Gicii thwang SV.NCKH-Eureka ldn thti 15 nam 2013 Ky yeru khoa hoc VIRUS GAY BENH VIEM GAN SIEU VI C: TAI XAC DINH KIEU GEN TREN VUNG TRINH TIINS5B VA XAC DINH CAC DOT BIEN DA HINH DON NUCLEOTIDE (SNP) TREN GEN IL28B NHAM TIEN LYONG PHUONG PHAP DIEU TRI THICH HOP CHO BENH NHAN Le Huyn Ai Thay*, Tic Thi Ngpc Ming Dung, Thi Toy, Nguyin Virang Minh Hoang, Triin Thi Diim Kau, Nguyin Thi Minh Hien Khoa Cong nghe Sinh hoc, Dai hoc MO; TP HCM 2Trung tam than clocin Y khoa Medic, TP HCM 3Cong ty co phan Cong nghe Viet A De tai act giai Nhi Gal tinging Sinh vien nghien ctiu khoa hoc — Eureka lciit XV ncim 2013 TOM TAT Viem gan Met, vi C la virus Hepatitis C (HCV) gay ra, lam inh Inning nghiem den sire khoe ngtrai NM-mg tong trinh nghien elm On day ghi nhan co sy lien quan gift kiEu gen, (genotype) va phan tuYp (subtype) dm HCV vai dinhen benh va kha Wang dap img dieu tri doi yen timg phac rieng Viing gen ma hi:5a protein khong eau true (Non- structural NS5B), RNA polymerase phi thuec RNA, vai * diem trinh to to phit lyyp cho tong tac phan biet kieu gen va phan tuSrp HCV Muc tieu nghien ciru cua chi:mg toi, truac het la xay dung ca so du lieu cyc be clang tin cay ve kieu gen va phan tuSrp HCV Thuc nghiem duce hen hanh duo ten phuong phip PCR- giii trinh hr met phan Wing NS5B cita cac dung virus tir cac mau benh pham mau va phan tich phi he phan tir de xac dinh kieu gen va phan tuyp HCV Doi von 12 mau benh pham, san pham giai trinh tinYen viing NS5B duct dua vao phan tich pha he phan tir va cho ket qua phan biet kieu gen, phan tujrp voi gia tri bootstrap >90% a m6i phan nh6m: nhom a (2 mau), lb (1 mau), 2m (4 mau), 6a (2 mau), 6e (2 mau); 6t (1 matt) Phuong phap ducyc xay dung to uu the han so sanh von phuong phap real-time PCR dtra tren yang 5'UTR Vi vay, nghien euu se dirge ma reng vai ca mau Ian hon thai gian sap tEri , Thong ke du lieu ve tan so xuat hien cac kieu gen nhiem HCV dac trimg theo dia ly, khu vut, cac kieu det bien da hinh, don nucleotide ten gen IL28B cac khao sat thyc nghiem cua cac nghien ciru trtrot day, danh gia co hay khong cac tinh chat quan (kieu gen not trei theo timg khu vtrc dia ly, cac kieu det bien SNP mang tinh dac hieu dia phuong ) Thiet ke be mei dac hieu cho phan img giai trinh to (sequencing) vung NS5B, vimg gin gen IL28B de tim dot bien Tu khoa: HCV, NS5B, phci he phcin tu, PCR- gidi trinh hi, RT-PCR MO DAU Hepatitis virus HCV la nguyen nhan chinh gay benh viem gan sieu vi C, la moi de doa hang du v&' sire khoe doi yen dan cu ten toan the giEri Viet Nam la met nhfmg nu& co tjr le mac benh viem gan sieu vi C cao teen the glen Pham DA, Leuangwutiwong P, Jittmittraphap A, Luplertlop N, Bach HK, Akkarathamrongsin S, Theamboonlers A Poovorawan Y(2009) High Prevalence of Hepatitis C Virus Genotype in Vietnam Asian Asian Pac J Allergy lmmunol,27(2-3): 153-160.) Viec xac dinh kieu gen (genotype), phan tuSfp (subtype) virus HCV la eat quan (Lu et al., 2007; Baclig et 2010) Da, c biet moi ki4u gen khac se cho phae dieu tri khac nhau, chang han nhu loai thuee, thai gian dieu tri, qua trinh dinbenh, (Tokita H, Okamoto H, lizuka H, Kishimoto J, Tsuda F, Lesmana LA, Miyakawa Y, Mayumi M (1996) Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes in the second (2e and 2f), tenth (10a) and eleventh (11 a) genetic groups J Gen Virol, 77(2): 293-301.Error! Reference