| Shrimp aquaculture is threatened by many diseases, among which white spot disease(WSD) caused by white spot syndrome virus(WSSV) is one of the major disease. With the prawn farming areas and scales expanding constantly, WSSV had emerged the variation between different strains accordingly. Deletions in the ORF14/15 and ORF23/24 variable regions have been used as molecular markers to study the epidemiology of the virus. The repeat units of ORF75, ORF94, and ORF125 vary in different isolates and the numbers of repeat units in these three genomic regions are a useful marker for epidemiological studies and for genotyping strains of WSSV. However, each repeat unit(RU)may contain single-nucleotide polymorphisms(SNPs) that are important for differentiating haplotypes with the same numbers of repeat units. The differences between different isolates may lead to WSSV virulence variation. This paper mainly analyzes the WSSV variation through the shrimp culture zone in 2013-2015, at the same time, we also make it clear that the miRNAs have differently expressed with different WSSV virulence infection. The mainly research contents and results are as follows:Chapter I: To understand the variations in WSSV ORF14/15 and ORF23/24 and the VNTR and SNPs located in ORF75, ORF94, and ORF125 in different regions of China, we investigated 64 samples that were WSSV-positive on PCR, collected in a disease outbreak area between March and December 2013. The tested samples were from Hainan, Guangdong, Guangxi, Zhejiang, Shandong, Hebei, and Tianjin. Using specific primers, WSSV-positive samples were genotyped with PCR and the amplified fragments were ligated to a T-vector and used to transform Top10 cells. The positive clones were selected and sequenced. After sequencing, the fragments deleted from ORF14/15 and ORF23/24 in different samples were compared with the sequences of Th-96-II and TW respectively, and the three WSSV VNTR regions and SNPs in ORF75, ORF94, and ORF125 from different samples were analyzed with the DNAMAN software. The results showed that there were five samples had the products of ORF14/15 amplification, and one sample had the products of ORF23/24 amplification. The lengths of the amplified fragments of ORF14/15 were 1267 bp, 1270 bp, and 1892 bp, corresponding to deletions of 6533 bp, 6530 bp, and 5908 bp, respectively, compared with Th-96-II. One type of ORF23/24 was confirmed in this experiment, and the amplified fragment was 1140 bp, corresponding to a deletion of 12070 bp compared with the TW isolate. No fragment was amplified from ORF75. Four different ORF94 VNTRs, including 0, 3, 4, or 12 repeat units, were identified. The ORF125 VNTR, which included 0 or 7 repeat units, was identified. The SNP analysis showed that the bases on the special site in this study were different from the reported types. The bases at site 48 in ORF94 were T, T, T(Type Ⅰ: 3 RUs), or the bases at site 48 were T, T, T, T(Type Ⅱ: 4 RUs), and the base were T and 11 A(Type Ⅲ: 12RUs), respectively. The bases at site 8, 18, 25, 66, and 69 in ORF125(contained 7 RUs), were G, G, G, G, and A, respectively.It is noteworthy that there were also some variations at sites 9, 50, 53, and 61 in ORF125. These results suggest that there are certain degrees of prevalence and variation in WSSV, which causes white spot disease in most parts of China, with clear variations between the different strains in ORF14/15, ORF23/24, ORF75, ORF94, and ORF125.Chapter II: In order to understand the WSSV ORF14/15、ORF23/24 、ORF75、ORF94 and ORF125 variation in different regions of China, 48 samples extracted from WSSV-infected shrimp from 7 different provinces of China since January 2014 to August 2014 were investigated in the disease outbreak area. Using the specific primers, WSSV-positive samples were genotyped by PCR and the amplified fragments were connected to T-vector and transformed to the Top10 cells. Furthermore, the positive clones were selected and sequenced. The results showed that there were 21 samples with the products of ORF14/15 amplification, and 16 samples had the products of ORF23/24 amplification. Only 4 different types of ORF14/15 were confirmed. The