Study On Etiology And Detection Methods Of Soft-shelled Turtle Iridovirus (STIV)

The soft-shelled turtle is an important economical aquatic product of China."Red neck disease" is a common illness in soft-shelled turtle cultivation,possibly caused by the soft-shelled turtle iridovirus（STIV） infection.STIV is isolated from sick soft shelled turtle（Trionyx sinensis） with "red neck disease" in a farm.After artificial infection with STIV,The mortality rate of morbidity Trionyx sinensis surpasses 40%. STIV can cause the soft-shelled turtle massive death,bring the heavy loss to the Trionyx sinensis breeding,so it is necessary to establish examination method of STIV.In this study,the biological characters were studied and one effective STIV examination method was established.The main results were summarized as follows:1.STIV isolation and analysis of the viral physical and chemical properties.The cell lines CO、FHM、and GCK cells were inoculated with supernatant extracted from the diseased Red-neck turtle’s organs and incubated.The cytopathic effect（CPE） was observed,The plaque forming test and the physico-chemical characteristics analysis were carried out.The results showed that the virus was unstable to heat,pH3 and pH9 treatment.It was sensitive to lipid solvent.STIV couldn’t proliferate at the effect of DNA inhibitor demonstrating the genome was DNA.Under the electron microscope,the virions were sphere particles with envelope. The diameter was 120～160nm.A higher magnification showed that the virions were in the polyhedron shape.Although STIV could replicate and produce CPE in CO、FHM、CK、GCK cell lines,it couldn’t cause CPE in CHSE、R1、PG cells.STIV had highest titer in CO cells and caused CPE more quickly.The temperature it replicated and caused CPE ranged from 15℃to 30℃in CO cells.2.Identification of the complete gene sequences of major caspid protein（MCP） of STIV.The MCP,classified as a late transcript by drug inhibition,encodes a protein of 463 aa with a predicted molecular weight of 50 kDa.Indirect immunofluorescence （ⅡF） and virus neutralization assay were developed to determine the sensitivity and virus neutralizing activity of MCP-specific antiserum.Furthermore,the MCP temporal expression pattern during STIV infection in vitro was characterized by Western-blot and Real-time RT-PCR assay.The results suggest that STIV could be classified as a member of genus Ranavirus in family Iridoviridae and has cell-type-specific programs of viral gene expression.3.To establish a real-time PCR method for rapid detection of STIV,the specific primers and probe were designed in the conserved region of STIV MCP.The primers, probe and the reactive condition were optimized to improve the sensitivity.It was found that the specificity of this assay was high for STIV without any cross-reactions with LCDV,RSIV,ISKNV,KHN and other commonly encountered viruses from aquatic animals.A good linear correlation was demonstrated in the standard curve for the real-time PCR assay.A minimum of 4.5×10^-3 pg/μL total DNA could be detected, indicating a good sensitivity of the assay.The coefficients of variance（CV） were 1.2%and 1.6%for the intra-assay and inter-assay tests respectively,indicating a good reliability.4.Establishment of Loop - Mediated Isothermal Amplification（LAMP） method of STIV.Two inner primers and two outer primers were designed in conserved region of thymidine kinase（TK） gene of Ranavirus.Detection to STIV was carried out by LAMP amplification technique.The amplification was carded out in 63℃for 60min after condition optimization.It was found that the specificity of this assay was high for STIV without any cross-reactions with LCDV,RSIV,VERV,PFR,VHSV,IHNV and IPNV.It showed that the STIV could be found in this condition.The sensitivity of this assay was same with that of common PCR,but one order of magnitude lower than real-time PCR.5.Preparation of Monoclonal Antibodies against STIV and establishment of pathogen detection ELISA for STIV.Balb/c mice were immunized by purified STIV, and the spleen cells were separated.After fusion with tumor cells（SP2/0）,the supernatant of cell culture and ascitic fluid induced by hybridoma cells were detected by indirect ELISA.As a result,4 hybridoma cell lines were obtained which could stably secrete specific monoclonal antibody against STIV and established pathogen detection ELISA for STIV.128 clinical samples were detected by this ELISA method with PCR method as the control.The result showed that the specificity and sensitivity of this ELISA method was 98.3%and 100%respectively;The overall agreement between ELISA and PCR was 98.4%.