source not found.Error! Reference source not found Cac phuong phap phan tich kieu gen HCV thirimg duce sir dung la phuong phap lai Innolipa, phuong phip giii trinh tin viing trinh to khong dich ma (5'-non untranslated region-5'-UTR) hoac phuong phap Real-time PCR, wv (Lu et at., 2007; Murphy et at., 2007; Baclig MO, Chan VF, Ramos JD, Gopez-Cervantes J, Natividad FF (2010) Correlation of the 5' untranslated region (5'UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping Int J Mol Epidemiol 134 Ky yen khoa hoc Giai thuring SV.NCKH-Eureka lan ilia 15 nam 2013 Genet, 1(3): 236-244.Xia X, Zhao W, Tee KK, Feng Y, Takebe Y, Li Q, Pybus OG, Lu L (2008) Complete genome sequencing and phylogenetic analysis of HCV isolates from China reveals a new subtype, designated 6u J Med Virol, 80(10): 1740-1746.0 Viet Nam, kieu gen HCV ph6 bien la 1, va (Pham DA, Leuangwutiwong P, Jittmittraphap A, Luplertlop N, Bach HK, Akkarathamrongsin S, Theamboonlers A, Poovorawan Y(2009) High Prevalence of Hepatitis C Virus Genotype in Vietnam Asian Asian Pac J Allergy Immunol,27(2-3): 153160.Pham DA, Leuangwutiwong P, Jittmittraphap A, Luplertlop N, Bach HK, Akkarathamrongsin S, Theamboonlers A, Poovorawan Y(2009) High Prevalence of Hepatitis C Virus Genotype in Vietnam Asian Asian Pac J Allergy Immuno1,27(2-3): 153-160.; Tanimoto T, Nguyen HC, Ishizaki A, Chung PT, Hoang TT, Nguyen VT, Kageyama S, Oka S, Pham VT, Ichimura H (2010) Multiple routes of hepatitis C virus transmission among injection drug users in Hai Phong, Northern Vietnam J Med Virol, 82(8): 1355-1363.) Theo cac nghien city truerc day, giai trinh t r vung gen kitting ma hoa a du 5' (5'NC 5' Non Coding) la thigh hop va da duoc ap,clong khac cho rang dinh kieu gen tren vung 5'UTR se cho ket qua lam sang Tuy nhien, gin day mOt so nghien khong chinh xac so vai viec sir dung viing gen dich ma Ma protein kh6ng eau true 5B (non structural - NS5B) (Nakatani SM, Santos CA, Riediger IN, Krieger MA, Duarte CA, Do Carmo Debur M, Carrilho FJ, Ono SK (2011) Comparative performance evalution of Hepatiris C virus genotyping based on the 5' untranslated region versus partial sequencing of the NS5B region of Brazilian patient with chronic hepatitis C Virol J, 8: 459 Lj la vung 5'NC c6 the nham Ian viec phan biet gifra kieu gen voi phan tujp I b hay giAa a va lb Trong do, vung NS5B voi nhang tinh chat ve trinh tu mang tinh bien Ong phi hop de phan biet giCia cac kieu gen va phan tujp tot nen duoc khuyen cao dung de thay the cho vung 5'UTR Gen interleukin-28B (IL28B) la mOt gen da hinh Gen ma hOa cho interferon loci I, ram tren nhiem sac the s6 19 dia ngtrai, a vi tri 19q13 Chinh sir da hinh cila gen IL28B nen mot so cac bien the da hinh dO da sira dOi viec sin xuat cac interferon da anh huemg den dap Ung mien dich cua vat chit Gan day, mot s6 cac bien the da hinh gin gen gang da duoc thing minh lien quan den viec kiem sok berth viem gan C, va tien doan kha [fang dap img diet' tri cua benh nhan virus C dieu tri vai PEG-INF/RBV.05 SNP dm IL28B dA duoc xac dinh, la SNP rs12979860, SNP rs8099917, va SNP rs12980275 SNP rs12979860 chinh la cong tic interferon: bien the C/C la ding tic bat (switch-on) va C/T la ding tic tit (switch-off) Bien the C/C cho phep tien doan hieu qua dieu tri cong IFN cao (tren 80%) vial it nguy co tai phat va tien trien man tinh hon so voi Wen the C/T tien doin hieu qua dieu tri that, voi nguy co tai Oat va tien trien man tinh cao.