A multifaceted study of White Spot Syndrome Virus (WSSV) a shrimp pathogen

xiii, 148 leavesThe white spot syndrome virus (WSSV), which has been associated with major viral epizootics, was first identified in China and Taiwan in 1992 and soon spread in shrimp farms all over the world. The mortality rates for shrimp population experiencing WSSV infections can reach IOO% within three to ten days from the onset of the symptoms. In this study, a WSSV strain was isolated from P. chinesis from Qindao, China. The morphology of this WSSV isolate was examined by electron microscope (EM) and compared with other WSSV isolates. A cDNA library was constructed from white spot syndrome virus (WSSV) infected penaeid shrimp. Among cDNA clones with WSSV sequence insert, twelve clones were sequenced and analyzed. By comparing with the DNA sequence in the GenBank, cDNA clones containing sequence identical to that of WSSV envelope protein VP28 and nucleoprotein VP15 were identified. Poly(A) sites on the mRNAs of VP28 and VP15 were identified. In the course of the study, a PCR strategy was developed for amplification of double-stranded cDNA and enrichment of abundant cDNA. A screening strategy was established to identify clones with viral cDNA insert from a cDNA library. The cDNA library approach does not require the isolation of virion, which usually is difficult, time-consuming and expensive. The cDNA library method is readily applicable to other viruses and the protocol used in this study can be used with little modification. Genes encoding major viral structural proteins VP28, VP26, VP24, VPl9 and VPl5 from five WSSV isolates were sequenced and compared with that of other sequences of WSSV strains available in the GenBank. The genes encoding these major viral structural proteins are identical among WSSV strains isolated from different shrimp species and/or geographical areas. Gene probes developed based on the DNA sequences of viral structural proteins can be used for WSSV diagnostic/identification. A truncated version of the white spot syndrome virus (WSSV) 27.5kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum from a rabbit immunized with the recombinant protein was found to recognize the 27.5kDa viral envelope protein of WSSV isolated from different geographic regions. The antiserum did not recognize any of the other known WSSV structural proteins. A sensitive immuno-dot assay for WSSV was developed using the specific rabbit polyclonal antiserum. A wet-format dipstick model was developed for simple and rapid WSSV detection. Different types of membranes were tested as solid support for dipstick assay. The conditions for antibody and microparticle coupling reaction were optimized. The present study would be of value for the development of a dry-format dipstick with detector reagent (antibody and microparticle complex) dried on a conjugate pad, which would be more desirable for field use