiko Yoshimatsu, Yasuhiro Kazuma, Shuya Mitoma, Gideon Schreiber, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Akifumi Takaori-Kondo, Jumpei Ito, Kotaro Shirakawa, Kazuo Takayama, Takashi Irie, Takao Hashiguchi, So Nakagawa, Takasuke Fukuhara, Akatsuki Saito, Terumasa Ikeda, Kei Sato

  Journal of virology e0101123  2023/10/05 [Refereed]
   

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

  Miyuki Kimura, Risa Matsuoka, Satoshi Taniguchi, Junki Maruyama, Slobodan Paessler, Saori Oka, Atsushi Yamashita, Takasuke Fukuhara, Yoshiharu Matsuura, Hideki Tani

  Virology 587 109867 - 109867 2023/08/10 

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

  Tomokazu Tamura, Daichi Yamasoba, Yoshitaka Oda, Jumpei Ito, Tomoko Kamasaki, Naganori Nao, Rina Hashimoto, Yoichiro Fujioka, Rigel Suzuki, Lei Wang, Hayato Ito, Yukie Kashima, Izumi Kimura, Mai Kishimoto, Masumi Tsuda, Hirofumi Sawa, Kumiko Yoshimatsu, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Yutaka Suzuki, Yusuke Ohba, Isao Yokota, Keita Matsuno, Kazuo Takayama, Shinya Tanaka, Kei Sato, Takasuke Fukuhara

  Communications biology 6 (1) 772 - 772 2023/07/24 

  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.
• Impact of imprinted immunity induced by mRNA vaccination in an experimental animal model.

  Shigeru Fujita, Keiya Uriu, Lin Pan, Naganori Nao, Koshiro Tabata, Mai Kishimoto, Yukari Itakura, Hirofumi Sawa, Izumi Kida, Tomokazu Tamura, Takasuke Fukuhara, Jumpei Ito, Keita Matsuno, Kei Sato

  The Journal of infectious diseases 2023/06/27 

  The emergence of 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. Here, we investigated this possibility using hamsters. While 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-LNP vaccine resulted in only faintly induced humoral immunity, supporting the possibility of imprinted immunity.
• saRNA vaccine expressing membrane-anchored RBD elicits broad and durable immunity against SARS-CoV-2 variants of concern.

  Mai Komori, Takuto Nogimori, Amber L Morey, Takashi Sekida, Keiko Ishimoto, Matthew R Hassett, Yuji Masuta, Hirotaka Ode, Tomokazu Tamura, Rigel Suzuki, Jeff Alexander, Yasutoshi Kido, Kenta Matsuda, Takasuke Fukuhara, Yasumasa Iwatani, Takuya Yamamoto, Jonathan F Smith, Wataru Akahata

  Nature communications 14 (1) 2810 - 2810 2023/05/19 

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

  Tomokazu Tamura, Jumpei Ito, Keiya Uriu, Jiri Zahradnik, Izumi Kida, Yuki Anraku, Hesham Nasser, Maya Shofa, Yoshitaka Oda, Spyros Lytras, Naganori Nao, Yukari Itakura, Sayaka Deguchi, Rigel Suzuki, Lei Wang, Mst Monira Begum, Shunsuke Kita, Hisano Yajima, Jiei Sasaki, Kaori Sasaki-Tabata, Ryo Shimizu, Masumi Tsuda, Yusuke Kosugi, Shigeru Fujita, Lin Pan, Daniel Sauter, Kumiko Yoshimatsu, Saori Suzuki, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Yuki Yamamoto, Tetsuharu Nagamoto, Gideon Schreiber, Katsumi Maenaka, Takao Hashiguchi, Terumasa Ikeda, Takasuke Fukuhara, Akatsuki Saito, Shinya Tanaka, Keita Matsuno, Kazuo Takayama, Kei Sato

  Nature communications 14 (1) 2800 - 2800 2023/05/16 

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

  Jumpei Ito, Rigel Suzuki, Keiya Uriu, Yukari Itakura, Jiri Zahradnik, Kanako Terakado Kimura, Sayaka Deguchi, Lei Wang, Spyros Lytras, Tomokazu Tamura, Izumi Kida, Hesham Nasser, Maya Shofa, Mst Monira Begum, Masumi Tsuda, Yoshitaka Oda, Tateki Suzuki, Jiei Sasaki, Kaori Sasaki-Tabata, Shigeru Fujita, Kumiko Yoshimatsu, Hayato Ito, Naganori Nao, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Yuki Yamamoto, Tetsuharu Nagamoto, Jin Kuramochi, Gideon Schreiber, Akatsuki Saito, Keita Matsuno, Kazuo Takayama, Takao Hashiguchi, Shinya Tanaka, Takasuke Fukuhara, Terumasa Ikeda, Kei Sato

  Nature communications 14 (1) 2671 - 2671 2023/05/11 

  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.
• Evaluation of broad anti-coronavirus activity of autophagy-related compounds using human airway organoids.

  Rina Hashimoto, Tomokazu Tamura, Yukio Watanabe, Ayaka Sakamoto, Naoko Yasuhara, Hayato Ito, Masahiro Nakano, Hiromitsu Fuse, Akira Ohta, Takeshi Noda, Yasufumi Matsumura, Miki Nagao, Takuya Yamamoto, Takasuke Fukuhara, Kazuo Takayama

  Molecular pharmaceutics 20 (4) 2276 - 2287 2023/04/03 

  To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 μM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.
• Increased flexibility of the SARS-CoV-2 RNA-binding site causes resistance to remdesivir

  Shiho Torii, Kwang Su Kim, Jun Koseki, Rigel Suzuki, Shoya Iwanami, Yasuhisa Fujita, Yong Dam Jeong, Jumpei Ito, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Kei Sato, Yoshiharu Matsuura, Teppei Shimamura, Shingo Iwami, Takasuke Fukuhara

  PLOS Pathogens 19 (3) e1011231 - e1011231 2023/03/27 

  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.
• Antiviral activity of micafungin and its derivatives against SARS-CoV-2 RNA replication.

  Shogo Nakajima, Hirofumi Ohashi, Daisuke Akazawa, Shiho Torii, Rigel Suzuki, Takasuke Fukuhara, Koichi Watashi

  Viruses 15 (2) 2023/02/06 

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

  Takahiro Tomiyama, Rigel Suzuki, Noboru Harada, Tomokazu Tamura, Katsuya Toshida, Yukiko- Kosai-Fujimoto, Takahiro Tomino, Shohei Yoshiya, Yoshihiro Nagao, Kazuki Takeishi, Shinji Itoh, Nobuhiro Kobayashi, Hayato Ito, Sachiyo Yoshio, Tatsuya Kanto, Tomoharu Yoshizumi, Takasuke Fukuhara

  Frontiers in cellular and infection microbiology 13 1197349 - 1197349 2023 

  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.
• Monitoring fusion kinetics of viral and target cell membranes in living cells using a SARS-CoV-2 spike-protein-mediated membrane fusion assay

  Hesham Nasser, Ryo Shimizu, Jumpei Ito, Akatsuki Saito, Kei Sato, Terumasa Ikeda, Keita Matsuno, Naganori Nao, Hirofumi Sawa, Mai Kishimoto, Shinya Tanaka, Masumi Tsuda, Lei Wang, Yoshikata Oda, Marie Kato, Zannatul Ferdous, Hiromi Mouri, Kenji Shishido, Takasuke Fukuhara, Tomokazu Tamura, Rigel Suzuki, Hayato Ito, Daichi Yamasoba, Izumi Kimura, Naoko Misawa, Keiya Uriu, Yusuke Kosugi, Shigeru Fujita, Mai Suganami, Mika Chiba, Ryo Yoshimura, So Nakagawa, Jiaqi Wu, Akifumi Takaori-Kondo, Kotaro Shirakawa, Kayoko Nagata, Yasuhiro Kazuma, Ryosuke Nomura, Yoshihito Horisawa, Yusuke Tashiro, Yugo Kawai, Takashi Irie, Ryoko Kawabata, MST Monira Begum, Otowa Takahashi, Kimiko Ichihara, Takamasa Ueno, Chihiro Motozono, Mako Toyoda, Yuri L. Tanaka, Erika P. Butlertanaka, Maya Shofa, Kazuo Takayama, Rina Hashimoto, Sayaka Deguchi, Takao Hashiguchi, Tateki Suzuki, Kanako Kimura, Jiei Sasaki, Yukari Nakajima, Kaori Tabata

  STAR Protocols 3 (4) 101773 - 101773 2666-1667 2022/12
• Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing anchored RBD: a randomised, observer-blind, phase 1 study

  Wataru Akahata, Takashi Sekida, Takuto Nogimori, Hirotaka Ode, Tomokazu Tamura, Kaoru Kono, Yoko Kazami, Ayaka Washizaki, Yuji Masuta, Rigel Suzuki, Kenta Matsuda, Mai Komori, Amber Morey, Keiko Ishimoto, Misako Nakata, Tomoko Hasunuma, Takasuke Fukuhara, Yasumasa Iwatani, Takuya Yamamoto, Jonathan F Smith, Nobuaki Sato

  2022/11/22 

  Summary BACKGROUND VLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying RNA (saRNA) vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the SARS-CoV-2 spike protein. METHODS A phase 1 study of VLPCOV-01 was conducted at Medical Corporation Heishinkai OPHAC Hospital, Japan. Participants aged 18 to 55 or ≥65 years who had completed two doses of the BNT162b2 mRNA vaccine 6 to 12 months previously were randomised to receive one intramuscular vaccination of 0·3, 1·0, or 3·0 μg VLPCOV-01, 30 μg BNT162b2, or placebo between February 16, 2022, and March 17, 2022. Solicited adverse events were collected up to 6 days post-administration. Interim immunogenicity analyses included SARS-CoV-2 IgG and neutralising antibody titres. Follow-up for safety and immunogenicity evaluation is ongoing. (The trial is registered: jRCT2051210164). FINDINGS 92 healthy adults were enrolled, with 60 participants receiving VLPCOV-01. No serious adverse events were reported up to 26 weeks, and no prespecified trial-halting events were met. VLPCOV-01 induced robust IgG titres against SARS-CoV-2 RBD protein that were maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5037 (95% CI 1272–19,940) at 0·3 μg to 12,873 (95% CI 937–17,686) at 3 μg, in comparison to 3166 (95% CI 1619–6191) with 30 μg BNT162b2. Among elderly participants, IgG titres at 26 weeks post-vaccination with 3 μg VLPCOV-01 were 9865 (95% CI 4396–22138) compared to 4183 (95% CI 1436–12180) following vaccination with 30 μg BNT162b2. Pseudovirus neutralising antibody responses were observed against multiple SARS-CoV-2 variants and strongly correlated with anti-SARS-CoV-2 IgG (r=0·950, p<0·001). INTERPRETATION VLPCOV-01 is immunogenic following low dose administration, with anti-SARS-CoV-2 immune responses comparable to BNT162b2. These findings support further development of VLPCOV-01 as a COVID-19 booster vaccine and the potential for saRNA vectors as a vaccine platform. FUNDING Supported by AMED, Grant No. JP21nf0101627.
• Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant.

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

  Cell host & microbe 30 (11) 1540 - 1555 2022/11/09 

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

  Kaoru Murakami, Sumio Iwasaki, Satoshi Oguri, Kumiko Tanaka, Rigel Suzuki, Kasumi Hayasaka, Shinichi Fujisawa, Chiaki Watanabe, Satoshi Konno, Isao Yokota, Takasuke Fukuhara, Masaaki Murakami, Takanori Teshima

  Journal of clinical virology plus 2 (4) 100109 - 100109 2022/11 

  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.
• Smoking Enhances the Expression of Angiotensin-Converting Enzyme 2 Involved in the Efficiency of Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

  Rigel Suzuki, Yuki Ono, Koji Noshita, Kwang Su Kim, Hayato Ito, Yuhei Morioka, Tomokazu Tamura, Daisuke Okuzaki, Tetsuzo Tagawa, Tomoyoshi Takenaka, Tomoharu Yoshizumi, Teppei Shimamura, Shingo Iwami, Takasuke Fukuhara

  Microbiology and immunology 67 (1) 22 - 31 2022/10/18 

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

  Izumi Kimura, Daichi Yamasoba, Tomokazu Tamura, Naganori Nao, Tateki Suzuki, Yoshitaka Oda, Shuya Mitoma, Jumpei Ito, Hesham Nasser, Jiri Zahradnik, Keiya Uriu, Shigeru Fujita, Yusuke Kosugi, Lei Wang, Masumi Tsuda, Mai Kishimoto, Hayato Ito, Rigel Suzuki, Ryo Shimizu, M.S.T. Monira Begum, Kumiko Yoshimatsu, Kanako Terakado Kimura, Jiei Sasaki, Kaori Sasaki-Tabata, Yuki Yamamoto, Tetsuharu Nagamoto, Jun Kanamune, Kouji Kobiyama, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Kotaro Shirakawa, Akifumi Takaori-Kondo, Jin Kuramochi, Gideon Schreiber, Ken J. Ishii, Takao Hashiguchi, Terumasa Ikeda, Akatsuki Saito, Takasuke Fukuhara, Shinya Tanaka, Keita Matsuno, Kei Sato

  Cell 0092-8674 2022/09
• Impact of JMJD6 on intrahepatic cholangiocarcinoma.

  Yukiko Kosai-Fujimoto, Shinji Itoh, Kyohei Yugawa, Takasuke Fukuhara, Daisuke Okuzaki, Takeo Toshima, Noboru Harada, Yoshinao Oda, Tomoharu Yoshizumi, Masaki Mori

  Molecular and clinical oncology 17 (2) 131 - 131 2022/08 

  The association of Jumonji domain-containing 6 (JMJD6) with the prognosis of various types of cancer has been demonstrated, except in intrahepatic cholangiocarcinoma (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.
• Hepatitis C virus NS5A protein promotes the lysosomal degradation of diacylglycerol O-acyltransferase 1 (DGAT1) via endosomal microautophagy

  Putu Yuliandari, Chieko Matsui, Lin Deng, Takayuki Abe, Hiroyuki Mori, Shuhei Taguwa, Chikako Ono, Takasuke Fukuhara, Yoshiharu Matsuura, Ikuo Shoji

  Autophagy Reports 1 (1) 264 - 285 2022/07/22
• Antiviral activity of ciclesonide acetal derivatives blocking SARS-CoV-2 RNA replication

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

  Journal of Pharmacological Sciences 149 (3) 81 - 84 1347-8613 2022/07 

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

  Tomokazu Tamura, Shiho Torii, Kentaro Kajiwara, Itsuki Anzai, Yoichiro Fujioka, Kisho Noda, Shuhei Taguwa, Yuhei Morioka, Rigel Suzuki, Yuzy Fauzyah, Chikako Ono, Yusuke Ohba, Masato Okada, Takasuke Fukuhara, Yoshiharu Matsuura

  PLoS pathogens 18 (6) e1010593  2022/06/03 

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

  Daichi Yamasoba, Izumi Kimura, Hesham Nasser, Yuhei Morioka, Naganori Nao, Jumpei Ito, Keiya Uriu, Masumi Tsuda, Jiri Zahradnik, Kotaro Shirakawa, Rigel Suzuki, Mai Kishimoto, Yusuke Kosugi, Kouji Kobiyama, Teppei Hara, Mako Toyoda, Yuri L Tanaka, Erika P Butlertanaka, Ryo Shimizu, Hayato Ito, Lei Wang, Yoshitaka Oda, Yasuko Orba, Michihito Sasaki, Kayoko Nagata, Kumiko Yoshimatsu, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Jin Kuramochi, Motoaki Seki, Ryoji Fujiki, Atsushi Kaneda, Tadanaga Shimada, Taka-Aki Nakada, Seiichiro Sakao, Takuji Suzuki, Takamasa Ueno, Akifumi Takaori-Kondo, Ken J Ishii, Gideon Schreiber, Hirofumi Sawa, Akatsuki Saito, Takashi Irie, Shinya Tanaka, Keita Matsuno, Takasuke Fukuhara, Terumasa Ikeda, Kei Sato

  Cell 185 (12) 2103 - 2115 2022/05/02 

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

  Rigel Suzuki, Daichi Yamasoba, Izumi Kimura, Lei Wang, Mai Kishimoto, Jumpei Ito, Yuhei Morioka, Naganori Nao, Hesham Nasser, Keiya Uriu, Yusuke Kosugi, Masumi Tsuda, Yasuko Orba, Michihito Sasaki, Ryo Shimizu, Ryoko Kawabata, Kumiko Yoshimatsu, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Hirofumi Sawa, Terumasa Ikeda, Takashi Irie, Keita Matsuno, Shinya Tanaka, Takasuke Fukuhara, Kei Sato

  Nature 603 (7902) 700 - 705 2022/03 

  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.
• Characterization of various remdesivir-resistant mutations of SARS-CoV-2 by mathematical modeling and molecular dynamics simulation

  Shiho Torii, Kwang Su Kim, Jun Koseki, Rigel Suzuki, Shoya Iwanami, Yasuhisa Fujita, Yong Dam Jeong, Yoshiharu Matsuura, Teppei Shimamura, Shingo Iwami, Takasuke Fukuhara

  2022/02/24
• Enhanced fusogenicity and pathogenicity of SARS-CoV-2 Delta P681R mutation.

  Akatsuki Saito, Takashi Irie, Rigel Suzuki, Tadashi Maemura, Hesham Nasser, Keiya Uriu, Yusuke Kosugi, Kotaro Shirakawa, Kenji Sadamasu, Izumi Kimura, Jumpei Ito, Jiaqi Wu, Kiyoko Iwatsuki-Horimoto, Mutsumi Ito, Seiya Yamayoshi, Samantha Loeber, Masumi Tsuda, Lei Wang, Seiya Ozono, Erika P Butlertanaka, Yuri L Tanaka, Ryo Shimizu, Kenta Shimizu, Kumiko Yoshimatsu, Ryoko Kawabata, Takemasa Sakaguchi, Kenzo Tokunaga, Isao Yoshida, Hiroyuki Asakura, Mami Nagashima, Yasuhiro Kazuma, Ryosuke Nomura, Yoshihito Horisawa, Kazuhisa Yoshimura, Akifumi Takaori-Kondo, Masaki Imai, Shinya Tanaka, So Nakagawa, Terumasa Ikeda, Takasuke Fukuhara, Yoshihiro Kawaoka, Kei Sato

  Nature 602 (7896) 300 - 306 2022/02 

  During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.
• DsRNA Sequencing for RNA Virus Surveillance Using Human Clinical Samples.

  Takuma Izumi, Yuhei Morioka, Syun-Ichi Urayama, Daisuke Motooka, Tomokazu Tamura, Takahiro Kawagishi, Yuta Kanai, Takeshi Kobayashi, Chikako Ono, Akinari Morinaga, Takahiro Tomiyama, Norifumi Iseda, Yukiko Kosai, Shoichi Inokuchi, Shota Nakamura, Tomohisa Tanaka, Kohji Moriishi, Hiroaki Kariwa, Tomoharu Yoshizumi, Masaki Mori, Yoshiharu Matsuura, Takasuke Fukuhara

  Viruses 13 (7) 2021/07/06 

  Although viruses infect various organs and are associated with diseases, there may be many unidentified pathogenic viruses. The recent development of next-generation sequencing technologies has facilitated the establishment of an environmental viral metagenomic approach targeting the intracellular viral genome. However, an efficient method for the detection of a viral genome derived from an RNA virus in animal or human samples has not been established. Here, we established a method for the efficient detection of RNA viruses in human clinical samples. We then tested the efficiency of the method compared to other conventional methods by using tissue samples collected from 57 recipients of living donor liver transplantations performed between June 2017 and February 2019 at Kyushu University Hospital. The viral read ratio in human clinical samples was higher by the new method than by the other conventional methods. In addition, the new method correctly identified viral RNA from liver tissues infected with hepatitis C virus. This new technique will be an effective tool for intracellular RNA virus surveillance in human clinical samples and may be useful for the detection of new RNA viruses associated with diseases.
• SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity.

  Chihiro Motozono, Mako Toyoda, Jiri Zahradnik, Akatsuki Saito, Hesham Nasser, Toong Seng Tan, Isaac Ngare, Izumi Kimura, Keiya Uriu, Yusuke Kosugi, Yuan Yue, Ryo Shimizu, Jumpei Ito, Shiho Torii, Akiko Yonekawa, Nobuyuki Shimono, Yoji Nagasaki, Rumi Minami, Takashi Toya, Noritaka Sekiya, Takasuke Fukuhara, Yoshiharu Matsuura, Gideon Schreiber, Terumasa Ikeda, So Nakagawa, Takamasa Ueno, Kei Sato

  Cell host & microbe 29 (7) 1124 - 1136 2021/06/15 

  Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.
• Phenotypic Characterization by Single-Cell Mass Cytometry of Human Intrahepatic and Peripheral NK Cells in Patients with Hepatocellular Carcinoma.

  Yuichi Yoshida, Sachiyo Yoshio, Taiji Yamazoe, Taizo Mori, Yuriko Tsustui, Hironari Kawai, Shiori Yoshikawa, Takasuke Fukuhara, Toru Okamoto, Yoshihiro Ono, Yu Takahashi, Ryuki Hashida, Takumi Kawaguchi, Akinobu Taketomi, Tatsuya Kanto

  Cells 10 (6) 2021/06/14 

  Overall response rates of systemic therapies against advanced hepatocellular carcinoma (HCC) remain unsatisfactory. Thus, searching for new immunotherapy targets is indispensable. NK cells are crucial effectors and regulators in the tumor microenvironment and a determinant of responsiveness to checkpoint inhibitors. We revealed the landscape of NK cell phenotypes in HCC patients to find potential immunotherapy targets. Using single cell mass cytometry, we analyzed 32 surface markers on CD56dim and CD56bright NK cells, which included Sialic acid-binding immunoglobulin-type lectins (Siglecs). We compared peripheral NK cells between HCC patients and healthy volunteers. We also compared NK cells, in terms of their localizations, on an individual patient bases between peripheral and intrahepatic NK cells from cancerous and noncancerous liver tissues. In the HCC patient periphery, CD160+CD56dim NK cells that expressed Siglec-7, NKp46, and NKp30 were reduced, while CD49a+CD56dim NK cells that expressed Siglec-10 were increased. CD160 and CD49a on CD56dim NK cells were significantly correlated to other NK-related markers in HCC patients, which suggested that CD160 and CD49a were signature molecules. CD49a+ CX3CR1+ Siglec-10+ NK cells had accumulated in HCC tissues. Considering further functional analyses, CD160, CD49a, CX3CR1, and Siglec-10 on CD56dim NK cells may be targets for immunotherapies of HCC patients.
• M Segment-Based Minigenome System of Severe Fever with Thrombocytopenia Syndrome Virus as a Tool for Antiviral Drug Screening.

  Hiroshi Yamada, Satoshi Taniguchi, Masayuki Shimojima, Long Tan, Miyuki Kimura, Yoshitomo Morinaga, Takasuke Fukuhara, Yoshiharu Matsuura, Takashi Komeno, Yousuke Furuta, Masayuki Saijo, Hideki Tani

  Viruses 13 (6) 2021/06/03 

  Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne bunyavirus that causes severe disease in humans with case fatality rates of approximately 30%. There are few treatment options for SFTSV infection. SFTSV RNA synthesis is conducted using a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are, therefore, potential antiviral targets. A library of small molecule compounds was processed using a high-throughput screening (HTS) based on an SFTSV minigenome assay (MGA) in a 96-well microplate format to identify potential lead inhibitors of SFTSV RNA synthesis. The assay confirmed inhibitory activities of previously reported SFTSV inhibitors, favipiravir and ribavirin. A small-scale screening using MGA identified four candidate inhibitors that inhibited SFTSV minigenome activity by more than 80% while exhibiting less than 20% cell cytotoxicity with selectivity index (SI) values of more than 100. These included mycophenolate mofetil, methotrexate, clofarabine, and bleomycin. Overall, these data demonstrate that the SFTSV MGA is useful for anti-SFTSV drug development research.
• Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction.

