Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells

Abstract

Background: Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G⁷¹⁰ to A⁷¹⁰) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively.

Results: Though all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID₅₀), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal.

Conclusions: These findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

Keywords: Infectious clone; PCV2 evolution; Porcine circovirus 2(PCV2); Rescued viruses; Truncated Rep protein; Virus replication.

Fig. 1

Diagram demonstrating the constructed infectious clone which includes a 1.1 copy of PCV2 genome, additionally, the location of the major ORFs encoding structural and non-structural proteins are also indicated

Fig. 2

The morphology and microstructure of the rescued viruses produced from PK15 cells, the representative image indicated that the rescued viruses have a spherical morphology with a size of 17–22 nm in diameter, as similar to those of PCV2-VLPs

Fig. 3

IFA results of PK cells infected by PCV2 rescued viruses. a Shown were representative IFA images (Magnification: 20 × 10, Olympus), NC, negative control; PC, positive control. b Quantification of PK15 cells infected by PCV2 viruses (n = 5). Results were expressed as mean ± SD. IFA assays were independently repeated three times. **represents statistical significance P<0.01 between indicated groups

Fig. 4

The virus load of PCV2 from 1st to 6th generation of serial passages was reflected by determining PCV2 copy number using quantitative real-time PCR. The X-axis shows the generations examined, while the Y-axis indicates the viral load as the logarithm of the copy number in the cell supernatants. Results were expressed as mean ± SD. Real-time PCR was repeated at least three times with each experiment performed in triplicate. **represents statistical significance P<0.01 among different groups

Fig. 5

The viral titers of PCV2 from 3rd to 6th generation of serial passages was determined by IFA assay. The X-axis indicates the generations tested, while the Y-axis shows the viral titer as the logarithm of the TCID₅₀ in the cell supernatants. **represents statistical significance P<0.01 among different groups

Fig. 6

The one-step growth curves of the rescued viruses (including the Positive control, YiY-3-2-3, YiY-3-2-1 and YiY-3-2-10) were performed by determining PCV2 genomic copy number using quantitative real-time PCR. a The content of the intracellular virus; b The content of the extracellular virus. The X-axis shows the incubation time, while the Y-axis indicates the viral load as the logarithm of the copy number of intracellular and extracellular virus. Results were expressed as mean ± SD. This assay was repeated three times. ** and *** represents statistical significance P<0.01 and P<0.001 relative to the YiY-3-2-3 group, respectively

Fig. 7

Primary amino acid sequence alignment and three-dimensional mapping of amino acids for the three PCV2 Rep proteins. a Comparison of the sequences of amino acid residues in the Rep proteins among the three PCV2 isolates. (GenBank accession numbers KU317473, KX831483 and KX831476). b The surface structures of PCV2 Rep proteins were generated based on the amino acid sequences, and displayed using PyMOL