(Pham et al,.) Muc tieu ctia nghien eau nay, truot het nham xay dkmg co so du lieu cue be) cho cac kieu gen IA phan tujp HCV Day la cong viec thiet thuc, lam co so cho viec so sanh, suy kieu gen, phan tujfp se duoc ap dung sau Phtrcrng phap RT-PCR ket hop voi giai trinh tu vung gen dich thus c vung NS5B dirge thiet lap de to trinh to giai duoc se &rot dung de phan tich phi he nham xac dinh kieu gen va phan tuyp HCV co mot so mau benh phim thu nhan to Trung tam chan doan Y khoa Medic, TP HCM.Btrac dau khao sat in silico vai trb cila SNP rs12979860 va rs8099917 doi vai dap Cmg dieu tri cua benh nhan viem gan C dieu tri voi PEG-IFNa/RBV tren the giai va Viet Nam Thiet ke va did nghiem kha nang hoot clOng ctla moi, he m6i (mau do) cho phan Ung Realtime-PCR nham xac dinh SNP quan tam NGUYEN LIEU VA PHIAING PHAP Nguyen lieu Cac matt benh pham thuOc 12 benh nhan bi viem gan C (duct cung cap bEri Trung tam chin doan Y khoa Medic, TP HCM) Phwang phap Xciy clung ca sa du lieu cuc 17 ye trinh ter vung NS5B virus HCV Dog tren co so da lieu ngan hang gen NCBI va Los Alamos Laboratory va ter nguan cac bai bao tham khao, co so du lieu cuc bo la tap hop nhAng trinh tu tham chieu thuOc viing NS5B.va trinh loci GBV-C/HGV lam nh6m ngoai (outgroup) da duoc tap hop Sau d6, thing toi nen hanh thong,ke so luting va phan yang dia ljt cho cac kieu gen va phan tujp HCV (vu tien chon cac trinh tu tir HCV co nguon goc phan lop tai Viet Nam) Khoo sat in silico ve SNP rs12979860 va rs8099917 135 Giai thwerng SV.NCKH-Eureka Idn tit& 15 nam 2013 Ky ydu khoa hoc Tien hanh khai thic dv lieu ten PubMed ve SNP rs12979860 va rs8099917 Ding tiled tien hanh thOng danh gia m6i quan he gicra cac loaf bien the veri chimg tOc, dia ly, dap ting dieu tri va kieu gen cua virus HCV nhiZm tren benh nhfin Tach chill RNA RNA duos tach chiet ter 12 mAu benh phfirn mau thuOc 12 benh nhan bi benh viem gan C bang phtrong phap mu dung phenol/chloroform, phong theo phuong phap cua Chomczynski va Sacchi (1987): 200 pl huyet duct them vao 900 pl Trizol, pH4 Nucleic acid sau de &rot trig bang isopropanol veri chfit try trig GlycoBlue Nucleic acid thu dirge sau trig duct gii dung dich TE X (Tris-EDTA) (deii v6i DNA) va H2O cat da qua xir ly bang DEPC (dOi voi RNA) vabio quirt a -20°C cho den sir dung Tong hop cDNA va khuech clgi vimg gen NS5B Thus hien phin ling Reverse transcriptase PCR voi bO kit RT-PCR cim ding ty co phfin cong nghe Viet A nham khuech dai cac ban cDNA 60 gen virus HCV (khoang 9.6 kb HCV) vai cac mau ngau nhien hexamer veri oligo d(A) (20 nucleotide) Chuang trinh luan nhiet tong hop cDNA: 25°C phin, 42°C 30 phut, 85°C• phtit, lam mat a 4°C Sau do, cac san pham cDNA duot thuc hien phan ting khuech dai yang gen NS5B veri cap mOi NS5B2F/NS5B2R tir ding trinh nghien ciru ctia tat gia Nakatani va dOng tic gii (2011): NS5B2F (5'TTCACGGAGGCTATGACYAGG), NS5B2R(5'- CGGGCATGMGACASGCTGTGA), cho san phim co kith think 688 bpveri Wing the tich phan ling PCR 25 pl theo chtrcrng trinh Juan nhiet phin (mg PCR: a 95°C phitt; ti6p theo 35 chu ky voi 94°C phut, 50°C I phin, 72°C phin; giai (Joan keo dai cu6i ming 72°C 10 phin va btrerc lam mat a 4°C Cac san pham khuech dai vimg NS5B &roc gin di giii trinh