MOT SO KET QUA NGHIEN CUfU BlTOfC OAU VE TEMBUS VIRUS tf VjTBENHTAIHANOI
Ddng Huu Anh, Nguyin Vdn Gidp, Huynh ThiMyLe Khoa Thuy. Hoc vien Nong nghiep VietNam
TOM TAT
Nghien ciiu nay dugc thuc hien nh5m xac dinh su c6 mat cua Tembusu vuus a dan vit benh tai khu vuc Ha Noi. Sir dung phan iing RT-PCR kit bgp voi kit qua giai trmh tu va phan tich trinh ttr gen NS5B da khang dinh su c6 mat ciia Tembusu virus trong cac miu vit benh thu thap tai Ha Ngi nam 2019. Trieu chimg Iam sang va benh tich dai tbi diln hinh cua vit duong tinh voi Tembusu virus la Men tugng mat dilu hoa van dgng, bai liet, giara de manh dot nggt, buong tning xuat huyet, lach stmg, gan simg. Buoc dSu phan tich, 3 chung Tembusu virus trong nghien ciiu nay dugc xep vao nhom Thailand strams va gan gQi (ve hinh tu gen NS5B) voi chung virus phat hien cr Thai Lan nam 2013 (KR061333).
Tic khoa: Tembusu virus, RT-PCR, NS5B, Ha Ngi.
Some initial result of study on Tembusu virus in the infected ducks in Ha Noi Dang Huu Anh, Nguyen Van Giap, Huynh Thi My Le
SUI\/II\/1ARY
This study was conducted to determine the presence of Tembusu virus in the duck flocks Ha Noi area. The result of RT-PCR combining with NS5B gene sequence analysis showed the presence of Tembusu virus In the Infected duck samples collected in Ha Noi in 2019. The typical clinic symptoms and gross lesions ofthe infected ducks with Tembusu virus included phenomena on loss of moving ataxia, paralysis, reduction of laying egg suddenly and severely hemorrhagic ovary, swollen spleen and liver. The initial analysis result indicated that the 3 Tembusu virus strains in this study were classified as Thailand Tembusu virus strain and closely related (NS5B gene sequence) with the Tembusu virus strain that was defected in Thai Land in 2013 (KR061333).
/keywords;Tembusu virus, RT-PCR, NS5B, Ha Noi.
L D A T V A N D E mgt ARN virus co do dai khoang 11 kilobases Tir thang 6 din thang 11 nam 2010, mgt hien ^"^ "^^^ **""§ 5gc mo (ORF) duy nhSt ma hoa nrgng benh a vit de dugc phat hien a Tnmg ^^° ^ protem cau triic ciing voi 7 protein khong Qulc voi bilu hien giam de dot nggt trong vong ^^'^ ^^'^ (Zhang va cs., 2017). Vuus khong chi 1-2 tu§n. Nhiing bieu hien khac bao gim giam t^ac dgng den vit de ma con co the lay nhiem cho an, uong, tieu chay phan xanh la cay, chay nuoc ^J^ ^^it, ga, ngong va chugt (Sun va cs., 2019).
