Construction And Investigation Of An EGFP Chimeric CDNA Infectious Clone Of Coxsackievirus A16

Coxsackievirus A16(CA16),which belongs to the Picornavirus virus family and the Enterovirus genus,is one of the major pathogens that cause hand,foot and mouth disease(HFMD)outbreaks globally.The main clinical symptoms of CA16 infection include a typical maculopapular rash and blisters on the hands and feet and in the mouth,with most cases being mild and self-limiting.However,a small number of individual cases present severe conditions like aseptic meningitis,brainstem encephalitis,pulmonary edema and pulmonary hemorrhage which ultimately lead to death.Its outbreak has become a major social and public health problem that endangers the health of infants and young children.Currently,there is still a paucity of drugs with special effects drugs and vaccines for the prevention and treatment of CA16 infection.The infectious clones constructed by using reverse genetics solves the problem of difficulty in operating RNA virus genome serve as a powerful tool for studying RNA viruses at the level of cDNA.The virus genomic RNA can be obtained from infectious clones via in vitro transcription after mutation,deficiency and insertion operations were performed on the virus genome in vitro,by transfecting into the susceptible cells and obtaining virus rescue.By comparing the effects of these operations on phenotypes and characteristics,we provide a new wayon studies on the structure and function,viral replication mechanisms and pathogenesis of RNA genes.In this study,we firstly constructed the full-length cDNA infectious clones of CA16 genome.With the CA16(G20 strain)genomic RNA as the template,we obtained the full-length cDNA of CA16 genome via amplification and cloned it into vector.Based on the T7 polymerase system andusing the linear recombinant plasmid as the template,we carried out in vitro transcription and obtained the CA16 virus genomic RNA,and used the CA16 genomic RNA to transfect Vero cell to for virusrescue.After identification using genomic sequence analysis,RT-PCR,immunofluorescence and transmission electron microscopy,we confirmed that the virusrescue was CA16.The charateristics of plaque morphology,replication kinetic curve and the pathogenicity induced by the CA16 rescued virus were consistent with those resulted from the CA16 wild-type virus,thereby these results suggested that the CA16 rescued virus possessed the overall biological features of CA16 wild-type virus.After we successfully constructed the CA16 genome full-length cDNA clones and obtained the CA16 viruses rescue,we,through the reverse genetics method,inserted the EGFP gene between the 5'UTR and VP4 genes of the CA16 virus genome by fusion PCR,and successfully constructed the full-length cDNA infectious clones of EGFP chimeric CA16 genome by introducing the 2A protease cleavage site between EGFP and VP4 genes.After genomic sequence analysis,RT-PCR,Western blot,immunofluorescence,transmission electron microscopy,in vitro Real-time PCR,fluorescence intensity observation,fluorescence quantitative analysis and frozen section fluorescence observation in vivo,we successfully obtained EGFP chimeric CA16 virus of green fluorescence protein reporter gene.The expressions of EGFP observed by fluorescence microscopy can directly indicate the growth of the virus.The plaque morphology,in vitro proliferation kinetic curveand the pathogenicity in neonatal mice of the EGFP chimeric CA16 virus were consistent with CA16 wild-type virus basically.However,the analysis of the survival of infected neonatal mice,theviral load in relevant tissues and pathological changes revealed thatthe pathogenicity of the EGFP chimeric CA16 virus rescuein neonatal mice was slightly lower than CA16 wild type virus.In this study,we applied reverse genetics to successfully obtain the CA16 resued virus and EGFP-CA16 virus,which not only proived a reasonable therotical support for further studying on the genetic function of CA16 virus,finding out the virulence locus,examining the infectious conditions of CA16 in cells,and fracking the localization of CA16 virus,but also further offered a novel strategy on studies of CA16 pathogenesis,screening of anti-CA16 drugs and development of vaccines.