. JOC CONG NGHE SINH HOC T O A N Q U O C 2013
NGHieN CO'U LyA CHON CHONG VI ROT PHUC VU SAN XUAT VAC-XIN
P H 6 N G BENH HOAI TO* THAN KINH CHO CA MU NU6l TAI VIET NAM Pham Thj Tam'', NguySn Thj Thu Hien\ Bilii Thj Hdi H6a\ Ph^m Cong Hoat^, Nguyen Quang Linh^'Wpn £>?/ hgc Md' Hi Ndi 'BC Khoa hgc va Cdng ngh$
' l^oa ThOy sin, tnrang D^i hgc Ndng lim HuS TbM TAT
B?nh ho^i tii than kinh {Nervous necrosis disease) li. benh gay t6n that n|ng nc cho ngh€ nuoi cd mii tren th6 gidi n6i chung va Vigt Nam n6i riSng. Phong bSnh bing v3c-xin ii giai phap hihi hieu de lam gidm thi€u thiet h^i ciia b ^ . De san xuat dirg'c v^c-xin phong benh hi?u quA cho c4 mu nuoi it Vi?t Nam, can phai c6 dupe chung vi rut vic-xin phan I§p trong niroc. Tu 26 chung NNV phan ISp 6 cdc tinh Quang Ninh, Hdi Phdng, Nam Binh, Khanh Hoa, Binh Thuan, bing phuang p h ^ trung hoa d3 xdc dinh dirge khdng nguyen ciia chiing KH05 tuong dSng v6i khdn^ nguyen ciia 25/26 chiing NNV phan Idp. Kifim tta khd nang t?io ddp iing mi§n dich ciia chung NNV KH05 cho thiy: virtitbdt hoat bang BEI vd beta-propiolacbme voi n6ng dO tuong ang la 0,002M vd 0,1% co th6 gdy bat ho9t hodn todn NNV nhung vdn ddm bao tinh khang nguyen ciia vi rUt. Vi nit KH 05 bit ho^t c6 khd nang tao d ^ iing mien dich trfin tho vdi hi§u gid khdng thi la 1/128, tuy nhien, khi bo sung chit b5 trg la AP, hi?u gid tdng len gap 64, khi bo sung AH hi^u gid tang len gap 32 ldn. Ve khd nang t ^ khang tbk tnmg hoa tren ca rnii b$t, hiSu gid khdng Ac tmng h6a 6 edc 16 cd dil n g h i ^ dfiu dat cao nhdt sau 30 ngdy ^ y iniin djch; vi nit v6 hoat tao khdng thk tmng hda vdi hi?u gid dat 1/8; khi bfi sung vdi AH, khang the tnmg hoa d ca tfing gap 16 ldn; b6 sung vdi AP, khans 4 6 trung hoa d cd iang gip 3 2 1 ^ . Phuong phdp cong cudng d ^ da chiing minh ring, chiing NNV KH 05 c6 khd nang t^io khdng tiiS bdo hd cho cd mil khi c6 chit bo trg AH ho|c AP, ty IS bdo h§ cd thi nghi?m d9t 75-88,33% vdi Uiu cong cudng dgc Id 0,2xTCID5o, 0,5xTCIDsova IxTCXDjo (TC1DSQ=10*^). Nhu v|y, chiing KH 05 c6 khd nang s'i dung ldm chiing vic-xm.
