Porcine circovirus 2 (PCV-2) genetic variability under natural infection scenario reveals a complex network of viral quasispecies

Abstract

Porcine circovirus 2 (PCV-2) is a virus characterized by a high evolutionary rate, promoting the potential emergence of different genotypes and strains. Despite the likely relevance in the emergence of new PCV-2 variants, the subtle evolutionary patterns of PCV-2 at the individual-host level or over short transmission chains are still largely unknown. This study aimed to analyze the within-host genetic variability of PCV-2 subpopulations to unravel the forces driving PCV-2 evolution. A longitudinal weekly sampling was conducted on individual animals located in three farms after the first PCV-2 detection. The analysis of polymorphisms evaluated throughout the full PCV-2 genome demonstrated the presence of several single nucleotide polymorphisms (SNPs) especially in the genome region encoding for the capsid gene. The global haplotype reconstruction allowed inferring the virus transmission network over time, suggesting a relevant within-farm circulation. Evidences of co-infection and recombination involving multiple PCV-2 genotypes were found after mixing with pigs originating from other sources. The present study demonstrates the remarkable within-host genetic variability of PCV-2 quasispecies, suggesting the role of the natural selection induced by the host immune response in driving PCV-2 evolution. Moreover, the effect of pig management in multiple genotype coinfections occurrence and recombination likelihood was demonstrated.

Figure 1

Maximum likelihood phylogenetic tree reconstructed using the ORF2 gene of estimated haplotypes and the reference sequences proposed by Franzo et al., 2015. Individual haplotypes have been named using the following scheme: AnimalID-SamplingWeek_HaplotypeNumber_HaplotypePrevalence. Farm A, B and C have been color-coded in green, blue and red, respectively.

Figure 2

Entropy value at each genome position are reported for farm A (top) and farm B (bottom). Data for different animal and week of age have been reported separately. Week post infection has been color coded. The ORF1 and ORF2 have been respectively represented as red and black line in the lower part of each graph.

Figure 3

Mean entropy (point) and 95 confidence intervals (error-bars; calculated using bootstrap) are reported for each animal at different wpi.

Figure 4

Estimate of selective pressures acting on the Cap gene. Normalized dN–dS is displayed for each codon position. Results of SLAC, FUBAR and FEL method have been color-coded.

Figure 5

Haplotype network drawn for each animal (animal ID is reported above the graph). The size of the circle is proportional to the prevalence of the haplotype while the different week post infection have been color coded. The five animals from farm A are represented on the left while the ones from farm B are represented on the right.

Figure 6

Summary of genotypes (color-coded) detected in different animals at different weeks of age. All considered animals originated from farm C.