Title 牛ロタウイルスBの疫学と分子生物学的性状に関する研究(本文(Fulltext) ) Author(s) 宮元, みち子 Report No.(Doctoral Degree) 博士(獣医学) 乙第153号 Issue Date 2017-09-22 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/73123 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
∵ࣟࢱ࢘ࣝࢫ
B ࡢᏛศᏊ⏕≀Ꮫⓗᛶ≧㛵ࡍࡿ◊✲
2017 ᖺ
ᒱ㜧ᏛᏛ㝔㐃ྜ⋇་Ꮫ◊✲⛉
ᐑඖ ࡳࡕᏊ
┠ ḟ ⥴ ゝ ... 1 ➨୍❶ ∵ࣟࢱ࢘ࣝࢫ B ࡀ㛵ࡋࡓங㔞ῶᑡࢆక࠺ᡂ∵ୗ⑩ࡢ㞟ᅋⓎ⏕ ᳨ฟ࢘ࣝࢫࡢ㑇ఏᏊゎᯒ ... 4 ᗎ ㄽ ... 5 ᮦᩱ࠾ࡼࡧ᪉ἲ ... 6 1. ㎰ሙࡢᴫせ࠾ࡼࡧ⛺࿌... 6 2. ᮦᩱࡢ᥇ྲྀ ... 6 3. RT-PCR ࠾ࡼࡧࢩ࣮ࢡ࢚ࣥࢫゎᯒ ... 6 4. 㟁Ꮚ㢧ᚤ㙾᳨ᰝ ... 7 5. ࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ㟁ẼὋື㸦RNA-PAGE㸧 ... 7 6. 㑇ఏᏊゎᯒ ... 7 7.࢘ࣝࢫศ㞳࠾ࡼࡧ BRVA ࡢࣛࢸࢵࢡࢫจ㞟ᛂ ... 8 8.⣽⳦᳨ᰝ࠾ࡼࡧᐤ⏕᳨ᰝ ... 8 9. ⾑Ύᢠయ᳨ᰝ ... 8 10.⤫ィゎᯒἲ ... 8 ⤖ ᯝ ... 10 1.⮫ᗋ⤒㐣࠾ࡼࡧᡤぢ ... 10 2. RT-PCR ... 10 3. 㟁Ꮚ㢧ᚤ㙾᳨ᰝ ... 11 4. RNA-PAGE... 11 5.㑇ఏᏊ┦ྠᛶゎᯒ ... 11 6. ࡑࡢࡢ᳨ᰝ ... 11 ⪃ ᐹ ... 13
ᅗ ⾲ ... 15 ➨❶ ᡂ∵ࡢ∵ࣟࢱ࢘ࣝࢫB ࡼࡿୗ⑩ࡢὶ⾜せᅉࡢ᳨ウ ... 20 ᗎ ㄽ ... 21 ᮦᩱ࠾ࡼࡧ᪉ἲ ... 22 1. BRVB ࡢὶ⾜ࡀ࠶ࡗࡓ㓗㎰ᅋᆅࡢᴫせ ... 22 2. ⣅౽࠾ࡼࡧ⾑Ύࢧࣥࣉࣝࡢ᥇ྲྀ ... 22 3. RT-PCR㸪ࢩ࣮ࢡ࢚ࣥࢫ࠾ࡼࡧ㑇ఏᏊゎᯒ ... 23 4. RNA-PAGE... 24 5. ⾑Ύᢠయ᳨ᰝ ... 24 6. ⣽⳦᳨ᰝ࠾ࡼࡧᐤ⏕᳨ᰝ ... 25 7. ⤫ィゎᯒ ... 25 8. ࢩ࣮ࢡ࢚ࣥࢫゎᯒ࡛ᚓࡽࢀࡓሷᇶ㓄ิࡢࢡࢭࢵࢩࣙࣥ␒ྕ... 25 ⤖ ᯝ ... 26 1. Ⓨ⏕≧ἣ ... 26 2. RT-PCR ... 26 3. RNA-PAGE ... 27 4. 㑇ఏᏊゎᯒ ... 27 5. ⾑Ύᢠయ᳨ᰝ ... 28 6. ⣽⳦᳨ᰝ࠾ࡼࡧᐤ⏕᳨ᰝ ... 29 ⪃ ᐹ ... 30 ᅗ ⾲ ... 32 ➨ ୕ ❶ ▼ᕝ┴᳨࡛ฟࡉࢀࡓBRVB ࡢ㑇ఏᏊゎᯒ... 39 ᗎ ㄽ ... 40 ᮦᩱ࠾ࡼࡧ᪉ἲ ... 42
1.㑇ఏᏊ㓄ิỴᐃࡢࡓࡵࡢ⣅౽ࢧࣥࣉࣝࡢ㑅ᐃ ... 42 2. RNA ᢳฟ䠈cDNA 䝷䜲䝤䝷䝸䞊స〇䛚䜘䜃ḟୡ௦䝅䞊䜽䜶䞁䝅䞁䜾 ... 42 3.㑇ఏᏊゎᯒ ... 43 ⤖ ᯝ ... 44 1. ࢭࢢ࣓ࣥࢺࡢ㑇ఏᏊ㓄ิࡢỴᐃ ... 44 2. ᮏ◊✲᳨࡛ฟࡉࢀࡓ BRVB ࡢ VP3㸪NSP ࠾ࡼࡧ NSP4 㑇ఏᏊ㛗 CDS ሷᇶ㓄ิࡢࣟࢱ࢘ࣝࢫ B ࡢࡑࢀࡽࡢẚ㍑ ... 45 3. BRVB ࡢ VP3㸪NSP3 ࠾ࡼࡧ NSP4 㑇ఏᏊࡢሷᇶ㓄ิ࡞ࡽࡧྛ⺮ⓑࡢ ࣑ࣀ㓟㓄ิࡢ┦ྠᛶࡢ᳨ウ ... 46 4. 㑇ఏᏊࢭࢢ࣓ࣥࢺ࠾ࡅࡿศᏊ⣔⤫ᶞゎᯒ ... 47 ⪃ ᐹ ... 49 ᅗ ⾲ ... 53 ⥲ ᣓ ... 71 ㅰ ㎡ ... 75 ཧ⪃ᩥ⊩ ... 76
1 ⥴ ゝ ᐙ␆ࡢୗ⑩ࡣ␆⏘⤒Ⴀᦆ┈ࢆࡶࡓࡽࡋ㸪≉࢘ࣝࢫᛶࡢୗ⑩ࡣ㎰ᐙ 㛫ఏࡋᦆኻࢆࡶࡓࡽࡍࠋ㇜࡛ࡣ㏆ᖺ㸪㇜ὶ⾜ᛶୗ⑩ࡀୡ⏺ⓗつᶍ࡛ὶ⾜ࡋ ࡓࠋࡑࡢⓎ⏕ࡣᡃࡀᅜ࡛ࡶᅜⓗᗈࡀࡾ㸪⨯ᝈ့ங㇜ࡀṚஸࡍࡿ➼ࡼࡾ㔜 ࡞⤒῭ⓗ⿕ᐖࢆࡶࡓࡽࡋࡓ㸦㎰ᯘỈ⏘┬࣮࣒࣮࣍࣌ࢪ㸸http://www.maff.go.jp㸧ࠋ ୍᪉㸪∵࡛ࡶ࢘ࣝࢫᛶࡢୗ⑩ࡀᏘࢆ୰ᚰᅜⓗⓎ⏕ࡋ㸪ᗂ∵࡛Ⓨ⫱ 㐜ᘏࡸ⬺ỈࡼࡿṚஸຍ࠼㸪ᦢங∵ࡢ⏕⏘ங㔞ࡢῶᑡࢆࡶࡓࡽࡍࡇ࡛㸪⤒ ῭ⓗ⿕ᐖࢆ࠼࡚࠸ࡿࠋ࢘ࣝࢫᛶୗ⑩ࡢ௦⾲ⓗ࡞ཎᅉࡋ࡚㸪ࢥࣟࢼ࢘ ࣝࢫ⛉ࡢ∵ࢥࣟࢼ࢘ࣝࢫ(BCoV)࠾ࡼࡧ∵ࢺࣟ࢘ࣝࢫ(BToV)㸪ࣞ࢜࢘ࣝࢫ ⛉ࡢ∵ࣟࢱ࢘ࣝࢫࡀᣲࡆࡽࢀࡿࠋBCoV ࡣㅖእᅜࡸᡃࡀᅜ࡛ࡶྂࡃࡼࡾࠕᏘ ㉥⑩ࠖࡤࢀࡿᡂ∵ࡢୗ⑩ࡢཎᅉ࢘ࣝࢫࡋ࡚▱ࡽࢀ࡚࠾ࡾ㸪ఏᰁຊࡀᙉ ࡃ㸪ᅜෆ࡛ࡣᡂ∵ୗ⑩ࡢཎᅉࡢ⣙༙ᩘࢆ༨ࡵ࡚࠸ࡿ㸦14,27,28,74,75,92㸧ࠋ BToV ࡣ㏆ᖺሗ࿌ࡉࢀࡓᗂ∵࠾ࡼࡧᡂ∵ࡢୗ⑩ཎᅉ࢘ࣝࢫ࡛࠶ࡾ㸪ᡃࡀᅜᗈ ࡃᾐ₶ࡋ࡚࠸ࡿࡇࡀ᫂ࡽࡉࢀ࡚࠸ࡿ㸦2, 45,46,49,77,79㸧ࠋ∵ࣟࢱ࢘ࣝ ࢫA (BRVA)㸪∵ࣟࢱ࢘ࣝࢫ B (BRVB)࠾ࡼࡧ∵ࣟࢱ࢘ࣝࢫ C (BRVC)ࡢ 3 ✀ࡀ∵ࡢୗ⑩ࡽ᳨ฟࡉࢀ࡚࠸ࡿ㸦11,15,21,61,89,90,91㸧ࠋBRVA ࡣ୍⯡ⓗ ᪂⏕Ꮚ∵ୗ⑩ࢆᘬࡁ㉳ࡋ㸪༢⊂࠶ࡿ࠸ࡣࡢཎᚤ⏕≀ࡢΰྜឤᰁࡼ ࡾ⮴Ṛⓗ࡞ᙳ㡪ࢆཬࡰࡍ(10,29,37,81)ࠋ∵ᗈࡃㄆࡵࡽࢀࡿ㑇ఏᏊᆺࡢ BRVA ຍ࠼㸪 ᕼ᭷࡞㑇ఏᏊᆺࡢ BRVA ࡼࡿᡂ∵ࡢୗ⑩ࡶሗ࿌ࡉࢀ࡚࠸ࡿ 㸦26,55,68,71㸧ࠋ୍᪉ࠊ㏆ᖺࠊBRVB ࠾ࡼࡧ BRVC ࢆཎᅉࡋࡓᡂ∵ࡢୗ⑩ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦11,61,66,67,73,89,90,91㸧ࠋࡋࡋ BRVB ࠾࠸࡚㸪ୗ⑩௨ እࡢ≧㛵ࡍࡿヲ⣽࡞ሗ࿌ࡣࡇࢀࡲ࡛ぢᙜࡓࡽ࡞ࡗࡓࠋ ࣟࢱ࢘ࣝࢫ B (RVB)ࡣ㸪ࣄࢺ㸪㇜㸪⨺㸪ࣛࢵࢺ࠾ࡼࡧ∵ࡽ᳨ฟࡉࢀ࡚࠸
2 ࡿ㸦5,9,12,47,73,76,80,81,98㸧ࠋࣄࢺ࠾ࡅࡿ RVB ឤᰁࡣ㸪1982 ᖺ୰ᅜ࠾ ࠸࡚ᡂே㞟ᅋୗ⑩ࢆᘬࡁ㉳ࡇࡍ᪂⯆ឤᰁࡋ࡚ฟ⌧ࡋ 㸦22,39,40,99㸧㸪 ࡑࡢᚋࣥࢻ㸪ࣂࣥࢢࣛࢹࢩࣗ࠾ࡼࡧ࣑࣐࣮ࣕࣥ࠾ࡅࡿⓎ⏕ࡀሗ࿌ࡉࢀ࡚࠸ ࡿ㸦5,47,52,76㸧ࠋ ୍᪉ BRVB ࡼࡿ∵ࡢୗ⑩ࡢⓎ⏕ࡣ㸪࣓ࣜ࢝㸪ⱥᅜ㸪ࣥࢻ࠾ࡼࡧᡃࡀ ᅜ࡛ࡢሗ࿌ࡀ࠶ࡿ㸦11,13,33,62,75,80,91㸧ࠋᾏእ࡛ࡣᏊ∵ࡢୗ⑩ࡢⓎ⏕ሗ࿌ࡀ ᩓぢࡉࢀࡿ୍᪉࡛㸪ᡃࡀᅜ࡛ࡣᦢங∵࠾ࡅࡿ㞟ᅋୗ⑩ࡢሗ࿌㝈ࡽࢀ࡚࠸ ࡿ㸦91㸧ࠋ1997 ᖺᾏ㐨࠾ࡼࡧ 1999 ᖺ▼ᕝ┴࠾ࡅࡿᡂ∵ࡢ㞟ᅋୗ⑩ ࡀሗ࿌ࡉࢀ࡚௨᮶㸪ᡂ∵ࡢୗ⑩ࡢཎᅉࡋ࡚BRVB ࡀὀ┠ࡉࢀ࡚࠸ࡿࠋ᭱㏆㸪 BRVB ࡼࡿᡂ∵ୗ⑩ࡢⓎ⏕௳ᩘࡣ㸪BCoV ࢆཎᅉࡍࡿࡶࡢḟ࠸࡛ከ࠸ ሗ࿌ࡉࢀ࡚࠸ࡿ㸦27㸧ࠋࡋࡋ㸪BRVB ࡀࡢࡼ࠺㎰ሙ㛫࠶ࡿ࠸ࡣಶయ㛫ࢆ ఏࡋ࡚࠸ࡃࡢ࡞㸪Ꮫⓗᐇែ᫂࡞Ⅼࡀከࡃṧࡉࢀ࡚࠸ࡓࠋ ࣟࢱ࢘ࣝࢫࡣ┤ᚄ⣙75 nm㸦ࢫࣃࢡ⺮ⓑࢆྵࡵࡿ⣙ 100 nm㸧ࡢṇ༑ 㠃యᵓ㐀ࢆᣢࡘ࢘ࣝࢫ࡛㸪ࣞ࢜࢘ࣝࢫ⛉ࣟࢱ࢘ࣝࢫᒓศ㢮ࡉࢀࡿ㸦21㸧ࠋ ࢚࣮ࣥ࣋ࣟࣉࡣಖ᭷ࡏࡎ㸪እẆ㸪ෆẆ࠾ࡼࡧࢥ⺮ⓑࡽ࡞ࡿ3 㔜ᵓ㐀ࢆᣢࡕ㸪 11 ศ⠇ࡢ 2 ᮏ㙐 RNA ࡼࡾ࡞ࡿࢤࣀ࣒ࢆ᭷ࡍࡿࠋศ⠇ RNA ࡣ 6 ✀ࡢ࢘ࣝࢫ ᵓ㐀⺮ⓑ㸦VP1~4㸪VP6 ࠾ࡼࡧ VP7㸧࠾ࡼࡧ 6 ✀ࡢ㠀ᵓ㐀⺮ⓑ㸦NSP1㹼6㸧ࢆ ࢥ࣮ࢻࡍࡿࠋࣟࢱ࢘ࣝࢫࡣ㸪ෆ᰾⺮ⓑVP6 ࡢ⾑Ύᆺᇶ࡙ࡁ A㹼H ࡢ 8 ⩌ ศ㢮ࡉࢀ࡚࠸ࡿ㸦58㸧ࠋࣟࢱ࢘ࣝࢫࡣศ㞳ࡉࢀࡿື≀✀ࡈ∵ࣟࢱ࢘ࣝࢫ㸪 ࣄࢺࣟࢱ࢘ࣝࢫ࠾ࡼࡧ㇜ࣟࢱ࢘ࣝࢫ࡞༊ศࡉࢀ㸪≉ᐃࡢᐟື≀✀ ឤᰁࡍࡿ୍⯡ⓗ⪃࠼ࡽࢀ࡚࠸ࡿ୍᪉㸪✀㛫ఏࡶ㉳ࡇࡍࡇࡀ▱ࡽࢀ࡚࠸ ࡿ㸦30,59㸧ࠋࣟࢱ࢘ࣝࢫࡣ୰ᢠయࢆㄏᑟࡍࡿ 2 ✀㢮ࡢᵓ㐀⺮ⓑ࡛࠶ࡿ VP7 ࠾ࡼࡧ VP4 ࡢ㑇ఏᏊ㓄ิࡼࡾ㸪G ᆺ࠾ࡼࡧ P ᆺศ㢮ࡉࢀࡿ㸦60㸧ࠋBRVA ࡶྵࡵ㸪ࣟࢱ࢘ࣝࢫA (RVA)ࡢ㑇ఏᏊゎᯒࡀ㐍ࢇ࡛࠾ࡾ㸪32 ✀ࡢ G ᆺ࠾ࡼࡧ
3 47 ✀ࡢ P ᆺࡀሗ࿌ࡉࢀ࡚࠸ࡿ㸦43㸧ࠋࡋࡋ RVB ࠾࠸࡚ࡣ 21 ✀ࡢ G ᆺࡀሗ ࿌ࡉࢀ࡚࠸ࡿࡶࡢࡢP ᆺࡢศ㢮ࡣ⾜ࢃࢀ࡚࠾ࡽࡎ㸦31,33㸧㸪ࡉࡽ BRVB ࠾ ࠸࡚ࡣヲ⣽࡞㑇ఏᏊᆺูࡸศᏊ⏕≀Ꮫⓗᛶ≧ࡣ᫂ࡽࡉࢀ࡚࠸࡞ࡗࡓࠋ ⴭ⪅ࡣ1999 ᖺ▼ᕝ┴࡛ BRVB ࡼࡿᡂ∵ࡢ㞟ᅋୗ⑩㐼㐝ᚋ㸪5 ௳ࡢ BRVB ឤᰁࢆ⤒㦂ࡋࡓࡇࡽ㸪BRVB ࡢᏛ࠾ࡼࡧศᏊ⏕≀Ꮫⓗᛶ≧ࢆ ᫂ࡽࡍࡿࡓࡵ◊✲ࢆ⾜ࡗࡓࠋᮏㄽᩥࡣḟࡢ 3 ❶ࡽᵓᡂࡉࢀ࡚࠸ࡿࠋ➨ ୍❶࡛ࡣ㸪1999 ᖺ▼ᕝ┴࡛Ⓨ⏕ࡋࡓ BRVB ࡼࡿங∵ࡢୗ⑩Ⓨ⏕࠾࠸ ࡚㸪ங⏕⏘ࡢᙳ㡪ཎᅉ࢘ࣝࢫࢆ᫂ࡽࡍࡿ┠ⓗ࡛ࠊୗ⑩Ⓨᮇ㛫࠾ ࡅࡿங㔞ῶᑡࡘ࠸࡚ࡢ⮫ᗋᏛⓗㄪᰝ㸪᳨ฟࡉࢀࡓ࢘ࣝࢫᰴࡢศᏊ⏕≀Ꮫ ⓗゎᯒࢆᐇࡋࡓࠋࡑࡢ⤖ᯝ㸪BRVB ࡼࡿୗ⑩࡛ங㔞ࡀῶᑡࡍࡿྍ⬟ᛶࢆ ୡ⏺࡛ึࡵ࡚♧ࡍࡇࡀ࡛ࡁࡓࠋࡲࡓ㸪ࡇࡢࡽ᳨ฟࡉࢀࡓBRVB ࡀ RNA 㟁ẼὋື࡛ᕼ᭷࡞ࢩ࣮ࣙࢺࣃࢱ࣮ࣥࢆ♧ࡍࡇࢆ☜ㄆࡋࡓࠋ➨❶࡛ࡣ㸪つ ᶍ㓗㎰ᆅᖏ࠾࠸࡚㸪3 ᖺ㛫㝸࡛ 2 ᅇࡢ BRVB ࡼࡿ㞟ᅋᛶୗ⑩ࡀⓎ⏕ࡋࡓ ᇶ࡙ࡁ㸪BRVB ࡼࡿୗ⑩ࡢὶ⾜ࡢせᅉࢆ᳨ウࡍࡿ┠ⓗ࡛㸪୧Ⓨ⏕ ࠾ࡅࡿ㎰ሙ࠾ࡼࡧಶయࡈࡢBRVB ᢠయㄪᰝࢆᐇࡋࡓࠋࡑࡢ⤖ᯝ㸪BRVB ࡼࡿᡂ∵ࡢୗ⑩ࡢⓎ⏕ࡀ∵⩌ࡢචࣞ࣋ࣝࡢᙳ㡪ࢆཷࡅࡿྍ⬟ᛶࢆ♧ࡍࡇ ࡀ࡛ࡁࡓࠋ➨୕❶࡛ࡣ㸪BRVB ࡢ㑇ఏሗࡢ✚ࢆ┠ⓗࡋ࡚㸪ࡇࢀࡲ࡛▼ ᕝ┴᳨࡛ฟࡉࢀࡓBRVB 9 ᰴࡢ㑇ఏᏊ㓄ิࢆỴᐃࡋ㸪ࡉࡽ㸪ࡇࢀࡲ࡛ሗ࿌ ࡢ࡞࠸VP3㸪NSP3 ࠾ࡼࡧ NSP4 㑇ఏᏊ㓄ิࢆỴᐃࡋࡓࠋࡑࡢ⤖ᯝ㸪VP3㸪NSP3 ࠾ࡼࡧNSP4 ࡀ RVB ࠾ࡅࡿ᪂ࡋ࠸㑇ఏᏊᆺ࡛࠶ࡿࡇࢆぢ࠸ࡔࡋࡓࠋࡋࡋ ࡞ࡀࡽ㸪ࡑࢀࡒࢀࡢࢭࢢ࣓ࣥࢺ࠾ࡅࡿ≉ᚩⓗ࡞ࣔࢳ࣮ࣇࡸศᏊ⏕≀Ꮫⓗ࡞ᛶ ㉁ࡣ㸪RVB ࡢᰴ㛫ಖᏑࡉࢀ࡚࠸ࡿࡇࢆド᫂ࡋࡓࠋ
4 ➨୍❶ ∵ࣟࢱ࢘ࣝࢫB ࡀ㛵ࡋࡓங㔞ῶᑡࢆక࠺ᡂ∵ୗ⑩ࡢ㞟ᅋⓎ⏕ ᳨ฟ࢘ࣝࢫࡢ㑇ఏᏊゎᯒ