Tu khoa: Bgnh hoai tii diin kinh, chung vdc-xin, vi nit vd boat, chat bd tig keo phen, khang the d|c h i ^ khang th^ trung hda, ty 1?
baohO Md'BAU
B§nh hogi ti> thin kinh (Nervous necnjsis disease) do Nenmus Necnjsis Virus- NNV la b?nh dp tlnh gay ra trfen nhilu lodi d bien. trong d6 c6 c6 mO (Phgm Thj Tam ef at., 2011). Bgnh hogi ti> than kinh ft c^ mO ttiu'd'ng xuat hi?n trong giai dogn cd gi6ng, vd'i t^ 1$ nhiSm ISn tdi 53/64 miu thu thgp {bjang irng 82,8%), ty 1$ ca ch6t do NNV khodng 90-100%
(Trin Vgn Hi'ch ef al, 2012). Hiiu hit nhu-ng nghifin ciki ve NNV deu cho thiy: m6 dich ci^a NNV la he thin kinh tnjng uwng (gom ndo vd tuj s6ng) vd v6ng mgc (Hegde et al., 2002). Vi nit ndy gdy hogi tCr cac noron than kinh din a^n nh&ng bieu ht§n b i t thuang nhu boi khfing djnh hu'dng, chu y l u theo hlnh trfln oc ho§c lao thing, nhanh vi phia tru'dc.
Vi§c ph6ng b#nh hogi tCr thin kinh (HTKT) duvc thyc hi^n theo 2 hu'dng: logi tri> con gi6ng mang mam b^nh vd sd dung vic-xin (Hegde ef ai.. 2002). Ct Vi§t Nam, hi$n tgi chu^a c6 vic-xin ph6ng bgnh HTTK, vt viy thit thoat trong khSu tgo gifing cd mii Id rat ldn (Tran Van Hich ef al., 2012). Sdn xuit vic-xin phflng b#nh Id giai phap hO'u hi§u d l ldm gidm thi^t hgi do bgnh gdy n§n. Nghi§n cdu ndy dirge thu'C hign nhdm muc dich xac ^nh chiing vdc-xin td cdc chung phdn Igp trong nu'dc vd ddnh gid khd nang gdy ddp dng miln djch de lam ca sd cho san xuit vIc-xin phong bfinh HTTK.
NGUYEN LI0U VA PHUtTNG P H A P Nguydn li$u
Cdc chOng vi njt gdy bfinh HTTK 6irgc phdn ldp td cd mCi mic bfinh tgi cdc finh Quang Ninh, Hdi Phong, Nam Djnh.
Khdnh Hoa, Binh Thugn. Hda chit, sinh phim chin doan sd dyng trcing nghifin cdu ndy diu dug'C mua td hang Sigma- Aldrich, Merck, invitrogen, Pierce, Thermo Science.
Phirang phap Nudi ciy vi nit tren te bio
NNV dirg'c nudi bang t l bao GS 1 (grouper spleen). R l bdo 6irgc hogt hoa trong mfli tnrdng Leibovitz's L-15 (L 15) cd 10% huyet thanh bdo thai bfi (PCS). Dieu kifin nufii t l bao: 27°C, 5% CO2, 2 ngdy (mdt dd 10^ t l bdo/ml). Gdy nhiem NNV vdo chai nuoi t l bdo GS 01. Ctiai d6i chdng khdng gdy nhilm NNV. Theo doi hifiu dng huy hoai t l bdo (CPE- cytopathic effect) mfii ngdy. Khi CPE dgt 90-95% thu djch vi mt khoi chai nufii cly rfii ly tdm thu djch noi cd chda vi nit Chuin dgvirOt
TCID50 (Tissue Culture infective Dose, 50%): Chuin dO vi nit du'oc thirc hifin tren dTa nhya 96 gilng nuoi te bdo GS 1.
Djch vi njt duyc pha lodng theo hfi s6 10 trong mdi tnjdng Hank's. Vi rut duvc hap phy vao t l bdo trong 2 gid. Tilp tyc nufli t l bdo bdng Leibovitz's L-15 cd 10% PCS. Quan sdt CPE trong vong 5 ngdy d l xdc djnh TCIDso theo phuwng phap cCia Reed vd Muench (Reed et al, 193S)
Phuvmg phap giy bit ho^t vi rut
Bat boat blno formalin (formaldehyde 35%). fonnalin ducrc bo sung vao djch vi nit de dgt nong dp 0,1 va 0,2%. H6n hgp duvc d d 37''C/ 24 gid. Fonmalin du'O'c logi khfii dich bat hogt bang cdch tmng hoa vdi PBS trong 12 gid.
