RESULTS: Six EV-71 isolates from an outbreak in Malaysia, in 1997, were sequenced completely. These isolates were identified as EV-71 subgenotypes, B3, B4 and C2. A phylogenetic tree that correlated well with the present enterovirus classification scheme was established using these full genome sequences and all other available full genome sequences of EV-71 and human enterovirus A (HEV-A). Using the 5' UTR, P2 and P3 genomic regions, however, isolates of EV-71 subgenotypes B3 and C4 segregated away from other EV-71 subgenotypes into a cluster together with coxsackievirus A16 (CV-A16/G10) and EV-71 subgenotype C2 clustered with CV-A8. Results from the similarity plot analyses supported the clustering of these isolates with other HEV-A. In contrast, at the same genomic regions, a CV-A16 isolate, Tainan5079, clustered with EV-71. This suggests that amongst EV-71 and CV-A16, only the structural genes were conserved. The 3' end of the virus genome varied and consisted of sequences highly similar to various HEV-A viruses. Numerous recombination crossover breakpoints were identified within the non-structural genes of some of these newer EV-71 subgenotypes.
CONCLUSION: Phylogenetic evidence obtained from analyses of the full genome sequence supports the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the most common causal agent of HFMD. It is suggested that these recombination events played important roles in the emergence of the various EV-71 subgenotypes.
RESULTS: In this study, several genetically modified sub-genotype B4 EV-A71 mutants were constructed by site-directed mutations at positions 158, 475, 486, 487 and 5262 or through partial deletion of the 5'-NTR region (∆ 11 bp from nt 475 to 486) to generate a deletion mutant (PD). EV-A71 mutants 475 and PD caused minimal cytopathic effects, produced lowest viral RNA copy number, viral particles as well as minimal amount of viral protein (VP1) in RD cells when compared to mutants 158, 486, 487 and 5262.
CONCLUSIONS: The molecular determinants of virulent phenotypes of EV-A71 sub-genotype B4 strain 41 (5865/Sin/000009) were found to differ from the C158 molecular determinant reported for the fatal EV-A71 sub-genotype B1 strain (clinical isolate 237). The site-directed mutations (SDM) introduced at various sites of the cDNA affected growth of the various mutants when compared to the wild type. Lowest viral RNA copy number, minimal number of plaques formed, higher infectious doses required for 50% lethality of RD cells and much reduced VP1 of the EV-A71 sub-genotype B4 strain 41 genome was attained in mutants carrying SDM at position 475 and through partial deletion of 11 bp at the 5'-NTR region.