Xac djnh va phan nhom cac chung virus gay hpi chufng roi loan sinh san va ho hap d Idn phan lap tai Viet Nam
Li/dng Nhan Tudn', Li/u Quang Minh', Iran Xuan Toan' Phqm Thi Phi/dng Mai', Tran Xuan Hoan', 0 6 Thj Hoa^
' l ^ n g th' n ^ m trpng di^m c8ng n ^ te bao i3$ng v^t, Vi^n ChSn tuoi
^Trmg t ^ OiSh ^okn tKt y Tnng u'ovig
Trong nghien cufu nay, 24 mau virus gay hgi chufng roi logn sinh sdn va ho ha'p d Idn (PRRSV) phan lap Xii 16 tinh d Viet Nam nam 2013 dU^c dilu tra va m^i quan he phat sinh loai cua nhuTng virus nay dUdc xay di^g dUa tren trinh ti^ axit amin cOa hai gen 0RF5 va Nsp2. Ket qua gidi trinh tif eac gen trin cho thay co si^ bien d6i Idn khi so sanh v6i trinh ti/ tren ehOng 07/QN (Genbank code:
FJ394029.1) phan lap 6 tinh Qudng Nam. MuTc dp tUdng d6ng eac trinh tU axit amin di/dc ma hda tij*
trinh tl/ nucleotide neu tren giiira 24 miu virus phan i^p vdi eh^ng 07/QN eung di/dc phan tieh va da phat hiin 19 vj trf thay doi tren gen 0RF5 va 66 vj tri thay doi trin gen Nsp2. D^ic biet, 3 mau phan l$p VN/QT-2013, VN/NA-2013, VN/ND-2013 di/dc phat hi#n mat di 3 axit amin liin tiep tai cac vj tri 869, 870 va 871. Phan tich moi quan he phat sinh loai da ehi ra ta't ci cac miu phan l|p di/dc deu thupc nhom II (bii'n the Bae My) va chDng ed dp ti/dng dong rat cao vdi cac chung PRRSV phan lap tai Trung Qudc.
TUkhda: gen ORF5 va Nsp2, giai trmh ttf, PRRSV, RT-PCR, virus.
Chi sdphin lo^l 4.3
Bat van de
Hoi chOfng roi loan sinh san va h6 h^p 6 Ipn (porcine reproductive and respiratory syndrome - PRRS) hay con dape gpi vdi ten pho bie'n la "Benh Idn tai xanh" la mot benh gay thiet hai ngtii§m trgng eho nganh ehan nuoi Idn tren the gi6i. O Viet Nam, Xii ttiang 3.2007, benh bat dau qky dich tai nhieu dja phirong, lam ton that Idn ve l<inh te cho ngudi ehan nuoi. Dac biet nam 2010, dich da xuat hien tai 1.978 xa, phudng thupc 286 quSn, huyen eua 49 tinh, thanh pho. Tdng s6 Idn mac benh la 812.947 eon, so Idn chet va bupe phai tieu huy la 442.699 con (Dang Van Ky, 2012).
Benh gay ra bdi porcine reproductive and respiratory syndrome virus (PRRSV). Ve mat phan loai, PRRSV thupc chi Arterivirus, hp Arteriviridae, bp Nidovirales (Cavanagh D Nidovirales va cs, 1997).
Co hai nguyen miu bien the eua PRRSV: type I bie'n t h i chau Au la lelystad virus (LV) va type II bien t h i Bae My la VR-2332. Cae ehCing Bae IVIy va chau Au eCia PRRSV gay ra eae hpi chijfng lam sang tLfdng tLf nhau, nhung chung co hai kieu gen rieng biit sai l<hac nhau xS'p xi 40% (Thiol H.J va es, 1993). V l
mat di truyen, he gen virus g§y benh tai xanh 6 Idn la loai virus nh6 eo he gen la ARN sdi dUdng eo Iti'eh thude khoang 15 Kbp. ARN virus cd v6 boc va ehUa it nhat 9 khung doc md (ppen reading frames - ORF). Nam ngay d vung khdng phien ma d dau 5' la 0RF1 a va 0RF1 b ma hda eho cac protein phi cau true tham gia vao qua trinh phiin ma va sao chep cua virus, tiep theo 0RF2a, 0RF2b va cae khung doc md tU 0RF3 de'n 0RF7 ma hda cho cae protein eau true eua virus (Kim H.K va cs, 2009; Kim S.H vacs, 2010).
