Attenuation of Human Enterovirus 71 High-Replication-Fidelity Variants in AG129 Mice

Abstract

In a screen for ribavirin resistance, a novel high-fidelity variant of human enterovirus 71 (EV71) with the single amino acid change L123F in its RNA-dependent RNA polymerase (RdRp or 3D) was identified. Based on the crystal structure of EV71 RdRp, L123 locates at the entrance of the RNA template binding channel, which might form a fidelity checkpoint. EV71 RdRp-L123F variants generated less progeny in a guanidine resistance assay and virus populations with lower mutation frequencies in cell culture passage due to their higher replication fidelity. However, compared with wild-type viruses, they did not show growth defects. In vivo infections further revealed that high-fidelity mutations L123F and G64R (previously reported) negatively impacted EV71 fitness and greatly reduced viral pathogenicity alone or together in AG129 mice. Interestingly, a variant with double mutations, RG/B4-G64R/L123F (where RG/B4 is an EV71 genotype B4 virus constructed by reverse genetics [RG])showed higher fidelity in vitro and less virulence in vivo than any one of the above two single mutants. The 50% lethal dose (LD 50 ) of the double mutant increased more than 500 times compared with the LD 50 of wild-type RG/B4 in mice. The results indicated that these high-fidelity variants exhibited an attenuated pathogenic phenotype in vivo and offer promise as a live attenuated EV71 vaccine. IMPORTANCEThe error-prone nature of the RNA-dependent RNA polymerase (RdRp) of RNA viruses during replication results in quasispecies and aids survival of virus populations under a wide range of selective pressures. Virus variants with higher replication fidelity exhibit lower genetic diversity and attenuated pathogenicity in vivo. Here, we identified a novel high-fidelity mutation L123F in the RdRp of human enterovirus 71 (EV71). We further elucidated that EV71 variants with the RdRp-L123F mutation and/or the previously identified high-fidelity mutation RdRp-G64R were attenuated in an AG129 mouse model. As EV71 has emerged as a serious worldwide health threat, especially in developing countries in the Asia-Pacific region, we urgently need EV71 vaccines. Learning from the poliovirus vaccination, we prefer live attenuated EV71 vaccines to inactivated EV71 vaccines in order to effectively control EV71 outbreaks at low cost. Our results imply a new means of attenuating EV71 and reducing its mutation rate at the same time.

Small plaque size variant of chikungunya primary isolate showed reduced virulence in mice

“…Cooperation of variants within quasispecies has been described in poliovirus and enterovirus 71 pathogenesis. 18,19 Several possible mechanisms could contribute to the small plaque phenotype of the virus. Previous study on West Nile virus showed amino acid substitutions in NS4B protein, a small poorly defined protein, associated with small plaque phenotype and the resulting virus induced less innate and adaptive immune responses in mice when compare to wild type virus.…”

Is a multivalent hand, foot, and mouth disease vaccine feasible?

“…151 However, the possibility to engineer attenuated high-fidelity-variants of EV-A71 with low pathogenicity could be a promising approach for future live vaccines. 152 In a comparative study of prototyptic EV-A71 vaccines produced by different technologies, formalin-inactivated EV-A71 virions adjuvanted in alum were found to be very immunogenic, to elicit strong cross-neutralization titers against different EV-A71 genotypes and subgenotypes in mice and non-human primates and to be the most potent and promising immunogens.…”

Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

“…Ribavirin has been used to select high-fidelity variants of EV71 (21,43), footand-mouth disease virus (FMDV) (44,45), and chikungunya virus (22), suggesting that these viruses occupy the lower end of the fidelity spectrum. One might expect that the need to retain crossspecies infectivity in zoonotic arboviruses necessitates higher-fidelity polymerases, as implied by yellow fever virus fidelity data (46), although data from dengue virus polymerase suggest that it has a fidelity comparable to those of PV and FMDV RdRPs based on single nucleotide incorporation assays (47).…”

“…Maintaining the proper genome heterogeneity is essential for virus fitness, as it allows rapid adaptation to different host environments, and viral pathogenesis can be attenuated by increasing or decreasing polymerase fidelity (16). This has been shown for poliovirus (PV), coxsackievirus B3 (CV), and enterovirus 71 (EV71) in mouse studies (17)(18)(19)(20)(21) and for chikungunya virus, where fidelity variants lose fitness in both vertebrate and invertebrate hosts (22,23).…”