Basic study for identification of common FMDV, CSFV IRES factors and its application

2022 Fiscal Year Annual Research Report

• Project/Area Number: 20H03164
• Research Institution: Kagoshima University
• Principal Investigator: 小原 恭子 鹿児島大学, 農水産獣医学域獣医学系, 教授 (20225478)
• Co-Investigator(Kenkyū-buntansha): 岡林 環樹 宮崎大学, 農学部, 教授 (10359995) ; 迫田 義博 北海道大学, 獣医学研究院, 教授 (40333637)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: FMDV / CSFV / IRES / shRNA / PYC / PKD1L3 / USP31 / KOマウス

Outline of Annual Research Achievements

口蹄疫、豚熱の原因ウイルスはいずれもプラス鎖RNAをゲノムにもち、5’側非翻訳領域(5’UTR)にリボゾームが内部認識して翻訳を開始する配列(IRES)を持つ。まず、口蹄疫ウイルス(FMDV)や豚熱ウイルス(CSFV)のIRES活性を測定できるジシストロニックベクターを持つ細胞株を樹立して、IRES阻害活性を持つ物質の探索を可能にした。その結果、FMDVの７血清型で保存したIRES配列に作用できるsiRNAを見出し、これを元に作成したshRNAを搭載したレンチウイルスベクターを作成して、２週間以上活性を抑制する事ができた。また、CSFV-IRESを標的とするshRNA発現ベクターも樹立し、IRES活性のみならず、CSFVの複製も抑制する事を明らかにした。さらに、これらのIRES活性を阻害する物質を探索したところ、フランス松の樹液から得られた健康食品であるピクノジュノール(PYC)がFMDV, CSFV-IRES活性を共に抑制する事を見出した。PYCがこれらのIRES活性に関与する共通な宿主因子に作用すると予測し、マイクロアレイで網羅的な発現解析を行なった。PYCの処理により発現が抑制される宿主因子のうち、polycystic kidney disease 1-like 3(PKD1L3)とubiquitin-specific peptidase 31(USP31)の発現抑制により、FMDV, CSFV-IRES活性が共に低下する事が明らかとなった。そこで、PKD1L3, USP31のノックアウト(KO)マウスを樹立した。これら遺伝子のノックアウトマウスは胎生致死を引き起こさず、これらのKOマウスを得る事ができた。今後は、KOマウス体内でFMDV, CSFV-IRES活性や複製活性が低下するか、解析を行ない、FMDV, CSFV抵抗性の動物の樹立に必要な知見を得る予定である。

