金井　亨輔 （カナイ　キョウスケ）

研究者情報

学位

• 博士（生命科学）（鳥取大学大学院）

ホームページURL

https://orcid.org/0000-0002-7984-207X

科研費研究者番号

• 20596621

ORCID ID

•  https://orcid.org/0000-0002-7984-207X

J-Global ID

• 201301027386223013

研究キーワード

• サイトカイン・ケモカイン   マウスγヘルペスウイルス68   ヒトヘルペスウイルス   エプスタイン・バーウイルス   ウイルス   

研究分野

• ライフサイエンス / ウイルス学

経歴

• 2023年10月 - 現在  鳥取大学医学部 感染制御学講座 ウイルス学分野准教授

所属学協会

• 日本免疫学会   日本ウイルス学会   

研究活動情報

論文

• Involvement of TLR4 in Acute Hepatitis Associated with Airway Infection of Murine γ-Herpesvirus 68.

  Kyosuke Kanai; Seiji Kageyama; Osamu Yoshie

  Journal of immunology (Baltimore, Md. : 1950) 2023年09月 

  Extrahepatic viral infections are often accompanied by acute hepatitis, as evidenced by elevated serum liver enzymes and intrasinusoidal infiltration of CD8+ T cells, without direct infection of the liver. An example is infectious mononucleosis caused by primary infection with EBV. Previously, we demonstrated that airway infection of mice with murine γ-herpesvirus 68 (MHV68), a murine model of EBV, caused liver inflammation with elevated serum liver enzymes and intrahepatic infiltration of IFN-γ-producing CD8+ T cells and NK cells. Mechanistically, the expression of the CXCR3-ligand chemokines, which are commonly induced by IFN-γ and attract IFN-γ-producing Th1-type cells via CXCR3, was upregulated in the liver. Importantly, the liver inflammation was suppressed by oral neomycin, an intestine-impermeable aminoglycoside, suggesting an involvement of some products from the intestinal microbiota. In this study, we showed that the liver inflammation and the expression of the CXCR3-ligand chemokines in the liver were effectively ameliorated by i.p. administration of anti-TLR4 mAb or C34, a TLR4 blocker, as well as in TLR4-deficient mice. Conversely, intrarectal inoculation of Escherichia coli as an extraintestinal source of LPS aggravated liver inflammation in MHV68-infected mice with increased expression of the CXCR3-ligand chemokines in the liver. In contrast, the lung inflammation in MHV68-infected mice was not affected by oral neomycin, i.p. administration of C34, or TLR4 deficiency. Collectively, the LPS-TLR4 pathway plays a pivotal role in the liver inflammation of MHV68-infected mice at least in part by upregulating the CXCR3-ligand chemokines in the liver.
• In Vitro Susceptibility of HIV Isolates with High Growth Capability to Antiretroviral Drugs.

  Alfredo A Hinay Jr; Kyosuke Kanai; Akeno Tsuneki-Tokunaga; Mizuki Komatsu; Elizabeth O Telan; Seiji Kageyama

  International journal of molecular sciences 23 23 2022年12月 

  It has been considered that reduced susceptibility to antiretroviral drugs is influenced by drug adherence, drug tolerance and drug-resistance-related mutations in the HIV genome. In the present study, we assessed the intrinsic high viral growth capability as a potential viral factor that may influence their susceptibility to antiretroviral drugs using an in vitro model. Phytohemagglutinin-activated peripheral blood mononuclear cells (1.5 × 106 cells) were infected with HIV isolates (106 copies/mL). The culture was carried out at different concentrations (0.001-20 μM) of 13 synthetic antiretroviral compounds (six nucleoside/nucleotide reverse transcriptase inhibitors, one non-nucleoside reverse transcriptase inhibitor, four integrase inhibitors, and two protease inhibitors), and HIV production was assessed using HIV-RNA copies in culture. The 90% inhibitory concentration (IC90) and pharmacokinetics of an antiretroviral agent were used as parameters to determine the reduced antiretroviral drug susceptibility of HIV isolates with high growth capability to synthetic antiretroviral compounds. The high growth capability of HIV isolates without any known drug resistance-related mutation affected their susceptibility to tenofovir (IC90 = 2.05 ± 0.40 μM), lamivudine (IC90 = 6.83 ± 3.96 μM), emtricitabine (IC90 = 0.68 ± 0.37 μM), and efavirenz (IC90 = 3.65 ± 0.77 μM). These antiretroviral drugs showed IC90 values close to or above the maximum plasma concentration against HIV isolates with high growth capability without any known drug resistance-related mutation. Our results may contribute to the development of effective strategies to tailor and individualize antiretroviral therapy in patients harboring HIV isolates with high growth capability.
• Pro-Inflammatory Cytokines and Interferon-Stimulated Gene Responses Induced by Seasonal Influenza A Virus with Varying Growth Capabilities in Human Lung Epithelial Cell Lines.

  Alfredo A Hinay Jr; Sosuke Kakee; Seiji Kageyama; Akeno Tsuneki-Tokunaga; Waldy Y Perdana; Yui Akena; Shota Nishiyama; Kyosuke Kanai

  Vaccines 10 9 2022年09月 

  In a previous study, we described the diverse growth capabilities of circulating seasonal influenza A viruses (IAVs) with low to high viral copy numbers in vitro. In this study, we analyzed the cause of differences in growth capability by evaluating pro-inflammatory cytokines (TNF-α, IL-6, IFN-β) and antiviral interferon-stimulated genes (ISG-15, IFIM1, and TRIM22). A549 cells (3.0 × 105 cells) were inoculated with circulating seasonal IAV strains and incubated for 6 and 24 h. In cells inoculated for 6 h, IAV production was assessed using IAV-RNA copies in the culture supernatant and cell pellets to evaluate gene expression. At 24 h post-infection, cells were collected for IFN-β and ISG-15 protein expression. A549 cells inoculated with seasonal IAV strains with a high growth capability expressed lower levels of IFN-β and ISGs than strains with low growth capabilities. Moreover, suppression of the JAK/STAT pathway enhanced the viral copies of seasonal IAV strains with a low growth capability. Our results suggest that the expression of ISG-15, IFIM1, and TRIM22 in seasonal IAV-inoculated A549 cells could influence the regulation of viral replication, indicating the existence of strains with high and low growth capability. Our results may contribute to the development of new and effective therapeutic strategies to reduce the risk of severe influenza infections.
• Local Spread of Influenza A (H1N1) Viruses Without A Mutation For The Maximum Duration of An Epidemic Season In Japan

  Seiji Kageyama; Akeno Tsuneki-Tokunaga; Kyosuke Kanai

  Archives of virology 167 1 195 - 199 2021年08月 

  Close observation of the local transmission of influenza A(H1N1) viruses enabled an estimation of the transmission period of the virus without a mutation. Of 4,448 isolates from 11 consecutive years, 237 isolates could be categorized into 57 strain groups with identical hemagglutinin genes. Transmission of these 57 strains was chased for the maximum duration of an epidemic season. In addition, 35 identical strains were recognized at the study site and other countries within 147 days. Consequently, it can be postulated that once an influenza virus enters a temperate country, the strain rarely mutates until the end of the season.
• Growth capability of epidemic influenza viruses in Japan since the 2009 H1N1 pandemic.

