A Novel Virus-Like Agent Originated From Genome Rearrangement of Porcine Circovirus Type 2 (PCV2) Enhances PCV2 Replication and Regulates Intracellular Redox Status In Vitro

Abstract

Genome rearrangement occurs to porcine circovirus type 2 (PCV2) during in vitro and in vivo infections, and a number of rearranged PCV2 genomes have been isolated and characterized. This study was conducted to investigate the role of the rearranged PCV2 (rPCV2) in PCV2 replication and the biological effect of rPCV2 in host cells. Two whole rPCV2 genome sequences (358 nt and 1125 nt in length) were synthesized and recombinant plasmids pBSK(+)-rPCV2 (pBSK(+)-1125 and pBSK(+)-358) were constructed. A novel virus-like agent (rPCV2-1125) was rescued by in vitro transfection of porcine kidney cell line (PK-15) and porcine alveolar macrophage 3D4/21 cells. The data indicate that rPCV2-1125 significantly enhanced PCV2 replication in vitro. Furthermore, rPCV2-1125 led to oxidative stress in host cells, as indicated by decreased intracellular glutathione (GSH) and total superoxide dismutase (SOD) activities, as well as increased malondialdehyde (MDA) levels. These results provide new insights into genome rearrangement of PCV2 and will contribute to future studies of PCV2 replication and associated mechanisms.

Keywords: genome rearrangement; porcine circovirus type 2; redox status; viral replication; virus-like agent.

Figure 1

Schematic diagrams of recombinant plasmids. Recombinant pBSK(+)-1125 and pBSK(+)-358 plasmids were constructed by inserting two tandem copies of the complete rPCV2-1125 or rPCV2-358 genome into the pBluescriptII SK(+) vector.

Figure 2

Detection of rPCV2 under microscope. (A) Rescued viruses examined by electron microscopy (bar=100 nm). PCV2 virus-like particles were observed using negative staining in rPCV2-1125 samples. (B) PCV2-based IFA only specifically detected wildtype PCV2 but not rPCV2. No fluorescence signal was detected in cells transfected with either pBSK(+)-1125 or pBSK(+)-358.

Figure 3

Effects of rPCV2 on PCV2 replication in vitro. Cells were seeded into 24-well tissue culture plates and grown to 60%-80% confluency, and then transfected with the recombinant pBSK(+)-1125 or pBSK(+)-358 plasmids. After 6 h, the cells were infected with PCV2 (multiplicity of infection (MOI) of 1.0) for 48 h. By IFA, PCV2-specific fluorescence was examined under a fluorescence microscope (A) (200× magnification). Samples from the cell extracts and the supernatants were harvested separately. PCV2 DNA copies (B, C) and virus tiers (D, E) were determined. Noninfected cells were considered as the mock control while the cells infected with PCV2 alone were used as PCV2-infected control. Data are presented as means ± SD from three independent experiments. In each cell line or in culture supernatant, ^** P < 0.01 and ^## P < 0.01 for transfected cells or culture supernatant vs the PCV2-infected control. Each experiment was performed in triplicate.

Figure 4

The intracellular redox status regulated by rPCV2. Cells were seeded into 24-well tissue culture plates and grown to 60%-80% confluency, and then transfected with the recombinant pBSK(+)-1125 or pBSK(+)-358 plasmids. 6 h post transfection, the cells were infected with PCV2 (1 MOI) for 48 h Cell culture supernatants were removed and the cell samples were washed with PBS, and then trypsinized and harvested separately. GSH (A), SOD (B) and MDA (C) were detected by the commercial kits. Data are presented as means ± SD from three independent experiments. In the same cell line, ^* P < 0.05, ^** P < 0.01, ^# P <0.05, and ^## P < 0.01 for transfected cells vs mock cells; ^& P < 0.05 and ^△ P < 0.05 for transfected cells vs the PCV2-infected control. Each experiment was performed in triplicate.