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Acute gastroenteritis (AGE) accounts for considerable morbidity and mortality in the paediatric population worldwide, especially in low-income countries. Human norovirus (HNoV), particularly GII.4 strains, are important agents of AGE. This study aimed to detect and characterise HNoV in children with and without AGE. Between 2019 and 2021, 300 stool samples (200 AGE and 100 without AGE) were collected from children below 5 years of age referred to the healthcare facilities of the rural communities of Vhembe District, South Africa. After detection using real-time RT-PCR, HNoV positive samples were subjected to RT-PCR and Sanger sequencing. Partial nucleotide sequences (capsid/RdRp) were aligned using the Muscle tool, and phylogenetic analysis was performed using MEGA 11. The nucleotides' percent identity among HNoV strains was compared using ClustalW software. A significant difference in HNoV prevalence between AGE children (37%; 74/200) and non-AGE (14%; 14/100) was confirmed (p < 0.0001). Genogroup II (GII) HNoV was predominant in AGE children (80%; 59/74), whereas most non-AGE children were infected by the GI norovirus genogroup (64%; 9/14). GII.4 Sydney 2012 [P31] strains were dominant (59%; 19/32) during the study period. A phylogenetic analysis revealed a close relationship between the HNoV strains identified in this study and those circulating worldwide; however, ClustalW showed less than 50% nucleotide similarity between strains from this study and those from previously reported norovirus studies in the same region. Our findings indicate significant changes over time in the circulation of HNoV strains, as well as the association between high HNoV prevalence and AGE symptoms within the study area. The monitoring of HuNoV epidemiology, along with stringent preventive measures to mitigate the viral spread and the burden of AGE, are warranted. [ABSTRACT FROM AUTHOR]