mui, mat dieu hoa van dong va bai het. Ty le Tembusu virus truoc kia dugc cho la nhom virus mac cao (Ien den 90%) va ty Ie chet dao dgng tir truyen lay thong qua vai tro cua dgng vat trung 5% den 30%. Cao va cong su nam 2011 da phan gian la muli giong nhu virus gay benh viem lap va nhan dien dugc virus co ten la Tembusu nao Nhat Ban. Hien tai nguai ta da phat hien ra vuus (TMU\0 (Cao va cs., 2011) Tembusu Tembusu virus da co the dugc truyin lay theo virus, thuoc chi Flavivirus. hg Flaviviridae nhirng duang khac ma khong cin thong qua vec- dugc phan lap Ian dau tien vao nam 1955 a ioai to truyin benb (Yan va cs.. 2018) Chan nuoi vit muli Culex iritaeniorhynchus tai Malaysia, la tai Viet Nam dang phat triln manli nhung hch
KHOA HOC KY THUAT THO Y TAP XXVII SO 1 - 2020
tiem phong vacxin cho dan vit nuoi chi tap trung vao benh viem gan vit, dich ta vit va ciim gia cSm. Thai gian gan day 6 nhieu dia phuong da xuat hien vit co bieu Men nghi nga do Tembusu virus gSy ra nhu giam de manh dot nggt, bai liet, buong triing xuat huyet. Tuy nhien chua thay c6 bao cao khoa hgc xac nhan su co mat va xep Ioai Tembusu virus gay benh. Nghien ciiu nay dugc thuc hien nhSm khang dinh su hien dien cua virus gay benh a dan vit, tii do giiip dinh huong cong tac chan doan va phong chong benh cho vit hieu qua,
n. NOI DUNG, VAT LIEU, PHU'ONG PHAPNGHlfeN ClTU
2.1. Ndi dung nghien cuu
- Nghien ciiu mgt so dac diem trieu chiing, benh tich dai the cua vit nghi mac Tembusu vims.
- Xac dinh sir co mat ciia Tembusu virus bang phan ling RT-PCR va giai trinh br gen NS5B.
- Vit 6m (2 - 7 thang tuoi) co trieu chting
nghi nga do Tembusu virus gay ra, thu thap tai cac trang trai d huyen Thach That va Gia Lam, Ha Ngi.
- Mlu ggp phu tang ciia vit benh bao gim:
nao, tim, phli, gan, lach, buing tning.
- Hoa chat diuig tach va tinh sach ARN ting si gom: (i) TRIzol reagent (15596026, Invih-ogen); (ii) chloroform (102445, Merck Millipore); (iii) 2-PropanoI (109634, Merck Millipore); (iv) cln 70%; (v) nuac ck da xir Iy bang 0,1 % diethylpyrocarbonate (IBS- BW004B, iNtRON Biotechnology).
- Tong hgp cDNA dugc thuc hien bing:
(i) random hexamers (N8080127, Invitrogen) va (ii) kit M-MLV Reverse Transcriptase (28025013, Invitrogen).
- Sinh pham, hoa chat dung cho phan ling PCR: (i) 2X PCR Master mix solution (i-MAXII, 25266, iNtRON Biotechnology); (ii) cap moi dac hieu gen NS5 cua virus dugc lay theo nghien ciru da cong bo (Liu va cs., 2012) voi thong tin a bang 1.
Ten moi NS5f NS5r
Bang 1. , Trinh tip moi cua plian ipng RT- PCR
Trinh tip moi ( 5 ' - 3 ' ) Kicli thirac (bp) TTTGGTACATGTGGCTCG
350 ACTGTTTTCCCATCACGTCC
2.3. Phirong phap nghien ciru 2.3.1. Phirffng phdp thu thdp mdu
Phucmg phap kiem tra trieu chimg Iam sang va ml kham kilm tra benh tich dai thi theo Tieu chuin Viet Nam TCVN 8420:2010. M k dugc thu thap theo Quy chuan ky thuat Qulc gia QCVN 01-83:2011.
2.3.2. Phuffng phdp tdch vd tinh sach ARN tong so
ARN ting s6 dugc tach bSng TRIzol reagent
(theo huang dan cua nha san xuat), vai cac buoc dugc torn tat nhu sau: (i) giai pbong ARN bang each bo sung 750 ^il TRIzol vao 250 fil huyen dich mau 10%, dong nhat bang each vortex a toe do toi da; (ii) tach pha ARN bang 200 pi chloroform, vortex dl ding nhat; (iii) tua ARN thu dugc a buac (ii) bang 500 nl 2- propanol, giu a -20''C/10 phut; (iv) rira hia ARN b5ng 1 ml cln 75%. Cac budc hi (ii) din (iv) duac ly tam o dilu kien 12.000 vong/ phiit trong 10 phiit a 4''C. Hoa tan tiia ARN bing 30 pi nuoc (da xii Iy Dnase/Rnase) va bao quan a -70''C.
