研究者データベース
| 板倉 友香里(イタクラ ユカリ) |
| 創成研究機構ワクチン研究開発拠点 |
| 特任助教 |
Last Updated :2023/12/05
研究活動情報
論文
- Koshiro Tabata, Yukari Itakura, Takuma Ariizumi, Manabu Igarashi, Hiroko Kobayashi, Kittiya Intaruck, Mai Kishimoto, Shintaro Kobayashi, William W Hall, Michihito Sasaki, Hirofumi Sawa, Yasuko OrbaApplied microbiology and biotechnology 2023年10月13日The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections. KEY POINTS: • The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs. • Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs. • Inoculation of mutated SVPs induces antibodies with low cross-reactivity.
- 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 SatoNature communications 14 1 2800 - 2800 2023年05月16日 [査読有り]
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. - 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 SatoNature communications 14 1 2671 - 2671 2023年05月11日 [査読有り]
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. - Yukari Itakura, Koshiro Tabata, Takeshi Saito, Kittiya Intaruck, Nijiho Kawaguchi, Mai Kishimoto, Shiho Torii, Shintaro Kobayashi, Naoto Ito, Michiko Harada, Satoshi Inoue, Ken Maeda, Ayato Takada, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito SasakiJournal of virology 97 5 e0043823 2023年04月12日 [査読有り]
Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein. - Mai Kishimoto, Masahiro Kajihara, Koshiro Tabata, Yukari Itakura, Shinsuke Toba, Seiya Ozono, Yuko Sato, Tadaki Suzuki, Naoto Ito, Katendi Changula, Yongjin Qiu, Akina Mori-Kajihara, Yoshiki Eto, Hayato Harima, Daniel Mwizabi, Bernard M Hang'ombe, William W Hall, Ayato Takada, Yasuko Orba, Hirofumi Sawa, Michihito SasakiJournal of virology 97 1 e0145522 2023年01月31日 [査読有り]
Rotavirus A (RVA) causes diarrheal disease in humans and various animals. Recent studies have identified bat and rodent RVAs with evidence of zoonotic transmission and genome reassortment. However, the virological properties of bat and rodent RVAs with currently identified genotypes still need to be better clarified. Here, we performed virus isolation-based screening for RVA in animal specimens and isolated RVAs (representative strains: 16-06 and MpR12) from Egyptian fruit bat and Natal multimammate mouse collected in Zambia. Whole-genome sequencing and phylogenetic analysis revealed that the genotypes of bat RVA 16-06 were identical to that of RVA BATp39 strain from the Kenyan fruit bat, which has not yet been characterized. Moreover, all segments of rodent RVA MpR12 were highly divergent and assigned to novel genotypes, but RVA MpR12 was phylogenetically closer to bat RVAs than to other rodent RVAs, indicating a unique evolutionary history. We further investigated the virological properties of the isolated RVAs. In brief, we found that 16-06 entered cells by binding to sialic acids on the cell surface, while MpR12 entered in a sialic acid-independent manner. Experimental inoculation of suckling mice with 16-06 and MpR12 revealed that these RVAs are causative agents of diarrhea. Moreover, 16-06 and MpR12 demonstrated an ability to infect and replicate in a 3D-reconstructed primary human intestinal epithelium with comparable efficiency to the human RVA. Taken together, our results detail the unique genetic and virological features of bat and rodent RVAs and demonstrate the need for further investigation of their zoonotic potential. IMPORTANCE Recent advances in nucleotide sequence detection methods have enabled the detection of RVA genomes from various animals. These studies have discovered multiple divergent RVAs and have resulted in proposals for the genetic classification of novel genotypes. However, most of these RVAs have been identified via dsRNA viral genomes and not from infectious viruses, and their virological properties, such as cell/host tropisms, transmissibility, and pathogenicity, are unclear and remain to be clarified. Here, we successfully isolated RVAs with novel genome constellations from three bats and one rodent in Zambia. In addition to whole-genome sequencing, the isolated RVAs were characterized by glycan-binding