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Human rhinoviruses (HRV) have been re-classified into three species (A-C), but the recently discovered HRV-C strains are not fully characterized yet. This study aimed to undertake a molecular and epidemiological characterization of HRV strains infecting children hospitalized over one year in two large research hospitals in Rome. Nasal washings from single HRV infections were retrospectively subjected to phylogenetic analysis on two genomic regions: the central part of the 5′Untranslated Region (5′UTR) and the Viral Protein (VP) 4 gene with the 5′ portion of the VP2 gene (VP4/2). Forty-five different strains were identified in 73 HRV-positive children: 55 % of the cases were HRV-A, 38 % HRV-C and only 7 % HRV-B. HRV-C cases were less frequent than HRV-A during summer months and more frequent in cases presenting wheezing with respect to HRV-A. Species distribution was similar with respect to patient age, and seasonality differed during summer months with fewer HRV-C than HRV-A cases. On admission, a significantly higher number of HRV-C cases presented with wheezing with respect to HRV-A. The inter- and intra-genotype variability in VP4/2 was higher than in 5′UTR; in particular, HRV-A patient VP4/2 sequences were highly divergent (8-14 %) at the nucleotide level from those of their reference strains, but VP4 amino acid sequence was highly conserved. In HRV-C isolates, the region preceding the initiator AUG, the amino acids involved in VP4 myristoylation, the VP4-VP2 cleavage site and the cis-acting replication element were highly conserved. Differently, VP4 amino acid conservation was significantly lower in HRV-C than in HRV-A strains, especially in the transiently exposed VP4 N-terminus. This study confirmed the high number of different HRV genotypes infecting hospitalized children over one year and reveals a greater than expected variability in HRV-C VP4 protein, potentially suggestive of differences in replication. [ABSTRACT FROM AUTHOR]