Strain KTM368 (G11P[25]) was isolated in Nepal in 2004 (26), and rotavirus strains Dhaka6 (G11P[25]), Dhaka22–01 (G11P[8]), and Matlab36–02 (G11P[8]) were isolated in Bangladesh in 2001, 2001, and 2002, respectively (25,28). Double-stranded RNA of tissue culture–adapted reference porcine strains YM (G11P[7]), isolated in Mexico in 1983 (23), and OSU (G5P[7]) and Gottfried (G4P[6]) isolated in the United States in 1976 (30), was used in our study.

Virus RNA was extracted by using a QIAamp Viral RNA Mini Kit (QIAGEN, Leusden, the Netherlands) according to the manufacturer’s instructions. Extracted RNA was denatured at 97°C for 5 min, and reverse transcription–PCR was performed by using the OneStep RT-PCR Kit (QIAGEN). Forward and reverse primers used for amplification of different gene segments were synthesized on the basis of alignments of known 5′ and 3′ sequences of respective gene segments found in GenBank (primers are available upon request from J.M.). PCRs were performed by using an initial reverse transcription step at 50°C for 30 min, followed by PCR activation at 95°C for 15 min, 40 cycles of amplification, and a final extension at 72°C for 10 min in a BiometraT3000 Thermocycler (Biometra, Westburg, the Netherlands). Cycle conditions for the amplification of VP1, VP2, VP3, and VP4 were 30 s at 94°C, 30 s at 50°C, and 6 min at 70°C. For other gene segments, conditions were 30 s at 94°C, 30 s at 45°C, and 3 min at 72°C.

PCR products were purified with the MSB Spin PCRapace Kit (Invitek, Berlin, Germany) and sequenced by using the dideoxy-nucleotide chain termination method with the ABI PRISM BigDye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems, Foster City, CA, USA) in an ABI PRISM 3100 automated sequencer (Applied Biosystems). Complete 5′ and 3′ terminal nucleotide sequences of the 11 gene segments were determined by using a modified rapid amplification of cDNA ends technique as described (15).

Chromatogram sequencing files were analyzed by using Chromas 2.3 (Technelysium, Helensvale, Queensland, Australia), and contigs were prepared by using SeqMan II (DNASTAR, Madison, WI, USA). Multiple sequence alignments were constructed in ClustalX 2 (31) and subsequently edited in MEGA4 (32).

Phylogenetic and molecular evolutionary analyses were conducted by using MEGA4 (32). Genetic distances were calculated by using the Kimura-2 correction parameter at the nucleotide level, and phylogenetic trees were constructed by using the neighbor-joining method with 500 bootstrap replicates.

Genotypes of each of the 11 genome segments for all the rotavirus strains under investigation were determined according to the genotyping recommendations of the RCWG by using the RotaC rotavirus genotyping tool (21,33). Because the sequence of the VP6 gene segment of strain KTM368 did not belong to any of the established VP6 I genotypes, it was submitted to the RCWG for appropriate genotype assignment. GenBank accession numbers for each of the gene segments of strains Dhaka6, KTM368, Dhaka22–01, Matlab36–02, YM, OSU, and Gottfried are shown in Technical Appendix 1.

According to guidelines of the RCWG, all gene segments belonged to established genotypes, except for the VP6 gene segment of KTM368 (21). This sequence was submitted to the RCWG, accepted as a new VP6 genotype, and designated I12. Complete genotype assignments of the 6 strains fully sequenced and reference strain Wa are shown in Table 1. All 3 human G11 strains have gene segments belonging to genotype 1 for the gene segments encoding VP1–VP3 and NSP1–NSP5. The VP4-encoding gene segment of human strains KTM368 and Dhaka6 had the unusual P[25] genotype, and human strains Matlab36–02 and Dhaka22–01 had the typical human P[8] genotype. With regard to VP6-encoding gene segments, only the human strain KTM368 from Nepal had the I12 genotype, and other human rotavirus strains isolated in Bangladesh had the typical human Wa-like VP6 genotype I1. The 3 porcine rotavirus strains YM, Gottfried, and OSU all had genotype 1 gene segments for VP1–VP3 and NSP2–NSP5 and the expected different G (G11, G4, and G5) and P (P[7] and P[6]) genotypes. The VP6 and NSP1 genotypes were either I1 or I5 and A1 or A8, respectively (Table 1).

For several gene segments of the partially sequenced strains Dhaka22–01, insufficient sequence data were available for a definitive classification according to the guidelines of the RCWG (21). However, when available sequence data of strain Dhaka22–01 were compared pairwise with those of the other human G11 strains, sequences of VP7, VP6, and VP4 of strain Dhaka22–01 were nearly identical with those of the strain Matlab36–02 (99.8%, 99.0% and 98.4% identity at the nucleotide level, respectively) (Technical Appendix 1). In addition, for the VP1–VP2 and NSP1–NSP5 gene segments of strain Dhaka22–01, high identities (range 97.9%–100%) at the nucleotide level were found between strain Dhaka22–01 and the other human Wa-like G11 strains. For the VP3 gene segment, identities between strain Dhaka22–01 and strains KTM368, Dhaka6, and Matlab36–02 were low (range 86.3%–86.7%), and the VP3 gene segment of strain Dhaka22–01 was closely related to strains in the human M1 subcluster (99.6% identity with strain Dhaka12–03). These findings suggest a typical human Wa-like origin for Dhaka22-01 (Technical Appendix 1).