affinity, pathogenicity in mice, and infectivity to the human gut using a 3D culture of primary intestinal epithelium. Our study reveals the first virological properties of bat and rodent RVAs with high genetic diversity and unique evolutional history and provides basic knowledge to begin estimating the potential of zoonotic transmission. - Michihito Sasaki, Koshiro Tabata, Mai Kishimoto, Yukari Itakura, Hiroko Kobayashi, Takuma Ariizumi, Kentaro Uemura, Shinsuke Toba, Shinji Kusakabe, Yuki Maruyama, Shun Iida, Noriko Nakajima, Tadaki Suzuki, Shinpei Yoshida, Haruaki Nobori, Takao Sanaki, Teruhisa Kato, Takao Shishido, William W Hall, Yasuko Orba, Akihiko Sato, Hirofumi SawaScience translational medicine 15 679 eabq4064 2023年01月18日 [査読有り]
In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (Mpro; also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to submicromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern, including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), has remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19. - Kittiya Intaruck, Yukari Itakura, Mai Kishimoto, Herman M Chambaro, Agus Setiyono, Ekowati Handharyani, Kentaro Uemura, Hayato Harima, Satoshi Taniguchi, Masayuki Saijo, Takashi Kimura, Yasuko Orba, Hirofumi Sawa, Michihito SasakiVirology 575 10 - 19 2022年10月 [査読有り]
Nelson Bay orthoreovirus (NBV) is an emerging bat-borne virus and causes respiratory tract infections in humans sporadically. Over the last two decades, several strains genetically related to NBV were isolated from humans and various bat species, predominantly in Southeast Asia (SEA), suggesting a high prevalence of the NBV species in this region. In this study, an orthoreovirus (ORV) belonging to the NBV species was isolated from Indonesian fruit bats' feces, tentatively named Paguyaman orthoreovirus (PgORV). Serological studies revealed that 81.2% (108/133) of Indonesian fruit bats sera had neutralizing antibodies against PgORV. Whole-genome sequencing and phylogenetic analysis of PgORV suggested the occurrence of past reassortments with other NBV strains isolated in SEA, indicating the dispersal and circulation of NBV species among bats in this region. Intranasal PgORV inoculation of laboratory mice caused severe pneumonia. Our study characterized PgORV's unique genetic background and highlighted the potential risk of PgORV-related diseases in Indonesia. - Koshiro Tabata, Yukari Itakura, Shinsuke Toba, Kentaro Uemura, Mai Kishimoto, Michihito Sasaki, Jessica J Harrison, Akihiko Sato, William W Hall, Roy A Hall, Hirofumi Sawa, Yasuko OrbaBiochemical and biophysical research communications 616 115 - 121 2022年08月06日 [査読有り]
The genus Flavivirus includes pathogenic tick- and mosquito-borne flaviviruses as well as non-pathogenic insect-specific flaviviruses (ISFVs). Phylogenetic analysis based on whole amino acid sequences has indicated that lineage II ISFVs have similarities to pathogenic flaviviruses. In this study, we used reactive analysis with immune serum against Psorophora flavivirus (PSFV) as a lineage IIa ISFV, and Barkeji virus (BJV) as a lineage IIb ISFV, to evaluate the antigenic similarity among lineage IIa and IIb ISFVs, and pathogenic mosquito-borne flaviviruses (MBFVs). Binding and antibody-dependent enhancement assays showed that anti-PSFV sera had broad cross-reactivity with MBFV antigens, while anti-BJV sera had low cross-reactivity. Both of the lineage II ISFV antisera were rarely observed to neutralize MBFVs. These results suggest that lineage IIa ISFV PSFV has more antigenic similarity to MBFVs than lineage IIb ISFV BJV. - Yukari Itakura, Koshiro Tabata, Kohei Morimoto, Naoto Ito, Herman M Chambaro, Ryota Eguchi, Ken-Ichi Otsuguro, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito SasakiiScience 25 4 104122 - 104122 2022年04月15日 [査読有り]
