Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment
Natural Host Range and Pathogenicity of White Spot Syndrome Virus Infection in Shrimp and Crab Species in China
Jian-guo He, Yong-gui Chen, Min Deng, Po Yao, Hua-ming Zhou, Shao-ping Weng, Shi-gui Jiang, Qing-xin Long, Siu-ming Chan
White spot syndrome (WSS) is a viral disease that causes serious problems in the global shrimp cultivation, especially in Asia (Inouye et al. 1994; Nakano et al. 1994; Takahashi et al. 1994; Chou et al. 1995; Huang et al. 1995; Wang et al. 1995; Wongteerasupaya et al. 1995). The causative agent of WSS has been named differently by various researchers (Takahashi et al. 1994; Huang et al. 1995; Wang et al. 1995; Wongteerasupaya et al. 1995; Lo et al. 1996; Inouye et al. 1996). Lightner (1996) consolidated some of those under the name “white spot syndrome baculovirus complex” (WSBV). The viral pathogen described in the present paper is white spot syndrome virus (WSSV).
The clinical symptom of infection was recorded in 1991 in Guangdong, China, as a white spot on the carapace of tiger prawn Penaeus monodon. Since 1993, WSS outbreaks have significantly decreased the production of cultivated shrimp in China. Many reports have expressed concern about the rapid spread of WSSV in China. Most of the studies focus on histopathology, pathogenicity, diagnosis, DNA sequence analysis, and morphology of WSSV (Hu 1994; Huang et al.1995; He et al. 1996, 1999, 2000; Weng et al. 1996; Lu et al. 1999; Deng et al. 2000; Zhan et al. 2000). However, only few studies have investigated the natural hosts for WSSV in China (Lu and Chen 1995).
We studied the natural host ranges and infection rate of WSSV as well as the relationship between WSSV pathogenicity to tiger prawn and WSSV dose.
For the polymerase chain reaction (PCR) template, various tissues from shrimp and crab (gill, muscle, and epidermal layer) were homogenized in 10 volumes of TN buffer (50 mM tris[hydroxymethyl] aminomethane [Tris]-HCl, pH 7.6, 0.4 M NaCl) on ice and centrifuged at 8,000 revolutions/ min (rpm) for 5 min at 4°C. The supernatant containing 1% digestion solution (50 mM KCl; 10 mM Tris-HCl, pH 8.3; 0.45% Nonidet P-40; 0.45% Tween 20; and 80 µg/µL proteinase K) was boiled for 15 min, then put on ice for 5 min and centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was used as template DNA for PCR.
For nested PCR, two pairs of primers (PCR: 496F1, 5’-CGT GCC TGA ATC AGT ATG TAC GC-3’; 496R1, 5’-GAC GTT ACA ATA GAC CCA TGT TCG AT-3’; 496F2, 5’-CTC ATG TAC CAA ATC TGG GTT ACG A-3’; 496R2, 5’-CGA TAG ACC ACA AGT TCC GTA GGA-3’) were designed based on the DNA sequence of WSSV from tiger prawn (Deng et al. 2000). The expected lengths of the amplified fragments are 328 base pairs (bp; 496F1 and 496R1) and 258 bp (496F2 and 496R2). A 25-µL reaction mixture contained 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200 µM of each deoxyribonucleoside triphosphate (dNTP), 10 pM of each primer, and 1 unit Taq DNA polymerase. The amplification was performed for one cycle of 94°C for 4 min, 55°C for 45 s, 72°C for 1 min; 34 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 1 min, plus a final 8-min extension at 72°C after 35 cycles.