To study the relationships between human G11 and porcine rotavirus strains in greater detail, we constructed phylogenetic trees by using entire open reading frame nucleotide sequences for the 11 gene segments (Technical Appendix 2). For VP7, strains KTM368, Dhaka6, and Matlab36–02 cluster closely within the G11 genotype, together with the G11P[8] strain CUK1 from South Korea; the porcine G11 rotavirus strains YM and A253 are more distantly related (Technical Appendix 2). For VP6, strain KTM368 (genotype I12) was only distantly related to strains belonging to genotype I1. Human strains Dhaka6 and Matlab36–02 cluster in a large I1 subcluster, which contains mainly human and a few porcine strains isolated in Bangladesh, Belgium, the United States, Thailand, India, Australia, and Japan (Technical Appendix 2). For VP4, strains KTM368 and Dhaka6 cluster closely in the rare P[25] genotype, whereas strain Matlab36–02 is closely related to recently isolated P[8] human strains from Bangladesh, Belgium, South Korea, and the Democratic Republic of the Congo (Technical Appendix 2).

In the phylogenetic trees of the remaining 8 gene segments (VP1–VP3 and NSP1–NSP5), at least 1 major human monophyletic subcluster could be distinguished within genotype 1. We also observed 1 major porcine genotype 1 subcluster, a finding that is consistent with the assumption that Wa-like human rotavirus strains and porcine rotaviruses have a common ancestor (Technical Appendix 2) (11). Several gene segments of human G11 strains cluster closely in human genotype 1 subcluster, but a few of them did not cluster closely with any known human or porcine rotavirus strains and formed a distinct (nonhuman, nonporcine) branch or subcluster within genotype 1.

For the VP2 and NSP1 gene segments, human strains KTM368, Dhaka6, and Matlab36–02 clustered closely within the genotype 1 (C1 and A1, respectively) human subcluster (Technical Appendix 2). Regarding the VP1, NSP2, NSP3, NSP4, and NSP5 gene segments, human rotavirus strains Dhaka6 and Matlab36–02 are localized within the human subcluster, whereas strain KTM368 formed a distinct branch inside genotype 1 and did not cluster with any known human or porcine rotavirus strain (Technical Appendix 2). For VP3, all 3 human G11 strains (KTM368, Dhaka6, and Matlab36–02) belonged to a distinct subcluster inside the VP3 M1 genotype (Technical Appendix 2).

The distinct genotype constellations (Table 2) differed in their degree of relatedness to typical human Wa genogroup rotavirus strains. With the exception of the G11 genotype, human strain Dhaka22–01 is nearly indistinguishable from other locally or more distantly circulating human Wa-genogroup rotavirus strains, such as strains Dhaka16–03 (G1P[8]) and Dhaka12–03 (G12P[6]) from Bangladesh or strain B4633–03 (G12P[8]) from Belgium, all isolated in 2003 (Technical Appendix 1) (19). Human strain Matlab36–02 is closely related to strain Dhaka22–01, except for the VP3 gene (Technical Appendix 2), for which human strains KTM368, Dhaka6, and Matlab36–02 form a distinct subcluster inside the M1 genotype. This finding suggests a recent reassortment event. Strain Dhaka6 is closely related to strain Matlab36–02 and only differs in the VP4 genotype (P[25] versus P[8]), respectively (Technical Appendix 2). This finding also suggests a recent reassortment event. Strain KTM368 from Nepal has VP2 and NSP1 gene segments that belong to the typical human subcluster within the C1 and A1 genotypes, respectively. All other gene segments belong to non–Wa-like genotypes (VP7: G11, VP4: P[25], and VP6: I12) or to a distinct subcluster or branch inside genotype 1 with an unknown origin.

There are 2 possible hypotheses for the observed acquisition of human rotavirus genes by G11 strains. The first hypothesis is that different human G11 virus strains described in this study may have originated from several unrelated interspecies transmission events of animal G11 strains to humans, followed by reassortment events that involved Wa-like human strains. The second hypothesis is that a gradual acquisition of human rotavirus genes occurred after 1 interspecies transmission event, followed by multiple successive reassortment events. The second hypothesis is that a currently unknown ancestral rotavirus, of probable porcine origin and having the G11-P[25]-I12 genotypes in a nonhuman Wa genogroup background, might have undergone multiple reassortment events with co-circulating human rotavirus strains, resulting in the different natural reassortant rotavirus strains described in this study. These reassortments resulted in a human G11P[8] rotavirus composed entirely of typical human genotype 1 (Wa-like) RNA segments.