5 ᗎ ㄽ BRVB ࡣ᭱ึ㸪⡿ᅜ࠾ࡼࡧⱥᅜ࡛Ꮚ∵ࡢୗ⑩ࡽᩓⓎⓗ᳨ฟࡉࢀࡿ㠀 A ⩌ࣟࢱ࢘ࣝࢫࡋ࡚ሗ࿌ࡉࢀ㸦3,12,62,80㸧㸪ࣀࢺࣂ࣮࢜ࢺࡢᏊ∵ᐇ㦂ⓗ ୗ⑩ࢆ㉳ࡇࡍࡇࡀド᫂ࡉࢀࡓ㸦11,73㸧ࠋࡑࡢᚋ㸪1990 ᖺ௦ึ㢌⡿ᅜࡢ࣡ ࢩࣥࢺࣥᕞ࠾ࡼࡧ࢜ࣁ࢜ᕞ࡛Ⓨ⏕ࡋࡓᡂ∵ࡢୗ⑩౽ࡽච㟁Ꮚ㢧ᚤ㙾ほᐹ ࡼࡾ BRVB ࡀ᳨ฟࡉࢀ㸪ᡂ∵ࡢୗ⑩㛵㐃ࡍࡿࡇࡀ♧ࡉࢀࡓ㸦75㸧ࠋᡃ ࡀᅜ࠾࠸࡚ࡣ 1997 ᖺᾏ㐨ࡢᦢங∵ࡢὶ⾜ᛶୗ⑩ࡼࡾึࡵ࡚ BRVB ࡀ ☜ㄆࡉࢀࡓࠋࡑࡢVP7 㑇ఏᏊࡢゎᯒࡢ⤖ᯝ㸪᳨ฟᰴ㸦Nemuro ᰴ㸧⡿ᅜࡢ BRVB ࡣሷᇶ࡛ࣞ࣋ࣝ 93-95%㸪࣑ࣀ㓟࡛ࣞ࣋ࣝ 96-97%ࡢ┦ྠᛶࡀ࠶ࡿࡇࡀ♧ ࡉࢀࡓ㸦91㸧ࠋ 1999 ᖺࡢ㸪▼ᕝ┴ࡢ୍㓗㎰ᐙ࡛ᡂ∵ୗ⑩ࡀⓎ⏕ࡋࡓࠋᛶ㚷ᐃࡢ⤖ᯝ㸪 BCoV㸪∵ࢹࣀ࢘ࣝࢫ 7 ᆺ࠾ࡼࡧ∵࢘ࣝࢫᛶୗ⑩࢘ࣝࢫ㸦BVDV㸧࡞ ࡢ࢘ࣝࢫ㸪⣽⳦࠾ࡼࡧᐤ⏕ࡢ㛵ࡣྰᐃࡉࢀ㸪BRVB ࡀ㏫㌿࣏࣓࣮ࣜࣛ ࢮ㐃㙐ᛂ㸦RT-PCR㸧࡛㝧ᛶ࡞ࡗࡓࠋᮏ࡛ࡣ㸪ᡂ∵ࡰ㢌ୗ⑩≧ ࡀㄆࡵࡽࢀࡓࡢຍ࠼㸪⏕ஙࡢ⏕⏘㔞ࡢῶᑡࡀ☜ㄆࡉࢀࡓࠋࡇࢀࡲ࡛ங㔞ῶ ᑡࢆᘬࡁ㉳ࡇࡍ࢘ࣝࢫࡋ࡚ࡣ BCoV ࡀᗈࡃ▱ࡽࢀ࡚࠸ࡓࡀ㸪ᮏࡀⓎ⏕ ࡋࡓⅬ࠾࠸࡚ࡣ㸪ࡑࢀ௨እࡢ࢘ࣝࢫࡀ༢⊂࡛ங㔞పୗࢆక࠺ୗ⑩ࢆᘬ ࡁ㉳ࡇࡋࡓሗ࿌ࡣぢᙜࡓࡽ࡞ࡗࡓࠋ ᮏ❶࡛ࡣ㸪BRVB ࡼࡿᡂ∵ࡢୗ⑩ࡢⓎ⏕ୗ⑩Ⓨᮇ㛫࠾ࡅࡿங㔞ῶ ᑡࡘ࠸࡚ࡢ⮫ᗋᏛⓗㄪᰝ㸪᳨ฟࡉࢀࡓ࢘ࣝࢫᰴࡢศᏊ⏕≀Ꮫⓗゎᯒࡢ⤖ ᯝࡘ࠸࡚㏙ࡿࠋ
6 ᮦᩱ࠾ࡼࡧ᪉ἲ 1. ㎰ሙࡢᴫせ࠾ࡼࡧ⛺࿌ Ⓨ⏕㎰ሙࡣ㸪▼ᕝ┴ࡢ୰⬟Ⓩᆅ༊⨨ࡍࡿ㓗㎰ᐙ࡛㸪࣍ࣝࢫࢱࣥ✀ࡢᦢ ங∵ 78 㢌ࢆᑐ㢌ᘧࡢࢱࢫࢺ࣮ࣝ㸪Ꮚ∵ 15 㢌ࢆ∵⯋ෆ✵ࡁࢫ࣮࣌ࢫ㣫㣴 ࡋ࡚࠸ࡓࠋ ⛺࿌࡛ࡣ㸪ୗ⑩Ⓨ⏕๓∵ࡢᑟධࡣ࡞ࡃ㸪⤥㣫ᩱࡢኚ᭦ࡸ࿘㎶㎰ᐙࡢ≀ ㈨➼ࡢ ᮶ࡣ☜ㄆ࡛ࡁ࡞ࡗࡓࡶࡢࡢ㸪1999 ᖺ 3 ᭶ 8 ᪥㹼4 ᭶ 11 ᪥ࡅ࡚ ┴እࡽࡢ∵⯋ෆసᴗဨࡀ▼ᕝ┴ෆࢆᕠᅇࡋ㸪14 ᡞࡢ㎰ሙ࡛సᴗࢆ⾜ࡗ࡚࠾ ࡾ㸪ᙜヱ㎰ሙ࡛ࡶ3 ᭶ 24㹼30 ᪥సᴗࢆᐇࡋࡓࠋ ⏕ஙࡢἪங㔞ࡣ⏕ங⏕⏘⪅ᅋయ࡚ㄪᰝࡋ㸪Ἢங㔞ࡢῶᑡࡘ࠸࡚ࡢゎᯒࡣ t ᳨ᐃ㸦p<0.05㸧࡚⤫ィฎ⌮ࢆᐇࡋࡓࠋ 2. ᮦᩱࡢ᥇ྲྀ 1999 ᖺ 4 ᭶ 14 ᪥Ỉᵝᛶୗ⑩ࡢ≧ࢆ࿊ࡋࡓᡂ∵ 6 㢌ࡼࡾ⣅౽ࢆ᥇ྲྀࡋ ࡓࠋࡲࡓ⣅౽ࢆ᥇ᮦࡋࡓ6 㢌ࡘ࠸࡚ࡣ⣅౽᥇ᮦ⾑Ύࢧࣥࣉࣝࡶྠ᥇ ྲྀࡋ㸪࣌⾑Ύࢆᚓࡿࡓࡵ㸪ࡑࡢ3 㐌㛫ᚋࡧ᥇⾑ࢆ⾜࡞ࡗࡓࠋ 3. RT-PCR ࠾ࡼࡧࢩ࣮ࢡ࢚ࣥࢫゎᯒ ⣅౽ࢆ⁛⳦ࣜࣥ㓟⦆⾪⏕⌮㣗ሷỈ࡛1㸸9 㸦w / v㸧ᕼ㔘ࡋ࡚ 10㸣ஙࢆ స〇ࡋ㸪10,000g࡛10 ศ㛫㐲ᚰศ㞳ࡋࡓୖΎࢆ᳨ᰝᮦᩱࡋࡓࠋRNA ᢳฟ
ࡣTRIZOL LS 㸦Gibco BRL㸪USA㸧ࢆ⏝ࡋ㸪ࣉࣟࢺࢥ࣮ࣝࡢ࠾ࡾᐇ
ࡋࡓࠋRT-PCR ࡣ TaKaRa RNA PCR Kit (AMV)Ver.2.1 (Takara, Shiga,
7
㸦13㸧࠾ࡼࡧ BCoV ࡢࢾࢡࣞ࢜࢝ࣉࢩࢻ㑇ఏᏊࢆᶆⓗࡍࡿࣉ࣐࣮ࣛ࣌
BCV N-F/BCV N-R㸦92㸧ࢆ⏝࠸࡚ᐇࡋࡓࠋୖグࡢࣉ࣐࣮ࣛ࣌ 9B3/9B4 ࢆ⏝࠸ࡓࢥࣟࢽ࣮PCR ἲࡼࡾࠊ㝧ᛶ࡞ࡗࡓ⭠⳦ࢡ࣮ࣟࣥ㸦TA ࢡ࣮ࣟࢽ ࣥࢢ࢟ࢵࢺ㸦Invitrogen㸪USA㸧㸧5 ࡘࡽࣉࣛࢫ࣑ࢻࢆ Plasmid Mini Kit
㸦QIAGEN, Germany㸧࡚ᢳฟࡋ㸪⮬ືࢩ࣮ࢡ࢚ࣥࢧ࣮ALFexpressϩ
㸦Amersham Pharmacia Biotech㸪USA㸧ࡼࡿࢩ࣮ࢡ࢚ࣥࢫゎᯒ౪ࡋࡓࠋ
4. 㟁Ꮚ㢧ᚤ㙾᳨ᰝ
⣅౽10%ஙࢆ 2%ࣔࣜࣈࢹࣥ㓟ࣥࣔࢽ࣒࡛࢘ᰁⰍࡋ㸪㟁Ꮚ㢧ᚤ㙾࡛ほᐹ
ࡋࡓࠋBRVA㸪BRVB㸪BRVC ࠾ࡼࡧ BCoV ࡘ࠸࡚ࡣ㸪ࡑࢀࡒࢀ BRVA Lincoln
ᰴ㸪BRVB ATI ᰴ㸪 BRVC Shintoku ᰴ࠾ࡼࡧ BCoV Mebus ᰴᑐࡍࡿච
⾑Ύࢆ⏝࠸ࡓᅛ┦ච㟁Ꮚ㢧ᚤ㙾᳨ᰝࢆ⾜ࡗࡓࠋ 5. ࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ㟁ẼὋື㸦RNA-PAGE㸧 ⣅౽ஙࡢ㐲ᚰୖΎࢆ 30㸣ࡢࢩࣙ⢾ᐦᗘ໙㓄ࡼࡿ㉸㐲ᚰ࡛⢭〇ᚋ㸪ࣇ࢙ ࣀ࣮ࣝ-ࢡ࣒ࣟࣟ࣍ࣝࢆ⏝࠸࡚ RNA ᢳฟࢆ⾜ࡗࡓࠋᚓࡽࢀࡓ RNA ࢧࣥࣉࣝࡣ Herring ࡽ㸦36㸧ࡢ᪉ἲ‽ࡌ㸪10㸣࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ㸦e-PAGEL;Atto㸪 Tokyo㸪Japan㸧ࢆ⏝࠸࡚㟁ẼὋືࢆࡋࡓᚋ㸪RNA ࣂࣥࢻࢆ㖟ᰁⰍ࢟ࢵࢺ㸦Bio Rad㸪USA㸧ࡼࡾྍどࡋࡓࠋ 6. 㑇ఏᏊゎᯒ BRVB VP7 ᶆⓗ RT-PCR ࡼࡾቑᖜࡉࢀࡓ cDNA ࢆ㸪ࢥࣥࣆ࣮ࣗࢱ࣮ࣉࣟࢢ
࣒ࣛGENETYX-MAC ver. 10 㸦ᰴᘧ♫ࢮࢿࢸࢵࢡࢫ㸪Japan㸧ࢆ⏝࠸㸪
8
㸦AB016818㸧㸪BRVB ATI ᰴ㸦U84472㸧㸪BRVB Mebus ᰴ㸦U84473㸧࠾ࡼࡧ
BRVB WD653 ᰴ㸦U84141㸧┦ྠᛶゎᯒࢆ⾜ࡗࡓࠋ 7.࢘ࣝࢫศ㞳࠾ࡼࡧ BRVA ࡢࣛࢸࢵࢡࢫจ㞟ᛂ ࢝ ࢤ ࢨ ࣝ ⭈ ⏤ ᮶ ᰴ ⣽ ⬊ 㸦MA104㸧࠾ࡼ ࡧ ࣄࢺ┤⭠⏤ ᮶ᰴ⣽⬊ 㸦HRT18㸧⣅౽ஙࢆ᥋✀ࡋ㸪37Υ࡛ᅇ㌿ᇵ㣴ࢆ⾜ࡗࡓࠋ⣽⬊ኚᛶຠᯝࡀ ㄆࡵࡽࢀ࡞ࡗࡓሙྜ㸪7 ᪥㛫㝸࡛ 3 ௦ࡢ┣⥅௦ᇵ㣴ࢆ⾜ࡗࡓࠋ ࢘ࣝࢫᇵ㣴ୖΎ࠾ࡼࡧ⣅౽ஙࡘ࠸࡚㸪ᕷ㈍ࡢࣛࢸࢵࢡࢫจ㞟ᛂ㸦ࣟ
ࢱࣞࢵࢡࢫࢻࣛ㸹Orion Diagnostica㸪Japan㸧ࡼࡿ RVA ᢠཎࡢ⡆᳨᫆
ᰝࢆ⾜ࡗࡓࠋ 8.⣽⳦᳨ᰝ࠾ࡼࡧᐤ⏕᳨ᰝ ࢧࣝࣔࢿࣛ⳦ࡢศ㞳ᇵ㣴᳨ᰝࡘ࠸࡚ࡣ㸪ࣁ࣮ࢼ࣮ࢸࢺࣛࢳ࢜ࢿ࣮ࢺᇵᆅ࡛ ⣅౽ࢆ 37Υ୍ᬌቑ⳦ᇵ㣴ࡋࡓᚋ㸪DHL ᐮኳᇵᆅ࡚ 37Υ 24 㛫ᇵ㣴ࢆᐇ ࡋࡓࠋཎᛶ⭠⳦ࡢศ㞳ᇵ㣴᳨ᰝࡘ࠸࡚ࡣ㸪⣅౽ࢆ106ಸࡲ࡛10 ಸẁ 㝵ᕼ㔘ࢆࡋࡓᚋ㸪DHL ᐮኳᇵᆅ᥋✀ࡋ 37Υ࡛ 24 㛫ᇵ㣴ࢆ⾜ࡗࡓࠋ ᐤ⏕᳨ᰝࡣࢩࣙ⢾㐲ᚰᾋ㐟ἲࡼࡾᐇࡋࡓࠋ 9. ⾑Ύᢠయ᳨ᰝ ᥇ྲྀࡋࡓ࣌⾑Ύࢆヨ㦂ᮦᩱࡋ࡚㸪BCoV Kakegawa ᰴ࠾ࡼࡧ∵ࢹࣀ࢘ ࣝࢫ㸵ᆺ⿄ᰴࢆ⏝࠸ࡓ㉥⾑⌫จ㞟ᢚไ㸦HI㸧ヨ㦂࠾ࡼࡧ BVDV 1 ᆺ Nose ᰴࢆ⏝࠸ࡓ୰ヨ㦂ࢆᐇࡋࡓࠋ ⤫ィゎᯒἲ ୗ⑩Ⓨ⏕๓ୗ⑩Ⓨ⏕ᮇ㛫୰ࡢἪங㔞ࡢᖹᆒ್ࡢᕪࡢ⤫ィᏛⓗゎᯒࡣࠊ࢚ࢡ
9
ࢭࣝ⤫ィ2013㸦Social Survey Research Information Co., Ltd., Tokyo, Japan㸧
10 ⤖ ᯝ 1.⮫ᗋ⤒㐣࠾ࡼࡧᡤぢ 1999 ᖺ 4 ᭶ 13 ᪥ࡼࡾ∵⯋ෆࡢᩘ㢌ࡢᦢங∵ࡀ✺↛ୗ⑩ࢆⓎࡋ㸪ࡑࡢᚋ 3 ᪥௨ෆࡰ㢌ࡀୗ⑩ࢆ࿊ࡋࡓࠋ౽ࡢᛶ≧ࡣⲔⰍࡶࡋࡃࡣ㯮ࡀࡗࡓỈᵝ ᛶ࡛࠶ࡗࡓࠋࡲࡓ㸪⾑ᵝ౽ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋୗ⑩≧ࢆ࿊ࡋࡓ∵ࡣⓎ⏕ ࡽ3㹼5 ᪥௨ෆᅇࡋࡓࡀ㸪∵⯋యࡢୗ⑩ࡢ⤊ᜥࡲ࡛ 1 㐌㛫ࢆせࡋࡓࠋ࡞ ࠾㸪Ⓨ∵ࡢ⒪ࡣ⾜ࢃ࡞ࡗࡓࠋ ᙜヱ㎰ሙ࡛ࡣୗ⑩Ⓨ⏕┤ᚋࡼࡾ⏕ஙࡢ1 ᪥Ἢங㔞ࡀῶᑡࡋ㸪5 ᪥┠ࡣࡑࡢῶ ᑡ㔞ࡣ᭱࡞ࡗࡓࠋࡲࡓ㸪Ἢங㔞ࡀⓎ⏕๓ࡢᖹᆒἪங㔞ࡲ࡛ᅇࡍࡿࡣ2 㐌㛫௨ୖࢆせࡋࡓ㸦ᅗ 1-1㸧ࠋୗ⑩Ⓨ⏕ᮇ㛫㸦7 ᪥㛫㸧ࡢᖹᆒἪங㔞ࡣୗ⑩Ⓨ⏕ ๓ࡢ7 ᪥㛫ࡢᖹᆒẚ㍑ࡋ㸪᭷ពῶᑡࡋ㸦p<0.05㸧㸪ᖹᆒἪஙῶᑡ㔞ࡣୗ⑩ Ⓨ⏕๓ࡢᖹᆒἪங㔞ࡢ⣙10%┦ᙜࡋࡓ㸦ᅗ 1-2㸧ࠋᏊ∵ࡘ࠸࡚ࡣ㸪ୗ⑩ࡸ ࡢ⮫ᗋⓗ࡞␗ᖖࡣ☜ㄆࡉࢀ࡞ࡗࡓࠋࡲࡓ㸪ୗ⑩Ⓨ⏕๓∵⯋ෆసᴗဨࡀᕠ ᅇࡋࡓ㎰ሙ⪺ࡁྲྀࡾㄪᰝࢆᐇࡋࡓࡇࢁ㸪4 ᡞࡢ㎰ሙ࡛๐㋟ᚋୗ⑩ࡀㄆࡵ ࡽࢀࡓࡀᐙ␆ಖ⾨⏕ᡤࡣ㏻ሗࡋ࡞ࡗࡓࡇࡀࢃࡗࡓࠋ 2. RT-PCR BCoV ࡢࢾࢡࣞ࢜࢝ࣉࢩࢻ㑇ఏᏊࢆᶆⓗࡋࡓ RT-PCR ἲ࡛ࡣ㸪࠸ࡎࢀࡢ⣅ ౽᳨య࠾࠸࡚ࡶ≉␗ⓗ࡞ྠ㑇ఏᏊࡢቑᖜࡣ☜ㄆࡉࢀ࡞ࡗࡓ㸦ࢹ࣮ࢱᮍᥖ㍕㸧ࠋ ୍᪉㸪BRVB ࡢ VP7 㑇ఏᏊࢆᶆⓗࡋࡓ RT-PCR ἲ࡛ࡣ㸪6 ᳨య୰ 5 ᳨య࠾ ࠸࡚㸪281 ሷᇶᑐ㸦bp㸧ࡽ࡞ࡿ≉␗ⓗ㑇ఏᏊ⏘≀ࡢቑᖜࡀ☜ㄆࡉࢀࡓ㸦ᅗ 1-3㸧ࠋ
11 3. 㟁Ꮚ㢧ᚤ㙾᳨ᰝ 㟁Ꮚ㢧ᚤ㙾᳨ᰝ࡛ࡣ࡚ࡢ⣅౽᳨య࢘ࣝࢫ⢏Ꮚࡣ☜ㄆࡉࢀ࡞ࡗࡓࡀ㸪 BRVB ච⾑Ύࢆ⏝࠸ࡓᅛ┦ච㟁Ꮚ㢧ᚤ㙾᳨ᰝ࡛ࡣ 3 ᳨య࡛┤ᚄ 75 ੈࡢ㌴㍯ ≧ࡢ࢘ࣝࢫ⢏Ꮚࡀ☜ㄆࡉࢀࡓࠋࡇࢀࡽࡢ⢏Ꮚࡣ㸪ࣟࢱ࢘ࣝࢫࡢ≉ᚩ࡛࠶ࡿ3 㔜࢝ࣉࢩࢻᵓ㐀ࢆ᭷ࡋ࡚࠸ࡓ㸦ᅗ1-4㸧ࠋ 4. RNA-PAGE RNA-PAGE ࡛ࡣ㸪⣅౽᳨య 11 ᮏࡢศ⠇ࡀほᐹࡉࢀࡓࠋ1㸪2㸪3 ࠾ࡼࡧ 4㸪 5 ࠾ࡼࡧ 6㸪7 ࠾ࡼࡧ 8㸪࠾ࡼࡧ 9㸪10 ࠾ࡼࡧ 11 ศ⠇ࡀ⤌࡞ࡾ㸪౪ヨ᳨యࡣ BRVB ≉᭷࡞ 4㸫2㸫2㸫3 ࡢ PAGE ࣃࢱ࣮ࣥࢆ♧ࡍࡇࡀุ᫂ࡋ㸪ᅇࡢ᳨ ฟᰴࢆBRVB IS-1 ᰴྡࡋࡓࠋࡲࡓ Nemuro ᰴࢆྠὋືࡋẚ㍑ࡋࡓࡇ ࢁ㸪Nemuro ᰴࡣ␗࡞ࡿࣃࢱ࣮ࣥࢆ♧ࡋࡓ㸦ᅗ 1-5㸧ࠋ 5.㑇ఏᏊ┦ྠᛶゎᯒ RT-PCR ࡛ᚓࡽࢀࡓ BRVB IS-1 ᰴࡢ VP7 㑇ఏᏊࡢሷᇶ㓄ิࢆỴᐃࡋ㸪
DDBJ/EMBL/GenBank ࢹ࣮ࢱ࣮࣋ࢫࡽධᡭࡋࡓ BRVB Nemuro ᰴ㸪ATI ᰴ㸪 Mebus ᰴ࠾ࡼࡧ WD653 ᰴࡢࡑࢀࡢ┦ྠᛶࢆ☜ㄆࡋࡓࡇࢁ㸪ࢩ࣮ࣙࢺࢤࣀ ࣒ࣃࢱ࣮ࣥࢆ♧ࡍWD653 ᰴࡣ 93.3㸣㸪ࣟࣥࢢࢤࣀ࣒ࣃࢱ࣮ࣥࡢ Nemuro ᰴ㸪 Mebus ᰴ࠾ࡼࡧ ATI ᰴࡣ 92.9%㹼93.4㸣㧗࠸┦ྠᛶࢆ♧ࡋࡓࡓࡵ㸪ᅇ᳨ ฟࡉࢀࡓ࢘ࣝࢫࡣBRVB ࡛࠶ࡿ☜ㄆ࡛ࡁࡓࠋ 6. ࡑࡢࡢ᳨ᰝ ࢘ࣝࢫศ㞳࡛ࡣMA104 ⣽⬊࠾ࡼࡧ HRT18 ⣽⬊ࡶ㸪3 ௦⥅௦ᚋࡶ⣽⬊ ኚᛶຠᯝࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡲࡓᇵ㣴ୖΎࢆ⏝࠸ࡓRVA ࡢࣛࢸࢵࢡࢫจ㞟
12
ᛂࡶ㝜ᛶ࡛࠶ࡗࡓࠋࡉࡽࠊ⣅౽ஙࢆ⏝࠸ࡓRVA ࡢࣛࢸࢵࢡࢫจ㞟ᛂࡶ㝜
ᛶ࡛࠶ࡗࡓࠋ࣌⾑Ύࢆ⏝࠸ࡓᢠయ᳨ᰝ࡛ࡣ㸪BCoV㸪∵ࢹࣀ࢘ࣝࢫ㸵ᆺ㸪 BVDV1 ᆺᑐࡍࡿ୰ᢠయࡢ᭷ព࡞ୖ᪼ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡲࡓ㸪⣽⳦᳨ᰝ ࠾ࡼࡧᐤ⏕᳨ᰝࡣ㝜ᛶ࡛࠶ࡗࡓࠋ