  Akeno Tsuneki-Tokunaga; Kyosuke Kanai; Asao Itagaki; Hideaki Tsuchie; Takayoshi Okada; Masaaki Kasagi; Kiyoshi Tanaka; Miho Aoki; Alfredo Jr A Hinay; Seiji Kageyama

  Archives of virology 166 4 1193 - 1196 2021年04月 

  The correlation of viral growth capability (n = 156) with the viral load in nasopharyngeal swabs (n = 76) was assessed. Epidemic influenza A/H1N1, A/H3N2, and B viruses showed a wide range of growth capability (104-1011 copies/mL) in Madin-Darby canine kidney cells. The growth was correlated with the nasopharyngeal viral load (r = 0.53). Six selected strains showed growth-dependent cell death (r = 0.96) in a growth kinetics assay. Epidemic influenza viruses exhibit a wide range of growth capability. Growth capability should be considered one of the key factors in disease prognosis.
• Intrinsic Replication Competences of HIV Strains After Zidovudine/Lamivudine/Nevirapine Treatment in the Philippines

  Seiji Kageyama; Alfredo Amolong Hinay; Elizabeth Freda Omengan Telan; Genesis May Jopson Samonte; Prisca Susan Agustin Leano; Akeno Tsuneki-Tokunaga; Kyosuke Kanai

  Journal of the International Association of Providers of AIDS Care (JIAPAC) 18 232595821985657 - 232595821985657 2019年01月 

  Although drug-resistant HIV variants are considered to be less fit than drug-susceptible viruses, replication competence of these variants harbored by patients has not yet been elucidated in detail. We herein assessed the replication competence of strains obtained from individuals receiving antiretroviral therapy. Among 11 306 participants in a drug resistance surveillance in the Philippines, 2629 plasma samples were obtained from individuals after a 12-month treatment with zidovudine (ZDV)/lamivudine (3TC)/nevirapine (NVP). The replication competence of HIV isolates was then assessed by reinoculation into seronegative peripheral blood mononuclear cells in the absence of drugs in vitro. The drug resistance rate was estimated to be 9.2%. Drug-resistant strains were still a minority of closely related strains in a phylogenetic cluster. Among the available 295 samples, 37 HIV strains were successfully isolated. Progeny viruses were produced at a wide range (5.1 × 10⁶ to 3.4 × 10⁹ copies/mL) in primary culture of peripheral blood mononuclear cells. The viral yields were higher than the corresponding plasma viral load (1300 to 3.4 × 10⁶ copies/mL) but correlated with those ( r = 0.4). These results suggest that strains with higher intrinsic replication competence are one of the primary targets of newly selected drugs at the increasing phase of the plasma viral load during antiretroviral therapy.
• Murine γ-Herpesvirus 68 Induces Severe Lung Inflammation in IL-27-Deficient Mice with Liver Dysfunction Preventable by Oral Neomycin.

  Kanai K; Park AM; Watanabe A; Arikawa T; Yasui T; Yoshida H; Tsunoda I; Yoshie O

  Journal of immunology (Baltimore, Md. : 1950) 200 8 2703 - 2713 2018年04月 [査読有り]
   

  IL-27 is an immunoregulatory cytokine consisting of p28 and EBI3. Its receptor also has two subunits, WSX1 and gp130. Although IL-27 promotes Th1 differentiation in naive T cells, it also induces IL-10 expression in effector Th1 cells to curtail excessive immune responses. By using p28-deficient mice and WSX1-deficient mice (collectively called IL-27–deficient mice), we examined the role of IL-27 in primary infection by murine g-herpesvirus 68 (MHV68), a murine model of EBV. Upon airway infection with MHV68, IL-27–deficient mice had more aggravated lung inflammation than wild-type mice, although MHV68 infection per se was better controlled in IL-27–deficient mice. Although epithelial cells and alveolar macrophages were primarily infected by MHV68, interstitial macrophages and dendritic cells were the major producers of IL-27. The lung inflammation of IL-27–deficient mice was characterized by more IFN-g–producing CD8+ T cells and fewer IL-10–producing CD8+ T cells than that of wild-type mice. An infectious mononucleosis–like disease was also aggravated in IL-27–deficient mice, with prominent splenomegaly and severe hepatitis. Infiltration of IFN-g–producing effector cells and upregulation of the CXCR3 ligand chemokines CXCL9, CXCL10, and CXCL11 were noted in the liver of MHV68-infected mice. Oral neomycin effectively ameliorated hepatitis, with decreased production of these chemokines in the liver, suggesting that the intestinal microbiota plays a role in liver inflammation through upregulation of these chemokines. Collectively, IL-27 is essential for the generation of IL-10–producing effector cells in primary infection by MHV68. Our findings may also provide new insight into the mechanism of hepatitis associated with infectious mononucleosis.
• IL-35 Suppresses Lipopolysaccharide-Induced Airway Eosinophilia in EBI3-Deficient Mice

  Kyosuke Kanai; Ah-Mee Park; Hiroki Yoshida; Ikuo Tsunoda; Osamu Yoshie

  JOURNAL OF IMMUNOLOGY 198 1 119 - 127 2017年01月 [査読有り]
   

  EBI3 functions as the subunit of immune-regulatory cytokines, such as IL-27 and IL-35, by pairing with p28 and p35, respectively. We treated wild-type and EBI3-deficient mice with intratracheal administration of LPS and obtained bronchoalveolar lavage fluid (BALF) 24 h later. Although neutrophils were the predominant cells in BALF from both groups of mice, eosinophils were highly enriched and there was increased production of eosinophil-attracting chemokines CCL11 and CCL24 in BALF of EBI3-deficient mice. The bronchial epithelial cells and alveolar macrophages were the major producers of CCL11 and CCL24. Because no such increases in eosinophils were seen in BALF of p28/IL-27-deficient mice or WSX-1/IL-27Ra subunit-deficient mice upon intratracheal stimulation with LPS, we considered that the lack of IL-35 was responsible for the enhanced airway eosinophilia in EBI3-deficient mice. In vitro, IL-35 potently suppressed production of CCL11 and CCL24 by human lung epithelial cell lines treated with TNF-alpha and IL-1 beta. IL-35 also suppressed phosphorylation of STAT1 and STAT3 and induced suppressor of cytokine signaling 3. In vivo, rIL-35 dramatically reduced LPS-induced airway eosinophilia in EBI3-deficient mice, with concomitant reduction of CCL11 and CCL24, whereas neutralization of IL-35 significantly increased airway eosinophils in LPS-treated wild-type mice. Collectively, our results suggest that IL-35 negatively regulates airway eosinophilia, at least in part by reducing the production of CCL11 and CCL24.
• Heat Shock Protein 27 Plays a Pivotal Role in Myofibroblast Differentiation and in the Development of Bleomycin-Induced Pulmonary Fibrosis.

