KHOA HQC CbNG NGHfi
XAC DIIMH V i R U T G A Y BEIMH H O A I TV T H A I V K I I V H [IMervaus necrosis t/iru^ T R £ | V t A CHIIXIH IXIUDi
T A I VIJIMG BIEIM MIEIM TRUIMG
Ph?m Thi Tam\ Pham Cdng Ho^t^, NguySn Thi Vui^ NguySn Thi ^hanh^ NguySn Quang Linh^
T6MTAT
Tu 30 mau ca chinh thu th§p d viing bi^n mi^n Trung nghi mac b^nh hoai tii thdn kinh da phat hi§n duoc 4 miu CO b^nh tich d md nSo cung nhu b^nh tich tr^n t^ bao mSn cam GS. Bang phuong phap giai tiinh tu vung bi^n ddi T4 ti-^n 2ARN da xic djnh dugc 04 chung A-HUIOl, A-HUI06, A-HUI12 va A-HUI17 thupc nhom vi nit gSy b^nh hoai tu th^n kinh tr^n ca chinh chau Au {European nervous necrosis virus).
Ti kh6a: Ca chinh, vung bi^'n ddi T4. nhdm vi nit gay ho^v ta tb,in kinh ca chinh cbiuAu.
LBATVllNDE
B^nh hoai tu thin kinh (Nervous necrosis disease) do vi rut gay hoai tu thin kinh (Nervous Necrosis Virus - NNV) la benh cip tinh, xuit hidn tren 22 loai ca, trong dd cd cic loai: mii (Epinephelus
^.), ca chinh (Anguilla anguilla), chdm (Lates calcarifed. gid (Racycentron canadum). Bdnh chu yeu tr^n ca gidng, ty le chet cd the len den 90 - 10096.
Hau het nhung nghien ciiu ve NNV ddu cho thiy:
mo dich cua NNV l i hd thin kinh trung uong (gdm nao va tuy sdng) vi vdng mac [5]. Vi nit niy giy hoai ti cac noron than kinh dan den nhiing bieu hidn bat thuong nhu boi khdng dinh hudng, chii yeu theo hinh tron 6c hoac lao diing, nhanh ve phia trude [5].
NNV dupe xic dinh loii dua vio trinh tu ciia viing bien ddi nim tren phan tu 2ARN ma hda protein VO ciia vi rut. Tai Viet Nam, vi nit nay da duge xic dmh tren ca mil [l], Trong nghien ciiu nay, NNV diroc kiem tra tren cic miu c i chinh cd trieu chiing lam sang cua benh hoai tii than kinh.
INGUYEN UEU VA PHUONG PHAP NGHEN CUU 1. Nguydn li^u
- 30 mau ca chinh cd tridu chiing dien hinh cua benh hoai tu than kinh.
2. Phuong phip nghidn ciiu a Phuong phap xuly miu
Thu nao nghi mac b^nh hoai tii than kinh. Mot phan diroc su dung lim tieu bin quan sit md benh hoc, phan con lai duoc diing de phan lap vi nit.
jKhoa Cong ngh? Sinh hpc, Vien fiai hoc Md Ha Npi J Bo Khoa hoc va Cong ngh?
^ Khoa Nong Lam Ngu, Trirdng D^i hpc Vinh Tnrong D^i hpc Ndng lam Hue
Nghidn cic md nay trong dung dich mudi can bing Hanks (HESS) vdi ti le 1:10. Sau dd ly tam d 10.000 vdng/phiit trong 15 phut, d 4''C. Djch ndi dugc hiit ra va Ipc qua mang loc 0,45 pm. Dich loc duoc cat trong tu Ignh cho d^n khi diing de phan lip vi nit tren td bao.
b. Phuong phip kiem tra md bdnh hoc Md mit va nao c i nghi mic bdnh hoai tu than kinh dupe cd dinh trong dung dich Davidson trong 24 gid. Sau dd. miu duoe lam mat nirdc bing con 95%
trong 4 gid, cdn 100% trong 4 gid, lim mSm bing metyl - sahxilat trong 12-24 gid. Ngam mau ti-ong dung dich parafm ndng chiy b nhidt do 65''C trong 10 gid rdi diic miu. Cic miu sau khi diic sg dugc cit lit mdng 5 nm. Nhudm miu bing dung dich hematoxiclin (hdng cau) va eozin, din miu bing bomcanada. Quan sat tieu ban h-dn kinh hien vi quang hpc.
