Objective  To understand the genetic characteristics and evolutionary relationships of the VP1 gene of enterovirus group A 71 (EV71) in Weifang, Shandong province, from 2014 to 2019, and further analyze the selective pressure on the VP1 region codon.

  Methods  Positive samples from the patients with hand foot and mouth disease (HFMD) caused by EV71 were randomly selected in Weifang from 2014 to 2019 and total viral RNA was extracted after human rhabdomyosarcoma (RD) cell isolation and culture. Sequence determination was performed after amplification of the VP1 region gene by reverse transcription-polymerase chain reaction (RT-PCR), then nucleotide and amino acid homologies with reference strains of each EV71 genotype were analyzed. The VP1 gene sequences of EV71 strains detected after 2008 in China were obtained from GenBank for the construction of a phylogenetic tree with the VP1 gene sequences of local EV71 strains. The natural selection pressure on VP1 codon of C4a subtype EV71 were analyzed by single-likelihood ancestor counting (SLAC) and fixed effects likelihood (FEL) method respectively on Datamonkey website service.

  Results  A total of 41 local EV71 strains were obtained, all of which had the highest homology with C4a genotype, with 94.9%−98.2% nucleotide homology and 98.3%−99.6% amino acid homology. The results of the phylogenetic tree showed that all the local EV71 strains belonged to C4a genotype, and formed two distinct clades, with clusters of strains and sporadic strains, and had close relationship with the strains in multiple areas. Totally 136 codons and 190 codons were identified as negative selected pressure sites by SLAC and FEL respectively, and no codon was under positive selection pressure.

  Conclusion  The EV71 strains circulating in Weifang from 2014 to 2019 belonged to sub-genotype C4a and multiple viral transmission chains existed. The strong negative selection pressure in the VP1 region indicated the stability of the antigen in the VP1 region, suggesting that the variation in the VP1 protein was not caused by selective pressure from the host immune system, which is favorable for the application of EV71 vaccine.