A reverse genetic study of the adaptation of human enterovirus 71 to growth in Chinese hamster ovary cell cultures

Abstract: We selected Chinese hamster ovary (CHO) cell-adapted strains of human enterovirus 71 (HEV71) belonging to sub-genogroups B5 (HEV71-B5) and C2 (HEV71-C2) by serial passage in CHO cells at a high multiplicity of infection. During the course of CHO cell passage, virus growth improved significantly, with increasing virus titres and the presence of cytopathic effect observed. A study of virus growth kinetics revealed that the CHO cell-adapted strains of HEV71-B5 (CHO-B5) and HEV71-C2 (CHO-C2) grew efficiently in CHO cells with maximum titres >100-fold higher than unadapted parental virus. Both CHO-B5 and CHO-C2 harboured single amino acid mutations within the VP2 capsid protein gene. CHO-B5 has an amino acid substitution of K149 →I in VP2 and CHO-C2 has an amino acid substitution of K149 →M in VP2. An isolate of sub-genogroup C4 (HEV71-C4) failed to adapt to CHO cells during serial passage. Infectious cDNA clone-derived populations of HEV71-C4 containing the mutations K149 →I or K149 →M in VP2 were generated by site-directed mutagenesis. Both mutations resulted in the ability of the virus to replicate efficiently in CHO cells, indicating that amino acid position 149 in VP2 is critical for the adaptation of HEV71 to growth in CHO cells. [Copyright &y& Elsevier]