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.
Porcine circovirus (PCV) belongs to the genus Circovirus in the family Circoviridae and contains a single-stranded 1.7-kb circular DNA1,2,3,4. There are three types of PCV: porcine circovirus type 1 (PCV1), PCV2, and PCV3. PCV1 is non-pathogenic and considered a contaminant of the porcine kidney cell line (PK-15)4,5. Recently, some groups reported that commercial human rotavirus vaccines and porcine-derived pepsin products were contaminated with PCV1 and PCV2 DNA5,6,7,8. Unexpectedly, it was found that PCV1 can infect human 293 T, HeLa, and Chang liver cells without causing any visible changes9. Infectious PCV1 was detected in the lysate of infected human hepatocellular carcinoma cells and was serially passaged in the cells5. Another group found that PCV1 infection caused ultrastructural alterations of infected human cells10. As the genomic sequence of PCV2 shows 80% overall nucleotide sequence identity with that of PCV111, it is easy to assume that PCV2 may infect human cells. Nevertheless, to date, there is controversy regarding the susceptibility of human cells to PCV2 infection.
PCV2 was first confirmed in 1982 and subsequently identified in pigs in the USA, France, Japan, Korea, China, and other countries1,4,12,13,14,15. PCV2 is the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD), which are widespread in swine-producing countries1,4,16,17. PCV2 DNA was amplified from PCV2-transfected 293 T, HeLa, Hep2, RH, and Chang liver cells, and the expression of viral antigen was observed in all cells9. A CPE was observed in PCV2-transfected cells 3 days post-infection (dpi); the cells were altered in morphology from stretched to round, and the number of dead cells and cell debris was increased in the supernatant9. However, the PCV2 signal was lost after 2 weeks, and viral particles were not produced9. Investigations performed by other groups showed no evidence for the existence of PCV2-specific antibodies in the sera of PCV2-exposed persons, indicating that PCV2 infection in human cells was non-productive18,19,20. Surprisingly, 235 (28.5%) samples of 826 stool swabs collected from 102 children who received a live rotavirus vaccine were positive for PCV-2 DNA21. Therefore, it is urgent to determine whether human cells are permissive for PCV2 infection and replication.
Human cell lines are susceptible to PCV2 infection
To investigate whether human cells are susceptible to PCV2 infection, twelve human cell lines, including six cancer cell lines and six normal cell lines, were infected with PCV2 at a multiplicity of infection (MOI) of 5 for 72 h. PCV2 genomic DNA was detected in all the human cells as well as the PK-15 cells (Fig. 1a). The PCV2 DNA copy numbers were approximately 106.5 to 108.5 copies/200 μL in the human cell lines examined in this study. Furthermore, Western blotting was performed to confirm viral expression. The viral Cap protein was detected in human cells as well as PK-15 cells infected with PCV2, while no protein was observed in non-infected cells (Fig. 1b).
Cap and Rep, which are the main proteins of PCV2, are a viral structural protein and a viral DNA replication-associated protein, respectively4. To further confirm infection of human cells with PCV2, viral growth was analysed by indirect immunofluorescence assay (IFA), using either rabbit anti-Cap antibody (Fig. 2a) or rabbit anti-Rep antibody (Fig. 2b) as the primary antibody, both of which were previously prepared in our lab22,23. The subcellular distributions of both Cap and Rep were observed 72 h after PCV2 infection. The fluorescence was significantly greater in PCV2-infected human cells and PK-15 cells than that of control cells (mock-infected cells), indicating that both the Cap and Rep proteins of PCV2 were expressed in human cells as well as PK-15 cells.
Taken together, these results demonstrated that the human cell lines used in the present study are susceptible to PCV2 infection.
