Studies On Responses To White Spot Syndrome Virus In Litopenaeus Vannamei

Over the past20years, white spot syndrome of shrimp has always been the spotlight of the biological research. Researchers from all over the world have devoted great efforts to the issue, from the initial morphology observation to the identification of various antiviral immune related genes. But so far it has no clues to uncover the "mystery". We believe that the immune responses are complicated processes; interpretation from the perspective of omics may give some clues to understand the interactions between prawns and virus. The rapid development of sequencing technology provides a good platform for us to realize our ideas.The transcriptome sequencing technology based on the Illumina/Solexa high-throughput sequencing platforms can detect the overall transcription of any species at the single-nucleotide level, and can also identify rare or novel transcripts, therefor providing the most comprehensive transcriptome information. Meanwhile, digital gene expression profile technology can analyze differential expressed genes under different conditions. In contrast to traditional sequencing technologies, deep sequencing has the characteristics of high-throughput, fast and accurate. Transcriptome studies can reveal gene function from the overall level, and elucidate the molecular mechanisms in specific biological processes and disease process.In the present study, transcriptomes of hemocytes in WSSV-infected white shrimp were sequenced using Illumina/Solexa sequencing technology. Transcriptome de novo assembly was carried out with short reads assembling program-SOAPdenovo; thus, sequence alignment, functional annotation and pathway analysis against databases including nr, Swiss-Prot, KEGG and COG were performed. Furthermore, we used the DGE technology screening the differential expressed genes in the early and late stage of WSSV infection. Finally, we analyzed the gene functions in the process of WSSV infection of some screened genes. The findings and conclusions are as follows.1. Based on these studies,2,581million high qulity90-bp short reads were generated. Through assembling, these short reads resulted in52,073unigenes with a mean size of520nt and an N50of745nt. Although the3’and5’ends of the assembled unigenes contained relatively fewer numbers of reads, the other positions of the assembled unigenes showed a greater and more even distribution. The findings are greatly enriched the transcriptome database of shrimp.2. Among the assembled unigenes,23,568ones can annotated into the four protein database. GO functional annotation showed that there were6,562unigenes were classified into50functional classifications including metabolic process, regulation of growth and development, apoptosis, biosynthesis, immune defense, molecular processing, signal transduction and transcriptional regulation. COG functional classification showed that there were7,822unigenes were classified into25functional classifications, mainly containing general function prediction only, translation, ribosomal structure and biogenesis and transcription factors. KEGG pathway analysis showed that there were14,941unigenes was annotated into240pathways and there were963unigenes were enriched in various immune-related pathways like MAPK pathway, jak-STAT pathway and calcium metabolic pathway. CDS prediction revealed that there were20,689unigenes had the predicted coding frames. The rest unigenes were analyzed using the software named ESTscan to predict the coding regions; the results indicated that there were5,669unigenes may represent new protein coding sequences. In addition,1,179unigenes were screened as the immune-related genes from the annotated unigenes. These analyses indicated that the antiviral immune system of shrimp was a complicated biological process.3. Using the DGE sequencing technologies, we analyzed the differential expressed genes of three samples containing control sample, early infection sample and late infection sample.60million high quality tags were obtained from the sequencing libraries of three samples. Through analyzing sequencing quality, saturation assessment and tags distribution, we concluded that the DGE data were good enough for further analysis. Through mapping tags to the transcriptome, we found that only315differentially expressed genes were detected with227up-regulated genes and88down regulated genes in the early infection sample, while a total of4226unigenes were mapped with2506unigenes up-regulated and1720ones down-regulated between the mock and late infection sample. GO functional annotation showed that specific enrichment of genes was observed in insoluble fraction, peptidase activity and cellular amino acid metabolic process terms in the late infection sample. KEGG annotation revealed that1232differentially expressed genes were identified in the late infection samples. Notably, enrichment of genes was observed for pathways including ECM-receptor interaction, complement and coagulation cascades, antigen processing and presentation etc. The results of qRT-PCR expression analysis had the same trend to the DGE data. We can concluded that the antiviral responses of shrimp were delayed and powerless in the early stage of WSSV infection; while in the late stage of virus infection, the immune system seemed fully activated.4. Based on the results of the transcriptome and DGE analysis, we investigated the transcriptional expression profiles of18key immune-related genes in shrimp which severely infected by WSSV. We found that the expression levels of6genes including chymotrypsin-like serine proteinase (CH-SPase), heat shock protein70cognate (HSP70), penaeidin (PEN), peroxinectin (PO), proliferating cell nuclear antigen (PCNA) and argonaute (AGO) changed significantly, while the expression of the other12genes had no significant changes compared to the control group. Among the6screened genes, CH-SPase showed significantly up-regulation, while the other5ones were significantly down-regulated. Knockdown of the expression of CH-SPase in WSSV-infected Chinese shrimp reduced the copy number of WSSV and delayed cumulative mortalities, suggesting that CH-SPase is an assist in the virus infection process.