MM' N^JHI f \ n u A t-iyu U U N U N I J H I ; bINR
12 gid. '
3rcM
Bit hoat bdnn Binary ethvlenlmine (BED: BEl 0,1 M bo sung vdo djch vi rut d l dgt ning dfi 0,001 va 0,002 M. HSnhjp di/^rc u d S/'C/ 36 gid. BEI duvc logi khdi djch bit hogt bing cdch tmng hda vdi 1 M Na-thiosulfate trong 12 gid.
Bit hoat bang beta-oropiolactone. chinh pH ciia djch vi nJt 7,3, b l sung bfita-propiolactone theo 19 Ig 1 -2000 {\
hon hgp bdng may khuly td trong 24 gid/4°C. Bfita-propiolactone diKTc logi bd bing cdch chlnh pH v l 7.0, ZT Phu-ong phap RT-PCR
ARN tong so cua NNV duoc tach bdng kit RNA extraction (Qiagen). Gen d$c tM#u T4 cua NNV ducrc x*c djnli.baiiebt kit RT-PCR (Invrtrogen) vd cgp m i l PCR F1-R3:(5'-GGATTTGGACGTGCGACCAArS; 5.
CGAGTC/^ACACGGGTG/V\GA-3') (Chi e( at.. 2003). Phan dng duvc thuc hifin qua cac giai dogn: RT: 50*^30 pni^
PCR' 30 chu ky theo cdc chu trinh nhi§t: 94''C/5 phut, 60''C/45 gidy, 72°C/1 phdt; 72°C/5 phiit. i Phirong phap trung hda tren ti bio
Phdn dng tnjng hda vi nit NNV iirgc thyc hifin trfin t l bdo GS01 theo hudng dan cua Vinalogy methods manual (Bha e(a/., 1996).
Phtrong phap giy mien djch
Phuang phap tgo khdng the dgc hifiu tren thd dugc Vngc hifin theo phuong phdp cua Yoshihito (Yoshihfto Kihiwaidii 2002)
Pftan img ELISA giin tiep (Yoshihito Kihrwaraki, 2002)
Phdn dng ELISA xdc dinh khdng t h i dgc hifiu duac thyc hifin theo phuang phdp cfla Yoshihito (Yoshihito Kihiwaalj, 2002)
Phuvng phap xic djnh muc dift hrong dong khing nguyen 1 Mdc dfi tuong dong khdng nguyfin duvc ddnh gid trfin ca sd thyc hifin phdn dng tung hoa gida cdc vi njt phdn I ^ J
cdc miu huyet thanh thu duvc td thd duvc gay miln dich vdi cdc mlu NNV bit hogt vd khdng the da dflng khdng NNM [Pierce, My). Dfi tuang dong khdng nguyen 'ri' duac tinh theo cfing thdc cua Rweyemamu vd Hingley (Rweyemamui chiing vi nit khfing cd tinh tuong dflng khdng nguyfin.
K^T QUA vA T H A O LUAN
K i t qud xac dinh chiing dai difin khang nguyfin
26 Chung NNV phdn Igp duvc trfin ca mii mac benh HTTK d cdc tTnh Qudng Ninh, Hdi Phdng, Nam Djnh Khdnh Hda vd Blnh Thugn dug'C sd dung d l gSy dap ung miln djch trfin thd phuc vy myc dich lya chgn dugi Chung dai difin khdng nguyfin d l sdn xuit vac-xin. Bang phuang phdp trung hda vdi khang t h i khdng Mil chuin cua Piece, My, chiing tfii da xdc djnh duvc 08 chung NNV phdn l^p bao g i m : QN 02, QN 04, KH 05. HPM HP 04. HP 07, KH 07 va KH 09 duac phdt hifin dac hifiu bdi khdrig t h i ndy. Ddnh gid tinh tuang ding khjfl|
nguydn ciia 8 chung ndy vdi 26 chung con Igi d l lya chpn chiing vdc-xin da thu dug'C k i t qua trong Bdng 1.