The amino acid residue at position 333 of the rabies virus (RABV) glycoprotein (G333) is a major determinant of RABV pathogenicity. Virulent RABV strains possess Arg333, whereas the attenuated strain HEP-Flury (HEP) possesses Glu333. To investigate the potential attenuation mechanism dependent on a single amino acid at G333, comparative analysis was performed between HEP and HEP333R mutant with Arg333. We examined their respective tropism for astrocytes and the subsequent immune responses in astrocytes. Virus replication and subsequent interferon (IFN) responses in astrocytes infected with HEP were increased compared with HEP333R both in vitro and in vivo. Furthermore, involvement of IFN in the avirulency of HEP was demonstrated in IFN-receptor knockout mice. These results indicate that Glu333 contributes to RABV attenuation by determining the ability of the virus to infect astrocytes and stimulate subsequent IFN responses. - Michihito Sasaki, Mai Kishimoto, Yukari Itakura, Koshiro Tabata, Kittiya Intaruck, Kentaro Uemura, Shinsuke Toba, Takao Sanaki, Akihiko Sato, William W Hall, Yasuko Orba, Hirofumi SawaBiochemical and biophysical research communications 577 146 - 151 2021年11月05日 [査読有り]
The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2. - Fumihiro Kodama, Hiroki Yamaguchi, Eunsil Park, Kango Tatemoto, Mariko Sashika, Ryo Nakao, Yurino Terauchi, Keita Mizuma, Yasuko Orba, Hiroaki Kariwa, Katsuro Hagiwara, Katsunori Okazaki, Akiko Goto, Rika Komagome, Masahiro Miyoshi, Takuya Ito, Kimiaki Yamano, Kentaro Yoshii, Chiaki Funaki, Mariko Ishizuka, Asako Shigeno, Yukari Itakura, Lesley Bell-Sakyi, Shunji Edagawa, Atsushi Nagasaka, Yoshihiro Sakoda, Hirofumi Sawa, Ken Maeda, Masayuki Saijo, Keita MatsunoNature communications 12 1 5539 - 5539 2021年09月20日 [査読有り]
The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014-2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan. - Michihito Sasaki, Shinsuke Toba, Yukari Itakura, Herman M Chambaro, Mai Kishimoto, Koshiro Tabata, Kittiya Intaruck, Kentaro Uemura, Takao Sanaki, Akihiko Sato, William W Hall, Yasuko Orba, Hirofumi SawamBio 12 4 e0141521 2021年08月31日 [査読有り]
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) possesses a discriminative polybasic cleavage motif in its spike protein that is recognized by the host furin protease. Proteolytic cleavage activates the spike protein, thereby affecting both the cellular entry pathway and cell tropism of SARS-CoV-2. Here, we investigated the impact of the furin cleavage site on viral growth and pathogenesis using a hamster animal model infected with SARS-CoV-2 variants bearing mutations at the furin cleavage site (S gene mutants). In the airway tissues of hamsters, the S gene mutants exhibited low growth properties. In contrast to parental pathogenic SARS-CoV-2, hamsters infected with the S gene mutants showed no body weight loss and only a mild inflammatory response, thereby indicating the attenuated variant nature of S gene mutants. This transient infection was sufficient for inducing protective neutralizing antibodies that cross-react with different SARS-CoV-2 lineages. Consequently, hamsters inoculated with S gene mutants showed resistance to subsequent infection with both the parental strain and the currently emerging SARS-CoV-2 variants belonging to lineages B.1.1.7 and P.1. Taken together, our findings revealed that the loss of the furin cleavage site causes attenuation in the airway tissues of hamsters and highlighted the potential benefits of S gene mutants as potential immunogens. IMPORTANCE SARS-CoV-2 uses its spike protein to enter target cells. The spike protein is cleaved by a host protease, and this event facilitates viral entry and broadens cell tropism. In this study, we employed SARS-CoV-2 mutants lacking the S protein cleavage site and characterized their growth and pathogenicity using hamsters, a laboratory animal model for SARS-CoV-2 infection. These mutants exerted low pathogenicity but induced sufficient levels of neutralizing antibodies in hamsters, which protected hamsters from rechallenge with pathogenic clinical SARS-CoV-2 strains. These virus mutants may be used as protective immunogens against SARS-CoV-2 infection. - Michihito Sasaki, Yukari Itakura, Mai Kishimoto, Koshiro Tabata, Kentaro Uemura, Naoto Ito, Makoto Sugiyama, Christida E Wastika, Yasuko Orba, Hirofumi SawaJournal of virology 95 11 2021年05月10日 [査読有り]