  Shiho Torii, Chikako Ono, Rigel Suzuki, Yuhei Morioka, Itsuki Anzai, Yuzy Fauzyah, Yusuke Maeda, Wataru Kamitani, Takasuke Fukuhara, Yoshiharu Matsuura

  Cell reports 35 (3) 109014 - 109014 2021/04/20 

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.
• Establishment of a Cell Culture Model Permissive for Infection by Hepatitis B and C Viruses.

  Teruhime Otoguro, Tomohisa Tanaka, Hirotake Kasai, Nobuhiro Kobayashi, Atsuya Yamashita, Takasuke Fukuhara, Akihide Ryo, Moto Fukai, Akinobu Taketomi, Yoshiharu Matsuura, Kohji Moriishi

  Hepatology communications 5 (4) 634 - 649 2021/04 

  Compared with each monoinfection, coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is well known to increase the risks of developing liver cirrhosis and hepatocellular carcinoma. However, the mechanism by which HBV/HCV coinfection is established in hepatocytes is not well understood. Common cell culture models for coinfection are required to examine viral propagation. In this study, we aimed to establish a cell line permissive for both HBV and HCV infection. We first prepared a HepG2 cell line expressing sodium taurocholate cotransporting polypeptide, an HBV receptor, and then selected a cell line highly permissive for HBV infection, G2/NT18-B. After transduction with a lentivirus-encoding microRNA-122, the cell line harboring the highest level of replicon RNA was selected and then treated with anti-HCV compounds to eliminate the replicon RNA. The resulting cured cell line was transduced with a plasmid-encoding CD81. The cell line permissive for HCV infection was cloned and then designated the G2BC-C2 cell line, which exhibited permissiveness for HBV and HCV propagation. JAK inhibitor I potentiated the HCV superinfection of HBV-infected cells, and fluorescence-activated cell-sorting analysis indicated that HBV/HCV double-positive cells accounted for approximately 30% of the coinfected cells. Among several host genes tested, cyclooxygenase-2 showed synergistic induction by coinfection compared with each monoinfection. Conclusion: These data indicate that our in vitro HBV/HCV coinfection system provides an easy-to-use platform for the study of host and viral responses against coinfection and the development of antiviral agents targeting HBV and HCV.
• Suppression of optineurin impairs the progression of hepatocellular carcinoma through regulating mitophagy.

  Shoichi Inokuchi, Tomoharu Yoshizumi, Takeo Toshima, Shinji Itoh, Kyohei Yugawa, Noboru Harada, Hiroyuki Mori, Takasuke Fukuhara, Yoshiharu Matsuura, Masaki Mori

  Cancer medicine 10 (5) 1501 - 1514 2021/03 

  Autophagy removes damaged organelles to inhibit malignant transformation during tumor initiation. Once a cancer matures, it uses the autophagic pathway as an energy source. Optineurin (OPTN) is an autophagy adaptor protein that recruits microtubule-associated protein 1 light chain 3, an autophagosome marker, to the autophagosome. Despite studies of the relation between cancer progression and autophagy adaptor proteins, there are no reports to our knowledge of a correlation between hepatocellular carcinoma (HCC) and OPTN. We aimed here to investigate the effects of OPTN expression on HCC progression through autophagy. Immunohistochemistry was used to measure the OPTN expression in the tissues of 141 Japanese patients with HCC. The effects of OPTN expression on HCC progression and mitophagy were assessed using an OPTN knockout (KO) cell line in vitro. We used this KO cell line to establish and exploit a mouse model of HCC to determine the effects of OPTN expression on tumor progression. Immunohistochemical analysis showed that patients with elevated expression of OPTN experienced shorter overall survival (OS) and recurrence-free survival (RFS). OPTN KO cells proliferated relatively slower versus wild-type (WT) cells in vitro. Western blot analysis showed that mitophagy was suppressed in OPTN KO cells, and ATP synthesis and beta-oxidation were reduced. The mouse model of HCC showed that OPTN KO cells formed smaller tumors versus WT cells less 10 weeks after implantation. Overall, the present findings suggest that OPTN is a key mediator of mitophagy that contributes to HCC progression through mitochondrial energy production.
• Ponesimod suppresses hepatitis B virus infection by inhibiting endosome maturation.

  Yuzy Fauzyah, Chikako Ono, Shiho Torii, Itsuki Anzai, Rigel Suzuki, Takuma Izumi, Yuhei Morioka, Yusuke Maeda, Toru Okamoto, Takasuke Fukuhara, Yoshiharu Matsuura

  Antiviral research 186 104999 - 104999 2021/02 

  The discovery of novel antivirals to treat hepatitis B virus (HBV) infection is urgently needed, as the currently available drugs mainly target viral proteins at replication step, whereas host factors also play significant roles in HBV infection. Although numerous studies have reported candidate drugs for HBV treatment, there remains a need to find a new drug that may target other steps of the HBV life cycle. In this study, by drug screening of a 533 G-protein-coupled receptors (GPCRs)-associated compound library, we identified ponesimod, a selective agonist of sphingosine-1-phosphate receptor 1 (S1P1), as a drug candidate for the suppression of HBV infection. However, the anti-HBV effect of ponesimod is independent of S1P1 and other sphingosine-1-phosphate receptors (S1PRs). Treatment with ponesimod at an early step of infection but not at a post-entry step significantly reduced the HBV relaxed circular DNA (rcDNA) level in a dose-dependent manner. Ponesimod treatment did not inhibit attachment, binding, or internalization of HBV particles via endocytosis through an interaction with sodium taurocholate cotransporting polypeptide (NTCP) or epidermal growth factor receptor (EGFR). Importantly, during the transportation of HBV particles to the nucleus, co-localization of HBV with early endosomes but not with late endosomes and lysosomes was induced by the treatment with ponesimod, suggesting that ponesimod interferes with the conversion of early endosomes to late endosomes without significant damage to cellular growth. Conclusion: Ponesimod is a promising anti-HBV drug targeting the endosome maturation of HBV. This finding can be applied to the development of novel antivirals that target the trafficking pathway of HBV particles.
• De novo hepatocellular carcinoma in living donor liver grafts: A Japanese multicenter experience.

  Ryoichi Goto, Yukiko Kosai-Fujimoto, Shintaro Yagi, Tsuyoshi Kobayashi, Nobuhisa Akamatsu, Tsuyoshi Shimamura, Satoru Imura, Satoshi Ogiso, Shugo Mizuno, Mitsuhisa Takatsuki, Takasuke Fukuhara, Tatsuya Kanto, Susumu Eguchi, Katsuhiko Yanaga, Yasuhiro Ogura, Takumi Fukumoto, Mitsuo Shimada, Kiyoshi Hasegawa, Hideki Ohdan, Shinji Uemoto, Yuji Soejima, Toru Ikegami, Tomoharu Yoshizumi, Akinobu Taketomi, Yoshihiko Maehara

  Hepatology research : the official journal of the Japan Society of Hepatology 50 (12) 1365 - 1374 2020/12 

  AIM: Direct-acting antivirals for hepatitis C virus have reduced the decompensation risk. Immunosuppressants for transplantation raise the risk of occurrence of de novo malignancies. We assessed the probabilities of and risk factors for de novo hepatocellular carcinoma (HCC) development post-living donor liver transplantation (LDLT). METHODS: We retrospectively evaluated the data of developed HCC in a graft including metastatic HCC post-LDLT from 2779 adult cases collected from nine major liver transplantation centers in Japan. RESULTS: Of 2779 LDLT adult recipients, 34 (1.2%) developed HCCs in their grafts. Of 34, five HCCs appeared to be de novo because of a longer period to tumor detection (9.7 [6.4-15.4] years) and no HCC within the native liver of the two recipients. The donor origin of three of five de novo HCCs was confirmed using microsatellite analysis in resected tissue. Primary disease of all five was hepatitis C virus-related cirrhosis, of which two were treated with direct-acting antivirals. Four of five developed HCC de novo in the hepatitis B core antibody-positive grafts. De novo HCCs had favorable prognosis; four of five were cured with complete remission. However, recurrent HCC (n = 29) in the graft had a poorer outcome, especially in patients with neutrophil to lymphocyte ratio scores above 4 (median survival time, 262 [19-463] days). CONCLUSION: Analysis of the database from major liver transplantation institutes in Japan revealed that de novo HCCs determined by microsatellite analysis were rarely detected, but the majority were successfully treated. LDLT recipients with higher risks of de novo HCC, including those with hepatitis B core antibody-positive grafts, should be carefully followed by surveillance of the liver graft.
• ISGylation of Hepatitis C Virus NS5A Protein Promotes Viral RNA Replication via Recruitment of Cyclophilin A.

  Takayuki Abe, Nanae Minami, Rheza Gandi Bawono, Chieko Matsui, Lin Deng, Takasuke Fukuhara, Yoshiharu Matsuura, Ikuo Shoji

  Journal of virology 94 (20) 2020/09/29 [Refereed][Not invited]
   

  Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is covalently conjugated to many substrate proteins in order to modulate their functions; this conjugation is called ISGylation. Several groups reported that the ISGylation of hepatitis C virus (HCV) NS5A protein affects HCV replication. However, the ISG15 conjugation sites on NS5A are not well determined, and it is unclear whether the role of NS5A ISGylation in HCV replication is proviral or antiviral. Here, we investigated the role of NS5A ISGylation in HCV replication by using HCV RNA replicons that encode a mutation at each lysine (Lys) residue of the NS5A protein. Immunoblot analyses revealed that 5 Lys residues (K44, K68, K166, K215, and K308) of the 14 Lys residues within NS5A (genotype 1b, Con1) have the potential to accept ISGylation. We tested the NS5A ISGylation among different HCV genotypes and observed that the NS5A proteins of all of the HCV genotypes accept ISGylation at multiple Lys residues. Using an HCV luciferase reporter replicon assay revealed that residue K308 of NS5A is important for HCV (1b, Con1) RNA replication. We observed that K308, one of the Lys residues for NS5A ISGylation, is located within the binding region of cyclophilin A (CypA), which is the critical host factor for HCV replication. We obtained evidence derived from all of the HCV genotypes suggesting that NS5A ISGylation enhances the interaction between NS5A and CypA. Taken together, these results suggest that NS5A ISGylation functions as a proviral factor and promotes HCV replication via the recruitment of CypA.IMPORTANCE Host cells have evolved host defense machinery (such as innate immunity) to eliminate viral infections. Viruses have evolved several counteracting strategies for achieving an immune escape from host defense machinery, including type I interferons (IFNs) and inflammatory cytokines. ISG15 is an IFN-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation. Here, we demonstrate that HCV NS5A protein accepts ISG15 conjugation at specific Lys residues and that the HERC5 E3 ligase specifically promotes NS5A ISGylation. We obtained evidence suggesting that NS5A ISGylation facilitates the recruitment of CypA, which is the critical host factor for HCV replication, thereby promoting HCV replication. These findings indicate that E3 ligase HERC5 is a potential therapeutic target for HCV infection. We propose that HCV hijacks an intracellular ISG15 function to escape the host defense machinery in order to establish a persistent infection.
• Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle.

  Shiho Torii, Yasuko Orba, Michihito Sasaki, Koshiro Tabata, Yuji Wada, Michael Carr, Jody Hobson-Peters, Roy A Hall, Ayato Takada, Takasuke Fukuhara, Yoshiharu Matsuura, William W Hall, Hirofumi Sawa

  The Journal of biological chemistry 295 (23) 7941 - 7957 2020/06/05 [Refereed][Not invited]
   

  Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.
• Various miRNAs compensate the role of miR-122 on HCV replication.

  Chikako Ono, Takasuke Fukuhara, Songling Li, Jian Wang, Asuka Sato, Takuma Izumi, Yuzy Fauzyah, Takuya Yamamoto, Yuhei Morioka, Nikolay V Dokholyan, Daron M Standley, Yoshiharu Matsuura

  PLoS pathogens 16 (6) e1008308  2020/06 [Refereed][Not invited]
   

  One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA. Recently, it has been reported that interaction of miR-122 to HCV RNA induces a conformational change of the 5'UTR internal ribosome entry site (IRES) structure to form stem-loop II structure (SLII) and hijack of translating 80S ribosome through the binding of SLIII to 40S subunit, which leads to efficient translation. On the other hand, low levels of HCV-RNA replication have also been detected in some non-hepatic cells; however, the details of extrahepatic replication remain unknown. These observations suggest the possibility that miRNAs other than miR-122 can support efficient replication of HCV-RNA in non-hepatic cells. Here, we identified a number of such miRNAs and show that they could be divided into two groups: those that bind HCV-RNA at two locations (miR-122 binding sites I and II), in a manner similar to miR-122 (miR-122-like), and those that target a single site that bridges sites I and II and masking both G28 and C29 in the 5'UTR (non-miR-122-like). Although the enhancing activity of these non-hepatic miRNAs were lower than those of miR-122, substantial expression was detected in various normal tissues. Furthermore, structural modeling indicated that both miR-122-like and non-miR-122-like miRNAs not only can facilitate the formation of an HCV IRES SLII but also can stabilize IRES 3D structure in order to facilitate binding of SLIII to the ribosome. Together, these results suggest that HCV facilitates miR-122-independent replication in non-hepatic cells through recruitment of miRNAs other than miR-122. And our findings can provide a more detailed mechanism of miR-122-dependent enhancement of HCV-RNA translation by focusing on IRES tertiary structure.
• Development of a High-Throughput Serum Neutralization Test Using Recombinant Pestiviruses Possessing a Small Reporter Tag.

  Madoka Tetsuo, Keita Matsuno, Tomokazu Tamura, Takasuke Fukuhara, Taksoo Kim, Masatoshi Okamatsu, Norbert Tautz, Yoshiharu Matsuura, Yoshihiro Sakoda

  Pathogens (Basel, Switzerland) 9 (3) 2020/03/04 [Refereed][Not invited]
   

  A serum neutralization test (SNT) is an essential method for the serological diagnosis of pestivirus infections, including classical swine fever, because of the cross reactivity of antibodies against pestiviruses and the non-quantitative properties of antibodies in an enzyme-linked immunosorbent assay. In conventional SNTs, an immunoperoxidase assay or observation of cytopathic effect after incubation for 3 to 7 days is needed to determine the SNT titer, which requires labor-intensive or time-consuming procedures. Therefore, a new SNT, based on the luciferase system and using classical swine fever virus, bovine viral diarrhea virus, and border disease virus possessing the 11-amino-acid subunit derived from NanoLuc luciferase was developed and evaluated; this approach enabled the rapid and easy determination of the SNT titer using a luminometer. In the new method, SNT titers can be determined tentatively at 2 days post-infection (dpi) and are comparable to those obtained by conventional SNTs at 3 or 4 dpi. In conclusion, the luciferase-based SNT can replace conventional SNTs as a high-throughput antibody test for pestivirus infections.
• Characterization of human pegivirus infection in liver transplantation recipients

  Takuma Izumi, Kazuhito Sakata, Daisuke Okuzaki, Shoichi Inokuchi, Tomokazu Tamura, Daisuke Motooka, Shota Nakamura, Chikako Ono, Masahiro Shimokawa, Yoshiharu Matsuura, Masaki Mori, Takasuke Fukuhara, Tomoharu Yoshizumi

  Journal of Medical Virology 91 (12) 2093 - 2100 0146-6615 2019/12/01 [Refereed][Not invited]
   

  © 2019 Wiley Periodicals, Inc. Approximately 2% of healthy persons are infected with human pegivirus (HPgV). HPgV is transmitted via vertical, sexual, and blood-borne routes. Recently, the association of HPgV infection with the risk of lymphoma was reported. Here, we examined the prevalence of chronic HPgV infection in liver transplantation (LT) recipients and patients with hepatectomy and the influence of HPgV infection after LT on clinical and perioperative factors. We enrolled 313 LT recipients and 187 patients with hepatectomy who received care at the Kyusyu University Hospital between May 1997 and September 2017. Of the 313 recipients and 187 patients enrolled in this study, 44 recipients (14.1%) and 2 patients (1.1%) had HPgV viremia, respectively. There was no significant association between HPgV infection and LT outcomes. Interestingly, one recipient was infected with HPgV during the peritransplant period, which was likely transmitted via blood transfusion because HPgV RNA was detected from the blood bag transfused to the recipient during LT. We reviewed the available literature on the prevalence HPgV infections in other organ-transplanted patients and whether they impacted clinical outcomes. They also had the higher prevalence of HPgV infection, while it appears to be of low or no consequences. In addition, HPgV infection induced the upregulation of interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells. LT recipients had higher HPgV viremia compared to patients with hepatectomy. Although HPgV infection was not associated with LT-related outcomes, it induced ISG expression in recipients.
• In Vivo Dynamics of Reporter Flaviviridae Viruses.

  Tomokazu Tamura, Manabu Igarashi, Bazarragchaa Enkhbold, Tatsuya Suzuki, Masatoshi Okamatsu, Chikako Ono, Hiroyuki Mori, Takuma Izumi, Asuka Sato, Yuzy Fauzyah, Toru Okamoto, Yoshihiro Sakoda, Takasuke Fukuhara, Yoshiharu Matsuura

  Journal of virology 93 (22) 0022-538X 2019/11/15 [Refereed][Not invited]
   

  Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals.IMPORTANCEIn vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.
• The evolution of surgical treatment for gastrointestinal cancers.

  Yoshihiko Maehara, Yuji Soejima, Tomoharu Yoshizumi, Naoyuki Kawahara, Eiji Oki, Hiroshi Saeki, Tomohiko Akahoshi, Toru Ikegami, Yo-Ichi Yamashita, Tadashi Furuyama, Keishi Sugimachi, Noboru Harada, Tetsuzo Tagawa, Norifumi Harimoto, Shinji Itoh, Hideto Sonoda, Koji Ando, Yuichiro Nakashima, Yoshihiro Nagao, Nami Yamashita, Yuta Kasagi, Takafumi Yukaya, Takeshi Kurihara, Ryosuke Tsutsumi, Shinkichi Takamori, Shun Sasaki, Tetsuo Ikeda, Yoshikazu Yonemitsu, Takasuke Fukuhara, Hiroyuki Kitao, Makoto Iimori, Yuki Kataoka, Takeshi Wakasa, Masami Suzuki, Koji Teraishi, Yasuto Yoshida, Masaki Mori

  International journal of clinical oncology 24 (11) 1333 - 1349 1341-9625 2019/11 [Refereed][Not invited]
   

  INTRODUCTION: According to the latest Japanese nationwide estimates, over a million Japanese people are newly diagnosed with cancer each year. Since gastrointestinal cancers account for more than 40% of all cancer-related deaths, it is imperative to formulate effective strategies to control them. MATERIALS AND METHODS, AND RESULTS: Basic drug discovery research Our research has revealed that the abnormal expression of regulators of chromosomal stability is a cause of cancers and identified an effective compound against cancers with chromosomal instability. We revealed the molecular mechanism of peritoneal dissemination of cancer cells via the CXCR4/CXCL12 axis to CAR-like cells and identified an MEK inhibitor effective against these tumors. Residual tumor cells after chemotherapy in colorectal cancer are LGR5-positive cancer stem cells and their ability to eliminate reactive oxygen species is elevated. The development of surgical procedures and devices In cases of gastric tube reconstruction for esophageal cancer, we determined the anastomotic line for evaluating the blood flow using ICG angiography and measuring the tissue O2 metabolism. We established a novel gastric reconstruction method (book-binding technique) for gastric cancer and a new rectal reconstruction method focusing on the intra-intestinal pressure resistance for rectal cancer. We established a novel tissue fusion method, which allows contact-free local heating and retains tissue viability with very little damage, and developed an understanding of the collagen-related processes that underpin laser-induced tissue fusion. Strategy to prevent carcinogenesis We succeeded in cleaving hepatitis B virus DNA integrated into the nucleus of hepatocytes using genome editing tools. The development of HCC from non-alcoholic steatohepatitis (NASH) may be prevented by metabolic surgery. CONCLUSION: We believe that these efforts will help to significantly improve the gastrointestinal cancer treatment and survival.
• Roles of secretory glycoproteins in particle formation of Flaviviridae viruses

  Takasuke Fukuhara, Yoshiharu Matsuura

  Microbiology and Immunology 63 (10) 401 - 406 0385-5600 2019/07 [Refereed][Not invited]
   

  The family Flaviviridae comprises four genera, namely, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus. These viruses have similar genome structures, but the genomes of Pestivirus and Flavivirus encode the secretory glycoproteins E and NS1, respectively. E plays an important role in virus particle formation and cell entry, whereas NS1 participates in the formation of replication complexes and virus particles. Conversely, apolipoproteins are known to participate in the formation of infectious particles of hepatitis C virus (HCV) and various secretory glycoproteins play a similar role in HCV particles formation, suggesting that there is no strong specificity for the function of secretory glycoproteins in infectious-particle formation. In addition, recent studies have shown that host-derived apolipoproteins and virus-derived E and NS1 play comparable roles in infectious-particle formation of both HCV and pestiviruses. In this review, we summarize the roles of secretory glycoproteins in the formation of Flaviviridae virus particles. rns rns rns
• USP15 Participates in Hepatitis C Virus Propagation through Regulation of Viral RNA Translation and Lipid Droplet Formation.

  Shinji Kusakabe, Tatsuya Suzuki, Yukari Sugiyama, Saori Haga, Kanako Horike, Makoto Tokunaga, Junki Hirano, He Zhang, David Virya Chen, Hanako Ishiga, Yasumasa Komoda, Chikako Ono, Takasuke Fukuhara, Masahiro Yamamoto, Masahito Ikawa, Takashi Satoh, Shizuo Akira, Tomohisa Tanaka, Kohji Moriishi, Moto Fukai, Akinobu Taketomi, Sachiyo Yoshio, Tatsuya Kanto, Tetsuro Suzuki, Toru Okamoto, Yoshiharu Matsuura

  Journal of virology 93 (6) 0022-538X 2019/03/15 [Refereed][Not invited]
   

  Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.
• Ectopic localization of autophagosome in fatty liver is a key factor for liver regeneration.

  Yoshihiro Matsumoto, Tomoharu Yoshizumi, Takeo Toshima, Kazuki Takeishi, Takasuke Fukuhara, Shinji Itoh, Toru Ikegami, Yuji Soejima, Masaki Mori

  Organogenesis 15 (1) 24 - 34 2019 [Refereed][Not invited]
   

  Autophagy has a critical role in liver regeneration. However, no studies have demonstrated autophagic flux in the regenerating fatty liver. The aim of this study was to clarify the dynamics of autophagy in the regeneration of the fatty liver. Following 70% partial hepatectomy (PH) in db/db fatty mice, which is a non-alcoholic fatty liver disease (NAFLD) model, we investigated the survival rate and recovery of liver volume. Histological examination of the regenerating liver was examined using electron microscopy. The 7-day survival rate after PH in db/db mice was 20%, which was significantly lower than that in control mice (P< .01). Liver regeneration within 48 h after PH was significantly impaired in db/db mice (P< .05). The number of proliferating cell nuclear antigen (PCNA) positive cells and the expression levels of cell-cycle markers cyclins D, E, and A were lower in db/db mice compared with controls. In the regenerating liver, LC3-II level was higher in db/db mice, but p62 expression was increased and cathepsin D expression, a marker of autophagolysosome proteolysis, was decreased compared with controls. Additionally, electronic microscopy revealed that autophagosomes during liver regeneration in db/db mice were mainly located in lipid droplets. Our findings indicate that the different localization of autophagosomes in db/db mice compared with controls led to impairment of liver regeneration in the fatty liver.
• Induction of selective autophagy in cells replicating hepatitis C virus genome.