13 ⪃ ᐹ ᮏ࡛ࡣ㸪ඛᡂ∵ࡢୗ⑩ࡀⓎ⏕ࡋ㸪Ꮚ∵ࢆ㝖ࡃ∵⩌యᛴ㏿ࡲ ࢇᘏࡋ㸪᭱ 10%ࡢἪங㔞ࡢῶᑡࡀㄆࡵࡽࢀࡓࠋⓎ⏕ᮇࡸ≧ࡽ຺ࡋ㸪 ᙜึࡣᮏࢆ BCoV ࡼࡿணࡋࡓࠋBCoV ࡼࡿୗ⑩ࡣ㸪ᡃࡀᅜ࡛ ᭱ࡶὶ⾜ࡋ࡚࠸ࡿᡂ∵ࡢୗ⑩࡛㸪Ꮨࡽඛࡅ࡚ᅜⓗⓎ⏕ࡀㄆࡵ ࡽࢀࡿ㸦85㸧ࠋࡋࡋ࡞ࡀࡽᮏ࡛ࡣ BCoV 㑇ఏᏊࡣ᳨ฟࡉࢀࡎ㸪BCoV ᑐ ࡍࡿᢠయࡢୖ᪼ࡶㄆࡵࡽࢀ࡞ࡗࡓࠋࡑࡢࡢཎయࡢ᳨ᰝ࡛ࡶBRVB ࡀ᳨ฟ ࡉࢀࡓ௨እ㔜せ࡞ཎయࡣ☜ㄆࡉࢀ࡞ࡗࡓࠋBCoV ࡼࡿୗ⑩࡛ࡣ⾑౽ࢆ క࠺ࡇࡀࡋࡤࡋࡤ࠶ࡿࡀ㸪ᮏ࠾࠸࡚⾑౽ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡑࢀ௨ እࡣ⮫ᗋⓗ BCoV ࡼࡿୗ⑩ࡢࡑࢀ㢮ఝࡋ࡚࠾ࡾ㸪⮫ᗋᡤぢ࡛ࡢ㚷ูࡣ ᅔ㞴⪃࠼ࡽࢀࡓࠋᡂ∵Ἢங㔞ࡢపୗࢆక࠺ୗ⑩ࢆᘬࡁ㉳ࡇࡍ࢘ࣝࢫࡣ㸪 ᮏࡀⓎ⏕ࡋࡓⅬ࡛ࡣBCoV ௨እ▱ࡽࢀ࡚࠾ࡽࡎ㸪BRVB ࡣ BCoV ྠ ᵝ㸪㓗㎰⤒Ⴀ㔜࡞ᦆ┈ࢆ࠼ࡿ࢘ࣝࢫ࡛࠶ࡿࡇࡀึࡵ࡚᫂ࡽ࡞ࡗ ࡓࠋ Ⓨ⏕㎰ሙ࡛ࡣ㸪ᡂ∵ࡢୗ⑩ࡀⓎ⏕ࡋ࡚࠸ࡿ∵⯋ෆᏊ∵ࡀ㣫㣴ࡉࢀ࡚࠸ࡓ ࡀ㸪࠸ࡎࢀࡢಶయࡶୗ⑩ࢆ♧ࡉࡎ㸪ࡑࡢࡢ␗ᖖࡶㄆࡵࡽࢀ࡞ࡗࡓࠋࣄࢺࡢ RVB ࡣᡂேୗ⑩ࢆᘬࡁ㉳ࡇࡋ㸪ᑠඣᑐࡍࡿୗ⑩ཎᛶࡣప࠸ࡉࢀ࡚࠸ࡿ 㸦101㸧ࠋ୍᪉㸪BRVB ࡣ᭱ึᏊ∵ࡢୗ⑩ࡽ᳨ฟࡉࢀ㸪ឤᰁᐇ㦂࠾࠸࡚ࡶ Ꮚ∵ୗ⑩ࢆᘬࡁ㉳ࡇࡍࡇࡀ☜ㄆࡉࢀ࡚࠸ࡿ㸦3,62,67,73㸧ࠋࡋࡋ㸪ᾏ 㐨ࡢᡂ∵ࡢୗ⑩ࡽ᳨ฟࡉࢀࡓ Nemuro ᰴࡣ㸪ࣀࢺࣂ࣮࢜ࢺᏊ∵ࡢᐇ㦂 ឤᰁ࡛ࡣୗ⑩ࡣ㉳ࡇࡍࡶࡢࡢ㸪ᡂ∵ࡢ≧ẚ⦆ࡸ࡞࡛࠶ࡿሗ࿌ࡉࢀ ࡚࠸ࡿ㸦91㸧ࠋࡇࡢࡇࡽᡃࡀᅜࡢ BRVB ࡣᏊ∵ࡼࡾࡶᡂ∵㔜⠜࡞≧ࢆ ㉳ࡇࡍྍ⬟ᛶࡀ⪃࠼ࡽࢀ㸪Ḣ⡿࡛ࡢሗ࿌㸦10,11,12㸧ࡢ㐪࠸ࡣὶ⾜ᰴࡢ㐪࠸
14 ࡼࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋ ᅇୗ⑩Ⓨ⏕๓∵ࡢᑟධࡀ࡞ࡗࡓࡇࡽ㸪ᅇࡢὶ⾜ࢆᘬࡁ㉳ࡇࡋ ࡓ BRVB ࡀ∵ࢆࡋ࡚ᣢࡕ㎸ࡲࢀࡓྍ⬟ᛶࡣప࠸⪃࠼ࡽࢀࡿࠋⓎ⏕๓ᙜヱ ㎰ሙ࡛∵⯋ෆసᴗဨࡀసᴗࢆ⾜ࡗ࡚࠸ࡿࡇࡽᮏࡢ㛵㐃ࡀࢃࢀࡓࡀ㸪 సᴗဨࡀᕠᅇࡋࡓࡢ㎰ሙࡢ࠺ࡕ㸪ୗ⑩ࡀⓎ⏕ࡋࡓ 4 ㎰ሙ࡛ࡣ᥇ᮦࡀ⾜ࢃࢀ ࡞ࡗࡓࡓࡵ㸪BRVB ࡢ㛵㐃ࡣ࡛᫂࠶ࡗࡓࠋࡋࡋⓎᮇࡽసᴗဨࡢ 㛵ࢆྰᐃࡍࡿࡇࡀ࡛ࡁࡎ㸪ᚋBRVB ࡀࡢࡼ࠺㎰ሙ㛫ࢆఏࡍࡿࢆ ࣄࢺࡼࡿᣢࡕ㎸ࡳࡶྵࡵ࡚ㄪᰝࡍࡿᚲせࡀ࠶ࡿ⪃࠼ࡽࢀࡓࠋ ᮏࡽ᳨ฟࡉࢀࡓBRVB IS-1 ᰴࡣ RNA-PAGE ࡽࢩ࣮ࣙࢺࢤࣀ࣒ࣃࢱ ࣮ࣥࢆ♧ࡍࡇࡀุ᫂ࡋࡓࠋᡃࡀᅜึࡢBRVB ᰴ࡛࠶ࡿ Nemuro ᰴࡣ㸪ࣟࣥࢢ ࢤࣀ࣒ࣃࢱ࣮࡛ࣥ࠶ࡿࡇࡽ㸪ᡃࡀᅜࡣᑡ࡞ࡃࡶ᪉ࡢᰴࡀᏑᅾࡋ࡚࠸ ࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋࣟࢱ࢘ࣝࢫ࡛ࡣࣟࣥࢢࢤࣀ࣒ࣃࢱ࣮ࣥࢆ♧ࡍᰴࡀ ᶆ‽࡛࠶ࡾ㸪ࢩ࣮ࣙࢺࢤࣀ࣒ࣃࢱ࣮ࣥࡣᕼሗ࿌ࡉࢀࡿࡢࡳ࡛࠶ࡿࠋࢩ࣮ࣙࢺ ࢤࣀ࣒ࣃࢱ࣮ࣥࡣ∵㸪ࣄࢺ㸪㇜࠾ࡼࡧ࢘ࢧࢠࡢRVA ࢘ࣝࢫࡶሗ࿌ࡉࢀ࡚࠸ ࡿ ࡀ 㸪RNA-PAGE ࣃ ࢱ ࣮ ࣥ ⮫ ᗋ ≧ ࡢ 㛵 㐃 ࡣ ሗ ࿌ ࡉ ࢀ ࡚ ࠸ ࡞ ࠸ 㸦17,35,57,64,67㸧ࠋ⡿ᅜ࠾ࡼࡧⱥᅜ࡛Ꮚ∵ୗ⑩ࢆ㉳ࡇࡋࡓ BRVB ᰴࡢ㑇ఏ Ꮚࡢሗࡣ࡞࠸ࡓࡵ㸪RNA-PAGE ࡢࣃࢱ࣮ࣥᏊ∵ࡢཎᛶࡣẚ㍑࡛ࡁ࡞࠸ ࡀ㸪ࢩ࣮ࣙࢺࢤࣀ࣒ࣃࢱ࣮ࣥࡢIS-1 ᰴࡶࣟࣥࢢࢤࣀ࣒ࣃࢱ࣮ࣥࡢ Nemuro ᰴࡶ ᡂ∵ࡢୗ⑩ࡽ᳨ฟࡉࢀ࡚࠸ࡿࡇࡽ㸪ᡂ∵ࡢཎᛶࡣRNA-PAGE ࣃࢱ ࣮ࣥ㛵㐃ࡋ࡞࠸ࡇࡀ♧၀ࡉࢀࡓࠋRVA ࡢࢩ࣮ࣙࢺࢤࣀ࣒ࣃࢱ࣮ࣥࡣ NSP5 㑇ఏᏊࡢ3’㠀⩻ヂ㡿ᇦ㸦UTR㸧ࡢ㓄ิ㔜」ࡀ࠶ࡾศᏊ㔞ࡀቑ࠼ࡿࡓࡵ㸪NSP5 ศ⠇ࡀ NSP4 ศ⠇ࡼࡾࡶ㐜ࢀ࡚Ὃືࡉࢀࡿࡓࡵ⏕ࡌࡿሗ࿌ࡉࢀ࡚࠸ࡿ (17,35,57)ࠋRVB ࡛࠶ࡿ IS-1 ᰴࡢ NSP5 㑇ఏᏊࡶྠᵝ࡞㔜」ࡀᏑᅾࡍࡿྍ⬟ ᛶࢆ᳨ウࡍࡿࡓࡵࡣࠊྠ㑇ఏᏊࡢሷᇶ㓄ิࢆỴᐃࡍࡿᚲせࡀ࠶ࡿࠋ
15
ᅗ⾲
ᅗ1-1. BRVB ࡼࡿୗ⑩Ⓨ⏕㎰ሙࡢୗ⑩Ⓨ⏕᭶ࡢἪங㔞ኚ
16 ᅗ1-2. BRVB ࡼࡿୗ⑩Ⓨ⏕㎰ሙࡢୗ⑩๓ୗ⑩ᮇ㛫ࡢἪங㔞ᖹᆒ್ࡢẚ㍑ ᖹᆒ್ࡢᕪࡢ᳨ᐃࡣ㹲᳨ᐃ࡚ᐇ㸦n㸻7㸧ࠋ ࢚࣮ࣛࣂ࣮ࡣᶆ‽೫ᕪࢆ♧ࡍࠋ Ⓨ⏕๓Ⓨ⏕ᮇ㛫࡛Ἢங㔞ࡢᖹᆒ್᭷ព࡞ᕪࡀぢࡽࢀࡓ㸦p=0.0069㸪 p<0.05㸧ࠋ 1550 1600 1650 1700 1750 1800 1850 Ⓨ⏕๓ Ⓨ⏕ᮇ㛫 Ἢங㔞(kg) P<0.05
17 ᅗ1-3. RT-PCR ἲࡼࡿ BRVB ࡢ VP7 㑇ఏᏊࡢ᳨ฟ M : ศᏊ㔞࣐࣮࣮࢝ NC : 㝜ᛶࢥࣥࢺ࣮ࣟࣝ 1~6 : ౪ヨᮦᩱ M NNC 11 22 3 4 5 6 281bp
18
ᅗ1-4. ᅛ┦ච㟁Ꮚ㢧ᚤ㙾᳨ᰝࡼࡾ᳨ฟࡉࢀࡓ BRVB ⢏Ꮚ
┤ᚄ⣙75nm ࡢ㌴㍯≧ࡢ 3 㔜࢝ࣉࢩࢻᵓ㐀ࢆ᭷ࡍࡿ
19 11 A B 6 10 9 8 7 5 3 4 2 1 1 2 3 4 6 5 11 7 8 10 9 ᅗ1-5. RNA-PAGE ࡼࡿ㟁ẼὋືࣃࢱ࣮ࣥ ࣮ࣞࣥA: BRVB IS-1 ᰴ ࣮ࣞࣥB: BRVB Nemuro ᰴ
20 ➨❶ ᡂ∵ࡢ∵ࣟࢱ࢘ࣝࢫB ࡼࡿୗ⑩ࡢὶ⾜せᅉࡢ᳨ウ
21 ᗎ ㄽ ➨୍❶࡛ BRVB ࡀᡂ∵ங㔞ࡢῶᑡࢆక࠺ὶ⾜ᛶୗ⑩ࢆᘬࡁ㉳ࡇࡋ㸪㓗㎰⤒ Ⴀᦆኻࢆ࠼ࡿࡢཎᅉ࡞ࡿࡇࢆ᫂ࡽࡋࡓࠋヲ⣽࡞ཎᏛⓗゎᯒ ࢆᐇࡍࡿࡓࡵࡣ㸪࢘ࣝࢫࢆศ㞳ࡍࡿࡇࡀ㔜せ࡛࠶ࡿࡀ㸪BRVB ࡢศ㞳 ᇵ㣴ࡣᴟࡵ࡚ᅔ㞴࡛࠶ࡾࠊࡑࡢᡂຌࡣ⌧ᅾ࡛ࡶሗ࿌ࡉࢀ࡚࠸࡞࠸ࠋࡇࡢࡇࡀ BRVB ࡢᏛᐇែࡢゎ᫂ࡢ㊊ࡏ࡞ࡾ㸪ࡑࡢᏛⓗᐇែࡣࢇ᫂ࡽ ࡉࢀ࡚࠸࡞ࡗࡓࠋ ࡋࡋTsunemitsu ࡽࡀ㸪⤌࠼ BRVB VP6 ࢆᢠཎࡋࡓᢠయ᳨ฟ⏝ ELISA ࢆ㛤Ⓨࡋ㸪࢘ࣝࢫศ㞳ࡀ࡛ࡁ࡞ࡃࡶ㸪ࡑࡢᾐ₶≧ἣㄪᰝࢆᐇࡍࡿࡇࡀ ྍ⬟࡞ࡗࡓ(93)ࠋ 2002 ᖺ 2005 ᖺࡢ 3 ᖺ㛫㝸࡛ 2 ᅇࢃࡓࡾ㸪▼ᕝ┴ෆࡢつᶍ㓗㎰ᅋᆅ ࠾࠸࡚ BRVB ࡼࡿᡂ∵ࡢ㞟ᅋᛶୗ⑩ࡀὶ⾜ࡋࡓࠋࡇࡢ୰ࡢ㎰ሙࡣ᪉ࡢὶ ⾜࡛ୗ⑩ࡀⓎ⏕ࡋࡓ㎰ሙ㸪࠶ࡿ࠸ࡣ 2 ᅇ┠ࡢὶ⾜ࡣୗ⑩ࡢⓎࢆㄆࡵ࡞ ࡗࡓ㎰ሙࡀ࠶ࡗࡓࠋ ࡑࡇ࡛➨2 ❶࡛ࡣ㸪ࡇࢀࡲ࡛ሗ࿌ࡢ࡞ࡗࡓ BRVB ࡼࡿୗ⑩ࡢὶ⾜ࡢ せᅉࢆ᳨ウࡍࡿࡓࡵ㸪୧Ⓨ⏕࠾ࡅࡿ㎰ሙ࠾ࡼࡧಶయࡈࡢBRVB ᢠయㄪᰝࢆ ELISA ἲࡼࡾᐇࡋ㸪ὶ⾜ᰴࡢ㑇ఏᏊゎᯒࡶేࡏ࡚ᐇࡋࡓࠋ
22 ᮦᩱ࠾ࡼࡧ᪉ἲ 1. BRVB ࡢὶ⾜ࡀ࠶ࡗࡓ㓗㎰ᅋᆅࡢᴫせ BRVB ࡼࡿୗ⑩ࡀⓎ⏕ࡋࡓつᶍ㓗㎰ᅋᆅࡣ㸪▼ᕝ┴ࡢຍ㈡ᆅ༊⨨ࡍ ࡿࠋᅋᆅࡣ 7 ࡘࡢ༊⏬ࡽᡂࡾ㸪ࡑࢀࡒࢀࡢ༊⏬ࡣ 4 ␆⯋ࡼࡾᵓᡂࡉࢀ࡚࠸ࡿ 㸦ᅗ2-1㸧ࠋࡲࡓࡑࢀࡒࢀࡢ༊⏬ࡣ 200 㹫ࡢ㛫㝸ࡀ࠶ࡾ㸪༊⏬ෆࡢ㎰ሙࡣ 15 m 㞳ࢀ࡚࠸ࡿࠋྛ㎰ᐙ࡛ࡣ࣍ࣝࢫࢱࣥ✀ࡢᦢங∵ࡀᑐᑼᘧࢱࢫࢺ࣮࡛ࣝ㣫㣴 ࡉࢀ࡚࠸ࡓࠋᏊ∵ࡣ㎰ሙࡼࡾ∵⯋ෆ✵ࡁࢫ࣮࣌ࢫࡲࡓࡣ∵⯋ෆタ⨨ࡋࡓ࣐ ࢫෆ㣫㣴ࡉࢀ࡚࠸ࡓࠋ 2002 ᖺࡣ㸪17 ㎰ሙ㸦A㹼Q ㎰ሙ㸧⣙ 1400 㢌ࡢᡂ∵㸦1 ㎰ሙ࠶ࡓࡾ 23㹼 275 㢌㸧࠾ࡼࡧ⣙ 200 㢌ࡢᏊ∵ࢆྵࡴ⫱ᡂ∵㸦1 ㎰ሙ࠶ࡓࡾ 0㹼60 㢌㸧ࡀ㣫㣴 ࡉࢀ࡚࠸ࡓࠋ2005 ᖺࡣ C ㎰ሙࡀᗫᴗࡋ 16 ㎰ሙ࡞ࡾ㸪⣙ 1500 㢌ࡢᡂ∵㸦1 ㎰ሙ࠶ࡓࡾ48 㢌㹼285 㢌㸧࠾ࡼࡧ⣙ 320 㢌ࡢᏊ∵ࢆྵࡴ⫱ᡂ∵㸦1 ㎰ሙ࠶ࡓࡾ 7㹼46 㢌㸧ࡀ㣫㣴ࡉࢀ࡚࠸ࡓࠋA ࠾ࡼࡧ F ㎰ሙࢆ㝖࠸ࡓ࡚ࡢ㎰ሙ࡛ྠ୍ࡢ㛤 ᴗ⋇་ᖌࡀデ⒪࠾ࡼࡧ⦾Ṫᴗົࢆᢸᙜࡋ࡚࠸ࡓࠋᖺ㛫ࡢᦢங∵ࡢ᭦᪂⋡ࡣ㓗㎰ ᅋᆅయ࡛⣙30㹼40㸣࡛࠶ࡗࡓࠋ᭦᪂∵ࡢ 2/3 ⛬ᗘࡣ┴ෆእࡢ㎰ሙࡼࡾᑟධ ࡉࢀ࡚࠸ࡓࠋ ࡲࡓ2002ᖺࡽ2005ᖺࡢ᪉ࡢⓎ⏕࠾ࡅࡿᖹᆒ㣫㣴㢌ᩘࡸᦢங∵ࡢᖺ㛫 ᖹᆒ᭦᪂ྜࡣ㸪Ⓨ⏕㎰ሙ㠀Ⓨ⏕㎰ሙࡢ㛫ᕪࡣ࡞ࡗࡓࠋ 2. ⣅౽࠾ࡼࡧ⾑Ύࢧࣥࣉࣝࡢ᥇ྲྀ 2002 ᖺࡢⓎ⏕࠾࠸࡚ࠊୗ⑩ࢆⓎࡋࡓᡂ∵ 20 㢌㸦E ㎰ሙ 10 㢌㸪D ㎰ሙ 5 㢌㸪J ㎰ሙ 3 㢌࠾ࡼࡧ L ㎰ሙ 2 㢌㸧ࡼࡾ⣅౽ࢆ᥇ྲྀࡋࡓࠋ2005 ᖺࡢⓎ⏕࡛ࡣ㸪 28 㢌ࡢୗ⑩Ⓨᡂ∵ 18 㢌ࡢᮍⓎᡂ∵ࡢ⣅౽ࢆ E ㎰ሙ(ୗ⑩Ⓨ 7 㢌㸪ᮍⓎ
23 12 㢌)㸪D ㎰ሙ(ୗ⑩Ⓨ 6 㢌㸪ᮍⓎ 6 㢌)㸪L ㎰ሙ㸦ୗ⑩Ⓨ 5 㢌㸧㸪Q ㎰ ሙ(ୗ⑩Ⓨ 5 㢌)࠾ࡼࡧ G ㎰ሙ㸦ୗ⑩Ⓨ 5 㢌㸧ࡽ᥇ᮦࡋࡓࠋୗ⑩Ⓨ∵ࡢ ⣅౽ࡣୗ⑩Ⓨぢࡼࡾ4 ᪥௨ෆࡢࡶࡢࢆ᥇ྲྀࡋࡓࠋ ⾑Ύࢧࣥࣉࣝࡣ⣅౽ࢆ᥇ྲྀࡋࡓ∵ࡽ᥇⾑ࢆ⾜࠸㸪Ⓨ⏕㸦ᛴᛶᮇ㸧࠾ࡼࡧ ᅇᮇ㸦19 ࡽ 28 ᪥ᚋ㸧᥇ྲྀࡋ࡚࣌⾑Ύࡋࡓࠋࡇࢀࡽࡢ∵ࡢ࠺ࡕࡢ㸵 㢌ࡣ㸪2002 ᖺ࠾ࡼࡧ 2005 ᖺࡢ 2 ᅇࡶ᥇ᮦࢆ⾜ࡗࡓࠋࡲࡓ 2002 ᖺⓎ⏕ࡢ 4 ᭶๓ᐙ␆ఏᰁண㜵ἲᇶ࡙ࡃᐃᮇ᳨ᰝࡼࡗ࡚ᚓࡽࢀࡓE ㎰ሙ 66 㢌㸪D ㎰ሙ69 㢌㸪A ㎰ሙ 118 㢌࠾ࡼࡧ O ㎰ሙ 71 㢌ࡢಖᏑ⾑Ύࡶ᳨ᰝ౪ࡋࡓࠋ 3. RT-PCR㸪ࢩ࣮ࢡ࢚ࣥࢫ࠾ࡼࡧ㑇ఏᏊゎᯒ RNA ᢳฟࡣ➨୍❶ྠᵝࡢ᪉ἲ࡛ᐇࡋࡓࠋRT-PCR ࡣ OneStep RT-PCR Kit㸦QIAGEN,Valencia㸪Caifolnia㸪USA㸧ࢆ⏝࠸ࡓࠋBRVB ࡢ㑇ఏᏊ᳨ฟࡣ VP7 ࢆᶆⓗࡋࡓ Chisangaram ࡽ㸦13㸧ࡢࣉ࣐࣮ࣛࢆ⏝࠸࡚ᐇࡋࡓࠋBRVA㸪 BRVC㸪BToV㸪BCoV ࠾ࡼࡧ BVDV ࡢ㑇ఏᏊ᳨ฟࡣ᪤ሗ㸦34,78,85,88,97㸧 ‽ࡌ࡚ᐇࡋࡓࠋ BRVB VP7 ࡢ㑇ఏᏊ㛗ࢆỴᐃࡍࡿ┠ⓗ࡛㸪 2002 ᖺࡢ 4 Ⓨ⏕㎰ሙ࠾ࡼࡧ 2005 ᖺࡢ 5 Ⓨ⏕㎰ሙࡽ 1 ㎰ሙ࠶ࡓࡾ 2 ᳨య㸪ྜィ 18 ᳨యࢆ᥇ྲྀࡋ㸪ࡇࢀࡽ ࢆ⏝࠸࡚BRVB VP7 㑇ఏᏊ㛗ࡢሷᇶ㓄ิࡢỴᐃࢆヨࡳࡓࠋࡑࡢ㝿ࠊVP7 㑇ఏ Ꮚ㛗ࢆቑᖜࡍࡿ Tsunemitsu ࡽ㸦91㸧ࡢࣉ࣐࣮ࣛࢆ⏝࠸࡚ RT-PCR ࢆ⾜ࡗ