Group A rotaviruses (RVAs) are representative enteric virus species and major causes of diarrhea in humans and animals. The RVA virion is a triple-layered particle, and the outermost layer consists of the glycoprotein VP7 and spike protein VP4. To increase the infectivity of RVA, VP4 is proteolytically cleaved into VP5* and VP8* subunits by trypsin; and these subunits form a rigid spike structure on the virion surface. In this study, we investigated the growth of RVAs in cells transduced with type II transmembrane serine proteases (TTSPs), which cleave fusion proteins and promote infection by respiratory viruses, such as influenza viruses, paramyxoviruses, and coronaviruses. We identified TMPRSS2 and TMPRSS11D as host TTSPs that mediate trypsin-independent and multi-cycle infection by human and animal RVA strains. In vitro cleavage assays revealed that recombinant TMPRSS11D cleaved RVA VP4. We also found that TMPRSS2 and TMPRSS11D promote the infectious entry of immature RVA virions, but they could not activate nascent progeny virions in the late phase of infection. This observation differed from the TTSP-mediated activation process of paramyxoviruses, revealing the existence of virus species-specific activation processes in TTSPs. Our study provides new insights into the interaction between RVAs and host factors, and TTSP-transduced cells offer potential advantages for RVA research and development.ImportanceProteolytic cleavage of the viral VP4 protein is essential for virion maturation and infectivity in group A rotaviruses (RVAs). In cell culture, RVAs are propagated in culture medium supplemented with the exogenous protease trypsin, which cleaves VP4 and induces the maturation of progeny RVA virions. In this study, we demonstrated that the host proteases TMPRSS2 and TMPRSS11D mediate the trypsin-independent infection and growth of RVA. Our data revealed that the proteolytic activation of RVAs by TMPRSS2 and TMPRSS11D occurs at the viral entry step. Because TMPRSS2 and TMPRSS11D gene expression induced similar or higher levels of RVA growth as trypsin-supplemented culture, this approach offers potential advantages for RVA research and development. - Yukari Itakura, Keita Matsuno, Asako Ito, Markus Gerber, Matthias Liniger, Yuri Fujimoto, Tomokazu Tamura, Ken-Ichiro Kameyama, Masatoshi Okamatsu, Nicolas Ruggli, Hiroshi Kida, Yoshihiro SakodaVirus research 276 197809 - 197809 2020年01月15日 [査読有り]
Classical swine fever viruses (CSFVs) do typically not show cytopathic effect (CPE) in cell culture, while some strains such as vaccine strain the GPE- induce CPE in the swine kidney-derived CPK-NS cell line cultured in serum-free medium. These latter strains commonly lack Npro-mediated inhibition of type-I interferon (IFN) induction. In order to explore the molecular mechanisms of GPE--induced CPE, we analyzed the cellular pathways involved. In CPK-NS cells infected with the attenuated-vaccine-derived vGPE- strain, both, apoptosis and necroptosis were induced. Necroptosis was type-I IFN-dependent and critical for visible CPE. In contrast, the parental virulent vALD-A76 strain did not induce any of these pathways nor CPE. We used reverse genetics to investigate which viral factors regulate these cell-death pathways. Interestingly, a mutant vGPE- in which the Npro function was restored to inhibit type-I IFN induction did not induce necroptosis nor CPE but still induced apoptosis, while an Npro-mutant vALD-A76 incapable of inhibiting type-I IFN production induced necroptosis and CPE. Although Erns of CSFV is reportedly involved in controlling apoptosis, apoptosis induction by vGPE- or apoptosis inhibition by vALD-A76 were independent of the unique amino acid difference found in Erns of these two strains. Altogether, these results demonstrate that type-I IFN-dependent necroptosis related to non-functional Npro is the main mechanism for CPE induction by vGPE-, and that viral factor(s) other than Erns may induce or inhibit apoptosis in vGPE- or vALD-A76 infected CPK-NS cells, respectively.
