Genetic Characterization And Suramin Sensitivity Analysis Of Enteroviruses A Circulating In Qingdao

HFMD is classified as a class C infectious disease,and the main affect crowd is children under 5 years old,especially infants aged 0-2 years.Two major pathogens of HFMD are EV-A71 and CV-A16,and other enteroviruses including coxsackievirus A2-A8,A10,A12,B2,B5,Echovirus can also cause HFMD.Usually EV-A71 infection is more likely to cause neurological disease,which can lead to death in severe cases.This study focused on EV-A71,CV-A16,CV-A6,CV-A10,all of which belong to Picornaviridae family,enteroviruses genus.The viral genome is a single-stranded,positive-sense RNA.HFMD is widely popular at home and abroad.For a long time,EV-A71 and CV-A16 are the main epidemic viruses and the dominant strain was alternation between these two genre.But in recent years,CV-A6 has become the third major pathogen of HFMD,and the incidence of HFMD caused by CV-A10 is on the rise.CV-A10 is the intestinal virus that can cause severe disease alone without EV-A71.At present,the progress of vaccine and antiviral compounds in hand,foot and mouth disease is slow,only enterovirus type 71 inactivated vaccine(Vero cell)is successfully listed.Vaccines of other enteroviruses are still in test or clinical research phase,and there is no efficacious drug on the market.The research has been divided into three parts.The first part studies the genetic characteristics of isolated strains in Qingdao.There are 12 strains of clinical samples coming from the Qingdao municipal center for disease control and prevention in this study.Through blind passage,we got 12 stable strains(3 strains of EV-A71 strain,3 strains CV-A6 strain,4 strains CV-A10 strain,2 strains of CV-A16 strain).Then by RNA extraction,reverse transcription,PCR amplification and sequencing,we obtained VP1 sequence.VP1 gene of EV-A71,CV-A6,CV-A10,CV-A16 was analyzed through phylogenetic analyses.Parts of isolated viruses were used for subsequent antiviral experiment.Evolution analysis found that the strains isolateddiffer greatly in different regions.For CV-A6 isolated strains of Qingdao,the sequence of VP1 is highly similar.1 strain of 4 CV-A10 strains had a distant evolutionary distance from other strains.In 2013,2015,2016,there was a certain distance from the evolution tree of CV-A16,indicating that CV-A16 has more popular subtypes in Qingdao.The second part is to compare the sensitivity to Suramin.All EV-A71 viruses with same MOI were inoculated in RD cells to compare the sensitivity of Suramin.A dose-response experiment was conducted to calculate the IC50.Then analysis the data,we found that it had no outstanding antiviral effect on epidemic strains in different regions.Previous studies found that Suramin also had certain inhibitory effect on partial coxsackieviruses serotype,so in the study,coxsackieviruses isolated from Qingdao clinical samples were used to analyze the sensitivity of Suramin.We concluded that all CV-A6 strains,part of CV-A10 strains had obvious inhibiting effect,but the inhibition effect of CV-A16 strain is not obvious.Compared with other CV-A strains,EV-A71 is more sensitive than CV-A.The third part is preliminary study of Suramin's mechanisms.In a previous study by Ren et al.(EMI,2014),the STD-NMR experiment indicated Suramin binds to the EV-A71 particle via the naphthalenetrisulfonic acid group.They found the Suramin napthalenetrisulfonic acid moiety H7 and H8 protons,displayed the strongest signal intensities,indicating close proximity of these protons and consequently that part of the molecule to the EV-A71 capsid.In this study,we compared the sensitivity of Sodium the naphthalene sulfonic acid and Suramin with EV-A71 FY 573 and we found only naphthalene sulfonic acid had no antiviral effect.The molecular structure of Suramin contains two three naphthalene sulfonate monomer,may be the two monomers' combined effect which confers its inhibiting function.At last,study of the mechanism of action by time-of-addition assay to analyze the inhibition process.The results were different from the previous studies,and the reasons were not taken into account that the virus entered cellular dynamics.In general,we got 2 strains of EV-A71 in 2015,1 strain EV-A71,3 strains of CV-A6 in 2015,4 strains of CV-A10 in 2014,1 strain of CV-A16 in 2015,and 1 strain of CV-A16 in 2016.For EV-A71,the epidemic strains in Qingdao were not in the same branch as those in Fuyang and Shanghai,and 3 strains of CV-A6 and the 4 strains of CV-10 had little evolutionary difference.The 2 strains of CV-A16 were significantly different.In the sensitivity analysis of Suramin in all strains,it was found that there was not a significant difference in sensitivity between the isolated strains of Qingdao and the other strains in other parts of the Chinese mainland.The inhibition mechanism of Suramin was preliminarily studied to predict the combined effect of the two naphthalene trisulfonate monomers in the structure.At present,the inhibition stage cannot be accurately guessed through the results.