Isolation and Biological Characteristics Study of Porcine Reproductive and Respiratory Syndrome Virus GZ2022 Strain

Abstract

PRRSV continues to evolve, complicating its epidemiological landscape in China. In this study, we isolated a novel PRRSV strain, GZ2022, from a swine farm in Guizhou Province. Subsequent analyses performed on this isolate included complete genome sequencing, phylogenetic analysis, recombination assessment, and characterization of its biological properties. Phylogenetic analysis revealed that GZ2022 clusters within Lineage 1 (NADC30-like) and features a 131-amino-acid deletion in NSP2, consistent with NADC30-derived strains. Recombination analysis identified NADC30 as the major parental strain (75% genomic contribution), with a minor recombinant region (25%) derived from the highly pathogenic HuN4 strain. In vitro growth kinetics revealed peak viral titers in Marc-145 cells at 72 h post infection (hpi). Pathogenicity was evaluated in 21-day-old piglets infected with GZ2022, the highly pathogenic PRRSV strain WUH3, or negative controls. Both infected groups exhibited typical PRRS clinical signs (fever, respiratory distress) and histopathological lesions (interstitial pneumonia, pulmonary consolidation). However, GZ2022-infected piglets exhibited attenuated virulence compared to WUH3, with reduced pulmonary hemorrhage and 0% mortality compared to 80% in the WUH3 group. Seroconversion (N-protein antibodies) was observed at 14 dpi (days post inoculation) in GZ2022-infected animals, persisting throughout the 28-day trial. Viral shedding dynamics aligned with moderate pathogenicity. These findings classify GZ2022 as a moderately virulent NADC30-like recombinant strain with partial HuN4-derived genomic regions. The emergence of such strains underscores the need for sustained surveillance of PRRSV genetic diversity and systematic evaluation of the biological properties of novel variants to refine control measures and inform vaccine development.

Keywords: GZ2022; PRRSV; complete genome sequencing; genetic variation; pathogenicity; phylogenetic analysis.

Figure 1

Isolation and identification of PRRSV GZ2022. (A) PCR identification of PRRSV GZ2022. PCR detection of PRRSV-ORF7 and PRRSV-ORF5 genes was performed on the cDNA of the samples. (B) CPE identification of the GZ2022 strain in PAM and Marc-145 cells. The GZ2022 strain was inoculated into PAM and Marc-145 cells, with a blank control group included. The red arrow indicates the site of cell lesion. Cytopathic effects were observed under an optical microscope at 3 to 4 dpi (CPE; 40 × magnification).

Figure 2

(A) Plaque assays of GZ2022 strain. Plaque assays were conducted using three experimental groups at dilutions of 10⁻⁴, 10⁻⁵, and 10⁻⁶, alongside a control group (Scale bar = 200 μm). (B) One step growth curve of PRRSV GZ2022 strain. The experiment was repeated three times at each time point.

Figure 3

Immunofluorescence analysis of PRRSV infection in Marc-145 cells at 48 hpi. (A) Marc-145 cells infected with the PRRSV GZ2022 strain exhibited positive immunofluorescence staining (green) accompanied by CPEs. (B) No specific immunofluorescence signal was detected in mock-inoculated cells (CPE; 40 × magnification).

Figure 4

(A) ORF5 phylogenetic tree. (B) NSP2 phylogenetic tree. (C) Full-length genome phylogenetic tree. (D) NSP2 region deletion pattern analysis. Multiple sequence alignment of NSP2 sequences was conducted to identify strain-specific deletions. (E) GP5 amino acid analysis. The asterisk (*) denotes B-cell epitopes, the circle (○) denotes T-cell epitopes, and the triangle (Δ) denotes linear antigenic epitopes. (F) Recombination breakpoints identified using the RDP5 algorithm. (G) SimPlot analysis showing genome-wide recombination signals.

Figure 4

(A) ORF5 phylogenetic tree. (B) NSP2 phylogenetic tree. (C) Full-length genome phylogenetic tree. (D) NSP2 region deletion pattern analysis. Multiple sequence alignment of NSP2 sequences was conducted to identify strain-specific deletions. (E) GP5 amino acid analysis. The asterisk (*) denotes B-cell epitopes, the circle (○) denotes T-cell epitopes, and the triangle (Δ) denotes linear antigenic epitopes. (F) Recombination breakpoints identified using the RDP5 algorithm. (G) SimPlot analysis showing genome-wide recombination signals.

Figure 4

(A) ORF5 phylogenetic tree. (B) NSP2 phylogenetic tree. (C) Full-length genome phylogenetic tree. (D) NSP2 region deletion pattern analysis. Multiple sequence alignment of NSP2 sequences was conducted to identify strain-specific deletions. (E) GP5 amino acid analysis. The asterisk (*) denotes B-cell epitopes, the circle (○) denotes T-cell epitopes, and the triangle (Δ) denotes linear antigenic epitopes. (F) Recombination breakpoints identified using the RDP5 algorithm. (G) SimPlot analysis showing genome-wide recombination signals.

Figure 5

(A) Clinical signs score. (B) Temperature changes of piglets. Note: At 17–22 dpi, only one piglet survived in the WUH3 group, and thus no variation could be calculated (n = 1). (C) Survival rate of PRRSV-infected piglets. (D) Weekly mean weight gain of PRRSV-infected piglets. Values in are shown as mean ± SD. *** p < 0.001; **** p < 0.0001; ns: no significance.

Figure 6

(A) Detection of virus shedding via nasal swabs. (B) Detection of virus shedding via rectal swabs. (C) Viral load in serum. (D) Viral load in tissues. Note: Viral loads in the WUH3-infected group represent individual measurements from both deceased and surviving piglets. (E) PRRSV antibody levels in serum. Values in are shown as mean ± SD.

Figure 7

Gross lesions in piglet lungs. At 28 dpi, all surviving piglets were euthanized and necropsied to observe gross pathological lesions in lungs among the GZ2022-infected, WUH3-infected, and control groups.