2.3.3. Phifffng phdp tdng hop cDNA ARN tong so dugc chuyen thanb cDNA bang random hexamers, sir dung bg kit M-MLV Reverse Transcriptase vai cac tbanh phan dugc phoi trgn theo hudng dan ciia nha san xuat. Phan ling tong bgp cDNA dugc thuc hien a 3T'C trong 60 phut.
2.3.4. Phirffng phdp PCR
Phan ling PCR phat hien Tembusu virus dugc thuc hien bang cap moi NS5f7NS5r, theo quy trinh da cong bo truac day (Liu va cs., 2012). Phan tich san phim PCR bSng dien di trong thach agarose 2% c6 bo stmg thuoc nhugm ADN(RedSafe) Ix.
2.3.5. Phifffng phdp phdn tich trinh tu-gen San pham PCR tinh sach dugc giai trinh tu theo hai chieu (xuoi va ngugc) bang phuang pbap Sanger (thuc hien tai cong ty Macrogen, Han Quoc). Trinh tu nucleotide dugc phan tich biing chuang trinh BioEdit v7.1.3.0 (Hall, 1999) tren co set doi chieu so sanh (i) giiia trinh tu nucleotide dugc giai trinh tu tbeo chieu xuoi va
chieu ngugc, va (ii) voi trinh nr gen NS5 cua chiing Tembusu virus tham chn;u (GenBank:
KY626659).
Trinh tu mgt ph^n gen NS5 thu dugc trong nghien ciiu nay dung de xac dinh moi lien he di truyen giira cac chung Tembusu virus ciia Viet Nam vai cac chiing tren the gioi cong bo o GenBank. Cac trinh tu tham chieu dugc chgn tbeo nghien ciiu truoc day (Yu va cs., 2018).
Trinh tu nucleotide dugc can chinh (alignment) bang phan mem MAFFT (Katob va Standley, 2013) voi cac tham so mac dinh. Cay phat sinh chiing Ioai (phylogenetic tree) dugc xay dung bang thuat toan neighbor-joining, tich hgp trong chuong trinh MEGA phien ban 7.0.26 (Kumar va cs., 2016). Mitc tin cay cua cac nhanh phan chia a mii niit (node) dugc bieu thi bang gia tri bootstrap. Bieu dien va hieu dinh cay phat smh chiing Ioai bang phan mem FigTree phien ban 1.4.4 (http://tree.bio.ed.ac.iik/software/figtree/).
m . KET QUA VA THAO LUAN
3.L Tinh hinh dich b|nh if mot so dan vit nuoi tai Ha Noi
Hinh 1. Trieu chirng lam sang va benh tich dai the cua vit dirong tfnh v&i xet nghiem phat hien Tembusu virus '
A: Vit CO ty IS mac cao, mat dieu hoa van dong, bai liet; B: Phan xanh la cay C- Lach ^ , m ^ -
D: Tui mat sung; E: Buong tnmg xuat huyet; F: Gan swng ^^'i9<
KHOA HOC KY THUAT THO Y TAP XXVII SO 1 - 2020
Trong khoang thai gian hi thang 3 den thang 10 nam 2019, mgt so dan vit nuoi tai Ha Ngi co bilu hien sot, giam an, uong. Vit bi mat dieu hoa van dgng, di diing xieu veo. Vit thit co hien tirong chet vcri so lugng lan trong vai ngay. Vit de giam de manh dot nggt. Khi mo kham vit thay CO bieu hien gan sung, lach sung, buong hiing xudt huyet.
Hmh 1 mo ta mgt si trieu chiing lam sang, benh tich dai the cua dan vit benb. Do 6 djch o vit CO the do ghep nhieu nguyen nhan nen ngoai nhimg dac diem tren, mgt so vit con co hien tugng tieu chay phan mau xanh la cay, gan hoai tii CO phii fibrin va tiii mat sung. Mgt so phac dl can thi6p dich da dugc ap dung gom sir dung khang sinh (Ceftiofur, Gentamycin, Doxycyclin, Florphenicol), bo trg bing dien giai va tiem lai vacxin dich ta vit nhimg chua thay hieu qua.
