Human cells are permissive for the productive infection of porcine circovirus type 2 in vitro

Abstract

Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases, which are widespread in swine-producing countries. However, there is controversy regarding the susceptibility of human cells to PCV2 infection. In this study, human cell lines were infected with PCV2 and blind passaged several times. PCV2 entered and replicated in human cells, and infectious virions were generated, indicating that human cell lines were permissive to PCV2 replication. Furthermore, PCV2 replication in human cell lines was enhanced by D-glucosamine or concanavalin A (ConA). However, the infection efficiency of PCV2 was lower in human cells than in PK-15 cells, suggesting that PCV2 infection was limited in human cells. Our study reveals that human cells are permissive for the productive infection of porcine circovirus type 2 in vitro.

Figure 1

Human cell lines are susceptible to PCV2 infection. Cancerous human cell lines (MCF-7, A549, HeLa, HepG2, U937, THP-1) and normal human cell lines (293 T, WI-38, HUVEC, WISH, HSAS4, HEH2) were infected with PCV2 at an MOI of 5 for 72 h. The viral DNA was quantified by SYBR Green quantitative real-time PCR, and viral proteins were detected by Western blot. Cells that were not infected with PCV2 were used as control cells. (a) SYBR Green quantitative real-time PCR. (b) Western blot. Western blot was performed using the porcine circovirus type 2/PCV2 Capsid antibody or mouse Beta actin Antibody (1:2000) as the primary antibody and HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG as the secondary antibody. Unprocessed original scans of the Western blots can be found in Supplementary Fig. S1.

Figure 2

Immunostaining of cells infected with the CC1 strain of PCV2 72 hpi. Cells were infected with PCV2 at an MOI of 5 for 72 h and stained with rabbit anti-Cap antibody (1:100) (a) or rabbit anti-Rep antibody (1:100) (b), followed by staining with FITC-labelled goat anti-rabbit IgG (H + L) (green, 1:1000). DAPI (blue) was used to stain nuclei. Cells were visualized using Eclipse TE2000-V (Nikon).

Figure 3

One-step growth curve of PCV2 in different cells. HeLa cells, HUVECs and PK-15 cells were infected with PCV2 strain CC1 at an MOI of 5 for 72 h at 37 °C in a 5% CO₂ atmosphere for 1 h. Thereafter, the infected cells were washed with PBS three times to remove potential free viruses that had not entered the cells, and then the PK-15 cells and human cells were cultured in fresh culture medium containing 2% FBS or 5% FBS, respectively (This time point was considered as 0 hpi.). Samples were collected at 0, 12, 24, 36, 48, 60 and 72 hpi. The viral DNA was quantified by SYBR Green quantitative real-time PCR. **p < 0·01; ***p < 0·001; ns, no significance.

Figure 4

Progeny viral particles are infectious in human cells. (a) PCV2 can replicate in serially passaged human cell lines. Cells were infected with PCV2 for 72 h, and the resultant cells were blind passaged four times, followed by SYBR Green quantitative real-time PCR. P1, the first passage; P2, the second passage; P3, the third passage; and P4, the fourth passage. (b) PCV2 collected from one cell line can infect other cell lines. Supernatants were collected from PCV2-infected cells (PK-15, HepG2, A549, or WI-38 cells or HUVECs) after three cycles of freezing and thawing. The supernatants were used to infect HeLa, HepG2, A549, WI-38, PK-15 cells and HUVECs. At 72 hpi, SYBR Green quantitative real-time PCR was performed to detect the PCV2 copy number in the cells. PCV2/PK-15, PCV2 collected from PK-15 cells; PCV2/HepG2, PCV2 collected from HepG2 cells; PCV2/A549, PCV2 collected from A549 cells; PCV2/WI-38, PCV2 collected from WI-38 cells; PCV2/HUVEC, PCV2 collected from HUVECs. (c) Indirect immunofluorescence assay (IFA). Supernatants were collected from PCV2-infected cells (293 T or HSAS4) after three cycles of freezing and thawing. The supernatants were used to infect HeLa, HepG2, PK-15 cells and HUVECs. At 72 hpi, the cells were stained with rabbit anti-Cap antibody (1:100), followed by staining with FITC-labelled goat anti-rabbit IgG (H + L) (green, Beyotime, 1:1000). DAPI (blue) was used to stain nuclei. Cells were visualized using Eclipse TE2000-V (Nikon). PCV2/293 T, PCV2 collected from 293 T cells; PCV2/HSAS4, PCV2 collected from HSAS4 cells. Supernatants collected from non-infected 293 T or HSAS4 cells were used as negative controls (mock).

Figure 5

PCR detection of the full-length PCV2 genome. Cells were infected with PCV2 for 72 h, and the resultant viruses were blind passaged five times, followed by the identification of PCV2 by PCR according to the protocol described by Gilliland et al.. The expected size of the PCR product from PCV2 was 1741 bp. H₂O and DNA from uninfected cells were used as negative controls. M, DNA marker. P1, the first passage; P3, the third passage; and P5, the fifth passage. NC, negative control. Unprocessed original scans of the gel can be found in Supplementary Fig. S2.

Figure 6

Transmission electron microscopy (TEM) ultrastructural analysis. PCV2 particles 20 nm in diameter were observed by TEM. Scale bar indicates 200 nm. P1, the first passage; P3, the third passage; P4, the fourth passage.

Figure 7

PCV2 infection in human cells is enhanced by D-glucosamine or ConA. Cells were inoculated with PCV2 for 1 h, washed with PBS three times and maintained in fresh culture medium containing 5 μg/mL ConA or 300 mM D-glucosamine and 2% FBS in the case of PK-15 cells or 5% FBS in the case of human cells. Control cells were inoculated with PCV2 for 1 h and maintained in fresh culture medium containing 2% FBS in the case of PK-15 cells or 5% FBS in the case of human cells. At 72 hpi, cells were lysed by freezing and thawing three times, and the viral DNA was quantified by SYBR Green quantitative real-time PCR. **p < 0·01; ***p < 0·001; ns, no significance.