Genome Characteristics And Cell Infectivity Of Coxsackievirus A19

Background:Hand,foot and mouth disease(HFMD)is an acute infectious disease that has been a serious health hazard to children.It can be caused by more than 20 types of enteroviruses(EVs),most of which are members of Enterovirus A Species(EV-A).In recent years,HFMD caused by EVs other than enterovirus 71 and coxsackievirus A16 increased.We previously detected gene fragments most similar to coxsackievirus A19(CV-A19)from the other EVs that caused HFMD locally.CV-A19 belongs to Enterovirus C Species(EV-C)and it is a member of difficult-to-cultivate coxsackieviruses A(CV-A).It had been isolated from specimens of respiratory and gastrointestinal diseases,and was associated with some serious neurological diseases.However,CV-A19 causing HFMD had not yet been reported.And there was no report of CV-A19 in China.Objectives:1.Sequencing the whole genome of the CV-A19 strain and analyzing the characteristics of the genome;2.Prokaryotic expression of recombinant CV-A19 VP1 protein and preparation of polyclonal antibodies;3.Infection of cells with CV-A19 and exploring the infectivity of CV-A19.Methods:1.Primers were designed with the primer walking method and reverse transcriptionpolymerase chain reaction(RT-PCR)was performed to amplify the intermediate sequence of the CV-A19 genome.Rapid amplification of c DNA ends(RACE)was used to amplify the3′ and 5′ ends of the genome.After sequencing analysis,the sequence of the whole genome was assembled,and the similarities between the sequence of the CV-A19 strain and the reference sequences were analyzed.The evolutionary trees were constructed,and the gene recombination was analyzed.2.The recombinant prokaryotic expression plasmid p ET-28a(+)-CV-A19 VP1 was constructed,transformed into E.coli BL21(DE3)competent cells.The cells were induced to express recombinant CV-A19 VP1 protein.After purification,the protein was used as antigen to immunize mice to prepare polyclonal antibodies,and determine the titer by Western blot.3.The human lung(bronchial)epithelial cells BEAS-2B,human rhabdomyosarcoma(RD)cells and the skeletal muscle cells of neonatal mice were infected with CV-A19.The cytopathic changes were observed.The CV-A19 VP1 protein in the cells showing cytopathic effect was detected by immunoperoxidase monolayer assay(IPMA),and the 3′ end of the CV-A19 VP1 gene was tested by RT-PCR.Results:1.The sequence of the whole genome(7409 bp)of the CV-A192019103106/XX/CHN/2019 strain was obtained,and submitted to the Gen Bank(Gen Bank No.: MT175706).By analyzing the sequence of this strain of CV-A19,the results showed that the similarity between the full-length nucleotide sequence of VP1 and that of the prototype strain NIH8663(Gen Bank No.: AF499641)was 86.4%,and the similarities among it and those of the existing strains of CV-A19 abroad were 76.0%～93.1%.The similarities among the full-length nucleotide sequence of its genome and those CV-A19 strains with full genome or full coding sequences(CDS)abroad were 79.8%～89.5%.CV-A19-BBD/BAN/2009(Gen Bank No.: KC785531),EV-C113-BBD-48/BAN/2006(Gen Bank No.:KC344833)and EV-C116-126/RUS/2010(Gen Bank No.: JX514942)might be the genetic donors of this strain of CV-A19.2.The recombinant prokaryotic expression plasmid was successfully constructed.The recombinant CV-A19 VP1 protein showed good antigenicity.The purified recombinant protein was used to immune mice,and the serum titer determined by Western blot was no less than 1:32000.3.BEAS-2B and RD cells were infected with CV-A19.After blind passage for three generations,there was no significant difference between the infected cells and the mockinfected cells.However,the skeletal muscle cells of neonatal mice infected with CV-A19 were significantly different from the mock-infected cells and the cells shrinked and aggregated.For the infected skeletal muscle cells of neonatal mice,both the results of IPMA using the mouse anti-CV-A19 VP1 polyclonal antibodies and the RT-PCR test with the primers for the 3′ end of CV-A19 VP1 gene were positive.Conclusions:1.The full genome of the CV-A19 2019103106 strain is 7409 bp,which is 79.8%～89.5%similar to the existing strain,and may be a recombinant of 3 strains of virus.2.The Western blot titer of the prepared mouse polyclonal antibodies against recombinant CV-A19 VP1 protein is no less than 1:32000.3.Infection of neonatal mouse skeletal muscle cells with CV-A19 can cause cytopathic effects.