2017YFD0500103), the National Natural Science Foundation of China (No

2017YFD0500103), the National Natural Science Foundation of China (No. 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 DNA5C8. 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 EVP-6124 (Encenicline) 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 EVP-6124 (Encenicline) USA, France, Japan, Korea, China, and other countries1,4,12C15. 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 EVP-6124 (Encenicline) 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-productive18C20. Surprisingly, 235 (28.5%) samples of 826 stool swabs collected from 102 children who received a live EVP-6124 (Encenicline) rotavirus vaccine were positive for PCV-2 DNA21. Therefore, it is urgent to determine whether human cells are permissive for PCV2 infection and replication. Results 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 PRKCB cells as well as the PK-15 cells (Fig.?1a). The PCV2 DNA copy numbers were approximately 106.5 to 108.5 copies/200?L 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). Open in a separate window 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.