Bang 1. H# so tuomg dong khdng nguyen cua cdc chung phdn lap VrnJt
QNZ QN4 HPZ HP4 HP6 HP7 HP8 HP10 MD1 ND3 ND4 NQ5 ND7
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0 0 I 0 ' 0 ' 0 K i t qud trong Bdng 1 cho thiy: chung KH 05 cfi tinh tuong dong khdng nguyfin cao nhlt, dat 25/26 chung F**Igp. Chiing nay se ducrc su dyng d l phyc vy cho cac nghifin cuu t i l p theo d l sdn xuat vac-xin phflng b ^ hogi td than kinh cho ca mu nudi trong nude.
K i t qua gdy bat boat NNV bang hoa chat
Chiing NNV KH05 duac nuoi cly trong te bao GS01 rli chuin dp d l dgt liiu TC1D50=10". Vi nit duiTc xd 1^ bit ho?t*
3 loai hda chit Id fonnaldehyde. beta-propiolactone vd BEl vdi cdc ndng dfi tuang dng la 0,1% vd 0,2%; 0.05%«*
: CONG NGHE SINH HQC TOAN QUOC 2013
0,1%; O.O01M vd 0.002 M. Sau khi b i t hogt vi njt duvc gdy nhiem trd lgi trfin dTa ^ bdo GS 01 d l ddnh gid mdc dfi bat hogt thfing qua sy hinh thdnh tifinh tich t l bao (CPE) va tgo t h i vdi trong te bdo bang phdn ung RT-PCR phdt hign gen ma hda protein vd oua NNV. K i t qua duvc trinh bdy trong Bdng 2.
Bang 2. K4t qud dinh giA mi>c dd bit hogt cua NNV
Cdc chl lifiu theo doi Mirc d$ hiiy hogi t6 bdo (CPE%) GenT4
Ndng dfi form^in 0 , 1 %
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30
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Kit qua trong Bdng 1 cho thiy: cdc hda chit formalin, beta-propiolactone vd BEl diu cd kha nang gay bit hogt vi nit NNV. cdc ning dd gdy bat hogt vi nit tuang dng Id 0,2%, 0,1% va 0.002 M.
v l n d l ddt ra Id, cd ba loai hfia chit formalin, beta-propiolactone vd BE) diu gdy b i t hoat NNV nhung cd anh hudng den khd ndng tgo ddp dng miln djch trfin cd hay khdng? D l tti Idi cho cdu hdi ndy, thf nghigm Sirgc bS tri nhu sau:
Cd mii bfit cd k(ch thudc 1,5cm dug'C chia thdnh 04 Id. moi id 100 con, trong dfi: Id 1 dug'C gay mien djch vdi NNV bit hogt bdng formalin nong dfi 0.2%; Id 2 dirge gdy miln djch vdi NNV bat hogt bdng BEl ning dd 0.002 M; lfi 3 dug-c gdy miln djch vdi NNV bat hogt bing beta-propiolactone nong dp 0.1%; liiu gdy miln djch cho 3 Id ndy Id: 0,2ml, TCID50=10^'^/ml, lfi 4 (dli chdng dm) khfing gdy miln djch vdi NNV. Mlu cd mii duac thu 6 dot, moi dgi lly nglu nhifin 5 cond cdc ngdy 0,10.15,30,45, 60 sau khi gdy miln djch. Cdc mau ca dug'C nghien trong dung dich PBS rli lytdm, tdch dich chilt de xdc djnh khdng t h i trung hda. Ket qua dugc trinh bdy d Hlnh 1.
