Application to infectious disease diagnosis by high-sensitivity in situ hybridization in formalin-fixed paraffin-embedded sections

• Project/Area Number: 26860251
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Human pathology
• Research Institution: Fujita Health University
• Principal Investigator: SHIOGAMA Kazuya 藤田保健衛生大学, 医学部, 助教 (10387699)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2015: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: in situ hybridization法 / LNA-ISH法 / AT tailing-ISH法 / 免疫染色 / SFTS virus / New Tofla virus / 電顕ISH法 / 免疫電顕法 / 高感度ISH法 / Tofla virus / ホルマリン固定パラフィン切片 / 感染症 / 病理診断 / ISH-LNA法 / ISH-AT tailing法 / SFTS / 日本紅斑熱

Outline of Final Research Achievements

We have succeeded to detect severe fever with thrombocytopenia syndrome (SFTS) virus and new Tofla virus by using high-sensitivity in situ hybridization (ISH) in formalin-fixed, paraffin-embedded (FFPE) sections. SFTS virus genome and mRNA were demonstrated on hemophagocytic macrophages in liver and spleen from 5 of 6 cases with autopsies. About 100 nm of viral particles were identified by using pre-embedded method with transmission electron microscope combined ISH and immunostaining. The clone 1C3 mouse monoclonal antibody was the best marker in among different SFTS virus targeting antibodies. Similar expression pattern was obtained in both high-sensitivity ISH as well as immunostaining. The new Tofla virus was visualized with high-sensitivity ISH in epithelial cells of stomach, duodenum esophagus, and germinal center in lymphoid follicle of colon in new TFL virus infected mice. Our high-sensitivity ISH technique made it possible to detect some infectious pathogens in FFPE sections.

Research Products

• [Journal Article] 18F-FDG PET imaging for identifying the dynamics of intestinal disease caused by SFTSV infection in a mouse model. (2016)
  • Author(s): Hayasaka D, Nishi K, Fuchigami T, Shiogama K, Onouchi T, Shimada S, Tsutsumi Y, Morita K.
  • Journal Title: Oncotarget Volume: 7 Issue: 1 Pages: 140-147
  • DOI: 10.18632/oncotarget.6645
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Tofla virus: A newly identified Nairovirus of the Crimean-Congo hemorrhagic fever group isolated from ticks in Japan. (2016)
  • Author(s): Shimada S, Aoki K, Nabeshima T, Fuxun Y, Kurosaki Y, Shiogama K, Onouchi T, Sakaguchi M, Fuchigami T, Ono H, Nishi K, Posadas-Herrera G, Uchida L, Takamatsu Y, Yasuda J, Tsutsumi Y, Fujita H, Morita K, Hayasaka D.
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 20213-20213
  • DOI: 10.1038/srep20213
  • NAID: 120006666534
  • Peer Reviewed / Open Access
• [Journal Article] Coated Glass Slides TACAS Are Applicable to Heat-Assisted Immunostaining and <i>In Situ</i> Hybridization at the Electron Microscopy Level (2015)
  • Author(s): Matsui T, Onouchi T, Shiogama K, Mizutani Y, Inada K, Yu F, Hayasaka D, Morita K, Ogawa H, Mahara F, Tsutsumi Y.
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 48 Issue: 5 Pages: 153-157
  • DOI: 10.1267/ahc.15012
  • NAID: 130005106598
  • ISSN: 0044-5991, 1347-5800
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Demonstration of SFTS virus with high-sensitivity in situ hybridization and immunostaining (2015)
  • Author(s): Shiogama K, Hayasaka D, Onouchi T, Mizutani Y, Inada KI, Morita K, and Tsutsumi Y
  • Organizer: 第104回日本病理学会総会
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2015-04-30
• [Presentation] SFTSの病理 (2015)
  • Author(s): 塩竈和也、堤 寬
  • Organizer: 第7回リケッチア症臨床研究会
  • Place of Presentation: 滋賀県立県民交流センター・ピアザ淡海
  • Year and Date: 2015-01-10 – 2015-01-11
• [Presentation] SFTS virusの組織化学的証明：高感度in situ hybridization法と免疫染色の応用 (2014)
  • Author(s): 塩竈和也、水谷泰嘉、尾之内高慶、稲田健一、堤 寬
  • Organizer: 第69回日本衛生動物学会西日本支部大会
  • Place of Presentation: 愛知医科大学
  • Year and Date: 2014-11-08 – 2014-11-09
• [Presentation] SFTS virusの組織化学的証明 (2014)
  • Author(s): 塩竈和也、堤 寬
  • Organizer: 第22回ダニと疾患のインターフェースに関するセミナー SADI太宰府大会
  • Place of Presentation: 太宰府館
  • Year and Date: 2014-07-04 – 2014-07-06