  Hiroyuki Mori, Takasuke Fukuhara, Chikako Ono, Tomokazu Tamura, Asuka Sato, Yuzy Fauzyah, Masami Wada, Toru Okamoto, Takeshi Noda, Tamotsu Yoshimori, Yoshiharu Matsuura

  The Journal of general virology 99 (12) 1643 - 1657 0022-1317 2018/12 [Refereed][Not invited]
   

  Hepatitis C virus (HCV) infection is known to induce autophagy, but the mechanism of autophagy induced by HCV remains controversial. Here, we investigated the characteristics of autophagy induced by HCV infection. First, to examine the involvement of autophagy-related gene (ATG) proteins in HCV-induced LC3 lipidation, we established ATG5, ATG13 or ATG14 knockout (KO) Huh7.5.1 cell lines and confirmed that the accumulation of lipidated LC3 was induced in an ATG13- and ATG14-independent manner. On the other hand, HCV infectivity was not influenced by deficiencies in these genes. We also confirmed that LC3-positive dots were co-localized with ubiquitinated aggregates, and deficiency of ATG5 or ATG14 enhanced the accumulation of ubiquitinated aggregates compared to that in the restored cells, suggesting that HCV infection induces ATG5- and ATG14-dependent selective autophagy. Moreover, LC3-positive ubiquitinated aggregates accumulated near the site of the replication complex. We further examined autophagy flux in cells replicating HCV RNA using bafilomycin or E64d, and found that the increase of LC3 lipidation by treatment with bafilomycin or E64d was impaired in HCV-replicating cells, suggesting that autophagy flux is inhibited by the progress of HCV infection. Our present study suggests that (1) HCV RNA replication induces selective autophagy and (2) the progress of HCV infection impairs autophagy flux.
• CEACAM1 Is Associated With the Suppression of Natural Killer Cell Function in Patients With Chronic Hepatitis C.

  Takahiro Suda, Tomohide Tatsumi, Akira Nishio, Tadashi Kegasawa, Teppei Yoshioka, Ryoko Yamada, Kunimaro Furuta, Takahiro Kodama, Minoru Shigekawa, Hayato Hikita, Ryotaro Sakamori, Takasuke Fukuhara, Yoshiharu Matsuura, Tetsuo Takehara

  Hepatology communications 2 (10) 1247 - 1258 2018/10 [Refereed][Not invited]
   

  Natural killer cells (NK cells) play an essential role in the immunological mechanism underlying chronic hepatitis C (CHC). Impairment of NK cell function facilitates persistent infection with hepatitis C virus (HCV) and hepatocellular carcinogenesis. However, the mechanism by which NK cell activity is suppressed in CHC is not completely understood. In this study, we focused on carcinoembryonic antigen-related cell-adhesion molecule 1 (CEACAM1). CEACAM1 is thought to suppress NK cell function. We examined the effect of CEACAM1 on NK cell function in CHC. We investigated the function of CEACAM1 in vitro using Huh7.5.1 cells and the HCV-Japanese fulminant hepatitis (JFH)-1 strain. We analyzed serum CEACAM1 level, NK cell function, and CEACAM1 messenger RNA (mRNA) level in human liver samples. Levels of CEACAM1 on the cell surface, CEACAM1 mRNA levels, and soluble CEACAM1 levels in supernatants were significantly higher in Huh7.5.1 cells infected with JFH-1 (Huh7.5.1/JFH-1 cells) than in Huh7.5.1 cells. Significantly higher NK cell cytotoxicity was observed toward K562 cells after coculture with CEACAM1 knockout Huh7.5.1/JFH-1 cells than after coculture with Huh7.5.1/JFH-1 cells. CEACAM1 expression was induced by the HCV E2 glycoprotein in HCV infection. Significantly higher serum CEACAM1 levels were detected in patients with CHC compared with healthy subjects and patients who achieved sustained virological responses. The expression of CD107a on NK cells from patients with CHC was negatively correlated with serum CEACAM1 levels. Significantly higher levels of CEACAM1 mRNA were detected in HCV-infected livers compared with uninfected livers. Conclusion: CEACAM1 expression was induced in hepatocytes following HCV infection and decreased NK cell cytotoxicity. These results demonstrate a possible role for CEACAM1 in the pathogenesis of CHC and hepatocellular carcinoma progression.
• Infection with flaviviruses requires BCLXL for cell survival.

  Tatsuya Suzuki, Toru Okamoto, Hiroshi Katoh, Yukari Sugiyama, Shinji Kusakabe, Makoto Tokunaga, Junki Hirano, Yuka Miyata, Takasuke Fukuhara, Masahito Ikawa, Takashi Satoh, Sachiyo Yoshio, Ryosuke Suzuki, Masayuki Saijo, David C S Huang, Tatsuya Kanto, Shizuo Akira, Yoshiharu Matsuura

  PLoS pathogens 14 (9) e1007299  1553-7366 2018/09 [Refereed][Not invited]
   

  BCL2 family proteins including pro-survival proteins, BH3-only proteins and BAX/BAK proteins control mitochondria-mediated apoptosis to maintain cell homeostasis via the removal of damaged cells and pathogen-infected cells. In this study, we examined the roles of BCL2 proteins in the induction of apoptosis in cells upon infection with flaviviruses, such as Japanese encephalitis virus, Dengue virus and Zika virus. We showed that survival of the infected cells depends on BCLXL, a pro-survival BCL2 protein due to suppression of the expression of another pro-survival protein, MCL1. Treatment with BCLXL inhibitors, as well as deficient BCLXL gene expression, induced BAX/BAK-dependent apoptosis upon infection with flaviviruses. Flavivirus infection attenuates cellular protein synthesis, which confers reduction of short-half-life proteins like MCL1. Inhibition of BCLXL increased phagocytosis of virus-infected cells by macrophages, thereby suppressing viral dissemination and chemokine production. Furthermore, we examined the roles of BCLXL in the death of JEV-infected cells during in vivo infection. Haploinsufficiency of the BCLXL gene, as well as administration of BH3 mimetic compounds, increased survival rate after challenge of JEV infection and suppressed inflammation. These results suggest that BCLXL plays a crucial role in the survival of cells infected with flaviviruses, and that BCLXL may provide a novel antiviral target to suppress propagation of the family of Flaviviridae viruses.
• Serum Asunaprevir and Daclatasvir Concentrations and Outcomes in Patients with Recurrent Hepatitis C Who Have Undergone Living Donor Liver Transplantation.

  Noboru Harada, Tomoharu Yoshizumi, Toru Ikegami, Shinji Itoh, Norihiro Furusho, Masaki Kato, Shinji Shimoda, Takasuke Fukuhara, Yuji Soejima, Yoshihiko Maehara

  Anticancer research 38 (9) 5513 - 5520 0250-7005 2018/09 [Refereed][Not invited]
   

  BACKGROUND/AIM: This study's aim was to investigate the safety and effectiveness of asunaprevir and daclatasvir treatment for recurrent hepatitis C virus (HCV) infection in transplant recipients. The study cohort comprised 14 transplant recipients with recurrent hepatitis C who were receiving asunaprevir and daclatasvir. PATIENTS AND METHODS: Serum concentrations of asunaprevir and daclatasvir, their therapeutic effects, trough concentrations/dose ratios of tacrolimus, and adverse effects were evaluated. RESULTS: Hepatitis C virus was still undetectable in 12 (85.7%) out of 14 patients 12 weeks after completing treatment. One week after starting treatment, asunaprevir concentrations were significantly higher in patients with baseline albumin concentrations ≤3.6 g/dl than in those with baseline albumin concentrations >3.6 g/dl. No marked fluctuations were identified in tacrolimus trough concentrations/dose ratios during the 24 weeks of therapy. CONCLUSION: Full doses of asunaprevir and daclatasvir-based treatment can be safely and effectively administered to liver transplant recipients for recurrent HCV genotype 1b after living donor liver transplantation (LDLT) with little effect on blood concentrations of tacrolimus.
• Roles of the 5' untranslated region of nonprimate hepacivirus in translation initiation and viral replication

  Tomohisa Tanaka, Teruhime Otoguro, Atsuya Yamashita, Hirotake Kasai, Takasuke Fukuhara, Yoshiharu Matsuura, Kohji Moriishi

  Journal of Virology 92 (7) 1098-5514 2018/04/01 [Refereed][Not invited]
   

  The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I) three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'- proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.
• A novel occludin-targeting monoclonal antibody prevents hepatitis C virus infection in vitro

  Ken Okai, Naoki Ichikawa-Tomikawa, Akira C. Saito, Tetsuya Watabe, Kotaro Sugimoto, Daiki Fujita, Chikako Ono, Takasuke Fukuhara, Yoshiharu Matsuura, Hiromasa Ohira, Hideki Chiba

  Oncotarget 9 (24) 16588 - 16598 1949-2553 2018/03/30 [Refereed][Not invited]
   

  Since hepatitis C virus (HCV) is thought to enter into host hepatocytes using the same cellular pathways regardless of the genotypes, the host factors are promising targets to prevent and treat HCV infection. Human occludin (hOCLN) is one representative entry factor, and its second extracellular loop (EC2) contributes to the species selectivity of HCV-susceptibility. However, the exact function of hOCLN during HCV entry remains unknown, and no hOCLN-targeting antibodies or synthetic drugs that prevent and treat HCV infection have yet been developed. Here we generated the anti-hOCLN-EC2 monoclonal antibody (mAb) 67-2, and demonstrated that it efficiently inhibited HCV infection in the HCV-permissive human cell line Huh7.5.1. We also showed, using three different culture systems of Huh7.5.1 cells, that this novel mAb is accessible to OCLN from the basolateral side of hepatocytes but not from the apical side. In addition, our Western blot analyses indicated that the established 67-2 mAb reacted not only with hOCLN but also with mouse OCLN, strongly suggesting that 67-2 does not recognize the human-specific amino acids in OCLN-EC2. Moreover, we revealed that the anti-hOCLN-EC2 mAb 67-2 showed no adverse effects on cell viability or the barrier function of tight junctions.
• Characterization of recombinant Flaviviridae viruses possessing a small reporter Tag

  Tomokazu Tamura, Takasuke Fukuhara, Takuro Uchida, Chikako Ono, Hiroyuki Mori, Asuka Sato, Yuzy Fauzyah, Toru Okamoto, Takeshi Kurosu, Yin Xiang Setoh, Michio Imamura, Norbert Tautz, Yoshihiro Sakoda, Alexander A. Khromykh, Kazuaki Chayama, Yoshiharu Matsuura

  Journal of Virology 92 (2) e01582-17  1098-5514 2018/01/01 [Refereed][Not invited]
   

  The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the Flaviviridae viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics in vivo. Taken together, our findings indicate that the recombinant Flaviviridae viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against Flaviviridae viruses.
• [Hepatic tropism of hepatitis C virus infection].

  Fukuhara T

  Uirusu 68 (1) 63 - 70 0042-6857 2018 [Refereed][Not invited]
  
• Characterization of SPP inhibitors suppressing propagation of HCV and protozoa

  Junki Hirano, Toru Okamoto, Yukari Sugiyama, Tatsuya Suzuki, Shinji Kusakabe, Makoto Tokunaga, Takasuke Fukuhara, Miwa Sasai, Takahiro Tougan, Yasue Matsunaga, Kazuo Yamashita, Yusuke Sakai, Masahiro Yamamoto, Toshihiro Horii, Daron M. Standley, Kohji Moriishi, Kyoji Moriya, Kazuhiko Koike, Yoshiharu Matsuura

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (50) E10782 - E10791 0027-8424 2017/12 [Refereed][Not invited]
   

  Signal peptide peptidase (SPP) is an intramembrane aspartic protease involved in the maturation of the core protein of hepatitis C virus (HCV). The processing of HCV core protein by SPP has been reported to be critical for the propagation and pathogenesis of HCV. Here we examined the inhibitory activity of inhibitors for gamma-secretase, another intramembrane cleaving protease, against SPP, and our findings revealed that the dibenzoazepine-type structure in the gamma-secretase inhibitors is critical for the inhibition of SPP. The spatial distribution showed that the gamma-secretase inhibitor compound YO-01027 with the dibenzoazepine structure exhibits potent inhibiting activity against SPP in vitro and in vivo through the interaction of Val223 in SPP. Treatment with this SPP inhibitor suppressed the maturation of core proteins of all HCV genotypes without the emergence of drug-resistant viruses, in contrast to the treatment with direct-acting antivirals. YO-01027 also efficiently inhibited the propagation of protozoa such as Plasmodium falciparum and Toxoplasma gondii. These data suggest that SPP is an ideal target for the development of therapeutics not only against chronic hepatitis C but also against protozoiasis.
• Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation

  Masahiko Ito, Suofeng Sun, Takasuke Fukuhara, Ryosuke Suzuki, Miho Tamai, Toyohiko Yamauchi, Kenji Nakashima, Yoh-Ichi Tagawa, Shigetoshi Okazaki, Yoshiharu Matsuura, Takaji Wakita, Tetsuro Suzuki

  ONCOTARGET 8 (33) 53899 - 53915 1949-2553 2017/08 [Refereed][Not invited]
   

  Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.
• Unconjugated interferon-stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I

  Nanae Minami, Takayuki Abe, Lin Deng, Chieko Matsui, Takasuke Fukuhara, Yoshiharu Matsuura, Ikuo Shoji

  MICROBIOLOGY AND IMMUNOLOGY 61 (7) 287 - 292 0385-5600 2017/07 [Refereed][Not invited]
   

  Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti- or pro-viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation-independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C-terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein-protein interaction.
• Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9

  Takeshi Kurihara, Takasuke Fukuhara, Chikako Ono, Satomi Yamamoto, Kentaro Uemura, Toru Okamoto, Masaya Sugiyama, Daisuke Motooka, Shota Nakamura, Masato Ikawa, Masashi Mizokami, Yoshihiko Maehara, Yoshiharu Matsuura

  SCIENTIFIC REPORTS 7 (1) 6122  2045-2322 2017/07 [Refereed][Not invited]
   

  Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.
• Host-derived apolipoproteins play comparable roles with viral secretory proteins E-rns and NS1 in the infectious particle formation of Flaviviridae

  Takasuke Fukuhara, Tomokazu Tamura, Chikako Ono, Mai Shiokawa, Hiroyuki Mori, Kentaro Uemura, Satomi Yamamoto, Takeshi Kurihara, Toru Okamoto, Ryosuke Suzuki, Kentaro Yoshii, Takeshi Kurosu, Manabu Igarashi, Hiroshi Aoki, Yoshihiro Sakoda, Yoshiharu Matsuura

  PLOS PATHOGENS 13 (6) e1006475  1553-7366 2017/06 [Refereed][Not invited]
   

  Amphipathic alpha-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein E-rns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic alpha-helices of E-rns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded E-rns and NS1 in the formation of infectious particles. We examined whether E-rns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and nonhepatic 293T cells. We found that exogenous expression of either E-rns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an E-rns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic alpha-helices of E-rns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host-and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while E-rns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.
• Host-derived apolipoproteins play comparable roles with viral secretory proteins E-rns and NS1 in the infectious particle formation of Flaviviridae

  Takasuke Fukuhara, Tomokazu Tamura, Chikako Ono, Mai Shiokawa, Hiroyuki Mori, Kentaro Uemura, Satomi Yamamoto, Takeshi Kurihara, Toru Okamoto, Ryosuke Suzuki, Kentaro Yoshii, Takeshi Kurosu, Manabu Igarashi, Hiroshi Aoki, Yoshihiro Sakoda, Yoshiharu Matsuura

  PLOS PATHOGENS 13 (6) e1006475  1553-7366 2017/06 [Refereed][Not invited]
   

  Amphipathic alpha-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein E-rns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic alpha-helices of E-rns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded E-rns and NS1 in the formation of infectious particles. We examined whether E-rns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and nonhepatic 293T cells. We found that exogenous expression of either E-rns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an E-rns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic alpha-helices of E-rns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host-and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while E-rns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.
• Characterization of miR-122-independent propagation of HCV

  Chikako Ono, Takasuke Fukuhara, Daisuke Motooka, Shota Nakamura, Daisuke Okuzaki, Satomi Yamamoto, Tomokazu Tamura, Hiroyuki Mori, Asuka Sato, Kentaro Uemura, Yuzy Fauzyah, Takeshi Kurihara, Takahiro Suda, Akira Nishio, Su Su Hmwe, Toru Okamoto, Tomohide Tatsumi, Tetsuo Takehara, Kazuaki Chayama, Takaji Wakita, Kazuhiko Koike, Yoshiharu Matsuura

  PLOS PATHOGENS 13 (5) e1006374  1553-7366 2017/05 [Refereed][Not invited]
   

  miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5' UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocytederived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.
• Characterization of miR-122-independent propagation of HCV

  Chikako Ono, Takasuke Fukuhara, Daisuke Motooka, Shota Nakamura, Daisuke Okuzaki, Satomi Yamamoto, Tomokazu Tamura, Hiroyuki Mori, Asuka Sato, Kentaro Uemura, Yuzy Fauzyah, Takeshi Kurihara, Takahiro Suda, Akira Nishio, Su Su Hmwe, Toru Okamoto, Tomohide Tatsumi, Tetsuo Takehara, Kazuaki Chayama, Takaji Wakita, Kazuhiko Koike, Yoshiharu Matsuura

  PLOS PATHOGENS 13 (5) e1006374  1553-7366 2017/05 [Refereed][Not invited]
   

  miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5' UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocytederived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.
• Quasispecies of Hepatitis C Virus Participate in Cell-Specific Infectivity

  Takasuke Fukuhara, Satomi Yamamoto, Chikako Ono, Shota Nakamura, Daisuke Motooka, Hiroyuki Mori, Takeshi Kurihara, Asuka Sato, Tomokazu Tamura, Takashi Motomura, Toru Okamoto, Michio Imamura, Toru Ikegami, Tomoharu Yoshizumi, Yuji Soejima, Yoshihiko Maehara, Kazuaki Chayama, Yoshiharu Matsuura

  SCIENTIFIC REPORTS 7 45228  2045-2322 2017/03 [Refereed][Not invited]
   

  It is well documented that a variety of viral quasispecies are found in the patients with chronic infection of hepatitis C virus (HCV). However, the significance of quasispecies in the specific infectivity to individual cell types remains unknown. In the present study, we analyzed the role of quasispecies of the genotype 2a clone, JFH1 (HCVcc), in specific infectivity to the hepatic cell lines, Huh7.5.1 and Hep3B. HCV RNA was electroporated into Huh7.5.1 cells and Hep3B/miR-122 cells expressing miR-122 at a high level. Then, we adapted the viruses to Huh7 and Hep3B/miR-122 cells by serial passages and termed the resulting viruses HCVcc/Huh7 and HCVcc/Hep3B, respectively. Interestingly, a higher viral load was obtained in the homologous combination of HCVcc/Huh7 in Huh7.5.1 cells or HCVcc/Hep3B in Hep3B/ miR-122 cells compared with the heterologous combination. By using a reverse genetics system and deep sequence analysis, we identified several adaptive mutations involved in the high affinity for each cell line, suggesting that quasispecies of HCV participate in cell-specific infectivity.
• CD14(+) Monocyte-Derived Galectin-9 Induces Natural Killer Cell Cytotoxicity in Chronic Hepatitis C

  Akira Nishio, Tomohide Tatsumi, Takatoshi Nawa, Takahiro Suda, Teppei Yoshioka, Yoshiki Onishi, Satoshi Aono, Minoru Shigekawa, Hayato Hikita, Ryotaro Sakamori, Daisuke Okuzaki, Takasuke Fukuhara, Yoshiharu Matsuura, Naoki Hiramatsu, Tetsuo Takehara

  HEPATOLOGY 65 (1) 18 - 31 0270-9139 2017/01 [Refereed][Not invited]
   

  Natural killer (NK) cell activation is associated with both liver injury and persistent infection in chronic hepatitis C (CHC); however, the detailed mechanism of this activation has not yet been fully elucidated. Because galectin-9 (Gal-9) has been reported to be increased in the serum and liver tissue of CHC patients, we investigated the function of Gal-9 in NK cell activation in CHC. First, we evaluated the function of Gal-9 on NK cytotoxicity in vitro. Gal-9 treatment resulted in increased cytotoxicity of naive NK cells, and the Gal-9-activated NK cells demonstrated cytotoxicity toward hepatoma cells and T cells. Additionally, coculturing peripheral blood mononuclear cells (PBMCs) with JFH-1/ Huh7.5.1 cells increased both Gal-9 production and NK cell cytotoxicity. Next, we investigated the source of Gal-9 and the mechanism of Gal-9 production. Deletion of CD14(+) monocytes from PBMCs resulted in reduced Gal-9 production in the coculture with JFH-1/ Huh7.5.1 cells. Gal-9 production was driven by coculturing of PBMCs with apoptotic hepatocytes. Blocking integrin avb3, a receptor for phosphatidylserine expressed on apoptotic cells, also resulted in decreased Gal-9 production. Finally, we found that serum Gal-9 levels were significantly higher in CHC patients than in healthy donors and patients who achieved sustained virologic response. Among CHC patients, serum Gal-9 levels were significantly higher in patients with elevated alanine aminotransferase (ALT) than in those with normal ALT. Conclusion: These results demonstrate that CD14(+) monocyte-derived Gal-9 increases NK cell cytotoxicity in HCV infection, which might be associated with liver injury and persistent infection.
• Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation

  Francesc Puig-Basagoiti, Takasuke Fukuhara, Tomokazu Tamura, Chikako Ono, Kentaro Uemura, Yukako Kawachi, Satomi Yamamoto, Hiroyuki Mori, Takeshi Kurihara, Toru Okamoto, Hideki Aizaki, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 90 (19) 8464 - 8477 0022-538X 2016/10 [Refereed][Not invited]
   

  Exchangeable apolipoproteins (ApoA, -C, and -E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it was shown that the exogenous expression of only short sequences containing amphipathic alpha-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene-knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic alpha-helix structures, we identified the human cathelicidin antimicrobial peptide (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue HCV infectious particle formation in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic alpha-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in nonhepatic 293T cells expressing claudin 1 and microRNA miR-122 confers complete propagation of HCV. These results suggest the possibility of extrahepatic propagation of HCV in cells with low-level or no expression of apolipoproteins but expressing secretory proteins containing amphipathic alpha-helices such as CAMP. IMPORTANCE Various exchangeable apolipoproteins play a pivotal role in the formation of infectious HCV during the assembly of viral particles, and amphipathic alpha-helix motifs in the apolipoproteins have been shown to be a key factor. To the best of our knowledge, we have identified for the first time the human cathelicidin CAMP as a cellular protein that can compensate for the role of apolipoproteins in the life cycle of HCV. We have also identified the domain in CAMP that contains amphipathic alpha-helices crucial for compensation and show that the expression of CAMP in nonhepatic cells expressing claudin 1 and miR-122 confers complete propagation of HCV. We speculate that low levels of HCV propagation might be possible in extrahepatic tissues expressing secretory proteins containing amphipathic alpha-helices without the expression of apolipoproteins.
• Lipoprotein Receptors Redundantly Participate in Entry of Hepatitis C Virus

  Satomi Yamamoto, Takasuke Fukuhara, Chikako Ono, Kentaro Uemura, Yukako Kawachi, Mai Shiokawa, Hiroyuki Mori, Masami Wada, Ryoichi Shima, Toru Okamoto, Nobuhiko Hiraga, Ryosuke Suzuki, Kazuaki Chayama, Takaji Wakita, Yoshiharu Matsuura

  PLOS PATHOGENS 12 (5) e1005610  1553-7366 2016/05 [Refereed][Invited]
   

  Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV.
• TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis

  Sayaka Aizawa, Toru Okamoto, Yukari Sugiyama, Takahisa Kouwaki, Ayano Ito, Tatsuya Suzuki, Chikako Ono, Takasuke Fukuhara, Masahiro Yamamoto, Masayasu Okochi, Nobuhiko Hiraga, Michio Imamura, Kazuaki Chayama, Ryosuke Suzuki, Ikuo Shoji, Kohji Moriishi, Kyoji Moriya, Kazuhiko Koike, Yoshiharu Matsuura

  NATURE COMMUNICATIONS 7 11379  2041-1723 2016/05 [Refereed][Not invited]
   

  Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin-proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCVcore protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells.
• Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx

  Takahisa Kouwaki, Toru Okamoto, Ayano Ito, Yukari Sugiyama, Kazuo Yamashita, Tatsuya Suzuki, Shinji Kusakabe, Junki Hirano, Takasuke Fukuhara, Atsuya Yamashita, Kazunobu Saito, Daisuke Okuzaki, Koichi Watashi, Masaya Sugiyama, Sachiyo Yoshio, Daron M. Standley, Tatsuya Kanto, Masashi Mizokami, Kohji Moriishi, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 90 (7) 3530 - 3542 0022-538X 2016/04 [Refereed][Not invited]
   

  Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly(135) with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5.
• Telaprevir versus simeprevir for the treatment of recurrent hepatitis C after living donor liver transplantation

  Toru Ikegami, Tomoharu Yoshizumi, Yoshihro Yoshida, Takeshi Kurihara, Norifumi Harimoto, Shinji Itoh, Masahiro Shimokawa, Takasuke Fukuhara, Ken Shirabe, Yoshihiko Maehara

  HEPATOLOGY RESEARCH 46 (3) E136 - E145 1386-6346 2016/03 [Refereed][Not invited]
   

  AimOur aim was to evaluate the clinical outcomes of telaprevir (TVR)- or simeprevir (SMV)-based triple therapy for recurrent hepatitis C after living donor liver transplantation. MethodsTwenty-six patients received antiviral therapy, consisting of either TVR (n=12) or SMV (n=14) in combination with pegylated interferon and ribavirin, plus cyclosporin. ResultsMore patients had a dose reduction of the direct-acting agent (36.3% vs 0.0%, P=0.02) or required blood transfusion for anemia (58.3% vs 7.1%, P<0.01) in the TVR group. The cyclosporin trough/dose ratio increased significantly from week 0 to week 4 in the TVR group (1.60.4 to 5.1 +/- 2.0, P<0.01), but not in the SMV group (1.2 +/- 0.3 to 1.3 +/- 0.2, P=0.68). The 24-week cumulative viral clearance rate was 91.7% and 85.7% in the TVR and in SMV groups, respectively. The early viral response and sustained viral response rates were 91.7% and 83.3%, respectively, in the TVR group, compared with 85.7% and 64.3%, respectively, in the SMV group. Interferon-mediated graft dysfunction occurred in four and five patients in the TVR and SMV groups, respectively; two patients were treated by oral steroids, five by steroid pulse and two by thymoglobulin, resulting in viral breakthrough in one case. ConclusionSMV-based triple therapy was associated with fewer adverse events and drug interactions with cyclosporin, and possibly less antiviral properties to TVR. Interferon-mediated graft dysfunction is a significant clinical problem that warrants particular caution following living donor liver transplantation.
• [Hepatitis virus related viruses].

