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Rotavirus A (RVA) remains a leading cause of acute gastroenteritis (AGE) hospitalizations in children worldwide. During the COVID-19 pandemic, a reduction in vaccination coverage in Brazil and elsewhere was observed, and some reports have demonstrated a reduction in AGE notifications during the pandemic. This study aims to investigate the diversity and prevalence of RVA genotypes in children and adults presenting with AGE symptoms in Brazil during the COVID-19 pandemic between 2020 and 2022. RVA was screened using RT-qPCR; then, G and P genotypes were characterized using one-step multiplex RT-PCR. A total of 2173 samples were investigated over the three-year period, and we detected RVA in 7.7% of samples (n = 167), being 15.5% in 2020, 0.5% in 2021, and 13.8% in 2022. Higher RVA prevalence was observed in the Northeastern region (19.3%) compared to the Southeastern (6.1%) and Southern regions (5.5%). The most affected age group was children aged between 0 and 6 months old; however, this was not statistically significant. Genotyping and phylogenetic analysis identified the emergence of G6P[8] during the period; moreover, it was detected in 10.6% of samples in 2020 and in 83.5% in 2022. In contrast, the prevalence of G3P[8], the previous dominant genotype, decreased from 72.3% in 2020 to 11.3% in 2022. We also identified unusual strains, such as G3P[9] and G9P[4], being sporadically detected during the period. This is the first report on the molecular epidemiology and surveillance of RVA during the COVID-19 pandemic period in Brazil. Our study provides evidence for the importance of maintaining high and sustainable levels of vaccine coverage to protect against RVA disease. Furthermore, it highlights the need to maintain nationwide surveillance in order to monitor future trends and changes in the epidemiology of RVA in Brazil.

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