Khung dpe md 0RF5 ma hda eho mpt phan X\i protein mang khdng bj dudng hda cd trpng IUdng 17 kD hoae mpt phan tCf protein dudng hoa trpng IUdng 25 kD (Mardassi H va cs, 1995). Hdn the nQa, vung 0RF5 eua PRRSV la vung gay dap Ung miSn djch chCi dao bdi nd lien quan tdi viec hinh thanh nhCfng khang the trung hda. Tren thuc te 5 PRRSV type II khung dgc 0RF5 cd chUa 3 vj tri epitope eua te bao B va 2 vj tri epitope cua te bao T. Ndi each khac, day ia vung di truyen quyet djnh chu yeu den dpc luc cija virus khi xam nhap vao ed the vat ehCi (Mardassi H va cs, 1995; Kim H.K va cs, 2009).
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33PHYIOGENETIC CEIATIONSHIP OF THE PODCINE DEPI30DUCTIVE AMD CEcSPIBATODY SYNDDOME VIDUSES
(PRDSV) ISOIATED IN VIETNAM
£inma-y
In this study, 24 porcine reproductive and respiratory syndrome virus (PRRSV) samples isolated from 16 provinces in Vietnam in 2013 were investigated, and the phylogenetie relationship of these viruses was constructed based on the amino acid sequences of 0RF5 and Nsp2 genes. The sequencing results of these genes indicated the high variation compared to the sequence of 07/QN strain (Genbank code: FJ394029.1) isolated in Quang Nam province. The identity of amino acid sequences deducted from the nucleotide sequences between the 24 isolates and 07/
QN strain was also analysed; 19 variations in 0RF5 gene and 66 variations in Nsp2 gene were found. Especially, three isolates VN/
QT-2013, VN/NA-2013, and VN/ND-2013 were found to possess three-continuous amino acid deletions (869-871) within Nsp2 gene.
Phylogenetie analyses based on the 0RF5 and Nsp2 amino acids indicated that all these isolates were grouped into PRRSV type II (North American genotype) and they were highly similar to PRRSV strains isolated in China.
Keywords: ORF5 and Nsp2 genes, PRRSV, RT- PCR, sequencing, virus.
Classification number 4.3
Gen Nsp2 la gen ma hda cho 1 protein phi cau true (non structural protein 2) tham gia vao qua trinh phien ma va sao chep cua PRRSV. Gen Nsp2 nam d vj trf khung dpc md ORFIa. Tian K va cs (2007) da phan tich trinh tU gen Nsp2 cua chung virus gay djch d Trung Qudc nam 2006 phat hien su thieu hut 3D axit amin so vdi cac chung phat hien trude dd va ehijng mdi phat hien nay cd ddc lUc cao hdn han cae chOng c6 diln.
Hieu biet ve sU da dang va tien hda cua PRRSV se dem lai hieu qu& cao trong qua trinh nghiin cUu vaccine dieu tri binh gay ra bdi virus nay. Do vay, Chung tdi dat myc tieu giai trinh tU 2 vung gen quan trpng 0RF5 va Nsp2 tU cac m i u PRRSV phan lap
tai Viet Nam nam 2013 nham danh gia si/ bii'n dil cac nucleotide, tU dd phin nhdm cac virus va danh gia mo'i quan he giQa cac m i u phan lip nay.
Difi titftfng va phuTtfno phap nghiin cilTu Ddi tU0ng nghien ciJti
Doi tUdng nghien cUu la cac mau PRRSV dupc phan lap tU cae mau benh pham thu thip Xii eac o djch d eac vung cua Viit Nam.
PhUdng phip nghien cUu
PhUdng phap tich chiit RNA virus tU cac miu benh pham thu dUgc: mau binh pham la md phii sau khi thu dupe bao goi can than, dua vao dung djch RNA-later va b&o quSn mat 6 2-7-8''C, sau do dupe ehuyin ngay v l phdng thf nghiem. Tgi diy, mau dupe bao quin d nhiit dp -70''C trUdc khi tien hanh tach ehiet RNA tong sd.