  Fukuhara T, Matsuura Y

  Nihon rinsho. Japanese journal of clinical medicine 73 Suppl 9 30 - 35 0047-1852 2015/12 [Refereed][Not invited]
  
• Roles of Lipoproteins and Apolipoproteins in Particle Formation of Hepatitis C Virus

  Takasuke Fukuhara, Chikako Ono, Francesc Puig-Basagoiti, Yoshiharu Matsuura

  TRENDS IN MICROBIOLOGY 23 (10) 618 - 629 0966-842X 2015/10 [Refereed][Not invited]
   

  More than 160 million people worldwide are infected with hepatitis C virus (HCV), and cirrhosis and hepatocellular carcinoma induced by HCV infection are life-threatening diseases. HCV takes advantage of many aspects of lipid metabolism for an efficient propagation in hepatocytes. Due to the morphological and physiological similarities of HCV particles to lipoproteins, lipid-associated HCV particles are named lipoviroparticles. Recent analyses have revealed that exchangeable apolipoproteins directly interact with the viral membrane to generate infectious HCV particles. In this review, we summarize the roles of lipid metabolism in the life cycle of HCV.
• Roles of lipoprotein and apolipoprotein in HCV infection.

  Fukuhara T, Matsuura Y

  Uirusu 65 (2) 269 - 276 0042-6857 2015 [Refereed][Not invited]
   

  Although chronic infection of hepatitis C virus (HCV) induces disorders of lipid metabolism, HCV is known to utilize lipid metabolism for efficient propagation in the liver. Due to the morphological and physiological similarities of HCV particles to lipoproteins, lipid-associated HCV particles are named lipoviroparticles. Previous reports have shown that lipoprotein receptors or cholesterol transporter participate in the entry of lipoviroparticles. In addition, recent analyses revealed that exchangeable apolipoproteins directly interact with the viral membrane to generate infectious HCV particles. In this review, we would like to discuss about involvement of lipoprotein and apolipoprotein in HCV lifecycle.
• Amphipathic alpha-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

  Takasuke Fukuhara, Masami Wada, Shota Nakamura, Chikako Ono, Mai Shiokawa, Satomi Yamamoto, Takashi Motomura, Toru Okamoto, Daisuke Okuzaki, Masahiro Yamamoto, Izumu Saito, Takaji Wakita, Kazuhiko Koike, Yoshiharu Matsuura

  PLOS PATHOGENS 10 (12) e1004534  1553-7366 2014/12 [Refereed][Invited]
   

  Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic alpha-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic alpha-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic alpha-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.
• Reduced-Dose Telaprevir-Based Triple Antiviral Therapy for Recurrent Hepatitis C After Living Donor Liver Transplantation

  Toru Ikegami, Tomoharu Yoshizumi, Masaki Kato, Satomi Yamamoto, Takasuke Fukuhara, Yoshiharu Matsuura, Shota Nakamura, Shinji Itoh, Ken Shirabe, Yoshihiko Maehara

  TRANSPLANTATION 98 (9) 994 - 999 0041-1337 2014/11 [Refereed][Not invited]
   

  Introduction The feasibility of telaprevir-based triple therapy for recurrent hepatitis C after liver transplantation (LT) has not been evaluated in Asian patients. Methods Eleven Japanese patients received reduced-dose telaprevir (1500 mg) and adjusted-dose cyclosporine after LT. Six patients were nonresponders and three were transient responders to dual therapy. Results Rapid viral response, early viral response, end of treatment response, and sustained viral response were achieved in 27.3%, 90.9%, 90.9%, and 81.8% of patients, respectively. One patient had viral breakthrough at week 8 with a T54A mutation in NS3. Deep sequence analysis showed that the T54A mutation reverted to wild-type after stopping telaprevir administration. Seven patients developed severe anemia, and six received blood transfusions (4-20 U). Their hemoglobin and estimated glomerular filtration rate remained significantly lower than pretreatment values at 36 weeks after treatment. Four patients developed plasma cell hepatitis after completing telaprevir treatment, and it was treated by increasing the immunosuppressants. Although the cyclosporine level/dose ratio was 2.7 times higher at week 4 than before treatment, it was 0.7 times lower at week 36. Conclusions Reduced-dosed telaprevir-based triple antiviral therapy achieved a high viral clearance rate in Japanese patients after LT. Major adverse events included severe anemia, renal dysfunction, and plasma cell hepatitis.
• Reduced-Dose Telaprevir-Based Triple Antiviral Therapy for Recurrent Hepatitis C After Living Donor Liver Transplantation

  Toru Ikegami, Tomoharu Yoshizumi, Masaki Kato, Satomi Yamamoto, Takasuke Fukuhara, Yoshiharu Matsuura, Shota Nakamura, Shinji Itoh, Ken Shirabe, Yoshihiko Maehara

  TRANSPLANTATION 98 (9) 994 - 999 0041-1337 2014/11 [Refereed][Not invited]
   

  Introduction The feasibility of telaprevir-based triple therapy for recurrent hepatitis C after liver transplantation (LT) has not been evaluated in Asian patients. Methods Eleven Japanese patients received reduced-dose telaprevir (1500 mg) and adjusted-dose cyclosporine after LT. Six patients were nonresponders and three were transient responders to dual therapy. Results Rapid viral response, early viral response, end of treatment response, and sustained viral response were achieved in 27.3%, 90.9%, 90.9%, and 81.8% of patients, respectively. One patient had viral breakthrough at week 8 with a T54A mutation in NS3. Deep sequence analysis showed that the T54A mutation reverted to wild-type after stopping telaprevir administration. Seven patients developed severe anemia, and six received blood transfusions (4-20 U). Their hemoglobin and estimated glomerular filtration rate remained significantly lower than pretreatment values at 36 weeks after treatment. Four patients developed plasma cell hepatitis after completing telaprevir treatment, and it was treated by increasing the immunosuppressants. Although the cyclosporine level/dose ratio was 2.7 times higher at week 4 than before treatment, it was 0.7 times lower at week 36. Conclusions Reduced-dosed telaprevir-based triple antiviral therapy achieved a high viral clearance rate in Japanese patients after LT. Major adverse events included severe anemia, renal dysfunction, and plasma cell hepatitis.
• Suppression of Autophagy During Liver Regeneration Impairs Energy Charge and Hepatocyte Senescence in Mice

  Takeo Toshima, Ken Shirabe, Takasuke Fukuhara, Toru Ikegami, Tomoharu Yoshizumi, Yuji Soejima, Tetsuo Ikeda, Shinji Okano, Yoshihiko Maehara

  HEPATOLOGY 60 (1) 290 - 300 0270-9139 2014/07 [Refereed][Not invited]
   

  Autophagy is a homeostatic mechanism that regulates protein and organelle turnover and uses the amino acids from degraded proteins to produce adenosine 5'-triphosphate (ATP). We investigated the activity of autophagy-associated pathways in liver regeneration after partial hepatectomy (PHx) in liver-specific autophagy-related gene 5 (Atg5) knockout (KO) mice. Liver regeneration was severely impaired by 70% PHx, with a reduction in postoperative mitosis, but a compensating increase in hepatocyte size. PHx induced intracellular adenosine triphosphate and beta-oxidation reduction as well as injured cellular mitochondria. Furthermore, PHx in Atg5 KO mice enhanced hepatic accumulation of p62 and ubiquitinated proteins. These results indicated that reorganization of intracellular proteins and organelles during autophagy was impaired in the regenerating liver of these mice. Up-regulation of p21 was associated with hepatocyte senescence, senescence-associated beta-galactosidase expression, irreversible growth arrest, and secretion of senescence-associated molecules, including interleukin (IL)-6 and IL-8. Conclusion: These findings indicate that autophagy plays a critical role in liver regeneration and in the preservation of cellular quality, preventing hepatocytes from becoming fully senescent and hypertrophic.
• Suppression of Autophagy During Liver Regeneration Impairs Energy Charge and Hepatocyte Senescence in Mice

  Takeo Toshima, Ken Shirabe, Takasuke Fukuhara, Toru Ikegami, Tomoharu Yoshizumi, Yuji Soejima, Tetsuo Ikeda, Shinji Okano, Yoshihiko Maehara

  HEPATOLOGY 60 (1) 290 - 300 0270-9139 2014/07 [Refereed][Not invited]
   

  Autophagy is a homeostatic mechanism that regulates protein and organelle turnover and uses the amino acids from degraded proteins to produce adenosine 5'-triphosphate (ATP). We investigated the activity of autophagy-associated pathways in liver regeneration after partial hepatectomy (PHx) in liver-specific autophagy-related gene 5 (Atg5) knockout (KO) mice. Liver regeneration was severely impaired by 70% PHx, with a reduction in postoperative mitosis, but a compensating increase in hepatocyte size. PHx induced intracellular adenosine triphosphate and beta-oxidation reduction as well as injured cellular mitochondria. Furthermore, PHx in Atg5 KO mice enhanced hepatic accumulation of p62 and ubiquitinated proteins. These results indicated that reorganization of intracellular proteins and organelles during autophagy was impaired in the regenerating liver of these mice. Up-regulation of p21 was associated with hepatocyte senescence, senescence-associated beta-galactosidase expression, irreversible growth arrest, and secretion of senescence-associated molecules, including interleukin (IL)-6 and IL-8. Conclusion: These findings indicate that autophagy plays a critical role in liver regeneration and in the preservation of cellular quality, preventing hepatocytes from becoming fully senescent and hypertrophic.
• Interferon-lambda4 genetic polymorphism is associated with the therapy response for hepatitis C virus recurrence after a living donor liver transplant

  H. Konishi, T. Motomura, Y. Matsumoto, N. Harimoto, T. Ikegami, T. Yoshizumi, Y. Soejima, K. Shirabe, T. Fukuhara, Y. Maehara

  JOURNAL OF VIRAL HEPATITIS 21 (6) 397 - 404 1352-0504 2014/06 [Refereed][Not invited]
   

  The standard therapy against hepatitis C virus (HCV) recurrence postliver transplantation includes interferon (IFN) and ribavirin. IFNL4 ss469415590 polymorphism has been reported as a novel predictor of the response to IFN therapy for chronic HCV infection. We examined the impact of IFNL4 polymorphism on the responsiveness to IFN therapy after liver transplantation. Tissue specimens were collected from 80 HCV-infected recipients and 78 liver donors, and their IFNL4 ss469415590 genotype, hepatic IFNL4 and interferon-stimulated genes' mRNA expression levels were examined. The association of the polymorphism and expression levels in terms of the IFN therapy response to HCV recurrence was analysed. Most individuals who had rs8099917 risk alleles also had ss469415590 risk alleles (R-2=0.9). Sustained virological response (SVR) rates were higher in both liver graft recipients and transplants with ss469415590 TT/TT alleles than in those with the risk G allele (P=0.003 and P=0.005, respectively). In recipients with ss469415590 TT/TT, IFNL4 TT mRNA levels showed no significant differences between livers of patients who responded to therapy and those who did not (P=0.4). In recipients with the risk G allele, IFNL4 G mRNA expression levels were significantly lower in SVR patients than in non-SVR patients (P=0.02). Hepatic interferon stimulable genes and IFNL4 mRNA expression were correlated. Our findings suggest that analysing the ss469415590 genotype and IFNL4 G expression provides a novel prediction strategy for the possible response to IFN therapy after liver transplantation.
• Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus

  Mai Shiokawa, Takasuke Fukuhara, Chikako Ono, Satomi Yamamoto, Toru Okamoto, Noriyuki Watanabe, Takaji Wakita, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 88 (10) 5578 - 5594 0022-538X 2014/05 [Refereed][Not invited]
   

  Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV life cycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (cell culture-adapted HCV [HCVcc]), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific alpha -fetoprotein by using a cDNA array database and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expression of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV.
• Strategies to treat interferon-induced graft dysfunction after living donor liver transplantation for hepatitis C

  Toru Ikegami, Huanlin Wang, Tomoharu Yoshizumi, Takeo Toshima, Shinichi Aishima, Takasuke Fukuhara, Norihiro Furusyo, Kazuhiro Kotoh, Shinji Shimoda, Ken Shirabe, Yoshihiko Maehara

  HEPATOLOGY INTERNATIONAL 8 (2) 285 - 292 1936-0533 2014/04 [Refereed][Not invited]
   

  Interferon-induced graft dysfunction (IGD) is a poorly defined, unrecognized, but potentially serious condition for patients receiving antiviral drugs after liver transplantation for hepatitis C. We evaluated the characteristics of 80 patients who received pegylated interferon-based antiviral treatment for hepatitis C after living donor liver transplantation (LDLT). Eight patients experienced IGD either during (n = 6) or after completing (n = 2) antiviral treatment. Pathological diagnosis included acute cellular rejection (ACR, n = 1), plasma cell hepatitis (PCH, n = 2), PCH plus ACR (n = 3), and chronic rejection (CR, n = 2). One patient with CR initially presented with PCH plus ACR and the other presented with ACR; both had apparent cholestasis. The six patients with ACR or PCH without cholestasis were successfully treated by discontinuing antiviral treatment and increasing immunosuppression, including steroids. By contrast, both of the patients with CR and cholestasis experienced graft loss, despite aggressive treatment. Univariate analysis showed that pegylated interferon-alpha 2a-based treatment (75 vs. 26.4 %, p < 0.01) was the only significant factor for IGD, and was associated with decreased 5-year graft survival (93.4 vs. 71.4 %, p = 0.04). IGD is a serious condition during or even after antiviral treatment for hepatitis C after LDLT. Early recognition, diagnosis, discontinuation of interferon, and introduction of steroid-based treatment may help to save the graft.
• Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells

  Chikako Ono, Akinori Ninomiya, Satomi Yamamoto, Takayuki Abe, Xiauyu Wen, Takasuke Fukuhara, Miwa Sasai, Masahiro Yamamoto, Tatsuya Saitoh, Takashi Satoh, Taro Kawai, Ken J. Ishii, Shizuo Akira, Toru Okamoto, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 88 (4) 2157 - 2167 0022-538X 2014/02 [Refereed][Not invited]
   

  The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-beta) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-beta promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIMS, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.
• Hepatic interferon-gamma-induced protein-10 expression is more strongly associated with liver fibrosis than interleukin-28B single nucleotide polymorphisms in hepatocellular carcinoma resected patients with chronic hepatitis C.

  Hideyuki Konishi, Ken Shirabe, Shohei Yoshiya, Tetsuo Ikeda, Toru Ikegami, Tomoharu Yoshizumi, Ayae Ikawa-Yoshida, Takashi Motomura, Takasuke Fukuhara, Yoshihiko Maehara

  Hepatology research : the official journal of the Japan Society of Hepatology 43 (11) 1139 - 47 1386-6346 2013/11 [Refereed][Not invited]
   

  AIM: Single nucleotide polymorphisms (SNP) around IL-28B and interferon (IFN)-stimulated gene (ISG) expression are predictors of response to standard therapy involving IFN for chronic hepatitis C virus (HCV) infection. We analyzed the association between these predictors to improve the prediction of the response to IFN therapy after liver resection for hepatocellular carcinoma (HCC). METHODS: Data were collected from 74 patients with HCV-induced HCC. The IL-28B genotype and hepatic ISG mRNA levels were analyzed to clarify their association, focusing on the progression of liver fibrosis. RESULTS: Fifty patients were identified as having major alleles (rs8099917 TT) and the remaining 24 patients had minor alleles (rs8099917 TG or GG). Hepatic ISG15 expression was lower in the IL-28B major group than that in the IL-28B minor group (P < 0.005). IP-10 expression was similar between the IL-28B major and minor groups (P = 0.44). IP-10 expression was elevated with advancing stages of liver fibrosis in HCV infected patients (P = 0.005). In patients with mild or no fibrosis, the IL-28B major group had lower IP-10 expression than the IL-28B minor group (P = 0.02). However, in patients with advanced fibrosis, IP-10 expression was not different between the IL-28B major and minor groups (P = 0.66). CONCLUSION: Hepatic ISG15 expression is associated with IL-28B polymorphisms, while IP-10 is strongly affected by liver fibrosis.
• Decreased roundabout 1 expression promotes development of intrahepatic cholangiocarcinoma

  Yohei Mano, Shinichi Aishima, Takasuke Fukuhara, Yuki Tanaka, Yuichiro Kubo, Takashi Motomura, Takeo Toshima, Tomohiro Iguchi, Ken Shirabe, Yoshihiko Maehara, Yoshinao Oda

  HUMAN PATHOLOGY 44 (11) 2419 - 2426 0046-8177 2013/11 [Refereed][Not invited]
   

  Roundabout 1 (Robol) is a transmembrane receptor of the immunoglobulin family. Slit2 is one of its ligands. The function of Slit2/Robol signaling in the development of intrahepatic cholangiocarcinoma (ICC) remains to be elucidated. We examined the immunohistochemical expression of Robol and Slit2 and their clinicopathologic implications in 132 cases of ICC. Also, small interfering RNA of Robol was transfected into a high-expression ICC cell line, and a Robol vector was transfected into a low Robol expression ICC cell line. The effect of Robol suppression and overexpression in cell proliferation and migration of cultured ICC cells with Slit2. stimulation was investigated. Immunohistochemical study of ICC in the low Robol expression group showed larger tumors (P=.015), a higher Ki-67 labeling index (P=.021), and low expression of Slit2 (P=.0005). The low Slit2 expression group frequently showed perineural invasion (P=.036) and lymph node metastases (P=.013). Low Robol expression was associated with a poor prognosis (P=.0207). Robol suppression in Huh28 cells tended to promote cell proliferation and migration, whereas Robol overexpression in RBE cells significantly suppressed cell proliferation and migration. Low Robol expression was associated with cell proliferation and migration in ICC and was one of the adverse prognostic factors in patients with these tumors. (C) 2013 Elsevier Inc. All rights reserved.
• Early extensive viremia, but not rs8099917 genotype, is the only predictor for cholestatic hepatitis C after living-donor liver transplantation

  Toru Ikegami, Ken Shirabe, Takasuke Fukuhara, Norihiro Furusyo, Kazuhiro Kotoh, Masaki Kato, Shinji Shimoda, Shinichi Aishima, Yuji Soejima, Tomoharu Yoshizumi, Yoshihiko Maehara

  HEPATOLOGY RESEARCH 43 (6) 621 - 629 1386-6346 2013/06 [Refereed][Not invited]
   

  Aim Cholestatic hepatitis C is one of the most serious but still unaddressed disorders after liver transplantation. Methods In this study, we analyzed 49 patients who underwent living-donor liver transplantation (LDLT) to treat hepatitis C virus (HCV) infection. Results Five patients developed cholestatic hepatitis C, with total bilirubin of 15.2 +/- 3.1mg/dL at diagnosis 6.2 +/- 1.0 weeks after LDLT. Univariate analysis showed that larger graft to standard liver volume ratio, higher HCV RNA titer at 2 weeks, earlier peak HCV RNA titer and cytomegalovirus infection were the significant risk factors. The development of cholestatic hepatitis C was not significantly associated with interleukin-28B genotype (rs8099917); four out of five affected patients had the T/T genotype. Multivariate analysis showed that higher HCV RNA titer at 2 weeks was the only significant factor (P=0.026) for the development of cholestatic hepatitis C. Receiver-operator curve analysis showed that that HCV RNA titer of more than 7.2log10IU/mL was the optimal cut-off for characterizing cholestatic hepatitis C. All of the patients were serum HCV RNA negative after treatment with pegylated interferon and ribavirin and all the patients are alive. Conclusion Early extensive viremia, but not the rs8099917 genotype, was the only predictor for cholestatic hepatitis C after LDLT.
• Impact of conversion from pegylated interferon-α2b to interferon-α2a for treating recurrent hepatitis C after liver transplantation

  Toru Ikegami, Ken Shirabe, Tomoharu Yoshizumi, Norihiro Furusyo, Kazuhiro Kotoh, Masaki Kato, Shinji Shimoda, Yuji Soejima, Takashi Motomura, Takasuke Fukuhara, Yoshihiko Maehara

  Transplantation 95 (6) e38 - e42 0041-1337 2013/05/27 [Refereed][Not invited]
  
• Human Blood Dendritic Cell Antigen 3 (BDCA3)(+) Dendritic Cells Are a Potent Producer of Interferon-lambda in Response to Hepatitis C Virus

  Sachiyo Yoshio, Tatsuya Kanto, Shoko Kuroda, Tokuhiro Matsubara, Koyo Higashitani, Naruyasu Kakita, Hisashi Ishida, Naoki Hiramatsu, Hiroaki Nagano, Masaya Sugiyama, Kazumoto Murata, Takasuke Fukuhara, Yoshiharu Matsuura, Norio Hayashi, Masashi Mizokami, Tetsuo Takehara

  HEPATOLOGY 57 (5) 1705 - 1715 0270-9139 2013/05 [Refereed][Not invited]
   

