Screening Of Enterovirus 71 VP1 Candidate Virulence Sites And Their Effects On Viral Replication

Hand,foot and mouth disease(HFMD),which caused by a variety of enterovirus,mainly infects infants and children fewer than 5 years old.The main clinical symptoms are fever and rash or herpes in hand,foot and mouth of patients.However,neurogenic complications such as sterile meningitis and pneumonedema can be found in severe cases and even lead to death.Hand foot and mouth disease(HFMD)has become a major public health concern around the world,especially in the Asia-Pacific region.Enterovirus 71(EV71),which belongs to family Picornaviridea,genus Enterovirus,is one of the major causative agent of HFMD,especially in severe and death cases.The neuropathogenesis of EV71 is still unclear,while several studies have revealed that sites which affect the viral virulence are exist in multiple regions of the viral genome.In this study,viruses from samples of HFMD patients and their close contacts were isolated,sequenced and analyzed in order to capture the variation during viral transmission.Two sites were screened in VP1 region and reverse genetics method was used to research the roles of VP1 candidates played in the pathogenesis of EV71.Objective:1.To isolate,sequence and analyze the virus from samples of HFMD cases and their close contacts,capture the variation during viral transmission and screen candidate virulence sites.2.To construct and rescue recombinant virus by replacing the VP1 region between mild and severe strains.3.To explore the role of VP1 candidate virulence sites played in the pathogenesis of EV71 by comparing the capacity of replication,pathogenicity and inducing host autophagy between original and recombinant strains.Methods:1.Samples of HFMD cases and their close contacts were pretreated and viruses were isolated by RD cells and detected by specific primers.2.Primers which designed to amplify complete genome of EV71 were used to amply isolated viruses,and the sequences of viruses were analyzed by software DNAstar 7.1 and MEGA6.3.Recombinant strain SDLY107-VP1 which VP1 region of mild strain was amplified and replaced into severe strain SDLY107,was constructed and rescued.Cytopathic effect assay,PCR and indirect immunofluorescence assay were used to identification the rescued virus,and CCID50 assay and plaque test were used to detect the titer of the rescued virus.4.The difference of the capacity of replication,pathogenicity and inducing host autophagy between original and recombinant strains were studied on SH-SY5Y,U87 and Vero cells.Results:1.Two EV71 strains were isolated successfully and named strain SDJN2015-01 and strain SDJN2015-01.1.Two strains were all belong to genotype C4a.2.Six amino acid mutation sites were found between two strains,among them two sites were located in VP1 protein(K98E and A133T)and four sites were located in 2C protein(D48N,V1261,1238V and N314Y).3.Recombinant strain SDLY107-VP1 was successfully rescued and the titer was detected.4.The capacity of replication,pathogenicity and inducing host autophagy between original and recombinant strains were similar in U87 and Vero cells,while significant differences were observed in SH-SY5Y cells.Cytopathic effect(CPE)cannot be induced by recombinant strain SDLY107-VP1,as well as significant cell injury and autophagy in SH-SY5Y cells.These were similar with mild strain SDJN2015-01,but shown obvious difference with severe strain SDLY107.Conclusion:1.New EV71 strains which belonged to genotype C4a were isolated from samples of HFMD cases and their close contacts.2.Successfully constructed and rescued recombinant strain SDLY107-VP1.Neurological virulence of EV71 might be influenced VP1 protein and the capacity of replication,pathogenicity and inducing host autophagy in nerve cells were affected by the mutation of 146th and 147th amino sites.