Human cell lines support PCV2 replication, and progeny viruses are infectious
To evaluate whether PCV2 can replicate in human cells, a one-step growth curve was performed in HeLa cells, HUVECs and PK-15 cells. Cells inoculated with PCV2 at an MOI of 5 were collected at intervals of 12 h. The viral DNA was quantified by SYBR Green quantitative real-time PCR. The amount of viral DNA gradually increased from 12 to 72 h (Fig. 3). PCV2 propagated more efficiently in PK-15 and HeLa cells and had a significantly higher copy number of viral DNA by 72 h post-infection (hpi) than in HUVECs, whereas the amount of viral DNA was lower at 24 hpi and increased from 36 to 72 hpi in HUVECs. Among the two human cell lines, viruses grown in HUVECs exhibited better growth than those grown in HeLa cells, as they exhibited viral DNA copy numbers of approximately 3.3 × 107 and 1.3 × 107 copies/200 μL by 72 hpi, respectively. Interestingly, although the cells were inoculated with similar copy numbers of PCV2 genomic DNA followed by attachment for one hour, PCV2 genomic DNA was detected at approximately 4.8 × 106 and 1.6 × 107 copies/200 μL in HeLa and HUVECs at 0 hpi, respectively, which were statistically higher than that in PK-15 cells (3.0 × 105 copies/200 μL).
To further investigate the growth characteristics of PCV2 in human cells, PCV2-infected cells were blind passaged four times, and PCV2 DNA copy numbers were analysed using real-time PCR. PCV2 proliferated consistently in four passages of human cells as well as PK-15 cells (Fig. 4a), suggesting that the virus can replicate in human cells and that the progeny viruses were infectious. To confirm this result, the PCV2-infected cells were lysed by three cycles of freezing and thawing, and the supernatants were transferred to other virus-free cells, followed by detection of the viral DNA at 72 hpi. Real-time PCR demonstrated that the progeny virions from one human cell and PK-15 cell can also replicate in virus-free PK-15 cells and other virus-free human cells (Fig. 4b). Moreover, supernatants collected from PCV2-infected or non-infected 293 T or HSAS4 cells were incubated with different human cells for 72 h, followed by IFA analysis. The results of IFA showed that the fluorescence was significantly increased in the human cells incubated with supernatants collected from PCV2-infected 293 T or HSAS4 (Fig. 4c) cells compared with that in non-infected cells.
Furthermore, to confirm the presence of PCV2 genomic DNA, conventional PCR using primers described by Gilliland et al.7 was performed to detect PCV2 DNA in the cell supernatants from passages 1, 3 and 5. A PCV2-specific product of the expected size, a 1741 bp product of the 1766 bp full-length PCV2 DNA genome, was amplified from all the cell supernatants from passages 1, 3 and 5 (Fig. 5). However, we did not detect PCV2-specific PCR products in the negative control. These results confirmed the presence of near full-length genomic PCV2 DNA in PCV2-infected cells, indicating that the viral DNA was replicated in human cells.
Moreover, electron microscopy was performed to visualize virions in human cells. Two human cell lines, HeLa and HSAS4, were randomly selected and infected with PCV2, and viruses were blind passaged in the respective cells. Cultures were then prepared and examined by transmission electron microscopy. Spherical virions with a diameter of 20 nm were observed in cells from each passage of the PCV2-infected human cells (Fig. 6).
Together, these results demonstrated that human cells are permissive for PCV2 infection and supported the in vitro replication of PCV2.
PCV2 infection in human cell lines is enhanced by D-glucosamine or concanavalin A (ConA)
Previously, we and other groups found that IL-2, ConA, and D-glucosamine significantly increased PCV2 yield24,25,26. Therefore, we assumed that treatment with ConA and D-glucosamine would enhance PCV2 infection in human cells. To confirm our hypothesis, three human cell lines and PK-15 cells were infected with PCV2 and treated with ConA or D-glucosamine. The amount of viral genomic DNA was significantly enhanced in PCV2-infected HeLa, HepG2, 293 T and PK-15 cells compared with that in untreated cells (Fig. 7). These results indicated that PCV2 infection in human cell lines can be enhanced by ConA or D-glucosamine, which is consistent with results obtained in PK-15 cells.
In the present study, we reported that human cells were permissive to productive PCV2 infection and that they supported viral replication in vitro. We infected twelve human cell lines with PCV2 and found that all human cells in the present study were susceptible to PCV2 infection, indicating that PCV2 has a broad cell tropism and can infect many types of human cells, including monocytes, epithelial cells and fibroblasts. However, the PCV2 DNA copy numbers were more than 107 copies/200 μL in most of the human cell lines, except for U937 and THP-1 cells (Fig. 1). It is possible that U937 and THP-1 cells are suspension cells, which may affect viral infection and alter it compared with that in adherent cells. In addition, both U937 and THP-1 cells are monocytes, which may interfere or suppress PCV2 infection. Further research is in progress to determine the reason for this difference.
