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1 ( 様式 10) 16fm j0002 平成 29 年 5 月 31 日 平成 28 年度医療研究開発推進事業費補助金 ( 感染症研究国際展開戦略プログラム (J-GRID)) 成果報告書 I. 基本情報 事業名 : ( 日本語 ) 感染症研究国際展開戦略プログラム (J-GRID) ( 英語 )Japan Initiative for Global Research Network on Infectious Diseases 補助事業課題名 :( 日本語 ) 下痢症病原体の遺伝的多様性解析に基づく病原解析と医療診断薬シーズの探索 ( 英語 Comparative genome analyses of enteric pathogens for exploring diagnostics 補助事業担当者 ( 日本語 ) 国立感染症研究所細菌第一部部長大西真 所属役職氏名 : ( 英語 )National Institute of Infectious Diseases Department of Bacteriology I, Director Makoto Ohnishi 実施期間 : 平成 28 年 4 月 1 日 ~ 平成 29 年 3 月 31 日 分担研究課題名 : ( 日本語 ) ( 英語 ) 補助事業分担者 ( 日本語 ) 所属役職氏名 : ( 英語 ) II. 成果の概要 ( 総括研究報告 ) 大西らはコレラ菌 139 株のゲノム情報を得た インド コルカタ分離株 ( 論文発表済 ) バングラデシュ ダッカ分離株の解析 ( 論文発表準備中 ) メコン流域諸国分離株( 論文発表準備中 ) の解析を行った また パンゲノム解析のために8 株の完全長ゲノムを取得した パンゲノム解析では V. cholerae は2つの大きなサブグループに分かれることが示された また オープンパンゲノムの特性が示され 水平伝播による遺伝子獲得が多量ある可能性が示唆された ICE (Integrative conjugative element) 領域 Super-Integron 領域について ORF プロファイルの解析を進めた 補助事業分担者である片山らは インド インドネシアにおける胃腸炎ウイルスの分子疫学解析を実施した インド NICEDの Dr. Mamta Chawla Sarkar らと の間 インドで検出された G9P [4] 株の全部のゲノムを決定して 世界的各地で流行している G9P [4] の正確な起源を理解することを目的として本研究を実施した インドの G9P [4] 株は G9-VP7による二重リアソータント株と DS-1 様の遺伝的バックボーンを有する (G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2) と (G9-P[4]- I2-R2-C2-M2-A2-N1-T2-E6-H2) の2 種類が観察された インドにおける G9 [4] 株の範囲内の複雑な 1
2 リアソートメントは インド国内での高い有病率と混合感染の頻度の高さに関連があると考えられた また 神戸大学 Airlangga 大学と NIID 研究群は DS-1 様の 9つの新しい G3P [8] を検出した さらに 急性胃腸炎で乳児および幼児から検出された2 株の G3P [6] は DS-1 バックボーンの遺伝子構成を有し G3セグメントがウマに由来する可能性が示唆された 我々は さらにインドネシアにおけるヒトノロウイルスの分子疫学調査をMiSeqを使用して行った にサンプリングされた検体を用いた調査の結果 重篤な小児下痢症入院症例から検出されるノロウイルスの遺伝子型が GII.4 であることが明らかになった 補助事業分担者である野崎らは アイルランガ大学寄生虫学講座の医師 Peni Kartika らとともにインドネシアスラバヤの赤痢アメーバ疑い症例 5 件から糞便を採取し 顕微鏡観察によりアメーバ様微生物を確認し Robinson 培地を用いて分離培養を実施した しかしながら Blastocystis 等の共雑により 赤痢アメーバの株の樹立に至らなかった 計画では 細菌共生で確立された株を随時無菌化 (axenization) し 順番に NGSによりゲノム配列取得 リードマッピング デノボアセンブリを行うことを計画していたが 実施することができなかった 海外分離株のゲノム解析を進める準備として 培養 分離 ゲノム採取の方法の標準化に取り組んだ その結果 国内の臨床株に関しては顕微鏡で原虫の形状 或いは ELISA で細胞表面 Galactose 特異的レクチンの抗原の存在が確認された症例に関して 便 或いは大腸内視鏡検査時の洗浄液から >30% の確立で赤痢アメーバ株の分離の成功が確認され 分離 培養法はほぼ標準化することができた 更に 海外標準株のゲノム比較をするために不可欠な国内の分離株 2 株の NGSデータを HiSeqにより取得した A group led by Dr. Ohnishi obtained Illumina MiSeq reads from 139 V. cholerae strains and PacBio reads from 8 V. cholerae. Comparative genome analyses of V. cholerae O1 Kolkata strains, Dhaka strains and Mekong Basin strains were done. Data from pan-genomic analysis of various serogroups of clinical and environment V. cholerae strains suggested that the species divided into two major sub-lineages, and the pan-genome might be open-pangenome, which has an unlimited large gene repertoire. As the open-pangenome indicates massive exchanging of genetc materials among mixed microbial community, we focus on analyses of super-variable genetic islands in V. cholerae genomes such as ICE and super-integrons. A group led by Co-PI, Dr. Katayama carried out a molecular epidemiological study of gastroenteric viruses in India. This study was undertaken to determine the whole genome of eleven unusual G9P[4] strains detected in India during , and to compare them with other human and animal global RVAs to understand the exact origin of unusual G9P[4] circulating in India and other countries worldwide. Of these 11 RVAs, four G9P[4] strains were double-reassortants with the G9-VP7 and E6-NSP4 genes on a DS-1-like genetic backbone (G9- P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2). The other strains showed a complex genetic constellation, likely derived from triple reassortment event with the G9-VP7, N1-NSP2 and E6-NSP4 on a DS- 1-like genetic backbone (G9-P[4]-I2-R2-C2-M2-A2-N1-T2-E6-H2). Presumably, these unusual G9P[4] strains were generated after several reassortment events between the contemporary cocirculating human rotavirus strains. The complex reassortment events within the G9[4] strains may be related to the high prevalence of mixed infections in India as reported in this study and other previous studies. According to a collaboration molecular epidemiological study conducuted by Kobe University, Airlangga University and NIID research group, 9 novel G3P[8] DS-1-like and 2 G3P[6] DS-1-like RVA strains with VP7 genes of likely equine origin from infants and 2
3 young children with acute gastroenteritis in Surabaya, Indonesia were identified. We also investigate human norovirus epidemiology in Indonesia using MiSeq for targetting on complete genome sequence analysis. This 1-year study highlights the continued important of GII.4 as the major genotypes of norovirus causing hospitalization due to diarrhea in children. However, interestingly the GII.13 strain which was regarded as a minor genotype has become the second important genotype (17%) in Indonesia. The novel GII.17-GII.17 strain has become third important genotype in Indonesia, while this strain only found sporadically in neighbor countries such as South Korea, Thailand, Vietnam. It can be expected that the currently dominant GII.4 will be replaced by novel GII.17 in next few years in Indonesia. A group led by Co-PI, Dr. Nozaki succeeded in identifying 5 cases with typical clinical symptoms associated with amebic dysentery from Surabaya in collaboration with Dr. Peni Kartika, University of Airlangga, Indonesia. Diagnosis was further made with antigen capture ELISA from Techlab. They attempted to established culture using Robinson medium, but failed due to overgrowth of Blastocystis which coexisted with possible Entamoeba in the culture. Although we initially planned to obtain sequence reads by next generation sequencing, followed by read mapping, we were unable to do it. Instead, we standardize the protocol for culture, isolation, and genomic DNA isolation from clinical isolated from Japan. Using the standardized protocols, we estimate we are able to establish clinical strains under xenic conditions (with bacterial flora from patients) using specimens such as stools and colon wash during colonoscopy with the probability of approximately 30%. We also obtained NGS HiSeq reads from two representative Japanese isolates. III. 成果の外部への発表 (1) 学会誌 雑誌等における論文一覧 ( 国内誌件 国際誌 1 件 ) 1. Daisuke Imamura, Masatomo Morita, Tsuyoshi Sekizuka, Tamaki Mizuno, Taichiro Takemura, Tetsu Yamashiro, Goutam Chowdhury, Gururaja P. Pazhani, Asish K. Mukhopadhyay, Thandavarayan Ramamurthy, Shin-ichi Miyoshi, Makoto Kuroda, Sumio Shinoda, Makoto Ohnishi. Comparative genome analysis of VSP-II and SNPs reveals heterogenic variation in contemporary strains of Vibrio cholerae O1 isolated from cholera patients in Kolkata, India (2) 学会 シンポジウム等における口頭 ポスター発表 1. ダッカ及びコルカタで分離されたコレラ菌流行株のゲノム疫学解析, 口頭, 森田昌知, 荒川英二, 泉谷秀昌, M. Alam, 今村大輔, 篠田純男, 大西真, 第 50 回腸炎ビブリオシンポジウム, 2016/10/20-21, 国内. 2. ベンガル地方で分離されたコレラ菌流行株の比較ゲノム解析, ポスター, 森田昌知, 荒川英二, 泉谷秀昌, M. Alam, 今村大輔, 篠田純男, 大西真, 第 90 回日本細菌学会総会, 2017/3/21-23, 国内. 3. コレラ菌の時空間系統ゲノミクスの試み 口頭 大西真 新興 再興感染症制御プロジェクト新興再興事業 J-GRID 合同シンポジウム 感染症研究連携のフロンティア 2017/3/16 3
