Objective To investigate the serotypes of human enterovirus B ( HEV-B) species cau-sing hand, foot and mouth disease ( HFMD) and to analyze the genetic characteristics of VP1 region in cox-sackievirus 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 pre-dominant 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 phylogenet-ic 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 cir-culating 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 speci-mens positive for HEV-B species accounted for 3. 06% of all enterovirus-positive specimens. The HFMD ca-ses 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 dem-onstrated 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 geno-type 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 se-rotypes. The VP1 region of CVB5 was more complex and active than that of CVB2 over the course of evolution.

Objective: To study the VP1 gene of coxsackievirus A6 (CVA6) and to reveal the molecular epidemiologic characteristics of CVA6 related to hand, foot and mouth disease (HFMD) in Anyang city from 2011 to 2015. Methods: Serotypes of human enterovirus (HEV) were detected with the real-time RT-PCR from the clinical specimens. Primers were designed and used to amply and sequence the VP1 region of CVA6. Phylogenetic tree was constructed and phylogenetic analysis was performed. Selection pressures of the VP1 gene were evaluated for different subgenotypes of CVA6 circulating in China in recent years. Results: A total of 365 specimens with CVA6 positivity were identified in Anyang city during 2011—2015. CVA6 specimens accounted for 19.59% of all HEV-positive specimens. Phylogenetic analysis showed that all CVA6 strains were divided into four genotypes and the genotype D was further divided into two subgenotypes. Among the twenty-two Anyang sequences involved in the phylogeny of CVA6, five sequences belonged to subgenotype D1 and the other seventeen sequences belonged to subgenotype D2. The values of selection pressure of the Chinese CVA6 strains within subgenotype D1 and subgenotype D2 were 0.031 and 0.075, respectively. Only one positively selected site was detected in the VP1 gene of subgenotype D1, meanwhile four were detected in the VP1 gene of subgenotype D2. Conclusions Subgenotype D1 and its successor, subgenotype D2, circulated in Anyang city during 2011—2015. The subgenotype replacement from CVA6 subgenotype D1 to CVA6 subgenotype D2 and the continuous transmission of CVA6 subgenotype D2 were the two major causes of HFMD epidemic related to CVA6 in Anyang city during 2013—2015.