Hl$u gid khdng t h i trung hda NNV cudng dfic thay dli theo xu hudng tang din qua cdc ngdy sau khi gdy dap dng miln djch vd dgt gid trj cao nhlt d ngdy thd 30 (logi tru Id doi chdng). Hifiu gid khdng the tmng hda tgo ra tu vi nit bat hogt vdi BEl vd beta-propiolactone luon cao han 4-8 lln so vdi vi nit bit hogt vdi fonnalin. Qua kit qud trong hlnh 1 vd bdng 2, formalin cd khd ndng gdy bat hogt vi nit nhung ldm gidm mdc dp tgo ddp dng miln d[ch d cd. Bfin cgnh dd, vi nit duvc bit hogt boi beta-propiolactone tgo ddp dng miln djch mgnh vd I n djnh han BEl. hifiu gid khang t h i d lfi cd gay miln djch vdi vi rdt bit hogt bdi beta-propiolactone dgt d mdc dg cao nhlt td ngdy thd 30 din ngdy thd 45 sau khi gdy miln djch, trong khi dd, hifiu gid khang t h i d Id d lfi cd gdy miln djch vdi vi nit bit hogt bdi BEl chl dgt d mdc dfi cao nhlt d ngdy thd 30 vd gidm di mfit nda td ngay thu 45. Mdc dii vdy. din ngdy thd 60, hifiu gid khdng t h i d cd hai lfi dgt bing nhau. bing 1/128. Thi nghifim ndy cht ra ring, BEl vd beta-preipiolactone vdi n i n g dfi tuang dng la 0,002M vd 0,1 % cd the gdy bit hogt hodn todn NNV nhung vln ddm bdo tlnh khdng nguyfin ciia vi nit.
300 2S0 200 130 100 50 0
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Hlnh 1. Ket qui xic dfnh khk ndng tpo d&p ung mien djch cua NNV bat hoat Xdc dinh khd ndng tgo ddp Ong mien djch cua vi rdt vIc-xin KH05
Trong thi nghifim ndy, chOng vIc-xin KH05 bit hogt bdi beta-propiolactone 0,1% duac phoi hgp vdi cdc chit b l trg- Id aluminum hydroxide (AH) k^ hifiu KH 05 + AH vd aluminum phosphate (AP) k^- hieu la KH 05+AP.
Kef qui xic dinh khi nang tfio khing the d^c hieu ciia vi rut vic-xin KH05
Khd ndng tgo khdng t h i d^c hifiu cCia vi rut NNV KH 05 dugc thf nghifim trfin thd dug-c tifim 0.5ml vac-xin vdi liiu TCIDso= lO'^'^/ml, sau hai l l n tifim, tgi ngdy thd 25 mdu thd duvc l l y de tdch huylt thanh, thuc hifin phdn dng ELISA d l phdt hifin khdng the ddc hifiu. Khi khong su dung c h i t bb tro, vi nit KH 05 b i t hogt cung cd kha ndng tao ddp dng miln djch trfin thd vdi hieu gid khdng t h i Id 1/128. Tuy nhifin, khi b l sung chit b l tro la AP, ddp dng miln djch ciia thd vdi vi riit vac-xin manh nhdt, hifiu gid khang t h i tang Ifin g i p 64 l l n so vdi lfi tifim NNV b i t hogt dan thuin; lfi vi nil vIc-xin bo sung AH, hifiu gia khdng t h i tang Ifin g i p 32 l l n so vdi 16 tifim NNV b i t hogt (Hinh 2).