  The polymorphisms in the interleukin (IL)-28B (interferon-lambda [IFN]-lambda 3) gene are strongly associated with the efficacy of hepatitis C virus (HCV) clearance. Dendritic cells (DCs) sense HCV and produce IFNs, thereby playing some cooperative roles with HCV-infected hepatocytes in the induction of interferon-stimulated genes (ISGs). Blood dendritic cell antigen 3 (BDCA3) 1 DCs were discovered as a producer of IFN-lambda upon Toll-like receptor 3 (TLR3) stimulation. We thus aimed to clarify the roles of BDCA3(+) DCs in anti-HCV innate immunity. Seventy healthy subjects and 20 patients with liver tumors were enrolled. BDCA3(+) DCs, in comparison with plasmacytoid DCs and myeloid DCs, were stimulated with TLR agonists, cell-cultured HCV (HCVcc), or Huh7.5.1 cells transfected with HCV/JFH-1. BDCA3(+) DCs were treated with anti-CD81 antibody, inhibitors of endosome acidification, TIR-domain-containing adapter-inducing interferon-beta (TRIF)-specific inhibitor, or ultraviolet-irradiated HCVcc. The amounts of IL-29/IFN-lambda 1, IL-28A/IFN-lambda 2, and IL-28B were quantified by subtype-specific enzyme-linked immunosorbent assay (ELISA). The frequency of BDCA3(+) DCs in peripheral blood mononuclear cell (PBMC) was extremely low but higher in the liver. BDCA3(+) DCs recovered from PBMC or the liver released large amounts of IFN-lambda s, when stimulated with HCVcc or HCV-transfected Huh7.5.1. BDCA3(+) DCs were able to induce ISGs in the coexisting JFH-1-positive Huh7.5.1 cells. The treatments of BDCA3(+) DCs with anti-CD81 antibody, cloroquine, or bafilomycin A1 reduced HCVcc-induced IL-28B release, whereas BDCA3(+) DCs comparably produced IL-28B upon replication-defective HCVcc. The TRIF-specific inhibitor reduced IL-28B release from HCVcc-stimulated BDCA3(+) DCs. In response to HCVcc or JFH-1-Huh7.5.1, BDCA3(+) DCs in healthy subjects with IL-28B major (rs8099917, TT) released more IL-28B than those with IL-28B minor genotype (TG). Conclusion: Human BDCA3(+) DCs, having a tendency to accumulate in the liver, recognize HCV in a CD81-, endosome-, and TRIF-dependent manner and produce substantial amounts of IL-28B/IFN-lambda 3, the ability of which is superior in subjects with IL-28B major genotype. (HEPATOLOGY 2013;57:1705-1715)
• Role of miR-122 and lipid metabolism in HCV infection

  Takasuke Fukuhara, Yoshiharu Matsuura

  Journal of Gastroenterology 48 (2) 169 - 176 0944-1174 2013/02 [Refereed][Not invited]
   

  Hepatitis C virus (HCV) exhibits a narrow host range and a specific tissue tropism. Mice expressing major entry receptors for HCV permit viral entry, and therefore the species tropism of HCV infection is considered to be reliant on the expression of the entry receptors. However, HCV receptor candidates are expressed and replication of HCV-RNA can be detected in several nonhepatic cell lines, suggesting that nonhepatic cells are also susceptible to HCV infection. Recently it was shown that the exogenous expression of a liver-specific microRNA, miR-122, facilitated the efficient replication of HCV not only in hepatic cell lines, including Hep3B and HepG2 cells, but also in nonhepatic cell lines, including Hec1B and HEK-293T cells, suggesting that miR-122 is required for the efficient replication of HCV in cultured cells. However, no infectious particle was detected in the nonhepatic cell lines, in spite of the efficient replication of HCV-RNA. In the nonhepatic cells, only small numbers of lipid droplets and low levels of very-low-density lipoprotein-associated proteins were observed compared with findings in the hepatic cell lines, suggesting that functional lipid metabolism participates in the assembly of HCV. Taken together, these findings indicate that miR-122 and functional lipid metabolism are involved in the tissue tropism of HCV infection. In this review, we would like to focus on the role of miR-122 and lipid metabolism in the cell tropism of HCV. © Springer 2012.
• Neutrophil-lymphocyte ratio reflects hepatocellular carcinoma recurrence after liver transplantation via inflammatory microenvironment

  Takashi Motomura, Ken Shirabe, Yohei Mano, Jun Muto, Takeo Toshima, Yuichiro Umemoto, Takasuke Fukuhara, Hideaki Uchiyama, Toru Ikegami, Tomoharu Yoshizumi, Yuji Soejima, Yoshihiko Maehara

  JOURNAL OF HEPATOLOGY 58 (1) 58 - 64 0168-8278 2013/01 [Refereed][Not invited]
   

  Background & Aims: Although the Milan criteria (MC) have been used to select liver transplantation candidates among patients with hepatocellular carcinoma (HCC), many patients exceeding the MC have shown good prognosis. Preoperative neutrophil-lymphocyte ratio (NLR) is a predictor of patient prognosis, but its mechanism has never been clarified. Methods: We assessed outcomes in 158 patients who had undergone living-donor liver transplantation (LDLT) for HCC. Recurrence-free survival (RFS) was determined in patients with high (>= 4) and low (<4) NLR. Levels of expression of vascular endothelial growth factor (VEGF), interleukin (IL)-8, IL-17, CD68, and CD163 were measured. Results: The 5-year RFS rate was significantly lower in patients with high (n = 26) than with low (n = 132) NLR (30.3% vs. 89.0%, p<0.0001), in patients with high (n = 15) than with low (n = 79) NLR who met the MC (73.6% vs. 100%, p = 0.0008) and in patients with high (n = 11) than with low (n = 53) NLR who exceeded the MC (0% vs. 76.1%, p = 0.0002). Tumor expression of VEGF, IL8, IL-17, CD68, and CD163 was similar in the high and low NLR groups, but serum and peritumoral IL-17 levels were significantly higher in the high-NLR group (p = 0.01 each). The density of peritumoral CD163 correlated with the density of peritumoral IL-17-producing cells (p = 0.04) and was significantly higher in the high-NLR group (p = 0.005). Conclusions: NLR predicts outcomes after LDLT for HCC via the inflammatory tumor microenvironment. Combined with the MC, NLR may be a new criterion for LDLT candidates with HCC. (C) 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
• Japanese Encephalitis Virus Core Protein Inhibits Stress Granule Formation through an Interaction with Caprin-1 and Facilitates Viral Propagation

  Hiroshi Katoh, Toru Okamoto, Takasuke Fukuhara, Hiroto Kambara, Eiji Morita, Yoshio Mori, Wataru Kamitani, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 87 (1) 489 - 502 0022-538X 2013/01 [Refereed][Not invited]
   

  Stress granules (SGs) are cytoplasmic foci composed of stalled translation preinitiation complexes induced by environmental stress stimuli, including viral infection. Since viral propagation completely depends on the host translational machinery, many viruses have evolved to circumvent the induction of SGs or co-opt SG components. In this study, we found that expression of Japanese encephalitis virus (JEV) core protein inhibits SG formation. Caprin-1 was identified as a binding partner of the core protein by an affinity capture mass spectrometry analysis. Alanine scanning mutagenesis revealed that Lys(97) and Arg(98) in the alpha-helix of the JEV core protein play a crucial role in the interaction with Caprin-1. In cells infected with a mutant JEV in which Lys(97) and Arg(98) were replaced with alanines in the core protein, the inhibition of SG formation was abrogated, and viral propagation was impaired. Furthermore, the mutant JEV exhibited attenuated virulence in mice. These results suggest that the JEV core protein circumvents translational shutoff by inhibiting SG formation through an interaction with Caprin-1 and facilitates viral propagation in vitro and in vivo.
• Effect of laparoscopic splenectomy in patients with Hepatitis C and cirrhosis carrying IL28B minor genotype

  Takashi Motomura, Ken Shirabe, Norihiro Furusyo, Tomoharu Yoshizumi, Toru Ikegami, Yuji Soejima, Tomohiko Akahoshi, Morimasa Tomikawa, Takasuke Fukuhara, Jun Hayashi, Yoshihiko Maehara

  BMC GASTROENTEROLOGY 12 158  1471-230X 2012/11 [Refereed][Not invited]
   

  Background: IL28B and ITPA genetic variants are associated with the outcome of pegylated-interferon and ribavirin (PEG-IFN/RBV) therapy. However, the significance of these genetic variants in cirrhotic patients following splenectomy has not been determined. Methods: Thirty-seven patients with HCV-induced cirrhosis who underwent laparoscopic splenectomy (Spx group) and 90 who did not (non-Spx group) were genotyped for IL28B and ITPA. The outcome or adverse effects were compared in each group. Interferon-stimulated gene 15 (ISG15) and protein kinase R expression in the spleen was measured using total RNA extracted from exenterate spleen. Results: Sustained virological response (SVR) rate was higher in patients carrying IL28B major genotype following splenectomy (50% vs 27.3%) and in patients carrying minor genotype in the Spx group compared to non-Spx group (27.3% vs 3.6%, P<0.05). Pretreatment splenic ISG expression was higher in patients carrying IL28B major. There was no difference in progression of anemia or thrombocytopenia between patients carrying each ITPA genotype in the Spx group. Although splenectomy did not increase hemoglobin (Hb) level, Hb decline tended to be greater in the non-Spx group. In contrast, splenectomy significantly increased platelet count (61.1 x 10(3)/mu l vs 168.7 x 10(3)/mu l, P<0.01), which was maintained during the course of PEG-IFN/RBV therapy. Conclusions: IL28B genetic variants correlated with response to PEG-IFN/RBV following splenectomy. Splenectomy improved SVR rate among patients carrying IL28B minor genotype and protected against anemia and thrombocytopenia during the course of PEG-IFN/RBV therapy regardless of ITPA genotype.
• Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus

  Takasuke Fukuhara, Hiroto Kambara, Mai Shiokawa, Chikako Ono, Hiroshi Katoh, Eiji Morita, Daisuke Okuzaki, Yoshihiko Maehara, Kazuhiko Koike, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 86 (15) 7918 - 7933 0022-538X 2012/08 [Refereed][Not invited]
   

  Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.
• CD44 Participates in IP-10 Induction in Cells in Which Hepatitis C Virus RNA Is Replicating, through an Interaction with Toll-Like Receptor 2 and Hyaluronan

  Takayuki Abe, Takasuke Fukuhara, Xiauyu Wen, Akinori Ninomiya, Kohji Moriishi, Yoshihiko Maehara, Osamu Takeuchi, Taro Kawai, Shizuo Akira, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 86 (11) 6159 - 6170 0022-538X 2012/06 [Refereed][Not invited]
   

  The mechanisms of induction of liver injury during chronic infection with hepatitis C virus (HCV) are not well understood. Gamma interferon (IFN-gamma)-inducible protein 10 (IP-10), a member of the CXC chemokine family, is expressed in the liver of chronic hepatitis C (CHC) patients and selectively recruits activated T cells to the sites of inflammation. Recently, it was shown that a low plasma concentration of IP-10 in CHC patients was closely associated with the outcome of antiviral therapy. In this study, we examined the role of the Toll-like receptor (TLR) pathway on IP-10 production in cells replicating HCV. Among the CXC chemokines, the expression of IP-10 was specifically increased in cells replicating HCV upon stimulation with conventional TLR2 ligands. The enhancement of IP-10 production upon stimulation with TLR2 ligands in cells replicating HCV induced CD44 expression. CD44 is a broadly distributed type I transmembrane glycoprotein and a receptor for the glycosaminoglycan hyaluronan (HA). In CHC patients, the expression of HA in serum has been shown to increase in accord with the progression of liver fibrosis, and HA also works as a ligand for TLR2. In the present study, IP-10 production upon HA stimulation was dependent on the expression of TLR2 and CD44, and a direct association between TLR2 and CD44 was observed. These results suggest that endogenous expression of HA in hepatocytes in CHC patients participates in IP-10 production through an engagement of TLR2 and CD44.
• [miR-122 participates in the cell tropism of hepatitis C virus].

  Fukuhara T, Matsuura Y

  Uirusu 62 (1) 1 - 8 0042-6857 2012/06 [Refereed][Not invited]
   

  Hepatitis C virus (HCV) exhibits a narrow host range and a specific tissue tropism. Studies on HCV life cycle have been progressed by the developments of in vitro replication and infection systems and an HCV laboratory strain (HCVcc) capable of propagating in human hepatoma cell line, Huh7 cells. Mice expressing four human entry receptor candidates for HCV permit entry of HCVcc, therefore tissue tropism of HCV was believed to be rely on the expression of the entry receptors. However, HCV infection is often associated with extra-hepatic manifestations and the determinants for cell tropism of HCV remain elusive. Recently, we have shown that several nonhepatic cell lines permit HCV-RNA replication through an expression of a liver-specific microRNA, miR-122, upon infection with HCVcc, while no infectious particle was produced. In the nonhepatic cells, only small numbers of lipid droplets and low levels of VLDL-associated proteins were observed in compared with Huh7 cells, suggesting that expression of miR-122 and functional lipid metabolism participates in the replication and assembly of HCVcc, respectively In this review, we would like to discuss about involvement of miR-122 and functional lipid metabolism in the determination of HCV cell tropism.
• V5-Drainage-Preserved Right Lobe Grafts Improve Graft Congestion for Living Donor Liver Transplantation

  Takeo Toshima, Akinobu Taketomi, Toru Ikegami, Takasuke Fukuhara, Hiroto Kayashima, Tomoharu Yoshizumi, Yuji Soejima, Ken Shirabe, Yoshihiko Maehara

  TRANSPLANTATION 93 (9) 929 - 935 0041-1337 2012/05 [Refereed][Not invited]
   

  Background. Right lobe (RL) grafts without middle hepatic vein for living donor liver transplantation (LDLT) result in congestion of recipients' livers and sometimes in unfavorable postoperative course. This study aimed to evaluate the feasibility of our new V5-drainage-preserved RL (VP-RL) graft. Methods. Based on a review of 49 donors' livers in a retrospective study using three-dimensional reconstruction-computed tomography volumetry, hepatic vein draining segment 4 (V4) anatomy was classified into three types: inferior V4 dominant (A); superior V4 dominant (B); and umbilical vein to left hepatic vein dominant (C). Differences in functional graft volume (GV) and remnant liver volume (RV) between VP-RL and modified RL (M-RL) grafts with all three types were evaluated. In a prospective study of actual 15 LDLT, the outcome of venous reconstruction and postoperative parameters with VP-RL grafts compared with M-RL grafts was analyzed. Results. In the retrospective study using three-dimensional reconstruction-computed tomography volumetry, in types B and C, functional GV of VP-RL was larger than that of M-RL (P<0.05) without impaired donors' functional RV, whereas functional RV in VP-RL was significantly decreased in type A (P<0.05). In the prospective study of actual 15 LDLT, using VP-RL with types B and C, size and number of venous reconstructions, and functional GV and postoperative parameters, such as postoperative serum total bilirubin levels and ascites volume, were significantly improved compared with those using M-RL (P<0.05). Conclusions. Using preoperative V4 anatomical classification, VP-RL graft procurement is a valuable strategy in RL-LDLT to improve postoperative course of both recipients and donors.
• Efficacy of splenectomy in preventing anemia in patients with recurrent hepatitis C following liver transplantation is not dependent on inosine triphosphate pyrophosphatase genotype

  Takashi Motomura, Erina Koga, Akinobu Taketomi, Takasuke Fukuhara, Yohei Mano, Jun Muto, Hideyuki Konishi, Takeo Toshima, Hideaki Uchiyama, Tomoharu Yoshizumi, Ken Shirabe, Yoshihiko Maehara

  HEPATOLOGY RESEARCH 42 (3) 288 - 295 1386-6346 2012/03 [Refereed][Not invited]
   

  Aim: A genetic polymorphism of inosine triphosphate pyrophosphatase (ITPA) has been associated with pegylated-interferon/ribavirin (PEG-IFN/RBV)-induced anemia in chronic hepatitis C patients. However, correlation of the genetic variant with anemia following liver transplantation has not been determined. Methods: Sixty-three hepatitis C virus (HCV)-positive patients who underwent liver transplantation and PEG-IFN/ RBV therapy were enrolled. The rs1127354 was determined for each individual. Results: There was no relationship with anemia or RBV dosage in patients carrying the CC allele (CC group, n = 43) and those carrying the CA allele (CA group, n = 20). The incidence of hemoglobin (Hb) decline > 3 g/dL (CC: 4.7%, CA: 0%) was relatively low, whereas the incidence of Hb levels < 10 g/dL (CC: 18.6%, CA: 30.0%) was high. Univariate analysis revealed that splenectomy inversely correlated with Hb levels < 10 g/dL at 4 weeks (P = 0.04). Among the 22 patients who did not undergo splenectomy, the incidence of Hb levels < 10 g/dL tended to be lower in the seven patients carrying the CA allele (28.6%) than in the 15 patients with the CC allele (60.0%). Conclusion: The ITPA genetic polymorphism does not correlate with post-transplant PEG-IFN/RBV-induced anemia. Splenectomy is useful in preventing anemia regardless of the ITPA genotype.
• Baculovirus GP64-Mediated Entry into Mammalian Cells

  Chikako Kataoka, Yuuki Kaname, Shuhei Taguwa, Takayuki Abe, Takasuke Fukuhara, Hideki Tani, Kohji Moriishi, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 86 (5) 2610 - 2620 0022-538X 2012/03 [Refereed][Not invited]
   

  The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-beta-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.
• Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122

  Hiroto Kambara, Takasuke Fukuhara, Mai Shiokawa, Chikako Ono, Yuri Ohara, Wataru Kamitani, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 86 (3) 1382 - 1393 0022-538X 2012/02 [Refereed][Not invited]
   

  The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of "cured" cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics.
• Neither MICA Nor DEPDC5 Genetic Polymorphisms Correlate with Hepatocellular Carcinoma Recurrence following Hepatectomy.

  Takashi Motomura, Yuki Ono, Ken Shirabe, Takasuke Fukuhara, Hideyuki Konishi, Yohei Mano, Takeo Toshima, Shohei Yoshiya, Jun Muto, Toru Ikegami, Tomoharu Yoshizumi, Yoshihiko Maehara

  HPB surgery : a world journal of hepatic, pancreatic and biliary surgery 2012 185496 - 185496 0894-8569 2012 [Refereed][Not invited]
   

  Purpose. Genetic polymorphisms of MICA and DEPDC5 have been reported to correlate with progression to hepatocellular carcinoma (HCC) in chronic hepatitis C patients. However, correlation of these genetic variants with HCC recurrence following hepatectomy has not yet been clarified. Methods. Ninety-six consecutive HCC patients who underwent hepatectomy, including 64 patients who were hepatitis C virus (HCV) positive, were genotyped for MICA (rs2596542) and DEPDC5 (rs1012068). Recurrence-free survival rates (RFS) were compared for each genotype. Results. Five-year HCC recurrence-free survival (RFS) rates following hepatectomy were 20.7% in MICA GG allele carriers, 38.7% in GA, and 20.8% in AA, respectively (P = 0.72). The five-year RFS rate was 23.8% in DEPDC5 TT allele carriers and 31.8% in TG/GG, respectively (P = 0.47). The survival rates in all (including HCV-negative) patients were also similar among each MICA and DEPDC5 genotype following hepatectomy. Among HCV-positive patients carrying the DEPDC5 TG/GG allele, low fibrosis stage (F0-2) occurred more often compared with TT carriers (P < 0.05). Conclusions. Neither MICA nor DEPDC5 genetic polymorphism correlates with HCC recurrence following hepatectomy. DEPDC5 minor genotype data suggest a high susceptibility for HCC development in livers, even those with low fibrosis stages.
• Heterogeneous Nuclear Ribonucleoprotein A2 Participates in the Replication of Japanese Encephalitis Virus through an Interaction with Viral Proteins and RNA

  Hiroshi Katoh, Yoshio Mori, Hiroto Kambara, Takayuki Abe, Takasuke Fukuhara, Eiji Morita, Kohji Moriishi, Wataru Kamitani, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 85 (21) 10976 - 10988 0022-538X 2011/11 [Refereed][Not invited]
   

  Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5'-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive-and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.
• De Novo Autoimmune Hepatitis Subsequent to Switching from Type 2b to Type 2a Alpha-Pegylated Interferon Treatment for Recurrent Hepatitis C After Liver Transplantation: Report of a Case

  Kazuki Takeishi, Ken Shirabe, Takeo Toshima, Toru Ikegami, Kazutoyo Morita, Takasuke Fukuhara, Takashi Motomura, Yohei Mano, Hideaki Uchiyama, Yuji Soejima, Akinobu Taketomi, Yoshihiko Maehara

  SURGERY TODAY 41 (7) 1016 - 1019 0941-1291 2011/07 [Refereed][Not invited]
   

  Interferon (IFN), which is the only possible agent for recurrent hepatitis C after liver transplantation, may cause serious immune-related disorders. We report a case of de novo autoimmune hepatitis (AIH), which developed subsequent to switching from 2b pegylated interferon-alpha (peg-IFN) to 2a peg-IFN after living donor liver transplantation (LDLT). A 51-year-old man with hepatitis C-associated liver cirrhosis underwent LDLT. About 13 months after the initiation of antiviral therapy, in the form of type 2b peg-IFN with ribavirin, a negative serum hepatitis C virus (HCV)-RNA titer was confirmed. Thereafter, the 2b peg-IFN was switched to 2a peg-IFN, 3 months after which severe liver dysfunction developed, despite a constantly negative HCV-RNA. Liver biopsy showed portal and periportal inflammatory infiltrates including numerous plasma cells, indicating AIH. He was treated with steroid pulse treatment, followed by high-level immunosuppression maintenance, but eventually died of Pneumocystis pneumonitis 4 months after the diagnosis of de novo AIH.
• Intracellular delivery of serum-derived hepatitis C virus

  Takasuke Fukuhara, Hideki Tani, Mai Shiokawa, Yukinori Goto, Takayuh Abe, Akinobu Taketomi, Ken Shirabe, Yoshihiko Maehara, Yoshiharu Matsuura

  MICROBES AND INFECTION 13 (4) 405 - 412 1286-4579 2011/04 [Refereed][Not invited]
   

  A robust and reliable cell culture system for serum-derived HCV (HCVser) has not been established yet because of the presence of neutralizing antibody and tropism for infection. To overcome this obstacle, we employed a lipid-mediated protein intracellular delivery reagent (PIDR) that permits internalization of proteins into cells. Although entry of HCVcc was not enhanced by the treatment with PIDR, entry of HCVser into hepatoma cell lines (Huh7 and HepG2) and immortalized primary hepatocytes (He and HuS/E2) was significantly enhanced by the PIDR treatment. The entry of HCVser into Huh7 cells in the presence of PIDR was resistant to the neutralization by an anti-hCD81 antibody, suggesting that PIDR is capable of internalizing HCVser in a receptor-independent manner. Interestingly, the PIDR-mediated entry of HCVser and HCVcc was enhanced by the addition of sera from chronic hepatitis C patients but not from healthy donors. In addition, neutralization of HCVcc infection by anti-E2 antibody was canceled by the treatment with PIDR. In conclusion, the PIDR is a valuable tool to get over the obstacle of neutralizing antibodies to internalize HCV into cells and might be useful for the establishment of in vitro propagation HCVser. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
• Involvement of cyclophilin B in the replication of Japanese encephalitis virus

  Hiroto Kambara, Hideki Tani, Yoshio Mori, Takayuki Abe, Hiroshi Katoh, Takasuke Fukuhara, Shuhei Taguwa, Kohji Moriishi, Yoshiharu Matsuura

  VIROLOGY 412 (1) 211 - 219 0042-6822 2011/03 [Refereed][Not invited]
   

  Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus that belongs to the Flaviviridae family. In this study, we have examined the effect of cyclosporin A (CsA) on the propagation of JEV. CsA exhibited potent anti-JEV activity in various mammalian cell lines through the inhibition of CypB. The propagation of JEV was impaired in the CypB-knockdown cells and this reduction was cancelled by the expression of wild-type but not of peptidylprolyl cis-trans isomerase (PPlase)-deficient CypB, indicating that PPlase activity of CypB is critical for JEV propagation. Infection of pseudotype viruses bearing JEV envelope proteins was not impaired by the knockdown of CypB, suggesting that CypB participates in the replication but not in the entry of JEV. CypB was colocalized and immunoprecipitated with JEV NS4A in infected cells. These results suggest that CypB plays a crucial role in the replication of JEV through an interaction with NS4A. (c) 2011 Elsevier Inc. All rights reserved.
• Variants in IL28B in Liver Recipients and Donors Correlate With Response to Peg-Interferon and Ribavirin Therapy for Recurrent Hepatitis C

