Heparan sulfate attachment receptor is a major selection factor for attenuated enterovirus 71 mutants during cell culture adaptation

PLoS Pathog. 2020 Mar 18;16(3):e1008428. doi: 10.1371/journal.ppat.1008428. eCollection 2020 Mar.


Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease (HFMD). However, this infection is sometimes associated with severe neurological complications. Identification of neurovirulence determinants is important to understand the pathogenesis of EV71. One of the problems in evaluating EV71 virulence is that its genome sequence changes rapidly during replication in cultured cells. The factors that induce rapid mutations in the EV71 genome in cultured cells are unclear. Here, we illustrate the population dynamics during adaptation to RD-A cells using EV71 strains isolated from HFMD patients. We identified a reproducible amino acid substitution from glutamic acid (E) to glycine (G) or glutamine (Q) in residue 145 of the VP1 protein (VP1-145) after adaptation to RD-A cells, which was associated with attenuation in human scavenger receptor B2 transgenic (hSCARB2 tg) mice. Because previous reports demonstrated that VP1-145G and Q mutants efficiently infect cultured cells by binding to heparan sulfate (HS), we hypothesized that HS expressed on the cell surface is a major factor for this selection. Supporting this hypothesis, selection of the VP1-145 mutant was prevented by depletion of HS and overexpression of hSCARB2 in RD-A cells. In addition, this mutation promotes the acquisition of secondary amino acid substitutions at various positions of the EV71 capsid to increase its fitness in cultured cells. These results indicate that attachment receptors, especially HS, are important factors for selection of VP1-145 mutants and subsequent capsid mutations. Moreover, we offer an efficient method for isolation and propagation of EV71 virulent strains with minimal selection pressure for attenuation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptation, Physiological*
  • Amino Acid Substitution
  • Animals
  • Capsid Proteins* / genetics
  • Capsid Proteins* / metabolism
  • Chlorocebus aethiops
  • Enterovirus A, Human* / genetics
  • Enterovirus A, Human* / metabolism
  • Genome, Viral*
  • Hand, Foot and Mouth Disease / genetics
  • Hand, Foot and Mouth Disease / metabolism
  • Hand, Foot and Mouth Disease / pathology
  • Humans
  • Mutation, Missense*
  • Receptors, Virus* / metabolism
  • Vero Cells


  • Capsid Proteins
  • Receptors, Virus

Grants and funding

This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI (https://www.jsps.go.jp/index.html), grant number JP18H02667 to SK and 19K07601 to KK and by Japan Agency for Medical Research and Development (AMED, https://www.amed.go.jp/), grant number 19fk0108084h1101 to SK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.