Epidemiologic Investigation and Genetic Variation Analysis of PRRSV, PCV2, and PCV3 in Guangdong Province, China from 2020 to 2022

Abstract

Recently, the emergence of HP-PRRSV (Highly Pathogenic porcine reproductive and respiratory syndrome virus) and the exacerbation of mixed infections of PRRSV and PCV have resulted in significant economic losses for the Chinese pig industry. This study collected a total of 226 samples suspected of infection with the aforementioned viruses from diverse pig farms in seven urban districts of central and northern Guangdong Province between 2020 and 2022. The positive rates of PRRSV, PCV2, and PCV3 in the samples were 33.2%, 37.6%, and 7.5%, respectively, and there were various mixed-infection scenarios present in the samples. This study successfully isolated multiple strains of PRRSV2 and PCV2 from their positive samples, and obtained the gene sequences of six PCV3 (ORF1 + ORF2) from samples. The associated sequences obtained were subjected to bioinformatic analysis and revealed the following:Predominantly prevalent strains of PRRSV in Guangdong Province include HP-PRRSV and NADC30-like variants, whereas PCV2 is primarily represented by the 2b and 2d subtypes. Specifically, the amino acid variation patterns exhibited by the PRRSV GP5 and NSP2 proteins of the strains sg_2108, qy_2008, and fs_2108 under environmental selective pressure are remarkably similar to the characteristics of Highly Pathogenic PRRSV; thus, it is inferred that they may possess higher virulence. The detected PCV3 strains were predominantly concentrated within the PCV3a-IM branch. All PRRSV strains involved in this study are wild-type-PRRSV (wt-PRRSV), comprising three recombinant strains and seven highly virulent strains. Among these strains, the ORF1a gene exhibited the highest variability in their genomes. Environmental selective pressure may enhance the virulence and immune evasion capabilities of PRRSV and drive mutations in the Cap proteins of PCV2 and PCV3. Conversely, PCV2 and PCV3 strains demonstrated greater stability in genetic evolution. In conclusion, this study enhances the epidemiological data regarding PRRSV, PCV2, and PCV3 in Guangdong Province, China, and is significant for the surveillance, prevention, and active control of these three diseases.

Keywords: epidemiological investigation; genetic variation analysis; porcine circovirus type 2 (PCV2); porcine circovirus type 3 (PCV3); porcine reproductive and respiratory syndrome virus (PRRSV).

Figure 1

Figure depicting the distribution of sample sources; the three quantities under the name of each district capital represent the number of samples collected by this laboratory in the respective region during the years 2020, 2021, and 2022.

Figure 2

Isolation of PRRSV and PCV2 strains; (A) The PRRSV strain was isolated in PAM cells, resulting in significant Cytopathic effects (72 hpi 100×); (B) The PRRSV strain was isolated in Marc-145 cells, resulting in significant Cytopathic effects (72 hpi 200×); (C) Isolation of PRRSV in Marc-145 cells: observations of lesions in Marc-145 cells post-infection and immunofluorescence assay (IFA) detection using pig-derived PRRSV-positive serum (72 hpi 100×); all isolated PRRSV strains induced similar results; (D) Isolation of porcine circovirus type 2 (PCV2) in PK-15 cells: observations of lesions in PK-15 cells post-infection and immunofluorescence assay (IFA) detection using PCV2-positive serum sourced from pigs (72 hpi 200×).

Figure 3

Phylogenetic analysis of PRRSV was conducted, wherein “▲” denotes the sequences of the strains obtained in this study. (A) Genetic evolution tree based on the whole genomes of PRRSV isolates and reference strains; (B) The phylogenetic tree was constructed based on the ORF5 gene sequences obtained in this study and the reference strain.

Figure 4

Phylogenetic analysis of PCV2 was conducted, wherein “▲” denotes the sequences of the strains obtained in this study. (A) Genetic evolution tree based on the whole genome of PCV2 isolates and reference strains; (B) Genetic evolution tree based on the ORF2 gene of PCV2 isolates and reference strains.

Figure 5

Phylogenetic analysis of PCV3 was conducted, wherein “▲” denotes the sequences of the strains obtained in this study. (A) Genetic evolution tree based on the complete coding sequences (ORF1 + ORF2)of PCV3 isolate variant strains and selected reference strains; (B) Genetic evolution tree based on the ORF2 genes of PCV3 isolate variant strains and selected reference strains.

Figure 6

PRRSV GP5 protein amino acid variation analysis, with the red box indicating the sequences of the strains obtained in this study. The black box area from left to right represents the signal peptide region, two hypervariable regions (HVRs), and three transmembrane regions (TMs). The gray area from left to right represents bait epitope region, primary neutralizing antigenic epitope region (PNE), two T-cell epitope regions, and one B-cell epitope region. The orange area indicates distinguishing sites on the GP5 protein for identifying PRRSV vaccine strains, field strains, and variations in virulence.

Figure 7

Analysis of amino acid variations in PRRSV Nsp2 protein reveals the presence of the sequences of the strains obtained in this study indicated by the red box, while the gray area denotes segments with amino acid deletions.

Figure 8

The analysis of amino acid variations in the PCV2 and PCV3 Cap proteins from the sequences of the strains obtained in this study, which are highlighted in red boxes. (A) Analysis of amino acid variation in Cap protein of PCV2; (B) Analysis of amino acid variation in Cap protein of PCV3.

Figure 9

SimPlot recombination analysis graphs of the full genome of the sequences of the PRRSV strains obtained in this study, where the gray area indicates the position of gene recombination. (A–F): SimPlot recombination analysis graphs corresponding to recombination events 1, 2, 3, 4, 5, and 8 in Table S16.

Figure 10

Similarity heat maps of the genome nucleotide sequences of the PCV2 and PCV3 strains obtained in this study, with the isolated strains highlighted in red font; (A) Heat map of nucleotide sequence similarity based on the genome sequences of PCV2; (B) Heat map of nucleotide sequence similarity based on the genome sequence of PCV3.

Figure 11

PRRSV GP5 protein model construction and visualization analysis of positively selected sites. The identified sites are presented as light green or dark green spheres. (A) Cartoon representation of the PRRSV GP5 protein monomer, site 203 was not displayed; (B,C,E): Microscopic display of positively selected sites and their hydrogen bonds on the GP5 protein model; (D) Overall display of the GP5 protein model and its positively selected sites.

Figure 12

Visualization analysis of the positively selected sites of the PCV2 Cap protein from the isolated strain; (A) Displaying the hydrophobic surface of the PCV2 Cap protein; (B) Visualizing the positively selected sites of the PCV2 Cap protein through cartoons and Atmos/Bonds display; (C,D) Analysis of the display of positively selected sites of the PCV2 Cap protein.