During uninterrupted surveillance for human rotaviruses in Palermo, Italy, which started in the mid-1980s, 3 strains (PAF96/94, PAH136/96, and PAI58/96) were detected that displayed AU-1–like features because they possessed long electropherotype, subgroup (SG) I (VP6 related) and G3P[9] genotypes. The viruses were identified from children <5 years of age who were hospitalized with acute gastroenteritis at the “G. Di Cristina” Children’s Hospital of Palermo in 1994 and 1996. Sequence analysis found all 3 strains to be genetically related to strains of either human or feline origin in the VP7, VP4, and VP6 genes. In contrast, the nonstructural protein (NSP) 4 gene of these viruses resembled that of G2P[4] human strains, suggesting a reassortment between AU-1–like and DS-1–like strains (5). To understand the evolution and origin of these viruses, we determined the full-length genome sequence of 2 such unusual G3P[9] viruses, strain PAH136/96 and PAI58/96, that appeared to be genetically distinct and for which enough material was available for additional analyses. The complete genome sequences were obtained as described elsewhere (12). Genome sequences were individually compared with cognate sequences of a variety of rotavirus strains by phylogenetic analysis by using MEGA4 software (www.megasoftware.net). In addition, the sequences were analyzed by using BLAST (www.ncbi.nlm.nih.gov/BLAST) with default search values. The GenBank nucleotide sequence accession numbers of PAH136/96 and PAI58/96, respectively, are GU296430 and GU296431 for VP7, GU296426 and GU296427 for VP4, GU296428 and GU296429 for VP6, GU296420 and GU296421 for VP1, GU296422 and GU296423 for VP2, GU296424 and GU296425 for VP3, GU296410 and GU296411 for NSP1, GU296412 and GU296413 for NSP2, GU296414 and GU296415 for NSP3, GU296416 and GU296417 for NSP4, GU296418 and GU296419 for NSP5.

The genomes of the PAH136/96 and PAI58/96 G3P[9] strains were 18,485 nt long. The 2 strains from Italy possessed the following genetic constellations: G3-P[9]-I2-R2-C2-M2-A3-N1-T6-E2-H3 for strain PAH136/96 and G3-P[9]-I2-R2-C2-M2-A3-N2-T6-E2-H3 for strain PAI58/96, differing only in the NSP2 gene (Tables 1, 2). Phylogenetic analysis showed that the VP2, VP3, VP4, VP7, NSP1, NSP3, NSP4, and NSP5 genomic segments of the 2 Italian G3P[9] viruses were closely related to each other, sharing high sequence similarity (Table 1; Technical Appendix). Although included in the same VP1 (R2) and VP6 (I2) genotypes, the 2 G3P[9] viruses in the VP1 and VP6 trees showed distinct patterns of segregation (Technical Appendix). Strains PAH136/96 and PAI58/96 shared only 83.2% nt and 86% nt identity in the VP1 and VP6 genes, respectively, i.e., values slightly above the proposed cutoff values for the VP1 (83%) and VP6 genotypes (85%) (Table 1).

We found that after sequence and phylogenetic analysis, each of the 11 genomic segments of the Italian G3P[9] viruses had a striking genetic similarity with the corresponding segment of G6/G8P[14] human or ruminant rotaviruses or to human/feline AU-1–like rotaviruses (Table 1; Technical Appendix). In particular, the PAH136/96 strain possesses VP1, VP2, VP3, VP6, NSP3, NSP4, and NSP5 gene segments closely related to G6P[14] human rotaviruses, and the PAI58/96 strain possesses VP1, VP2, VP3, VP6, NSP2, NSP3, and NSP4 gene segments clustering with P[14] human rotaviruses, bovine rotaviruses, and other ruminant rotaviruses. The NSP1 genes of both strains segregated in a distinct branch within the A3 genotype containing feline, human/feline-like rotaviruses, and a G8P[14] rotavirus strain isolated from a guanaco (Technical Appendix). Matthijnssens et al. demonstrated that human G6P[14] rotaviruses were closely related to bovine and G6/G8P[14] ovine, antelope, and guanaco rotavirus strains because they shared a consensus genomic constellation (G6/G8)-P[14]-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-(E2/E12)-H3 (13). The NSP2 gene of PAH136/96 and the NSP5 of PAI58/96 were strictly related to the cognate sequences of the feline Cat2 strain. The VP7 and VP4 genes of PAH136/96 and PAI58/96 human rotaviruses were also highly similar to the Cat2 strain and the feline/human-like AU-1 strain. The Cat2 strain displayed a puzzling genomic composition (G3/P[9]-I3-R3-C2-M3-A3-N1-T6-E3-H3), which we hypothesize resulted from multiple reassortment events involving canine, feline, human, and bovine rotaviruses (14). No genetic correlation was found in the VP7 gene between the G3P[9] strains of this study and the human G3 strains circulating in Palermo over a 20-year surveillance period (15).

Full-genome sequencing of 2 unusual G3P[9] human rotavirus strains identified in Italy indicated that 1) viruses with a genetic makeup different from the Wa-, DS-1-, and AU-1-like gene pools may circulate in humans; 2) these viruses appear to have a relatively stable genetic constellation originating from reassortment events among feline/human AU-1–like rotaviruses, feline Cat2-like rotaviruses, and either ruminant rotaviruses or G6P[14] human rotaviruses; 3) these viruses, although retaining a stable genetic constellation, do not appear to have a clonal origin but are more likely to result from multiple introductions of particular genome segments from currently unknown animal rotavirus reservoirs.

Investigating the genetic features of human rotaviruses with unusual genetic/antigenic makeup is pivotal to gather information on the mechanisms by which some rotavirus strains may emerge in human populations. In addition, because of the possible animal origin of G3P[9] viruses, epidemiologic studies are warranted to identify the animal reservoir.