Comparative whole genome analysis reveals re-emergence of typical human Wa-like and DS-1-like G3 rotaviruses after Rotarix vaccine introduction in Malawi

Genotype G3 rotaviruses rank among the most common rotavirus strains worldwide in humans and animals. However, despite a robust long-term rotavirus surveillance system from 1997 in Blantyre, Malawi, these strains were only detected from 1997 to 1999 and then disappeared and re-emerged in 2017, five years after the introduction of the Rotarix rotavirus vaccine. Here we analysed 27 whole genome sequences to understand how G3 strains re-emerged in Malawi. We randomly selected samples each month between November 2017 and August 2019 from stool samples of children hospitalised with acute diarrhoea at the Queen Elizabeth Hospital in Blantyre, Malawi. We found three genotypes namely G3P[4] ( n =20), G3P[6] ( n =1) and G3P[8] ( n =6) associated with the re-emergence of G3 strains in Malawi post-Rotarix vaccine introduction. The identified genotypes co-circulated at different time points and were associated with three typical human G3 strains consisting of either a Wa-like or DS-1-like genetic constellation and reassortant strains possessing Wa-like and DS-1-like genetic backbones. Time-resolved phylogenetic trees demonstrated that the most recent common ancestor for each segment of the re-emerged G3 strains emerged between 1996 and 2012, possibly through introductions from outside the country due to the limited genetic similarity with G3 strains which circulated before their disappearance in the late 1990s. Further genomic analysis revealed that the reassortant DS-1-like G3P[4] strains acquired a Wa-like NSP2 genome segment (N1 genotype) through intergenogroup reassortment; an artiodactyl-like VP3 through intragenogroup interspecies reassortment; and VP6, NSP1 and NSP4 segments through intragenogroup reassortment likely before importation into Malawi. Additionally, the re-emerged G3 strains contain amino acid substitutions within the antigenic regions of the VP4 proteins which could potentially impact the binding of rotavirus vaccine-induced antibodies. Altogether, our findings shows that multiple rather than a single genotype have driven the re-emergence of G3 strains likely from other countries highlighting the role of human mobility and genome reassortment events in the dissemination and evolution of rotavirus strains in Malawi necessitating the need for long-term genomic surveillance of rotavirus in high disease burden settings to inform disease prevention and control.