ࡓࠋᚓࡽࢀࡓ⏘≀ࡣpCR2.1 TOPO vector (Life Technologies㸪USA) ࡚ࢡࣟ
࣮ ࢽ ࣥ ࢢ ࡋ 㸪 ࢧ ࢡ ࣝ ࢩ ࣮ ࢡ ࢚ ࣥ ࢫ ࢆ ⾜ ࠸ 㸪 ⮬ ື ࢩ ࣮ ࢡ ࢚ ࣥ ࢧ ࣮ABI
PRISM3100㸦Life Technologies㸪USA㸧ࢆ⏝࠸࡚㓄ิࢆゎㄞࡋࡓࠋVP4 㑇ఏᏊ 5’ഃࡢ⣙ 1300 bp ࡢሷᇶ㓄ิࡢỴᐃࢆ┠ⓗࡋ࡚㸪2002 ᖺࡢ 4 Ⓨ⏕㎰ሙ࠾ࡼࡧ 2005 ᖺࡢ 3 Ⓨ⏕㎰ሙ㸪ࡑࢀࡒࢀ 1 ㎰ሙࡘࡁ 1 ᳨యࡎࡘࡘ࠸࡚㸪ࣉ࣐࣮ࣛ
24
1F 㸦 5’- GGTATTTAATCACTAGGC-3’ 㸧 ࠾ ࡼ ࡧ 1295R
㸦5’-GGATTCAAACTGTTGTCAACTGG-3’㸧ࡼࡿ RT-PCR ࢆ⾜࠸㸪㓄ิࢆỴ
ᐃࡋࡓࠋ࡞࠾㸪RT-PCR ࡣ PrimeScript One Step RT-PCR Kit ver.2 㸦Takara,
Shiga, Japan㸧ࢆ⏝࠸ࡓࠋࢩ࣮ࢡ࢚ࣥࢫࢹ࣮ࢱࢆ Clustal W 㸦87㸧 ࡚ᩚิ
ࡋࡓᚋ㸪 Lasergene software 㸦DNASTAR㸪WI㸪USA㸧ࡢ MegAlign program
Ver 11.2.1 ࢆ⏝࠸ࠊDDBJ/EMBL/GenBank ࢹ࣮ࢱ࣮࣋ࢫⓏ㘓ࡉࢀ࡚࠸ࡿࡢ BRVB ࡸࣄࢺ⏤᮶ RVB ඹ⣔⤫ᶞゎᯒ࠾ࡼࡧ┦ྠᛶゎᯒࢆ⾜ࡗࡓࠋ 4. RNA-PAGE ➨୍❶ྠᵝࡢᡭἲ࡛⣅౽ஙୖΎࢆ⢭〇ࡋࡓᚋ㸪RNA ᢳฟࢆᐇࡋࡓࠋᚓࡽ ࢀࡓ RNA ࢧࣥࣉࣝࡘ࠸࡚ࡣ㸪➨୍❶ྠᵝ Herring ࡽ㸦36㸧ࡢ᪉ἲ‽ࡌ㸪 7.5㸣ࡢ࣏ࣜࢡ࣑ࣜࣝࢻࢤࣝ㸦e-PAGEL;Atto㸪Tokyo㸪Japan㸧ࢆ⏝࠸࡚Ὃ
ືࡋࡓᚋ㸪RNA ࣂࣥࢻࢆ㖟ᰁⰍ࢟ࢵࢺ㸦Bio Rad㸪USA㸧ࡼࡾྍどࡋࡓࠋ
5. ⾑Ύᢠయ᳨ᰝ BRVB ᢠయࡢ᳨ฟࡣ,Tsunemistu ࡽ㸦89㸧ࡢ VP6 ⺮ⓑࡢࣂ࢟ࣗࣟ࢘ࣝࢫⓎ ⌧⤌ࡳ࠼⺮ⓑࢆ⏝࠸ࡓELISA ࡚ᐇࡋࡓࠋBRVB ᢠయ౯ࡣ㸪ᢠཎࡀࢥ࣮ࢸ ࣥࢢࡉࢀࡓ࢙࢘ࣝࡢ OD ್ࡼࡾᮍࢥ࣮ࢸࣥࢢ࢙࢘ࣝࡢ OD ್ࢆᘬ࠸ࡓ್࡛ ⟬ฟࡋࡓࠋ࣌⾑Ύ࡛ 0.2 ࡶࡋࡃࡣࡑࢀ௨ୖࡢ OD ್ୖ᪼ࢆ᭷ព࡞ᢠయୖ᪼ ุ᩿ࡋࡓࠋࡲࡓⓎ∵ᮍⓎ∵ࡢ࣌⾑Ύࢆ⏝࠸ BVDV1 ᆺ 㸦Nose ᰴ㸧࠾ ࡼࡧ 2 ᆺ㸦KZ-91cp ᰴ㸧ࢆ⏝࠸ࡓ୰ヨ㦂࠾ࡼࡧ BCoV㸦Kakegawa ᰴ㸧࠾ࡼ ࡧ∵ࢹࣀ࢘ࣝࢫ7 ᆺ㸦⿄ᰴ㸧ࢆ⏝࠸ࡓ HI ヨ㦂ࢆᐇࡋࡓࠋ᳨ᰝࡋࡓ࢘ ࣝࢫᑐࡍࡿ࣌⾑Ύࡢᢠయࡀ㸪2 ಸ㝵ẁᕼ㔘࡛ 4 ⟶ࡶࡋࡃࡣࡑࢀ௨ୖࡢୖ᪼ࡀ ぢࡽࢀࡓሙྜ㸪᭷ព࡞ୖุ᩿᪼ࡋࡓ㸦100㸧ࠋ
25 6. ⣽⳦᳨ᰝ࠾ࡼࡧᐤ⏕᳨ᰝ ࢧࣝࣔࢿࣛ⳦࣭ཎᛶ⭠⳦ࡢศ㞳ᇵ㣴᳨ᰝ㸪ࢥࢡࢩࢪ࣒࢘࠾ࡼࡧࢡࣜࣉࢺ ࢫ࣏ࣜࢪ᳨࣒࢘ᰝࢆ➨୍❶ྠᵝࡢ᪉ἲ࡛ᐇࡋࡓࠋ 7. ⤫ィゎᯒ ㎰ᐙ㛫࠶ࡿ࠸ࡣ∵㛫ࡢRVB ᢠయ㝧ᛶ⋡ࡢ⤫ィᏛⓗゎᯒࡣ㸪࢚ࢡࢭࣝ⤫ィ 2012
㸦Social Survey Research Information Co., Ltd., Tokyo, Japan㸧ࢆ⏝࠸㸪࢝
᳨ᐃࡶࡋࡃࡣࣇࢵࢩ࣮ࣕࡢ┤᥋᳨ᐃ࡛ᐇࡋࡓࠋ
8. ࢩ࣮ࢡ࢚ࣥࢫゎᯒ࡛ᚓࡽࢀࡓሷᇶ㓄ิࡢࢡࢭࢵࢩࣙࣥ␒ྕ
Ỵᐃࡋࡓሷᇶ㓄ิࡣࢡࢭࢵࢩࣙࣥ␒ྕ LC005523㹼LC005538ࢆࡾࡾ㸪
26 ⤖ ᯝ 1. Ⓨ⏕≧ἣ 2002 ᖺ 10 ᭶㸪G ㎰ሙࡢᦢங∵࡛᭱ึୗ⑩ࡀⓎ⏕ࡋ㸪ᩘ᪥࡛∵⯋ෆࡢᦢங ∵ᗈࡀࡗࡓࠋࡑࡢᚋ2 㐌㛫௨ෆᅋᆅࡢ 17 ᡞ୰ 9 ᡞ㸦D㸪E㸪F㸪G㸪J㸪K㸪 L㸪P ࠾ࡼࡧ Q ㎰ሙ㸧㸪ᡂ∵⣙ 660 㢌୰⣙ 550 㢌㸦83㸣㸧ࡲࢇᘏࡋࡓ㸦ᅗ 2-1㸧ࠋ ᭱ึࡢୗ⑩Ⓨ⏕ 2 ᪥๓㸪G ㎰ሙ 4 㢌ࡢ㞤∵ࡀ┴እࡼࡾᑟධࡉࢀ࡚࠸ࡓࠋࡇ ࢀࡽࡢ∵ࡣୗ⑩≧ࢆ࿊ࡋ࡚࠾ࡽࡎ㸪ᑟධ⣅౽ࡢ᥇ྲྀࡣ⾜ࢃࢀ࡞ࡗࡓࠋ 2005 ᖺ 3 ᭶ࡣ㸪ྠ୍㓗㎰ᅋᆅ 16 ᡞ୰ 9 ᡞ㸦D㸪E㸪G㸪J㸪K㸪L㸪M㸪P ࠾ ࡼࡧQ ㎰ሙ㸧ࡢᡂ∵⣙ 700 㢌୰⣙ 450 㢌㸦64㸣㸧࡛ୗ⑩ࡀⓎ⏕ࡋࡓ㸦ᅗ 2-1㸧ࠋ 2002 ᖺྠᵝ㸪ᩘ᪥௨ෆ∵⯋ෆୗ⑩ࡀᗈࡀࡾ㸪ึⓎࡽ 2 㐌㛫௨ෆ㎰ሙ㛫 ࡲࢇᘏࡋࡓࠋᅇࡢୗ⑩ࡀⓎ⏕ࡍࡿ๓∵ࡢᑟධࡣ࡞ࡗࡓࠋ9 ㎰ሙ୰ M ㎰ሙࢆ㝖ࡃ8 ㎰ሙࡣ 2002 ᖺࡢⓎ⏕ࢆ⤒㦂ࡋࡓ㎰ሙ࡛࠶ࡗࡓࠋ 2002 ᖺ 2005 ᖺࡢ᪉ࡢⓎ⏕ඹ㏻ࡋࡓ⮫ᗋᡤぢࡀ࠸ࡃࡘㄆࡵࡽࢀࡓࠋ ࠼ࡤឤᰁ∵ࡣỈᵝᛶⲔⰍࡢୗ⑩ࢆ࿊ࡋ㸪⾑౽࠾ࡼࡧⓎ⇕≧ࡣㄆࡵࡽࢀࡎ㸪ୗ ⑩ࡢⓎ⏕ᚋ3㹼5 ᪥࡛≧ࡀᅇࡋࡓⅬ࡛࠶ࡿࠋࡲࡓ⏕ஙࡢἪங㔞ࡣⓎ⏕๓ࡢᖹ ᆒἪங㔞ẚ㍑ࡋ࡚⣙ 10㸣ῶᑡࡋࡓࠋࡉࡽᡂ∵ࡢୗ⑩ࡀὶ⾜ࡋ࡚࠸ࡿᮇ㛫㸪 ⫱ᡂ∵࠾ࡼࡧᏊ∵ୗ⑩࡞ࡢ⮫ᗋ≧ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ 2. RT-PCR 2002ᖺⓎ⏕ࡀ࠶ࡗࡓ4㎰ሙ20᳨యࡢ࠺ࡕ16᳨య㸦80.0%㸧㸪࠾ࡼࡧ2005ᖺ Ⓨ⏕ࡀ࠶ࡗࡓ5㎰ሙࡢ28᳨యࡢ࠺ࡕ22᳨య㸦78.6%㸧࡛BRVBࡢVP7㑇ఏᏊࡀ ᳨ฟࡉࢀࡓࠋࡲࡓ㸪2005ᖺⓎ⏕㎰ሙࡢୗ⑩౽ྠ᥇ྲྀࡉࢀࡓṇᖖ౽18᳨య ࡢ࠺ࡕ8᳨యࡽࡶBRVBࡢVP7㑇ఏᏊࡀ᳨ฟࡉࢀࡓࠋ୍᪉㸪࠸ࡎࢀࡢ᳨యࡽ ࡶBRVA㸪BRVC㸪BCoV㸪BoTV࠾ࡼࡧBVDVࡢ㑇ఏᏊࡣ᳨ฟࡉࢀ࡞ࡗࡓࠋ
27 3. RNA-PAGE RT-PCRἲ࡛BRVB㝧ᛶ࡛࠶ࡗࡓ᳨యࡢ୍㒊㸪 BRVB≉᭷࡞RNA-PAGE ࣃࢱ࣮ࣥ㸦4-2-2-3㸧ࢆ☜ㄆࡋࡓࠋࡲࡓ2005ᖺࡢὋືࣃࢱ࣮࡛ࣥࡣ㸪5~6ศ⠇ࡢ 㛫㸪࠾ࡼࡧ7~8ศ⠇ࡢ㛫㝸ࡀ2002ᖺࡢࡑࢀࡽࡢ㛫㝸ࡼࡾࢃࡎᗈࡀࡗ࡚࠸ࡿ ࡇࡽ㸪Ὃືࣃࢱ࣮ࣥࡣ㢮ఝࡋ࡚ࡣ࠸ࡿࡶࡢࡢ㸪ࡃྠ୍࡛ࡣ࡞࠸ุ᩿ࡉ ࢀࡓ㸦ᅗ2-2㸧ࠋ 4. 㑇ఏᏊゎᯒ VP7㑇ఏᏊࡢ㛗ゎᯒࡢ⤖ᯝ㸪2002ᖺ࠾ࡼࡧ2005ᖺࡢྛᖺ࠾ࡅࡿྠ୍㎰ሙ ⏤᮶ᰴࡢ㑇ఏᏊሷᇶ㓄ิࡢ┦ྠᛶࡣ100㸣࡛࠶ࡗࡓࠋࡼࡗ࡚㸪ࡑࢀࡒࢀࡢⓎ⏕࡛ 1㎰ሙࡘࡁ 1ᰴ㸪ྜィ 9ᰴࡢሷᇶ㓄ิࢆỴᐃࡋ㸪ࡑࢀࡒࢀࡢᰴD-2002㸪 E-2002㸪J-2002㸪L-2002㸪D-2005㸪E-2005㸪G-2005㸪K-2005࠾ࡼࡧL-2005 ྡࡅࡓࠋࡇࢀࡽࡢᰴࡢVP7㑇ఏᏊ㛗㸦UTRྵࡴ㸧ࡣ816bp࡛㸪ゎᯒ⏝ ࠸ࡓᰴࡢ୰࡛ࡣ㸪1997ᖺ᳨ฟࡉࢀࡓBRVB Nemuroᰴ㸦AB016818㸧᭱ࡶ㏆ ⦕࡛࠶ࡗࡓࠋ2002ᖺⓎ⏕ࡢᰴࡣ࡚ྠ୍࡛࠶ࡾ㸪 2005ᖺⓎ⏕ࡢᰴࡶ࡚ ྠ୍࡛࠶ࡗࡓࠋ2002ᖺ2005ᖺࡢᰴࢆẚ㍑ࡍࡿ5㹼6ሷᇶࡢ㐪࠸ࡀ࠶ࡾ㸪┦ྠ ᛶࡣ99.3~99.4㸣࡛࠶ࡗࡓࠋࡉࡽ⣔⤫ᶞ࡛ࡣ2002ᖺ2005ᖺࡢᰴࡣ㸪␗࡞ࡿ ࢡࣛࢫࢱ࣮ࢆᙧᡂࡋࡓ(ᅗ2-3a)ࠋ VP4㑇ఏᏊࡢ5'ᮎ➃ࡢ1295 bpࡽ࡞ࡿሷᇶ㓄ิࡣ㸪 D-2002㸪E-2002㸪J-2002㸪 L2002㸪G-2005㸪K-2005࠾ࡼࡧL-2005ࡢ7ᰴࡘ࠸࡚ゎᯒࡋࡓࠋࡇࢀࡽࢆࢹ࣮ ࢱ࣮࣋ࢫୖࡢ∵࠾ࡼࡧࣄࢺࡢࡑࢀẚ㍑ࡋࡓࡇࢁ㸪ࣄࢺ⏤᮶ࡢRVBࡼࡾࡶ ࣥࢻࡢ∵ࡼࡾ᳨ฟࡉࢀࡓBRVBᰴ࡛࠶ࡿRUBV226ᰴ㸪RUBV282ᰴ࠾ࡼࡧ DB176ᰴ㏆⦕࡛࠶ࡿࡀ㸪┦ྠᛶࡣ79㹼80㸣ẚ㍑ⓗప࠸ࡇࡀ☜ㄆࡉࢀࡓࠋ VP4㑇ఏᏊࡶVP7㑇ఏᏊྠᵝ㸪2002ᖺ2005ᖺࡢᰴࡣ⣔⤫ᶞ࡛␗࡞ࡿࢡࣛࢫࢱ
28 ࣮ᒓࡋࡓ㸦ᅗ2-3b)ࠋ 5. ⾑Ύᢠయ᳨ᰝ BCoV㸪∵ࢹࣀ࢘ࣝࢫ7ᆺ࠾ࡼࡧBVDV-1ᆺ 2ᆺᑐࡍࡿᢠయ౯ࡣ㸪ྛᖺ ࡢࣉࣞ࠾ࡼࡧ࣏ࢫࢺ⾑Ύࡢẚ㍑࠾࠸࡚᭷ព࡞ୖ᪼ࢆㄆࡵ࡞ࡗࡓࠋBRVB࠾ ࠸࡚ࡣ㸪2002ᖺࡣ20㢌ࡢ࠺ࡕ16㢌㸦80㸣㸧࠾ࡼࡧ2005ᖺࡣ28㢌ࡢ࠺ࡕ24㢌 㸦86㸣㸧࡛᭷ព࡞ᢠయୖ᪼㸦OD್ 0.2௨ୖ㸧ࢆㄆࡵࡓ㸦ᅗ2-4a࠾ࡼࡧb㸧ࠋࡇ ࢀࡽࡢ⤖ᯝࡽ㸪2002ᖺ࠾ࡼࡧ2005ᖺࡢὶ⾜ᛶୗ⑩ࡣBRVB༢⊂ࡼࡿࡶࡢ ⪃࠼ࡽࢀࡓࠋࡲࡓ㸪Ⓨ⏕㎰ሙࡢᮍⓎ∵18㢌ࡢ࠺ࡕ13㢌ࡢ࣌⾑Ύ࡛BRVB ᑐࡍࡿⴭࡋ࠸ᢠయୖ᪼ࡀㄆࡵࡽࢀࡓ㸦ᅗ2-4c㸧ࠋ 2002ᖺࡢୗ⑩Ⓨ⏕4᭶๓ࡢⓎ⏕㎰ሙD࠾ࡼࡧE㎰ሙࡢBRVBᢠయ㝧ᛶ⋡ࡣࡑ ࢀࡒࢀ32㸣࠾ࡼࡧ30㸣࡛࠶ࡗࡓࠋࡑࢀᑐࡋBRVBࡼࡿୗ⑩Ⓨ⏕ࡀ☜ㄆࡉࢀ ࡞ࡗࡓA㎰ሙ࠾ࡼࡧO㎰ሙࡢᢠయ㝧ᛶ⋡ࡣࡑࢀࡒࢀ67㸣࠾ࡼࡧ61㸣࡛࠶ࡾ㸪Ⓨ ⏕㎰ሙࡢᢠయ㝧ᛶ⋡ẚ㍑ࡋ࡚᭷ព㧗ࡗࡓ㸦p<0.01㸪⾲2-2㸧ࠋ ࡲࡓ㸪2002ᖺⓎ⏕ࡢⓎ⏕㎰ሙD㸪E㸪J࠾ࡼࡧLࡢᛴᛶᮇࡢᢠయ㝧ᛶ⋡ࡣ15㸣 ࡛࠶ࡗࡓ㸦ᅗ2-4a㸧ࠋ2005ᖺⓎ⏕ࡢ㎰ሙD㸪E㸪G㸪L࠾ࡼࡧQ㎰ሙࡢᛴᛶᮇ ࡢᢠయ㝧ᛶ⋡ࡣ26㸣࡛࠶ࡾ㸪㎰ሙD࠾ࡼࡧEࡢ㠀Ⓨ∵ࡢ67㸣ẚ㍑ࡋⴭࡋࡃప ࡗࡓ(p<0.01㸪ᅗ2-4b࠾ࡼࡧc)ࠋ 2002ᖺⓎࡋࡓ7㢌୰4㢌㸦57㸣㸧ࡀ2005ᖺࡢⓎ⏕ࡶBRVB㑇ఏᏊࡀ㝧 ᛶ࡞ࡗࡓࠋࡉࡽࡇࢀࡽࡢ∵7㢌୰5㢌㸦71㸣㸧ࡣ2005ᖺࡢ࣌⾑Ύ࡛᫂ࡽ ࡞ᢠయୖ᪼ࡀㄆࡵࡽࢀࡓ㸦ᅗ2-5㸧ࠋࡋࡋࡇࢀࡽࡢ∵ࡣ2005ᖺୗ⑩≧ࢆ♧ ࡉ࡞ࡗࡓࠋ
29 6. ⣽⳦᳨ᰝ࠾ࡼࡧᐤ⏕᳨ᰝ
ࢧࣝࣔࢿࣛ⳦࣭ཎᛶ⭠⳦ࡢศ㞳ᇵ㣴᳨ᰝ,ࢥࢡࢩࢪ࣒࣭࢘ࢡࣜࣉࢺࢫ࣏ࣜ ࢪ᳨࣒࢘ᰝࡣ࠸ࡎࢀࡶ㝜ᛶ࡛࠶ࡗࡓࠋ
30 ⪃ ᐹ 2002ᖺBRVBࡼࡿୗ⑩ࡢὶ⾜ࡀⓎ⏕ࡋࡓ㎰ሙ࠾࠸࡚㸪ୗ⑩ὶ⾜๓ࡢ∵ ⩌ࡢᢠయ㝧ᛶ⋡ࡣ㠀Ⓨ⏕㎰ሙࡢ∵⩌ࡼࡾࡶ᭷ពపࡗࡓ㸦⾲2-2㸧ࠋࡇࡢࡇ ࡼࡾ㸪BRVBࡼࡿᡂ∵ࡢὶ⾜ᛶୗ⑩ࡢⓎ⏕ࡣ㸪ྠ࢘ࣝࢫᑐࡍࡿ⩌චࡢ పୗࡀ㛵ࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋࣄࢺࡢRVAឤᰁ࡛ࡣ㸪⾑Ύ୰ࡢRVAᑐࡍ ࡿᢠయ౯ࡣ㸪Ⓨ㜵ᚚࡢᣦᶆ࡞ࡿሗ࿌ࡉࢀ࡚࠸ࡿ㸦42,70,95㸧ࠋBRVBᑐ ࡍࡿᢠయࡶྠᵝⓎண㜵ຠᯝࢆᣢࡘணࡉࢀࡿࡇࡽ㸪㠀Ⓨ⏕㎰ሙࡢ∵ ⩌࡛ࡣ㸪Ⓨ㜵ᚚ༑ศ࡞ᢠయࢆᣢࡘಶయࡀከᩘᏑᅾࡋࡓࡓࡵ㸪BRVBࡼࡿὶ ⾜ᛶୗ⑩ࡢⓎ⏕ࡀ㜼Ṇࡉࢀࡓ⪃࠼ࡽࢀࡓࠋ⯆῝࠸ࡇ㸪ᗂඣࡢࣄࢺRVA ࣡ࢡࢳࣥࡢ㞟ᅋ᥋✀ࡣ㸪࣡ࢡࢳࣥ᥋✀ࢆཷࡅ࡚࠸࡞࠸ᗂඣࡢⓎࢆண㜵ࡍࡿ ࡉࢀ࡚࠸ࡿ㸦53㸧ࠋ௨ୖࡼࡾ㸪㠀Ⓨ⏕㎰ሙࡢ∵࠾ࡅࡿ⩌චࡀࠊBRVBᑐ ࡍࡿᢠయࢆಖ᭷ࡋ࡞࠸ಶయࡢⓎࢆண㜵ࡋࡓྍ⬟ᛶࡶ⪃࠼ࡽࢀࡓ(24,88)ࠋ 2005ᖺⓎ⏕ࡋࡓ2ᅇ┠ࡢὶ⾜ࡣ2002ᖺࡢὶ⾜ࢆ⤒㦂ࡋࡓࢇࡢ㎰ሙ࡛ Ⓨ⏕ࡋࡓࠋ㓗㎰ᅋᆅࡢྛ㎰ሙ࡛ࡣᦢங∵ࡢ⣙30㹼40㸣ࡀẖᖺ᭦᪂ࡉࢀ㸪᭦᪂ࡉ ࢀࡓ㞤∵ࡢ⣙2/3ࡣ┴ෆእࡢ㎰ሙࡼࡾᑟධࡉࢀ࡚࠸ࡓࠋࡇࢀࡣ2ᅇ┠ࡢⓎ⏕๓ ࡢ2004ᖺ㡭ࡲ࡛㸪㎰ሙ࡛ᦢங∵ࡢ⣙༙ศ㏆ࡃࡀ2002ᖺࡢὶ⾜ࢆ⤒㦂ࡋ࡚࠸ ࡞࠸ಶయ᭦᪂ࡉࢀ࡚࠸ࡓࡇࢆ♧ࡋ࡚࠸ࡿࠋࡋࡓࡀࡗ࡚㸪ᦢங∵ࡢ᭦᪂క ࠺⩌චࡢపୗࡀ2ᅇ┠ࡢୗ⑩ࡢⓎ⏕ࡘ࡞ࡀࡗࡓ⪃࠼ࡽࢀࡓࠋ ࡲࡓ㸪2002ᖺ࠾ࡼࡧ2005ᖺὶ⾜ࡋࡓBRVBࡢVP7࠾ࡼࡧVP4㑇ఏᏊゎᯒࡢ ⤖ᯝ㸪ྛᖺࡢὶ⾜ᰴࡣ⣔⤫ᶞ࡛ࡣ␗࡞ࡿࢡࣛࢫࢱ࣮ࢆᙧᡂࡋࡓࡶࡢࡢ㸪ሷᇶ㓄 ิࡢ┦ྠᛶゎᯒ࡛ࡣ99.3㹼99.4㸣㧗࠸┦ྠᛶࢆ♧ࡋࡓࡇࡽ㸪2002ᖺ࠾ ࡅࡿὶ⾜ᰴࡀఱࡽࡢᙧ࡛㎰ሙෆᐃ╔ࡋ㸪2005ᖺ࠾ࡅࡿᗘ┠ࡢὶ⾜㛵 ࡋࡓྍ⬟ᛶࡀ㧗࠸⪃࠼ࡽࢀࡓࠋ