その他活動・業績
- 板倉友香里, 板倉友香里, 松野啓太, 松野啓太, 伊藤麻子, 藤本悠理, 田村友和, 亀山健一郎, 岡松正敏, NICOLAS Ruggli, NICOLAS Ruggli, 喜田宏, 喜田宏, 澤洋文, 澤洋文, 迫田義博, 迫田義博 日本ウイルス学会学術集会プログラム・予稿集(Web) 67th 2019年
- 板倉友香里, 松野啓太, 岡松正敏, 迫田義博, 迫田義博 日本獣医学会学術集会講演要旨集 161st 345 -345 2018年08月21日 [査読無し][通常論文]
受賞
- 2023年03月 北海道大学 大塚賞
- 2022年09月 The 10th Sapporo Summer Symposium for One Health Best Oral Presentation Award
「Budding and virion morphogenesis of rabies virus regulated by ESCRT-I component TSG101」 - 2021年10月 日本獣医学会公衆衛生学分科会 奨励賞
「G蛋白質333位のアミノ酸が規定する狂犬病ウイルスの病原性発現機構の解析」 - 2019年10月 トガ・フラビ・ペスチウイルス研究会 奨励賞
「I型インターフェロン産生抑制機構を欠く豚コレラウイルスが豚腎臓由来無血清培養細胞に誘導する細胞変性効果のメカニズム」 - 2018年09月 日本獣医学会微生物学分科会 若手奨励賞
「豚コレラウイルス弱毒生ワクチン株GPF−を感染させたCPK-NS細胞における細胞変性効果のメカニズム」
共同研究・競争的資金等の研究課題
- 日本学術振興会：科学研究費助成事業 特別研究員奨励費研究期間 : 2021年04月 -2023年03月代表者 : 板倉 友香里【RABV免疫抗原の作出とマウスへの免疫】 RABVゲノム複製を担う因子の一つであるP蛋白質を抗体の標的候補とし、免疫抗原の作出を試みた。Hisタグ融合P蛋白質を発現、精製後、マウスに免疫した。また、HAタグ融合P蛋白質の発現細胞を用いて、抗P蛋白質抗体を検出するためのELISAを構築し、免疫したマウスにおける抗体産生を確認した。 【抗体クローンの選抜方法の検討】 今後取得するモノクローナル抗体の中から、P蛋白質に結合し、RABVのゲノム複製を阻害する抗体クローンを選抜する。第一スクリーニングとして、構築したELISAを実施し、P蛋白質に対する結合性を抗体クローンごとに評価する。また、第二スクリーニングとして、in vitro RNAポリメラーゼアッセイの構築を試みた。その結果、ウイルスゲノム複製を担う蛋白質の存在下で、RNA合成のシグナル増加が認められた。今後、構築したin vitro RNAポリメラーゼアッセイの至適条件を検討する。 【細胞膜透過性ペプチドの選抜】 抗体の細胞内移行に適した細胞膜透過性ペプチド（CPP: Cell Penetrating Peptide）とその付加部位を検討した。IgG抗体の、重鎖あるいは軽鎖のC末端に、6種類のCPPを付加し、計12種類のCPP付加IgG抗体を組換え蛋白質として作出した。標的蛋白質に対する結合性をELISAで評価した結果、全てのCPP付加IgG抗体は、CPPの付加により結合性が変化しなかった。また、免疫染色法、及びイムノブロット法により、CPPを付加した抗体が細胞内に移行することが確かめられた。