Tiln hanh tach ehiet RNA: khoang 3 mg mau phoi, eat nhd roi nghien trong coi chay sU v5 trCing trong dung dieh nitd Idng. Hda m l u vdi 3 ml dung dieh PBS (pH = 7,4) thanh huyin djch 10% roi ly t i m 2000 g trong 15 phut. Hut lay dich nude trong d phia trin xCf ly vdi khang sinh penicillin (200 UI/
ml) va Streptomycin (200 pg/ml), hoae cd t h i Ipc vo trung qua mang lpe cd kfch thUdc Id Ipc 0,45 pm.
Sau dd SLf dung bd kit Rneasy mini Kit cua QIagen d l tach ehiet RNA.
Pftt/i?nfifp/jap ch^y RT-PCR-theo kit superscript III first - strand synthesis system ci!ia hang Invitrogen.
PhUdng phip PCR: ch^y theo protocol Hot Start Taq DNA Polymerase cija Fermentas: trinh X[f cap moi nhan len dogn gen Nsp2 nhU sau: Nsp2- F: 5'- AAA GAC CAG ATG GAG GAG GA-3', Nsp2-R:
5'-GAG CTG AGT ATT TTG GGO GTG-3; trinh ti/
cap moi nhan lin toan bd khung doc gen 0RF5 nfiU sau: 0RF5-F: 5'-ATG TTG GGG AAG TGC TTG ACC-3', 0RF5-R: 5'-CTA GAG ACG ACC CCA TTG TTC CGC-3'.
PhUdng phap dien di: diin di trin gel agarose 1,5%.
PhUdng phip tinh sach ADN tUgel:X\nh sgch theo kit Free UV Gel Extraction cua hang Invitrogen.
Giai trinh tU gen: tien hanh giSi trinh tif trin may ABI-3130, kit sCf dung cho phlin Ung Terminator V 3.1 Cycle Sequencung Kitcita hang ABI, kit lam sach sau phan Ung BigDye^ XTerminator"^ - Purification Kit cda hang ABI.
PhUdng phip xO iy so li$u: bang phan mem Bioedit version 7.0.5.3 va MEGA 5.1.
SII«IlGrtViaNam^<''"°^=
34Kat qui va thao In^n
Kit qua nhin PCR hai do^n gen Nsp2 vi 0RF5 Da tach dUde 24 m l u mRNA tif 24 m l u binh pham vk tong hpp thanh cdng 24 mau cDNA trin may realtime PCR. T i l n hanh ph^n Ung PCR tU 24 m l u cDNA nhan l i n 2 gen Nsp2 va 0RF5 (sCf dyng 2 cap moi nghien cUu). Ket quIi kilm tra trin dien di do eho thay hai gen Nsp2 va 0RF5 da dupe nhan len thanh edng ed kich thude tUdng Ung la 666 bp va 603 bp, phu hpp vdi kieh thUdc \y thuylt (hinh 1).
Marker sCf dung la 1 kb DNA ladder.
2008). TU ke't quk ta thay, ed tong so 167 vi trf sai khae trin chudi trinh tu 603 nucleotide gen 0RF5 (ty l i 27,7%). Dilu nay eho thay gen 0RF5 cd kha nang dot bien cao.
Khi so sanh trinh tU axit amin gen 0RF5 cOa cac mau thu dupe va ehijng 07/QN, ta tha'y cd 19 vj tri axit amin bi bie'n doi trin toan b i chudi polypeptide 201 axit amin eua gen 0RF5 (hinh 2). NhU vay, so vdi 167 vj trf sai khac nucleotide thi cd rat nhieu vi tri ddt bii'n c i m , khdng anh hudng den trinh tif chu5i polypeptide gen 0RF5. B i n cgnh dd, trong c^c m l u thu dupc ta thay mau VN/YB1 -2013 la mSu cd nhilu vj tri axit amin sai khae nhat (13 vj tri) va m l u VN/
HD-2013 la mau cd ft sai khac nha't (2 vi tn").
Hinh 1: k^t qua dien di sin pham PCR hai gen Nsp2 vi ORF5 trin gel agarose
SSn pham PCR sau dd dUdc tien hanh tinh sgch bang kit Purelink"" PCR Purification Kit c^a hang Invitrogen. Ket qu& cho thay, s^n pham PCR dupe thu h i i sau tinh sgch cd ham IUdng du cho cac nghien cUu tiep theo.