  Ah-Mee Park; Kyosuke Kanai; Tatsuki Itoh; Takao Sato; Tatsuya Tsukui; Yutaka Inagaki; Moises Selman; Kouji Matsushima; Osamu Yoshie

  PloS one 11 2 e0148998  2016年 [査読有り]
   

  Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.
• Reactivation of persistent Epstein-Barr virus (EBV) causes secretion of thyrotropin receptor antibodies (TRAbs) in EBV-infected B lymphocytes with TRAbs on their surface

  Keiko Nagata; Yuji Nakayama; Katsumi Higaki; Marika Ochi; Kyosuke Kanai; Michiko Matsushita; Satoshi Kuwamoto; Masako Kato; Ichiro Murakami; Takeshi Iwasaki; Eiji Nanba; Hiroshi Kimura; Kazuhiko Hayashi

  AUTOIMMUNITY 48 5 328 - 335 2015年08月 [査読有り]
   

  Epstein-Barr virus (EBV) is a ubiquitous virus that infects most adults latently. It persists in B lymphocytes and reactivates occasionally. Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). We have reported that Graves' disease patients and healthy controls have EBV-infected lymphocytes that have TRAbs on their surface (TRAb(+)EBV(+) cells) in peripheral blood mononuclear cells (PBMCs). EBV reactivation is known to be associated with plasma cell differentiation and antibody production of B cells. In this study, we investigated whether TRAb(+) EBV(+) cells really produce TRAbs or not when persistent EBV is reactivated. We cultured PBMCs from 12 Graves' disease patients and 12 healthy controls for several days with cyclosporine A to expand the EBV-infected cell population, and then compared TRAb levels between EBV reactivation by 33 degrees C culture and EBV nonreactivation by 37 degrees C culture of PBMCs. Flow cytometry confirmed that all samples at day 0 (reactivation starting point) contained TRAb(+) EBV(+) cells. During 33 degrees C culture, EBV-reactivated cells with EBV-gp350/220 expression increased from about 1 to 4%. We quantified TRAb levels in culture fluids by radio-receptor assay, and detected an increased concentration for at least one sampling point at 33 degrees C (from days 0 to 12) for all patients and healthy controls. TRAb levels were significantly higher in supernatants of 33 degrees C culture than of 37 degrees C culture, and also significantly higher in supernatants from patients than those from controls. This study revealed TRAb production from TRAb(+) EBV(+) cells in response to reactivation induction of persistent EBV in different efficiencies between patients and controls.
• Presence of Epstein-Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves' disease patients and in healthy individuals

  Keiko Nagata; Katsumi Higaki; Yuji Nakayama; Hiromi Miyauchi; Yui Kiritani; Kyosuke Kanai; Michiko Matsushita; Takeshi Iwasaki; Hirotsugu Sugihara; Satoshi Kuwamoto; Masako Kato; Ichiro Murakami; Eiji Nanba; Hiroshi Kimura; Kazuhiko Hayashi

  AUTOIMMUNITY 47 3 193 - 200 2014年05月 [査読有り]
   

  Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves' disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves' disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves' disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves' disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves' disease.
• EBNA-2-Deleted Epstein-Barr Virus from P3HR-1 Can Infect Rabbits with Lower Efficiency than Prototype Epstein-Barr Virus from B95-8

  Hitoshi Sano; Keiko Nagata; Kaoru Kato; Kyousuke Kanai; Kiyoshige Yamamoto; Keisuke Okuno; Satoshi Kuwamoto; Hiromi Higaki-Mori; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Susumu Kanzaki; Kazuhiko Hayashi

  INTERVIROLOGY 56 2 114 - 121 2013年 [査読有り]
   

  Objectives: To clarify characteristics on rabbit in vivo infection with type 2 EBV nuclear antigen (EBNA-2)-deleted Epstein-Barr virus (P3HR-1-EBV) and compare infectious efficacy of P3HR-1-EBV with previously reported prototype type 1 EBV from B95-8. Methods: Twelve Japanese White rabbits were inoculated with P3HR-1-EBV via intranasal or intravenous routes and autopsied on day 70-84. Results: In only 2 of 12 P3HR-1-EBV-inoculated rabbits, EBV-DNA was detected in peripheral blood mononuclear cells (PBMCs). BamHI M rightward reading frame (BMRF)-1, EBNA-1 and BamHI Z leftward reading frame (BZLF)-1-mRNA were intermittently expressed in PBMCs. In 1 infected rabbit with continuous detection of EBV-DNA in PBMCs, many EBER-1-positive lymphocytes were observed in germinal centers and/or marginal zones in some follicles of the appendix, and for the first time a lymphocyte with EBER-1 expression infiltrating in the squamous cell layer of the tonsils was found. The other rabbit with a transient detection of EBV-DNA in PBMCs had no EBER-1-positive lymphocytes in the tissues examined. Few EBER-1-positive lymphocytes were detected in some rabbits without detection of EBV-DNA in PBMCs. Conclusions: P3HR-1-EBV showed less efficient infection in rabbits than EBV from the B95-8 cell line. However, a P3HR-1-EBV-inoculated animal model is meaningful because this is the first study of EBNA-2 function on in vivo EBV infection and it demonstrated the in vivo infectivity with lytic-type infection by EBNA-2-deleted EBV. Copyright (c) 2013 S. Karger AG, Basel
• Synthetic Peptides of Epstein-Barr virus-major envelope glycoprotein-350/220 do not prevent infection in a rabbit Epstein-Barr virus infection model

  Kaoru Kato; Hitoshi Sano; Keiko Nagata; Hirotsugu Sugihara; Kyosuke Kanai; Satoshi Kuwamoto; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi

  Journal of Vaccines and Vaccination 3 6 2012年10月 [査読有り]
   