c. Phuong phip nudi ciy ti'bio
Ddng te bio GS (Grouper Spleen- Td bio ddng lich ca mu) cung cap bdi Cdng ty Invitrogen duge nudi cay trong mdi tnidng Leibovitz L15 bo sung 5- 10% huyet thanh bio thai bd, 0,1% L^glutamin- penixilin - sti-eptomyxin Onvitrogen). Te bio duge nudi ti-ong chai nhua cd nut van the tich 25 cm^ cho den khi te bao mgc tiiinh mdt lop rdi bo sung dich benh pham. Nudi ciy vi nit d nhidt dd 25-30°C, hing ngiy thay mdi trudng nudi cay te bao v i quan sat CPE.
d. Phuong phap xic dinh lidu giy chet 50% te bio (TClDso}
Te bao GS dupe nudi cay de dat mat dp khoang 5xl0^/ml trong dia 96 gi^ng, rdi bd sung 20 pi dich vi nit duoc pha loang d cac ndng dd khic nhau theo hd
NONG NGHIEP VA PHAT TRIEN N 6 N G THON - KY 1 - THANG 6/2012 71
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so 10. Giir dia nudi cay trong tu i m 37*'C trong 1 gid dc vi nit hap phu len td bao i ni licp tuc bd sung 80 j.il mdi li-utmg Ix'ibovif 1,15 5-10% luiyet Ihanh bao thai.
Nudi cay vi nit b nhid do lirvliO'C. hang ngj'iy thay mdi tnnmg nudi c.iy te bao va qu;m sat CPI']. TCIDSO duoe xac dinh theo cdiig thiic cua Reed vn Miicnch (1938).
//. PInitmg phnp PT-PCR
ARN long sd ciia NNV dinn- tach bing kil Quick Prep Total RN.A, Isolation (Pharmacia bioledi). ADN l)n suiij,' i\\nK tao thanh tir |)han img KV- PCR vin bp kit RT-PCR O'haniiacia biotech) v;i c?ip mdi i'CR F2 - R3 (Invitrogen) Phan img dupe thuc hiCn vdi 30 cliu ky thoo cac chu trinh nhid 90'C/5 phiit, 55"C/-l.'t giay, 72"C/1 phiit. Sau do. pli.iii img dune tidp luc (7 72"C/10 phiit.
• . K E T QUA VA THAO LUAN
1. Ki^m tra md bdnh hpc v i phin lip vi nit trdn cic mau c i chinh nghi mic bOnh h o ^ tu thdn kinh
Trong nghidn cim niy da thu thap 30 mau ca chinh nudi dvung bien midn Trung cd cic birii hidn:
boi bat Ihmmg theo hinh trdn dc hoac boi khdng dinh hudng, cac trieu chimg lim sang nay diroe nghi la do vi rut giy benh b ca quan than kinh gay nen.
Ket qui chin doin md benh hoc vcVi md nao c i cho thiy: 2 mau ky hieu A-HUlOl va A-HU117 cd xuat hien khdng bio va the vui b nao vdi mat dd day dac (hinh 1). Su cd mat cua vi nit ciing nhu dang giy tdn thuong b td chiic thin kinh ciia 02 mau benh pham budc dau cd the ket luan li ca mdc benh do vi nit hoai tu thin kinh.
Cic mau cdn lai tuy khdng phit hi^n khdng bao nhung khdng nim ngoai k h i nSng dang df thdi ky tien nhidm. Do dd, da tiep tuc tien hinh phan lap vi nit tir cic mau c i niy bing t^ b i o GS. Kdt qua Amrc trinh bay trong hinh 1.
Hi0n tuf.mg vi nit giy bdnh tren te bao nudi cay dupe dinh gii bang chi sd hidu img huy hoai td bao (CPE) (hinh 2) Sau 6 ngiy nudi cay, nhiing dau hieu CPE bii dau xuat hidn b cic gieng te bao gay nhiSm 04 miu A-llUlOl. A-HUI06, A-HU112 v i A-HUI17.
