Characterization of a triple-recombinant, reassortant rotavirus strain from the Dominican Republic

Abstract

We report the genome of a novel human triple-recombinant G4P[6-8_R] mono-reassortant strain identified in a stool sample from the Dominican Republic during routine facility-based rotavirus strain surveillance. The strain was designated as RVA/Human-wt/DOM/2013840364/2013/G4P[6-8_R], with a genomic constellation of G4-P[6-8_R]-I1-R1-C1-M1-(A1-A8_R)-N1-(T1-T7_R)-E1-H1. Recombinant gene segments NSP1 and NSP3 were generated as a result of recombination between genogroup 1 rotavirus A1 human strain and a genotype A8 porcine strain and between genogroup 1 rotavirus T1 human strain and a genotype T7 bovine strain, respectively. Analyses of the RNA secondary structures of gene segment VP4, NSP1 and NSP3 showed that all the recombinant regions appear to start in a loop (single-stranded) region and terminate in a stem (double-stranded) structure. Also, the VP7 gene occupied lineage VII within the G4 genotypes consisting of mostly porcine or porcine-like G4 strains, suggesting the occurrence of reassortment. The remaining gene segments clustered phylogenetically with genogroup 1 strains. This exchange of whole or partial genetic materials between rotaviruses by recombination and reassortment contributes directly to their diversification, adaptation and evolution.

Fig. 1

Phylogenetic trees based on the nucleotide sequences (ORF) of the VP7(a), VP4(b), NSP1(c) and NSP3(d) gene segments. Strain RVA/Human-wt/DOM/2013840364/2013/G4P[6–8_R] is identified by the black filled box. Approximate-likelihood ratio test values >70 are shown adjacent to each node. The GenBank strain names, and G- and P-type associations are shown where available. Each scale bar indicates the number of nucleotide substitutions per site.

Fig. 2

Identification of recombinant VP4, NSP1 and NSP3 genes. (a) Boot-scanning plot of the RVA/Human-wt/DOM/2013840364/2013/G4P[6–8_R] VP4 gene sequence against VP4 derived from strains E1911/2009/G1P[8] and CHN/R479/2004/G4P[6] is depicted, with region from nucleotides 228 to 778 (purple) and 1685–1866 (pink) highlighted. Plot is modelled on a pairwise alignment with a step size of 20 and 2000 bootstrap replicates. ML trees showing phylogenetic relationships of the recombinant strain 2013840364/2013/G4P[6–8_R], strains E1911/2009/G1P[8] and CHN/R479/2004/G4P[6] in the sequences prior to the breakpoint, nucleotides 9–227; within the recombined gene segment, nucleotides 228–778 and 1685–1866; and after the breakpoint, nucleotides 779–1684 and 1867–2328. The VP4 genes from strains Wa/1974/G1P[8] and ST3/1975/G4P[6] were used as outgroups. (b) Boot-scanning plot of the RVA/Human-wt/DOM/2013840364/2013/G4P[6–8_R] NSP1 gene sequence against NSP1 derived from strains B3458/2003/G9P[8] and CMP45/08/2008/G9P[20] is depicted, with region from nucleotides 379 to 502 and 1152–1458 highlighted in pink and purple, respectively. Plot is modelled on a pairwise alignment with a step size of 20 and 2000 bootstrap replicates. ML trees showing phylogenetic relationships of the recombinant strain 2013840364/2013/G4P[6–8_R], strains B3458/2003/G9P[8] and CMP45/08/2008/G9P[20] in the sequences prior to the breakpoint, nucleotides 1–378; within the recombined gene segment, nucleotides 379–502 and 1152–1458; and after the breakpoint, nucleotides 503–1151. The NSP1 gene from strain Wa/1974/G1P[8] was used as an outgroup. (c) Boot-scanning plot of the RVA/Human-wt/DOM/2013840364/2013/G4P[6–8_R] NSP3 gene sequence against NSP3 derived from strains Dhaka25/2002/G12P[8] and UK/1973/G6P[5] is depicted, with region from nucleotides 400 to 806 highlighted in pink. Plot is modelled on a pairwise alignment with a step size of 20 and 2000 bootstrap replicates. ML trees showing phylogenetic relationships of the recombinant strain 2013840364/2013/G4P[6–8_R], strains Dhaka25/2002/G12P[8] and UK/1973/G6P[5] in the sequences prior to the breakpoint, nucleotides 1–399; within the recombined gene segment, nucleotides 400–806; and after the breakpoint, nucleotides 807–933. The NSP3 genes from strain Wa/1974/G1P[8] were used as outgroups.

Fig. 2

Identification of recombinant VP4, NSP1 and NSP3 genes. (a) Boot-scanning plot of the RVA/Human-wt/DOM/2013840364/2013/G4P[6–8_R] VP4 gene sequence against VP4 derived from strains E1911/2009/G1P[8] and CHN/R479/2004/G4P[6] is depicted, with region from nucleotides 228 to 778 (purple) and 1685–1866 (pink) highlighted. Plot is modelled on a pairwise alignment with a step size of 20 and 2000 bootstrap replicates. ML trees showing phylogenetic relationships of the recombinant strain 2013840364/2013/G4P[6–8_R], strains E1911/2009/G1P[8] and CHN/R479/2004/G4P[6] in the sequences prior to the breakpoint, nucleotides 9–227; within the recombined gene segment, nucleotides 228–778 and 1685–1866; and after the breakpoint, nucleotides 779–1684 and 1867–2328. The VP4 genes from strains Wa/1974/G1P[8] and ST3/1975/G4P[6] were used as outgroups. (b) Boot-scanning plot of the RVA/Human-wt/DOM/2013840364/2013/G4P[6–8_R] NSP1 gene sequence against NSP1 derived from strains B3458/2003/G9P[8] and CMP45/08/2008/G9P[20] is depicted, with region from nucleotides 379 to 502 and 1152–1458 highlighted in pink and purple, respectively. Plot is modelled on a pairwise alignment with a step size of 20 and 2000 bootstrap replicates. ML trees showing phylogenetic relationships of the recombinant strain 2013840364/2013/G4P[6–8_R], strains B3458/2003/G9P[8] and CMP45/08/2008/G9P[20] in the sequences prior to the breakpoint, nucleotides 1–378; within the recombined gene segment, nucleotides 379–502 and 1152–1458; and after the breakpoint, nucleotides 503–1151. The NSP1 gene from strain Wa/1974/G1P[8] was used as an outgroup. (c) Boot-scanning plot of the RVA/Human-wt/DOM/2013840364/2013/G4P[6–8_R] NSP3 gene sequence against NSP3 derived from strains Dhaka25/2002/G12P[8] and UK/1973/G6P[5] is depicted, with region from nucleotides 400 to 806 highlighted in pink. Plot is modelled on a pairwise alignment with a step size of 20 and 2000 bootstrap replicates. ML trees showing phylogenetic relationships of the recombinant strain 2013840364/2013/G4P[6–8_R], strains Dhaka25/2002/G12P[8] and UK/1973/G6P[5] in the sequences prior to the breakpoint, nucleotides 1–399; within the recombined gene segment, nucleotides 400–806; and after the breakpoint, nucleotides 807–933. The NSP3 genes from strain Wa/1974/G1P[8] were used as outgroups.