  Epstein-Barr virus (EBV) is a ubiquitous herpes virus that usually infects humans asymptomatically, occasionally inducing various EBV-associated diseases, including infectious mononucleosis (IM), chronic active EBV infection, and different types of malignant tumors. A history of IM is associated with a 3-fold increased risk for subsequent EBV-related Hodgkin lymphoma. In immunocompromised individuals or transplant patients, EBV presents a high risk for morbidity and mortality, although prophylactic vaccination against EBV might contribute to reduce this risk. In this study, we used a rabbit EBV infection model to determine whether vaccination with synthesized peptides based on gp350/220 sequences could effectively prevent EBV infection or decrease the rate or degree of EBV infection. Six rabbits vaccinated with EBV gp350-peptides and four controls were challenged with a minimum dose of EBV infection EBV-DNAs or EBV-RNAs were detected in 5/6 and 4/4 rabbits, respectively. This suggested that a gp350-peptide vaccine could not prevent primary EBV infections in rabbits and indicated the presence of EBV infection routes or mechanisms in rabbits other than the gp350-CD21 interaction observed in EBV infection of human B-cells. However, this vaccine probably has the efficacy to control the viral loads in inoculated rabbits, because 5 out of 6 vaccinated rabbits showed no detectable EBV-DNA in their blood and either no or only few EBER-1-positive lymphocytes in the lymphoid tissues. This vaccine was immunogenic however, developing other improved vaccines or adjuvants will be necessary to reduce EBV-related diseases. © 2012 Kato K, et al.
• Re-evaluation of the genome sequence of guinea pig cytomegalovirus

  Kyosuke Kanai; Souichi Yamada; Yumiko Yamamoto; Yoshiko Fukui; Ichiro Kurane; Naoki Inoue

  JOURNAL OF GENERAL VIROLOGY 92 Pt 5 1005 - 1020 2011年05月 [査読有り]
   

  Congenital infection by human cytomegalovirus (HCMV) is a major cause of birth defects and developmental abnormalities. Since guinea pig cytomegalovirus (GPCMV) crosses the placenta and causes infection in utero, GPCMV models are useful for studies of the mechanisms of transplacental transmission. During our characterization of a genomic locus required for GPCMV dissemination in animals, we found that the nucleotide sequence in and around the nearby immediate early genes in our lineage of GPCMV strain 22122 [designated GPCMV (ATCC-P5)] showed clear differences from that reported previously for the same strain [designated GPCMV (UMN)] passaged extensively in vitro. Since in vitro passaging of HCMV is known to result in genetic alterations, especially in the UL128-UL131A locus, and loss of growth ability in particular cell types, in this study we determined the complete genome sequence of GPCMV (ATCC-P5), which grows efficiently in animals. A total of 359 differences were identified between the genome. sequences of GPCMV (UMN) and GPCMV (ATCC-P5), and these resulted in structural differences in 29 protein-encoding regions. In addition, some genes predicted from our analysis but not from GPCMV (UMN) are well conserved among cytomegaloviruses. An additional 18 passages of GPCMV (ATCC-P5) in vitro generated no further marked alterations in these genes or in the locus corresponding to the HCMV UL128-UL131A. Our analyses indicate that the published sequence of GPCMV (UMN) contains a substantial number of sequencing errors and, possibly, some mutations resulting from a long history of passaging in vitro. Our re-evaluation of the genetic content of GPCMV will provide a solid foundation for future studies.
• Association of merkel cell polyomavirus infection with morphologic differences in merkel cell carcinoma

  Satoshi Kuwamoto; Hiromi Higaki; Kyosuke Kanai; Takeshi Iwasaki; Hitoshi Sano; Keiko Nagata; Kaoru Kato; Masako Kato; Ichiro Murakami; Yasushi Horie; Osamu Yamamoto; Kazuhiko Hayashi

  HUMAN PATHOLOGY 42 5 632 - 640 2011年05月 [査読有り]
   

  Recently, it has been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus, thought to be a carcinogenic agent. However, it is not fully elucidated whether Merkel cell carcinomas differ with regard to the presence or absence of Merkel cell polyomavirus. To address this, we investigated morphologic differences between Merkel cell polyomavirus positive and negative Merkel cell carcinomas by morphometry. Using polymerase chain reaction and real-time quantitative polymerase chain reaction, Merkel cell polyomavirus was detected in 20 (77%) of 26 Merkel cell carcinoma cases, including 4 Merkel cell carcinomas combined with squamous cell carcinomas. Interestingly, Merkel cell polyomavirus was detected only in ordinary (pure) Merkel cell carcinomas; none of the 4 combined Merkel cell carcinomas + squamous cell carcinomas was positive for Merkel cell polyomavirus (P = .001). Morphometric analyses revealed that Merkel cell polyomavirus negative Merkel cell carcinomas had more irregular nuclei (P < .001) and more abundant cytoplasm (P = .001) than Merkel cell polyomavirus positive Merkel cell carcinomas, which had uniform round nuclei and scant cytoplasm. Reliability of the morphometry was confirmed using intraobserver and interobserver reliability tests. These results demonstrated statistically significant differences in tumor cell morphology between Merkel cell polyomavirus positive and negative Merkel cell carcinomas and reconfirmed the absence of Merkel cell polyomavirus in combined tumors. Furthermore, the results strongly suggest fundamental biological differences between Merkel cell polyomavirus positive and negative Merkel cell carcinomas, supporting that Merkel cell polyomavirus plays an important role in the pathogenesis of Merkel cell polyomavirus positive Merkel cell carcinoma. (C) 2011 Elsevier Inc. All rights reserved.
• The Influence of Epstein-Barr Virus Reactivation in Patients with Graves' Disease

  Keiko Nagata; Shuji Fukata; Kyosuke Kanai; Yukio Satoh; Takaya Segawa; Satoshi Kuwamoto; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi; Takeshi Sairenji

  VIRAL IMMUNOLOGY 24 2 143 - 149 2011年04月 [査読有り]
   

  In Graves' disease, the IgG class autoantibody against thyrotropin receptor (TRAb) is produced excessively and induces hyperthyroidism. Epstein-Barr virus (EBV) is one of the human herpesviruses that persists for life, mainly in B lymphocytes, and is occasionally reactivated. Therefore, EBV may affect the antibody production of B lymphocytes that would normally produce TRAb. The purpose of the present study was to evaluate the association of EBV reactivation with the etiology of Graves' disease. Serum levels of EBV antibodies and IgE were determined by ELISA. TRAb levels were determined by radioreceptor assay. We performed in-situ hybridization (ISH) of EBV-encoded small RNA (EBER)1 on the thyroid tissue of one of our patients. In Graves' disease patients with TRAb levels >= 10%, EA antibody levels, which indicate EBV reactivation, were moderately but significantly correlated with the levels of TRAb, and weakly but significantly correlated with IgE. EBER1-ISH revealed that one of our patients had EBV-infected lymphocytes infiltrating the thyroid gland. EBV reactivation may stimulate antibody-producing B lymphocytes predisposed to make TRAb, and this may contribute to or exacerbate the disease.
• In vitro Epstein-Barr Virus Infection Model of Rabbit Lymphocytes from Peripheral Blood or Spleen

  Kyosuke Kanai; Kaoru Kato; Hitoshi Sano; Keiko Nagata; Keisuke Okuno; Satoshi Kuwamoto; Hiromi Higaki; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi

