Isolation And Identification Of HEV71 Strains From Mild Or Severe Children With Hand-foot-and-mouth Disease And Analysis Of Their VP1 Genotypes

Background:Human enterovirus 71 (HEV71), a single positive stranded RNA virus in EV-A virus group, is the major pathogen of human hand-foot-and-mouth disease (HFMD). The capsid of HEV71 is composed with four viral proteins named VP1, VP2, VP3 and VP4. The VP1 contains the celllular receptor-binding sites, epitopes of neutralizing antibody and drug-binding sites. Therefore, the VP1 plays important roles in viral infection, anti-virus immune response and drug treatment effect. When the VP1 function-associated important sites are mutated, the virulence and drug resistance of HEV71 are subsequently changed, which will have a significant influence on the severity of HFMD patients’ conditions. In the VP1 sequence of HEV71, there is a picornavirus capsid protein domain-like (rhv_like) domain that contains nine drug-binding sites. According to the diversity of VP1 sequences, HEV71 can be divided into A, B0～5 and C1～5 genotypes, and the predominant HEV71-VP1 genotypes may be various in differect areas. Thus, investigation of the predominant VP1 genotyoes of HEV71 in differect areas and the mutation of drug-binding sites in their rhv_like domain is significant for prevention, controlling and therapy of HFMD.Objective:The aim of this study was to isolate the HEV71 strains from the HFMD-suffering children with different pathogenetic conditions, and then determine the VP1 genotypes of the viral isolates and the mutation of drug-binding sites in the rhv like domain.Methods:The HEV71 strains were isolated from the samples of four mild and four severe HFMD children in Shaoxing area of Zhejiang province in 2014, and then the viral isolates were identified by RT-PCR. The entire VP1 genes in all the HEV71 isolates were amplified by RT-PCR, followed by sequencing of PCR products. Using professional bioinformatic softwares, the VP1 genotypes of HEV71 isolates and the mutation of drug-binding sites in the rhv_like domain of VP1 genes were determined. Moreover, VP1 gene-based phylogenetic evolution tree of the HEV71 isolates was generated.Results:All the viral strains isolated from the samples of eight HFMD children were identified as HEV71 according to the results of HEV and HEV71 RT-PCRs. Compared with the VP1 genotypes of reference standard strains of HEV71 in GenBank, the highest identities of nucleotide and amino acid sequences of the viral isolates (97.2%-98.8% and 99.3%-99.7%) were shown with the C4a genotype. The mutation at the 179th drug-binding site in the two HEV71 isolates from two severe cases of the HFMD children was found. Compared with the reported VP1 genotypes of HEV71 from domestic and overseas areas, five of the eight HEV71 isolates presented the higher genetic relationship with those from Anhui and Hunan areas of China, while another three HEV71 isolates presented the higher genetic relationship with those from Shanghai and Shandong areas of China.Conclusions:All the HEV71 strains isolated from the HFMD children in the local area with different pathogenetic conditions belong to VP1-C4a genotype, and the mutation at the 179th drug-binding site in the rhv_like domain of VP1 genes may be associated with severity of the disease.