Group A rotavirus is the main cause of non-bacterial acute diarrhoea in children worldwide [1, 2]. Rotaviruses possess a segmented genome of double-stranded RNA (dsRNA) that is surrounded by a triple layered capsid protein. The two outermost proteins (VP7 and VP4) of the capsid elicit most of the neutralizing antibodies and therefore represent the major targets for vaccine development. Antigenic and genetic differences within these two proteins have been used to assess the G and P type, respectively . Despite the great diversity presented by rotaviruses, only five strains, (G1P, G2P, G3P, G4P and G9P), largely predominate in humans worldwide. Rotaviruses bearing uncommon G or P types have been mostly found in developing countries, and their increasing frequency has raised concerns regarding massive vaccination programmes . Recently, a novel classification system was proposed based on the characterization of the 11 genome segments to analyse rotavirus evolutionary relationships, frequency of gene reassortment events, and track the emergence of uncommon rotaviruses thought to result from interspecies transmission [4, 5]. Thus, a genotype was described for each genome segment and the presence of association of genes (genome constellations) determined for multiple strains .
In anticipation of the massive rotavirus vaccination in Argentina, a rotavirus surveillance system based on sentinel units distributed across the country was set-up to monitor the strain distribution [6, 7]. In 2006, two G4P rotavirus strains, Arg4605 and Arg4671, were reported in the Santa Fe sentinel unit (c. 470 km North of Buenos Aires, Capital city). Because G4P strains have been found to be prevalent in swine [8-10], we conducted sequence analysis of VP7, VP8* and NSP4 gene segments of these strains, which revealed genetic relatedness to porcine rotavirus . Thus, to further characterize and determine the origin of these strains, nearly the full length (>95%) of their genome was sequenced and compared with human and porcine strains.
Stool samples containing both G4P rotaviruses were retrieved from our archival collection. RNA extraction was performed with AxyPrep Body Fluid Viral DNA/RNA MiniprepKit (Axygen Scientific, Union City, CA, USA) and the gene segments were amplified by RT-PCR using the Qiagen One Step RT-PCR kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. PCR products were purified with a QIAQuick PCR purification kit (Qiagen) and the sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) on a 3500Dx Genetic Analyzer automated sequencer (Applied Biosystems). Primers targeting the conserved 5- and 3-end regions as well as internal primers were used for RT-PCR and sequence reactions (primer sequences are available upon request).The nucleotide sequences have been deposited in GenBank under accession numbers FJ712692-FJ712693 (from a previous study) and KC412033-KC412052.
To analyse the complete genome constellation and the possible origin of these strains, phylogenetic trees were reconstructed for each of the 11 gene segments using the neighbour-joining method, Tamura-Nei substitution model (TN93) and gamma-distributed rate variation among sites as implemented in MEGA version 5 . Argentinean strains were compared against several human and animal prototype strains (Fig. 1). Both strains presented a Wa-like genome constellation and were found to be closer to porcine than to human strains (Table 1) [12-14]. The Argentinean strain Arg4605 revealed a genome constellation G4P-I1-R1-C1-M1-A8-N1-T7-E1-H1 and presented 10 genes of porcine origin, six of them being closely related to the porcine prototype strain Gottfried (Fig. 1). Interestingly, the NSP3 gene segment was more closely related to bovine strain UK (T7) than other porcine-like strains. The Argentinean strain Arg4671 presented a different genome constellation, G4P-I1-R1-C1-M1-A8-N1-T1-E1-H1, with 10 genes of porcine origin (Fig. 1). Comparative distance analysis of deduced amino acid sequences between these two strains and relevant human, porcine and porcine-like human rotavirus strains confirmed the possible porcine origin of multiple segments in both strains (Supporting information). Recently, the genome analyses of two porcine-like rotavirus strains detected in humans, one in Belgium (G9P) and one in Brazil (G4P), have shown the same genome constellation as Arg4605, thus expanding the presence of the genotypes A8 and T7 in porcine-like strains [12, 13]. Despite the two Argentinean strains presenting similar genome constellations, four (VP1, VP7, NSP3 and NSP5) gene segments grouped in different phylogenetic clusters (Fig. 1) and presented different electropherotype patterns of their dsRNA genomes (data not shown). It is noteworthy that the phylogenetic analysis of the VP7 gene has shown that these strains also grouped in different clusters, Arg4605 being more related to the Gottfried strain (Lineage III) and Arg4671 to the porcine Argentinean strains (Lineage VIII) . This, overall, suggests that either two independent interspecies transmission events or one followed by posterior reassortment of genes could have originated these unusual human strains.
When epidemiological data were consulted, the Arg4605 strain was detected in January from a 9-month-old girl who was admitted to the hospital for intravenous rehydration therapy because of multiple episodes of vomiting. No co-infection with bacterial enteric pathogens or parasites was detected. On the other hand, the Arg4671 strain was detected in April in a 1-year-old boy who also presented with vomiting but the case did not require hospitalization and he was treated with oral rehydration as an outpatient. He did not present with bacterial or parasitic co-infection either. Because the typical rotavirus season in Argentina starts in May and finishes in August , these were considered two sporadic cases of rotavirus infection. It is noteworthy that spatial analysis showed that these two patients lived in the same neighbourhood in Santa Fe (Santa Rosa de Lima neighbourhood). This is an area where people live in precarious housing and several families deprived of basic human needs were severely affected during the floods that occurred between 2003 and 2007. These adverse living conditions in the suburban areas promote a common environment for animals and humans, and the results presented here emphasize the role that might be played by farm animals as a reservoir for emerging rotavirus strains in humans.
Although these strains did not spread efficiently in the population, this work reinforces the ability of certain rotavirus strains to infect different species. Rotavirus genotype surveillance as well as complete characterization of unusual strains is needed to further understand rotavirus evolutionary dynamics and its impact on current and future vaccination programmes.