Interspecies transmission of porcine-originated G4P[6] rotavirus A between pigs and humans: a synchronized spatiotemporal approach

Abstract

As a leading viral cause of acute gastroenteritis in both humans and pigs, rotavirus A (RVA) poses a potential public health concern. Although zoonotic spillover of porcine RVA strains to humans is sporadic, it has been detected worldwide. The origin of chimeric human-animal strains of RVA is closely linked to the crucial role of mixed genotypes in driving reassortment and homologous recombination, which play a major role in shaping the genetic diversity of RVA. To better understand how genetically intertwined porcine and zoonotic human-derived G4P[6] RVA strains are, the present study employed a spatiotemporal approach to whole-genome characterization of RVA strains collected during three consecutive RVA seasons in Croatia (2018-2021). Notably, sampled children under 2 years of age and weanling piglets with diarrhea were included in the study. In addition to samples tested by real-time RT-PCR, genotyping of VP7 and VP4 gene segments was conducted. The unusual genotype combinations detected in the initial screening, including three human and three porcine G4P[6] strains, were subjected to next-generation sequencing, followed by phylogenetic analysis of all gene segments, and intragenic recombination analysis. Results showed a porcine or porcine-like origin for each of the eleven gene segments in all six RVA strains. The G4P[6] RVA strains detected in children most likely resulted from porcine-to-human interspecies transmission. Furthermore, the genetic diversity of Croatian porcine and porcine-like human G4P[6] strains was propelled by reassortment events between porcine and porcine-like human G4P[6] RVA strains, along with homologous intragenotype and intergenotype recombinations in VP4, NSP1, and NSP3 segments. Described concurrent spatiotemporal approach in investigating autochthonous human and animal RVA strains is essential in drawing relevant conclusions about their phylogeographical relationship. Therefore, continuous surveillance of RVA, following the One Health principles, may provide relevant data for assessing the impact on the protectiveness of currently available vaccines.

Keywords: Croatia; G4P[6]; domestic pig; human; reassortment; recombination; rotavirus A; zoonosis.

Figure 1

The phylogenetic tree of the full-length VP7 segment G4 genotype CDS sequences. The strains from the present study were bolded and marked in purple (for pig-derived strains) and blue (for human-derived strains). Accession numbers of all strains are included in the taxa labels. The tree was generated by the ML method and T92+G+I model in MEGA 11 software. The bootstrap analysis with 1,000 replicates was used to assess the branching support (showed values > 0.7). The scale bar represents the number of substitutions per site. Rotarix G1 strain as an outgroup.

Figure 2

The phylogenetic tree of the full-length VP4 segment P[6] genotype CDS sequences. The strains from the present study were bolded and marked in purple (pig-derived strains) and in blue (human-derived strains). Accession numbers of all strains are included in the taxa labels. The tree was generated by the ML method and the HYK+G+I model in MEGA 11 software. The bootstrap analysis with 1,000 replicates was used to assess the branching support (showed values > 0.7). The scale bar represents the number of substitutions per site. RotaTeq P[5] strain stands as an outgroup.

Figure 3

The phylogenetic tree of the full-length backbone viral proteins CDS sequences: VP1 (A), VP2 (B), VP3 (C), and VP6 (D). The strains from the present study were bolded and marked in purple (pig-derived strains) and in blue (human-derived strains). Accession numbers of all strains are included in the taxa labels. The branches colored in red are designated to typically human RVA strains to accentuate the separation between porcine and human-originated RVAs of the same genotype. The tree was generated in MEGA 11 software by the ML method, models TN93+G+I, and T92+G, respectively. The bootstrap analysis with 1,000 replicates was used to assess the branching support (showed values > 0.7). The scale bar represents the number of substitutions per site. RotaTeq genogroup 2 strains stand as an outgroup, besides VP3 (C), where it shares the M1 genotype.

Figure 4

The phylogenetic tree of the full-length backbone non-structural proteins CDS sequences, NSP1 (A), NSP2 (B), NSP3 (C), NSP4 (D), and NSP5 (E). The strains from the present study were bolded and marked in purple (for pig-derived strains) and blue (for human-derived strains). Accession numbers of all strains are included in the taxa labels. The branches colored in red are designated to typically human RVA strains to accentuate the separation between porcine and human-originated RVAs of the same genotype. The tree was generated in MEGA 11 software by the ML method, models T92+G+I (A, C) and T92+G (B, D, E). The bootstrap analysis with 1,000 replicates was used to assess the branching support (showed values > 0.7). The scale bar represents the number of substitutions per site. RotaTeq genogroup 2 strains stand as an outgroup.