First molecular detection and genetic analysis of porcine circovirus 4 in the Southwest of China during 2021-2022

Abstract

Porcine circovirus 4 (PCV4) was identified in 2019 as a novel circovirus species and then proved to be pathogenic to piglets. However, there is a lack of its prevalence in the Southwest of China. To investigate whether PCV4 DNA existed in the Southwest of China, 374 samples were collected from diseased pigs during 2021-2022 and detected by a real-time PCR assay. The results showed that the positive rate of PCV4 was 1.34% (5/374) at sample level, and PCV4 was detected in two of 12 cities, demonstrating that PCV4 could be detected in pig farms in the Southwest of China, but its prevalence was low. Furthermore, one PCV4 strain (SC-GA2022ABTC) was sequenced in this study and shared a high identity (98.1-99.7%) with reference strains at the genome level. Combining genetic evolution analysis with amino acid sequence analysis, three genotypes PCV4a, PCV4b, and PCV4c were temporarily identified, and the SC-GA2022ABTC strain belonged to PCV4c with a specific amino acid pattern (239V for Rep protein, 27N, 28R, and 212M for Cap protein). Phylogenetic tree and amino acid alignment showed that PCV4 had an ancient ancestor with mink circovirus. In conclusion, the present study was the first to report the discovery and the evolutionary analysis of the PCV4 genome in pig herds of the Southwest of China and provide insight into the molecular epidemiology of PCV4.

Keywords: genetic analysis; genotype; molecular detection; porcine circovirus 4; the Southwest of China.

FIGURE 1

The geographical distribution of the 374 samples in the Southwest of China. In China, provinces with PCV4 complete genome amplified are filled with orange. In the Southwest of China, cities with sample collections are filled with light green, and cities with positive sample detection are marked with red solid circles.

FIGURE 2

Neighbor-joining trees were constructed with a p-distance model and bootstrapping at 1,000 replicates. Phylogenetic tree was constructed based on the complete genome of 42 PCV4 strains. SC-GA2022ABTC strain was marked with the black solid triangle (▲). Scale bar indicates nucleotide substitutions per site.

FIGURE 3

Neighbor-joining trees were constructed with a p-distance model and bootstrapping at 1,000 replicates. (A) Phylogenetic tree was constructed based on amino acids of Rep of 63 CRESS DNA virus strains. (B) Phylogenetic tree was constructed based on amino acids of Rep of 27 Circoviridae strains. SC-GA2022ABTC strain was marked with the black solid triangle (▲). The other three Porcine circovirus 4 (PCV4) reference strains were marked with black filled circles (⬤). Scale bar indicates nucleotide substitutions per site.

FIGURE 4

All amino acid mutation sites of Rep protein and Cap of 42 Porcine circovirus 4 (PCV4) strains. PCV4 strains were divided into three genotypes, comprising PCV4a (light orange), PCV4b (light green), PCV4c (light blue). The specific amino acid patterns of genotype were displayed in the red dotted box. Amino acid residues contained in potential linear B-cell epitopes are highlighted in light grey. SC-GA2022ABTC strain investigated in this study. SC-GA2022ABTC strain sequenced in this study was marked with the black solid triangle (▲).