Serotypes of HFMD-associated HEV-B species and genetic characteristics of the VP1 gene in coxsackievirus B2 and B5 strains isolated in Anyang area from 2011 to 2015

Abstract

Objective To investigate the serotypes of human enterovirus B (HEV-B) species causing hand, foot and mouth disease (HFMD) and to analyze the genetic characteristics of VP1 region in coxsackievirus B2 (CVB2) and coxsackievirus B5 (CVB5) strains circulating in Anyang area during 2011 to 2015. Methods Real-time RT-PCR and semi-nested RT-PCR were performed to identify coxsackievirus A16 (CVA16), enterovirus 71 (EV71) and other serotypes of enterovirus in order to obtain the complete etiologic composition of HFMD. The numbers of HEV-B serotypes and the percentages of specimens positive for every serotype in all enterovirus-positive specimens were calculated. As CVB2 and CVB5 were the predominant serotypes of HEV-B species, five pairs of primers targeting the VP1 regions of CVB2 and CVB5 were designed to obtain the complete nucleotide sequences of CVB2 and CVB5 VP1 regions. The phylogenetic trees were constructed based on the VP1 sequences obtained in this study and those submitted to GenBank by using MEGA7.0 and BioEdit7.2. The selection pressures on VP1 regions of CVB2 and CVB5 strains circulating in China in recent years were evaluated with the online program of DataMonkey. Results A total of 57 specimens that belonged to 14 serotypes of HEV-B species were detected in Anyang area from 2011 to 2015. The 14 serotypes of HEV-B species accounted for 56% of all serotypes of enterovirus and the specimens positive for HEV-B species accounted for 3.06% of all enterovirus-positive specimens. The HFMD cases caused by most of the HEV-B serotypes were sporadic cases. Small outbreaks of HFMD could also be caused by some serotypes of HEV-B such as CVB2 and CVB5. The complete sequences of VP1 region were obtained from 8 CVB2 strains and 9 CVB5 strains. The phylogenetic trees based on the VP1 sequences demonstrated that the CVB2 strains were classified into four genotypes (A-D). The mean evolutionary distances between different genotypes ranged from 0.191 to 0.208 and the similarities in nucleotide sequences ranged from 79.7% to 85.8%. The CVB5 strains were classified into 6 genotypes (A-F). The mean evolutionary distances and the similarities in nucleotide sequences between different genotypes of CVB5 strains ranged from 0.170 to 0.285 and 76.0% to 86.8%, respectively. Strains of different genotypes varied significantly in the residues on positons 157 and 263 in the VP1 region of CVB2 strains and on positions 75, 90 and 95 in the VP1 region of CVB5 strains. All of the CVB2 strains isolated in Anyang area belonged to D genotype and located intensively in one lineage. The CVB5 strains circulated in Anyang area belonged to F genotype and located in two lineages. The selection pressures on CVB2 strains of D genotype and CVB5 strains of F genotype circulating in China in recent years were 0.037 and 0.036, respectively. Six positively selected amino acid sites were found in the VP1 region of CVB5 strains, but no positively selected amino acid site was found in the VP1 region of CVB2 strains. Conclusion HEV-B species was an essential component of the etiologic spectrum of HFMD in Anyang area during 2011 to 2015, of which CVB5 and CVB2 were the predominant serotypes. The VP1 region of CVB5 was more complex and active than that of CVB2 over the course of evolution. Key words: Hand, foot and mouth disease; Enterovirus; Coxsackievirus B2; Coxsackievirus B5; Phylogenetic tree; Positive selection pressure