Complete genome sequencing and molecular characterization of indigenous enterovirus A71 genogroups D and G of India

Enterovirus 71 (EV-A71), a neurotropic member of the Picornaviridae family, is a positive-strand RNA virus comprising of seven recognized genogroups (A-G) and has caused significant outbreaks of hand, foot, and mouth disease worldwide. Despite prior identification of D and G genogroups in India, comprehensive full-genome characterization of these indigenous lineages remains unreported.

We performed next-generation sequencing (NGS) and molecular phylogenetic analyses on two Indian isolates from acute flaccid paralysis cases: R13223-IND-02 (genogroup D) and V11-2209-01 (genogroup G). The complete genomes generated through NGS were aligned with publicly available full-length and partial EV-A71 sequences using MAFFT, and phylogenetic trees were constructed employing the maximum-likelihood method. Amino acid variations were then compared across the genogroups, as well as with prototype and neurovirulent reference strains.

Phylogenetic reconstruction revealed robust clustering of these strains within distinct clades (bootstrap support ≥ 99%)", divergent from globally prevalent genogroups B and C. Both D and G genogroups appear geographically restricted to India, while genogroup D persists as an endemic lineage. Comparative genomic analyses demonstrated extensive nucleotide divergence, including numerous non-synonymous substitutions across both structural (VP1-VP4) and non-structural (2A-3D) coding regions. An E-Q substitution at amino acid position 145 in the VP1 structural protein was detected in both D and G genogroups, a site functionally linked to receptor binding and mouse virulence.

The findings highlight the evolutionary independence and sustained circulation of D and G genogroups in India, suggesting a unique regional evolutionary trajectory. The implications hold significant global relevance, offering valuable insights for molecular surveillance, advancing pathogenesis research, and supporting the development of genogroup-specific vaccine strategies tailored to diverse epidemiological landscapes. This study is the first to report the complete genome of EV-A71 D and G genotypes in India, and its unique mutation characteristics provide a direct target for the development of region-specific vaccines.