31 ࣄࢺ࡛ࡣ㏻ᖖ㸪RVAࡢึឤᰁ࡛ࡣ⤊⏕චࡀᡂ❧ࡏࡎ㸪ࡑࡢᚋࡶឤᰁࡍࡿ ࡀ㸪≧ࡣ୍⯡ⓗ㔜⠜࡞ࡽ࡞࠸ሗ࿌ࡉࢀ࡚࠸ࡿ(21,25,56,94)ࠋࡉࡽ㸪 ࣄࢺRVAឤᰁ㛵ࡍࡿࢥ࣮࣍ࢺ◊✲࡛ࡣ㸪ྠࡌᆺࡢ࢘ࣝࢫࡢឤᰁᑐࡍࡿ 㜵ᚚචࡀ2ᖺ⥆ࡃࡇࡀ♧ࡉࢀࡓ㸦8㸧ࠋ୍᪉㸪RVAࡣᑐ↷ⓗRVBឤᰁࡢ ච㛵ࡍࡿሗࡣࢇ࡞࠸ࠋᅇ㸪VP7ࡸVP4㑇ఏᏊ㓄ิࡀᴟࡵ࡚㏆⦕࡞ BRVBࡀ⣙2ᖺ༙ࡢ㛫㝸࡛ᡂ∵ឤᰁࡋࡓࡇࡀ᫂ࡽ࡞ࡗࡓࠋࡋࡋ BRVBࡀ᳨ฟࡉࢀࡓࡶࢃࡽࡎ㸪ࡇࢀࡽࡢ∵ࡣ2ᅇ┠ࡢឤᰁୗ⑩≧ࢆ ♧ࡉ࡞ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡽ㸪BRVBࡣᡂ∵ឤᰁࡍࡿࡀ㸪ឤᰁࡣ ࡑࢀ㔜⠜࡞≧ࢆࡶࡓࡽࡉ࡞࠸ࡇࡀ⪃࠼ࡽࢀࡓࠋࡇࡢࡇࡣࣄࢺࡢRVA ឤᰁ㠀ᖖ㢮ఝࡋࡓ⌧㇟࡛࠶ࡾ㸪BRVBࡼࡿୗ⑩ண㜵⟇ࢆㅮࡌࡿୖ࡛ࡢཧ⪃ ࡞ࡿᛮࢃࢀࡓࠋ ᅇ㸪BRVBࡣྠ୍㎰ሙෆ࡛ࡶᩘᖺ㛫㝸࡛ᡂ∵ࡢἪங㔞ࡢῶᑡὶ⾜ᛶୗ⑩ࢆ ᘬࡁ㉳ࡇࡋࡓࠋࡑࡢせᅉࡋ࡚㸪⩌චࡀపୗࡋ࡚࠸ࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋ ࡉࡽྠ୍∵࡛ࡶRVBឤᰁࢆ⧞ࡾ㏉ࡍࡇࡀุ᫂ࡋࡓࠋᚋ㸪BRVB᳨ฟࡢ⡆ ᫆ⓗ᳨ᰝἲࡀ㛤Ⓨࡉࢀ㸪BRVBࡢࡉࡽ࡞ࡿᏛᐇែࡀゎ᫂ࡉࢀࡿࡇࢆᮇᚅࡋࡓ ࠸ࠋ
32
ᅗ ⾲
⾲2-1. ゎᯒࡋࡓ BRVB ࡢ DDBJ/EMBL/GenBank ࢡࢭࢵࢩࣙࣥ␒ྕ
Accession No. Strain Region
LC005523 D-2002 VP7 LC005524 E-2002 VP7 LC005525 J-2002 VP7 LC005526 L-2002 VP7 LC005527 D-2005 VP7 LC005528 E-2005 VP7 LC005529 G-2005 VP7 LC005530 K-2005 VP7 LC005531 L-2005 VP7 LC005532 D-2002 VP4 LC005533 E-2002 VP4 LC005534 J-2002 VP4 LC005535 L-2002 VP4 LC005536 G-2005 VP4 LC005537 K-2005 VP4 LC005538 L-2005 VP4
33 ᅗ2-1. 2002ᖺ࠾ࡼࡧ2005ᖺBRVBࡀὶ⾜ࡋࡓ㓗㎰ᅋᆅࡢᴫ␎ᅗ ࣝࣇ࣋ࢵࢺࡣ㎰ሙྡࢆ♧ࡍࠋ ⰍࡁࡣBRVBࡼࡿୗ⑩Ⓨ⏕㎰ᐙࢆ♧ࡍࠋ ✵ⓑࡣ✵ࡁ␆⯋ࢆ♧ࡍࠋ 15 m 200 m 15 m In 2002 In 2005 A A B B B B I J K y
empty empty empty
C D L M E N F G H O P Q K D L M E N F G H O P Q Outbreak
34 ᅗ2-2㸬2002ᖺ2005ᖺࡢBRVB RNA㟁ẼὋືࣃࢱ࣮ࣥ ࣮ࣞࣥࡢࣝࣇ࣋ࢵࢺࡣ㎰ሙྡࢆ♧ࡍࠋ E࠾ࡼࡧDࡣ2002ᖺ㸪L㸪K㸪G࠾ࡼࡧQࡣ2005ᖺࡢ᳨ฟᰴࠋ RVAࡣRVAཧ↷ᰴ(OSUᰴ)ࠋ ᩘᏐ▮༳ࡣࣟࢱ࢘ࣝࢫࡢ㑇ఏᏊศ⠇ࢆ♧ࡍࠋ
E D L K G Q
2002
2005
RVA
Fig. 2 1 2 34 56 78 9 10 11 1 23 4 5 6 789 10 11 10 11 1 2 3 4 9 10 11 9 7 8 5 635 ⾲2-2. 2002 ᖺࡢୗ⑩Ⓨ⏕ 4 ᭶๓ࡢᡂ∵ࡢ BRVB ᢠయ᳨ᰝᡂ⦼ 㝧ᛶᩘ ᳨యᩘ ྜ(%) Da 21 66 31.8A Ea 21 69 30.4A Ab 79 118 66.9B Ob 43 71 60.6B ㎰ሙ RVBᢠయ᳨ฟ a 4ࣨ᭶ᚋὶ⾜ᛶRVBࡼࡿୗ⑩Ⓨ⏕ b 4ࣨ᭶ᚋὶ⾜ᛶRVBࡼࡿୗ⑩Ⓨ⏕࡞ࡋ A,B ␗➢ྕ㛫࡛᭷ពᕪ࠶ࡾ (P<0.01)
36 ᅗ2-3. ∵࠾ࡼࡧࣄࢺ⏤᮶ RVB ࡢ VP7 㑇ఏᏊ㸦a㸧࠾ࡼࡧ VP4 㑇ఏᏊ 5'ᮎ➃㡿 ᇦ㸦b㸧ࡢሷᇶ㓄ิᇶ࡙ࡃศᏊ⣔⤫ᶞ ࣈ࣮ࢺࢫࢺࣛࢵࣉ್ࢆศᒱࡢ⫪♧ࡍࠋ 㻜 㻟㻠㻚㻠 㻡 㻝㻜 㻝㻡 㻞㻜 㻞㻡 㻟㻜 RVB/Cattle-wt/JPN/G-2005/2005/G3PX RVB/Cattle-wt/JPN/K-2005/2005/G3PX RVB/Cattle-wt/JPN/D-2005/2005/G3PX RVB/Cattle-wt/JPN/L-2005/2005/G3PX RVB/Cattle-wt/JPN/E-2005/2005/G3PX RVB/Cattle-wt/JPN/D-2002/2002/G3PX RVB/Cattle-wt/JPN/L-2002/2002/G3PX RVB/Cattle-wt/JPN/E-2002/2002/G3PX RVB/Cattle-wt/JPN/J-2002/2002/G3PX 㻥㻡 RVB/Cattle-wt/JPN/Nemuro/1997/G3PX 㻝㻜㻜 RVB/Cattle-wt/USA/MN10-1/2010/G3PX RVB/Cattle-wt/USA/ATI/19XX/G3PX RVB/Cattle-wt/USA/WD653/19XX/G3PX RVB/Cattle-wt/USA/Mebus/19XX/G3PX RVB/Cattle-wt/IND/DB101/2001/G5PX RVB/Cattle-wt/IND/DB180/2001/G5PX 㻤㻥 RVB/Cattle-wt/IND/DB176/2001/G5PX 㻝㻜㻜 㻝㻜㻜 RVB/Human-wt/IND/CAL/1998/G2PX RVB/Human-wt/BGD/Bang373/2000/G5PX 㻝㻜㻜 RVB/Human-wt/CHN/WH-1/2002/G5PX RVB/Human-wt/CHN/ADRV/1986/G5PX 㻝㻜㻜 㻝㻜㻜 㻥㻟
a
b
㻜 㻟㻡㻚㻣 㻡 㻝㻜 㻝㻡 㻞㻜 㻞㻡 㻟㻜 㻟㻡 RVB/Cattle-wt/JPN/D-2002/2002/G3PX RVB/Cattle-wt/JPN/L-2002/2002/G3PX RVB/Cattle-wt/JPN/E-2002/2002/G3PX RVB/Cattle-wt/JPN/J-2002/2002/G3PX RVB/Cattle-wt/JPN/K-2005/2005/G3PX RVB/Cattle-wt/JPN/L-2005/2005/G3PX RVB/Cattle-wt/JPN/G-2005/2005/G3PX RVB/Cattle-wt/IND/RUBV226/2004/G5PX RVB/Cattle-wt/IND/RUBV282/2005/G5PX RVB/Cattle-wt/IND/DB176/2001/G5PX RVB/Human-wt/IND/IC-008/2008/G2PX RVB/Human-wt-/IND/NIV-094456/2009/G2PX RVB/Human-wt/IND/CAL/1998/G2PX RVB/Human-wt/BGD//Bang373/2000/G2PX RVB/Human-wt/CHN/ADRV/1986/G2PX RVB/Human-wt/CHN/WH-1/2002/G2PX 㻣㻣 㻣㻜 㻥㻣 㻤㻤 㻝㻜㻜 㻝㻜㻜 㻝㻜㻜 㻝㻜㻜 㻝㻜㻜㻤㻢 㻝㻜㻜 㻝㻜㻜 Fig. 537 ᅗ2-4㸬2002ᖺ㸦a㸧࠾ࡼࡧ2005ᖺ㸦b࠾ࡼࡧc㸧ࡢୗ⑩ⓎࡶࡋࡃࡣᮍⓎ∵ࡢ ࣌⾑ΎࡢBRVBᢠయࡢ᥎⛣ Ⅼ⥺ࡣOD್0.2ࢆ♧ࡋࠊࡇࢀࡼࡾୖ࡛BRVBᢠయ㝧ᛶ࡛࠶ࡿุᐃࡍࡿࠋ ࣌⾑Ύ࡛OD್0.2௨ୖࡢᕪࡀぢࡽࢀࡓሙྜࢆ⾑Ύᛂࡀ࠶ࡗࡓぢ࡞ࡍࠋ 2002ᖺⓎ⏕ࡢⓎ⏕㎰ሙࡢᛴᛶᮇࡢᢠయ㝧ᛶ⋡ࡣ15㸣ࠋ 2005ᖺⓎ⏕ࡢⓎ⏕㎰ሙࡢᛴᛶᮇࡢୗ⑩Ⓨ∵ࡢᢠయ㝧ᛶ⋡ࡣ26㸣㸪 㠀Ⓨ∵ࡢ67㸣ẚ㍑ࡋⴭࡋࡃపࡗࡓ(p<0.01)ࠋ
38
ᅗ2-5. 2002ᖺ࠾ࡼࡧ2005ᖺࡢྠ୍∵㸦7㢌㸧࡛ࡢBRVBᢠయࡢ᥎⛣ୗ⑩ࡢ
≧ࡢ᭷↓࠾ࡼࡧBRVB≉␗㑇ఏᏊ᳨ฟࡢ㛵ಀ
Ⅼ⥺ࡣOD್0.2ࢆ♧ࡋࠊࡇࢀࡼࡾୖ࡛BRVBᢠయ㝧ᛶ࡛࠶ࡿุᐃࡍࡿࠋ
39 ➨ ୕ ❶
40 ᗎ ㄽ ࣟࢱ࢘ࣝࢫࡣ11 ᮏࡢ 2 ᮏ㙐 RNA ࢭࢢ࣓ࣥࢺࢆ㑇ఏᏊࡋ࡚ಖ᭷ࡍࡿࠋࡇ ࢀࡲ࡛ࣟࢱ࢘ࣝࢫ࡛ࡣ㜵ᚚචࢆㄏᑟࡍࡿእẆ⺮ⓑ࡛࠶ࡿVP7 ࠾ࡼࡧࢫࣃ ࢡ⺮ⓑ࡛࠶ࡿ VP4 ࡢ㑇ఏᏊ㓄ิࡼࡿ GX-P[X] 㸦X ࡣ㑇ఏᏊᆺ␒ྕࢆ♧ࡍ㸧 㑇ఏᏊᆺูࡀ⾜ࢃࢀ࡚ࡁࡓ㸦20㸧ࠋ୍᪉㸪」ᩘࡢࣟࢱ࢘ࣝࢫᰴࡀྠ୍ࡢ⣽⬊ ឤᰁࡋࡓሙྜ㸪㑇ఏᏊࢭࢢ࣓ࣥࢺࡢ㸦㑇ఏᏊ㞟ྜ㸧ࡀ㉳ࡇࡾ㸪⤖ᯝࡋ ࡚ぶᰴࡣ␗࡞ࡿࢭࢢ࣓ࣥࢺࡢ⤌ࡳྜࢃࡏࢆᣢࡘᏊᏞ࢘ࣝࢫᰴࡀㄌ⏕ࡍࡿࠋ ࣟࢱ࢘ࣝࢫࡣᰴ㛫࡛㑇ఏᏊࢭࢢ࣓ࣥࢺࢆࡍࡿ㑇ఏᏊ㞟ྜࢆ㉳ࡇࡋ㸪ࡑ ࡢ⤖ᯝ」㞧࡞㑇ఏᏊᆺࢆᣢࡘᰴࡀㄌ⏕ࡍࡿࠋᚑ᮶ࡢGX-P[X]ᆺู࡛ࡣ VP7 ࠾ࡼ ࡧVP4 㑇ఏᏊ௨እࡢࢭࢢ࣓ࣥࢺࡢሗࡀḞⴠࡍࡿࡓࡵ㸪ࡢࢭࢢ࣓ࣥࢺࡀ㛵 ࡍࡿ㑇ఏᏊ㞟ྜᰴࢆ༊ูࡍࡿࡇࡀ࡛ࡁ࡞࠸㸦30,32,59,68㸧ࠋࡇࢀࡽࡢࡇ
ࡽ㸪Rotavirus Classification Working Group ࡽࣟࢱ࢘ࣝࢫࡢࢭࢢ࣓ࣥࢺ
