Clinical and genomic surveillance of rotavirus causing acute gastroenteritis in the community of Madrid, Spain, 2021-2023: predominance of equine-like G3 strains.

Spain has recently included rotavirus vaccines (RV1 [Rotarix] and RV5 [RotaTeq]) into the National Immunization Programme, following a period of exclusive private availability. This study aimed to analyze rotavirus molecular epidemiology and clinical outcomes during the period preceding routine nationwide vaccination, when vaccinated and unvaccinated children coexisted. This prospective study analyzed 647 rotavirus-positive stool samples with associated clinical-epidemiological data from patients with rotavirus gastroenteritis (RVGE) collected between December 2021 and June 2023. Among children aged 0-4 years with RVGE, incomplete or absent vaccination (adjusted-OR:3.92) and having pre-existing medical conditions (adjusted-OR: 17.67) were significant risk factors for RVGE-related hospitalisation (p < 0.001). Equine-like-G3P[8] (EQL-G3) was the predominant genotype (62.4%). Despite its predominance, EQL-G3 was not associated with vaccination status or an increased risk of hospitalisation among RV-positive cases. Among vaccinated children, EQL-G3 strains were more frequent in RV1 recipients (p = 0.012), while typical G3P[8] strains predominated in RV5 recipients (p < 0.001). Whole-genome analyses identified NSP2 mono-reassortants, bovine-like NSP4 genes and potentially immunologically relevant mutations at antigenic epitopes, highlighting substantial genomic diversity. Overall, genotype distribution was largely similar between vaccinated and unvaccinated children, providing no evidence that viral genotype drives disease severity among RV-positive cases. Our findings reinforce the protective effect of full rotavirus vaccination in reducing hospitalizations, regardless of circulating genotypes. Nonetheless, given the observed genetic diversity and signals of reduced cross-protection of RV1 against EQL-G3 strains, continued and expanded molecular surveillance is essential to improve understanding of rotavirus diversity and to contextualize circulating genotypes within their clinical relevance over time.