Construction Of An Infectious CDNA Clone Of Encephalomyocarditis Virus NJ08 Strain From Pigs And Biological Characteristics Of The Rescued Virus

Encephalomyocarditis virus?EMCV?belonging to a member of the genus Cardiovirus in the family Picomaviridae,is a single-stranded positive-sense and non-enveloped RNA virus,which has been recognized worldwide as an important zoonotic pathogen.EMCV infection was able to result in acute fatal myocarditis in piglets,whose mortality rate could be up to 100%.In this study,on the base of EMCV NJ08 strain from the middle east in china,we had established successfully reverse genetic system with the use of transcription method in vivo and achieved recombinant virus.Subsequently,the biological characteristics of rescued virus was also identified,and the immunogenicity of which has been researched further.The system laid a good foundation for the future in-depth study of the molecular mechanism involving in pathogenicity,gene function,the development of new vaccines.The specific studies were as following:1.Construction and characterization of infectious cDNA clone of encephalomyocarditis virus from pigsIn this study,the full-length genomic cDNA of EMCV NJ08?GenBank HM641897?was amplified by RT-PCR in three overlapped fragments?A/B/C?.the genetic marker?EcoRI?has been introduced into fragment C by a silent mutation at nucleotide position 6243?T?A?.Then three fragments was cloned into pEasy-simple Blunt cloning vector to acquire real sequence,which were assembled sequentially in CMV-p plasmid to construct a full-length cDNA clone pCMV-NJ08 with PacI?XhoI and NheI.Direct transfection of BHK-21 cells with both the pCMV-NJ08 and lipofectamine allowed the discovery of obvious cytopathic effect in BHK-21 cells.The recombinant virus has been obtained through indirect immunofluorescence assay?IFA?and the identification of a unique marker sequence.The rescued virus had similar growth characteristics with parental virus in terms of the TCID₅₀,plaque assay and growth kinetics curve on BHK-21 cells.However,the virulence in mice significantly reduced.The availability of the infectious cDNA clone provided a genetic platform for studying gene function and the rational design of the virus vaccines.2.The immunological characteristics of killed recombinant encephalomyocarditis virus in miceIn this study,BEI?final concentration 0.002mM?was used for the inactivation of recombinant virus?rNJ08?and parental virus?NJ08?.Two kinds of adjuvants containing ISA15A and ISA206 were used to prepare the inactivated rNJ08 and NJ08 vaccines.Forty six-week-old ICR mice were randomly divided into eight groups.Groups 1-2 were subcutaneously injected with killed rNJ08 and NJ08 vaccine(10⁷TCID₅₀/0.2ml)prepared with ISA15A.Groups 3-4 were subcutaneously injected with inactivated rNJ08 and NJ08 vaccine(10⁷TCID₅₀/0.2ml)prepared with ISA206.Groups 5-7 were respectively vaccinated inactivated rNJ08 vaccine containing various amount of antigen(10⁶TCID₅₀/0.2ml,10⁵TCID₅₀/0.2ml,10⁴TCID₅₀/0.2ml)prepared with ISA15A.The group 8 was injected with 2%DMEM as the negative control.The ELISA antibody titers were detected at 3 and 6 weeks post vaccination,and neutralizing antibody and lymphocyte proliferation were detected at 6 weeks post vaccination.The results showed the ELISA antibody levels and neutralizing antibody titers shared significant antigen dose-dependent manner in rNJ08 vaccine-inoculated groups,which increased significantly in all groups after booster immunization?above 1:16?.Lymphocyte proliferation conversion experiments indicated that the stimulation index among four groups inoculated same doses of antigen were approximately 3.0.The results indicated that rNJ08 and NJ08 had similar immunogenicity,which were able to effectively induce a good immune response and the production of neutralizing antibody.Inacti-vated rNJ08 was more secure due to its less virulence.