  INTERVIROLOGY 54 1 17 - 24 2011年 [査読有り]
   

  Most humans become lifelong carriers of Epstein-Barr virus (EBV) by adulthood. Primary EBV infection in adolescents causes infectious mononucleosis. EBV infection is associated with various diseases, neoplasms and hematological disorders. Recently, we reported that EBV can infect rabbits by intravenous, intranasal and/or peroral inoculation, which caused primary EBV infection in rabbits with heterogeneous host reactions. Some rabbits showed chronic and lifelong EBV infection with hemophagocytosis. In this study, to reveal detailed mechanisms in rabbit EBV infection, an in vitro investigation was performed. We elucidated that: (1) EBV can infect rabbit peripheral blood mononuclear cells and splenic lymphocytes in vitro, because EBV gene expressions were confirmed. (2) It is highly likely that the B cell is the main target cell of rabbit EBV infection and is immortalized similar to humans. (3) CD8+ T cells increased in the rabbit in vivo model after EBV inoculation, whereas an increase of B cells occurred after their transient decrease. These data suggest that EBV-infected B cells were proliferated, while CD8+ T cells increased to recognize and kill them. This system may explain the paths of rabbit EBV infection and host reaction, simulating human EBV infection. In vitro studies will be helpful to reveal the pathogenesis of rabbit EBV infection and EBV-associated diseases. Copyright (c) 2010 S. Karger AG, Basel
• [Varicella-zoster virus (VZV)].

  Kanai K; Yamada S; Inoue N

  Uirusu 60 2 197 - 207 2010年12月
• Lifelong persistent EBV infection of rabbits with EBER1-positive lymphocyte infiltration and mild sublethal hemophagocytosis

  Kyosuke Kanai; Kazuaki Takashima; Keisuke Okuno; Kaoru Kato; Hitoshi Sano; Satoshi Kuwamoto; Hiromi Higaki; Keiko Nagata; Hirotsugu Sugihara; Masako Kato; Ichiro Murakami; Kazuhiko Hayashi

  VIRUS RESEARCH 153 1 172 - 178 2010年10月 [査読有り]
   

  Most humans become lifelong carriers of Epstein-Barr virus (EBV) by adulthood. Primary EBV infection in adolescents causes in one to two-third of cases infectious mononucleosis. EBV infection is associated with various diseases, neoplasms and hematological disorders. Recently we reported that EBV can infect rabbits frequently by intravenous, intranasal or/and peroral inoculation, which caused primary EBV infection in rabbits with heterogeneous host reactions. Here we presented follow up data that of six primary EBV-infected rabbits out of seven inoculated intravenously with EBV, two out of six EBV-infected rabbits showed lifetime EBV infection. (1) EBV-DNA were detected in blood through life. (2) High antibody titers against EA-D were maintained over 1000 days. (3) A focal mass lesion was transiently observed by ultrasonography in the spleen of one rabbit. (4) Two lifelong EBV-detected rabbits died on day 1522 or 1400, and autopsy revealed proliferation of lymphocytes expressing EBER1 or LMP1 accompanied with mild hemophagocytosis in the spleen or lymph nodes. We hypothesized some EBV-infected rabbits could not eliminate EBV for life and showed somewhat similar features to persistent EBV infection, mild CAEBV and/or mild sublethal hemophagocytosis. These lifelong EBV-infected rabbits might be a new useful animal model for studying lifelong persistent EBV infection, taking place in almost all adults. (C) 2010 Elsevier B.V. All rights reserved.
• Epstein-Barr Virus Can Infect Rabbits by the Intranasal or Peroral Route: An Animal Model for Natural Primary EBV Infection in Humans

  Keisuke Okuno; Kazuaki Takashima; Kyosuke Kanai; Makoto Ohashi; Ryosuke Hyuga; Hirotsugu Sugihara; Satoshi Kuwamoto; Masako Kato; Hitoshi Sano; Takeshi Sairenji; Susumu Kanzaki; Kazuhiko Hayashi

  JOURNAL OF MEDICAL VIROLOGY 82 6 977 - 986 2010年06月 [査読有り]
   

  Epstein Barr virus (EBV) is spread universally in humans, and it causes infectious mononucleosis and sometimes induces serious EBV-associated disease. The detailed mechanism of primary infection in humans has remained unclear, because it is difficult to examine the dynamics of EBV in vivo. In this study, a natural EBV-infection rabbit model by intranasal or peroral inoculation is described. Ten male rabbits were examined for EBV-DNA or mRNA expression and anti-EBV antibodies in blood. Four of 10 rabbits showed the evidence of EBV infection; detection of EBV-DNA or EBV-related genes mRNA in peripheral blood mononuclear cells, increased EBV antibodies in the plasma, and the presence of lymphocytes expressing EBER1 and EBV-related gene proteins in the lymphoid tissues of a rabbit. Three of four infected rabbits were detected transiently EBV-DNA and/or mRNA of EBV-related genes such as EBNA1, EBNA2, BZLF1, and EA in blood, while in one of four, EBV-DNA and/or mRNA were detected for more than 200 days after viral inoculation The level of EA-IgG increased and its level was maintained in all infected rabbits, whereas those of VCA-IgM and VCA-IgG increased transiently, and EBNA-IgG was not elevated. Pathological examination of a rabbit infected transiently revealed some scattered lymphocytes expressing EBER1, LMP1, and EBNA2 in the spleen and lymph nodes. EA expression was also observed in the spleen. These findings suggest that EBV can infect the rabbit by the intranasal or peroral route, and that this rabbit model is useful for examining the pathophysiology of natural primary EBV infection in humans. J. Med. Virol. 82:977-986, 2010. (C) 2010 Wiley-Liss. Inc
• Epstein-Barr virus associated post-transplant Hodgkin lymphoma in an adult patient after cord blood stem cell transplantation for acute lymphoblastic leukemia.

  Okuno K; Horie Y; Kanai K; Kato M; Kuwamoto S; Okazaki T; Hayashi K

  Journal of clinical and experimental hematopathology : JCEH 49 1 45 - 51 2009年05月 [査読有り]
   

  Post-transplant lymphoproliferative disorder (PTLD) is one of the most important complications of solid organ transplantation or hematopoietic stem cell transplantation. Most PTLDs are associated with Epstein-Barr virus (EBV) infection. Although post-transplant Hodgkin lymphoma (HL) is included in PTLD, there have been no studies in the literature on adult cases of post-transplant HL after cord blood stem cell transplantation (CBSCT). This is due to the fact that EBV infection of cord blood cells usually does not occur, and EBV-infected lymphocytes of the recipient should be eradicated by preconditioning therapy. We report a 26-year-old woman case of post-transplant HL, which occurred after CBSCT for relapsed acute lymphoblastic leukemia. Three years and eight months after CBSCT, the enlarged cervical lymph node was histologically diagnosed as EBV associated post-transplant HL, which showed immunophenotypes of classical HL and latency type II EBV infection. She underwent chemotherapy, and has survived 4 years and 6 months after CBSCT. Differential diagnosis of post-transplant HL with good prognosis and HL-like PTLD with aggressive behavior is important, and immunohistochemical methods were useful and essential for it. The source of EBV associated HL in this case will be discussed.
• The evolution of Epstein-Barr virus inferred from the conservation and mutation of the virus glycoprotein gp350/220 gene

