Shrimp White Spot Baculovirus Genome-wide Transcription Map Analysis And Functional Genomics Research

Shrimp white spot bacilliform virus (WSBV), also called white spot syndrome virus (WSSV), is the heaviest pathogen to shrimp culture. It is an enveloped, non-occluded, bacilliform and circular dsDNA virus. WSBV is unclassified for the lack of molecular information and considered at a specific taxonomic position. Infection of penaeid shrimp by WSBV can result in mortality up to 90 to 100% within 3 to 7 days. Prevention and inhibition of this virus can be difficult due to its extremely virulence and a wide host range including most of aquatic crustacean and targets various tissues. The virus has spread into all over the world. It is also a threat to entironment. The complete genome of WSBV is 305,107bp. The pertinent study for import genes may be the most important for WSBV study. To obtain overall information of gene transcription, we studied the transcriptional profile of WSBV with DNA microarray. Some important gene would be further studied.The viral proliferation was studied at 2-7hr postinfection by competitive quantitative PCR. After infection, the quantity of WSBV was declined before 4 hrs and increased gradually thereafter. The accumulation of WSBV was more than 10n/mg tissue in dead infected shrimps. There was no correlation between survival time and weight of prawns ranging from 4.6g to 11.6g. As indicated in the proliferation study, 6 hr postinfection was putatively selected as early stage of infection and moribund as late stage in subsequent microarray assay.For the filter microarray (membrane) assay, 259 primers sets were synthesized to amplify WSBV DNA fragments by PCR. Most of putative open reading frames (ORFs) of WSBV could be detected by these amplicons. RNA extracted from negative control, samples of 2hr postinfection, 6hr postinfection and moribund stage was analyzed respectively. Most of putative ORFs are transcriped. Fifty-one of ORFs were found at early stage. For high frequency of expression at early stage, gene of Wsv238 gene selected for further study.This report described a spontaneous deletion in the WSBV genome. The size of the deleted fragment was approximately 4.6, 4.8 and 8.1 kb in different species. The deletion was later confirmed to be associated with the virulence of WSBV. There arefive ORFs (WSBV489, WSBV492, WSBV493, WSBV495 and WSBV497) in the deleted fragment. They might be responsible for the virulence but not for its infectivity and finally leading to the attenuation of the virus.WP228 (Wsv492) was expressed in E.coli vector as soluble fusion protein. The recombinant protein was shown nonspecific binding with DNA that mean relating to the gene regulation function. WP136 (Wsv493) was expressed in same vector but the recombinant protein was insoluble and the function research could not be processed.There are nucleus localization signals (NLS) and rich of glycines in the sequence of WP486 protein analyzed by biological informatics. The fragment of WP486 (376Aa at c-terminal) was expressed and the anti-WP486 antibody was raised for localization of WP486 with immune electron microscopy and western blot analysis. Natural protein of WP486 is shown about 32kD. The WP486 was determined localizing in the nucleus of infected shrimp cell in localization study. It was assumed as an important regulatory protein of WSBV.