Molecular Epidemiology of Rotavirus A in Calves: Evolutionary Analysis of a Bovine G8P[11] Strain and Spatio-Temporal Dynamics of G6 Lineages in the Americas

Figure 1. Distribution of fecal scores among sampled calves according to the production system (beef and dairy farms) and clinical condition (diarrheic and non-diarrheic).

Figure 2. (a–f). Maximum likelihood phylogenetic trees of VP7, VP4, VP6, VP1, VP2, VP3, NSP1, NSP2, NSP3, NSP4, and NSP5 genes. The best fitting models of nucleotide substitution were: GTR + F + G4 (VP7, VP1, VP2, NSP2, NSP3), GTR + F + I + G4 (VP4, VP6, VP3, NSP1), TPM2 + F + G4 (NSP4), and TIM2 + F + I + G4 (NSP5). The analysis included similar sequences retrieved from GenBank as well as those from G8 and/or P[11] RVA strains (Table 3). Trees were rooted using genotypes other than those of strain 4385VT_D_BA, as follows: G11 (VP7); P[1], P[14], and P[17] (VP4); I1, I3, and I10 (VP6); R2 (VP1); C3 and C5 (VP2); M3 (VP3); A3, A11, and A18 (NSP1); N1 (NSP2); T1, T7, and T9 (NSP3); E1, E2, E3, and E5 (NSP4); H1 and H5 (NSP5). Ultrafast bootstrap values (10,000 replicates) ≥ 90% are shown as branch nodes. The bovine strain 4385VT_D_BA (white circle) is highlighted using a grey shadow. Other Argentinean strains from cattle (black circle), goat (black square), guanaco (black triangle), horse (black diamond), and vicugna (black inverted triangle) are indicated. Scale bars indicate nucleotide substitutions per site.

Figure 3. Phylogenetic analyses of G8 RVA strains. (A) Phylogenetic tree of the G8 genotype. The phylogram was reconstructed using the Kimura 2-parameter substitution model and the neighbor-joining method. For better visualization, lineages are surrounded by black lines and strain names are abbreviated. The G8 linages are designated according to previous studies [38,40] using Roman numbers. Argentinean G8 strains from this and previous studies are highlighted with an irregular blue pentagon. (B) Comparison of genetic distances within (intra-lineage) and between (inter-lineage) the G8 lineages. Genetic distance values, expressed as percentages, were calculated using the pairwise distance algorithm and the p-distance model (MEGA 7.0). (C) Frequency graph of pairwise genetic distances. The percentage values of genetic distances are indicated in the abscissa (x-axis) and the frequencies of these values are indicated in the ordinate (y-axis). The cutoff value to best discriminate lineages is shown by a dotted line.

Figure 4. Phylogenetic trees of bovine RVA strains from Buenos Aires province, Argentina. (A) VP7 sequences of RVA strains corresponding to G6(III) and G6(IV) lineages. (B) VP8* sequences of RVA strains with P[5] and P[11] genotypes. Phylograms were reconstructed using the Kimura 2-parameter substitution model and the neighbor-joining method (MEGA 7.0 software). The phylogram of VP7-G6 lineages was rooted using strain RVA/Cow-wt/ARG/B383/1998/G15P[11]. The analysis only included similar sequences and with available information. Each strain is represented with a number and symbol; white symbols correspond to beef farms, whereas black symbols correspond to dairy farms, as follows: □ “Cuenca del Salado”; △ “Depresion de Laprida”; ■ “Mar y Sierras”; ▲ “Oeste”; ● “Abasto Norte”; ▼ “Abasto Sur”. Bootstrap values (1000 replicates) >70% are shown as branch nodes. The name of the strain, type of production system, and province of collection are indicated. Abbreviations: D/B, Dairy or Beef; BA, Buenos Aires. (C) The map of Buenos Aires province shows RVA strains according to the production system (dairy or beef) and the location of farms in districts and production regions; each strain is…

Figure 5. Phylodynamic and phylogeographic reconstruction of bovine G6 RVA strains in American countries. (A) Maximum clade credibility tree (MCCT) of G6 lineage III [G6(III)] and G6 lineage IV [G6(IV)] strains together. The color of the branches represents the most probable country of origin. Posterior probability values of main clades are shown. Time of the most recent common ancestor (TMRCA) and the 95% highest posterior density (95% HPD) intervals are indicated below the tree nodes. Country abbreviations using the three-letter code (alpha-3) (ISO 3166): ARG, Argentina; BRA, Brazil; CAN, Canada; MEX, Mexico; URY, Uruguay; USA, the United States of America; VEN, Venezuela. (B) Inferred spread pattern of G6(III) and G6(IV) lineage strains in American countries. Line color represents the relative strength of connection between countries according to the Bayes Factor (BF) test: red arrows, decisive support with BF > 1000; blue arrows, strong support with 100 < BF < 1000; and green arrows, supported rates with 3 < BF <100.