  Asako Kawaguchi; Kyosuke Kanai; Yukio Satoh; Chizu Touge; Keiko Nagata; Takeshi Sairenji; Yoshitsugu Inoue

  VIRUS GENES 38 2 215 - 223 2009年04月 [査読有り]
   

  To study variations of Epstein-Barr virus (EBV), we analyzed the gp350/220 gene for several cell lines and Japanese wild isolates using direct sequencing. The N-terminal region was highly conserved in all EBVs except for Jijoye/P3HR-1 and a few isolates. The variation of the region coincided with EBV types A and B (also referred to as types 1 and 2) and were, respectively, designated as the types a and b. The type A/a was detected in most Japanese cell lines and wild isolates, and was classified as China1 type with latent membrane protein (LMP) 1 gene. The type B/b was detected in only a few wild isolates with the Med and China2 types. The C-terminus had more diversity than the N-terminus and lacked the divergence between types A/a and B/b. The phylogenetic analyses of the gp350/220 and LMP1 genes may suggest a mode of EBV evolution into types A/a and B/b and then to LMP1 subtypes.
• Epstein-Barr virus (EBV) 感染ウサギにおけるEBV抗体レスポンス

  長嶋賢典; 高島一昭; 安藤健介; 奥野啓介; 金井亨輔; 西連寺剛; 林一彦

  米子医学雑誌 59 2 53 - 61 2008年03月 [査読有り]
  
• Basedow病患者におけるEBウイルス抗体価の上昇

  長田佳子; 大橋誠; 金井亨輔; 長嶋賢典; 西連寺剛

  日本内分泌学会雑誌 83 4 1077 - 1077 2008年03月
• The vIL-10 gene of the Epstein-Barr virus (EBV) is conserved in a stable manner except for a few point mutations in various EBV isolates

  Kyosuke Kanai; Yukio Satoh; Hiroyuki Yamanaka; Asako Kawaguchi; Kazutaka Horie; Kenji Sugata; Yoshiko Hoshikawa; Tetsutaro Sata; Takeshi Sairenji

  VIRUS GENES 35 3 563 - 569 2007年12月 [査読有り]
   

  A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame I (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vlL-10) gene was analyzed using PCR-direct sequencing in African Burkitt's lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had Chinal-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.
• Difference of Epstein-Barr virus isolates from Japanese patients and African Burkitt's lymphoma cell lines based on the sequence of latent membrane protein 1

  Kyosuke Kanai; Yukio Satoh; Yuriko Saiki; Haruo Ohtani; Takeshi Sairenji

  VIRUS GENES 34 1 55 - 61 2007年01月 [査読有り]
   

  Sequence variations in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene have been described in many EBV-isolates. To characterize the genomic relationship between Japanese EBV and the EBV isolates of other countries, we analyzed the LMP1 nucleotide sequences in EBV positive cell lines and clinical specimens, including five African Burkitt's lymphoma (BL) cell lines, a Japanese BL cell line, a B-lymphoblastoid cell line, a nasopharyngeal carcinoma hybrid cell line, six gastric carcinoma tissues, two peripheral blood mononuclear cells, and a B95-8 cell line, which contained the prototype EBV genome. We determined the C-terminal nucleotide sequences of LMP1 by PCR-direct sequencing analysis and characterized the sequence variation of Japanese isolates, made a phylogenetic tree from the sequence patterns of LMP1 by a neighbor-joining method. The results indicate that the Japanese EBV isolates are greatly different from the African BL isolates but are closely related to the China 1, which is a strain of Chinese EBV isolates.
• Carboxyl-terminal sequence variation of latent membrane protein 1 gene in Epstein-Barr virus-associated gastric carcinomas from eastern China and Japan

  Yun Wang; Kyosuke Kanai; Yukio Satoh; Bing Luo; Takeshi Sairenji

  INTERVIROLOGY 50 3 229 - 236 2007年 [査読有り]
   

  Objectives: To elucidate variations of latent membrane protein 1 (LMP1) in Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) and explore the LMP1 variations of neighboring countries, China and Japan. Methods: In 12 and 8 EBVaGCs from eastern China and Japan, respectively, the C-termini of LMP1 were analyzed using PCR and sequencing. The sequences were compared with previously published strains and were characterized on a phylogenetic tree. The difference between Chinese and Japanese isolates was characterized. Results: Ten of 12 Chinese GC isolates (83.3%) and all of the 8 (100%) Japanese GC isolates belonged to the China 1 strain. Also, B95-8 type isolates were found in 2 of 12 Chinese GC. In the 18 China 1 type isolates, additional mutations outside the signature sequence changes were found. All Japanese isolates (100%) had two or more additional mutations, whereas only 5 of 10 (50%) Chinese isolates had two or more additional mutations. The difference was statistically significant (p = 0.0359). Conclusions: China 1 is the dominant strain in GC from eastern China and Japan. The similarity to that of nasopharyngeal carcinoma (NPC) from China supports the view that China 1 strain represents a geographic-associated polymorphism rather than an NPC-associated polymorphism. Japanese isolates show more mutations than Chinese isolates, suggesting a geographic difference between Chinese and Japanese isolates in GC.

MISC

• インフルエンザウイルス分離株の増殖能解析

  景山誠二; 徳永朱乃; A HINAI Alfredo, Jr.; 金井亨輔 日本予防医学会学術総会プログラム・抄録集 18th (CD-ROM) 2021年
• IL‐35は気道における好酸球浸潤を抑制する

  金井亨輔; 朴雅美; 角田郁生; 吉田裕樹; 義江修 日本インターフェロン・サイトカイン学会学術集会抄録集 81st 88 2016年04月
• 水痘帯状疱疹ウイルス特異的細胞性免疫を誘導する抗原の探索

  金井亨輔; 谷口留美; 谷口留美; 湯華民; 森康子; 井上直樹 日本ワクチン学会学術集会プログラム・抄録集 16th 2012年
• 胎盤組織培養系の樹立とウイルス感染動態の解析

  山田壮一; 橋本楓; 橋本楓; 福地早希; 福地早希; 片野晴隆; 佐藤由子; 金井亨輔; 井上直樹 日本ウイルス学会学術集会プログラム・抄録集 60th 2012年
• 小児臓器移植前後におけるワクチン接種の安全性と有効性に関する研究

