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1 2011 9, 18(5): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J JX-0902 刘永奎 1,2, 王庆 1, 曾伟伟 1, 石存斌 1, 张超 3, 陈道印 4, 吴淑勤 1., ; 2., ; 3., ; 4., 摘要 :,,,,,, 70 nm, (grass carp reovirus, GCRV),,,, ; (CIK), 6,,, SDS-PAGE, ( JX-0902) 11 dsrna,, GCRV 873, HZ08 GenBank S6 (GQ896337), JX-0902 cdna, S6, JX-0902 GCRV HZ08 GD108 S6 98% 99%, : ; ; ; ; 中图分类号 : S941 文献标志码 : A 文章编号 : (2011) , [1] 20 70,, [2 4] 20 80, [5 9], [7], [10 12] 1991, [13], (Grass Carp Hemorrhage Virus, GCHV), (Grass Carp Reovirus, GCRV),,,,, JX-0902,,, 收稿日期 : ; 修订日期 : 基金项目 : ( ); (nycytx-49-14). 作者简介 : (1983 ),,,. lyk1015@yahoo.cn 通信作者 :,,,. wushuqin001@21cn.com

2 (CIK), (PSF), GCRV873 HZ08 ; M199 (FBS) Hanks Trizol Reagent Invitrogen ; Ex Taq DNA dntps DNA Marker TaKaRa ; Qiagen ; , ;, ( 1),, % 1.3 (0.65%) 3, ( ),, 3, r/min 30 min,, 0.22 μm 20 (12 0.5) cm 4 ( 2 ), 20, (100 cm 50 cm 40 cm), 1, (27±1) 0.2 ml, 0.2 ml, 4, 1.4 [14], CIK,, Hanks 2, 1.3, 28 1 h,, ( 2%FBS), 28, 3, 1.3, 6 2.5% GCRV873 HZ %,,, (JEM-2010HR) SDS-PAGE [11] Qiu [15] RNA(dsRNA) Trizol Reagent, SDS-PAGE [16], SDS-PAGE 5%, 12%, 60 V 2 h, 100 V 20 h S6 GenBank GCRV-HZ : 1(F5 -GTAATTTAAGCATAGCGT GGAGA-3 ; R5 -CTCATTACGCGTCACACCTTC- 3 ) 2(F5 -CAGCGTCTGGAGTGAGTATGG- 3 ; R5 -AGGCGTTACCTAGCACAT-3 ) 3 (F5 -GCAGGCAATTACTATCTATGG-3 ; R5 -GA TGAAAATGCAAAGCGGGGGTCGG-3 ) RNA PCR, PCR 1%, pmd18-t h, DH5α, LB, 5 PCR SEQMAN(DNAstar) BLAST ( CLUSTAL-X version 1.83, S6 MEGA : GD108(GCRV GD108, HQ231208), HZ08(GCRV HZ08, GQ896337), (Golden Shiner Reovirus,

3 5 : JX GSV, NC_005171), 873 (GCRV 873, AF403392), (Threadfin reovirus, TFV, AY235428), A(Aquareovirus A, Aqu A, NC_007592), (American Grass Carp Reovirus, AGCRV, NC_010589), (Mammalian orthoreovirus, MRV)T1L(NC004262), T2J(AF461683) T3D(M19408), (Avian Reovirus, ARV)ARV176(AY750053) ARV918 (AY635945) ARV919(AY635939) S1133(AY635934) 2 2.1, 1,, :, ;, ( 1, 2) 2.2 CIK, 7 d,, 6, ( 3), 70 nm,,, CIK ( 4) SDS-PAGE,, JX-0902 GCRV873 HZ08 SDS-PAGE 20 h,, 10, S1 S2, 2 ( 5) JX-0902 HZ08, GCRV S6, JX-0902 S6 RT-PCR GCRV JX-0902 S (A530, T470, C508, G522), 1 ORF 4, 26.11% (A), 25.72% (G), 23.15% (U) 25.02% (C) 5 42, 3 35 ORF AUG, (UAG) AUG 3 A, +4 G(AUCAUGG), Kozak [17] JX GUAAU UU UUCAUC-3 : 5 5 -GCAUAGCGUGG-3 ( 10 20) 3 Tab.1 表 1 病料组织液人工感染试验结果 Results of artificially infecting experiment with filtered liquid from diseased sample group item 1 test group 1 2 test group 2 1 control group 1 2 control group 2 /cm body length 12±0.5 12±0.5 12±0.5 12±0.5 /ml infection dose /h hours from infection to disease 72±1 63±1 0 0 /h hours from infection to death 82±1 75± dead nos. in 268 hours after infection /% mortality