length of amplification fragments was 1260 bp, 1270 bp, 1892 bp and 2662 bp respectively, which correspond to 6540 bp, 6530 bp, 5908 bp and 5138 bp deletion respectively compared with Th-96-II. Two different ORF23/24 types were confirmed. The amplicon fragments were 1140 bp and 1146 bp, which correspond to the length of 12070 bp and 12064 bp deletion respectively compared with TW isolate. In addtion, 5 different ORF75 VNTR types were confirmed, comprising 4, 10, 11, 12, 13 RUs, 2 different ORF94 VNTR including 4 and 14 RUs were identified, 5 different ORF125 VNTR including 0, 3, 5, 6, and 7RUs were identified.It was showed that there was a certain degree of prevalence and variation of WSSV which caused white spot disease in most parts of China. The variation between different isolates in 0RF14/15 was obviously,and the variation between different strains in ORF23/24 had the slight difference, and the variation between different strains in 0RF75, ORF94 and ORF125 was obviously. It is suggested that the deletion fragments variants of different region may correlate with the WSSV virulence and it is need to investigate furtherly, the specific VNTR variants of different region may correlate with the disease outbreaks and it is need to investigate furtherly.Chapter III: To understand the variations in WSSV ORF14/15 and ORF23/24 and the VNTR in different regions of China in 2015, we investigated 57 samples that were WSSV-positive on PCR, collected in a disease outbreak area between April and October 2015. The tested samples were from Shandong, Guangdong, Hainan, Zhejiang, Tianjin and Jiangsu. Using specific primers, WSSV-positive samples were genotyped with PCR and the amplified fragments were ligated to a T-vector and used to transform Top10 cells. The positive clones were selected and sequenced. After sequencing, the fragments deleted from ORF14/15 and ORF23/24 in different samples were compared with the sequences of Th-96-II and TW respectively, and the three WSSV VNTR regions in ORF75, ORF94, and ORF125 from different samples were analyzed with the DNAMAN software. The results showed that there were 35 samples had the products of ORF14/15 amplification, and 14 sample had the products of ORF23/24 amplification. The lengths of the amplified fragments of ORF14/15 were 1270 bp, 1892 bp and 2075 bp, corresponding to deletions of 6530 bp, 5908 bp and 5725 bp, respectively, compared with Th-96-II. One type of ORF23/24 was confirmed in this experiment, and the amplified fragment was 1140 bp, corresponding to a deletion of 12070 bp compared with the TW isolate. 10 fragments were amplified from ORF75, including 1, 2, 3 repeat units with 45 bp, and 1 with 102 bp. Four different ORF94 VNTRs, including 4, 5, 10, or 12 repeat units, were identified. The ORF125 VNTR, which included 3, 5 or 6 repeat units, was identified.Chapter IV: WSSV is one of the most harmful pathogeny in the pacific white shrimp, and genetic variations caused the strains of different virulence. MicroRNAs(miRNAs) involved in the regulation of virus defense. To understand the different virulence of WSSV on mi RNA expression in Litopeneaus vannamei, the deep sequencing was performed to compare two small RNA libraries prepared from hepatopancreas of Litopeneaus vannamei infected with high-virulence or low-virulence WSSV. Approximately 29,398,623 raw reads from high-virulence library and 35,291,803 raw reads from low-virulence library were obtained. There were about 37 functional miRNAs were identified. Sixteen miRNAs were significantly up-regulated and twenty-one miRNAs were significantly down-regulated in high-virulence infection library compared with low-virulence infection library. Of these, Igi-miR-1175-3p was the most significant different miRNA, followed by bmo-miR-1175-3p and ipu-miR-26 b, respectively. The putative target genes for differentially expressed miRNAs were concerned with biological processes, signal meditated, cell differentiation and apoptosis, immune recognition and other more functions. The results will help to understand the miRNAs response to different virulence WSSV infection. |
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