tu er cOng ty Macrogen (Hin QuOc) Phan tich ket giai trinh to Cac ket qui giai trinh tu vimg NS5B cua cac dong virus HCV &roc hieu chinh (proof-reading) va so sanh voi hai ngutm co ser dir lieu: (1) ten ngan hang gen NCBI, (2): co ser du lieu cue be): Tir do, chung toi chon loc ket qua va tien hanh phfin tich su phit sinh loci ctia virus HCV c6 12 mail benh phfim voi cac phuong phap Neighborjoining (NJ), Maximum likelihood (ML) va Maximum Parsimony (MP), sir dung trinh tu tham chieu tir co so dir lieu cue 13O not tren Khoo sat sy hoat &Ong caa m6i khuech dal yang trinh to chat! SNP tren thyr nghiem Tien hanh kiem tra su hoat Ong cim cap m6i da chon bang phuong phap PCR ved chimg duong la mau benh pham cim benh nhan viem gan C &roc cung cap bed Trung tam y khoa Medic Sau tien hanh tip cac thi nghiem khao sat nhiet dO lai toi tru cap mei va dr) dac hieu san phfim khue'ch dai KET QUA VA THAO LUAN Xfiy dung err so' du lieu cue b6 ve trinh ty yang NS5B virus HCV Qua phan tich chi tiet tren may tinh, chung toi ghi nhfin veng NS5B vira mang tinh bao ton lai c6 tinh bien Ong rat phi, hop de phan biet de clang cac phan tuyp HCV so voi sir dung Wing 5' khong dich ma (Baclig et al., 2010; Nakatani et al., 2011) Cac trinh to v6ng NS5B (lucre thu thap nhieu chung a nhieu vimg dia ly khic (chau Au, chau A, chau Phi ) (Tokita et al., 1994; Tokita et al., 1996; Lu et al., 2007; Pham et al., 2009; Xia et al., 2008) dac biet co ser lieu bao ham cac chung virus co ngu6n g6c phan lap a Viet Nam DM vai kieu gen 1, chimg tbi thu dirge 39 trinh voi cac phan tuyp tir la den 11 Kieu gen co 30 trinh hr voi phan tuyp tir 2a den 2r D61 vOi kieu gen co tat ca 83 trinh hr thu6c phiin tuyp 6a den 6w Kieu gen co 10 trinh tu veri phan tuyp 3a den 3k, kieu gen co 15 trinh to tir 4a den 4t, kieu gen c6 hai trinh to va trinh to GBV thuOc nhOm ngoai (outgroup) Khito sat in silico ye SNP rs12979860 vi rs8099917 Dji vai rs12979860 Bie'n the C/C chi4m tjt le cao nhat (40,99 %), bien the TIT chiem t' le thap nhat (13,01%) Ngueri da tang co ty le C/C cao nhat (42,45%), ngtriri da den thi ty' le C/T cao nhat (41,89%) Su phan bO cac bign thg theo dia lY }thong co su chenh lech clang kg: Bien the C/C luon chiem ty' le cao nhat va T/T luon chigm ty' le thgp nhfit Qua ket qua thong Ice kha nang dap Crng virus keo dai (SVR) cua tong bien the chung toi nhan thay hien the C/C c6 SVR cao nhat (80%), bien the T/T co SVR thap nhat (12%) Doi vai rs8099917 136 Ky yiu khoa hoc Giai thuimg SV.NC1CH-Eureka lain thir 15 nam 2013 CO so khac biet giaa cac khu voc dia ly nhung nhin chung bien the T/T luon c6 tan so xuat hien cao nhit (60%), bien thEG/G co ten s6 xuat hien it nhat (5%).Bien the T/T c6 SVR cao nhat (37%), G/T va G/G c6 dap irng dieu tri thap han (20%) Khio sat sir hoyt dfing cua moi khuech dyi Wing trinh tv chink SNP tren thirc nghiem Qua qua trinh tham khao va danh gia he mai - mau d6 sir dung phuang phap real-time PCR chung da chon &roc cap mai toi tru nhat xac dinh SNP quan tam Trinh to mai sau de da duqc chung tei chinh sira bang phan mem bioedit nham phis hop han yeti nghien ciru Buac clau kiem tra so hoot deng, de dac hieu va nhia de lai tai tru dm cap mei ten thoc nghiem Ket qua cho thay ca cap mei deu hoot dang tat, san phirn khuech dai co kith thuac bang vai kith thuot dy kien (71bp clei voi rs12979860 