Sequencing And Analysis Of Partial Genome Of Scylla Serrata Reovirus And RT-LAMP Detection Methods Development

Scylla serrata reovirus (SsRV) is one of the most prevalent viral pathogens of the mud crab (Scylla serrata). This pathogen is widespread in east China and causes severe economic losses to the nation’s mud crab industry. In this study, histopathology and ultrastructural pathology examination of S.Serrata infected reovirus were observed by paraffin section and ultrathin section technique. The results of histopathogy examination showed that the obviously histopathological changes found in hepatopancreas, gills, intestine, heart, stomach and muscle tissues. Among them, hepatopancreas, gill and intestine were the main target tissues with the most obvious pathological changes, which included damaged basal membrane of hepatopancreas, hepatocyte necrosis, clogging gill cavity, shedding epithelium of intestine, muscular fibers derangement, fragmentation. The results of ultrastructural pathology examination showed that SsRV widely distributed in liver, pancreas, gills, heart tissue and can reproduce in these cells. So, SsRV virus apparently can infect a variety of target organs, moreover, SsRV infection causes a series of pathological chagens in the target tissue cells, such as the condensation of nuclear chromatin, the increasing of electron density, the formation of myelin bodies and lysosome, the destruction of mitochondrial structure and the expansion and distortion of rough endoplasmic reticulum. SsRV virus was purifiedby differential centrifugation and sucrose density gradient centrifugation method. The complete virus particles are 70 nm in diameter, spherical, icosahedral and no envelop present. The genome is consisted of 13 dsRNA segments with the electrophoretic pattern of 1/5/7, which molecular weight is 25 kb.We sequenced and analyzed SsRV genome segments SI, S2, S3, S7, X1, X2, X3 and X4 using single primer amplification technique (SPAT), which were 4327,2721,2715, 1517,2460,1255,1244 and 1155 nucleotides long, respectively. All eight segments share six conserved nucleotides at the 5’ends and six conserved nucleotides at the 3" ends (5’-AUAAAU......AACGAU-3’). As with most members of the family Reoviridae, the first and last nucleotides of all eight segments of SsRV are inverted complements.. RNA segments S1, S2, S3, S7, X2, X3, and X4 each contained a single open reading frame (ORF) that encoded predicted proteins of 160 kDa,100 kDa,96 kDa,46 kDa,40 kDa,36 kDa and 30kDa, respectively, while X1 contained two ORFs, which encoded putative proteins of 32 and 25 kDa. A. Analysis those sequences using the BLASTp program and bioinformatic software, the results showed that Segments S1 and S2 was the viral RNA dependent RNA pylmerase (RDRP and uanylyltransferase, respectively. whereas the ORFs of other segments had no homologies with any other viral genes.. The phylogenetic analysis of SsRV polymerase shows that it has a distant, origin with the viruses in the genera of the family Reoviridae. Based on these observations, we propose that SsRV should be considered a member of a new genus in the family Reoviridae.To obtain expressed proteins of S7, X2, X3, and X4 gene in vitro, the open reading frame (ORF) of segment S7, X2, X3, and X4 was produced by PCR amplification, and the amplified fragment was cloned into prokaryotic expression vector pE30a(+), respectively. The recombinant plasimid, which was named as pET30/1517-ORF, pET30/1255-ORF, pET30/1244-ORF and pET30/1155-ORF, was then transformed into E.coli BL21(DE3) host cells, respectively. The nucleotide sequence of the recombinated plasmid pET30/1517-ORF, pET30/1255-ORF, pET30/1244-ORF and pET30/1155-ORF were sequenced and the results indicated that the target genes had been inserted into the BamHⅠ/HindⅢsite of the expression vector in right reading frame. For pET30/1255-ORF and pET30/1155-ORF,48 kDa protein and 36 kDa protein were expressed respectively with the induction of IPTG. The molecular weight of express products accorded with the anticipated molecular weight of pET30/255-ORF and pET30/1155-ORF fusion protein by SDS-PAGE, and the express products can be recognized by rabbit anti-SsRV polyantibody indicated by western-blot. However, pET30/1517-ORF and pET30/1244-ORF was not expressed in E.coli BL21(DE3).In the present study, a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for the rapid and sensitive detection of SsRV was developed and evaluated. No cross-reactivity was found with the other two tested viruses and SsRV-negative animals. The sensitivity of the RT-LAMP assay was determined to be 0.8 fg SsRV dsRNA, which was 1000-fold higher than that of one-step reverse transcription polymerase chain reaction (RT-PCR). In summary, the RT-LAMP assay is a simple, cost-effective, sensitive, and specific tool for the rapid detection of SsRV infection.