Mgt so trang trai da gui mau xet nghiem dich ta vit va ciim gia cam nhung ket qua deu am tinh (ket qua khong hao cao). Theo thong tin ghi nhan tir bilu hien lam sang va kit hgp voi tinh hinh dich tl benh do Tembusu virus a vit trong
khu vLtc, chiing toi da su dung phuong phap RT- PCR de xac dinh xem lieu co su hien dien ciia Tembusu virus o nhiing dan vit nay hay khong.
3.2. Kit qua RT- PCR phat hien Tembusu virus
Kit qua RT-PCR phat hien Tembusu virus trong 12 mlu benh pham (hinh 2A) cho thiy mlu doi chiing am ciia phan iing khong co vach dac hieu; c6 9/12 mau xet nghiem co vach san pham PCR trong khoang kich thuoc 350 bp. Mlu benli pham so 3, 10 khong c6 vach san pham va dugc danh gia la am tinh. Vi Tembusu virus chua timg dugc phat hien o Viet Nam, de khang dinh tinh dac hieu cua phan iing, san pham RT-PCR da dugc giai trinh tu gen (hinh 2B). Ket qua tim kiem tririh tu tuang dong tu ca so dir lieu cua GenBank (https://blast.ncbi.nlm.nih.gov/
Blast.cgi) cho biet: tririh tu nucleotide dau vao (350 nucleotide) gilng 97,13% (JQ627864) din 98,28% (KY623423) voi cac chung Tembusu virus da biet. Nhu vay, phan iing RT-PCR dac hieu va co 9/12 mau xet nghiem duong tinh vai Tembusu virus.
- 220 230 2-:0 250 26C B ATeG.A.A,CCAAT GC.'^.C.A.GAGC TG TC GC.A,C.A.A.CCAC T C.^TT.AAAT T
MM
Hinh 2. Ket qua RT- PCR phat hien Tembusu virus A- giing 1-12 la cite miu xet nghiem, M la 100bp DNA ladder, mau dii chirng am (-):
fi." gian do giai trinh ttr gen san phim PCR (mau s6 6)
3.3. Mire do bilu hien lam sang ciia dan vit diTffng tinh voi Tembusu virus
Kit qua hoi ciiu tu nhirng vit co mau ducmg tinh vai Tembusu virus bang phucmg phap RT- PCR dugc trinh bay a bang 2. Ket qua cho thiy nhirng bieu hien co ty le cao a vit benh gim: Vit mat dieu boa van dgng (9/9 con), bai liet (8/9 con), lach sung (6/9 con). Ci vit de co hien tugng giam de manh (tir 90% xuong con 10% h:ong vong 1 tuan theo thong tin tir trang trai) va xuat huyet buIng tning chiem ty Ie 5/5 con khi kilm tra benh tich dai the. Nam
2018, mgt nghien ciiu hoi ciru man benh pham duong tinh vai Tembusu \ inis tai Thai Lan da cong bo nhu-ng mlu vit benh tir nam 2007 co trieu chimg thin kinh, giam de manh dot nggt (Ninvilai va cs., 2018). Lu va cs. da gay benh thuc nghiem Tembusu virus cho vit va ghi nhan benh tich dai thi a vit 7, 18,21 hian tuoi gom gan sung, xuat huylt co tim, xuat huyet rugt va phu phoi (Lu vacs., 2016). Ket qua hoi ciiu cua chiing toi ve mau henh pham duong tinh Tembusu virus pbu hgp voi nhirng nghien cuu ciia cac tac gia tren.
Bang 2. Ty le trieu chu-ng va benh tich cua vjt dtpcng tinh vo-i Tembusu virus
Bleu hien Vjt de (con) Vit thjt (con) So co bieu hien (con) Tyle Mat aieu hoa van dong
Bai liet
BuIng tning xuat huylt Lacfi sung Gan sung
5 6 7
5/5 6/9 7/9
3.4. Ket qua phan tich moi lien he di truyen ciia cac chung Tembusu virus
Dua vao trinh tu mgt phan gen NS5B (350 nucleotide), cay phat sinh chiing loai da dugc xay dung voi 3 chiing cua Viet Nam va 137 trinh tu tuang iing cong bo tren GenBank (hinh 3).