Phan tich trinh ttf vi so sinh su sai khic trinh ti/gen ORF5
Siin pham PCR gen 0RF5 sau khi tinh saeh dUdc gi&i trinh tu 2 chilu, sQ dyng may giai trinh tu ABI 3130, sau dd Chung tdi da tien hanh so sanh (blast) tren ngan hang gen (http://blast.ncbi.nlm.nih.
gov/Blast.cgi) va ket qua xac dinh trinh tU thu dupe tUdng dong vdi trinh tU 0RF5 cdng bd. Bang phan mIm Bioedit, ehung tdi da so sanh va chi ra mdt s6 mau cd dp tUdng d6ng hoan toan vdi nhau v l trinh tu gen, cy the la: eac m l u VN/HB1-2013, VN/HN- 2013, VN/HB2-2013, VN/ND-2013 va VN/PT2-2013 tUdng ddng; eac mau VN/YB2-2013, VN/YB3-2013, VN/YB4-2013 tUdng ding; cae m l u VN/DB1-2013, VN/DB2-2013 va VN/BN-2013 tUdng ddng.
Trinh tu gen 0RF5 cCia cac mlu sau dd dUdc so sanh vdi trinh tU ci^ia chiing 07/QN [Genbank code:
FJ394029.1) la chCing PRRSV thu dupe 6 Qu&ng Nam, Viet Nam nSm 2007 (Youjun Feng va cs.
Hinh 2. so sanh sU sai khic trinh tU axit amin gen ORF5 eua cac miu thu dUdc vdi ehung 07/QN (phan mem Bioedit)
Phin tich trinh ttf va so sinh su sai khic trinh tugen Nsp2
Sau khi giai trinh ty gen Nsp2 va so sanh (blast) eae mlu giai trinh tU tren ngan hang gen (http://
blast.ncbi.nim nih.gov/Blast.cgl), chung tdi th^y ki't qua xae djnh trinh ty cd do tUdng dong cao vdi trinh ty gen Nsp2 eua PRRSV cdng bo. Khi phan tfch mUc do tUdng ding giQa cac mlu thu dUdc, sil dyng phan mem Bioedit, chung tdi thu dupe ki't qu& mpt so mlu cd trinh tu tUdng dong hoan toan: mlu VN/
HBI -2013, VN/HN-2013, VN/HB2-2013 tUdng dong;
m l u VN/YB2-2013, VN/YB4-2013 tUdng dong.
te2?i*ViaN.m 1(3)3-15
So sanh trinh tu cac mau vdi chung so sanh la 07/QN {Genbank code: FJ394029.1), ket quk thu dupc cho thay cd 127 vi tri sai khac nucleotide d cac mlu thu dUpc va chung 07/QN. Dac biet, trong 3 mlu VN/QT-2013, VN/NA-2013 va VN/ND-2013 cd su thii'u hyt 9 nucleotide d vi tri 296-305 so vdi cac mlu khae va chung 07/QN doi ehUng.
Tien hanh so sanh trinh tU axit amin gen Nsp2 cCia cac mlu thu dupe va chung 07/QN ta thay xuat hiin 66 v\ trf axit amin bi bie'n doi (hinh 3). NhU viy, so vdi 127 vj tri sai khae nucleotide thi cd nhieu vi tri dot bie'n cam, khdng anh hudng di'n trinh tU chuoi polypeptide gen Nsp2. Trong eae mau thi mlu VN/BD2-2013 la mlu cd nhieu vi trf axit amin sai khac nhi't (20 vj trO va mau VN/HD-2013 la mlu cd ft VI trf sai khac nhat (2 vj tri). 6 mdt s i vi tri axit amin hau he't eac mau dlu cd sai khae vdi chCing 07QNnhUR793W, A811T, S814P, D833G,L877P, V887M, F892R, P896L, V928A, K935E, L942S, A955V, M974V. Dac biit, 3 mlu VN/QT-2013, VN/
NA-2013, VN/ND-2013 bj mat 3 axit amin 6 vi tri 869, 870 va 871.
nam 2013 d l u thugc type il va cd khoang each di truyin g i n vdi cae chung thu dupe d Trung Quoc nhu JX 2012, JXwnOe (hinh 4). DUa vao quan h i di truyen cua cac mlu thu dUdc, ta cd the chia cac mlu thanh cae nhdm, ket qua dUdc trinh bay trong bang 1.
