Next generation sequencing of near-full length genome of norovirus GII.4 from Botswana.

Noroviruses are highly diverse, with genotype GII.4 causing most epidemics. This study aimed to investigate the evolutionary dynamics of norovirus genogroup GII strains among acutely infected children under 5 years in Botswana, between 2016 and 2018. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify the whole norovirus genome, followed by next-generation sequencing using Oxford Nanopore technology. Twelve samples were successfully analyzed, with 11 identified as norovirus GII.4 Sydney [P31] and one as GII.4 Sydney [P13]. This study generated the first near-full length norovirus sequences in Botswana (93-95% coverage). Our results show that the norovirus GII.4 strains circulating in Botswana are under evolution through recombination and antigenic drift. Recombination in the GII.4 Sydney [P31] and GII.4 Sydney [P13] strains occurred in the ORF1/ORF2 junction and within ORF1, respectively. This study provides the first description of the GII.4 Sydney [P13] recombinant. Amino acid variation in the immunogenic sites was analyzed. Mutations in epitope A correlate with the emergence of novel norovirus GII.4 strains with altered antigenicity. In this study, we identified 43 unique amino acid substitutions in the VP1 region, with six occurring in epitopes, A (G295N, and E368Q) and E (S40T, N412D, N412K and T413H). The shell subdomain of the GII.4 Sydney [P13] variant was closely related to norovirus GII.17. Lastly, we also observed several mutations in the T cell restricted epitopes of both strains. Our study has made a novel contribution to understanding the evolution of norovirus GII.4 in Botswana.