Replacement of the replication factors of porcine circovirus (PCV) type 2 with those of PCV type 1 greatly enhances viral replication in vitro

Abstract

Porcine circovirus type 1 (PCV1), originally isolated as a contaminant of PK-15 cells, is nonpathogenic, whereas porcine circovirus type 2 (PCV2) causes an economically important disease in pigs. To determine the factors affecting virus replication, we constructed chimeric viruses by swapping open reading frame 1 (ORF1) (rep) or the origin of replication (Ori) between PCV1 and PCV2 and compared the replication efficiencies of the chimeric viruses in PK-15 cells. The results showed that the replication factors of PCV1 and PCV2 are fully exchangeable and, most importantly, that both the Ori and rep of PCV1 enhance the virus replication efficiencies of the chimeric viruses with the PCV2 backbone.

FIG. 1.

Schematic diagram of wild-type and chimeric PCVs and viral genomic DNA titer changes during in vitro replication. (A) Organization of wild-type and chimeric PCV infectious DNA clones. Genes encoding the viral capsid (cap) and replication-associated proteins (rep) are shown, along with the origin of replication (Ori). Genetic elements from wild-type PCV1 are shown in black, while those from wild-type PCV2 are white. Chimeric PCV clones were constructed by overlap extension PCR, using primers 1 to 16 in Table 1, and were cloned into plasmid vectors, using unique KpnI and SacII restriction enzyme sites located within rep. (B) Net changes of viral genomic DNA copy numbers during replication in PK-15 cells from 12 hpi to 96 hpi. The net change in viral genomic copies was calculated by subtracting the viral DNA genome copy numbers present at 12 hpi from those present at 96 hpi. The mean changes in log₁₀ viral genomic copies/ml ± the standard errors of the means (SEM) were compared among different viruses, using one-way ANOVA followed by Tukey's procedure for multiple comparisons, and are plotted for each virus. An asterisk (*) indicates a significant difference between the results for chimeras PCV1-Ori2 and PCV1-rep2 (P = 0.027).

FIG. 2.

One-step growth curve of wild-type and chimeric PCVs in PK-15 cells. PK-15 cells were infected with PCV1 (•), PCV2 (○), chimera PCV1-Ori2 (▪), chimera PCV2-Ori1 (□), chimera PCV1-rep2 (▴), or chimera PCV2-rep1 (▵) at a multiplicity of infection of 0.5. Cells in triplicate wells were harvested every 12 h through 96 hpi. The median 50% tissue culture infectious dose (TCID₅₀) for each virus was plotted, with bars showing the range of the data. (A) The growth kinetics of wild-type PCV1 and PCV2 were compared; an asterisk (*) indicates a significant difference. PCV1 replicates more efficiently than PCV2. (B) Comparison of the growth characteristics of wild-type PCV2 and two chimeric PCV2 viruses containing the Ori (□) or Rep (▵) of PCV1. The TCID₅₀ for each virus was plotted, with bars showing the range of the data and asterisks indicating significant differences. Chimeric PCV2 viruses containing PCV1 Ori or Rep replicate more efficiently than wild-type PCV2. (C) Comparison of the growth characteristics of wild-type PCV1 and two chimeric PCV1 viruses containing the Rep (▴) or Ori (▪) of PCV2. The two chimeric PCV1 viruses containing the Rep or Ori of PCV2 had decreased replication efficiencies compared to that of wild-type PCV1, although the difference was not statistically significant.