Receptors Capture And Preliminary Screening Of Spring Viremia Of Carp Virus

Spring Viraemia of Carp(SVC)is a highly infectious disease with high mortality in fish.The causative pathogen of this disease is spring viraemia of carp virus(SVCV),which is a member of the genus Vesiculovirus of the family Rhabdoviridae.SVCV predominantly affects cyprinid fishes,among which carp(Cyprinus carpio)is the most susceptible species.So far,the diagnostic methods of SVCV have been intensively studied.Howerver,the mechanism of the virus infection is still poorly explored and the identity of the receptor of SVCV to enter the host cells is unknown.Identification of the receptor of SVCV can help to understand the mechanism of the virus infection and provide a theoretical basis for the further study on vaccine and treatment.In this study,SVCV was cultured in a host cell line,epithelioma papulosum cyprinid(EPC).Then SVCV particles were isolated and purified by sucrose density gradient centrifugation.The virions were found to enrich between 30% and 45% sucrose layers.After labelled by trifunctional crosslinkers Sulfo-SBED or ASB,SVCV was incubated with the membrane proteins of EPC,or living cells of EPC.Binding proteins of SVCV were captured with the crosslinkers and purified with affinity chromatography using streptavidin packed column.Binding proteins of SVCV were analyzed by reversed phase nano ESI-LC-MS an d submitted to perform the database search analysis.The customized protein sequence database was generated RNA-Seq data of EPC in this study.As a result,578 proteins were captured by Sulfo-SBED,comparing with 159 in the control group,and 415 pr oteins were captured by ASB,comparing with 287 in the control group.The proteins captured only in the experimental groups were picked out for the further study.Trans membrane helices in proteins were predicted using online service of TMHMM-2.0(htt ps://services.healthtech.dtu.dk/service.php?TMHMM-2.0).The result showed that 29 prot eins and 21 proteins might be the membrane proteins in the above SVCV binding proteins captured by Sulfo-SBED and by ASB respectively.Six of these proteins were captured in both methods.In these membrane proteins,36 proteins showed high homo logy with proteins that located on mitochondria or ribosomes and other organelle mem branes by blast analysis.Eight proteins showed high homology with plasma membrane proteins which were supposed to be the potential SVCV receptors and were used to be verified in the following RNA interference(RNAi)experiments.The 8 proteins wer e Unigene12835,Unigene13423,Unigene19308,Unigene7122,CL1989.Contig1,CL3290.Contig2,CL3871.Contig1 and CL51.Contig3,respectively.Small interference RNA(siRNA)was obtained by digestion of double-stranded RNA(ds RNA)using enzyme RNase Ⅲ in vitro.Si RNA mixture(siRNA mix),the enzyme-digested product,was isolated and purified by agarose gel electrophoresis and gel extraction.Then siRNA mix was transfected into EPC,and EPC cells were sampled at 24 h post-transfection.Reverse real-time quantitative PCR(RT-q PCR)was carried out to determine the relative expression level of target genes.The results showed that the expression of the target genes was significantly down-regulated about 50% after both 0.1 nmol/L and 0.5 nmol/L siRNA mix transfection(P<0.05).The siRNA mix of eight research genes was combined in pairs,with a total of 28 combinations.The siRNA mix of the two genes in the combination was transfected into EPC.SVCV was added to infect the EPC cells at 36 h post-transfection.Cells and the culture medium were collected at 48 h post SVCV challenge.The proliferation of SVCV was determined by RT-q PCR.The results showed that only the combination of CL3290.Contig2 and CL51.Contig2 had significantly inhibited the proliferation of SVCV comparing with the Mock group,which indicated that CL3290.Contig2 and CL51.Contig2 could be the receptors or co-receptors of SVCV.The two genes were transfected and checked seperately.As a result,proliferation of SVCV was inhibited not only by the knock-down of CL3290.Contig2 but also by knock-down of CL51.Contig2,suggesting the high possibility that CL3290.Contig2 and CL51.Contig2 are the receptors of SVCV to enter the EPC cells.