va 118bp dej vai rs8099917) Khuech dai dac hieu Wing trinh to chira SNP quan tam va nhiet dcj lai toi uu caa cap mei Ian !trot la 52°C va 54°C Hinh B Binh A 200 bp _„ 118 by Hinh Ket qua cli0i di sari phAm PCR db tini Tin toi thictl cho clip ni6i RS860 (With A) va inOi RS917 (hinh B) Thang chuin 100 by RT-PCR, giii trinh ty va phfin tich phi hg phfin tin 12 mau benh pham mau deu cho ket qua RT-PCR dot chat Itrang tat, du de gai giai trinh to (da lieu khOng trinh bay) Ket qua giai trinh to dirge doc tren phan mem Chromas Pro 2.0.4, phan tich vai tong co Blast Ket qua truy xuat to tong cu BLAST cho thay trinh to cac mau san pham RT-PCR thuec clang cac kieu gen HCV (du lieu khong trinh bay) Trinh to NS5B tir 12 mau benh pham va cac trinh to tham chieu (khoang 340 nucleotide/ trinh tu) yang NS5B cua cac kieu gen 1, 2, 3, 4, va HCV va trinh tin thuec virus GBVC, HGV (NC_001710) doge dung cay pha he theo ca phuong phap Neighbor-joining (NJ), Maximum likelihood (ML) va Maximum parsimony (MP) vOl gia tri bootstrap lap lai 1000 Ian Ka qua cho thay co so phan.b6 hop ljr giira cac kieu gen va phan tujp dia hinh hoc (topology) cha ca cay phi he Gia tri bootstrap deu the hien tin cay cao (Hinh 1) Tinh hop nhat ye' so phan be cha kieu gen va cac phan tuyp deu dirge the hien tren ca cay pha he cho ca nhOm trinh Or tham chieu Ian 12 trinh to giai &roc nghien ciru Ket qua duor tai kiem tra bang each chung toi that hien Blast vai 12 trinh to not tren va sir dung ccr sa din lieu caa GenBank, di, day kh6ng phai la phucmg An hoan town tai iru Tuy nhien, ca hai ket qua deu the hien tinh phis hop (da lieu khong trinh bay) 137 Giai thitemg SV.NCKH-Eureka kin thir 15 num 2013 (A) K.))yiu khoa hoc (C) (B) _11 —0 = r _v.—ammo -.1 • 911.-1 • • _kw -•- -•-•-4- • Hinh C5y pha he HCV du:crc clung theo phuang phial): (A) Neighbor-joining, (B) Maximum likelihood (C) Maximum parsimony Tr ket qua nay, chimg toi xác dinh kieu gen va phan tuyp HCV tir 12 miu berth pham nhu sau: (1) Trinh ti,r NS5B a mill thuec kieu gen (m5u X3601, X3601-6-9 thuec phan tuyp la, mau VA-1 thuec phial tuyp lb) mau thuec kieu gen (4 mu X3602-7-9, 1317, X3602-6-9 X3602-14-8 thuec pilau tuyp 2m), m5-11 duce xac dinh la kieu gen gen (inau X3603 va VA-6 thuec phan tuyp 6a mau X3602 va X3604 dtrac xep vao pilau tuyp 6e, trinh tu X3604-15-8 duric xep vao pilau tuyp 6t) 138 1C)).014 khoa hoc Giai thuzing SV.NCKH-Eureka ldn thti 15 ncim 2013 Bing So sanh phiyang phap giai trinh ttr met phhn ming NS5B va Real-time RT PCR tren vung 5'UTR KM qui phan tuyp HCV vai phu'o'ng phip NS5B Dinh ki6u gen bang phu'o'ng phi', Real - time RT PCR (1 Kik' gen Ki6u gen Kitting dinh duvc ki6u gen Kik, gen hoic Tong miu 1a lb 2m 6a 6e 6t 1 12 1 2 Tong mau Trong qua trinh tien hanh thuc nghiem buErc au nay, chimg toi so sinh kat qua xac dinh kieu gen va phan tuyp day vEri met phtrang phap khac sir dung be kit ctia ding ty Viet A dua tren kjr thuat real-time RT PCR dua tren vimg 5'UTR Ket qui so sanh nhu sau (Bang 1): phtrong phap real-time RT-PCR va phtrang phap cho kat qua xac dinh kieu gen diing nhit a mau (3 mau — kieu gen 1, mau — kieu gen 2) Co mau &rot xac dinh thuec kieu gen I beri phtrong phap real-time RT-PCR nhtmg phuang phap giii trinh hr cho ket qua thuec kieu gen Ngoai ra, co mau (VA-6) real-time RT-PCR khong xac dinh chinh xac kieu