Porcine circovirus 2 (PCV2) population study in experimentally infected pigs developing PCV2-systemic disease or a subclinical infection

Abstract

Porcine circovirus 2 (PCV2) is a single stranded DNA virus with one of the highest mutation rates among DNA viruses. This ability allows it to generate a cloud of mutants constantly providing new opportunities to adapt and evade the immune system. This pig pathogen is associated to many diseases, globally called porcine circovirus diseases (PCVD) and has been a threat to pig industry since its discovery in the early 90's. Although 11 ORFs have been predicted from its genome, only two main proteins have been deeply characterized, i.e. Rep and Cap. The structural Cap protein possesses the majority of the epitopic determinants of this non-enveloped virus. The evolution of PCV2 is affected by both natural and vaccine-induced immune responses, which enhances the genetic variability, especially in the most immunogenic Cap region. Intra-host variability has been also demonstrated in infected animals where long-lasting infections can take place. However, the association between this intra-host variability and pathogenesis has never been studied for this virus. Here, the within-host PCV2 variability was monitored over time by next generation sequencing during an experimental infection, demonstrating the presence of large heterogeneity. Remarkably, the level of quasispecies diversity, affecting particularly the Cap coding region, was statistically different depending on viremia levels and clinical signs detected after infection. Moreover, we proved the existence of hyper mutant subjects harboring a remarkably higher number of genetic variants. Altogether, these results suggest an interaction between genetic diversity, host immune system and disease severity.

Figure 1

The average and 95% confidence intervals of log₂ antibody titers (IPMA) measured in sera (a) and log₁₀ PCV2 copies in 1 ml of PBS in nasal (b) and rectal swab (c) suspensions and 1 ml of serum (d) are reported for each wpi. Different groups have been color-coded.

Figure 2

The SNV frequency is reported for each genome position. The regions corresponding to the ORF1 and ORF2 have been represented as red and black lines, respectively. Error bars indicate the presence of multiple subjects harboring the same SNV and describe the frequency variability. The wpi are reported as columns while the different groups are reported in separate rows.

Figure 3

The entropy average and 95% confidence intervals of the complete genome and Cap and Rep coding regions are reported for each wpi. The different groups have been color-coded. The entropy values are reported for the complete dataset (a) and after excluding the ‘hypermutant’ subjects (i.e. 153 and 180) (b). Asterisks indicate the presence of significant differences among groups.

Figure 4

The entropy average and 95% confidence intervals of the complete genome are reported for each subject and wpi. Different groups have been color-coded.