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, Elsevier BV, 2024年06月
研究論文（学術雑誌）

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年02月14日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年02月14日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年02月13日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年02月08日, ［国際誌］
英語, 研究論文（学術雑誌）, 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
Research Square Platform LLC, 2024年01月19日
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, Elsevier BV, 2024年01月
研究論文（学術雑誌）

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年11月07日, ［国際誌］
英語, 研究論文（学術雑誌）, 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, American Society for Microbiology, 2023年10月31日
研究論文（学術雑誌）, 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
Cold Spring Harbor Laboratory, 2023年10月13日
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.

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年10月05日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

牛ウイルス性下痢ウイルスの迅速な組換えウイルスの作出　　　　　　　　　　　　　　　
田村 友和, 荻野 紗帆, 日尾野 隆大, 鈴木 理滋, 鈴木 紗織, 磯田 典和, 迫田 義博, 福原 崇介
日本獣医学会学術集会講演要旨集, 166回, 134, 134, (公社)日本獣医学会, 2023年09月
日本語

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年07月24日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

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年05月19日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年05月16日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年05月11日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Protein quality control machinery supports primary ciliogenesis by eliminating GDP-bound Rab8-family GTPases.
Takahashi, T, Shirai, J, Matsuda, M, Nakanaga, S, Matsushita, S, Wakita, K, Hayashishita, M, Suzuki, R, Noguchi, A, Yokota, N, Kawahara, H
iScience, 2023年04月, ［査読有り］
英語, 研究論文（学術雑誌）

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, Public Library of Science (PLoS), 2023年03月27日
研究論文（学術雑誌）, 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年02月06日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年, ［国際誌］
英語, 研究論文（学術雑誌）, 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, Elsevier BV, 2022年12月
研究論文（学術雑誌）

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
Cold Spring Harbor Laboratory, 2022年11月22日
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年11月09日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年11月, ［国際誌］
英語, 研究論文（学術雑誌）, 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年10月18日, ［国際誌］
英語, 研究論文（学術雑誌）, Smoking is one of the risk factors most closely related to the severity of COVID-19. However, the relationship between smoking history and SARS-CoV-2 infectivity is unknown. In this study, we evaluated the ACE2 expression level in the lungs of current smokers, ex-smokers, and non-smokers. The ACE2 expression level of ex-smokers who smoked cigarettes until recently (cessation period shorter than 6 months) was higher than that of non-smokers and ex-smokers with a long history of non-smoking (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 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. This article is protected by copyright. All rights reserved.

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, Mst 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, 185, 21, 3992, 4007, 2022年10月13日, ［国際誌］
英語, 研究論文（学術雑誌）, After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.

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, 2022年07月
研究論文（学術雑誌）, 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年06月03日, ［国際誌］
英語, 研究論文（学術雑誌）, 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年05月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

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
Cold Spring Harbor Laboratory, 2022年02月24日
研究論文（学術雑誌）

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年02月01日, ［国際誌］
英語, 研究論文（学術雑誌）, The emergence of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, Omicron, is an urgent global health concern (ref.1). Our statistical modelling suggests that Omicron has spread more rapidly than the Delta variant in several countries including South Africa. Cell culture experiments show that Omicron is less fusogenic than Delta and an ancestral SARS-CoV-2 strain. Although the spike (S) protein of Delta is efficiently cleaved into two subunits, which facilitates cell-cell fusion2,3, Omicron S is less efficiently cleaved compared to Delta S and ancestral SARS-CoV-2 S. Furthermore, in a hamster model, Omicron shows decreased lung infectivity and is less pathogenic compared to Delta and ancestral SARS-CoV-2.

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, 2021年11月25日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

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年04月01日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

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, 2020年12月18日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation.
Rigel Suzuki, Hiroyuki Kawahara
EMBO reports, 17, 6, 842, 57, 2016年06月, ［査読有り］, ［国際誌］
英語, The majority of transmembrane proteins are integrated into the endoplasmic reticulum (ER) by virtue of a signal sequence-mediated co-translational process. However, a substantial portion of transmembrane proteins fails to reach the ER, leading to mislocalized cytosolic polypeptides. Their appropriate recognition and removal are of the utmost importance to avoid proteotoxic stress. Here, we identified UBQLN4 as a BAG6-binding factor that eliminates newly synthesized defective polypeptides. Using a truncated transmembrane domain protein whose degradation occurs during a pre-ER incorporation process as a model, we show that UBQLN4 recognizes misassembled proteins in the cytoplasm and targets these to the proteasome. We suggest that the exposed transmembrane segment of the defective polypeptides is essential for the UBQLN4-mediated substrate discrimination. Importantly, UBQLN4 recognizes not only the defective model substrate but also a pool of endogenous defective proteins that were induced by the depletion of the SRP54 subunit of the signal recognition particle. This study identifies a novel quality control mechanism for newly synthesized and defective transmembrane domain polypeptides that fail to reach their correct destination at the ER membrane.

A conserved island of BAG6/Scythe is related to ubiquitin domains and participates in short hydrophobicity recognition.
Hirofumi Tanaka, Toshiki Takahashi, Yiming Xie, Ryosuke Minami, Yuko Yanagi, Mizuki Hayashishita, Rigel Suzuki, Naoto Yokota, Masumi Shimada, Tsunehiro Mizushima, Naoyuki Kuwabara, Ryuichi Kato, Hiroyuki Kawahara
The FEBS journal, 283, 4, 662, 77, 2016年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BAG6 (also called Scythe) interacts with the exposed hydrophobic regions of newly synthesized proteins and escorts them to the degradation machinery through mechanisms that remain to be elucidated. In this study, we provide evidence that BAG6 physically interacts with the model defective protein substrate CL1 in a manner that depends directly on its short hydrophobicity. We found that the N terminus of BAG6 contains an evolutionarily conserved island tentatively designated the BAG6 ubiquitin-linked domain. Partial deletion of this domain in the BAG6 N-terminal fragment abolished in cell recognition of polyubiquitinated polypeptides as well as the hydrophobicity-mediated recognition of the CL1 degron in cell and in vitro. These observations suggest a mechanism whereby the BAG6 ubiquitin-linked domain provides a platform for discriminating substrates with shorter hydrophobicity stretches as a signal for defective proteins.

