Rotavirus A infection in hospitalized children in Croatia during three seasons: the predominance of G3 genotype and the emergence of intergenogroup reassortant strains

Rotavirus A (RVA), in countries like Croatia with low vaccination coverage, represents an important healthcare burden due to frequent hospitalizations. The aim of the study was to investigate rotavirus diversity in hospitalized children in Croatia and to evaluate the potential zoonotic background of circulating strains. During three consecutive RVA seasons (2018-2021), we have collected 602 RVA positive samples from children admitted to three regional hospitals. Multiplex semi-nested RT-PCR and Sanger sequencing were applied for VP7/VP4 genotyping. For G1 and G3 strains, NSP5 and VP6 segments were additionally genotyped and selected strains were further analyzed by NGS on Illumina platform. Overall, we detected eight and five different G and P genotypes, respectively. In descending order, G3, G9, G2, G1, G4, G6, G8, G10 and P[8], P[4], P[9], P[6], P[14]. The G3 genotype was predominant (54%) with rising season-to-season prevalence of G3 equine-like (G3e) lineage. Furthermore, P[8] genotype was detected in 79% of samples. Interestingly, a high share (15%) of mixed G genotypes was observed. On the contrary, strains with zoonotic background were infrequent (1.6%). The NSP5/VP6 genotyping and NGS confirmed the emergence of intergenogroup reassortant strains ; G1P[8] and G3eP[8], both in DS-1-like and Wa-like constellations. Notable genetic heterogeneity and sporadic detection of RVA strains with zoonotic background were reported in hospitalized children in Croatia. A significant finding is the circulation of intergenogroup reassortant strains. These results provide a valuable source of knowledge prerequisite in considering potential introduction of vaccination in the national immunization program.