Development Of A Chinese Perch Cell LineSusceptible To ISKNV And Mechanism Of VirusRepication Relying On Glutamine

The Chinese perch or mandarin fish Siniperca chuatsi?Basilewsky 1855?is one of the popular cultured fish species in China.But since 1994 infectious spleen and kidney necrosis virus disease?ISKNVD?has been the major disease causative factor causing high mortality and resulting in significant economic losses to S.chuatsi cultured in China.Thus it is necessary to study deeply on ISKNV.However,because of lack of susceptible cell line for ISKNV propagation,studies on ISKNV are limited,such as pathogenic mechanism,vaccine development,and so on.Thus susceptible cell line has been the technical bottleneck of ISKNV research.In this study,a continuous cell line from Chinese perch brain?CPB?was developed and it is high susceptible to ISKNV.Based on the CPB cell line,two rapid detecting methods were developed,including qPCR method for virus titer testing of ISKNV,monoclonal antibody against MCP of ISKNV.After that the relation between ISKNV replication with glutamine was deeply investigated.The results obtained in this work were as follows:?1?Development and characterization of a novel cell line derived from the brain of Chinese perch Siniperca chuatsiAccording to the brain character,primary cell cultures were initiated from brain tissue of S.chuatsi by the collagenase digestion method.A continuous cell line named chinese perch cell line?CPB?was developed,and has been subcultured over 140 passages up to now.CPB cell line predominantly consisted of fibroblast-like cells that could grow better in Leibovitz's L-15supplemented with 10%fetal bovine serum at 28?.Heterologous genes could be expressed in the CPB cell line.The CPB cells were highly susceptible to infectious spleen and kidney necrosis virus?ISKNV?with a titre of 6.58–6.62 log TCID50 ml-1 and numerous ISKNV particles were observed in the cytoplasm by transmission electron microscopy.At the same time,ISKNV infection was confirmed by reverse transcriptase polymerase chain reaction,immunodot blot and individual challenge experiments.The results of individual challenge experiments showed that a positive correlation with the cultured water temperature and mortality and disease time was found in those cultured with high water temperature at the same time interval.?2?qPCR method for virus titer testing of infectious spleen and kidney necrosisThe development and validation of a TaqMan qPCR assay for determination of ISKNV titers is reported in the present study.ORF007 gene of ISKNV was successfully cloned into the eukaryotic expression system pcDNA3.1+,and named pcDNA007.The specific primers and probe designed to amplify a small genomic fragment of ISKNV 007 gene.The method was specific and reproducible as well as sensitive with the minimum detectable limit of 10viral copies.At the same time cytopathic effect?CPE?method was compared and analyzed to qPCR.The results showed that the titers obtained by qPCR correlated well with TCID₅₀ as indicated by linear regression and linear equation was y=1.076x+0.545?R²=0.9986?.Y meaned Lg gene copies concentration and X means Lg TCID₅₀ of virus.The results approved that the real-time PCR method can replace the TCID₅₀ method in evaluating the titer of ISKNV.?3?Development and identification of monoclonal antibody against major capsid protein of ISKNVThe monoclonal antibody?MAb?against recombinant MCP protein was developed and the properties were identified.The purified recombinant MCP was injected into BALB/c mice through subcutaneous route for three times.Then myeloma cells SP2/0 were fused with the spleen cells of the immunized BALB/c mice.Three hybridoma cell lines against ISKNV MCP were screened using indirect ELISA and were identified to be IgG1 subtype,designated as5F1,3D9 and 5B4,respectively.The three McAbs had no reaction with SCRV and STIV except ISKNV by the indirect ELISA.The hybridoma cell line 5F1 was selected for ascites preparation.When the recombinant MCP and ISKNV supernatant was used as detective antigen respectively,the titer of ascites was 1?51 200 and 1?400 respectively.Indirect immunofluorescence and Western-blot analysis showed that the McAb 5F1 could recognize authentic MCP protein of ISKNV and working concentration of McAb 5F1 was confirmed.From above,the MAb against MCP of ISKNV was successfully prepared,which laid a foundation for further study.?4?Mechanism of efficient replication of infectious spleen and kidney necrosis virus dependent on glutamineIn the previous study in our lab,we observed that glutamine metabolism in Chinese perch brain?CPB?cells is altered during ISKNV infection,and ISKNV replication was decreased in CPB cells cultured in the glutamine-depleted medium.In this section,we further studied that ISKNV replication rely on the glutamine for supplying virus with TCA cycle intermediates,the ATP and glutathione synthesis.The results showed that ISKNV replication was inhibited in CPB cells cultured in the presence of?–?-epigallocatechinmo nogallate?an inhibitor of glutamate dehydrogenase?or L-buthionine sulfoximine?an inhibitor of glutathione synthesis?.However,virus replication was rescued by the addition of multiple tricarboxylic acid cycle intermediates,ATP,or glutathione reduced ethyl ester.ATP and reduced glutathione/oxidized glutathione levels were increased in CPB cells infected with ISKNV,but were decreased in CPB cells cultured in glutamine-depleted medium.These results indicate ISKNV infection induces glutaminolysis to accommodate the biosynthetic and energy needs as well as GSH with suitable dose for its efficient virus replication.