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Hinh 3. Hl|u gia khdng the trung hda cua c i d i f ^ x u I f vd'i vi rut vac-xm vdi cac chat bo trg- khdc nhau Hinh 2. Hi^u gid khang the trong mau tho dirov tifim
NNV vd hogt vdi cac chat bo tr? khdc nhau (ELISA ODts6>0,e)
Ket qua xac dfnh khi nang t§io khing thi trung hda vi khi nang bio h^ ciia vi rOt vic-xIn KH05 Thi nghifim xdc djnh khd ndng tgo khdng t h i trung hda dug'C thyc hi|n trfin cd mii cd kich thudc 1,5cm. Sau khi gjy miln djch 10, 15. 30. 45, 60 ngay, bat nglu nhifin cd d cdc lfl th( nghiem, nghien nhd, thu djch chiet Idm phdn ung trung hfia vdi chdng NNV QN02 (TCID^=10^'^) roi gdy nhiem d Igi tren te bdo GS01. 0 lfi ddi chung cho thiy, trong c^ khii^
cd khdng thi trung hda vi nit gdy bfinh HTTK. Nhin chung, hifiu gid khdng t h i tmng hda cdc Id cd thi nghifim deu (^
cao nhlt sau 30 ngdy gdy miln djch. Vi nit vd hogt cfl ddp dng tgo khdng ^ 1 tmng hfla nhung d mdc dfi thip (HGKT dgt 1/2-1/8). Tuy nhifin, khi cd chit bo trp, khdng t h i tmng hfla d cd tdng gip 8-16 lan, dgc bifit, sau 30 ngdy su dyngif nit phii hap vdi AP, hifiu gid tmng hfia tdng gip 32 lan (Hinh 3).
Thf nghifim ddnh gid khd ndng bdo hfi cd vdi bfinh HTTK ciia vi nit KH05 dupe ddnh gid bang phuong phdp cflii||:
cudng dfic. Sau khi gdy bat hogt. vi nit KH 05 dupe phii trfin vdi AH AP rli gdy miln djch cho cd. Tgi ngdy thd 30,d!
dugc phdn lfi vd cfing cudng dpc vdi chGng NNV QN 02 (TClD5o=10"'^) vdi cdc lieu: 0,2xTCID5o, O.SxTClDso. IxTClDnj 2xTClD5o. 3xTC!Dso, 4xTClD5o, Lfi dli chung, cd khflng duvc gdy miln djch vdi b i t ky loai khdng nguyfin ndo nhunuj vln cong cudng dfic vdi liiu nhu tren. Tilp tuc theo doi cd trong 15 ngay d l ddnh gid mdc dfi bdo hd TJ 1$ bdoh) (RPS%) dugc tinh bdng cdng thdc: 1 - (ty lfi chit d lfi thi nghiem-ty lfi chet d 16 dli chung am)/ty lfi chit d lfi dli chiin duang).
Hinh 4. So sanh mdc do bao hfi tren ca duvc gSy m i l n djch vdi NNV KH05 vo hoft c6 ch3t bS try Ithdc nhau Vi nJt bit hogt dupe b l sung AP vd AH co the tgo khdng the bao vfi td 75-83.33% ca thf nghifim vdi liiu cflng dfic la 0,2xTCID5o. O.SxTClDso, IxTCIDso.Ty lfi bdo hfi d cac 16 ca dupe cong cudng dfic lieu 0,2xTCID5o va 0,5x1- la tuong duong nhau, tuy nhifin khi cfing cudng dpc vdi liiu IxTCIDso, ty lfi bdo hd d 16 cd duac gay mien djch v«
bdt hogt bo sung AP cao hon so vdi 16 ca dupe gdy mien dich vdi vi nJt bit hogt b l sung AP Cde kit qud ndy cho
- K; C O N G N G H ? SINH Hpc TOAN QUOC 2013
g e l t ludn r i n g , k e o p h f i n la chat b l t r p phu h p p d l s a n x u i t vdc-xin b i t hogt phfing bfinh HTTK. K i t q u d nay p h u h p p v d i
^ H nghifin c u u c u a J i a o e t a l , 2 0 1 0 v d V i n a y et a l , 2 0 1 3 . K ^ T L U A N
