Construction And Application Of An Encephalomyocarditis Virus-HB10 Infectious CDNA Clone

Encephalomyocarditis virus(EMCV) can infect a wide range of animal species, including rodents, pigs, and elephants, nonhuman primates, and human infections have also been reported. EMCV is a nonenveloped, positive single-stranded RNA virus, belonging to genus Cardiovirus within the family Picornaviridae. It has an approximately 7.8 kb viral genome which is composed of a unique coding sequence flanked by untranslated regions(UTR). The 5’UTR comprises a poly(C) tract and an internal ribosome entry site(IRES), which allows direct initiation of translation of the single viral polyprotein. The 3’UTR terminates with a heterogeneous poly(A) tail. The viral genome encodes a polyprotein precursor, which is divided into the P1, P2 and P3 regions. Polyprotein cleavage is carried out mainly by the 3C protease of EMCV. However, the first “cleavage” occurs during elongation, prior to the synthesis of the 3C protease, between the 2A and 2B proteins, due to an inherent instability of the peptide chain at the NPG(P) sequence.The aim of this study is to construct the infectious c DNA clone of EMCV-HB10 strain using reverse genetic technique. The full length c DNA of EMCV-HB10 strain was obtained by RT-PCR, and inserted into a low-copy plasmid vector p OK12. In order to rescue the recombinant virus, the recombinant plasmid was linearized with Bam H I and the transcript RNA was synthesized in vitro. The transcripts were transfected into BHK-21 cells and typical cell pathogenic effect(CPE) was observed after 36 h. The full length genome was amplified by RT-PCR to identify the rescued recombinant EMCV virus(r EMCV-C9). In compared with the 5’UTR of parental virus, the results indicated that the recombinant EMCV virus contains 9 bp poly(C) tract(C9) in comparison with the C7TCTC3TC10 poly(C) of the parental virus EMCV-HB10, which in turn served as a genetic marker to identify the c DNA-derived virus r EMCV-C9. All nucleotide sequence except the short poly(C) tract(C9), was authentic to that of the wild-type EMCV-HB10. The expression of VP1 was detected by indirect immunofluorescence assay(IFA) and the rescued virus had similar growth characteristics compared with its parental virus, suggesting the the short poly(C) tracts did not affect the expression of VP1 and viral rescue. In vivo, LD50 values of r EMCV-C9 is 2×104 TCID50, with 213 times the LD50 of wild type EMCV-HB10, showing a lower pathogenicity in mice. Our dates proved that the length of the poly(C) tract of EMCV-HB10 does not play a significant restrictive role for viral infectivity in BHK-21 cells, while related to its pathogenicity in mice. Our results showed that r EMCV-C9 was partially attenuated in mice.The ability to express heterologous antigens from the attenuated mengo strain suggests that EMCV strain could be used as a live vectored vaccines. Here, short poly(C) tract(C9) c DNA clone of the EMCV-HB10 strain provided an essential tool for development of novel engineering vaccine against EMCV. To obtaina an avirulent clone of EMCV-HB10, we passed the EMCV 190 times in BHK-21 and isolated a clone EMCV-F190-C25 by plaque assay. Sequence the EMCV-F190-C25 clone revealed some amino acid mutations and a deletion of 127 amino acids in the 2A protein compared with its parental EMCV-HB10. We cloned the partial genome of EMCV-P190-C25 into p EMCV-C9 and obtained a novel recombinant virus vector, designated as p C9-P190-?2A. In addition, a plasmid was constructed by introduction sequence of a duplicated primary cleavage cassette and a multiply clone site at the junction between VP1 and Δ2A genes. A flag tag was also inserted to the virus vector, facilitating detection of the heterologous protein expression. The recombinant plasmid was designated as p C9-P190-Flag-MCS-?2A. The plasmid was linearized with Bam H I and the transcript RNA was infectious in BHK-21 cells. In vivo, mice infected with this recombinant viral vectored r C9-P190-Flag-MCS-?2A did not die and exhibited no clinical signs. We inserted the c DNA of e GFP protein into the MCS of p C9-P190-Flag-MCS-?2A and the obtained a rescued recombinant virus, designated as r C9-P190-Flag-e GFP-?2A. The recombinant virus could express the e GFP protein at least within five passages in BHK-21 cells. We found that e GFP was expressed in two forms: VP1 fused e GFP and free e GFP. Taken together, we successfully constructed a full-length c DNA infectious clone of EMCV-HB10 and isolated an EMCV HB10-derived virus which harbors the 127 aa deletion in the 2A protein. The novel established avirulent viral vectors with duplicated primary cleavage cassette provided an effective system for the expression of exogenous proteins and potential for viral tracking.