Chimwemwe Mhango, Malawi Liverpool Wellcome programmeEnd Chinyama, Malawi Liverpool Wellcome Trust programmeLandilani Gauti, Malawi University of Science and TechnologyFlywell Kawonga, Malawi Liverpool Wellcome programmeErnest Matambo, Malawi Liverpool Wellcome programmeBenjamin Kumwenda, Kamuzu University of Health SciencesArox Kamng'ona, Kamuzu University of Health SciencesCeleste Donato, doherty instituteChrispin Chaguza, St Jude Children's Research HospitalKhuzwayo Jere, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool

Malawi Liverpool Wellcome programme Malawi University of Science and Technology Kamuzu University of Health Sciences Doherty Institute St Jude Children's Research Hospital Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool

Epidemiology, Evolution, and Diversity

Rotavirus remains a leading cause of gastroenteritis in children under five, especially in low- and middle-income countries (LMICs). In Malawi, G9 strains circulated before rotavirus vaccine introduction but re-emerged in 2017, becoming predominant by 2019. Here, we investigate genetic diversity, evolutionary origins, and public health implications of G9 rotaviruses in Malawi.

We performed whole-genome sequencing on pre-vaccine (n=15; 2001–2008) and post-vaccine (n=13; 2017–2022) samples. Genotype constellations were determined, and phylogeographic and temporal-resolved phylogenies were used to assess diversity and infer evolutionary pathways.

Pre-vaccine G9 strains showed four variants: Wa-like (G9P[8], G9P[6]), DS-1-like (G9P[6], G9P[8]), and reassortant Wa-like (G9P[6]) constellations. Post-vaccine G9 strains included G9P[8] with Wa-like and G9P[6] with DS-1-like constellations. VP7 phylogeography showed diverse pre-vaccine clades versus two post-vaccine monophyletic clusters linked to rotaviruses detected in Mozambique (G9P[6]) and Japan (G9P[8]). Greater diversity was observed in pre-vaccine Wa-like and post-vaccine DS-1-like strains. Evolutionary analyses revealed high similarity between post-vaccine G9P[6] and earlier G2/G3 DS-1-like strains, while pre-vaccine Wa-like segments resembled co-circulating G1P[8] strains, suggesting local reassortment. Estimated tMRCA ranged from 2001–2004 (pre-vaccine Wa-like) to 2009–2015 (post-vaccine DS-1-like).

G9 rotaviruses in Malawi show dynamic evolution shaped by intra-genogroup reassortment. The post-vaccine predominance of DS-1-like G9P[6] strains highlights their adaptability to existing genomic backbones. These findings reveal a heightened propensity of G9 strains to reassort and emphasize the need for continued genomic surveillance to monitor emerging strains and guide vaccine strategies in LMICs.