PCV2 is an extremely slow-growing virus, and PCV2 infection and replication in PK-15 cells yielded very low viral titers11,24. Viral antigens were only observed in a few cells at 18 hpi, and cell-free progeny viruses began to appear at approximately 30 hpi27. Previously, we and other groups found that IL-2, ConA, and D-glucosamine significantly increased PCV2 yield24,25,26. However, the infection efficiency of PCV2 was lower in all human cell lines than that in PK-15 cells (Fig. 4a,b), even though the human cells were treated with ConA or D-glucosamine, which significantly enhanced PCV2 infection in PK-15 cells (Fig. 7). One possible reason is that human cells are not the natural host cells of PCV2 and cannot provide a suitable cellular environment for the sustainable replication of PCV2. Moreover, although the cells were inoculated with similar copy numbers of PCV2 genomic DNA followed by attachment for one hour, PCV2 genomic DNA was detected at a concentration of approximately 4.8 × 106 and 1.6 × 107 copies/200 μL in HeLa and HUVECs at 0 hpi, respectively, and these concentrations were statistically higher than that in PK-15 cells (3.0 × 105 copies/200 μL) (Fig. 3), indicating that PCV2 can attach more easily to human cells than PK-15 cells. Analysis of the interactions between PCV2 and human cells is ongoing. Additionally, in vitro and in vivo experiments are very different, and animal experiments (e.g., primates) will be used to confirm these results in our future research.
PCV2 has immunosuppressive properties17, which may increase susceptibility to opportunistic infection. Moreover, PCV2 infection can downregulate immune cell functions during recall antigen responses and induce IL-10 secretion by monocytic cells, leading to the effective repression of IL-12 in porcine PBMCs28. Therefore, whether PCV2 infection in human cells affects the cellular immune response needs to be elucidated. Furthermore, it was reported that singular PCV2 infection in pigs rarely results in clinical disease, while PCVD/PCVAD is often enhanced in severity and prolonged in duration with concurrent viral or bacterial infections4,29. Thus, whether co-infection of PCV2 with other pathogens occurs in human cells needs to be investigated.
In conclusion, the present study demonstrated that PCV2 is capable of infecting human cells in vitro. PCV2 can enter and replicate in human cells, resulting in infectious virions. It is noteworthy that the infection efficiency of PCV2 was lower in human cell lines than in PK-15 cells, indicating that PCV2 infection was limited in human cells under the current experimental conditions. Therefore, whether PCV2 is a zoonotic pathogen needs to be further investigated.
Virus and cells
PCV2 strain CC1 (GenBank accession no. JQ955679) was isolated and stored in our lab30.
Six cancerous human cell lines (MCF-7, A549, HeLa, HepG2, U937, THP-1) and six normal human cell lines (293 T, WI-38, HUVEC, WISH, HSAS4, HEH2) were purchased from Shanghai Tong Wei Biological Technology Co., Ltd. (China) and used for PCV2 infection. PCV-free PK-15 cells were stored in our lab and used as a control. For information on the cells used in this study, please see Supplementary Table S1.
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% foetal bovine serum (FBS, Gibco, USA) at 37 °C in a 5% CO2 atmosphere. At 30–50% confluency, cells were infected with PCV2 strain CC1 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 and human cells were cultured in fresh culture medium containing 2% FBS or 5% FBS, respectively. Seventy-two hours post-infection (hpi), cells were lysed by freezing and thawing three times while shaking the half-frozen medium and stored at −80 °C. The viral DNA was quantified by SYBR Green quantitative real-time PCR, and the viral protein was detected by Western blot.
To determine whether human cell lines support productive PCV2, serial passages were performed in PCV2-infected human cells and PK-15 cells. Briefly, PCV2-infected cells were washed with PBS three times and then trypsinized by elution with 0.25% trypsin. The resuspended cells were maintained 1:1 in fresh culture medium containing 4% FBS in the case of PK-15 cells or 10% FBS in the case of human cells for 24 h, and the resultant cells were blind passaged five times. The viral DNA at each passage was quantified by SYBR Green quantitative real-time PCR.