Chting virus g d y bfinh H T T K K H 0 5 d u p e l u a chpn t u 2 6 chung v i nat phdn lap d u p e trfin c d m i j m i c b e n h d cdc finh Qudng Ninh, Hdi P h d n g , N a m D j n h , Khdnh Hda, Blnh T h u g n . Beta-propiolactone 0 , 1 % gdy b i t hogt N N V n h u n g k h & i g dnh h u d n g d i n tfnh k h d n g nguyfin cda vi nit. D e cfi c o s d s d n x u i t vac-jun phflng bfinh, vi n i t KH05 b i t hogt d u p e b l sung thfim c h i t Ijo t r p A H , A P d l d d n h gid k h a ndng g d y d d p d n g mifin djch. K h i c d c h i t b l t r p , h i f i u g i d k h a n g t h i d g c hifiu t d n g Ifin g i p 3 2 v a 6 4 l a n . t u o n g u n g v d i Id t h d t i f i m N N V b a t h o g t b o s u n g A P v d A H ; h i f i u g i d k h d n g t m n g hda d ed t d n g g d p 8 - 6 4 lan s a u 3 0 ngdy. V i n i t b i t hogt d u o c b l s u n g A P , A H cfi t h i t a o khdng t h i b d o h d t u 7 5 - 83.33% c d t h i nghifim v o i lieu c d n g c u f i n g dfic Id 0,2xTClD5o, 0,5xTClD5o, IxTCIDso Chat b l t r o A P kich thich t g o d d p dng m i l n djch t o h o n A H . N h d n g k i t q u d nghifin c d u v l t i n h t u o n g dong khdng nguyfin, k h d ndng tgo khdng t h i dgc hifiu, khd ndng tgo k h d n g t h i t m n g hda, khd n d n g bdo hfi Id c o s d d l nghi&n c d u l a o vdc-xin phfing bfinh H T T K c h o c a mu nudi tgi Vifit N a m .
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SELECTION STRAIN OF NERVOUS NECROSIS VIRUS FOR PRODUCING A VACCINE USING FOR GROUPER AQUACULTURE IN VIETNAM
Pham T h i T a m ^ *, N g u y e n T h i T h u H i e n , B u i T h i H a i H o a \ P h a m C o n g H o a t ^ N g u y e n Q u a n g L i n h ^ ' Hanoi Open University
^ Ministry of Science and Technology
^ Faculty of Fineries. Hue University of Agriculture and Forestry
SUMMARY
Nervous necrosis vims caused of heavy losses for grouper aquaculture in the world and Vietnam. Prevention by vaccination is an effective solution to minimize the damage of this disease. To produce an effective vaccine for prevention nervous necrosis disease infected in grouper farming in Vietnam thai the vaccine vims strain need to selected from fields isolations. From 26 strains isolated in the provinces as Quang Ninh, Hai Phong, Nam Dinh, Khanh Hoa, Binh Thuan, the strain namely KH 05 was identified Iliat its antigen is similar to 25/26 NNV isolated trams. KH05 strain can he inactivated by 0,1% beta-propiolactone and 0.002 M BEI but still keep entire the ability to generate the immune response. Inactivated vims can stimulate a specific immunity in rabbits with antibody tiler was 1/128, however, KH 05 combined with the AP adjuvant, antibody titers increased 64 fold, whereas combine to AH, antibody titer increased 32 times Evaluation the ability to generate neutralizing antibodies in grouper, neutralizing antibody titers achieved m experimental fish peaked at day 30 after immunization, antibody titers obtained from the inactivated NNV is 1/8, increased to 16-fold in fish belong to trial of immunization of inactivated virus combine to AH and 32-fold in fish immunized with inactivated vims combine to AP By challenge with bight virulent NNV strain, antibodies producing by immunization of KH 05 NNV can protect grouper with a challenge dose as 0,2 xTCIDjo, 0 5 xTCIDjo and IxTCIDso (TCID50 = 10**), protection rate is 75- 88,33%. Thus, KH 05 strain capable of use as a vaccme strain.
Ke)'words: nervous necrosis disease , vaccine strain, inactivated vims, aluminum adjuvant, specificity antibody, neutralizing aniibodies, protection rate
* Author for con-esspondence Tel (04)3 8693639 Email; tamanphami5)vahoo com