  Takasuke Fukuhara, Akinobu Taketomi, Takashi Motomura, Shinji Okano, Akinori Ninomiya, Takayuki Abe, Hideaki Uchiyama, Yuji Soejima, Ken Shirabe, Yoshiharu Matsuura, Yoshihiko Maehara

  GASTROENTEROLOGY 139 (5) 1577 - + 0016-5085 2010/11 [Refereed][Not invited]
   

  BACKGROUND & AIMS: Patients with hepatitis C virus (HCV)-related liver disease frequently undergo orthotopic liver transplantation, but recurrent hepatitis C is still a major cause of morbidity. Patients are treated with peg-interferon and ribavirin (PEG-IFN/RBV), which has substantial side effects and is costly. We investigated genetic factors of host, liver donor, and virus that might predict sensitivity of patients with recurrent hepatitis C to PEG-IFN/RBV. METHODS: Liver samples were analyzed from 67 HCV-infected recipients and 41 liver donors. Liver recipient and donor DNA samples were screened for single nucleotide polymorphisms near the IL28B genes (rs12980275 and rs8099917) that affect sensitivity to PEG-IFN/RBV. HCV RNA was isolated from patients and analyzed for mutations in the core, the IFN sensitivity-determining region, and IFN/RBV resistance-determining regions in nonstructural protein 5A. RESULTS: In liver recipients and donors, the IL28B single nucleotide polymorphism rs8099917 was significantly associated with a sustained viral response (SVR; P = 0.003 and P = .025, respectively). Intrahepatic expression of IL28 messenger RNA was significantly lower in recipients and donors that carried the minor alleles (T/G or T/T) in rs8099917 (P = .010 and .009, respectively). Genetic analyses of IL28B in patients and donors and of the core and nonstructural protein 5A regions encoded by HCV RNA predicted an SVR with 83% sensitivity and 82% specificity; this was more effective than analysis of any single genetic feature. CONCLUSIONS: In patients with recurrent HCV infection after orthotopic liver transplantation, combination analyses of single nucleotide polymorphisms of IL28B in recipient and donor tissues and mutations in HCV RNA allow prediction of SVR to PEG-IFN/RBV therapy.
• Early Diagnosis and Treatment Resolved Cholestatic Hepatitis C Without Fibrosis After Living Donor Liver Transplantation: Report of a Case

  Takasuke Fukuhara, Kazutoyo Morita, Kazuki Takeishi, Takeo Toshima, Kenji Umeda, Shigeyuki Nagata, Keishi Sugimachi, Toru Ikegami, Tomonobu Gion, Yuji Soejima, Akinobu Taketomi, Yoshihiko Maehara

  SURGERY TODAY 40 (10) 982 - 985 0941-1291 2010/10 [Refereed][Not invited]
   

  Cholestatic hepatitis is a life-threatening recurrent pattern of hepatitis C virus (HCV) in immunosuppressed patients, for which curative treatment has not yet been established. We report the successful treatment of cholestatic hepatitis in a 59-year-old man who had undergone right lobe living donor liver transplantation (LDLT) for liver cirrhosis (LC) caused by HCV. Following uneventful surgery and an uncomplicated post-transplant clinical course, there was an abrupt increase in total bilirubin in comparison to aminotransferase on postoperative day (POD) 60 (total bilirubin 16.2 mg/dl, alanine aminotransferase 100 U/l, HCV-RNA 390 kIU/ml). The histological findings of the liver tissue showed lymphocyte infiltration in the periportal zone and severe cholestasis. Considering the clinical course, cholestatic hepatitis was strongly suspected and pegylated interferon and ribavirin therapy was started immediately, resulting in not only a viral response, but minimal progression of fibrosis. This case serves to demonstrate that early diagnosis and timely initiation of optimal antiviral therapy is essential for the resolution of cholestatic hepatitis C.
• Improved Results of a Surgical Resection for the Recurrence of Hepatocellular Carcinoma After Living Donor Liver Transplantation

  Akinobu Taketomi, Takasuke Fukuhara, Kazutoyo Morita, Hiroto Kayashima, Mizuki Ninomiya, Yoichi Yamashita, Toru Ikegami, Hideaki Uchiyama, Tomoharu Yoshizumi, Yuji Soejima, Ken Shirabe, Yoshihko Maehara

  ANNALS OF SURGICAL ONCOLOGY 17 (9) 2283 - 2289 1068-9265 2010/09 [Refereed][Not invited]
   

  Purpose. This study was designed to analyze the clinical outcomes of the recurrence of hepatocellular carcinoma (HCC) after living donor liver transplantation (LDLT) and to evaluate the efficacy of a surgical resection in treating such a recurrence. Methods. A total of 101 adult LDLT recipients with HCC between 1996 and 2007, including 17 who had recurrent HCC, were reviewed. The endpoints analyzed were survival from time of transplant and survival from time of recurrence. Recipient demographics, laboratory valuables, and tumor characteristics were analyzed. Any medical or surgical treatments that had been administered for any recurrence also were considered. Results. The mean duration until the initial recurrence after LDLT and the mean duration until death after the initial recurrence were 12.9 months and 12.0 months, respectively. A univariate analysis showed that gender, interferon therapy, early posttransplant tumor recurrence, and eligibility for a surgical resection all had a beneficial impact on survival from tumor recurrence. A surgical resection of tumor relapse was the most important variable in our study, and therefore the patients were divided into two groups: surgical therapy group (n = 9), and nonsurgical therapy group (n = 7). Interestingly, the overall survival rates of the surgical group were significantly better than those of the nonsurgical group and were similar to that of the patients without HCC recurrence. Conclusions. Surgical therapy might be useful for patients who experience a recurrence of HCC after LDLT to improve their outcome, when such treatment is available.
• Mutations in hepatitis C virus genotype 1b and the sensitivity of interferon-ribavirin therapy after liver transplantation

  Takasuke Fukuhara, Akinobu Taketomi, Shinji Okano, Toru Ikegami, Yuji Soejima, Ken Shirabe, Yoshihiko Maehara

  JOURNAL OF HEPATOLOGY 52 (5) 672 - 680 0168-8278 2010/05 [Refereed][Not invited]
   

  Background & Aims: The results of post-transplant antiviral therapy for recurrent hepatitis C virus (HCV) are poor, and significant pre-transplant predictors for sustained viral response (SVR) have not yet been identified. Methods: Pegylated interferon/ribavirin therapy was performed for more than 48 weeks in 50 patients who underwent liver transplantation (LT) for HCV genotype 1-related liver disease. Of these, 22 patients achieved SVR. The predictive potential of the viral mutations, including amino acids (aa) 70 and 91 in the Core region, interferon sensitivity-determining region (ISDR, aa 2209-2248) and interferon/ribavirin resistance-determining region (IRRDR, aa 2334-2379) in NS5A, was evaluated. Results: In 16 patients, the sequences in the pre- and post-transplant samples were the same, except for aa 70 in the Core of 1 patient. The SVR achievement percentage was significantly lower in the Non-double wild (DW) at aa 70 and 91, the ISDR < 2 and IRRDR < 6 groups than in the DW (30% vs. 65%, p = 0.015), the ISDR >= 2 (35% vs. 69%, p = 0.035) and IRRDR >= 6 (25% vs. 78%, p<0.001) groups, respectively. Predictive scoring with these three items provides a newly established and significant predictor for SVR after LT (p = 0.015). Conclusion: DW, ISDR >= 2 and IRRDR >= 6 were found to be significant predictors for SVR after LT. In addition, it is possible that the establishment of a new scoring system consisting of these three factors may be a useful marker to predict interferon sensitivity for recurrent HCV after LT. (C) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
• Impact of preoperative serum sodium concentration in living donor liver transplantation

  Takasuke Fukuhara, Toru Ikegami, Kazutoyo Morita, Kenji Umeda, Shigeru Ueda, Shigeyuki Nagata, Keishi Sugimachi, Tomonobu Gion, Tomoharu Yoshizumi, Yuji Soejima, Akinobu Taketomi, Yoshihiko Maehara

  JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY 25 (5) 978 - 984 0815-9319 2010/05 [Refereed][Not invited]
   

  Background and Aims: The importance of hyponatremia in deceased donor liver transplantation (DDLT) has been recently discussed frequently. However, its impact on the outcomes in living donor liver transplantation (LDLT) has not yet been elucidated. The current study was designed to demonstrate the impact of pre-transplant sodium concentration on postoperative clinical outcomes. Methods: One hundred and thirty-four patients who underwent LDLT for end-stage liver diseases were examined to evaluate the significance of pre-transplant hyponatremia (Na < 130 mEq/L) on the short-term clinical outcomes and the efficacy of the Model for End-Stage Liver Disease and serum sodium (MELD-Na) score using the sodium concentration and original MELD score. Results: The preoperative sodium and MELD score for all patients were 133.9 mEq/L (range: 109-142) and 16.2 (range: 6-38), respectively. According to a multivariate analysis, not only the MELD score (P = 0.030) but also the sodium concentration (P = 0.005) were found to be significant predictive factors for short-term graft survival. Preoperative hyponatremia was a significant risk factor for the occurrence of sepsis (P < 0.001), renal dysfunction (P < 0.001) and encephalopathy (P = 0.026). The MELD-Na score was 19.6 (range: 6-51) and the area under the receiver-operator curve of that (c-statistics: 0.867) was higher than MELD score and sodium concentration (c-statistics: 0.820 and 0.842, respectively). Conclusion: Preoperative hyponatremia was a significant risk for postoperative complications and short-term graft loss. The addition of sodium concentration to MELD score might therefore be an effective predictor for post-transplant short-term mortality in LDLT.
• Impact of amino acid substitutions in the core region of HCV on multistep hepatocarcinogenesis

  Takasuke Fukuhara, Kazuki Takeishi, Takeo Toshima, Kazutoyo Morita, Shigeru Ueda, Tomohiro Iguchi, Shigeyuki Nagata, Keishi Sugimachi, Toru Ikegami, Tomonobu Gion, Yuji Soejima, Akinobu Taketomi, Yoshihiko Maehara

  HEPATOLOGY RESEARCH 40 (2) 171 - 178 1386-6346 2010/02 [Refereed][Not invited]
   

  Aim: The core protein of hepatitis C virus (HCV) has multiple functions for not only viral replication but also hepatocellular carcinogenesis. A significant association of the substitutions in the core region with hepatocarcinogenesis has recently been reported. In this report, we evaluated the association of the substitutions in the core region with multistep hepatocarcinogenesis. Methods: Sixty-nine non-cancerous and cancerous liver tissues were obtained from the patients with primary developed hepatocellular carcinoma (HCC) due to HCV and 17 cirrhotic liver tissues were obtained from the patients without HCC. A sequence analysis of the core protein of HCV was performed and the association between the substitution rates in the core gene and the degree of fibrosis or steatosis during the primary development of HCC and tumor differentiation was analyzed. Results: The substitution rates of amino acid 70, 75, 91 and 147 exceeded 25% (amino acid 70, 51%; 75, 45%; 91, 36%; 147, 30%). All substitution rates were comparable among cancerous and non-cancerous region of patients with HCC and non-cancerous region without HCC. The substitution rates of these four amino acids were not associated with the degree of fibrosis, steatosis or tumor differentiation during the primary development of HCC. In addition, the substitution rates were comparable between the patients with or without HCC. The cumulative substitution numbers in the core region were also not associated with the degree of fibrosis and steatosis. Conclusions: It is possible that the substitutions in the core region are not associated with HCV-related multistep hepatocarcinogenesis.
• Phase I/II study of the lipiodolization using DDP-H (CDDP powder; IA-call(A (R))) in patients with unresectable hepatocellular carcinoma

  Yo-ichi Yamashita, Akinobu Taketomi, Shinji Itoh, Norifumi Harimoto, Kazutoyo Morita, Takasuke Fukuhara, Shigeru Ueda, Kensaku Sanefuji, Keishi Sugimachi, Tsuyoshi Tajima, Yoshihiko Maehara

  CANCER CHEMOTHERAPY AND PHARMACOLOGY 65 (2) 301 - 307 0344-5704 2010/01 [Refereed][Not invited]
   

  Lipiodol Ultra-Fluid (Lipiodol(A (R))), an oily contrast medium, is selectively retained in hepatocellular carcinoma (HCC) through hepatic arterial infusion. DDP-H (IA-call(A (R))) developed as a CDDP powder, and may be a possible chemotherapeutic agent with lipiodol. We carried out a phase I/II study of the lipiodolization using DPP-H in patients with unresectable HCC. Phase I and pharmacokinetic study: The dose-limiting toxicity (DLT), the maximum tolerance dose (MTD), and the recommended dose (RD) were determined using a modified Fibonacci scheme. The concentration-time profile of total platinum in plasma was analyzed. Phase II study: Thirty-five patients with unresectable HCC received lipiodolization using DDP-H under RD, and the efficacy and safety were assessed. DLT was grade 3 vomiting at 40 mg/m(2). Therefore, MTD and RD were 35 mg/m(2). The peak of total platinum in plasma was over 1.0 mu g/ml at 40 mg/m(2) at 30 min after infusion. Of the 35 patients, 16 (45.7%) demonstrated complete responses, and 4 (11.4%) demonstrated partial responses with an additional 9 patients (25.7%) having stable diseases, as assessed by RECIST. Grade 3 thrombocytopenia was found in 1 patient (2.9%), grade 2 hyperbilirubinemia was found in 2 patients (5.7%), and grade 2 vomiting was found in 4 patients (11.4%). Lipiodolization using DDP-H at 35 mg/m(2) is effective and well tolerated in patients with unresectable HCC.
• Solitary asymptomatic thyroid metastasis from hepatocellular carcinoma detected by FDG-PET/CT

  Takeo Toshima, Akinobu Taketomi, Ken Shirabe, Kazuki Takeishi, Takashi Motomura, Youhei Mano, Kazutoyo Morita, Takasuke Fukuhara, Keishi Sugimachi, Yasuhiro Maruoka, Koichiro Abe, Tsuyoshi Tajima, Yoshihiko Maehara

  Case Reports in Gastroenterology 4 (2) 279 - 285 1662-0631 2010 [Refereed][Not invited]
   

  Thyroid metastases from hepatocellular carcinoma (HCC) seldom occur and are often difficult to diagnose because of their asymptomatic clinical course. We evaluated a very rare case of solitary thyroid metastasis from HCC that showed high uptake of fluorine-18 fluorodeoxyglucose (FDG), when imaged using fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT). The patient was a 74-year-old man and presented with a remarkably elevated des-gamma-carboxy prothrombin level of 1,157 mAU/ml 22 months after hepatic lobectomy. FDG-PET/CT imaging revealed a hypodense tumor with high FDG uptake, with a maximum standardized uptake value of 5.2 in the thyroid left lobe. Solitary thyroid metastasis from HCC was suspected and subsequent fine needle aspiration did indeed reveal HCC. The patient received left thyroidectomy with left regional lymph node dissection. Two months after left thyroidectomy, contrast-enhanced computed tomography showed local recurrence, and the patient received ongoing radiotherapy treatment. To our knowledge, the present study is the first to demonstrate the feasibility of FDG-PET/CT in the diagnosis and management of clinically diagnosed, asymptomatic, solitary thyroid metastasis from HCC. © 2010 S. Karger AG, Basel.
• The Benefits of Interferon Treatment in Patients Without Sustained Viral Response After Living Donor Liver Transplantation for Hepatitis C

  T. Ikegami, A. Taketomi, Y. Soejima, T. Yoshizumi, T. Fukuhara, K. Kotoh, S. Shimoda, M. Kato, Y. Maehara

  TRANSPLANTATION PROCEEDINGS 41 (10) 4246 - 4252 0041-1345 2009/12 [Refereed][Not invited]
   

  Although it has been recognized that interferon (IFN) treatment is crucial for recurrent hepatitis C after liver transplantation, its benefits have not been determined among patients without a sustained viral response (SVR). Methods. Eighty patients who received IFN plus ribavirin treatment after living donor liver transplantation were grouped as follows: group I (n = 18) SVR; group II (n = 25) no-SVR but viral response [VR] positive; Group III (n = 13) no-VR but biochemical response [BR] positive; and group IV (n = 24) no-VR and no-BR. Results. In groups II and III, not only the histological activity grade and fibrosis stage, but also the serum parameters including transaminases and type IV collagen were stable for 3 years after induction of IFN-based treatment. In group 1, the activity grade and fibrosis stage significantly improved (P < .01). In group IV, the fibrosis stage significantly deteriorated (P < .01); the serum transaminases and type IV collagen were significantly higher than the other groups (P < .01). The mean duration of IFN treatment was significantly longer among group II (96 weeks) compared with the other cohorts (P < .05). The 5-year graft survival rate in groups II (91%) and III (100%) were comparable to those of group I (100%); group IV (62%) was significantly lower than the other groups (P < .05). Conclusion. IFN treatment was beneficial even among subjects with IFN-dependent VR or BR, although they did not achieve SVR.
• De Novo Hepatocellular Carcinoma in a Liver Graft with Sustained Hepatitis C Virus Clearance After Living Donor Liver Transplantation

  Kazutoyo Morita, Akinobu Taketomi, Yuji Soejima, Toru Ikegami, Takasuke Fukuhara, Tomohiro Iguchi, Shigeyuki Nagata, Keishi Sugimachi, Tomonobu Gion, Ken Shirabe, Yoshihiko Maehara

  LIVER TRANSPLANTATION 15 (11) 1412 - 1416 1527-6465 2009/11 [Refereed][Not invited]
   

  The occurrence of de novo hepatocellular carcinoma (HCC) after liver transplantation (LT) for advanced HCCs has been extremely limited. In this article, a case of de novo HCC in a liver graft with sustained hepatitis C virus clearance after living donor liver transplantation (LDLT) for multiple HCCs and hepatitis C cirrhosis is reported. The recipient was a 58-year-old female, and the left lobe living donor was the 30-year-old healthy daughter of the recipient. Three years after LDLT, the patient received 48 weeks of interferon treatment for recurrent hepatitis C with advanced fibrosis. The patient has shown successful viral clearance since then. However, an HCC was recognized in the liver graft during a follow-up computed tomography scan performed 6 years after LDLT, and it was surgically resected. To analyze its origin [either from the patient (metastatic) or from the living donor (de novo)], genotyping by microsatellite analysis of tissue and blood samples from the donor and recipient was performed, and it revealed that the HCC originated from the donor. To the best of our knowledge, this is the first report of de novo HCC in a liver graft with sustained hepatitis C virus clearance after LT for advanced HCCs and hepatitis C cirrhosis. Liver Transpl 15:1412-1416, 2009. (C) 2009 AASLD.
• Congestion of the donor remnant right liver after extended left lobe donation

  Takasuke Fukuhara, Kenji Umeda, Takeo Toshima, Kazuki Takeishi, Kazutoyo Morita, Shigeyuki Nagata, Keishi Sugimachi, Toru Ikegami, Tomonobu Gion, Yuji Soejima, Akinobu Taketomi, Yoshihiko Maehara

  TRANSPLANT INTERNATIONAL 22 (8) 837 - 844 0934-0874 2009/08 [Refereed][Not invited]
   

  P>The clinical importance of congestion of the remnant right lobe has not yet been fully elucidated in donors who donate their left lobe with the middle hepatic vein. The impact of congestion on clinical course and liver regeneration in 52 donor remnant livers were evaluated. The donors were divided into three groups according to the degree of the congestion: the mild [congestion ratio (CR) < 10%], moderate (CR ranged from 10% to 25%) and severe congestion groups (CR > 25%). The regeneration ratio of the graft at postoperative day 7 (7 POD) was 22.0 +/- 14.3% and inversely correlated with the CR in the remnant right lobe (P = 0.003). Aspartate aminotransferase and alanine aminotransferase at 7 POD were significantly higher in the severe CR group in comparison to the mild CR group (P = 0.003 and 0.019, respectively), but those of the three groups were comparable at 30 POD. The hospital stays were significantly longer in the severe CR group (P = 0.010). In conclusion, the congestion of the donors' remnant right liver can lead the transient liver dysfunction and poor regeneration. Therefore, the conversion of the graft from the left to right lobe might be appropriate according to the degree of the congestion.
• Human VAP-C Negatively Regulates Hepatitis C Virus Propagation

  Hiroshi Kukihara, Kohji Moriishi, Shuhei Taguwa, Hideki Tani, Takayuki Abe, Yoshio Mori, Tetsuro Suzuki, Takasuke Fukuhara, Akinobu Taketomi, Yoshihiko Maehara, Yoshiharu Matsuura

  JOURNAL OF VIROLOGY 83 (16) 7959 - 7969 0022-538X 2009/08 [Refereed][Not invited]
   

  Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) and subtype B (VAP-B) are involved in the regulation of membrane trafficking, lipid transport and metabolism, and the unfolded protein response. VAP-A and VAP-B consist of the major sperm protein (MSP) domain, the coiled-coil motif, and the C-terminal transmembrane anchor and form homo- and heterodimers through the transmembrane domain. VAP-A and VAP-B interact with NS5B and NS5A of hepatitis C virus (HCV) through the MSP domain and the coiled-coil motif, respectively, and participate in the replication of HCV. VAP-C is a splicing variant of VAP-B consisting of the N-terminal half of the MSP domain of VAP-B followed by the subtype-specific frameshift sequences, and its biological function has not been well characterized. In this study, we have examined the biological functions of VAP-C in the propagation of HCV. VAP-C interacted with NS5B but not with VAP-A, VAP-B, or NS5A in immunoprecipitation analyses, and the expression of VAP-C inhibited the interaction of NS5B with VAP-A or VAP-B. Overexpression of VAP-C impaired the RNA replication of the HCV replicon and the propagation of the HCV JFH1 strain, whereas overexpression of VAP-A and VAP-B enhanced the replication. Furthermore, the expression of VAP-C was observed in various tissues, whereas it was barely detected in the liver. These results suggest that VAP-C acts as a negative regulator of HCV propagation and that the expression of VAP-C may participate in the determination of tissue tropism of HCV propagation.
• Rapid Normalization of Portopulmonary Hypertension After Living Donor Liver Transplantation

  K. Sugimachi, Y. Soejima, K. Morita, S. Ueda, T. Fukuhara, S. Nagata, T. Ikegami, A. Taketomi, Y. Maehara

  TRANSPLANTATION PROCEEDINGS 41 (5) 1976 - 1978 0041-1345 2009/06 [Refereed][Not invited]
   

  Portopulmonary hypertension (PPHTN) is a relatively rare complication of end-stage liver disease, and a serious problem in the context of liver transplantation. Herein we have reported a case of decompensated liver cirrhosis with PPHTN, which rapidly resolved after adult-to-adult living donor liver transplantation (LDLT). A 54-year-old man was referred to our hospital with end-stage liver cirrhosis owing to chronic hepatitis C. Preoperative mean pulmonary artery pressure (mPAP), as assessed by right heart catheterization, was 38 mm Hg. Continuous infusion of epoprostenol decreased the mPAP to 24 mm Hg over 44 days. He underwent LDLT using a right hepatic lobe graft donated by his son. The postoperative course was uneventful, epoprostenol was weaned by postoperative day (POD) 21, and the mPAP normalized to 21 mm Hg on POD 28. The patient was discharged on POD 31 without any vasodilators. Our case revealed that liver transplantation can rapidly resolve PPTHN.
• Proctocolectomy for colon cancer associated with ulcerative colitis a few months after living donor liver transplantation for primary sclerosing cholangitis: Report of a case

  Takasuke Fukuhara, Akinori Egashira, Masakazu Imamura, Toru Ikegami, Eiji Oki, Yuji Soejima, Noriaki Sadanaga, Akinobu Taketomi, Takashi Yao, Masaru Morita, Yoshihiro Kakeji, Masazumi Tsuneyoshi, Yoshihiko Maehara

  SURGERY TODAY 39 (1) 59 - 63 0941-1291 2009/01 [Refereed][Not invited]
   