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年

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年

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年

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年

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年

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年

RNA結合タンパク質が司るHBVの新しい複製機構の解明
科学研究費助成事業
2024年04月01日 - 2026年03月31日
鈴木 理滋
日本学術振興会, 若手研究, 北海道大学, 24K18446

オートファジーを介した新型コロナウイルスの新しい病原性機構の解明
科学研究費助成事業 若手研究
2021年04月01日 - 2023年03月31日
鈴木 理滋
新型コロナウイルス感染症（COVID-19）の症状は多岐に渡り、無症状感染者もいれば重篤な急性呼吸器症状を呈する感染者も存在する。しかしながら、なぜ症状に違いが生じるのか、その原因は不明のままである。最近、無症状患者と重症患者から単離された新型コロナウイルス（SARS-CoV-2）のゲノム解析が行われ、重症患者ではnsp6タンパク質に変異（T11083G変異）があることが明らかになった。この結果からnsp6の変異がSARS-CoV-2の病原性発現と深く関与していることが示唆されるが、そのメカニズムは明らかにされていない。nsp6は細胞内で合成されるとオートファジーを誘導し、それを自身のゲノムRNA複製に利用する。このことから、nsp6の変異がオートファジーに何かしらの影響を与えた結果、病原性を発現した可能性が考えられる。そこで本申請研究では当研究室で新たに開発されたSARS-CoV-2のリバースジェネティクス法を駆使して、nsp6変異株を作出し、nsp6によるオートファジー誘導機構を解明することでSARS-Cov-2の病原性発現メカニズムの理解を目指す。本年度はまずSARS-CoV-2に感染した細胞でオートファジーが誘導されていることをオートファジーマーカーであるLC3を指標に調べた結果、感染細胞でオートファジーが誘導されていることを確認した。また、リバースジェネテクス法を用いてT11083G変異を有するSARS-CoV-2の作製に成功した。更に、この変異ウイルスと野生株の増殖性について培養細胞を用いて検討した所、変異ウイルスの増殖性が高い可能性を示した。今後は他の培養細胞を用いた増殖能の検討やハムスターを用いたin vivo系の実験を行う予定である。
日本学術振興会, 若手研究, 北海道大学, 21K15452

HBc抗体陽性非ウイルス性肝癌のHBVゲノム挿入部位の同定及び発癌機構の解明
科学研究費助成事業 研究活動スタート支援
2020年09月11日 - 2022年03月31日
鈴木 理滋
近年、非ウイルス性肝癌患者の中にHBV既往感染マーカーであるHBc抗体陽性の患者がいることが明らかになった。これはHBV既往感染と肝癌発症に関係性がある事を示しているが、どのようにHBV既往感染者は肝癌を発症するのか謎に満ちていた。そこで、実際の検体を用いてHBVゲノム挿入による発癌の影響を調べるため、次世代シーケンス解析を試みた。新型コロナウイルスの影響により次世代シーケンス解析ができなかったが、HBVゲノムの挿入が発癌に関与することを明らかにするため、既に培養細胞で報告されているHBVゲノムの挿入部位を欠損した時の影響を調べた結果、HBVゲノムの挿入は腫瘍形成に関与することを明らかにした。
日本学術振興会, 研究活動スタート支援, 大阪大学, 20K22951

ＵＢＱＬＮ４複合体が司る膜タンパク質の新しい品質管理機構
科学研究費助成事業 特別研究員奨励費
2014年04月25日 - 2017年03月31日
鈴木 理滋
本研究の目的は、埋め込みに失敗した不良膜タンパク質の代謝メカニズムの解明である。この目的を達成するため、本研究者はこれまで不良膜タンパク質モデルをデザインし、このモデル基質の認識・分解に関わる因子としてUBALN4を同定した。しかしながら、UBQLN4が不良膜タンパク質モデルだけでなく、細胞内に生じうる内在性不良膜タンパク質の分解に関与するかは謎に満ちていた。そこで本研究者は内在性不良膜タンパク質を誘導するため、トランスロコン特異的阻害剤を用いて検証した。UBQLN4 KD条件下かつトランスロコン阻害剤を用いると、コントロールKD条件下に比べ、細胞内に蓄積するユビキチン化タンパク質量が増加した。また、免疫染色法を行った結果、ユビキチン陽性凝集体が観察された。この結果はUBQLN4が細胞内に生じる内在性不良膜タンパク質の分解に関わることを示唆している。次に本研究者は蓄積したユビキチン化タンパク質の中に膜タンパク質が含まれていることを検証するため、一回膜貫通型タンパク質VCAM1の挙動を調査した。VCAM1はトランスロコン阻害剤存在下で不良膜タンパク質として速やかに分解されるタンパク質である。UBQLN4 KD条件下かつトランスロコン阻害剤存在下で、不良VCAM1が安定化すること、また可溶性・不溶性分画を行った結果、不溶性画分に不良VCAM1が多く存在することを見出した。これらの結果から、UBQLN4は内在性不良膜タンパク質の分解に関与することを初めて明らかにした。
日本学術振興会, 特別研究員奨励費, 首都大学東京, 14J05957