Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment
New Open Reading Frame of White Spot Syndrome Virus from Penaeid Shrimps and Related Detection of the Virus by Multiplex Polymerase Chain Reaction
Chun Xia, Jin Liu
Outbreak of virus infection with heavy mortality in cultured shrimp has occurred at many farms in China (Huang et al. 1995). White spot syndrome virus (WSSV; previously called HHNBV) was the causative agent because the infected shrimp has gross signs of infection in the form of white inclusions embedded in the carapace at the late stages. In Japan, the causative agent was called penaeid rod-shaped DNA virus (PRDV; Kimura et al. 1996). WSSV and PRDV are similar in morphology and characteristic lesions and are a kind of nonoccluded rod-shaped virus, classified as nonoccluded Baculovirus under the subfamily Nudibaculovirinae of family Baculoviride (Lo et al. 1996; NRIA 1997).
To identify and compare the viruses, an open reading frame (ORF) was cloned from WSSV. To detect this virus rapidly and efficiently, a multiplex polymerase chain reaction (MuPCR) method was established.
The WSSV was sampled from the south Pacific coast of China (Xia and Liu 1999). The genomic DNA of WSSV was extracted from the internal organs of virus-positive shrimps using the method of Xia and Huang (1999). According to the sequence of PRDV, the forward primer and reverse primer were designed. The cloned DNA fragment was sequenced and designated as WSSV-ORFx.
The gene and amino acid (aa) sequences were analyzed using Genetyx software (SDC Software Development Co., Tokyo, Japan).
According to the WSSV-ORFx sequence, four primers (P1, P2, P3, P4) were designed for MuPCR (Figure 1). Each primer set was completed among these forward and reverse primers, which were labeled as I (P1/P3/P4), II (P2/P3/P4), III (P2/P1/P4), IV (P1/P2/P3), and V (P1/P2/P3/P4). To amplify the WSSV-ORFx gene, MuPCR was performed using the five primer sets. Optimal primer set IV was selected to continue amplifying WSSV-ORFx. Then, the optimal MgCl2 concentrations, PCR cycles, and the detection limit of MuPCR were determined.
Hepatopancreas were collected from WSSV-infected shrimps and homogenized. With primer set IV and 32 cycles, the MuPCR was performed to amplify the WSSV-ORFx gene. As experimental controls, template DNA of PRDV (Kimura et al. 1996) and genomic DNA from negative shrimp were established.