ࡢ 㑇 ఏ Ꮚ 㓄 ิ ᇶ ࡙ ࡃ 㑇 ఏ Ꮚ ᆺ ู ࡀ ᥦ ၐ ࡉ ࢀ 㸪 ࢭ ࢢ ࣓ ࣥ ࢺ VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 ࡢྛ㑇ఏᏊᆺ ࢆ GX-P[X]-IX-RX-CX-MX-AX-NX-TX-EX-HX㸦X ࡣ㑇ఏᏊᆺ␒ྕࢆ♧ࡍ㸧 ♧ࡍࡇ࡞ࡗࡓ㸦59,60㸧ࠋࡇࡢࢩࢫࢸ࣒ࡀ 2008 ᖺᥦၐࡉࢀ࡚௨᮶㸪RVA ࡛ࡣᩘከࡃࡢ㑇ఏᏊࢹ࣮ࢱࡀ DDBJ/EMBL/GenBank ࢹ࣮ࢱ࣮࣋ࢫⓏ㘓ࡉࢀ ࡓࠋࡋࡋ㸪RVB ࡘ࠸࡚ࡣࣄࢺ㸪㇜࠾ࡼࡧࣛࢵࢺ⏤᮶ᰴࡢᩘᰴࡘ࠸࡚㑇 ఏᏊ㓄ิࡀⓏ㘓ࡉࢀ࡚࠸ࡿ㐣ࡂࡎ㸪BRVB 㛵ࡋ࡚ࡣ VP7㸪VP4㸪VP6 ࠾ࡼ ࡧNSP5 ࡢ㓄ิࡀᩘᰴ㸪VP1㸪VP2㸪NSP1-࣌ࣉࢳࢻ 1࣭࣌ࣉࢳࢻ 2 ࠾ࡼࡧ NSP2 ࡢ㓄ิࡣ 2 ᰴࡋⓏ㘓ࡉࢀ࡚࠸࡞࠸㸦31,33,91,93㸧ࠋࡉࡽ VP3㸪NSP3 ࠾ࡼ ࡧNSP4 ࡢ㑇ఏᏊ㓄ิ㛵ࡋ࡚ࡢⓏ㘓ࡣⓙ↓࡛࠶ࡿࠋ ࡑࡇ࡛▼ᕝ┴᳨࡛ฟࡉࢀࡓ BRVB ࡢࢤࣀ࣒ RNA ࢆḟୡ௦ࢩ࣮ࢡ࢚ࣥࢧ࣮࡛ ゎᯒࡋ㸪ࡇࢀࡲ࡛᫂ࡽ࡞ࡗ࡚࠸࡞࠸VP3㸪NSP3 ࠾ࡼࡧ NSP4 㑇ఏᏊ㓄ิ
41
ࢆỴᐃࡋࡓࠋࡲࡓ㸪ᚓࡽࢀࡓྛ㑇ఏᏊࡢሷᇶ㓄ิࢆศᏊ⣔⤫Ꮫⓗゎᯒࡸࡑࡢ ࡢ㑇ఏᏊゎᯒ౪ࡋࡓୖ࡛ࠊྛ࢘ࣝࢫ⺮ⓑ㉁ࡢศᏊ⏕≀Ꮫⓗᛶ≧ࡘ࠸࡚⪃ ᐹࡋࡓࠋ
42 ᮦᩱ࠾ࡼࡧ᪉ἲ 1.㑇ఏᏊ㓄ิỴᐃࡢࡓࡵࡢ⣅౽ࢧࣥࣉࣝࡢ㑅ᐃ ➨୍❶࠾ࡼࡧ➨❶࡛⏝࠸ࡓ⣅౽ࡢ࠺ࡕ㸪BRVB ᶆⓗ RT-PCR ࡛㝧ᛶ࡞ࡗ ࡓ㸪ࡑࢀࡒࢀ1 ᡞ 1 ᳨య࠾ࡼࡧ 3 ᡞ 5 ᳨యࢆ㑅ᐃࡋࡓࠋࡉࡽ㸪BRVB ࡼࡿ ᡂ∵ୗ⑩ࡢⓎ⏕ࡀ࠶ࡗࡓ㸪2002 ᖺ▼ᕝ┴ H ᕷࡢ 1 ᡞ 3 ᳨య㸪࠾ࡼࡧ 2006 ᖺ ࠾ࡼࡧ2011 ᖺ➨❶ྠࡌ㓗㎰ᅋᆅࡢ 3 ᡞ 7 ᳨యࢆ㑅ࢇࡔࠋࡍ࡞ࢃࡕ㸪ྜィ 16 ᳨యࡢ⣅౽ஙࢆ⏝࠸㸪ḟୡ௦ࢩ࣮ࢡ࢚ࣥࢫࡢࡓࡵࡢࣛࣈ࣮ࣛࣜస〇ࢆ⾜ ࡗࡓࠋ 2. RNA ᢳฟ䠈cDNA 䝷䜲䝤䝷䝸䞊స〇䛚䜘䜃ḟୡ௦䝅䞊䜽䜶䞁䝅䞁䜾㻌 ➨୍❶࠾ࡼࡧ➨❶ྠᵝ࡞᪉ἲ࡛⣅౽ࡽ RNA ࢆᢳฟࡋ㸪DNase
I(Takara, Shiga, Japan)ࡼࡾ᳨య୰ࡢ DNA ࢆ㝖ཤᚋ㸪S1 ࢾࢡ࣮ࣞࢮ (Takara, Shiga, Japan)ࢆ⏝࠸࡚ 1 ᮏ㙐 RNA ࢆ㝖ཤࡋࡓࠋ࢚ࢱࣀ࣮ࣝỿẊἲ࡛ RNA ࢆ⢭〇ࡋࡓᚋ㸪ᢳฟ RNA ⃰ᗘࢆ Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, USA)࡛ ᐃࡋࡓࠋ1 ᳨యᙜࡓࡾ 10㹼100 ng/ul ࡞ࡿࡼ࠺ RNA ⃰
ᗘࢆㄪᩚࡋ㸪NEBNext® Ultra RNA Library Prep Kit for Illumina Version 2.0
㸦New England Biolabs, Ipswich, USA㸧ࢆ⏝࠸࡚ྲྀࡾᢅ࠸ㄝ᫂᭩ᚑ࠸ cDNA
ࣛࣈ࣮ࣛࣜࢆస〇ࡋࡓࠋ࡞࠾㸪ࣛࣈ࣮ࣛࣜ࠾ࡅࡿ᳨య cDNA ࡢᤄධࢧ
ࢬࡣ⣙300㹼450 bp ࡞ࡿࡼ࠺ㄪᩚࡋࡓࠋస〇ࡋࡓ cDNA ࣛࣈ࣮ࣛࣜࡣ᭱
⤊⃰ᗘࢆ Qubit® 2.0 Fluorometer ࢆ⏝࠸࡚ㄪᩚࡋࡓᚋ㸪MiSeq bench-top
sequencer㸦Illumina, San Diego, USA㸧ࣉࣛࡋࡓࠋࢩ࣮ࢡ࢚ࣥࢫࡣ 151 ࢧࢡࣝࡢ࢚࣌ࣥࢻ࣮ࣜࢻࡼࡿ࣮ࣔࢻ࡛⾜ࡗࡓࠋ
43 3.㑇ఏᏊゎᯒ
ᚓࡽࢀࡓࢹ࣮ࢱࡣMiSeq reporter program㸦Illumina㸪USA㸧ࢆ⏝࠸࡚ fastaQ
ࣇࣝኚࡋ㸪CLC Genomics Workbench 7.5.5㸦CLC bio, Aarhus, ࢹࣥ
࣐࣮ࢡ㸧࣏࣮ࣥࢺࡋࡓࠋస〇ࡉࢀࡓࢩ࣮ࢡ࢚ࣥࢫ࣮ࣜࢻࡣCLC Genomics
Workbench ࡢde novo assembly ࢥ࣐ࣥࢻࡼࡾ 300 ሷᇶ௨ୖࡢࢥࣥࢸࢢࢆ
ᵓ⠏ࡋ㸪NCBI BLAST ࣉࣟࢢ࣒ࣛࢆ⏝࠸࡚ BLAST ゎᯒࢆᐇࡋࡓࠋBRVB 㑇
ఏᏊุ᫂ࡋࡓࢥࣥࢸࢢࡘ࠸࡚CLC Genomics Workbench ࡛ࢩ࣮ࢡ࢚ࣥ ࢫࢹࣉࢫࢆㄪ㸪༑ศಙ㢗ᛶࡀ࠶ࡿุ᩿ࡉࢀࡓ㓄ิ௨እࢆ㝖ཤࡋࡓࠋሷᇶ 㓄ิࡣClustalW㸦87㸧ࢆ⏝࠸࡚࣓ࣛࣥࢺࡋ㸪MEGA5.22㸦86㸧ࢆ⏝࠸࡚ ㏆㞄᥋ྜἲࡼࡿ⣔⤫ᶞゎᯒࢆᐇࡋࡓࠋ⣔⤫ᶞゎᯒ࡛ࡣ᭱ࡶ㐺ᛂࡋࡓ㐍ࣔ ࢹࣝࢆMEGA5.22 ࡼࡾ㑅ᐃࡋ㸪VP1㹼VP3㸪 VP4㸪VP6㸪NSP1㸪 NSP2 ࠾ࡼࡧNSP5 ࡘ࠸࡚ࡣ cpREV + G + I + F ἲ㸪NSP3 ࡣ WAG + I + F ἲ㸪 NSP4 ࡣ WAG + G + I + F ἲ࠾ࡼࡧ VP7 ࡣ cpREV + G + F ἲࢆ᥇⏝ࡋࡓࠋࣈ ࣮ࢶࢫࢺࣛࢵࣉゎᯒ㸦23㸧ࡘ࠸࡚ࡣ 1000 ᅇ⧞ࡾ㏉ࡋᐇࡋࡓࠋ࣌࣡ࢫ┦ ྠᛶࡢゎᯒࡣCLC Genomics Workbench ࡛⾜ࡗࡓࠋ㑇ఏᏊᆺࡢ┦㐪ࡢᣦᶆࡍ ࡿሷᇶ㓄ิ࠾ࡼࡧ࣑ࣀ㓟㓄ิࡢ┦ྠᛶࡘ࠸࡚ࡣ㸪┦ྠᛶศᕸᅗ㸦59㸧ࢆ⏝ ࠸࡚⟬ฟࡋࡓࠋࡲࡓ㸪ぶỈᛶ/Ỉᛶࣉࣟࢵࢺศᯒࢆ SOSUI ࣉࣟࢢ࣒ࣛ㸦63㸧 ࡼࡾ⾜ࡗࡓࠋ⭷㈏㏻㡿ᇦࡢண ࡘ࠸࡚ࡣSOSUI ࣉࣟࢢ࣒ࣛ࠾ࡼࡧ TMHMM ࣉࣟࢢ࣒ࣛ㸦48㸧ࢆ⏝࠸࡚ᐇࡋࡓࠋ
44 ⤖ ᯝ 1. ࢭࢢ࣓ࣥࢺࡢ㑇ఏᏊ㓄ิࡢỴᐃ ࢩ࣮ࢡ࢚ࣥࢫ౪ࡋࡓ16 ᰴ୰㸪RVB/Cow-wt/JPN/IS-1/1999/G3P[X] (IS-1)㸪 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] (IS-2)㸪 RVB/Cow-wt/JPN/E-2002/2002/G3P[X] (E-2002)㸪 RVB/Cow-wt/JPN/E-2005/2005/G3P[X] (E-2005)㸪 RVB/Cow-wt/JPN/D-2002/2002/G3P[X] (D-2002)㸪 RVB/Cow-wt/JPN/D-2005/2005/G3P[X] (D-2005)㸪 RVB/Cow-wt/JPN/D-2006/2006/G3P[X] (D-2006)㸪 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] (G-2006)࠾ࡼࡧ RVB/Cow-wt/JPN/P-2011/2011/G3P[X] (P-2011)ࡢ 9 ᰴࡘ࠸࡚ࡣ VP3㸪NSP3 ࠾ࡼࡧNSP4 㑇ఏᏊࡢ࣑ࣀ㓟ࢥ࣮ࢻ㡿ᇦ㸦Coding Sequence:CDS㸧ࡢ㛗 㑇ఏᏊ㓄ิࡀᚓࡽࢀࡓࠋࡋࡋࡢ7 ᰴࡘ࠸࡚ࡣ㸪ࡇࢀࡽࡢ㑇ఏᏊࡢ㛗 ሷᇶ㓄ิࢆᚓࡽࢀ࡞ࡗࡓࡓࡵ㸪௨ᚋࡢゎᯒࡽ㝖እࡋࡓࠋゎᯒࡋࡓ9 ᰴࡢ࠺ ࡕVP7㸪NSP1㸪NSP2 ࠾ࡼࡧ NSP5 ࡘ࠸࡚ࡣ୍㒊ࡢᰴ࡛㛗ࡣᚓࡽࢀ࡞ ࡗࡓࠋVP4㸪VP6㸪VP1 ࠾ࡼࡧ VP2 ࡘ࠸࡚ࡣ㒊ศⓗ࡞㑇ఏᏊ㓄ิࡋᚓࡿ ࡇࡀ࡛ࡁ࡞ࡗࡓࠋ ᚓࡽࢀࡓVP7㸪NSP1-࣌ࣉࢳࢻ 1㸪NSP1-࣌ࣉࢳࢻ 2㸪NSP2 ࠾ࡼࡧ NSP5 ࡢ㛗CDS 㓄ิࡣࡑࢀࡒࢀ 744 ሷᇶ㸦247 ࣑ࣀ㓟㸧㸪306 ሷᇶ㸦102 ࣑ ࣀ㓟㸧㸪963 ሷᇶ㸦320 ࣑ࣀ㓟㸧㸪903 ሷᇶ㸦300 ࣑ࣀ㓟㸧࠾ࡼࡧ 501 ሷᇶ㸦166 ࣑ࣀ㓟㸧࡛࠶ࡾ㸪ࣥࢻ᳨࡛ฟࡉࢀࡓBRVB㸦31㸧ࡢࡑࢀࡽྠࡌ㛗ࡉ࡛࠶ ࡗࡓ㸦ࢹ࣮ࢱᮍᥖ㍕㸧ࠋ 9 ᰴࡢ VP3㸪NSP3 ࠾ࡼࡧ NSP4 㑇ఏᏊ㓄ิ࠾ࡼࡧ୍㒊ࡢᰴࡢ VP4㸪VP6㸪
45 VP1㸪VP2㸪NSP1㸪NSP2 ࠾ࡼࡧ NSP5 㑇ఏᏊ㓄ิࢡࢭࢵࢩࣙࣥ␒ྕ LC185660㹼LC185712 ࢆࡾࡾ㸪DDBJ/EMBL/GenBank ࢹ࣮ࢱ࣮࣋ࢫⓏ 㘓ࡋࡓ㸦⾲3-1㸧ࠋ 2. ᮏ◊✲᳨࡛ฟࡉࢀࡓ BRVB ࡢ VP3㸪NSP ࠾ࡼࡧ NSP4 㑇ఏᏊ㛗 CDS ሷᇶ㓄ิࡢࣟࢱ࢘ࣝࢫB ࡢࡑࢀࡽࡢẚ㍑ ᮏ◊✲࡛ゎᯒࡋࡓBRVB ࡢ VP3 㑇ఏᏊࡢ CDS ࡣ 2292 ሷᇶ㸦763 ࣑ࣀ㓟㸧 ࡛࠶ࡾ㸪ࣛࢵࢺ㸪ࣄࢺ࠾ࡼࡧ㇜ࡢ RVB ࡢࡑࢀྠࡌ㛗ࡉ࡛࠶ࡗࡓ(72,101)ࠋ9 ᰴࡢ࠺ࡕIS-1㸪IS-2 ࠾ࡼࡧ G-2006 ᰴࡢ 5’࠾ࡼࡧ 3’UTR ሷᇶ㓄ิࡢ㛗ࢆỴ ᐃࡋࡓࡇࢁ㸪ࡑࢀࡒࢀ 8 ࠾ࡼࡧ 63 ሷᇶ࡛ᵓᡂࡉࢀ࡚࠸ࡓࠋBRBV ࡢ VP3 ࡢ 㛗 ࣑ ࣀ 㓟 㓄 ิ ࢆ ࣄ ࢺ ࠾ ࡼ ࡧ ㇜ ࡢ RVB ẚ ㍑ ࡋ ࡓ ࡇ ࢁ ,
(guanine-N7)-methyltransferase (N7-MTase)ࢻ࣓ࣥࢆᵓᡂࡍࡿ GxxxE(S/T)
࠾ࡼࡧLΩxL(S/T)NxxN ࣔࢳ࣮ࣇ㸦x ࡣ௵ពࡢ࣑ࣀ㓟ࠊȐࡣⰾ㤶᪘࣑ࣀ㓟㸧 ࡀBRVB ࡢ VP3 ࣑ࣀ㓟㓄ิࡢࡑࢀࡒࢀ 216ࠥ221 㸦GpefES㸧࠾ࡼࡧ 416-424 㸦 LYsLSNviN 㸧 ಖ Ꮡ ࡉ ࢀ ࡚ ࠸ ࡿ ࡇ ࡀ ☜ ㄆ ࡉ ࢀ ࡓ ࠋ ࡲ ࡓ , ribose-2'-O-methyltransferase ࢻ ࣓ ࣥ ࠾ ࡼ ࡧ guanylyltransferase/RNA 5'-triphosphatase (GTase/RTPase)ࢻ࣓ࣥྵࡲࢀࡿ SGHȍࣔࢳ࣮ࣇ㸦ȍࡣ Ỉᛶ࣑ࣀ㓟㸧ࡀBRVB ࡢ VP3 ࡢ 573-576 㸦SGHL㸧ࡋ࡚Ꮡᅾࡍࡿࡇ ࡀ☜ㄆࡉࢀࡓ㸦ᅗ3-1㸧ࠋ BRVB ࡢ NSP3 㑇ఏᏊࡢ CDS ࡣ 891 ሷᇶ㸦296 ࣑ࣀ㓟㸧ࡽᡂࡾ㸪ࣛࢵ ࢺRVB ࡢ 1173 ሷᇶ㸦390 ࣑ࣀ㓟㸧࠾ࡼࡧࣄࢺ RVB ࡢ 1044 ሷᇶ㸦347 ࣑ ࣀ㓟㸧ࡼࡾ▷ࡃ㸪㇜ RVB ࡢ 657 ሷᇶ㸦282 ࣑ࣀ㓟㸧ࡼࡾࡶ㛗ࡗࡓ 㸦1,19,44,50,101,102㸧ࠋNSP3 ࠾ࡼࡧ NSP4 ࡢ 5’࠾ࡼࡧ 3’UTR ࡣ㸪ゎᯒࡋࡓ ࡚ࡢ BRVB ᰴ࠾࠸࡚㛗ࡢ㓄ิࡣᚓࡽࢀ࡞ࡗࡓࠋBRVB㸪ࣄࢺ࠾ࡼࡧࣛ
