( Epi nephel us lanceolat us)

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1 43 4 ( ) Vol. 43 No Journal of Xiamen University (Natural Science) J ul : (2004) ( Epi nephel us lanceolat us) 1, 2, 1, 1 (1., ; 2., ) : RT2PCR,, ; RT2PCR, 97 %.,2,..,,,,. : ; ; ; : S 941 :A ( Epi nephel us spp. ) ( Serranidae) ( Epi nephel us), 100,50.,,,. 10,.,,,. ( E. : : (2002N009), (3502Z ) :( ),,,.. lanceola2 t us),,,,,,.,, (4 5cm),,,,,,, 1, 60 %80 %., (Nervous necro2 sis virus,nnv),( V ibrio algi2 nolyticus) ( Aeromonas caviae),. 1. 1) 2) PCR (OIE) [1 ]. : 5 2CGA GTC AAC ACG GGT GAA GA23, 5 2CGT GTC A GT CA T GTG TCG CT23,NNV RNA2 (421bp). 3) Sangon. DNA Ladder Promarg. 4) V ITEK J R GN I ; ; SPF, g ;. 5) NNV RT2PCR OIE [1 ],.

2 558 ( ) 2004 / 100 mg 1. 5 ml, 500LDEPC, r/ min 10 min., 40L K(1 mg/ ml) 40 L 1 %SDS,37 30 min. 600L / / (25g24g1),30 s, r/ min 5 min,. / (24g1),30 s, r/ min 5 min,. 1. 5,- 20 1h, r/ min 1 min,, 10L, PCR. cdna 10L PCR 200 L PCR,90 5 min, 5L 5 1L M2mLV (20 U/L ) 0. 5 L RNA (20 U/ L) 2. 5L (40mol) 2L dn TP 5L 25 mmol MgCL 2,42 30 min min,,. PCR 3. 5L (40mol) 1L (40mol) Taq 5 U 1 L dn TP,10 L 10 Taq 5 L 25 mmol MgCl 2 50 L, PCR, :72 10 min 95 2 min, s s s 25, 72 5 min,4. TBE 2 % (0. 5g / ml EB),6L 2 L,DNA Marker bp DNA. 6) PCR PCR Sangon,, DNAsis BLAST. 7) (3. 5 % NaCl),25 24 h, DHL TCBS, h,. Grop % Grop02,37 18 h,, 5 SPF :1 2, 0. 2 ml r/ min 10 min,,,0. 2 ml ml ml 3. 5 %.,37 18 h,,,37 24 h ,.,, 22.,,,,,,.,.,,.,,,. () , 1. 6 (16), bp, 6, NNV ; 3,NNV., NNV, NNV PCR RT2PCR,ContigExpress,2. Internet Genbank/ NCB I,BLAST, NNV ( E. m alabaricus) NNV % [2 ],NNV % [2 ]., ( E. aw oara) NNV 97 %, NNV,NNV

3 4 : ( Epinephelus lanceolatus) RT2PCR M :100 bp DNA, 16 : Fig. 1 Electrophoresis map of RT2PCR amplicon from E. lanceolatus 8 %, NNV AMS, ( Grop01) ( Grop02). AMS h, ;,7 d.,,;, 2 RT2PCR GROP21,NNV21 ( E. m alabaricus) ( AF ),NNV22 ( AF ) Fig. 2 Comparison of the amplicon nucleotide sequence against the NCBI nucleotide databases

4 560 ( ) Crop01 Crop02 Tab. 1 Result of biochemical test of strain Grop01 and Grop02 Grop01 Grop02 Grop01 Grop02 Grop01 Grop02 DP B ONPG % % % % V2P - 10 % + 2 Grop01 Grop02 Tab. 2 Pathogenic test of strain Grop01 and Grop02 on mice 1224 h 2448 h h / % 1 Grop Grop Grop Grop Grop01 + Grop ,,. 3 ( Viral encephalopathy and retinopa2 thy),.,,.,,1 100 %, 50 %80 %, [36 ]., [6 ].,,NNV.,,, NNV.,, NNV. PCR. RT2PCR OIE NNV,. RT2PCR,,,,