  Colorectal cancer (CRC) frequently develops in patients with ulcerative colitis (UC). We report a case of CRC treated successfully by proctocolectomy 8 months after living donor liver transplantation (LDLT) for primary sclerosing cholangitis (PSC). The lesion was detected early, probably as a result of colonoscopic surveillance after LDLT. Thus, patients with a long history of UC, who undergo LDLT for PSC, should be followed up with regular surveillance colonoscopy. Moreover, surgery, such as radical resection of the colon and rectum should be performed without delay, even shortly after LDLT. To our knowledge, this is the first report of a patient undergoing proctocolectomy after LDLT.
• A Simple Hilar Dissection Technique Preserving Maximum Blood Supply to the Bile Duct in Living Donor Liver Transplantation

  Yuji Soejima, Takasuke Fukuhara, Kazutoyo Morita, Tomoharu Yoshizumi, Toru Ikegami, Yoichi Yamashita, Keishi Sugimachi, Akinobu Taketomi, Yoshihiko Maehara

  TRANSPLANTATION 86 (10) 1468 - 1469 0041-1337 2008/11 [Refereed][Not invited]
   

  Duct-to-duct reconstruction is associated with a higher incidence in biliary strictures in living donor liver transplantation (LDLT). However, a standard dissection technique for the recipient's bile duct has not been established. Here, we describe a simple bile duct dissection technique preserving maximum vascular integrity during total hepatectomy of the recipient. The present technique might facilitate duct-to-duct bile duct reconstruction in both right and left lobe LDLT and, thus, contribute to reduce biliary complications such as biliary strictures. We believe that this technique can be a standard in the field of LDLT.
• The impact of surgical treatment and poor prognostic factors for patients with intrahepatic cholangiocarcinoma: Retrospective analysis of 60 patients

  Yo-Ichi Yamashita, Akinobu Taketomi, Kazutoyo Morita, Takasuke Fukuhara, Shigeru Ueda, Kensaku Sanefuji, Tomohiro Iguchi, Hiroto Kayashima, Keishi Sugimachi, Yoshihiko Maehara

  ANTICANCER RESEARCH 28 (4C) 2353 - 2359 0250-7005 2008/07 [Refereed][Not invited]
   

  Background: Intrahepatic cholangiocarcinoma (ICC) is a primary adenocarcinoma of the liver arising from the intrahepatic bile duct. Hepatectomy with extensive lymph node dissection is the standard treatment for ICC. Patients and Methods: Sixty patients with ICC who underwent hepatectomy in our institution between 1986 and 2005 were investigated to determine prognostic factors and to evaluate the impact of surgical treatment for ICC using univariate and multivariate analyses. Results: The overall survival rate of the RO resection group (n=43) was significantly higher than that of the RI/2 group (n=17). However, in patients with lymph node metastasis (n=24), RO resection had no survival impact. According to multivariate analysis, the independent factors of poor prognosis were: the presence of lymph node metastasis, lymphatic invasion, poor differentiation and RI/2 resection. Conclusion: RO resection can provide prolonged survival for patients with ICC. Patients with lymph node metastasis, lymphatic invasion, or poorly differentiated ICC have poor prognosis after operation and additional treatment, such as adjuvant chemotherapy, is recommended.
• One-step venous reconstruction for a right lobe graft with multiple venous orifices in living donor liver transplantation

  Yuji Soejima, Naoyuki Ueda, Takasuke Fukuhara, Tomoharu Yoshizumi, Toru Ikegami, Yoichi Yamashita, Keishi Sugimachi, Akinobu Taketomi, Yoshihiko Maehara

  LIVER TRANSPLANTATION 14 (5) 706 - 708 1527-6465 2008/05 [Refereed][Not invited]
  
• A case of hepatocellular carcinoma resected after improvement of liver function by balloon-occluded retrograde transvenous obliteration

  Kazutoyo Morita, Akinobu Taketomi, Yo-Ichi Yamashita, Takasuke Fukuhara, Hiroto Kayashima, Yousuke Kuroda, Shinji Itoh, Kouzou Konishi, Hirofumi Kawanaka, Yoshihiko Maehara

  Japanese Journal of Gastroenterological Surgery 41 (4) 418 - 423 1348-9372 2008 [Refereed][Not invited]
   

  Hepatocellular carcinoma with liver cirrhosis is often unresectable due to liver dysfunction. A 82-year-old man had two hepatocellular carcinomas measuring 2.5cm at S6 and 1cm at S8 of the liver. Despite transcatheter arterial chemoembolization (TACE) for these carcinomas, local recurrence was seen at S6, TACE repeated, and local recurrence seen again. He had severe liver dysfunction (Child-Pugh 8 Grade B, and liver damage C) and a huge gastro-renal shunt. Balloon-occluded retrograde transvenous obliteration (B-RTO) was conducted to increase portal flow to the liver and improve liver function. After B-RTO, liver function improved to Child-Pugh 6 Grade A, and liver damage B. Partial hepatic resection (S6) was successful and the man was discharged on postoperative day 14 without postoperative complications. The obliteration of portosystemic shunt using B-RTO makes it possible to conduct hepatic resection for hepatocellular carcinoma in patients with severe liver dysfunction. This strategy is especially useful in the treatment of TACE resistant carcinoma. ©2008 The Japanese Society of Gastroenterological Surgery.
• Reduction in the incidence of Helicobacter pylori-associated carcinoma in Japanese young adults

  M Ito, K Haruma, T Kamada, Y Kitadai, T Hidaka, T Tsuda, H Komatsu, T Fukuhara, M Yoshihara, K Chayama

  ONCOLOGY REPORTS 8 (3) 633 - 636 1021-335X 2001/05 [Refereed][Not invited]
   

  Helicobacter pylori (Hp) is a major risk factor for gastric carcinogenesis. In Japan, the incidence of Hp infection in young adults has declined markedly. The purpose of this study was to clarify this trend in the incidence of Hp-associated gastric carcinoma (GCa) in young (under 30 years of age) Japanese patients. We enrolled 53 such patients who underwent surgical resection of GCa in one of 18 hospitals in the Hiroshima prefecture between 1976 and 1999. The patients were classified into groups based on three 8-year periods (1976-83, 1984-91, and 1992-1999) in which their cases occurred. We compared the numbers of patients and estimated the histology of carcinomas, grades of gastritis and Hp infection histologically. Of the 53 patients, 49 (92%) showed Hp infection. The frequency of GCa in young adults has decreased gradually (21, 18, and 14 patients in 1976-83, 1984-91 and 1992-99, respectively). The numbers of Hp-positive carcinomas decreased radically (21, 17 and 11 patients, respectively). This trend was associated with improvement in the degree of gastritis in non-neoplastic mucosa. Of the four Hp-negative patients, three had signer ring cell carcinoma. Moreover, the numbers of patients with non-signet ring cell carcinoma also decreased (18, 12 and 7 patients in each period, respectively). These results suggest that Hp-associated carcinoma has declined gradually in young Japanese.

MISC

• Cryo-EM structure of SARS-CoV-2 XBB.1.5 spike protein and its ACE2 receptor recognition mechanism and structural comparison with XBB.1

  矢島久乃, 安楽佑樹, 喜多俊介, 佐々木慈英, 木村(寺角)香菜子, 前仲勝実, 佐藤佳, 福原崇介, 橋口隆生  日本ウイルス学会学術集会プログラム・予稿集(Web)  70th-  2023
• Reverse genetics systems for severe acute respiratory syndrome coronavirus 2

  田村友和, 福原崇介  日本血栓止血学会誌  34-  (4)  2023
• SARS-CoV-2BA.2株に由来する亜株の性状解析

  木村出海, 山岨大智, 山岨大智, 田村友和, 直亨則, 直亨則, 伊東潤平, 松野啓太, 松野啓太, 松野啓太, 澤洋文, 澤洋文, 澤洋文, 福原崇介, 佐藤佳  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• 高速リーバースジェネティクスのキャップ非依存性+ssRNAウイルスへの応用

  山本紘嵩, 田村友和, 伊藤駿, 鈴木理滋, 福原崇介  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• HBV感染に関与するRNA結合タンパク質の同定とその機能解析

  小林展大, 小林展大, 齋藤智哉, 齋藤智哉, 鈴木理滋, 田村友和, 武冨紹信, 福原崇介  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• 数理モデルと分子動力学シミュレーションを用いたレムデシビル耐性SARS-CoV-2変異株の解析

  鈴木理滋, 鳥居志保, 鳥居志保, キム クァンス, 小関準, 岩波翔也, 藤田泰久, チョン ヨンダム, 田村友和, 松浦善治, 島村徹平, 岩見真吾, 岩見真吾, 岩見真吾, 岩見真吾, 岩見真吾, 岩見真吾, 福原崇介  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• フラビウイルスの粒子産生における分泌型NS1タンパク質の役割

  田村友和, 田村友和, 鳥居志保, 鳥居志保, 梶原健太郎, 安齋樹, 藤岡容一朗, 野田暉翔, 田鍬修平, 森岡佑平, 森岡佑平, 鈴木理滋, YUZY Fauzyah, 小野慎子, 小野慎子, 大場雄介, 岡田雅人, 福原崇介, 福原崇介, 松浦善治, 松浦善治  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• 肝疾患患者の変異株に対するワクチン効果の評価系の確立と免疫抑制の影響

  伊藤駿, 冨山貴央, 鈴木理滋, 田村友和, 吉住朋晴, 福原崇介  日本ウイルス学会学術集会プログラム・予稿集(Web)  69th-  2022
• Generation of recombinant highly virulent classical swine fever virus strain possessing a luminescent HiBiT tag as a reporter

  LOC Huynh Tan, 廣瀬静香, 金琢洙, 田村友和, 福原崇介, 松浦善治, 日尾野隆大, 磯田典和, 磯田典和, 迫田義博, 迫田義博  日本獣医学会学術集会講演要旨集  164th (CD-ROM)-  2021
• Rimonabant suppresses RNA transcription of hepatitis B virus by inhibiting hepatocyte nuclear factor 4α.

  Asuka Sato, Chikako Ono, Tomokazu Tamura, Hiroyuki Mori, Takuma Izumi, Shiho Torii, Yuzy Fauzyah, Takuya Yamamoto, Yuhei Morioka, Daisuke Okuzaki, Takasuke Fukuhara, Yoshiharu Matsuura  Microbiology and immunology  64-  (5)  345  -355  2020/05  [Not refereed][Not invited]
   

  Chronic infection with hepatitis B virus (HBV) sometime induces lethal cirrhosis and hepatocellular carcinoma. Although nucleot(s)ide analogs are used as main treatment for HBV infection, the emergence of the drug-resistant viruses has become a problem. To discover novel antivirals with low side effects and low risk of emergence of resistant viruses, screening for anti-HBV compounds was performed with compound libraries of inhibitors targeting G-protein-coupled receptors (GPCRs). HepG2-hNTCP C4 cells infected with HBV were treated with various GPCR inhibitors and harvested at 14 day postinfection for quantification of core protein in the first screening or relaxed circular DNA in the second screening. Finally, we identified a cannabinoid receptor 1 inhibitor, rimonabant, as a candidate showing anti-HBV effect. In HepG2-hNTCP C4 cells, treatment with rimonabant suppressed HBV propagation at the viral RNA transcription step but had no effect on entry or covalently closed circular DNA level. The values of half maximal inhibitory concentration, half maximal effective concentration, and selectivity index of rimonabant in primary human hepatocyte (PHH) are 2.77 μm, 40.4 μm, and 14.6, respectively. Transcriptome analysis of rimonabant-treated primary hepatocytes by RNA sequencing revealed that the transcriptional activity of hepatocyte nuclear factor 4α (HNF4α), which is known to stimulate viral RNA synthesis, was depressed. By treatment of PHH with rimonabant, the expression level of HNF4α protein and the production of the messenger RNAs (mRNAs) of downstream factors promoted by HNF4α were reduced while the amount of HNF4α mRNA was not altered. These results suggest that treatment with rimonabant suppresses HBV propagation through the inhibition of HNF4α activity.
• SARS-CoV-2の新規リバースジェネティクス法の確立

  鳥居志保, 小野慎子, 鈴木理滋, 森岡佑平, 安齋樹, FAUZYAH Yuzy, 前田裕輔, 神谷亘, 福原崇介, 福原崇介, 松浦善治  日本分子生物学会年会プログラム・要旨集(Web)  43rd-  2020
• C型肝炎ウイルスNS5A蛋白質のISGylation修飾反応におけるウイルス遺伝子型間の解析

  阿部隆之, 松井千絵子, LIN Deng, 福原崇介, 松浦善治, 勝二郁夫  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• 高効率な非レトロRNAウイルス網羅探索手法FLDSのヒト臓器への適用

  浦山俊一, 浦山俊一, 浦山俊一, 田村友和, 泉琢磨, 中村昇太, 元岡大祐, 吉住朋晴, 平井美穂, 高木善弘, 布浦拓郎, 福原崇介  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• NS1蛋白質はフラビウイルスの感染性粒子形成に重要である

  福原崇介, 田村友和, 鳥居志保, 小野慎子, 梶原健太郎, 森岡佑平, 山本拓弥, ファウジャー ユージー, 泉琢磨, 迫田義博, 松浦善治  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• ミニゲノムを用いた重症熱性血小板減少症候群ウイルスのゲノム複製・転写を抑制する低分子化合物のスクリーニング

  山田博司, 木村美幸, 龍龍, 谷口怜, 下島昌幸, 福原崇介, 松浦善治, 米納孝, 中嶋希, 古田要介, 西條政幸, 谷英樹  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• 肝臓移植レシピエントにおけるペギウイルス感染の特徴

  泉琢磨, 坂田一仁, 奥崎大介, 井口昭一, 田村友和, 元岡大祐, 中村昇太, 小野慎子, 下川雅弘, 松浦善治, 森正樹, 福原崇介, 吉住朋晴  日本ウイルス学会学術集会プログラム・予稿集(Web)  67th-  2019
• Research frontier of hepatitis C virus propagation and pathogenesis

  福原 崇介, 松浦 善治  医学のあゆみ  260-  (6)  518  -521  2017/02/11
• 肝細胞癌におけるAdenomatous polyposis coli結合蛋白EB1の機能解析

  相山健, 横尾英樹, 折茂達也, 大畑多嘉宣, 畑中佳奈子, 畑中豊, 松野吉宏, 福原崇介, 高橋秀徳, 若山顕治, 敦賀陽介, 蒲池浩文, 神山俊哉, 武冨紹信  日本外科学会定期学術集会(Web)  116th-  PS‐107‐1 (WEB ONLY)  -1  2016  [Not refereed][Not invited]
  
• HuH‐7由来オーバル細胞様細胞株HdoによるHCV生活環の調節に関わる分化関連宿主因子の同定

  伊藤昌彦, 福原崇介, 鈴木亮介, 田川陽一, 松浦善治, 脇田隆字, 鈴木哲朗  日本ウイルス学会学術集会プログラム・抄録集  62nd-  193  2014/10/31  [Not refereed][Not invited]
  
• 肝炎ウイルス研究における遺伝子改変技術の有用性

  福原崇介, 和田真実, 小野慎子, 中村昇太, 山本聡美, 森寛之, 岡本徹, 奥崎大介, 山本卓, 松浦善治  日本分子生物学会年会プログラム・要旨集(Web)  37th-  2014
• 肝細胞癌株のリプログラミングによるHCV感受性および腫瘍原性の低下

  伊藤昌彦, 伊藤徳臣, 福原崇介, 鈴木亮介, 田川陽一, 松浦善治, 脇田隆字, 鈴木哲朗  日本ウイルス学会学術集会プログラム・抄録集  61st-  281  2013/10/29  [Not refereed][Not invited]
  
• Human BDCA3+DCs Contribute to the Induction of Intrahepatic ISGs as a Potent IFN-lambda Producer in HCV Infection

  Sachiyo Yoshio, Tatsuya Kanto, Tokuhiro Matsubara, Masaya Sugiyama, Kazumoto Murata, Takasuke Fukuhara, Yoshiharu Matsuura, Masashi Mizokami, Norio Hayashi, Tetsuo Takehara  HEPATOLOGY  58-  1184A  -1184A  2013/10  [Not refereed][Not invited]
  
• The roles of autophagy in HCV life cycle

  岡本 徹, 福原 崇介, 田鍬 修平  細胞  45-  (1)  44  -47  2013/01
• HuH‐7由来オーバル様細胞におけるHCV感受性の解析

  伊藤昌彦, 鈴木亮介, 福原崇介, 松浦善治, 脇田隆字, 鈴木哲朗  日本ウイルス学会学術集会プログラム・抄録集  60th-  192  2012/10/31  [Not refereed][Not invited]
  
• Human BDCA3+dendritic cells in blood and in the liver are a potent producer of IFN-lambda in response to hepatitis C virus

  Sachiyo Yoshio, Tatsuya Kanto, Shoko Kuroda, Tokuhiro Matsubara, Koyo Higashitani, Hisashi Ishida, Naoki Hiramatsu, Hiroaki Nagano, Masaya Sugiyama, Kazumoto Murata, Takasuke Fukuhara, Yoshiharu Matsuura, Masashi Mizokami, Norio Hayashi, Tetsuo Takehara  HEPATOLOGY  56-  285A  -285A  2012/10  [Not refereed][Not invited]
  
• 生体肝移植術後早期のVirtual Touch Tissue Quantification(VTTQ)によるグラフト肝硬度評価の有用性

  伊地知 秀樹, 調 憲, 武冨 紹信, 吉住 朋晴, 池上 徹, 萱島 寛人, 森田 和豊, 福原 崇介, 戸島 剛男, 間野 洋平, 本村 貴志, 武藤 純, 的野 る美, 吉屋 匠平, 前原 喜彦  日本外科学会雑誌  113-  (臨増2)  382  -382  2012/03  [Not refereed][Not invited]
  
• C型肝炎ウイルスの細胞親和性はmiR-122の発現と脂質代謝系によって規定される

  福原崇介, 塩川舞, 小野慎子, 山本聡美, 寒原裕登, 加藤大志, 岡本徹, 奥崎大介, 松浦善治  日本ウイルス学会学術集会プログラム・抄録集  60th-  2012
• 【C型肝炎のすべて2012】 インターフェロンの上手な使い方 肝移植後のHCV感染に対するPEG-IFN/Ribavirin療法

  武冨 紹信, 本村 貴志, 福原 崇介, 池上 徹, 吉住 朋晴, 調 憲, 前原 喜彦  肝・胆・膵  63-  (6)  1158  -1164  2011/12  [Not refereed][Not invited]
  
• 宿主IL28B/ITPA遺伝子多型解析から見た移植後再発HCV治療戦略

  本村 貴志, 武冨 紹信, 福原 崇介, 武藤 純, 間野 洋平, 戸島 剛男, 森田 和豊, 萱島 寛人, 池上 徹, 吉住 朋晴, 調 憲, 松浦 善治, 前原 喜彦  移植  46-  (6)  658  -658  2011/12  [Not refereed][Not invited]
  
• 生体肝移植後の肝癌再発予防を目指したIL12/DC併用Neoadjuvant immunotherapyの展開

  萱島 寛人, 戸島 剛男, 岡野 慎士, 吉屋 匠平, 武藤 純, 的野 る美, 本村 貴志, 間野 洋平, 森田 和豊, 福原 崇介, 池上 徹, 吉住 朋晴, 武冨 紹信, 調 憲, 前原 喜彦  日本癌治療学会誌  46-  (2)  686  -686  2011/09  [Not refereed][Not invited]
  
• 宿主IL28B/ITPA遺伝子多型解析から見た移植後再発HCVに対する治療戦略

  本村 貴志, 武冨 紹信, 福原 崇介, 吉屋 匠平, 武藤 純, 的野 る美, 間野 洋平, 戸島 剛男, 森田 和豊, 萱島 寛人, 池上 徹, 吉住 朋晴, 調 憲, 前原 喜彦  肝臓  52-  (Suppl.2)  A651  -A651  2011/09  [Not refereed][Not invited]
  
• 【Changed!?臓器移植法改正施行後1年の現況】 さらに新しいチャレンジ DonorとRecipientの宿主遺伝子背景からみた再発HCVに対するIFN感受性予測

  本村 貴志, 武冨 紹信, 福原 崇介, 池上 徹, 吉住 朋晴, 調 憲, 前原 喜彦  肝・胆・膵  63-  (1)  135  -142  2011/07  [Not refereed][Not invited]
  
• Noticeable infection with a virus and control measures (6) Hepatitis C

  福原 崇介, 松浦 善治  防菌防黴 = Journal of antibacterial and antifungal agents  39-  (6)  363  -374  2011/06/10
• The Impact of IL28B Genetic Variants on Recurrent Hepatitis C in Liver Transplantation: Significant Lessons from a Dual Graft Case

  T. Motomura, A. Taketomi, T. Fukuhara, Y. Mano, K. Takeishi, T. Toshima, N. Harada, H. Uchiyama, T. Yoshizumi, Y. Soejima, K. Shirabe, Y. Matsuura, Y. Maehara  AMERICAN JOURNAL OF TRANSPLANTATION  11-  (6)  1325  -1329  2011/06  [Not refereed][Not invited]
   

  IL28B genetic polymorphism is related to interferon-sensitivity in chronic hepatitis C, but the significance of grafts carrying different genotypes from recipients is still unclear in liver transplantation. A 51-year-old Japanese male carrying a minor genotype underwent dual liver transplantation for liver cirrhosis due to hepatitis C virus (HCV). The left lobe graft carried a major genotype, and the right a minor genotype. He achieved virological response during the course of pegylated-interferon and ribavirin therapy against recurrent hepatitis C for 2 years, but HCV relapsed immediately at the end of the therapy. Two years after antiviral therapy, liver biopsy was performed from each graft. The specimens showed A1F0 in the left lobe graft and A2F2 in the right. Moreover, quantitative polymerase chain reaction was performed using RNA extracted from each specimen to see there was no HCV RNA in the left lobe whereas there was in the right. This case provides clear evidence that IL28B genetic variants determine interferon sensitivity in recurrent hepatitis C following liver transplantation, which could result in new strategies for donor selection or for posttransplant antiviral therapy to HCV positive recipients.
• 外科と輸血 肝細胞癌に対する肝切除後輸血の予後に与える影響 20年間の解析

  原田 昇, 調 憲, 武冨 紹信, 吉住 朋晴, 内山 秀昭, 伊地知 秀樹, 吉松 正憲, 福原 崇介, 戸島 剛男, 武石 一樹, 前原 喜彦  日本外科学会雑誌  112-  (臨増1-2)  280  -280  2011/05  [Not refereed][Not invited]
  
• ゲノム解析による肝移植後再発C型肝炎に対する高精度IFN治療効果予測法の開発

  武冨 紹信, 福原 崇介, 本村 貴志, 武藤 純, 的野 る美, 戸島 剛男, 武石 一樹, 吉松 正憲, 伊地知 秀樹, 原田 昇, 内山 秀昭, 吉住 朋晴, 調 憲, 松浦 善治, 前原 喜彦  日本外科学会雑誌  112-  (臨増1-2)  357  -357  2011/05  [Not refereed][Not invited]
  
• C型肝硬変に対するIFN導入目的の脾摘における、IL28B、ITPA遺伝子多型解析 脾摘の有用性

  本村 貴志, 武冨 紹信, 福原 崇介, 間野 洋平, 武藤 純, 武石 一樹, 戸島 剛男, 伊地知 秀樹, 原田 昇, 内山 秀昭, 吉住 朋晴, 調 憲, 富川 盛雅, 前原 喜彦  肝臓  52-  (Suppl.1)  A217  -A217  2011/04  [Not refereed][Not invited]
  
• Evolving Strategies to Prevent Biliary Strictures After Living Donor Liver Transplantation

  T. Ikegami, Y. Soejima, K. Shirabe, A. Taketomi, T. Yoshizumi, H. Uchiyama, T. Fukuhara, T. Ikeda, Y. Maehara  TRANSPLANTATION PROCEEDINGS  42-  (9)  3624  -3629  2010/11  [Not refereed][Not invited]
   