2 2 The symptom of dead grass carps artificially infected 4

4 Viral particles in cell plasma of grass carp CIK cell Fig.

3 Viral particles in spleen of diseased grass carp 5

4 Fig.1 1 The symptom of spontaneously diseased grass carps Fig.2 2 The symptom of dead grass carps artificially infected 4 (CIK) Fig.4 Viral particles in cell plasma of grass carp CIK cell Fig SDS-PAGE SDS-PAGE of genome of three virus strains 3 Fig.3 Viral particles in spleen of diseased grass carp 5 -CCCGCUUUGC-3 ( ) 11 GCRV JX S , kd, 5.863, Blast, GCRV GD108 HZ08, (GSV) GCRV873(S6 ) (TFV, S6 ) A(Aqua A) (AGCRV) MRV 2 MRV 3 MRV 1 ARV, 99% 98% 34% 34% 32% 32% 31% 25% 25% 25% 24% GenBank GCRV JX-0902 S6, ( 6), JX-0902 GCRV GD108 HZ JX-0902, 2, : 8, 11, 9 11, 40%; 5 7, 10 11, 25%

5 5 : JX JX-0902 S6, GSV: (NC_005171); GCRV873: 873 (AF403392); TFV: (AY235428); Aqua A: A(NC_007592); GCRV-GD108: GD108 (HQ231208); JX-0902: ; HZ08: HZ08 (GQ896337); AGCRV: (NC_010589); T2J: MRV 2(AF461683); T3D: MRV 3(M19408); T1L: MRV 1; ARV918, ARV919, ARV176: (AY635945, AY635939, AY750053); S1133: S1133 (AY635934). Fig.6 Phylogenetic tree based on the amino acid sequence of JX-0902 S6 with counterpart segments from other reoviruses The tree was constructed by Neighbor-Joining menthod. The frequencies at which the branches cluster were tested by bootstrap analysis (1 000 replicates). GSV: Golden Shiner Reovirus(NC_005171); GCRV873: grass carp reovirus 873 strain (AF403392); TFV: Threadfin reovirus(ay235428); Aqu A: Aquareovirus A(NC_007592); GCRV GD108: grass carp reovirus GD108 strain(hq231208); JX-0902: strain isolated in the study; HZ08: grass carp reovirus HZ08 strain(gq896337); AGCRV: American Grass Carp Reovirus(NC_010589); T1L: mammalian orthoreovirus(nc004262), T2J(AF461683), T3D(M19408), ARV176: Avian Reovirus 176 strain(ay750053), ARV918 (AY635945), ARV919(AY635939); S1133: Avian Reovirus S1133 strain (AY635934).,,, [18], 19~33, 6 9,,,, [19],,, 1 JX-0902, JX-0902 S6 JX-0902,,, 90%, 3.2 JX-0902, 26~28,,, ;, 19~23,,, 19~23, :, ;, ; ( ),,,,, GCRV H962 [20], JX-0902 CIK, (CPE), HZ08 [21] GCRV861 [22], GCRV873 CIK CPE [23], CIK,,, [24], 3.3 SDS-PAGE, JX-0902 GCRV873 GSV TFV AGCRV Aqua A, HZ08 GD108,, HZ08 GD108 JX-0902