  竹田誠; 井上直樹; 金井亨輔; 染谷健二; 岡本貴世子 小児臓器移植前後におけるワクチン接種の安全性と有効性に関する研究 平成23年度 総括・分担研究報告書 2012年
• 水痘帯状疱疹ウイルス特異的細胞性免疫を誘導する抗原の探索

  金井亨輔; 湯華民; 森康子; 井上直樹 日本ウイルス学会学術集会プログラム・抄録集 60th 2012年
• EBウイルス感染B細胞株の再活性化後期におけるEBER1とgp350/220の局在:72A1抗体を用いたP3HR-1細胞株の蛍光染色

  長田佳子; 檜垣克美; 中山祐二; 金井亨輔; 宮内裕美; 桐谷唯; 佐野仁志; 加藤郁; 木村宏; 林一彦 日本ウイルス学会学術集会プログラム・抄録集 60th 2012年
• 小児臓器移植前後におけるワクチン接種の安全性と有効性に関する研究

  竹田誠; 井上直樹; 金井亨輔; 染谷健二; 岡本貴世子 小児臓器移植前後におけるワクチン接種の安全性と有効性に関する研究 平成22年度 総括・分担研究報告書 2011年
• EBV長期感染疾患ウサギモデル:EBV静注で感染したウサギの死亡まで(4年以上)の経過観察結果

  林一彦; 高島一昭; 金井亨輔; 加藤郁; 佐野仁志; 桑本聡史; 加藤雅子; 村上一郎 日本病理学会会誌 99 (1) 2010年
• メルケル細胞癌+扁平上皮癌混合腫瘍(MCC+SqCC)におけるMCPyVの検出率の検討

  桑本聡史; 金井亨輔; 檜垣裕美; 加藤雅子; 村上一郎; 堀江靖; 林一彦 日本病理学会会誌 99 (1) 2010年
• P3HR-1株産生EBVの経鼻投与におけるEBウイルス感染ウサギの解析

  佐野仁志; 金井亨輔; 金井亨輔; 加藤郁; 長田佳子; 林一彦 日本ウイルス学会学術集会プログラム・抄録集 58th 2010年
• メルケル細胞ポリオーマウイルス感染の有無によるメルケル細胞癌の形態学的相違の検討

  桑本聡史; 金井亨輔; 金井亨輔; 檜垣裕美; 檜垣裕美; 岩崎健; 加藤雅子; 村上一郎; 堀江靖; 山元修; 林一彦 日本ウイルス学会学術集会プログラム・抄録集 58th 2010年
• In vivoで増殖可能なGuinea pig CMV(GPCMV)のゲノム配列の解析

  金井亨輔; 山田壮一; 山本由美子; 福井良子; 倉根一郎; 井上直樹 日本ウイルス学会学術集会プログラム・抄録集 58th 2010年
• EBVの終生持続感染ウサギモデル

  林一彦; 高島一昭; 金井亨輔; 長田佳子; 佐野仁志; 桑本聡史; 加藤郁 日本ウイルス学会学術集会プログラム・抄録集 58th 2010年
• バセドウ病手術例におけるEBER1で陽性リンパ球の検出

  長田佳子; 石黒清介; 金井亨輔; 杉原弘貢; 加藤郁; 佐野仁志; 林一彦 日本ウイルス学会学術集会プログラム・抄録集 58th 2010年
• 経鼻・経口投与によるEBウイルス感染ウサギ動物モデルの解析

  林一彦; 奥野啓介; 高島一昭; 金井亨輔; 金井亨輔; 金井亨輔; 加藤郁; 日向亮輔; 桑本聡史; 加藤雅子; 佐野仁志; 西連寺剛 日本ウイルス学会学術集会プログラム・抄録集 57th 2009年
• 経口/経鼻投与によるEBウイルス感染ウサギ動物モデルの検討

  奥野啓介; 奥野啓介; 高島一昭; 金井亨輔; 金井亨輔; 大橋誠; 長島賢典; 日向亮輔; 杉原弘貢; 佐野仁志; 佐野仁志; 桑本聡史; 加藤雅子; 西連寺剛; 林一彦; 神崎晋 臨床血液 50 (9) 2009年
• ウサギ単核球へのEBV感染の分子基盤の検討

  金井亨輔; 金井亨輔; 金井亨輔; 加藤郁; 奥野啓介; 佐野仁志; 長田佳子; 長田佳子; 西連寺剛; 林一彦 日本ウイルス学会学術集会プログラム・抄録集 57th 2009年
• バセドウ病患者におけるEBウイルス再活性化

  長田佳子; 長田佳子; 深田修司; 金井亨輔; 金井亨輔; 瀬川孝耶; 佐藤幸夫; 林一彦; 西連寺剛 日本ウイルス学会学術集会プログラム・抄録集 57th 2009年
• 経口・経鼻感染によるEBウイルス感染ウサギ動物モデルの解析

  奥野啓介; 高島一昭; 金井亨輔; 日向亮輔; 長島賢典; 西連寺剛; 林一彦 日本ウイルス学会学術集会プログラム・抄録集 56th 2008年
• Epstein-Barr virus(EBV)特異的Fluorescence in situ hybridization(FISH)プローブの作製とEBVゲノムの解析

  菅田謙治; 金井亨輔; 中村泰之; 長島賢典; 安藤健介; 長田佳子 日本ウイルス学会学術集会プログラム・抄録集 56th 2008年
• 臍帯血移植後の頸部リンパ節腫大を契機に発見されたHodgkin病様の移植後リンパ増殖性疾患(PT-LPD)の1例

  奥野啓介; 堀江靖; 加藤雅子; 金井亨輔; 北村幸郷; 林一彦 日本リンパ網内系学会会誌 48 2008年
• EBウイルスエンベロープタンパクgp350/220遺伝子配列からのウイルスのタイプ及びサブタイプへの進化

  金井亨輔; 川口亜佐子; 佐藤幸夫; 長田佳子; 唐下千寿; 西連寺剛; 井上幸次 日本ウイルス学会学術集会プログラム・抄録集 56th 2008年
• EBV再活性化におけるBMRF1タンパク質の細胞内局在の電子顕微鏡での観察

  長田佳子; 森野慎一; 大橋誠; 長島賢典; 金井亨輔; 佐藤幸夫; 林一彦; 西連寺剛 日本ウイルス学会学術集会プログラム・抄録集 56th 2008年
• in vitroにおけるウサギリンパ球へのEBV感染の検討

  金井亨輔; 金井亨輔; 奥野啓介; 日向亮輔; 安藤健介; 長島賢典; 菅田謙治; 長田佳子; 西連寺剛; 林一彦 日本ウイルス学会学術集会プログラム・抄録集 56th 2008年
• ヒト末梢血におけるEBV encoded small RNAの発現の検討

