Isolation And Identification Of Encephalomyocarditis Virus And Its Genomic Sequencing And Analysis

Encephalomyocarditis?EMC?is an infectious disease caused by Encephalomyocarditis virus?EMCV?that can infect a variety of animals.EMCV is a non-enveloped single-stranded positive-sense small RNA virus.Pig is the most susceptible animal,pig infection can lead to acute myocarditis and respiratory failure in piglets,pregnant sows reproductive disorders.To date,approximately 40 EMCV strains have been isolated from different animals worldwide,and have caused great harm to the pig industry.At present,there are no specific therapeutic drugs and effective vaccines for prevention and control of the disease.Therefore,the isolation and identification of wild-type strains of EMCV and its whole genome sequence analysis research can lay a theoretical and material basis for the development of a new high-performance vaccine for the disease.In this study,heart samples of suspected EMCV abortion from a pig farm in Harbin,Heilongjiang Province,was treated and then cultured on hamster kidney cells?BHK-21?.The virus strain quickly showed typical cytopathic effect.The lesions showed cell rounding and opacification,and partial shedding and disintegration.The identification by RT-PCR and electron microscopy demonstrated of an EMCV field strain was successfully isolated and named EMCV HLJ strain.According to the EMCV sequence information uploaded on the GeneBank,four pairs of overlapping specific primers were designed to amplify the entire genome sequence of the EMCV HLJ isolate using primer design and sequence alignment software.Following the 5'RACE kit instructions,the 5'UTR end sequence of the virus was obtained too.Then the amplified PCR product was ligated into the pMD18-T vector.After successful identification,all the sequence was determined and spliced into the whole genome sequence of the virus.The genome of EMCV HLJ strain is 7,734 bp in length,in which the 5'UTR is 717 bp long and contains 16 bp of Ploy?C?.The 3'UTR is 138 bp in length and contains a 16 bp Ploy?A?tail,structural protein?VP4,VP2,VP3,VP1?and non-structural proteins?2A,2B,2C,3A,3B,3C,3D?,and these gene length is 210bp,768 bp,693 bp,831 bp,429 bp,450 bp,975 bp,264 bp,60 bp,615 bp,and 1384 bp,respectively.EMCV HLJ strain has high homology with the virus of domestic pig and mouse sources.Among them,99.7%homology with BD2 strain of domestic porcine isolate,and reached99.5%homology with domestic mouse isolate GX0602,and only 71.5%homology with the RD1338 isolates of German mouse.A phylogenetic tree was established based on the entire genome of EMCV,the major antigen protein VP1 and the most conserved 3D gene sequence,respectively.The phylogenetic tree of the whole genome sequence and the 3D genome sequence showed that the EMCV HLJ isolates were in group I and both has close relationship with GS01strain.The phylogenetic tree of the VP1 gene is different,although it is in group I,but it is more closely related to HN13 strain.This may be related to the higher antigenicity and variability of VP1 protein.In order to further understand the biological activity of the EMCV HLJ strain,the molecular characteristics and antigenicity of the major antigenic protein VP1 were analyzed.It was found that the main mutation in VP1 protein of EMCV HLJ strain was also concentrated in the 3-63 aa position,in which 20 aa?from valine to leucine?and 54 aa?from glutamic acid to lysine?were uniquely mutations.This might be related to the need for the virus to adapt to the new environment and cause mutations in the VP1 protein.The DNA Star software was used to analyze its hydrophobicity and antigenicity,and then used the 3D structural pattern map to find the antigenic determinants located on the VP1 protein.The five dominant epitopes were A₅EKGVTENTDATADFL₂₀,T₁₆ADFLAQPVYLP₂₇,P₁₅₄TTQVLHEVSS₁₆₄,T₁₇₀PQVYSAG₁₇₇,and N₂₄₄MRVFCPRPTVFFP₂₅₇,respectively.In order to further study the characteristics of the EMCV HLJ strain,the rabbits were immunized to produce EMCV whole virus polyclonal antibodies.The ELISA results showed that the antibody titer could reach 1:51200,IFA showed that the prepared antibody had good reaction with EMCV specifically,which provides a material basis for further research on EMCV characteristics and diagnostic methods in the future.