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

Author summary Viruses must overcome various setbacks in a variety of tissues and cells during transmission from the initial replication site to the final target site. To achieve this, RNA viruses employ a strategy to adapt to different environments by creating a diverse viral population using low-fidelity RNA-dependent RNA polymerases. On the other hand, when the viruses are propagated in clonal cell cultures, in vitro adaptation occurs. The viruses may acquire new properties or lose some properties they had in vivo. In vitro adaptation is often associated with attenuation. Therefore, the selection pressures imposed on viruses replicating in vitro and in vivo are quite different. It is unclear how this environmental difference affects viral populations. Clinical isolates of EV71 replicate in cultured cells poorly. However, after a few passages, the viruses adapt to this condition and replicate efficiently. In this study, we demonstrate that attachment receptor usage is a major selection pressure for in vitro adaptation of EV71 by analyzing the population dynamics of cell culture-adapted viruses. This mechanism appears to be a major mode of attenuation. 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.