IIi<'n lu(,mg quan sat i\myc trdn dia te bao nudi cay la su bidn dni hinh dang cOa mdt sd td bio tir dang thudn (lai sang tnin. ddng Ihiri cd cac dam te bao tap trung, CO cyni. Sang ngay ihir 7, hidu img hiiy hoai te bio bi(-ii hiin n> hon bdi su xuat hidn nhimg diem tan liao, nluiu te bii(» crtng phdng, bong iroc khdi bd xniA giong nudi cay. Cho di-n ngiy thir 9, didn tich bi t;in bao (> hai man A-MUlOl va A-HU117 len tin 95%, cdn d hai mau A-Hl'IlKi va A-HUI12, di^n Uch lan bao klioing 20^ nhimg phin Ion c i c te bao nudi cay bi bicn dang. Cac bien ddi benh Uch tren te bao GS ciia vi rut phi'm lap dufa" tir ca chinh li tmmg tu voi nghien cuu tnroc day ve dac diem benh Uch ciia N.W gay bdnh hogi tir thiin kinh tren c i mu (Ij. Chuan dp TClDi,/niI cua cac mau A-HUIOl, A-HUI06, A-HU112 va A-HUI17 lin Imrt la 10*', 10- '. 10^^. 10^^
Nhu viy, 01 m i u c i chinh ky hieu A-HUIOl, A- HUI06, A-HU112 va A-HUI17 bi nhiem vi nit, 26 miu bdnh pham cdn lai khdng g i y benh tich te- bio va cd the kit luan cic mau nay khdng cd Betanodavirus.
'A . ; - - ' • ' ' • • ' . . * . . •
Hmh 1. Hinh inh kl^m tra md b^oh hpc ctia nao c i b^nh p h ^
> - H - . . l . , . > ^ V ' - " • " ' '
:.• *"
' - • > , • - . v ^ V ^ • t ^ • - , , .
• • • . ti'. li' -. •. J • * '' h : -' Hinh 2. BOnh tich cua ti bdo GS sau khi gay nhiem
cAc raSu ci b^nh
2. Xdc dinh lodi vi nit giy b§nh trin ci chinh b i n g phirong phdp xdc djnh trinh tii gien ddc hi^u
Ciiu tnic genom {hi gien) ciia NNV gom 02 phan tu lARN vd 2ARN, trong do lARN ma hoa enzym ARN polymeraza co tinh ddc tnmg cho ho Betanodavirus. Con 2AIW md hoa glycoprotein cau tao v6 VI nit mang tinh dac tnmg cho timg loai NNV.
Doan gien nay co kich thuoc 1,410 bazo, trong do chiia mpt viing bao thu co trinh tu xdc dmh 93* va mdt viing bien ddi cd trinh tu xac dinh la 62%. Trong nghiSn ciiu ndy, dd xdc dinh trinh tu viing bien ddi
72 N 6 N G NGHIEP VA PHAT TRIEN N O N G THON - KY 1 - THANG 6/2012
KHOA HOC C 6 N G NGHE
T4 cua gien m a h d a p r o t e i n vd c u a 0 4 c h u n g vi riit
C§p moi F 2 ( 5 ' - C G T G T C A G T C A T G T G T C G C T - 3')vaR3 (5'-CGAGTCAACACGGGTGAAGA-3') d u p e su dung d e t i c h d d n g d o g n g i e n T 4 k i c h t h u d e khoang 400 b p d 0 4 c h u n g vi n i t k y h i d u A-HUlOl, A- HUI06, A-HUI12 v a A-HUI17. C i c u-jnh tir gien d u p e xac dinh bdi h e d i d n g giai t r i n h t u ABI 3110 PRISM (Perkin- Elmer) d d n g t h d i t h a m k h i o 0 3 U'inh t i r g i e n sin cd CLia G e n e B a n k ( b a n g 1) d e s o s a n h m d i t u t m g quan cheo ve m a t di t r u y d n c u a c i c c h i i n g c h o k e l qua nhu sau:
Bang 1. Mdi q u a n h d c h d o v ^ t y Id t r i n h bf g i e n T 4 ciia 04 chiing vi n i t p h i n l i p tir c i c h i n h n g h i m i c
b d n h h o a i tii t h i n k i n h
»-Hir«
A- HUj)
' • " • '
A- A
HLi;;
A ENN\ GNN\-
« n i : S" ; j '
« n i " i.' ;•:< ;•
EOT « ?i * « Gwv r s" ' ' •' ^•
i v n •; -1 -4 -J 60 s :
Ghi chti:. A-HUIOl, A-HUI06. A-HU112 va A- HU117: cac chting vi nit phin lip tti ca chinh nghi ink benh hoai tti thin kmh: ENNV: European Netvous Necro.sis Virus (Chi, va cdng sir, 2003);
GNNV; Grouper Nervous Necrosis Virus (Chi va mngsu, 2003): JFNNV: Japanese Flounder Nervous Necmsis Virus (Nishizawa va cong str, 1995).
