Unusual Assortment of Segments in 2 Rare Human Rotavirus Genomes

Using full-length genome sequence analysis, we investigated 2 rare G3P[9] human rotavirus strains isolated from children with diarrhea. The genomes were recognized as assortments of genes closely related to rotaviruses originating from cats, ruminants, and humans. Results suggest multiple transmissions of genes from animal to human strains of rotaviruses.

G roup A rotaviruses possess a genome of 11 segments of double-stranded RNA (1).Rotaviruses are associated with acute gastroenteritis in humans and a wide variety of other mammalian and avian species (1).The evolution and diversity of rotaviruses is driven by genomic reassortment, accumulation of point mutations, intragenic recombination, and interspecies transmission (2,3).At least 23 G genotypes (structural viral protein [VP] 7 related) and 32 P genotypes (VP4 related) have been identifi ed thus far in rotaviruses (4).Unlike other G and P types, G3 has been identifi ed in rotavirus strains from humans and from almost all other susceptible mammalian species, including dogs, cats, monkeys, horses, rabbits, pigs, and ruminants, in association with various P types, thus exhibiting a broad host range (1).G3 human rotaviruses are usually associated with P [8] or P [6] and, rarely, with P [9] (5,6).
Historically, RNA-RNA hybridization has been used to study the genetic relationships among rotavirus strains and has shown 2 major pools among human rotaviruses, named Wa-like and DS-1-like (7).Recently, a new classifi cation system based on whole-genome sequence analysis enabled researchers to better understand the complex interactions between human and animal rotaviruses (8,9).Application of this new classifi cation system showed a close evolutionary relationship between human Wa-like and porcine rotavirus strains and between human DS-1-like and bovine rotavirus strains, suggesting that the 2 major human rotavirus G and P types might have an animal origin (8).A third human rotavirus family, designated AU-1-like, comprises a group of globally circulating but overall rare strains, mainly with the G3P [9] combination.Early RNA-RNA hybridization studies suggested a genetic relationship of particular human G3P [9] strains with feline rotaviruses (10).Later, feline-bovine reassortant G3P [9] rotaviruses were also identifi ed in humans (11).However, because of the limits of resolution of the RNA-RNA hybridization method, determining the exact origin of individual genome segments in these strains was not possible.

The Study
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 identifi ed 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.

Conclusions
Full-genome sequencing of 2 unusual G3P [9] human rotavirus strains identifi ed 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 human/ feline 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.
S.D.G. was supported by the grant "Variabilità genetica di ceppi di rotavirus umani e animali-Fondi di Ateneo 2006."J.M. was supported by a Fonds voor Wetenschappelijk Onderzoek postdoctoral fellowship.V.M. was supported by the grant "Infezioni virali del cane a carattere zoonosico-Fondi Ateneo 2008."Dr De Grazia is a research assistant at the University of Palermo, Department of Health Promotion Sciences "G. D'Alessandro," Palermo, Italy.Her primary research interests are microbial typing, viral enteric pathogens, and viral epidemiology.Unusual Assortment of Segments in 2 Rare Human Rotavirus Genomes Technical Appendix Phylogenetic trees based on the full-length nucleotide sequences of 11 rotavirus genes: viral proteins (VP) 1-5, 6, and 7, and nonstructural proteins (NSP) 1-5.The trees were generated by using the neighbor-joining method and Kimura 2-parameter model.Bootstrap values (500 replicates) >80 are shown.The various genotypes are bo, bovine; hu, human; fe, feline; ca, canine; gu, guanacos; si, simian; ov, ovine; av, avian.The Italian G3P[9] viruses are in boldface.Scale bars indicate genetic distance between sequences of segments analyzed.

Table 1 .
Nucleotide identity of 11 genome segments of 2 human rotavirus strains, Italy, 1994 and 1996* [9]ay shading indicates genetic relationships with respect to the G3P[9]Italian viruses, according to the patterns of segregation displayed in the phylogenetic analyses in the online Technical Appendix (www.cdc.gov/EID/content/15/5/859-Techapp.pdf).Boldface indicates complete genomic constellations of the 2 G3P[9]Italian viruses sequenced in this study.VP, structural protein; NSP, nonstructural protein.