Phylogenetic Analysis and Serological Investigation of Porcine Circovirus Indicates Frequent Infection with Various Subtypes

Abstract

Porcine circoviruses (PCVs) are notorious for triggering severe diseases in pigs and causing serious economic losses to the swine industry. In the present study, we undertook a comprehensive approach for the investigation of PCV prevalence, including the phylogenetic analysis of obtained PCV sequences, the determination of major circulating genotypes and serological screening based on different recombinant Cap proteins with specific immunoreactivity. Epidemiological surveillance data indicate that PCV2d and PCV3a are widely distributed in Southwest China, while PCV4 has only sporadic circulation. Meanwhile, serological investigations showed high PCV2 antibody positivity in collected serum samples (>50%), followed by PCV4 (nearly 50%) and PCV3 (30-35%). The analysis supports different circulation patterns of PCV2, PCV3 and PCV4 and illustrates the PCV2/PCV3 genetic evolution characteristics on a nationwide basis. Taken together, our findings add up to the current understanding of PCV epidemiology and provide new tools and insight for PCV antiviral intervention.

Keywords: Cap; PCV; phylogeny; prevalence; serology.

Figure 1

The geographical distribution of samples from Sichuan Province. The number of samples ranged from 3 to 211 (A). The number of positive samples detected via SYBR Green-based real-time PCR assay (B).

Figure 2

ML trees of PCV2 ORF2 gene. Scale bar: 0.1. (A) ML tree of ORF2 from PCV2 strains before 2009 (model: TN93 + G4). (B) ML tree of ORF2 from PCV2 strains during 2009–2016 (model: TIM3 + R4). (C) ML tree of ORF2 from PCV2 strains during 2017–2018 (model: TIM3 + R3). (D) ML tree of ORF2 from PCV2 strains after 2019 (model: TIM3 + R3). (E) ML tree of PCV3 ORF2 gene. Scale bar: 0.01 (model: TN93 + R3).

Figure 3

Amino acid sequence alignment of PCV2 (A) and PCV3 (B) in Cap protein. The black boxes show the residues’ deletion or frameshift mutation. The conserved amino acid sites are marked with the red background and blue boxes.

Figure 4

Expression and identification of PCV2, PCV3 and PCV4 Cap protein. PCV2a, 2b, 2d, PCV3a, PCV3b and PCV4 Cap proteins were identified via Western blot using anti-Strep tag antibody (A) and PCV2-, PCV3- and PCV4-positive sera (B). −: negative control, a, b, d: sub genotype of PCV2 and PCV3.

Figure 5

Seroprevalence of antibodies in 2019 and 2021. The OD450 values of all serum samples (n = 165) are displayed as a heatmap (A), and the total number of positive samples are shown in a Venn diagram (B). The distribution dot plot showed the positive rate of each antigen (C), while the annual positive rate is shown in a bar chart (D).