6 JX , GCRV873 [19],,,,, 参考文献 : [1]. [J]., 1994, 9 (3) : 1 4. [2]. [J]., 1977(6) : [3]. [J]., 1977, 6(2): [4]. [J]., 1978, 6(6) : [5],. II. [J]., 1980, 7(1): [6] (, ). [J]., 1983, 23(3): 39. [7] 燊,. [J]., 1983(18): [8],. [J]., 1984, 24(2): [9],. [J]., 1984 (3): 48. [10],. 854 SDS-PAGE [J]., 1991, (5): [11],,,. [J]., 1989, 13(1): 1 5. [12],,,. [J]., 1991, 6(3): [13] Francki R I B, Fanquet C M, Knudson D L, et al. Classification and nomenclature of viruses: Fifth report of the interuational committee on taxonomy of viruses[j]. Arch Virol, 1991, 2(Supp1): [14],,,. (GCRV991) [J]., 2002, 17(2): [15] Qiu T, Lu R H, Zhang J, et al. Molecular characterization and expression of the M6 gene of grass carp hemorrhagevirus (GCHV), anaquareovirus[j]. Arch Virol, 2001, 146: [16]., cdna,,, RT-PCR,, [D]. :, [17] Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes[j]. Cell, 1986, 44: [18]. [J]., 1980, 20(1): [19],,,. [J]., 2010, 40(3): [20],,,. H962 [J]., 1998, 24(6): [21],,,. HZ08 [J]., 2010, 17(6): [22],,,. [J]., 1998, 5(3): [23],,,. [J]., 2002, 17(2): [24],,,. 873 [J]., 2001, 17( 2):

7 5 : JX Isolation and identification of grass carp reovirus strain JX-0902 LIU Yongkui 1,2, WANG Qing 1, ZENG Weiwei 1, SHI Cunbin 1, ZHANG Chao 3, CHEN Daoyin 4, WU Shuqin 1 1. Pearl River Fisheries Research Institute of Chinese Academy of Fishery Sciences, Guangzhou , China 2. Shanghai Ocean University, Shanghai , China 3. Jining Fishery Monitoring Center, Jining , China 4. Fish Disease Control Center in Liantang Town of Nanchang, Nanchang , China Abstract: We identified a new strain of reovirus in grass carp from Nanchang city, Jiangxi Province. We collected diseased yearling grass carp, Ctenopharyngodon idellus exhibiting typical hemorrhage symptoms from Nanchang city and observed large numbers of viral cells in ultra-thin sections of the spleen and kidney. The virus had a non-enveloped structure with a diameter of approximately 70 nm, very similar to the characteristics of the grass carp reovirus (GCRV). We isolated samples of the virus by homogenizing the tissue, centrifuging the homogenate, then conducting sterilizing filtration. We were able to replicate the hemorrhaging by artificially infecting grass carp with the purified sample. Artificial infection of grass carp kidney cell line (CIK) revealed no obvious cytopathic effects (CPE) after six passages. However, observations under the electron microscope revealed that the virus was propagated in the cytoplasm, and had the same shape as the virus observed in the tissue sections. The newly isolated virus (temporarily named GCRV JX-0902) consisted of 11 segments of dsrna, a typical characteristic of aquareovirus. There were significant differences in the genome banding pattern of GCRV873 and JX-0902, but no difference between HZ08 and JX We designed PCR primers based on sequence segment 6 (GQ896337) of GCRV in GenBank to amplify specific bands using JX-0902 cdna as the template. The amino acid sequence of JX-0902 S6 had 98% and 99% identity with GCRV-HZ08 and GD108, respectively. Thus, we conclude that the new isolate is a member of the genus of aquareovirus. Key words: grass carp hemorrhage; reovirus; Jiangxi isolate; separation; identification Corresponding author: WU Shuqin. wushuqin001@21cn.com

2月5期aa

2月5期aa 中国农学通报 212,28():2-27 Chinese Agricultural cience Bulletin 血清 11 型鸭疫里默氏菌的分离鉴定 1,2 2, 2, 2, 2, 2, 2, 杨晓辉, 程龙飞, 陈红梅, 施少华, 傅光华, 万春和, 黄瑜 ( 1 福建省农业科学院畜牧兽医研究所, 福州