Hinh 4: ciy quan hS phit sinh cic miu PRRSV dUa tren trinh tU axit amin 2 gen 0RF5 va Nsp2 (phin mdm MEGA 5.1, phUdng phip neighbor -joining. Bootstrap 1000, maximum
composite likelihood)
) 1: phin nham cac mau virus phan lip dUOc dUa vio moi quan h$ di truyen
Hinh 3: so sanh sU sai khac trinh tU axit amin gen Nsp2 gida cac miu thu dUac va ehijng 07/QN (phan mem Bioedit)
Xic dinh quan hg di truyen vi phin nhdm cac miu virus nghien cdu
Tien hanh dung cay quan he phat sinh (neighbor - joining) cua eac chung da phin lap vdi dQ lieu trinh tu axit amin hai gen 0RF5 va [sisp2 cCia PRRSV trin NCBI bang phan mem MEGA 5.1, chung tdi phat hien theiy cac chijng virus phan lap d Viet Nam
STT 1
2
3
4
5 Nhom L i k e - J X 2 0 1 2
VN - Group 1
VN - Group 2
VN - Group 3
Like - JXw2006
Mlu VN/HT-2013
VN/PT2-2013, V N / H B I - 2 0 1 3 , V N / HN-2013, VN/HB2-2013, VN/ND- 2013, VN/HP-2013, VNH'Q-aOIS, VN/NA-2013, VN/QT-2013 VN/PT1-2013, VN/QN-2013, VN/BN- 2013, VN/BD1-2013, VN/DB1-2013, VN/DB2-2013
VN/BD2-2013, VN/YB2-2013, V N / YB3-2013, VN/YB4-2013 VN/HY-2013, VN/NB-20013, V N / YB1-2013, VN/HD-2013
SSsNCTiViaNam'^'^-^'''^
3EKet luan va kien nghi Ket luin
Hai dogn gen 0RF5 va Nsp2 phan tfch ed tfnh da hinh cao. So vdi ehCing 07/QN {Genbank code:
FJ394029.1) phan lap tai tTnh Quang Nam nam 2007, trinh tu axit amin cac gen 0RF5 va Nsp2 cua cac m l u phan tfch ed nhieu bii'n d l i , cy the la trong trinh tu axit amin gen 0RF5 va Nsp2 xu^t hien tuong Ung 19 va 66 vi tri sai khac. Dae biit, 3 m l u VN/QT- 2013, VN/NA-2013, VN/ND-2bl 3 l3J mat 3 axit amin S vi tri 869, 870 va 871 tren gen Nsp2. Ki't qua nay la ed sd cho nhOng danh gia v l sU bie'n chung cua PRRSV 6 V i i t Nam sau nay.
Ta't ca eac ehung PRRSV phan tfch deu thude type II (ehCing Bae My) va cd quan he di truyen gan vdi cac Chung PRRSV tU Trung Qudc va chCing 07/
QN phan lgp d Quang Nam/Viet Nam nam 2007.
Kien nghj
• PRRSV cd kha nang bien doi cao, vi vay can theo ddi l i i n tyc nham danh gia kip thdi cac bien doi cua virus de cd cac bien phap ddi phd kjp thdi va hieu qua.
- Dua vao k i t qua nghien cQu tha'y rang, chung virus gay b i n h d Viet Nam ed quan he di truyen gan gui vdi cac chung cOa Trung Qudc, do dd sif dyng vacxin san xui't tU Trung Quoc se cd h i i u qua tdt hdn.
Tai Ii6u tham lihao
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9. Youjun Feng, Tiezhu Zhao, Tung Nguyen, Ken Inui, Ying Ma, Thi Hoa Nguyen, Van Cam Nguyen, Di Liu. Quang Anh Bui, Long Thanh To, Chuanbin Wang, Kegong Tian, and George F.
Gao (2008), "Porcine Respiratory and Reproductive Syndrome Virus Variants, Vietnam and China", Emerging Infectious Diseses, VoL14, No.ll.