46 ࢵࢺࡢNSP3 ࡢ࣑ࣀ㓟㓄ิࢆ࣓ࣛࣥࢺࡋࡓࡇࢁ㸪Ș-helices (H1㹼H8) ࠾ࡼࡧș-strands㸦S1㹼S3㸧ࢆྵࡴ RNA ⤖ྜ㡿ᇦࡣ࡚ࡢᰴ㛫ಖᏑࡉࢀ࡚ ࠸ࡓ㸦ᅗ3-2㸧ࠋ BRVB ࡢ NSP4 㑇ఏᏊࡢ CDS 㓄ิࡣ 627 ሷᇶ㸦208 ࣑ࣀ㓟㸧࡛࠶ࡾ㸪ࣛ ࢵࢺRVB ࡢ 657 ሷᇶ㸦218 ࣑ࣀ㓟㸧㸪ࣄࢺ RVB ࡢ 660 ሷᇶ㸦219 ࣑ࣀ㓟㸧 ࠾ࡼࡧ㇜RVB ࡢ 657 ሷᇶ㸦218 ࣑ࣀ㓟㸧ࡢ࠸ࡎࢀࡼࡾࡶ▷ࡗࡓ㸦5,50,101㸧ࠋ BRVB ࡢ NSP4 ࣑ࣀ㓟㓄ิࡣࣄࢺ࠾ࡼࡧ㇜ RVB ࡢࡑࢀࡣࡁࡃ␗࡞ࡗ࡚࠾ ࡾ㸪RVA ࡛ㄆࡵࡽࢀࡿ࢚ࣥࢸࣟࢺ࢟ࢩࣥࢻ࣓ࣥࡶࣄࢺ RVB ࡛☜ㄆࡉࢀࡿ࢚ࣥ ࢸࣟࢺ࢟ࢩࣥᵝࢻ࣓ࣥࡶྵࡲࢀ࡚࠸࡞ࡗࡓ㸦ᅗ3-3㸧ࠋSOSUI ࣉࣟࢢ࣒ࣛࢆ ⏝࠸࡚⾜ࡗࡓぶỈᛶ/Ỉᛶࣉࣟࣇࣝࡢ᥎ᐃ࡛ࡣ㸪ࣄࢺ࠾ࡼࡧ㇜ RVB ࠾ࡼࡧ BRVA ྠᵝ㸪BRVB ࡢ NSP4 ࡢ N ᮎ➃࣑ࣀ㓟㡿ᇦ h1㹼h3 ࡢỈᛶ㡿 ᇦࡀ☜ㄆࡉࢀࡓ㸦ᅗ3-4 A㸧ࠋSOSUI ࠾ࡼࡧ TMHMM ࣉࣟࢢ࣒ࣛࢆ⏝࠸ࡓ NSP4 ࡢ⭷㈏㏻㡿ᇦࡢ᥎ᐃ࡛ࡣ㸪BRVB IS-1 ᰴࡢ NSP4 ࡢ 32ࠥ53 ࠊ࡞ࡽࡧࣄࢺ RVB ࡛࠶ࡿ RVB/Human-wt/CHN/ADRV/1982/G2P[X] ᰴࡢNSP4 ࡢ 39ࠥ60 ࠊࡑࢀࡒࢀ22 ࣑ࣀ㓟࡛ᵓᡂࡉࢀࡿỈᛶ⭷㈏㏻㡿ᇦࡀ☜ㄆࡉࢀࠊࡉࡽ BRVA ࡛࠶ࡿ RVA/bovine-tc/USA/NCDV/1971/G6P[1]ᰴࡢ NSP4 ࡢ 29ࠥ51 ࡶ⭷㈏㏻㡿ᇦࡀㄆࡵࡽࢀࡓ㸦ᅗ3-4 B㸧ࠋ 3. BRVB ࡢ VP3㸪NSP3 ࠾ࡼࡧ NSP4 㑇ఏᏊࡢሷᇶ㓄ิ࡞ࡽࡧྛ⺮ⓑࡢ࣑ ࣀ㓟㓄ิࡢ┦ྠᛶࡢ᳨ウ BRVB ࡢ VP3㸪NSP3 ࠾ࡼࡧ NSP4 ࡢ㛗 CDS ࡢሷᇶ࠾ࡼࡧ࣑ࣀ㓟㓄 ิࢆࡢRVB ᰴẚ㍑ࡋࡓࠋBRVB ࢆࡢ RVB ẚ㍑ࡋࡓࡁ㸪VP3㸪NSP3 ࠾ࡼࡧ NSP4 㡿ᇦࡢ┦ྠᛶࡣሷᇶ㓄ิ࡛ 63.3–64.9%㸪65.9–68.2%࠾ࡼࡧ 52.6–56.2%㸪࣑ࣀ㓟㓄ิ࡛ 60.9–63.0%㸪56.4–60.3%࠾ࡼࡧ 36.1–39.3%࡛࠶
47 ࡗࡓ㸦⾲ 3-2㹼3-4㸧ࠋVP3㸪NSP3 ࠾ࡼࡧ NSP4 㡿ᇦࡢࣛࢵࢺ㸪ࣄࢺ࠾ࡼࡧ㇜ RVB ࡢ㑇ఏᏊᆺࢆ M1-T1-E1㸪M2-T2-M2 ࠾ࡼࡧ M4-T4-E4 ࡋ㸪BRVB ࡢ㑇 ఏᏊᆺࢆ M3-T3-E3 ௬ྡࡋࡓࠋᆺู᭱ࡶ㐺ᙜ࡞࢝ࢵࢺ࢜ࣇ್ࢆ㸪┦ྠ ᛶศᕸᅗ࠾࠸࡚㑇ఏᏊᆺእ┦ྠᛶࡢฟ⌧㢖ᗘࢆ㑇ఏᏊෆ┦ྠᛶࡢฟ⌧㢖ᗘ࡛ 㝖ࡋࡓ್ࡀ㸯ࢆୗᅇࡿ⨨࠾ࡅࡿ┦ྠᛶࡢ್ࡋࡓࠋࡇࢀᚑࡗ࡚ィ⟬ࡍࡿ 㸪ሷᇶ㓄ิ࠾ࡼࡧ࣑ࣀ㓟ࡢ࢝ࢵࢺ࢜ࣇ್ࡣ㸪VP3 ࡛ࡑࢀࡒࢀ 70%࠾ࡼࡧ 73%㸪 NSP3 ࡛ 78%࠾ࡼࡧ 74%㸪NSP4 ࡛ 70%࠾ࡼࡧ 69%࡛࠶ࡗࡓ㸦ᅗ 3-5㹼3-7㸧ࠋ 4. 㑇ఏᏊࢭࢢ࣓ࣥࢺ࠾ࡅࡿศᏊ⣔⤫ᶞゎᯒ 㛗ࡢ VP3㸪NSP3 ࠾ࡼࡧ NSP4 ࡢ࣑ࣀ㓟㓄ิࡼࡿ⣔⤫ᶞゎᯒ࡛ࡣ㸪 BRVB ࡣ M3㸪T3 ࠾ࡼࡧ E3 ௬ྡࡋࡓࢢ࣮ࣝࣉࢆᙧᡂࡋ㸪ࣄࢺ㸦M2-T2-E2㸧㸪 ㇜㸦M4-T4-E4㸧࠾ࡼࡧࣛࢵࢺ㸦M1-T1-E1㸧ࡢ RVB ࡣ㞳ࢀ࡚ࢡࣛࢫࢱ࣮ࢆ ᙧᡂࡋࡓ㸦ᅗ3-8 a, b, c㸧ࠋ ḟ㒊ศⓗ࡞VP7㸪VP4㸪VP6㸪VP1㸪VP2㸪NSP1㸪NSP2 ࠾ࡼࡧ NSP5 ࣑ࣀ㓟㓄ิࢆ⏝࠸࡚⣔⤫ᶞゎᯒࢆ⾜ࡗࡓࠋVP7 ࡼࡿ⣔⤫ᶞゎᯒ࡛ࡣ㸪ᡃࡀᅜ ࡢBRVB ᰴࡣ⡿ᅜࡢ BRVB ࣑ࣀ㓟㓄ิ࡛ 95.8㸫99.2%ࡢ┦ྠᛶࢆ♧ࡋ㸪 ࡶG3 㑇ఏᏊᆺศ㢮ࡉࢀࡓࠋࣥࢻࡢ BRVB ࡣ㸪ᡃࡀᅜࡢ BRVB ࣑ࣀ 㓟㓄ิ࡛85.0㹼88.8㸣ࡢ┦ྠᛶࢆ♧ࡋࡓࡀ㸪ᡃࡀᅜࡢ BRVB ࡣᑡࡋ␗࡞ࡗࡓ ศᒱࢆ♧ࡋ㸪G5 㑇ఏᏊᆺࢆᙧᡂࡋࡓ㸦ᅗ 3-9 A㸪⾲ 3-5㸧ࠋNSP2 ࡘ࠸࡚ࡣ㸪 ᡃࡀᅜࡢBRVB ࡣࣥࢻࡢ BRVB ࡣ࣑ࣀ㓟࡛ࣞ࣋ࣝ 89.1-92.0㸣㸪㇜ RVB ࡣ 86.2-89.1㸣ࡢ┦ྠᛶࢆ♧ࡋ㸪ࣥࢻࡢ BRVB ࠾ࡼࡧ㇜ RVB ࡶ N2 㑇ఏᏊᆺศ㢮ࡉࢀࡓ㸦ᅗ3-9 G㸪⾲ 3-11㸧ࠋNSP5 ࡛ࡣ㸪ᡃࡀᅜࡢ BRVB ࡣ ࣥࢻࡢBRVB ࣑ࣀ㓟㓄ิ࡛ 85.6-88.6㸣ࡢ┦ྠᛶࢆ♧ࡋ㸪ࡶ H6 㑇ఏᏊ ᆺศ㢮ࡉࢀࡓ㸦ᅗ3-9 H㸪⾲ 3-12㸧ࠋVP6 ࡛ࡣ㸪ᡃࡀᅜࡢ BRVB ࡣࣥࢻࡢ
48 BRVB ࡣ 92.1-95.2㸣ࡢ┦ྠᛶࢆ♧ࡋ㸪ഹศᒱࡀ␗࡞ࡿࡀ㸪ࡶ I3 ࢡ ࣛࢫࢱ࣮ࢆᙧᡂࡋࡓ㸦ᅗ3-9 C㸪⾲ 3-7㸧ࠋNSP1 ࣌ࣉࢳࢻ 2 ࡘ࠸࡚ࡣ㸪ᡃࡀᅜ ࡢBRVB ࡣ⊂❧ࡋࡓࢡࣛࢫࢱ࣮ࢆᙧᡂࡋࡓࡀ㸪ࣄࢶࢪࡢ RVB 67.9–68.8%ࡢ ࣑ࣀ㓟┦ྠᛶࢆ♧ࡋ㸪ࡶ I3 㑇ఏᏊᆺࢆᙧᡂࡋࡓ㸦ᅗ 3-9 F㸪⾲ 3-10㸧ࠋ NSP1 ࣌ࣉࢳࢻ 1 ࡘ࠸࡚ࡣ G-2006 ࡢࡳࡢ㓄ิࡋᚓࡽࢀࡎ㸪ࡇࡢ㓄ิࡣࣄࢶ ࢪ RVB ࡢࡑࢀ 75.2%ࡢ࣑ࣀ㓟㓄ิ┦ྠᛶࢆ♧ࡋࡓ㸦ࢹ࣮ࢱᮍᥖ㍕㸧ࠋ㑇ఏ Ꮚᆺࡀᥦၐࡉࢀ࡚࠸࡞࠸ VP4㸪VP1 ࠾ࡼࡧ VP2 ࡘ࠸࡚ࡣ㸪ᡃࡀᅜࡢ BRVB ࡣࣥࢻࡢ BRVB ࣑ࣀ㓟࡛ࣞ࣋ࣝࡑࢀࡒࢀ 90.0–91.7%㸪85.4–87.6%࠾ࡼ ࡧ94.9–97.1%ࡢ┦ྠᛶࢆ♧ࡋ㸪ྠࡌࢡࣛࢫࢱ࣮ᒓࡋࡓ㸦ᅗ 3-9 B, D, E㸪⾲ 3-6, 3-8, 3-9㸧ࠋ
49 ⪃ ᐹ ᮏ❶࠾ࡅࡿゎᯒ࡛ࡣ࣓ࢱࢤࣀ࣒ࡢᡭἲࢆ⏝࠸ࡓࢩ࣮ࢡ࢚ࣥࢫἲࡼࡾ㸪⣅ ౽ࡽ┤᥋ᢳฟࡋࡓ RNA ࢆࡗ࡚ BRVB ࢤࣀ࣒㓄ิࢆỴᐃࡋࡓࠋ⣅౽୰ࡣ ᐟ⏤᮶ࡸ⣽⳦࡞ࡢ㑇ఏᏊࡀከࡃྵࡲࢀ࡚࠸ࡿࡇࡽ㸪ࡑࢀࡽ⏤᮶ࡍࡿ1 ᮏ㙐RNA ࢆ㝖ཤࡍࡿࡓࡵ S1 ࢾࢡ࣮ࣞࢮࢆ⏝ࡋࡓࠋS1 ࢾࢡ࣮ࣞࢮࡣ࣓ࢱ ࢤࣀ࣒ࡼࡿࢹ࣮ࣉࢩ࣮ࢡ࢚ࣥࢫࢆ┠ⓗࡋࡓゎᯒ⏝ࡢࣛࣈ࣮ࣛࣜࢆస〇 ࡍࡿ㝿᭷ຠ࡛࠶ࡿࡇࡀド᫂ࡉࢀ࡚࠸ࡿ㸦78㸧ࠋࡇࡢᡭἲࢆᛂ⏝ࡍࡿࡇ࡛ VP3㸪VP7ࠊNSP1㸪NSP3㸪NSP4 ࠾ࡼࡧ NSP5 ࡢ㛗ࡢ CDS 㓄ิࢆᚓࡿ ࡇࡀ࡛ࡁࡓࠋ୍᪉㸪VP4㸪VP6㸪VP1㸪VP2 ࠾ࡼࡧ NSP2 ࡣ㒊ศⓗ࡞㓄ิࡋ ᚓࡿࡇࡀ࡛ࡁ࡞ࡗࡓࠋࡇࢀࡣ࢘ࣝࢫ㑇ఏᏊࡢ㔞ࡀ༑ศ࡛࡞ࡗࡓࡇ ࡸࢩ࣮ࢡ࢚ࣥࢫࡋࡃ࠸㓄ิࢆ᭷ࡋ࡚࠸ࡿࡇ㉳ᅉࡋ࡚࠸ࡿ⪃࠼ࡽࢀࡓࠋ ࡋࡋࡇࢀࡽࡢࢭࢢ࣓ࣥࢺࡶ⣔⤫ᶞゎᯒ౪ࡍࡿ༑ศ࡞㑇ఏᏊ㛗ࡀᚓࡽࢀ㸪 11 ࢭࢢ࣓ࣥࢺ࡚ࡘ࠸࡚ศᏊ⣔⤫ᶞゎᯒࢆ⾜࠺ࡇࡀྍ⬟࡛࠶ࡗࡓࠋBRVB ࡢVP3㸪NSP3 ࠾ࡼࡧ NSP4 㑇ఏᏊࡢሗࡣ DDBJ/EMBL/GenBank ࢹ࣮ࢱ࣋ ࣮ࢫⓏ㘓ࡀ࡞ࡗࡓࡇࡽ㸪ᮏ❶ࡢ◊✲࡛᫂ࡽࡉࢀࡓࡇࢀࡽࡢ㓄ิࡣ ୡ⏺ึࡢሗ࿌࡛࠶ࡿࠋ ࣟࢱ࢘ࣝࢫࡢ VP3 ࡣ N7-MTase ࡸ GTase/RTPase ࡞ከᶵ⬟ᛶࡢ࢟ࣕࢵ ࣆࣥࢢ㓝⣲ᶵ⬟ࢆ᭷ࡍࡿ㸦65㸧ࠋBRVB ࡢ VP3 ࡢ࣑ࣀ㓟㓄ิࡣ 60.9–63.0%㸪 ࡢRVB ࡣప࠸┦ྠᛶࢆ♧ࡋࡓࡀ㸪N7-MTase ࡸ GTase/RTPase ࢻ࣓ࣥࢆ ☜ㄆࡍࡿࡇࡀ࡛ࡁࡓࠋࡍ࡞ࢃࡕࠊᅇゎᯒࡋࡓBRVB ᰴࡢ VP3 ࡶࠊ࢘ࣝࢫ mRNA ྜᡂࡢ࢟ࣕࢵࣆࣥࢢᛂ㔜せ࡞ᶵ⬟ࢆ᭷ࡍࡿࡇࡀᙉࡃ♧၀ࡉࢀࡓࠋ ࣟࢱ࢘ࣝࢫࡢNSP3 ࡢ N ᮎ➃㡿ᇦࡣࣟࢱ࢘ࣝࢫ mRNA ࡢ 3’UTR Ꮡᅾ ࡍࡿ≉ᐃࡢሷᇶ㓄ิ⤖ྜࡍࡿ୍᪉㸪ࡑࡢ C ᮎ➃㡿ᇦࡣ⩻ヂ㛤ጞᅉᏊࡋ࡚▱
50 ࡽࢀࡿeIF4G ⤖ྜࡍࡿ㸦16㸧ࠋNSP3 ࡢࡇࢀࡽࡢ⤖ྜάᛶࡣ࢘ࣝࢫ mRNA ࡢ⩻ヂᙳ㡪ࡍࡿ㸦96㸧ࠋBRVB ࡢ NSP3 ࠾ࡅࡿ RNA ⤖ྜ㡿ᇦࡣಖᏑࡉࢀ࡚ ࠸ࡓࡀ㸪eIF4G ⤖ྜ㡿ᇦࡣࡢ RVB ࡸ RVA ࡢྠ㡿ᇦࡣ࡞ࡾ␗࡞ࡗ࡚࠸ࡓ (16)ࠋBLAST ゎᯒ࡛ࡣ BRVB ࡢ NSP3 ᭱ࡶ┦ྠᛶࡀ㧗ࡗࡓࡢࡣ㇜ RVB ࡢ RVB/Pig-wt/VNM/14250/2012/GXP[X] ᰴ࡛࠶ࡗࡓ(70)ࠋࡋࡋࡇࡢᰴࡢ㓄ิ 㸦ࢡࢭࢵࢩࣙࣥ␒ྕ KX362419㸧ࡣ 242 ሷᇶᑐ▷ࡍࡂࡿࡓࡵ㸪⣔⤫ᶞゎᯒ ຍ࠼ࡿࡇࡣ࡛ࡁ࡞ࡗࡓ㸦ࢹ࣮ࢱᮍᥖ㍕㸧ࠋ RVA ࡢ NSP4 ࡣ᭱ึ㸪⭠⟶ࡢ⣽⬊ෆࡢ࢝ࣝࢩ࣒࢘ࣞ࣋ࣝࢆኚࡉࡏࡿࡇ ࡼࡾஙࡢࡳ࣐࢘ࢫᖺ㱋౫Ꮡᛶࡢୗ⑩ࢆᘬࡁ㉳ࡇࡍ࢘ࣝࢫᛶ࢚ࣥࢸࣟࢺ࢟ ࢩࣥࡋ࡚ሗ࿌ࡉࢀࡓ㸦7,18,38㸧ࠋBRVB ࡢ NSP4 ࣑ࣀ㓟㓄ิࡣࣄࢺ㸪㇜࠾ ࡼࡧࣛࢵࢺ RVB ࡢࡑࢀࡣ┦ྠᛶࡀ 36.1–39.3%ⴭࡋࡃపࡗࡓࠋRVA ࡢ NSP4 ࣑ࣀ㓟㓄ิ 113–135 ┦ᙜࡍࡿ࢚ࣥࢸࣟࢺ࢟ࢩࣥࢻ࣓ࣥࡣ caveolin-1 ⤖ྜࡍࡿ㸦6㸧ࠋࣄࢺ RVB CAL-1 ᰴࡢ NSP ࣑ࣀ㓟㓄ิ 106–127 ࡢ࢚ࣥࢸࣟࢺ࢟ࢩࣥᵝࢻ࣓ࣥࡣ㸪RVA ࡢ࢚ࣥࢸࣟࢺ࢟ࢩࣥࢻ࣓ࣥ 27% ࡢ┦ྠᛶ࡛࠶ࡿ㸦41㸧ࠋࡋࡋ࡞ࡀࡽᅇゎᯒࡋࡓ BRVB ࡢ NSP4 ࡣ࢚ࣥࢸ ࣟࢺ࢟ࢩࣥᵝࢻ࣓ࣥ┦ᙜࡍࡿ࣑ࣀ㓟㓄ิࡣぢࡘࡽ࡞ࡗࡓࠋࡼࡗ࡚㸪 ᅇ᳨ฟࡉࢀࡓBRVB ࡣ NSP4 ࡢ࢚ࣥࢸࣟࢺ࢟ࢩࣥάᛶ౫Ꮡࡍࡿࡇ࡞ࡃୗ ⑩ࢆⓎࡉࡏࡿ⬟ຊࢆ᭷ࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋRVA ࡢ NSP4 ࡢ N ᮎ➃㡿ᇦ ࠶ࡿ⣙20 ࣑ࣀ㓟ࡽᵓᡂࡉࢀࡿ 3 ࢝ᡤࡢỈᛶࢻ࣓ࣥ h1-h3 ࡢ h2 ࡣ㸪࢘ ࣝࢫࡢᙧែᙧᡂࡍࡿ㝿⭷㏱㐣ᛶࢆㄪᩚࡍࡿᙺࢆᢸࡗ࡚࠸ࡿ㸦41㸧ࠋSOSUI ࣉࣟࢢ࣒ࣛࡼࡾ㸪ᅇゎᯒࡋࡓ IS-1 ࠾ࡼࡧ᪤▱ࡢࣄࢺ RVB ࡢ ADRV ᰴ NSP4 ࡢ⭷㈏㏻㡿ᇦࡀᏑᅾࡍࡿࡇࡀド᫂ࡉࢀࡓࠋࡲࡓ㸪∵ RVA ࡢ NCDV ᰴ ࠾࠸࡚ࡶ MTHMM ࣉࣟࢢ࣒ࣛࡼࡾ NSP4 ⭷㈏㏻㡿ᇦࡀ♧ࡉࢀࡓࠋࡍ࡞ ࢃࡕ㸪SOSUI MTHMM ࣉࣟࢢ࣒ࣛࡢ㛫୍࡛⮴ࡋࡓ⤖ᯝࡣᚓࡽࢀ࡞