4 国内 (3) 国民との科学 技術対話社会 に対する取り組み 1. 特記なし (4) 特許出願 該当なし 4
5 ( 様式 10) 16fm j0002 平成 29 年 5 月 17 日 平成 28 年度医療研究開発推進事業費補助金 ( 感染症研究国際展開戦略プログラム (J-GRID)) 成果報告書 I. 基本情報 事業名 : ( 日本語 ) 新興再興感染症に対する革新的医薬品開発推進研究事業 ( 英語 )Research Program on Emerging and Re-emerging Infectious Diseases 補助事業課題名 : ( 日本語 ) 下痢症病原体の遺伝的多様性解析に基づく病原解析と医療診断薬シーズの探索 ( 英語 )Comparative genome analyses of enteric pathogens for exploring diagnostics 補助事業担当者 ( 日本語 ) 国立感染症研究所ウイルス第二部第一室室長片山和彦 所属役職氏名 : ( 英語 )Department of Virology II, National Institute of Infectious Diseases. Section Chief. Kazuhiko Katayama 実施期間 : 平成 28 年 4 月 1 日 ~ 平成 29 年 3 月 31 日 分担研究課題名 : ( 日本語 ) ( 英語 ) 補助事業分担者 ( 日本語 ) 所属役職氏名 : ( 英語 ) II. 成果の概要 ( 総括研究報告 ) 補助事業代表者 : 国立感染症研究所 細菌第一部 部長 大西真総括研究報告を参照 III. 成果の外部への発表 (1) 学会誌 雑誌等における論文一覧 ( 国内誌 0 件 国際誌 1 件 ) 1. Doan HY, Haga K, Fujimoto A, Fujii Y, Takai-Todaka R, Oka T, Kimura H, Yoshizumi S, Shigemoto N, Okamoto-Nakagawa R, Shirabe K, Shinomiya H, Sakon-Tanaka N, and Katayama K. Genetic analysis of human rotavirus C: the appearance of Indian-Bangladeshi strain in Far East Asian countries. Infect Genet Evol Apr 9;41: doi: /j.meegid [Epub ahead of print] (2) 学会 シンポジウム等における口頭 ポスター発表 5
6 1. Soetjipto, Takako Utsumi, Maria Inge Lusida, Rury Mega Wahyuni, Zayyin Dinana, Juniastuti, Laura Navika Yamani, Subijanto Marto Sudarmo, Alpha Fardah Athiyyah, Andi Darma, Chieko Matsui, Lin Deng, Yen Hai Doan, Kazuhiko Katayama, and Ikuo Shoji. A preliminary survey of norovirus infections among children in Surabaya, Indonesia. International seminar Global Strategy to Combat Emerging Infectious Diseases in Borderless Era (GSEID). August 8-9, 2016, Surabaya, Indonesia. 2. Zayyin Dinana, Takako Utsumi, Rury Mega Wahuni, Laura Navika Yamani, Chieko Matsui, Lin Deng, Maria Inge Lusida, Soetjipto, Yen Hai Doan, Kazuhiko Katayama, and Ikuo Shoji. Occurrence of norovirus infection an asymptomatic population in Indonesia. International seminar Global Strategy to Combat Emerging Infectious Diseases in Borderless Era (GSEID). August 8-9, 2016, Surabaya, Indonesia (3) 国民との科学 技術対話社会 に対する取り組み 特記なし (4) 特許出願 無し 6
7 ( 様式 10) 16fm j0002 平成 29 年 5 月 31 日 平成 28 年度医療研究開発推進事業費補助金 ( 感染症研究国際展開戦略プログラム (J-GRID)) 成果報告書 I. 基本情報 事業名 : ( 日本語 ) 感染症研究国際展開戦略プログラム (J-GRID) ( 英語 )Japan Initiative for Global Research Network on Infectious Diseases 補助事業課題名 : ( 日本語 ) 下痢症病原体の遺伝的多様性解析に基づく病原解析と医療診断薬シーズの探索 ( 英語 )Comparative genome analyses of enteric pathogens for exploring diagnostics 補助事業担当者 ( 日本語 ) 寄生動物部 部長 野崎智義 所属役職氏名 : ( 英語 )Department of Parasitology Director Tomoyoshi Nozaki 実施期間 : 平成 28 年 4 月 1 日 ~ 平成 29 年 3 月 31 日 分担研究課題名 : ( 日本語 ) ( 英語 ) 補助事業分担者 ( 日本語 ) 所属役職氏名 : ( 英語 ) II. 成果の概要 ( 総括研究報告 ) 補助事業代表者 : 国立感染症研究所 大西真 総括研究報告を参照 III. 成果の外部への発表 (1) 学会誌 雑誌等における論文一覧 ( 国内誌 0 件 国際誌 0 件 ) なし (2) 学会 シンポジウム等における口頭 ポスター発表 Kumiko Nakada-Tsukui, Tsuyoshi Sekizuka, Emi Sato-Ebine, Aleyla Escueta-de Cadiz, Dar-der Ji, Makoto Kuroda and Tomoyoshi Nozaki. Identification of an AIG1 gene as a novel virulence-associated gene by comparative genomics of Entamoeba histolytica clinical isolates. U.S.-Japan Cooperative Medical Sciences Program (USJCMSP), 19th International Conference on Emerging Infectious Diseases in the Pacific Rim. Seoul, South Korea. 7
8 February 7-10, 2017, 国外 (3) 国民との科学 技術対話社会 に対する取り組み なし (4) 特許出願 なし 8
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