Research Progress Status

令和4年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和4年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Development of short hairpin RNA expression vectors targeting the internal ribosomal entry site of the classical swine fever virus genomic RNA. (2023)
  • Author(s): Okamoto R, Ito N, Ide Y, Kitab B, Sakoda Y, Tsukiyama-Kohara K
  • Journal Title: BMC Biotechnol. Volume: 23 Pages: 37
  • DOI: 10.1186/s12896-023-00805-6
  • Peer Reviewed / Open Access
• [Journal Article] Novel cytochrome P450 1 (CYP1) genes in tree shrews are expressed and encode functional drug-metabolizing enzymes. (2023)
  • Author(s): Uno Y, Noda Y, Murayama N, Tsukiyama-Kohara K, Yamazaki H.
  • Journal Title: Physiol C Toxicol Pharmacol. Volume: 265 Pages: 109534
  • DOI: 10.1016/j.cbpc.2022.109534.
  • Peer Reviewed
• [Journal Article] Increasing Dengue Burden and Severe Dengue Risk in Bangladesh: An Overview. (2023)
  • Author(s): Kayesh MEH, Khalil I, Kohara M, Tsukiyama-Kohara K.
  • Journal Title: Trop Med Infect Dis. Volume: 8 Pages: 32
  • DOI: 10.3390/tropicalmed8010032.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Regulatory Role of Ribonucleotide Reductase Subunit M2 in Hepatocyte Growth and Pathogenesis of Hepatitis C Virus. (2023)
  • Author(s): Kitab B, Tsukiyama-Kohara K
  • Journal Title: Int J Mol Sci. Volume: 24 Pages: 2619
  • DOI: 10.3390/ijms24032619
  • Peer Reviewed
• [Journal Article] Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig (2023)
  • Author(s): Ushirozako G, Noda Y, Murayama N, Kawaguchi H, Tsukiyama-Kohara K, Yamazaki H, Uno Y.
  • Journal Title: Drug Metab Dispos. Volume: 51 Pages: 610-617
  • DOI: 10.1124/dmd.122.001152
  • Peer Reviewed
• [Journal Article] Newly identified cytochrome P450 3A genes of tree shrews and pigs are expressed and encode functional enzymes. (2023)
  • Author(s): Uno Y, Jikuya S, Noda Y, Oguchi A, Murayama N, Kawaguchi H, Tsukiyama-Kohara K, Yamazaki H.
  • Journal Title: Comp Biochem Physiol C Toxicol Pharmacol. Volume: 267 Pages: 109579
  • DOI: 10.1016/j.cbpc.2023.109579.
  • Peer Reviewed
• [Journal Article] Molecular insights of a CBP/β-catenin-signaling inhibitor on nonalcoholic steatohepatitis-induced liver fibrosis and disorder. (2023)
  • Author(s): Yamaji K, Iwabuchi S, Tokunaga Y, Hashimoto S, Yamane D, Toyama S, Kono R, Kitab B, Tsukiyama-Kohara K, Osawa Y, Hayashi Y, Hishima T, Tateno C, Kimura K, Okanoue T, Kohara M.
  • Journal Title: Biomed Pharmacother. Volume: 166 Pages: 115379
  • DOI: 10.1016/j.biopha.2023.115379.
  • Peer Reviewed
• [Journal Article] Tree shrew cytochrome P450 2E1 is a functional enzyme that metabolises chlorzoxazone and p-nitrophenol. (2023)
  • Author(s): Ushirozako G, Murayama N, Tsukiyama-Kohara K, Yamazaki H, Uno Y
  • Journal Title: Xenobiotica. Volume: 53 Pages: 573-580
  • DOI: 10.1080/00498254.2023.2280996
  • Peer Reviewed
• [Journal Article] Investigation of koala retrovirus in captive koalas with pneumonia and comparative analysis of subtype distribution. (2023)
  • Author(s): Akter L, Hashem MA, Rakib TM, Rashid MHO, Hossain KA, Akhter R, Utsunomiya M, Kitab B, Hifumi T, Miyoshi N, Maetani F, Tsukiyama-Kohara K.
  • Journal Title: Arch Virol. Volume: 168 Pages: 298
  • DOI: 10.1007/s00705-023-05928-x
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Effects of neddylation on viral infection: an overview. (2023)
  • Author(s): Kayesh MEH, Kohara M, Tsukiyama-Kohara K
  • Journal Title: Arch Virol. Volume: 169 Pages: 6
  • DOI: 10.1007/s00705-023-05930-3.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A Novel, Comprehensive A129 Mouse Model for Investigating Dengue Vaccines and Evaluating Pathogenesis. (2023)
  • Author(s): Ngwe Tun MM, Nwe KM, Balingit JC, Takamatsu Y, Inoue S, Pandey BD, Urano T, Kohara M, Tsukiyama-Kohara K, Morita K
  • Journal Title: Vaccines (Basel). Volume: 11 Pages: 1857
  • DOI: 10.3390/vaccines11121857
