Analyses Of Diabetic Virulence Determinant On Encephalomyocarditis Virus VP1 Protein And The Construction Of EGFP Virus

Encephalomyocarditis virus(EMCV)is a zoonotic virus that belongs to Picronaviridae,Cardiovirus Genus.It is an enteric virus that transmits via fecal-oral route like other picornaviruses including Poliovirus,EV-71 and Coxsackievirus.EMCV has a wide range of hosts including rodents,pigs and primates and causes multi-systematic diseases including encephalitis,myocarditis,reproductive failure and diabetes.In the present study,we first constructed a novel CMV promotor-driven EMCV reverse genetic system and introduced a single mutation at the 152 amino acid threonine of capsid protein VP1 and analyzed its role of rendering diabetes post infection.Meanwhile,we also inserted the gene of enhanced green fluorescent protein(EGFP)prior to the 2A gene in the EMCV infectious clone to establish a chimeric virus that secrets EGFP,which will provide a powerful tool to EMCV investigation both in vitro and in vivo.In this study,we have cloned the full-length genome of an EMCV isolate BJC3 derived from an aborted swine fetus into the vector pRK5 that contains a CMV promotor to form a new EMCV reverse genetic system,and rescued the wild-type cloned EMCV progenies by transfecting the recombinant plasmid into BHK-21 cells.The growth kinetics of cloned virus on BHK-21 cells demonstrated that it shared similar proliferation properties with its parental virus and the full-length genomic sequencing results had confirmed the genetic stability of the cloned virus through passaging.Subsequently,we further analyzed the neuropathgenicity and tissue tropism of the cloned virus using mouse model and it indicated that cloned virus was less neurovirulent than parental virus BJC3 whereas maintained sound replication capability in vivo.It suggested that the novel CMV-driven infectious clone can efficiently and reliably provide cloned EMCV that resembles the in vitro and in vivo biological characteristics of wild type parental virus.It was previously acknowledged that the Alanine at 776 position of the EMC-D genome is the key virulent determinant that determines its diabetogenicity,however little is known about whether the mutations at the 776 amino acid are sufficient to change the encephalitic outcome into diabetic phenotype during the infection of neurotropic EMCV strains.Thus,we mutated the threonine into alanine at the 776 amino acid site of the genome and tested the infection and proliferation characteristics of mutant virus EMCV-T776A in pancreatic cell line ?-TC-6.It was exhibited that T77A mutant virus showed comparable growth kinetics with the recombinant parental EMCV-RV in BHK-21 and ?-TC-6 cells,indicating this mutation does not alter the pancreatic cell tropism of EMCV in vitro.We than inoculated C57BL/6 and ICR mice with indicated doses of T776A and looked at whether the alanine is the viral determinant that results in the gain or loss of diabetogenicity of BJC3.However,the results showed that the blood glucose level of inoculated mice was mildly elevated at 7 dpi compared to the PBS treated mice,while it was still much lower than the blood glucose threshold of diabetic criteria.It was implied that the 776 amino acid does not enhance the pancreatic tropism of neuropathogenic EMCV stain in vivo.Chimeric EMCV with EGFP insertion is a powerful tool that can be utilized in the investigation of biological characteristics both in vitro and in vivo.Therefore,we introduced the EGFP coding gene at the downstream flank of the genome sequence that encodes EMCV 2A protein on the basis of previously constructed CMV-driven infectious clone.We obtained the EGFP chimera by transfecting BHK-21 cells with the recombinant plasmid and tested the genomic replication,protein synthesis and the infectivity of chimeric viral particles by real-time PCR,IFA and TCID50 assay.The collective data demonstrated that recombinant plasmid transfection can produce viable chimeric virus progenies expressing EGFP in BHK-21 cells,whereas the chimeric virus exhibited hampered replication capacity and produced gradually diminished green fluorescence signals through passing,indicating that EGFP genetic insertion impaired the assembly and release of chimeric virus particles and the accumulated depletion and mutation during continuous passaging gradually led to the malfunction of EGFP.