gen ma ghi nhan hoac 6, thi giii trinh to khAng dinh la kieu gen Nhu vay, rO rang la giii trinh to to hieu qua hon: khong nhting tra IEri &rot kieu gen ma cho biet phan tuyp; ngoai ra, khong the gay nhim Ian gitra kieu gen va (va day ding la van de da &roc cac ding bo tren the giEri ghi nhan, xac dinh kieu gen dua ten vimg trinh to 5'UTR) Viet Nam da c6 nhieu ding trinh nghien cua va img dung cac k'y thuat sinh hoc phan tir (RT-PCR va giii trinh tu) vies phat hien va xac dinh kieu gen HCV Wing NS5B cung da ducm met so tat gia nu& khai thac nham xac dinh kieu gen HCV Tuy nghien ctin chi mai ducrc thus hien vEri so luting mau benh khong nhieu nhtmg diem khac biet la chung toi di )(Ay dung met phtrong phap luan nghien ciru, met ca sa dir lieu cue be dang tin cay claw xac lap dung lam tham chieu ba cay pha he phan tir neu ten de xac dinh kieu gen HCV Chinh dieu da dua den kat luan chinh xac ve kieu gen va phan tuyp HCV 12 mau benh phim KET LUAN Qua thuc hien nghien ciru nay, chung CM rig ducrc met se, kat luan nhtr sau: (I) Da xay dung ca s6 dit lieu cue be va thiet lap quy trinh thuc nghiem bang phtrong phip giii trinh to met phan yang NS5B HCV va buErc au da cho kat qua dinh kieu gen va phfin tuyp ctia cac dung HCV trong12 mau benh pham real-time RT-PCR chi xac dinh dugc a mire kieu gen (2) Du that nghiem chi mai hen hanh tren 12 mau nhung,chang toi cung ghi nhan kieu gen 1, vi la 05 Wen nhiem HCV Viet Nam Nghien dm se duostiep tut trien khai vol di Mau Ion hon thEri gian tot DOi voi SNP rs12979860 va rs8099917 tren gen IL28B co kha nang tien doan kat qua dieu tri benh nhan viem gan sieu vi C Su phan bo cUa cac 'Jet bien c6 str khic gift cac eking tic, khu vac dia ly nhtmg nhin chung bien the C/C cua rs12979860 va Trr ctia rs8099917 luen co tan so xuAt hien va kha nang dap mg dieu tri cao nhat TAI LIEU THAM KHAO Baclig MO, Chan VF, Ramos JD, Gopez-Cervantes J, Natividad FF (2010) Correlation of the 5' untranslated region (5'UTR) and non-structural 5B (NS5B) nucleotide sequences in hepatitis C virus subtyping Int J Mol Epidemiol Genet, 1(3): 236-244 Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanatephenol-chloroform extraction Anal Biochem, 162(1): 156-9 Lu L Li C, Fu Y, Gao F, Pybus OG, Abe K, Okamoto H, Hagedorn CH Murphy D (2007) Complete genomes of hepatitis C virus (HCV) subtypes 6c, 61, 6o, 6p and 6q: completion of a full panel of genomes for HCV genotype J Gen Virol, 88(5): 1519-1525 Murphy DG, Willems B, Deschenes M, Hilzenrat N, Mousseau R, Sabbah S (2007) Use os sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/El and 5' untranslated region sequences J Clin Microbiol 45(4):1102-1112 139 Giai thtrang SV.NCKH-Eureka kin tine 15 natn 2013 K5i yin khoa hoc Nakatani SM, Santos CA, Riediger IN, Krieger MA, Duarte CA, Do Carmo Debur M, Carrilho FJ, Ono SK (2011) Comparative performance evalution of Hepatiris C virus genotyping based on the 5' untranslated region versus partial sequencing of the NS5B region of Brazilian patient with chronic hepatitis C Virol J 8: 459 Pham DA, Leuangwutiwong P, Jittmittraphap A, Luplertlop N, Bach HK, Akkarathamrongsin S, Theamboonlers A, Poovorawan Y(2009) High Prevalence of Hepatitis C Virus Genotype in Vietnam Asian Asian Pac J Allergy Immuno1,27(2-3): 153-160 Tanimoto T, Nguyen HC, Ishizaki A, Chung PT, Hoang TT, Nguyen