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1 946 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 殷嵘等 / 不同途径感染白斑综合征病毒 (WSSV) 对日本沼虾的致病性 June 25, 2017, 33(6): Chin J Biotech, All rights reserved 海洋生物技术 (WSSV) 殷嵘 1,2, 郭媛媛 1,2, 韦章良 3, 施定基 4, 何培民 1,2, 贾睿 ,2,,,. (WSSV)., 2017, 33(6): Yin R, Guo YY, Wei ZL, et al. Pathogenicity of white-spot syndrome virus in Macrobrachium nipponensis via different infection routes. Chin J Biotech, 2017, 33(6): 摘要 : 日本沼虾肉质鲜美 经济价值高, 但其对白斑综合征病毒 (White spot syndrome virus, WSSV) 的易感性尚不明确 采用流行病学调查 感染试验和荧光定量 PCR (qpcr) 等方法, 研究了日本沼虾 Macrobrachium nipponense 对 WSSV 的易感性 感染发病率以及 WSSV 在其体内的增殖情况 结果表明 : 日本沼虾是 WSSV 的自然宿主, 上海市附近省份地区 120 个样本的自然携带率为 8.33% 日本沼虾可以通过注射 摄食 浸泡途径感染 WSSV,10 d 内累计感染率均为 100%, 累计死亡率分别为 100% 75% 和 0% 其中注射途径感染日本沼虾, 感染后 5 d 肌肉病毒含量达到感染后 1 d 的 倍,8 d 死亡率达 100% 用注射 WSSV 感染日本沼虾死亡率来测量病毒致死日本沼虾的半数致死剂量 (LD 50 ), 病毒粒子 /µl 能使日本沼虾死亡率达到 50% 以上 在养殖生产中, 日本沼虾可以通过摄食感染 WSSV 的病虾或死虾而感染 WSSV, 也能通过浸泡在含 WSSV 的水体中而感染, 并因此成为一种传播媒介, 从而影响了 WSSV 的迅速传播与致病 : 日本沼虾, 河虾, 白斑综合征病毒, 增殖, 半数致死剂量 Received: January 6, 2017; Accepted: March 23, 2017 Supported by: National High Technology Research and Development Program of China (863 Program) (No. 2014AA093506), Shanghai Science and Technology Committee (No ). Corresponding author: Rui Jia. Tel: ; rjia@shou.edu.cn (863 ) (No. 2014AA093506) (No )

2 殷嵘等 / 不同途径感染白斑综合征病毒 (WSSV) 对日本沼虾的致病性 947 Pathogenicity of white-spot syndrome virus in Macrobrachium nipponensis via different infection routes Rong Yin 1, 2, Yuanyuan Guo 1, 2, Zhangliang Wei 3, Dingji Shi 4, Peimin He 1, 2, and Rui Jia 1, 2 1 College of Marine Ecology and Environment, Shanghai Ocean University, Shanghai , China 2 College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai , China 3 Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou , Guangdong, China 4 Institute of Botany, Chinese Academy of Sciences, Beijing , China Abstract: Macrobrachium nipponensis is delicious and has high economic value, but its susceptibility to white-spot syndrome virus (WSSV) is unknown. Susceptibility, morbidity, and multiplication of WSSV in M. nipponense were studied by epidemiological survey, infection experiment and qpcr. M. nipponense was the natural host of WSSV, and the natural carrying rate was about 8.33%. M. nipponense could be infected with WSSV via oral administration, muscle injection and immersion, and the cumulative infection rate of 10 d exposure was 100%, and the cumulative mortality rates were 100%, 75% and 0%, respectively. The infection of WSSV is fast by muscle injection. The virus content after 5 day s injection is times higher than that of the first day of infection, and the mortality rate reached 100% after 8 days. The median lethal dose (LD 50 ) measured as the mortality of infected M. nipponense via injection indicated the LD 50 in the concentration of WSSV of virions/µl. In shrimp farming, M. nipponense can be infected by ingesting WSSV infected shrimp or dead shrimp, and also by soaking in WSSV-containing water and thus become a vector, consequently affecting the spread and pathogenicity of WSSV. Keywords: Macrobrachium nipponense, river prawn, white spot syndrome virus (WSSV), proliferation, median lethal dose Macrobrachium nipponense (Arthropoda) (Crustacea) (Decapoda) (Palaemonidae) (Macrobrachium) [1] [2-4] [5-7] t [8] [9-12] (White spot syndrome virus WSSV) (Taura syndrome virus TSV) (Infectious hypodermal and hematopoietic necrosis virus IHHNV) [13] WSSV 100% WSSV WSSV WSSV [14] 1992 WSSV [15-17] [18] WSSV cjb@im.ac.cn

