Reverse Genetic Analysis of Adaptive Mutations within the Capsid Proteins of Enterovirus 71 (EV-A71) Strains Necessary for Infection of CHO-K1 Cells

Enterovirus 71 (EV-A71) is a human pathogen that does not naturally infect rodent cells. However, virus strains that productively infect rodent cells could be produced by serial passage in a rodent cell line. The resulting viruses are useful tools to identify molecular determinants of adaptation to a new host species. We adapted an EV-A71 clinical isolate (EV71:BS) in Chinese hamster ovary (CHO-K1) cells and sequenced the genome of the resulting virus. We explored the contribution of viral capsid mutations that may have contributed to the CHO-adapted phenotype by introducing the mutations surrounding the receptor-binding canyon region into the genome of EV71:BS by reverse genetics tools. We subsequently assessed whether the resulting mutant viruses productively infected CHO-K1 cells. Contrary to previous reports in the literature, our findings demonstrate that a single amino acid substitution in capsid VP2149 K→I is not sufficient for the resulting mutant virus to infect CHO-K1 cells. Moreover, a mutant virus harbouring other capsid mutations we evaluated (VP1 K98→E, E145→A, and L169→F) also did not infect CHO-K1 cells. These findings strongly suggest that cooperation of various adaptive mutations in the capsid is necessary to enable successful EV-A71 infection of a different host species.