VT, Kageyama S, Oka S, Pham VT, Ichimura H (2010) Multiple routes of hepatitis C virus transmission among injection drug users in Hai Phong, Northern Vietnam J Med Virol, 82(8): 1355-1363 Tokita H, Okamoto H, lizuka H, Kishimoto J, Tsuda F, Lesmana LA, Miyakawa Y, Mayumi M (1996) Hepatitis C virus variants from Jakarta, Indonesia classifiable into novel genotypes in the second (2e and 20, tenth (1 Oa) and eleventh (11a) genetic groups J Gen Virol, 77(2): 293-301 Tokita H, Okamoto H, Tsuda F, Song P, Nakata S, Chosa T, lizuka H, Mishiro S, Miyakawa Y, Mayumi M (1994) Hepatitis C virus variants from Vietnam are classifiable into the seventh, eighth, and ninth major genetic groups Proc Natl Acad Sci U S A, 91(23): 11022-11026 Xia X, Zhao W, Tee KK, Feng Y, Takebe Y, Li Q, Pybus OG, Lu L (2008) Complete genome sequencing and phylogenetic analysis of HCV isolates from China reveals a new subtype, designated 6u J Med Virol, 80(10): 17401746 140 lq yea khoa hoc Gidi thtrerng SV.NCKH-Eurika ldn thin 15 nean 2013 GENOTYPING HEPATITIS C VIRUS BASED ON SEQUENCEANALYSIS OF THE NS5B REGION Le Huyen Ai Thuy' * 'Faculty of Biotechnology, Ho Chi Minh city Open University 2Medic central HCM city Viet A Technology -Corporation Company SUMMARY Hepatitis C virus (HCV) is blood-brone pathogen leading global liver diseases and possess significant threat to public heatlh worldwide Recent studies showed that different HCV genotypes are associated with pathogenicity, infectivity and response to treatment regimens in special situations The non-structural 5B (NS5B) region encodes the RNA-dependent RNA polymerase, this is the variable sequence which is considered as the reference sequence for indentifying both genotypes and subtypes of HCV The objective was to collect a source of information and data about genotypes and subtypes in Vietnam The experimental study develop a method to amplify and sequence, phylogenetic analyse base on NS5B region by using HCV RNA isolated from the plasma samples of patients chronically infected with HCV For the 12 samples with NS5B sequencing, phylogenetic analysis showed that concordance of genotypes and subtypes with a bootstrap > 90% for each cluster Those were subtyped as genotype la in two samples, genotype b in one sample, genotype 61 in one sample, genotype 2m in four samples and genotype 6e in others Our resutls suggest that HCV NS5B region genotyping method has more advantaged and preferable than realtime PCR method for epidemilogical investigation of HCV Thus, our study should be further tested with the large sample sizes Keywords: Hepatitis C virus (HCV) NS5B region, sequencing, RT-PCR phylogeny *Author for corresspondence: Email: lhathuy@gmail.com 141 ... concordance of genotypes and subtypes with a bootstrap > 90% for each cluster Those were subtyped as genotype la in two samples, genotype b in one sample, genotype 61 in one sample, genotype 2m in four... SEQUENCEANALYSIS OF THE NS5B REGION Le Huyen Ai Thuy' * 'Faculty of Biotechnology, Ho Chi Minh city Open University 2Medic central HCM city Viet A Technology -Corporation Company SUMMARY Hepatitis... reference sequence for indentifying both genotypes and subtypes of HCV The objective was to collect a source of information and data about genotypes and subtypes in Vietnam The experimental study