To evaluate whether ConA and D-glucosamine enhanced PCV2 infection in human cells, PCV2-infected cells were cultured in fresh culture medium containing 5 μg/mL ConA (Sigma, USA) or 300 mM D-glucosamine (Sigma, USA) 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. Seventy-two hours post-infection, cells were lysed by freezing and thawing three times, and the viral DNA was quantified by SYBR Green quantitative real-time PCR.
Polymerase chain reaction (PCR) and quantitative real-time PCR
PCV2 DNA was extracted from infected cells using a TIANamp Virus DNA/RNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. The genomic DNA was dissolved in 50 μL of DNase-free ddH2O and stored at −20 °C. PCR was performed to detect the full-length PCV2 genome according to the protocol described by Gilliland et al.7. SYBR Green quantitative real-time PCR was performed according to the protocol described by Yang et al.24 using the Bio-Rad IQTM Multicolor Real-Time PCR Detection System and the Luna® Universal qPCR Master Mix (New England Biolabs, USA). The primers used in this study are listed in Supplemental Table 1 (Table S1). The experiments were repeated at least three times.
Western blot (WB)
Western blot was performed according to the protocol described by Yang et al.31. Briefly, whole-cell lysates were separated by SDS-PAGE, electro-transferred to PVDF membranes (Millipore), blocked for 1 h with 5% skim milk in TBS-T buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween-20), and then incubated with the porcine circovirus type 2/PCV2 Capsid antibody (1:500, GeneTex, Texas, USA) or mouse Beta actin Antibody (1:2000, Proteintech Group, USA) at 4 °C overnight. Subsequently, the PVDF membranes were washed with 5% skim milk in TBS-T buffer three times, followed by blotting for 1.5 h with HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (1:5000, Beyotime, Jiangsu, China). The immunoreactive bands were visualized using the BeyoECL Plus Western blot detection system (Beyotime, Jiangsu, China) according to the manufacturer’s instructions. The experiments were repeated at least three times.
Indirect immunofluorescence assaNy (IFA)
The IFA assay was performed as described by Yang et al.24. Briefly, cells in 24-well plates were infected with PCV2 for 72 h and fixed with 80% ice-cold acetone at −20 °C overnight. The cells were then washed three times with PBS-Tween (PBS-T) and incubated with rabbit anti-Cap antibody or rabbit anti-Rep antibody (1:100) at 4 °C for 12 h. After an additional five washes with PBS-T, the cells were incubated with FITC-labelled goat anti-rabbit IgG (H + L) (1:1000, Beyotime, Jiangsu, China) at 37 °C for 30 min. After an additional five PBS-T washes, the cells were incubated with DAPI (Beyotime, Jiangsu, China) at 37 °C for 8 min, followed by washing with PBS-T three times. Thereafter, the cells were examined using an Eclipse TE2000-V (Nikon). Antibodies against PCV2 proteins, rabbit anti-Cap antibody or rabbit anti-Rep antibody, were previously prepared and stored in our lab22,23.
One-step growth curve
HeLa cells, HUVECs and PK-15 cells were infected with PCV2 at an MOI of 5 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 at different time points was quantified by SYBR Green quantitative real-time PCR.
Electron microscopy (EM) analyses
Cells were infected with PCV2 for 48 h and blind passaged four times. PCV2-infected cells at each passage were lysed by freezing and thawing three times while shaking the half-frozen medium. Cell fractions containing viral particles were collected and examined by negative staining transmission electron microscopy (Hitachi TEM system).
Statistical analysis was performed using GraphPad Prism software, version 5 (GraphPad Software, San Diego, CA). For each separate set of assays, at least 3 independent experiments were evaluated. The results are expressed as the mean ± standard deviation (SD). The results were considered statistically significant at p < 0.05.
The data used and/or analysed in the current study are available from the corresponding author on reasonable request.
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This work was financially supported by the National Key Research and Development Program of China (No. 2017YFD0500103), the National Natural Science Foundation of China (No. 31772747), the Science and Technology Research Program during the 13th Five-year Plan Period of Jilin Educational Committee [JJKH20190172KJ], the Program for JLU Science and Technology Innovative Research Team (JLUSTIRT, No. 2017TD-28), the Fundamental Research Funds for the Central Universities, and the Graduate Innovation Fund of Jilin University.