Prokaryotic Expression Of VP1 Gene Of Swine Encephalomyocarditis Virus, Serological Survey And Identification Of EMCV Isolates

Porcine encephalomyocarditis is an acute infectious disease of swine caused by encephalomyocarditis virus (EMCV). This disease is clinically characterized by encephalitis, myocarditis or inflammation around cardiac muscles. In this study, the structural protein VPl gene of EMCV was successfully cloned and expressed. And the expressed recombinant protein VPl was used as the antigen to develop an indirect ELISA for detection of antibody against EMCV. A serological survey was carried out for pig farms from some areas of China by using the established assay. Five encephalomyocarditis virus (EMCV) strains in pigs were isolated and identified from clinical tissue samples. The cDNA of VPl and partial 3D genes for all the isolates were sequenced and analysed.The complete VPl gene of EMCV was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-6P-l. The recombinant protein VPl could amount to 27.1% of the total mass of bacterial proteins after induction with 0.1 mmol/L IPTG. Analysis by SDS-PAGE showed that the molecular weight of the expressed product was 60.0 kDa. And Western Blotting analysis indicated that the purified recombinant protein could be recognized by polyclonal anti-serum of swine prepared with EMCV.Using purified recombinant protein VPl as coating antigen, an indirect ELISA was developed for detection of antibody in serum to EMCV. The optimal reaction conditions for the assay were determined. The results indicated that the optimal coating concentration of recombinant protein VPl was 0.625 μg/mL, the dilution of serum sample was 1:50, the working concentration of HRP-labeled rabbit anti-porcine IgG was 1:800, serum sample for detecting and HRP-labeled rabbit anti-porcine IgG should be incubated at 37℃ for 45 min, the threshold for serum sample was 0.3. The indirect ELISA had good specificity and sensitivity compared with IFA. 1786 serum samples collected from intensive pig farms in 13 regions of China were examined using the ELISA, showing that the average positive rate of EMCV antibody was 75.84%. It was concluded that EMCV infection has prevailed in China.Five EMCV strains were isolated from the clinical tissue samples orignated from pig farms in Beijing, Tianjin, Hubei, Hebei and Liaoning by using BHK-21 cells. These isolates were designated EMCV BJC3, EMCV HB1, EMCV HuB1, EMCV LN1 and EMCV TJ1 respectively and identified using IFA. The virus particles with diameter of 27 nm were observed by immune electron microscopy (IEM). Results of physicochemical characteristic tests indicated that EMCV BJC3 and EMCV HB1 were not able to endure acid and not sensitive to chloroform either. However, the two isolates had different sensitivity to trypsin. Exposed under 60℃ for 1 h, the viruses could be inactivated and protection of bivalent cation at temperature of 50℃ was negligible.The VPl genes and partial 3D genes of the isolates were sequenced. Sequence analysis revealed that the nucleotide identity of VPl gene among the isolates was above 99% and the predicted amino acid identity was above 97.5%. The nucleotide identity of VPl genes between EMCV strains inGenBank database and the five isolates ranged from 81.61% to 99.69%. Correspondingly, amino acid identity was above 95.5%. The identity of 3D gene amplified of both the nucleotide and amino acid was 100% among the isolates and others. A phylogenetic tree was constructed based on the nucleotide sequences of VP1 gene of EMCV, presenting that the five isolates are located on the backbone of the tree with lesser variation.