  Introduction. The optimal surgical technique has not been elucidated that reduces the occurrence of biliary strictures after living donor liver transplantation (LDLT). Method. We performed retrospective analysis of 193 consecutive LDLTs, including 78 right and 115 left lobe grafts. An external biliary stent was used for all of the cases. Results. The overall 1-, 3-, and 5-year biliary stricture free survival rates were 87.5%, 85.3%, and 85.3%, respectively. The 1- and 3-year biliary stricture free survival rates for duct-to-duct reconstruction were 86.9% and 84.9%, and those for hepaticojejunostomy were 90.1% and 80.8%, respectively. A multivariate analysis revealed that the original number of graft bile ducts greater than the number of external stents, right lobe grafts, bile leaks, and recipient age older than 60 years represented the significant risk factors (P < .05) to develop a biliary stricture after LDLT. All cases with biliary strictures (n = 61) were initially managed nonsurgically, but 4 patients ultimately required interventions. Conclusions. Biliary reconstruction in LDLT using an appropriate number of external stents resulted in fairly acceptable outcomes. However, not only the proper use of stents or graft selection, but also nonsurgical factors, are important factors that determine the incidence of biliary strictures after LDLT.
• 肝移植後早期合併症 劇症肝炎に対する生体肝移植後に門脈ガス血症を来した1例

  福原 崇介, 本村 貴志, 武石 一樹, 戸島 剛男, 内山 秀昭, 副島 雄二, 武冨 紹信, 調 憲, 前原 喜彦  臨牀と研究  87-  (9)  1302  -1303  2010/09  [Not refereed][Not invited]
   

  54歳女。全身倦怠感・食欲不振・白色便が出現し、近医で著明な肝酵素上昇を指摘された。重症急性肝炎の診断で入院加療を受けたが、PTが37%まで低下し当院転院となった。第13病日より血漿交換を開始したが、PTは改善せず、第15病日に意識障害を来たして肝性脳症III度に至り、劇症肝炎亜急性型と診断した。その後も改善がないため、第20病日に長女の右葉グラフトを用いた生体肝移植術を行った。切除肝は病理学的に典型的な劇症肝炎の所見を呈していた。移植術は問題なく終了したが、術後16日目に創部離開を認め、開放創修復術を行った。その翌日に著明な肝酵素上昇、急速なアシドーシス進行、白血球・血小板の低下を認め、DICと診断した。腹部CTでは門脈本幹から肝内門脈にガスを認めたが、腸管には虚血や壁在ガスは認めなかった。門脈ガス血症に対し抗生剤投与、輸血、CHDFを行ったが、DICの改善傾向はなく、突然の腹部膨満、アシドーシスの急速な悪化により、再手術後23日目に死亡した。剖検で1.5cm大の小腸穿孔を認めた。
• ChildB肝硬変肝癌に対する肝移植の適応と成績

  原田 昇, 調 憲, 武冨 紹信, 吉住 朋晴, 内山 秀昭, 伊地知 秀樹, 福原 崇介, 戸島 剛男, 武石 一樹, 本村 貴志, 間野 洋平, 池田 泰治, 是永 大輔, 竹中 賢治, 前原 喜彦  日本癌治療学会誌  45-  (2)  588  -588  2010/09  [Not refereed][Not invited]
  
• レシピエント自家下大静脈・門脈を用いた"all-in-one"肝右葉グラフト肝静脈再建法

  副島 雄二, 調 憲, 武冨 紹信, 内山 秀昭, 二宮 瑞樹, 萱島 寛人, 森田 和豊, 福原 崇介, 前原 喜彦  日本消化器外科学会総会  65回-  60  -60  2010/07  [Not refereed][Not invited]
  
• バーチャル画像に基づいた手術計画 バーチャル画像に基づいた生体肝移植右葉グラフト摘出術 とくに肝静脈解剖から見た合理的中肝静脈分枝V5切離の検討について

  萱島 寛人, 戸島 剛男, 本村 貴志, 間野 洋平, 武石 一樹, 梅田 健二, 森田 和豊, 福原 崇介, 二宮 瑞樹, 内山 秀昭, 副島 雄二, 武冨 紹信, 調 憲, 前原 喜彦  日本肝胆膵外科学会・学術集会プログラム・抄録集  22回-  143  -143  2010/05  [Not refereed][Not invited]
  
• C型肝炎に対する肝移植 ウイルスおよび宿主の遺伝子解析による肝移植後の再発C型肝炎に対するIFN治療効果の予測

  福原 崇介, 武冨 紹信, 本村 貴志, 戸島 剛男, 武石 一樹, 森田 和豊, 萱島 寛人, 二宮 瑞樹, 内山 秀昭, 副島 雄二, 調 憲, 松浦 善治, 前原 喜彦  肝臓  51-  (Suppl.1)  A108  -A108  2010/04  [Not refereed][Not invited]
  
• IS-3-2 Extended indication for living donor liver transplantation in patients with hepatocellular carcinoma : Kyushu University experience using newly developed risk score(Liver transplantation in the treatment of hepatocellular carcinoma:Current status and future perspectives in the Japanese society,International Symposium(3))

  Soejima Yuji, Shirabe Ken, Taketomi Akinobu, Uchiyama Hideaki, Ninomiya Mizuki, Kayashima Hiroto, Fukuhara Takasuke, Morita Kazutoyo, Takeishi Kazuki, Toshima Takeo, Motomura Takashi, Maehara Yoshihiko  Journal of Japan Surgical Society  111-  (2)  39  -39  2010/03/05
• 生体肝移植右葉グラフト摘出における3次元構築による肝静脈解剖から見た合理的中肝静脈分枝V5切離法の確立

  萱島 寛人, 戸島 剛男, 調 憲, 間野 洋平, 本村 貴志, 武石 一樹, 梅田 健二, 森田 和豊, 福原 崇介, 二宮 瑞樹, 内山 秀昭, 副島 雄二, 武冨 紹信, 前原 喜彦  日本外科学会雑誌  111-  (臨増2)  290  -290  2010/03  [Not refereed][Not invited]
  
• 肝細胞癌におけるERCC1発現とCDDP感受性に関する研究

  植田 茂, 調 憲, 福原 崇介, 森田 和豊, 梅田 健二, 萱島 寛人, 内山 秀昭, 副島 雄二, 武冨 紹信, 前原 喜彦  日本外科学会雑誌  111-  (臨増2)  570  -570  2010/03  [Not refereed][Not invited]
  
• P-3-233 肝移植後再発HCVに対するIFN治療におけるウィルスRNA変異による効果予測(肝・肝移植,一般演題(ポスター),第64回日本消化器外科学会総会)

  福原 崇介, 武冨 紹信, 副島 雄二, 森田 和豊, 植田 茂, 永田 茂行, 杉町 圭史, 池上 徹, 祇園 智信, 前原 喜彦  日本消化器外科学会雑誌  42-  (7)  1324  -1324  2009/07/01
• HP-134-7 C型肝炎陽性肝細胞癌術後における初回IFN治療の有効性(肝臓(肝がん外科治療1),ハイブリッドポスター,第109回日本外科学会定期学術集会)

  戸島 剛男, 武冨 紹信, 武石 一樹, 梅田 健二, 福原 崇介, 森田 和豊, 植田 茂, 井口 友宏, 實藤 健作, 永田 茂行, 杉町 圭史, 池上 徹, 祇園 智信, 副島 雄二, 前原 喜彦  日本外科学会雑誌  110-  (2)  623  -623  2009/02/25
• HP-134-3 糖尿病合併肝細胞癌の臨床的特徴からみた生活習慣病と肝細胞癌の密接な関連に関する検討(肝臓(肝がん外科治療1),ハイブリッドポスター,第109回日本外科学会定期学術集会)

  杉町 圭史, 武冨 紹信, 副島 雄二, 祇園 智信, 池上 徹, 永田 茂行, 實藤 健作, 井口 友宏, 植田 茂, 森田 和豊, 福原 崇介, 戸島 剛男, 武石 一樹, 前原 喜彦  日本外科学会雑誌  110-  (2)  622  -622  2009/02/25
• HP-215-2 肝細胞癌におけるERCC 1発現とCDDP感受性に関する研究(肝臓(肝がん治療1),ハイブリッドポスター,第109回日本外科学会定期学術集会)

  植田 茂, 武冨 紹信, 戸島 剛男, 武石 一樹, 福原 崇介, 森田 和豊, 梅田 健二, 實藤 健作, 井口 友弘, 永田 茂之, 杉町 圭史, 池上 徹, 祇園 智信, 副島 雄二, 前原 喜彦  日本外科学会雑誌  110-  (2)  749  -749  2009/02/25
• SF-057-4 肝細胞癌におけるDNA polymeraseδ発現の生物学的意義に関する研究(肝(基礎研究3),サージカルフォーラム,第109回日本外科学会定期学術集会)

  實藤 健作, 武冨 紹信, 副島 雄二, 池上 徹, 杉町 圭史, 永田 茂行, 井口 友宏, 植田 茂, 森田 和豊, 福原 崇介, 前原 喜彦  日本外科学会雑誌  110-  (2)  295  -295  2009/02/25
• SF-019-3 肝肺症候群を合併した肝硬変症例に対する生体肝移植の治療成績(肝移植1,サージカルフォーラム,第109回日本外科学会定期学術集会)

  永田 茂行, 武冨 紹信, 副島 雄二, 池上 徹, 武石 一樹, 戸島 剛男, 福原 崇介, 森田 和豊, 植田 茂, 実藤 健作, 井口 友宏, 杉町 圭史, 祇園 智信, 前原 喜彦  日本外科学会雑誌  110-  (2)  247  -247  2009/02/25
• VW-5-8 生体肝移植における3D-CTによる中肝静脈灌流域術前評価と手術成績(出血の少ない肝切除-肝静脈灌流域を考慮した肝切除の実際,ビデオワークショップ,第109回日本外科学会定期学術集会)

  武冨 紹信, 福原 崇介, 戸島 剛男, 武石 一樹, 梅田 健二, 植田 茂, 森田 和豊, 實藤 健作, 井口 友宏, 永田 茂行, 杉町 圭史, 池上 徹, 祇園 智信, 副島 雄二, 前原 喜彦  日本外科学会雑誌  110-  (2)  191  -191  2009/02/25
• HP-174-1 生体肝移植後における術前血清ナトリウム値の臨床的意義(肝移植2,ハイブリッドポスター,第109回日本外科学会定期学術集会)

  福原 崇介, 武冨 紹信, 池上 徹, 武石 一樹, 戸島 剛男, 梅田 健二, 森田 和豊, 植田 茂, 實藤 健作, 井口 友宏, 永田 茂行, 杉町 圭史, 祇園 智信, 吉住 朋晴, 副島 雄二, 前原 喜彦  日本外科学会雑誌  110-  (2)  686  -686  2009/02/25
• HP-174-2 生体肝移植ドナー適応境界症例における手術成績の検討(肝移植2,ハイブリッドポスター,第109回日本外科学会定期学術集会)

  祇園 智信, 武冨 紹信, 武石 一樹, 戸島 剛男, 福原 崇介, 森田 和豊, 植田 茂, 井口 友宏, 實藤 健作, 永田 茂行, 杉町 圭史, 池上 徹, 副島 雄二, 前原 喜彦  日本外科学会雑誌  110-  (2)  686  -686  2009/02/25
• HP-174-4 60歳以上のレシピエントに対する生体肝移植の治療成績(肝移植2,ハイブリッドポスター,第109回日本外科学会定期学術集会)

  梅田 健二, 武冨 紹信, 副島 雄二, 井口 友宏, 福原 崇介, 森田 和豊, 植田 茂, 實藤 健作, 永田 茂行, 池上 徹, 前原 喜彦  日本外科学会雑誌  110-  (2)  686  -686  2009/02/25
• O-1-178 成人間生体肝移植左葉ドナーにおける右葉残肝うっ血領域の検討(肝 移植1,一般演題(口演),第63回日本消化器外科学会総会)

  福原 崇介, 武冨 紹信, 副島 雄二, 萱島 寛人, 丸山 晴司, 原田 昇, 杉町 圭史, 山下 洋市, 住吉 朋晴, 前原 喜彦  日本消化器外科学会雑誌  41-  (7)  1175  -1175  2008/07/01
• SY-2-1-3 肝切除後胆汁瘻の防止対策 : 術中胆汁瘻試験とドレーンT-bil濃度測定によるモニタリング(シンポジウム2-1 消化器外科手術式別の重症合併症とその発生防止対策 肝胆膵,第63回日本消化器外科学会総会)

  山下 洋市, 武冨 紹信, 森田 和豊, 福原 崇介, 植田 茂, 實藤 健作, 萱島 寛人, 丸山 清司, 杉町 圭史, 前原 喜彦  日本消化器外科学会雑誌  41-  (7)  996  -996  2008/07/01
• DP-114-2 肝細胞癌におけるDNA polymeraseδ発現の生物学的意義に関する研究(第108回日本外科学会定期学術集会)

  實藤 健作, 武冨 紹信, 副島 雄二, 吉住 朋晴, 山下 洋市, 杉町 圭史, 丸山 晴司, 原田 昇, 萱島 寛人, 井口 友宏, 植田 茂, 森田 和豊, 福原 崇介, 梅田 健二, 前原 喜彦  日本外科学会雑誌  109-  (2)  572  -572  2008/04/25
• VS-5-1 硬変合併肝細胞癌に対するLimited resectionの最近の工夫(第108回日本外科学会定期学術集会)

  山下 洋市, 武冨 紹信, 森田 和豊, 福原 崇介, 植田 茂, 實藤 健作, 原田 昇, 杉町 圭史, 丸山 晴司, 吉住 朋晴, 副島 雄二, 前田 貴司, 前原 喜彦  日本外科学会雑誌  109-  (2)  110  -110  2008/04/25
• VD-007-2 肝切除中の偶発的肝外胆管損傷に対するマネージメント : 生体肝移植の経験を活かして(第108回日本外科学会定期学術集会)

  武冨 紹信, 森田 和豊, 福原 崇介, 梅田 健二, 植田 茂, 實藤 健作, 井口 友宏, 萱島 寛人, 原田 昇, 丸山 清司, 杉町 圭史, 山下 洋市, 吉住 朋晴, 副島 雄二, 前原 喜彦  日本外科学会雑誌  109-  (2)  329  -329  2008/04/25
• DP-137-4 肝内胆管癌におけるFocal Adhesion Kinase (FAK)発現の臨床的意義(第108回日本外科学会定期学術集会)

  大田 隆司, 武冨 紹信, 森田 和豊, 福原 崇介, 植田 茂, 實藤 健作, 萱島 寛人, 丸山 晴司, 杉町 圭史, 山下 洋市, 吉住 朋晴, 副島 雄二, 相島 慎一, 前原 喜彦  日本外科学会雑誌  109-  (2)  618  -618  2008/04/25
• SF-062-4 生体肝移植後の肝細胞癌再発予防を目的としたIL-12/DC併用療法によるNeoadjuvant immunotherapyの検討(第108回日本外科学会定期学術集会)

  萱島 寛人, 武冨 紹信, 岡野 慎二, 福原 崇介, 森田 和豊, 植田 茂, 實藤 健作, 井口 友宏, 原田 昇, 杉町 圭史, 丸山 晴司, 山下 洋市, 吉住 朋晴, 副島 雄二, 前原 喜彦  日本外科学会雑誌  109-  (2)  264  -264  2008/04/25
• SF-055-4 生体肝移植における摘出肝門脈グラフトを用いた中肝静脈分枝再建の意義(第108回日本外科学会定期学術集会)

  梅田 健二, 武冨 紹信, 副島 雄二, 吉住 朋晴, 池上 徹, 福原 崇介, 森田 和豊, 植田 茂, 實藤 健作, 井口 友宏, 萱島 寛人, 原田 昇, 杉町 圭史, 丸山 晴司, 山下 洋市, 前原 喜彦  日本外科学会雑誌  109-  (2)  255  -255  2008/04/25
• SF-055-3 成人間生体肝移植における過小グラフト症候群に対する同時性脾摘術の有効性(第108回日本外科学会定期学術集会)

  福原 崇介, 武冨 紹信, 吉住 朋晴, 梅田 健二, 森田 和豊, 植田 茂, 實藤 健作, 井口 友宏, 萱島 寛人, 丸山 晴司, 原田 昇, 杉町 圭史, 山下 洋市, 副島 雄二, 前原 喜彦  日本外科学会雑誌  109-  (2)  255  -255  2008/04/25
• SF-056-4 生体肝移植におけるB型肝炎関連ドナーグラフト使用の長期成績とその妥当性(第108回日本外科学会定期学術集会)

  副島 雄二, 武冨 紹信, 吉住 朋晴, 池上 徹, 山下 洋市, 杉町 圭史, 丸山 晴司, 原田 昇, 萱島 寛人, 實藤 健作, 井口 友宏, 植田 茂, 福原 崇介, 森田 和豊, 前原 喜彦  日本外科学会雑誌  109-  (2)  256  -256  2008/04/25
• DP-170-2 生体肝移植ドナー脂肪肝のMRIによる評価(第108回日本外科学会定期学術集会)

  森田 和豊, 武冨 紹信, 杉町 圭史, 福原 崇介, 植田 茂, 實藤 健作, 井口 友宏, 萱島 寛人, 原田 昇, 丸山 晴司, 山下 洋市, 吉住 朋晴, 副島 雄二, 吉満 研吾, 前原 喜彦  日本外科学会雑誌  109-  (2)  684  -684  2008/04/25
• DP-168-1 右葉グラフトを用いた生体肝移植における術前グラフト肝うっ血域評価の有用性(第108回日本外科学会定期学術集会)

  吉住 朋晴, 武冨 紹信, 副島 雄二, 山下 洋市, 杉町 圭史, 丸山 晴司, 萱島 寛人, 福原 崇介, 森田 和豊, 植田 茂, 前原 喜彦  日本外科学会雑誌  109-  (2)  679  -679  2008/04/25
• DP-168-7 生体肝移植後肝癌再発のスコア予測に関する研究(第108回日本外科学会定期学術集会)

  原田 昇, 武冨 紹信, 副島 雄二, 吉住 朋晴, 池上 徹, 山下 洋市, 杉町 圭史, 萱島 寛人, 實藤 健作, 井口 友宏, 植田 茂, 森田 和豊, 丸山 晴司, 福原 崇介, 前原 喜彦  日本外科学会雑誌  109-  (2)  681  -681  2008/04/25

Research Projects

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

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/11 -2030/03 

  Author : 佐藤 佳, 福原 崇介, 松野 啓太, 齊藤 暁, 木村 香菜子
• Development of novel anti-viral therapeuticing an advanced screening technologys us

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/10 -2025/03 

  Author : 小林 弘一, 田村 友和, 福原 崇介, 應田 涼太
• The role of MHC class I transactivator NLRC5 in COVID-19

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2025/03 

  Author : 小林 弘一, 澤 洋文, 福原 崇介, 應田 涼太
• Promotion of emerging and re-emerging virus research using high-speed reverse genetics

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2024/03 

  Author : 福原 崇介, 小野 慎子, 津田 祥美

   

  これまでに申請者は高速リバースジェネティクス系による組換えSARS-CoV-2の簡便かつ迅速な作製系を確立している。この技術を用いて、SARS-CoV-2に認められた変異の中で、L452Rに着目し、この変異を持つ組換えウイルスを作製した。この変異を持つことによって、ワクチンおよび感染に伴って誘導される獲得免疫からウイルスが逃避できることを明らかにした。 次に、SARS-CoV-2のデルタ株に特徴的に存在するP681R変異を持った組換えウイルスを作製し、その性状解析を行なった。P681R変異はスパイクタンパク質の開裂効率に関与し、それが細胞の融合能に関わることを明らかにした。P681R変異はin vitroおよびin vivoでのウイルスの感染拡大効率に関与しており、この1アミノ酸置換によりウイルスの病原性が有意に変化することが明らかになった。さらに肺組織における炎症も強くなることが明らかになった。この結果をNature誌に公表した。さらに、オミクロン株（BA.1株）の意義を解明するために、臨床分離株および組換えウイルスを用いた解析を行なった。オミクロン株のスパイクはデルタ株や従来株と比べて開裂効率が悪く、感染拡大効率が悪いことが明らかになった。in vivoにおいても気管支上皮から肺胞に感染が広がりにくいことがわかり、逆にオミクロン株は気管支上皮細胞に感染細胞が持続的に残存することが明らかになった。オミクロン株の病原性および炎症の程度は弱毒の方向に進化していることがわかった。この研究成果をNature誌に公表した。
• Characterization of SPP involved in pathogenesis of HCV

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2015/04 -2018/03 

  Author : Matsuura Yoshiharu, OKAMOTO Toru, FUKUHARA Takasuke

   

  We have previously reported that the maturation of core protein of hepatitis C virus (HCV) by the cleavage with signal peptide peptidase (SPP) is essential for propagation of HCV. In this study, we examined the inhibitory activity of inhibitors for γ-secretase, another intramembrane cleaving protease, to SPP and revealed that interaction of Val223 in SPP with dibenzoazepine structure in the γ-secretase inhibitors is critical for inhibition of SPP. Treatment with SPP suppressed maturation of HCV core proteins of all genotypes of HCV and did not induce emergence of drug-resistant viruses. These results suggest that SPP is an ideal target for the development of therapeutics against chronic hepatitis C.
• Genome Editing system in HCV research

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2016/03 

  Author : Fukuhara Takasuke

   

  In this study, for the detailed and reliable analyses about the interaction of HCV with host factors, we established gene knockout (KO) Huh7 cells. By using zinc finger nuclease (ZFN), TALEN and CRISPR/Cas9 system, several gene KO Huh7 cells were established. In the analyses about HCV assembly using ApoB, ApoE or MTTP KO cells, we clarified that apolipoproteins participate in the formation of infectious HCV particles. In the analyses about autophagy and HCV infection suggest that HCV-RNA replication inhibits de novo autophagy through the induction of LC3 lipidation around the replication complex. In addition, we revealed that miR-122 expression strongly enhances not replication but translation of viral RNA through the direct interaction with 5'UTR of HCV-RNA. The results in this study will provide clues to the life cycle of HCV and assist in the development of novel antivirals targeting the assembly process of HCV.
• Treatment strategies of recurrent hepatitis C after liver transplantation based on IL28B singlencleotide polymorphism.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011/04 -2014/03 

  Author : TAKETOMI Akinobu, MAEHARA Yoshihiko, SHIRABE Ken, MATSUURA Yoshiharu, FUKUHARA Takasuke, YOSHIZUMI Tomoharu, UCHIYAMA Hideaki

   

  A nationwide survey of living donor liver transplantation (LDLT) for hepatitis C virus-positive recipients was performed in Japan. Of 514 recipients collected in the study, 361 patients underwent antiviral treatment after liver transplantation. VR rate was 64.1%, and SVR rate was 38.3%. The 5-yr and 10-yr cumulative patient survival with SVR (94.1% and 83.0%) were significantly superior than those without SVR (79.7% and 60.8%). Presence of the major allele (TT) in both the recipient and the donor corresponded to SVR of 58.2%. Presence of the minor allele (TG or GG) in either the recipient or the donor corresponded to SVR of 15.7%, 16.7% and 28.5%. Multivariate analysis revealed that genotype of IL28B polymorphisms in either the recipient or donor was an independent factor for achieving SVR. In conclusion, our study demonstrated that both donor and recipient IL28B genotype were strongly associated with graft survival and response to IFN/RBV therapy for LDLT recipients.
• Mechanistic analysis about the association of IL28B genetic polymorphism with IFN sensitivity in HCV infected patients

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011 -2012 

  Author : FUKUHARA Takasuke

   

  In this study, we evaluated the importance of IL28B genetic polymorphism in IFN sensitivity for recurrent hepatitis C after liver transplantation. Interestingly, genetic variability in both recipients and donors was significantly associated with IFN sensitivity, suggesting crosstalk between lymphocyte and HCV-infected hepatocyte participates in the IFN sensitivity. In addition, we established allele-specific IL28B knockout Huh7 cells by using chimeric nucleases.