Molecular characterization of emerging G9P[4] rotavirus strains possessing a rare E6 NSP4 or T1 NSP3 genotype on a genogroup-2 backbone using a refined classification framework.

Rotavirus infections associated with unusual strains are an emerging concern in rotavirus vaccination programmes. Recently, an increase in circulation of unusual G9P[4] strains was reported from different regions of India, placing this genotype in third position, after G1P[8] and G2P[4], of the most common rotavirus strains. The aim of the present study was to analyse the complete genomic constellation of three G9P[4] strains (RV09, RV10 and RV11), determine their genetic relatedness to other genogroup-2 strains and understand the evolution of a rare E6 and other NSP4 genotypes. All strains revealed the presence of a genogroup-2 backbone, with RV09 constituting the NSP3 T1 genotype and RV10 and RV11 bearing the NSP4 E6 genotype. A refined criterion adopted to classify the nine internal gene segments of G2P[4] and non-G2P[4] strains with the genogroup-2 backbone into lineages and sub-lineages indicated divergence of >8 % (except NSP1: >5.5 %) for lineages and >3 % for sub-lineages. The VP1 and/or VP3 genes of study strains showed close relationships with animal-like human rotaviruses. The estimated evolutionary rate for the NSP4 E6 genotype was marginally higher (3.78×10-3 substitutions per site per year) than that of genotypes E1 (2.6×10-3 substitutions per site per year) and E2 (3.06×10-3 substitutions per site per year), suggesting a step towards adaptation of E6 on a genogroup-2 backbone. The time and origin of the most recent common ancestor of E6 genotype were estimated to be 1981 and South Asia, respectively. Full-genome and evolutionary analyses performed in this study for G9P[4] strains will help better understand the extent of gene reassortment and origin in unusual rotavirus strains that may remain viable and cause infections in humans.