5 4 : ( Epinephelus lanceolatus) Grop01 Grop02 Tab. 3 Result of antibiotic sensitivity test in strain Grop01 and Grop02 Grop01 / mm Grop02 / mm Grop01 / mm Grop02 / mm G SMZ/ TMP , 3 NNV,NNV,.,, (Striped jack) [7 ]., NNV NNV,, 99 %. NNV. Chi [8,9 ] (1999) NNV. 2432,NNV GF21 ; 20 37, 28,20 32, 37, GNNV.,,4 9, 68, 3032,. 1 2, 22,,,. Lee [10 ] (1999),,,.,.,NNV,,.,,,. :

6 562 ( ) 2004 [1 ] (OIE),. (3 ) [ S].,. :, [2 ] Lin C S, Lu M W, Tang L, et al. Characterization of virus2like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus [J ]. Virology,2001,290 (1) : [3 ] Mori K, Nakai T, Negahara M,et al. A viral disease in hatchery2reared larvae and juveniles of redspotted grouper [J ]. Fish Pathol,1991,26 : [4 ] Naikai T,Nguyen H D,Nishizawa T,et al. Occurrence of viral nervous necrosis in kelp grouper and tiger puffer[j ]. Fish Pathol,1994,29 : [ 5 ] Fukuda Y,Nguyen H D,Furuhashi M,et al. Mass mortal2 ity of cultured sevenband grouper, Epinephelus septem 2 f asciatus, associated with viral nervous necrosis [J ]. Fish Pathol,1996,31 (3) : [6 ] Chi S C,Lo C F, Kou G H,et al. Mass mortalities associ2 ated with viral nervous necrosis ( VNN) disease in two species of hatchery2reared grouper, Epinephelus f us2 cugutatus and Epinephelus akaara [ J ]. J. Fish Dis., 1997,20 : [7 ] Iwamoto T, Mise K, Mori K,et al. Establishment of an infectious RNA transcription system for Striped jack ner2 vous necrosis virus, the type species of the betanodavirus [J ].J. Gen. Virol,2001,82 (11) : [8 ] Chi S C, Hu W W,Lo B J,et al. Temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae[j ]. Vir. Res.,1999,63 : [9 ] Chi S C,Establishment and characterization of continuous cell line ( GF21 ) derived from grouper, Epinephelus coioides :a cell line susceptible to grouper nervous necrosis virus ( GNNV) [J ].J. Fish Dis.,1999,22 :1-10. [ 10 ] Lee K K, Yang T I,Liu P C,et al. Duai challenges of in2 fectious pancreatic necrosis virus and V ibrio carchariae in the grouper, Epinephelus sp[j ]. Vir. Res.,1999,63 : Study on the Pathogen of the Fulminant Infectious Disease in the Fry of Epinephel us lanceolat us in Southern Fujian CHEN Xin2zhong 1, GON G Yan2qing 2, SU Yong2quan 1,WAN G J un 1 (1. College of Oceanography and Enviroment, Xiamen Univ., Xiamen , China ; 2. Xiamen Ent ry2exit Inspection and Quarantine Bureau, Xiamen ,China) Abstract : In recent years, some cultured fry of grouper Epi nephel us lanceolat us in southern Fujian Province, China had suffered an epidemical disease which caused heavy mortalit y. The average deat h rate was as high as 60 to 80 percent. The sick or died groupers did not show any pat holgical signs ot her t han neurological abnormalities in the early time of the disease, no significant external or intermal lesions or parasites were observed. An initial st udy was taken on t he pat hogen infected by t he fishes. The reverse t ranscription polymerase chain reaction (RT2PCR) was used to detect nervous necrosis virus. It was found that the juvenile grouper had been infected wit h nervous necrosis virus. The f ragment of 421bp of NNV was amplified and sequenced. It showed more t han 97 percent homologue with the sequences of other NNV strains from grouper in Gene Bank. This result also in2 dicated that the amplified sequence was the conservative area of the gene in NNV. Two strain of pathogen as V ibrio algi nolyticus and Aeromonas caviae were separated from the sick grouper. These germs had virulence to t he experimental animal and did not sensitive to most antibiotics. All of t he clinic symptoms and examinations indicated that the NNV was the major cause of the disease and the second2infection of V. al gi nolyticus and A. caviae had high morbidity and more mortality from the infection in these fries. Key words : Epi nephel us lanceolat us ; nervous necrosis virus ; V ibrio al gi nolyticus ; A erom onas caviae