Sd lieu trong bang 1 the hien trinh tu cua vung bien doi T4 tren 2A1?N cua 04 chung A-HUlOl, k- HUI06, A-HU112 va A-HUI17 la tuong ddng tuyet ddi, cac trinh tu nay cQng cd mdi tuong dong cao (99%) doi vdi chiing vi nit gay benh tren ca chinh chau Au tMnropean Nervous Necrosis Virus) trong khi dd gia tnnay thap hon (6082%) khi so sanh vdi 02 chung ('fouper Nervous Necro.sis Virus va Japanese floaaderNervous Necrosis Virus.
B i l g p h u o n g p h a p U P G M A ( U n w e i g h t e d Pair Group Method with A r i t h m e t i c M e a n ) ( I M u r t a g h . IW) ii xac dinh c a y p h a n loai, 0 7 c h u n g s o s a n h
% chia thanh 03 n h d m , t r o n g dd 04 c h u n g A- KJIOl, A-HUI06, A-HUI12 vd A-HUI17 p h a n l,ip d u o c w ca chinh duoc x d p v a o n h d m European Nervous
* i T O S K K r a s ( H i n h 3 ) .
Hinh 3. Cdy p h d n lodi d u a trSn trinh t y viing T 4 cua c d c c h i i n g N N V phdn Idp W. KET LUiiiy
- Kiem tra m d bdnh p h a m 30 m a u ca chinh nghi m d c b d n h hoai t u thdn kinh d a phdt hien 02 m a u cd b d n h tich d m d nao.
- Kiem tra b a n g p h u o n g p h a p gay nhidm U-en te b a o m a n c a m d 3 p h a t hidn 04 mdu gay b e n h tich te b a o vdi bieu hien t u o n g t u vdi bieu hien b e n h tich d o N N V gay r a t h e o c a c n g h i e n ciru trude day.
- T h d n g q u a trinh t u gien T 4 ndm h e n 2ARN d a xdc dinh d u o c Ot c h u n g vi n i t phan lap thuoc n h d m European Neivtws Necrosis Vans.
TAI UEU THAM KHAO
1. P h a m T h i T.im, P h a m C o n g Hoat (2011). P h a t hien, p h a n lap va x a c dinh m o t sd dac tinh cua vi rut g a y b e n h hoai tir than kinh {Nervous Necrosis Virus) t r e n c d n u i t u n h i e n tai viing bien Q u a n g Ninh. T,^p chi Ndng nghiep va PTNT, ISSN 0866-7020, sd 20, 2011.
2. Chi S. C , Shieh J. R , Lin, S. J. (2003). Dis.
Aquat. O r g . 55 (3), 221-228.
3. I M u r t a g h F . (1984). ComplexiUes of H i e r a r c h i c C l u s t e r i n g Algorithms: t h e state of t h e art. Computational Statistics Quarterlyl: 101-113.
4. Nishizawa T , M o r i K., F u r u h a s h i M., Nakai T , F u n i s a w a 1. M u r o g a K. J. (1995). Gen. Virol 76 ( P T 7 ) , 1563-1569.
5. Renault, T , Haffner, P., Baudin, L. F., Breuil, G., B o n a m i , J. R., 1991. M a s s mortalities in hatchery- r e a r e d s e a b a s s iMtes calcarifer larvae associated with t h e p r e s e n c e in t h e brain a n d retina of virus-like particles. BuU. Eur Assoc. Fish Pathol 1 1 , 6 8 - 7 3 .
NONG NGHIEP VA PHAT TRIEN N 6 N G THON - Kt 1 - THANG 6/2012 73
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IDENTIFICATION VIRAL NERVOUS NECROSIS DISEASE IN Anguilla anguilla FARMING IN CENTRAL OFVIErnMAM
Phara Thi Tam, Pham Cong Hoat, i.guyen Thi Vui, Nguyen Thi Thanh, Nguyen Quang Linh
Summary
04 strains of vims infected In neurvous central system namely A-HUIOl, A-HUI06, A-HUI12 and A-HUI17 were detected from 30 samples of Anguilla anguilla collected in Central of Vietnam, these strains also can be innoculated to GS cells. By nucleoUde sequencing method to identiiy sequence of variable region T4 belong to RNA2, all of four this trains were identical in European nervous necrosis w/r/s genotype.
Keywords: Anguilla anguilla, European nervous necrosis virus, genotype, variable gene T4.
Ngudi phdn bifn: PGS.TS. Nguyfa VIA Khdng Ngdy nhjn bdi: 12/03/2012
Ngdy thdng qua phin bi$n: 12/4/2012 Ngdy duyf t ding: 20/4/2012
N O N G NGHlgP VA PHAT TRIEN N O N G T H O N - KV 1 - THANG 6/2012