  金井亨輔; 佐藤幸夫; 西連寺剛 日本ウイルス学会学術集会プログラム・抄録集 54th 2006年
• 我が国のEBウイルスLMP1遺伝子に見られる多様性とEBV系統樹の解析

  金井亨輔; 佐藤幸夫; 西連寺剛 日本ウイルス学会学術集会プログラム・抄録集 53rd 2005年

共同研究・競争的資金等の研究課題

• マウスヘルペスウイルス68初感染マウスに生じる肝炎における腸内細菌の役割の解析

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2019年04月 -2022年03月 

  代表者 : 金井 亨輔

   

  我々はヒトがEpstein-Barr Virus (EBV)に初感染した際に生じる肝炎の発症機序を解明するために、EBVと同じγヘルペスウイルス亜科に属するMurine herpesvirus 68 (MHV68)に初感染したマウスに生じる肝炎を動物実験モデル系として用いて解析してきた。これまでに我々はMHV68感染マウスに肝炎が生じること、肝炎が腸内細菌の働きにより生じている可能性を示してきた。 本年度我々は、腸内細菌産物であるLipopolysaccharide (LPS) やPeptidoglycan (PGN) は腸管から門脈を介し肝臓へ送られる (Porter, Physiol Behav. 1998) こと、これら産物を認識するToll-like Receptor (TLR) からのシグナルとIFNγとの共刺激でCXCR3リガンドケモカイン産生が亢進する (Proost, J Leukoc Biol. 2004) ことが示されていることから、LPS・PGNを有力候補とした解析を行った。LPS受容体であるTLR4の阻害剤であるC34や，PGNの受容体であるTLR2の中和抗体を投与して解析した結果、C34腹腔内投与マウスでは体重減少の抑制、肝組織へ浸潤CD8陽性T細胞数の減少、CXCR3リガンドケモカイン産生の減少といったデータが得られた。一方でTLR2の中和抗体を投与したマウスでは未投与マウスとの相違は見られなかった。これらの結果からMHV68肝炎はPDGではなくLPSによって引き起こされている可能性が示された。 本研究の核心的な問いは「どの腸内細菌がどのようにMHV68を引き起こすのか」というものであり、本年度における解析の結果「グラム陰性菌がTLR4の経路を介して肝炎を生じさせている」可能性を示すことができたことからおおむね順調に進捗していると考えられる。
• HIV流行阻止・エイズ発症予防のための臨床分離株の増殖能と増殖因子の検討

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 景山 誠二; 金井 亨輔; 常城 朱乃

   

  2015年・16年のフィリピンにおける薬剤耐性ヒト免疫不全ウイルス（HIV）の流行頻度を9.2%と推定した。この集団において、薬剤未使用者に12ヶ月間治療した後に得た血清・血漿142検体のうち、37株について分離に成功した。この株の増殖能を検討したところ、抹消血液のHIV量と比例したが、試験管内での増殖能が全てについて血漿中より多かった。その増殖能には株による大きな幅があった。このように、内在性のHIVの増殖能は、血漿ウイルス量では判断できない。したがって、内在性ウイルスの増殖能を判定・モニターする意義は大きい。特に耐性ウイルスの出現に伴い薬剤を変更する場合は、この指標に注目する必要がある。
• 粘膜免疫における CCL28 の役割の解明

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2014年04月 -2017年03月 

  代表者 : 義江 修; 中山 隆志; 角田 郁生; 樋口 智紀; 金井 亨輔; 藤田 貢

   

  CCL28遺伝子欠損マウスでは、大腸粘膜でのIgA産生細胞の減少と分布異常、糞便中のIgA量の著明な減少、および個々のIgA産生細胞のIgA産生量の低下が見いだされ、CCL28は大腸粘膜でのIgA産生に重要な役割を担っていることが確認された。さらにCCL28遺伝子欠損マウスの糞便ではバシラス綱が相対的に増加していた。大腸でのIgA産生量の低下と細菌叢の変化と関連して、CCL28遺伝子欠損マウスではデキストラン硫酸誘発腸炎の増悪が見られ、それは抗菌薬投与で軽減された。
• 水痘帯状疱疹ウイルス特異的ＣＴＬ抗原の探索とワクチン接種に伴う細胞性免疫の解析

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2012年04月 -2014年03月 

  代表者 : 金井 亨輔

   

  水痘帯状疱疹ウイルス (VZV) の感染防御には,細胞性免疫 (CMI) が大きく関わっている. CMIを誘導するVZVの主要なタンパクを明らかにするため, エリスポット法を用いた抗原の探索を行った. 核に分布するVZVタンパクを検索対象とし, 健常人の末梢血単核球に共通してCMIを誘導するタンパクを探索した. その結果, ORF8, 61, 66がCMIを誘導することを明らかにした. このうち, ORF61についてはエピトープ配列の同定を行った. 今回新たに明らかにされた抗原ペプチドは, 標準化した抗原を用いたCMI定量系の樹立に寄与できると考えられる.
• EBウイルス感染日本白ウサギにおける感染状態の解析

  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 : 2009年 -2009年 

  代表者 : 金井 亨輔

   

  EBウイルス関連疾患の病態・病因解明および治療法開発のため、EBウイルス感染モデル動物による実験系の構築を目的とした研究が、様々な動物種を用いてなされている。ウサギ(Japanese White種)は有望な動物種候補のひとつであり、EBウイルスが静脈注射により感染することがこれまでに知られている。EBウイルス感染ウサギは一過性の脾腫、末梢血中でのEBV-DNAの検出がなされ、これらの症状は次第に緩和する。本研究は、EBウイルス感染ウサギにおけるEBウイルスの感染状態のより詳細な解析を目的とした以下の検討を行った. EBウイルス感染ウサギの末梢血におけるEBV遺伝子発現の経時的解析を行ったところ、EBウイルス遺伝子の発現が長期間継続して確認された。また、死後の剖検で脾臓からウイルス遺伝子の発現が確認されただけでなく、血球貪食像や異型リンパ球が認められた。このことから、ウサギにおけるEBウイルス感染症状は一時的なものだけではなく、終生継続していることが示され、またEBウイルスが長期的な影響を宿主に及ぼしている可能性が示唆された。 試験管内における末梢血リンパ球、脾臓リンパ球のEBV感染の解析を行ったところ、EBウイルス遺伝子発現が確認され、試験管内でも感染が成立することが示された。また、これまで検討に用いられたB95-8株EBVとは異なるEBV株であるP3HR-1株EBVを用いてウサギへの感染の検討を行い、P3HR-1株EBVでもウサギへの感染が成立することが明らかにした。これらの事実は、ウサギを用いてのEBウイルスの病態・病因解析が、試験管内で行える可能性を示す。 本研究により得られた事実は、EBウイルス感染モデル動物としてのウサギの有用性が改めて支持した。今後、これらEBウイルス感染ウサギが、EBウイルス関連疾患の病態・病因の解析に役立つことが期待される。

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