3 948 ISSN CN /Q Chin J Biotech June 25, 2017 Vol.33 No d 100% [19-20] [21-22] [23] WSSV [24-27] WSSV Macrobrachium rosenbergii Macrobrachium idella [28] WSSV qpcr WSSV WSSV WSSV WSSV 1 材料与方法 1.1 野生日本沼虾体内 WSSV 携带率调查 40 / PCR WSSV WSSV 1.2 材料 ( (5.5±0.5) cm (1.8±0.2) g/ ) 55 cm 45 cm 20 cm PCR WSSV 2 d (28±0.5) 12 h 12 h 2 (9:00 21:00) 1/3 24 h PCR qpcr ( ) ( ) 1.3 病毒制备与定量 WSSV WSSV [29] copies/µl µl/ [30] 3 80 WSSV Procambarus clarkii 3 ( 6 7 d ) PBS (ph 7.4) 1 10 (W/V) r/min 5 s g 5 min μm

4 殷嵘等 / 不同途径感染白斑综合征病毒 (WSSV) 对日本沼虾的致病性 949 WSSV WSSV Procambarus clarkii 6 ( 6 7 d ) [31] WSSV WSSV WSSV OD 600 C=OD /µl WSSV 1.4 感染实验 d 4 d (OD 600 =0.4) µl/ TM (50 mmol/l Tris-HCl 5 mmol/l MgCl mmol/l NaCl ph 7.4) ( ) d 4 d 4 30 WSSV WSSV 12 h copies/mg 5% [32] 2 2 d d 4 d WSSV 10 5 /L 4 h TM 1.5 养殖管理 2 1/3 24 h 12 h 12 h 病毒 qpcr 检测 [33] WSSV DNA 20 mg DNA qpcr qpcr [34] VP28-140Fw (5 -AGG-TGT-GGA-ACA-ACA-CAT-CAA- G-3 ) VP28-140Rv (5 -TGC-CAA-CTT-CAT- CCT-CAT-CA-3 ) 141 bp qpcr 20 µl 2 µl DNA 10 µl SYBR Green 0.6 µl (300 nmol/l) 6.8 µl RT-PCR FTC min s s s min 1.7 病毒感染日本沼虾的半数致死剂量 (LD 50 ) WSSV cjb@im.ac.cn

5 950 ISSN CN /Q Chin J Biotech June 25, 2017 Vol.33 No.6 (LD 50 ) PCR WSSV WSSV µl/ TM 10 d 数据处理 [35] P=(P' C)/(1 C) P P' C ± (x ±s) Origin Pro8.0 SPSS 19.0 (One-Way ANOVA) 表 1 野生日本沼虾体内感染 WSSV 的调查 Table 1 A survey on the WSSV in wild M. nipponense Sample size WSSV Infection rate (ind) (%) Notes: : negative; +: weak positive; ++: positive; +++: strong positive. 2.2 日本沼虾口服感染 WSSV WSSV 48 h 72 h 84 h h 192 h 10 d 76% 80% 1 2 ( 1) DNA PCR WSSV ( 2) P< 结果与分析 2.1 野生日本沼虾 WSSV 检测 PCR ( 1) WSSV 8.33% WSSV (C t 20 ) WSSV 图 1 口服感染 WSSV 后日本沼虾累计死亡数 Fig. 1 Cumulative deaths of M. nipponense post oral infection.

6 殷嵘等 / 不同途径感染白斑综合征病毒 (WSSV) 对日本沼虾的致病性 951 图 2 口服 WSSV 病虾后日本沼虾 WSSV 检测为阳性 Fig. 2 Detection of M. nipponense in the shrimp orally infected with WSSV. 1 10: death; 11 17: survival; 18 21: negative control; M: marker. WSSV WSSV 2.3 日本沼虾注射感染 WSSV WSSV 24 h 48 h 48 h 72 h 96 h 50% 192 h 100% 2 4 ( 3) PCR 24 h WSSV 96 h 120 h WSSV 12 h WSSV 图 3 注射感染 WSSV 后日本沼虾累计死亡数 Fig. 3 Cumulative deaths of M. nipponense post intramuscular infection. 2.4 日本沼虾浸泡感染 WSSV 10 5 /L 10 d 2 10 d PCR ( 4) WSSV 2 ( ) cjb@im.ac.cn

7 952 ISSN CN /Q Chin J Biotech June 25, 2017 Vol.33 No WSSV 的半数致死剂量 (LD 50 ) WSSV % % % ( 5) 100% LD /µl % /µl 图 4 浸泡 WSSV 粒子 10 d 后日本沼虾 WSSV 检测为阳性 Fig. 4 The detection of M. nipponense was positive after 10 days of immersion of WSSV particles. 1 10: infection; 11 15: negative control; M: marker. 图 5 日本沼虾注射不同 WSSV 稀释液后累计死亡率 Fig. 5 Cumulative mortality of M. nipponense intramuscularly infected with different dilutions of WSSV. 3 讨论 [36] WSSV WSSV WSSV WSSV WSSV WSSV [37-38] WSSV WSSV WSSV WSSV 4 (84 h) 8 (192 h) 75% 3 (60 h) 8 (192 h) 100% 5% 10% WSSV WSSV WSSV [39] WSSV 10 3 /L 10 d [40-41] 1 d 100% (10 5 /L)

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