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] TLR agonists as vaccine adjuvants in the prevention of viral infections: an overview. (2023)
  • Author(s): Kayesh MEH, Kohara M, Tsukiyama-Kohara K.
  • Journal Title: Front Microbiol. Volume: 14 Pages: 1249718
  • DOI: 10.3389/fmicb.2023.1249718.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Characterization of host factors associated with the internal ribosomal entry sites of foot-and-mouth disease and classical swine fever viruses. (2022)
  • Author(s): Ide Y, Kitab B, Ito N, Okamoto R, Tamura Y, Matsui T, Sakoda Y, Tsukiyama-Kohara K.
  • Journal Title: Scientific Reports Volume: 12 Pages: 6709
  • DOI: 10.1038/s41598-022-10437-z.
  • Peer Reviewed / Open Access
• [Journal Article] Subtype distribution and expression of the koala retrovirus in the Japanese zoo koala population. (2022)
  • Author(s): Hashem MA, Kayesh MEH, Maetani F, Goto A, Nagata N, Kasori A, Imanishi T, Tsukiyama-Kohara K
  • Journal Title: Infect Genet Evol. Volume: 102 Pages: 105297
  • DOI: 10.1016/j.meegid.2022.105297.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Toll-like Receptor Response to Hepatitis C Virus Infection: A Recent Overview. (2022)
  • Author(s): Kayesh MEH, Kohara M, Tsukiyama-Kohara K.
  • Journal Title: Int J Mol Sci. Volume: 23 Pages: 5457
  • DOI: 10.3390/ijms23105475
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Molecular Insights into Innate Immune Response in Captive Koala Peripheral Blood Mononuclear Cells Co-Infected with Multiple Koala Retrovirus Subtypes. (2022)
  • Author(s): Kayesh MEH, Hashem MA, Maetani F, Goto A, Nagata N, Kasori A, Imanishi T, Tsukiyama-Kohara K.
  • Journal Title: Pathogens Volume: 11 Pages: 911
  • DOI: 10.3390/pathogens11080911.
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Newly identified tree shrew cytochrome P450 2B6 (CYP2B6) and pig CYP2B6b are functional drug-metabolising enzymes. (2022)
  • Author(s): Uno Y, Ushirozako G, Uehara S, Murayama N, Fujiki Y, Kawaguchi H, Tsukiyama-Kohara K, Yamazaki H
  • Journal Title: Xenobiotica. Volume: 52 Pages: 687-696
  • DOI: 10.1080/00498254.2022.2141153.
  • Peer Reviewed
• [Presentation] IRES共通因子PKD1L3、USP31の生体内機能の解析 (2023)
  • Author(s): 宇都宮雅史、小原恭子
  • Organizer: 第１６6 回日本獣医学会学術集会
• [Presentation] Suppression of dengue virus replication by the French maritime pine extract Pycnogenol (2023)
  • Author(s): Kazi Anowar Hossain1, Rupaly Akhter, Md Haroon Or Rashid, Lipi Akter, Masashi Utsunomiya, Bouchra Kitab, NgweTun MM, Michinori Kohara, Kouichi Morita, Kyoko Tsukiyama-Kohara
  • Organizer: 第１６6 回日本獣医学会学術集会
• [Presentation] ツパイCYP2E1の同定と解析 (2023)
  • Author(s): 後迫玄城，村山典恵，小原恭子，山崎浩史，宇野泰広
  • Organizer: 第１６6 回日本獣医学会学術集会
• [Presentation] 口蹄疫ウイルスおよび豚熱ウイルスRNAのIRES機能に関連する宿主因子の特性について (2022)
  • Author(s): 井手優太郎、Bouchra Kitab, 迫田義博、小原恭子
  • Organizer: 第１６５回日本獣医学会学術集会
• [Presentation] Characterization of host factors associated with the internal ribosomal entry sites of foot-and-mouth disease and classical swine fever viruses (2022)
  • Author(s): Yutaro Ide, Bouchra Kitab, Nobumasa Ito, Riai Okamoto, Yui Tamura, Takafumi Matsui, Yoshihiro Sakoda, and Kyoko Tsukiyama-Kohara
  • Organizer: IUMS2022
  • Int'l Joint Research
• [Presentation] Subtype distribution and expression of the koala retrovirus in koala from the Japanese zoo (2022)
  • Author(s): Md Abul Hashem, Mohammad Enamul Hoque Kayesh, Kyoko Tsukiyama-Kohara
  • Organizer: 第１６５回日本獣医学会学術集会
• [Presentation] ツパイCYP2A13 の同定と解析 (2022)
  • Author(s): 後迫玄城，村山典恵，小原恭子，山崎浩史，宇野泰広
  • Organizer: 第１６５回日本獣医学会学術集会
• [Book] 動物衛生学 (2022)
  • Author(s): 高井伸二 末吉益雄 永幡肇 編集 （獣医衛生学教育研修協議会）
  • Total Pages: 423
  • Publisher: 文英堂出版
  • ISBN: 978-4-8300-3269-1
• [Book] 獣医微生物学 第４版 (2022)
  • Author(s): 公益財団法人 日本獣医学会 微生物分科会 編
  • Total Pages: 514
  • Publisher: 文英堂出版
  • ISBN: 978-4-8300-3270-7
• [Remarks] 鹿児島大学共同獣医学部附属越境性動物疾病制御研究センター
  • URL: https://www.vet.kagoshima-u.ac.jp/TAD/