Trinh tu gen NS5B thuang dugc dimg trong nghien ciiu moi lien he di truyen ciia giong Flavivirus (Kuno va cs., 1998), trong do Tembusu virus la mgt tbanh vien. Kit qua xay dung cay phat sinb chung loai trong nghien cuu nay giong vai cong bo truoc day (Yu va cs., 2018) khi ciing chi ra co 5 nh6m di truyen ciia Tembusu virus voi gia tri bootstrap > 90% (hinh 3).
Dira vao dac diem phan nh6m, cac chiing
Tembusu virus cua Trung Quoc tao thanh 2 nhanh lan rieng biet (Chinese strains I, II) va tach biet voi chiing virus phat hien dugc a Thai Lan va Malaysia. Ciing dua vao dac diem tren, 3 chiing Tembusu virus trong nghien ciiu nay dugc xep vao nhom Thailand strains va gin gui (vl trinh tu gen NS5B) voi chung virus phat hien a Thai Lan nam 2013 (KR061333).
Ket qua phan tich trinh tu gen cung cho thay, phan irng RT-PCR da dugc t i i uu va dat do rin cay cao trong chin doan benh do Tembusu virus gay ra. Trong so nhung mlu duong tinh voi phan ung RT-PCR, c6 ca mlu cua dan vit thit bi benh a giai doan 2 thang tuoi, chirng to Tembusu virus ciing co kha nang gay nhilm cho vit thit a Viet Nam.
KHOA HOC KY THUAT THU Y TAP XXVII SO 1 - 2020
Thailand strains
Malaysia strains II
Malaysia strains I Aroinoet^Mms
Hinh 3. Cay phat sinh chiing loai cua Tembusu virus
Ghi chu: mot phan trinh tu NS5B (350 nucleotide) duac dung de xay dirng cay, gia tri bootstrap duuc trinh bay cho cac nhanh chinh. Tembusu virus duac phan thanh 5 nhom chinh dua vao nghien c&u cua Yu va cs. 2018. Nhanh cua cay phat sinh chung loai din tai 3 chung cua Viet Nam (mui ten) du^c danh mau xanh.
IV. K £ T LUAN
Nhu vay, ket qua phat Men virus bang phan ling RT-PCR kit hgp voi kit qua giai
khang dinh su co mat ciia Tembusu virus trong cac mau vit benh thu thap tai Ha Noi nam 2019. Vit benh thucrng co bieu hien giam de manh dot nggt (voi vit de), mat dilu trinh tu va phan tich trinh tu gen NS5B da boa van dgng, bai het. Benb tich dai the khi
mo kham Ihay buong trung xuat huyet, gan sung, lach strng. Ba chiing Tembusu virus trong nghien ciru nay dugc xep vao nhom Thailand strains va gan giii (ve trinh tu gen NS5B) vai chiing virus phat hien o Thai Lan nam 2013 (KR061333). Nhtrng nghien ciiu ke tiep can thtrc hien la xac tiinh dac diem dich te hoc, dich te hoc phan tu cua benh, phan lap virus gay benh va huang tai giai phap phong chong benh hieu qua.
TAI LIEU THAM KHAO 1. Cao Z., Zhang C , Liu Y., Ye W., Han J.,
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30(4): 772-780.
4. Kumar S., Stechcr G. & Tamura K. (2016).
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Phylogeny of the Genus Flavivirus. Journal of Virology. 72(1): 13.
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Evolution of Tembusu Virus in Ducks, Chickens, Geese, Sparrows, and Mosquitoes in Northern China. Viruses. 10(9): 485.
I2.Zhang W., Chen S., Mahalingam S., Wang M. & Cheng A. (2017). An updated review of avian-origin Tembusu vims: a newly emerging avian Flavivirus. Joumal of General Firology. 98(10): 2413-2420.
Ngay nhan 5-12-2019 Ngay phan bien 2I-I2-20I9 Ngay dang 1-1-2020