51 ࡗࡓࡶࡢࡢ㸪㢮ఝࡋࡓỈࣉࣟࣇࣝࣃࢱ࣮ࣥࡀBRVB㸪ࣄࢺ RVB ࠾ࡼࡧ∵ RVA ☜ㄆࡉࢀࡓࠋ ⌧ᅾ㸪RVB ࡣ 21 ✀ࡢ VP7 G 㑇ఏᏊᆺ㸦51㸧㸪13 ✀ࡢ VP6 I 㑇ఏᏊᆺ㸦54㸧㸪 7 ✀ࡢ NSP1 A 㑇ఏᏊᆺ㸦82㸧㸪4 ✀ࡢ NSP2 N 㑇ఏᏊᆺ㸦84㸧࠾ࡼࡧ 6 ✀ࡢ NSP5 H 㑇ఏᏊᆺࡀሗ࿌ࡉࢀ࡚࠸ࡿ(83,84)ࠋࡲࡓ NSP1 ࡣ࣌ࣉࢳࢻ 1 ࠾ࡼࡧ࣌ ࣉࢳࢻ2 ศࢀ࡚࠾ࡾ㸪ࡑࢀࡒࢀ 7 ࡘࡢ A 㑇ఏᏊᆺศ㢮ࡉࢀ࡚࠸ࡿ㸦82㸧ࠋ ᮏ❶࡛ࡢ⣔⤫ᶞゎᯒࡢ⤖ᯝ㸪ᡃࡀᅜࡢBRVB ࡣ G3㸪I3㸪N2 ࠾ࡼࡧ H6 㑇ఏᏊ ᆺࢆ♧ࡍࡇࡀࢃࡗࡓࠋࡲࡓ㸪ࣥࢻࡢBRVB ࡣ G5㸪I3㸪N2 ࠾ࡼࡧ H6 ࡛ ࠶ࡗࡓࡇࡼࡾࠊBRVB ࡢ VP7 ࡣᑡ࡞ࡃࡶ G3㸪G5 ࡢ 2 ࡘࡢ㑇ఏᏊᆺࡀᏑ ᅾࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ VP6 ࡢ࣑ࣀ㓟㓄ิᇶ࡙ࡃ⣔⤫ᶞ࡛ࡣ㸪ᡃࡀᅜࡢ BRVB ࡣ I3 㑇ఏᏊᆺ ศ㢮ࡉࢀࡓࡀ㸪ࣥࢻࡢBRVB ࡣᑡࡋ␗࡞ࡿศᒱࢆ♧ࡋࡓࠋࡋࡋ㸪VP6 ࡢ ⣔⤫ゎᯒ⏝࠸ࡓ࣑ࣀ㓟㓄ิࡣ 126 ࣑ࣀ㓟࡛࠶ࡾ㸪㑇ఏᏊᆺูࢆ⾜࠺ࡢ ࡣ༑ศᛮࢃࢀࡓࡓࡵ㸪⣔⤫ᶞࡢసᡂ༑ศ࡞㸪ࡼࡾ㛗࠸࣑ࣀ㓟㓄ิࢆᚓ ࡚ゎᯒࡍࡁ⪃࠼ࡽࢀࡓࠋ NSP5 ࡘ࠸࡚ࡣ㸪ᡃࡀᅜࡢ BRVB ࡣࣥࢻࡢ BRVB ␗࡞ࡿࢡࣛࢫࢱ࣮ࢆ ᙧᡂࡋࡓࡀ㸪᪤ሗ࠶ࡿNSP5 ᆺูࡢ࢝ࢵࢺ࢜ࣇ್ࡣሷᇶ࡛ࣞ࣋ࣝ 78%࡛࠶ࡾ 㸦84㸧㸪ࡑࢀࢆᑡࡋୖᅇࡗࡓࡓࡵ㸪ࣥࢻࡢ BRVB ࡶ H6 㑇ఏᏊᆺᒓࡍ ࡿุ᩿ࡉࢀࡓࠋNSP1 ࡢ㑇ఏᏊᆺูࡣ㸪࣌ࣉࢳࢻ 1 ࡛ࡣ࣑ࣀ㓟࡛ࣞ࣋ࣝ 72㸣 ࠾ࡼࡧ࣌ࣉࢳࢻ2 ࡛ࡣ࣑ࣀ㓟࡛ࣞ࣋ࣝ 80㸣ࡢ࢝ࢵࢺ࢜ࣇ್ࡀタᐃࡉࢀ࡚࠸ࡿ ࡀ㸦82㸧㸪ᡃࡀᅜࡢ BRVB ࡣ࣌ࣉࢳࢻ 2 ࡛ࣄࢶࢪࡢ RVB ࡢ A3 㑇ఏᏊᆺ 67.9 㹼68.8%ࡢ┦ྠᛶࢆ♧ࡋࡓࠋࡋࡋ㸪BRVB G-2006 ᰴࡣࣄࢶࢪᰴ࣌ࣉࢳࢻ 1 ࡛ 75.2%ࡢ┦ྠᛶࢆ♧ࡋࡓࠋࡇࡢࡇࡽ㸪ᡃࡀᅜࡢ BRVB ࡣ A3 㑇ఏᏊᆺ ศ㢮ࡉࢀࡿࡢࡶࡋࢀ࡞࠸ࠋᮏ❶࡛ࡣࠊゎᯒࡋࡓBRVB ᰴࡢ VP3ࠊNSP3 ࠾ࡼ
52 ࡧ NSP4 ࡢ࣑ࣀ㓟㓄ิᇶ࡙ࡁࠊࡑࢀࡒࢀࢆ M1-M4ࠊT1-T4 ࠾ࡼࡧ E1-E4 ࡢ㑇ఏᏊᆺ௬ศ㢮ࡋࡓࠋࡑࡢୖ࡛ࠊྠศ㢮ᇶ࡙ࡁࠊྛ㑇ఏᏊᆺࢆ༊ูࡍ ࡿ┦ྠᛶࡢᇶ‽࡞ࡿ࢝ࢵࢺ࢜ࣇ್ࢆୖグࡢྛ⺮ⓑ㉁ࡘ࠸࡚ᥦၐࡋࡓࠋࡋ ࡋ⌧ᅾࡢࡇࢁࢹ࣮ࢱ࣮࣋ࢫࡽධᡭ࡛ࡁࡿVP3㸪NSP3 ࠾ࡼࡧ NSP4 ࡢ㑇ఏ Ꮚ㓄ิࡢᩘࡣᑡ࡞ࡃ㸪࢝ࢵࢺ࢜ࣇ್ࢆᐃࡵࡿࡣ༑ศ⪃࠼ࡽࢀࡿࡓࡵ㸪 ᚋከࡃࡢ㑇ఏᏊࢹ࣮ࢱࡢ✚ࡀᚲせ⪃࠼ࡽࢀࡓࠋ ᅇ㸪BRVB ࡢ VP3㸪NSP3 ࠾ࡼࡧ NSP4 ࡣࡢ RVB ࡢ㑇ఏᏊ㓄ิࡣ ࡞ࡾ␗࡞ࡿࡇࡀุ᫂ࡋࡓࠋࡋࡋ࡞ࡀࡽ㸪ࡑࢀࡒࢀࡢ࢘ࣝࢫ⺮ⓑ㉁࠾ࡅ ࡿ≉ᚩⓗ࡞ࣔࢳ࣮ࣇࡸศᏊ⏕≀Ꮫⓗ࡞ᛶ㉁ࡀ㸪RVB ࡢᰴ㛫ಖᏑࡉࢀ࡚࠸ࡿࡇ ࡀド᫂ࡉࢀࡓࠋࡲࡓ㸪ᡃࡀᅜ࠾ࡼࡧࣥࢻࡢ BRVB ࡣ, ࡚ࡢ㑇ఏᏊࢭࢢ࣓ ࣥࢺ࠾࠸࡚ࡢRVB ࡣ␗࡞ࡿ⣔⤫ࢢ࣮ࣝࣉࢆᙧᡂࡍࡿࡇࡀࢃࡗࡓࠋ ᅇ㸪᪂ࡓᚓࡽࢀࡓBRVB ࡢ㑇ఏᏊሗࡣ㸪RVB ࡢศᏊ⏕≀Ꮫⓗゎ᫂ࡍࡿୖ ࡛᭷⏝࡛࠶ࡿ⪃࠼ࡽࢀࡓࠋ
53
Accession No. Strain Region
LC185660 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] VP3 LC185661 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] VP3 LC185662 RVB/Cow-wt/JPN/D-2002/2002/G3P[X] VP3 LC185663 RVB/Cow-wt/JPN/E-2002/2002/G3P[X] VP3 LC185664 RVB/Cow-wt/JPN/D-2005/2005/G3P[X] VP3 LC185665 RVB/Cow-wt/JPN/E-2005/2005/G3P[X] VP3 LC185666 RVB/Cow-wt/JPN/D-2006/2006/G3P[X] VP3 LC185667 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] VP3 LC185668 RVB/Cow-wt/JPN/P-2011/2011/G3P[X] VP3 LC185669 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] NSP3 LC185670 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] NSP3 LC185671 RVB/Cow-wt/JPN/D-2002/2002/G3P[X] NSP3 LC185672 RVB/Cow-wt/JPN/E-2002/2002/G3P[X] NSP3 LC185673 RVB/Cow-wt/JPN/D-2005/2005/G3P[X] NSP3 LC185674 RVB/Cow-wt/JPN/E-2005/2005/G3P[X] NSP3 LC185675 RVB/Cow-wt/JPN/D-2006/2006/G3P[X] NSP3 LC185676 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] NSP3 LC185677 RVB/Cow-wt/JPN/P-2011/2011/G3P[X] NSP3 LC185678 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] NSP4 LC185679 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] NSP4 LC185680 RVB/Cow-wt/JPN/D-2002/2002/G3P[X] NSP4 LC185681 RVB/Cow-wt/JPN/E-2002/2002/G3P[X] NSP4 LC185682 RVB/Cow-wt/JPN/D-2005/2005/G3P[X] NSP4 LC185683 RVB/Cow-wt/JPN/E-2005/2005/G3P[X] NSP4 LC185684 RVB/Cow-wt/JPN/D-2006/2006/G3P[X] NSP4 LC185685 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] NSP4 LC185686 RVB/Cow-wt/JPN/P-2011/2011/G3P[X] NSP4 LC185687 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] VP7 LC185688 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] VP7 LC185689 RVB/Cow-wt/JPN/D-2006/2006/G3P[X] VP7 LC185690 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] VP7 LC185691 RVB/Cow-wt/JPN/P-2011/2011/G3P[X] VP7 LC185692 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] VP4 LC185693 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] VP4 LC185694 RVB/Cow-wt/JPN/D-2002/2002/G3P[X] VP4 LC185695 RVB/Cow-wt/JPN/E-2002/2002/G3P[X] VP4 LC185696 RVB/Cow-wt/JPN/D-2005/2005/G3P[X] VP4 LC185697 RVB/Cow-wt/JPN/E-2005/2005/G3P[X] VP4 LC185698 RVB/Cow-wt/JPN/D-2006/2006/G3P[X] VP4 LC185699 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] VP4 LC185700 RVB/Cow-wt/JPN/P-2011/2011/G3P[X] VP4 LC185701 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] VP6 LC185702 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] VP6 LC185703 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] VP1 LC185704 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] VP1 LC185705 RVB/Cow-wt/JPN/IS-2/2002/G3P[X] VP2 LC185706 RVB/Cow-wt/JPN/D-2005/2005/G3P[X] VP2 LC185707 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] NSP1 LC185708 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] NSP1 LC185709 RVB/Cow-wt/JPN/IS-1/1999/G3P[X] NSP2 LC185710 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] NSP2 LC185711 RVB/Cow-wt/JPN/G-2006/2006/G3P[X] NSP5 LC185712 RVB/Cow-wt/JPN/P-2011/2011/G3P[X] NSP5 ᅗ⾲ ⾲3-1 ▼ᕝ┴᳨࡛ฟࡉࢀࡓ BRVB ࡢྛ㑇ఏᏊࢭࢢ࣓ࣥࢺ DDBJ/EMBL/GenBank ࢡࢭࢵࢩࣙࣥ␒ྕ
