Porcine circovirus 2 (PCV2) replicates in vivo in a wide variety of cells, including cells of the monocyte /macrophage lineage and cells of epithelial origin. PCV2 binding and internalization into these cells are not known.

Experiments were performed in cultures of the porcine 3D4/31 monocytic and PK-15, SK and ST epithelial cell lines to investigate the entry of PCV2 into its host cells. It was demonstrated by fluorescence confocal microscopy that PCV2 quickly bound to all cells of each cell type giving rise to a random distribution over the entire cell surface. PCV2 was slowly internalized in a time-dependent manner.

Effect of IFN-g and ELS acidification inhibitors on PCV2 infection of PK-15 cells. Representative light microscopic images of PK-15 cells treated with or without IFN-g and/or chloroquine diphosphate after performing an IPMA staining to identify PCV2-infected cells. A) No treatment. B) 500 U/ml IFN-g. C) 125 µM chloroquine diphosphate. D) 500 U/ml IFN-g combined with 125 µM chloroquine diphosphate. Bar, 200 µm.

The role of glycosaminoglycans (GAG) as receptors for PCV2 attachment to host cells was also investigated. Different soluble GAG or GAG combinations were mixed with PCV2 and these mixtures were allowed to competitively bind onto 3D4/31 cells. Heparan sulfate (HS) and chondroitin sulfate B (CS-B) reduced PCV2 binding to 3D4/31 cells, thereby reducing PCV2 infection. Removing HS and CS-B from the cell surface also reduced PCV2 infection, confirming HS and CS-B as receptors for PCV2 attachment. Further, it was shown that both PCV2 field isolates and PCV2 laboratory strains directly bind to GAG.

PCV2-inoculated cells were treated with different chemical agents known to inhibit certain endocytosis pathways and subsequent co-localization experiments were performed.

In 3D4/31 cells, PCV2 was internalized via clathrin-mediated endocytosis and PCV2 infection required endosomallysosomal system (ELS) acidification.

In epithelial cells, depletion of membrane cholesterol enhanced PCV2 infection. PCV2 was internalized via clathrinmediated endocytosis and a dynaminindependent clathrin- and caveolaeindependent pathway (CCIP). The former trapped PCV2 while the latter was efficient for PCV2 replication. Further, it was demonstrated that inhibiting ELS acidification increased PCV2 replication by increasing the disassembly of internalized PCV2 and that a cellular serine protease was involved in this process.

Treatment of PK-15 cells with interferon-gamma (IFN-g) or ELS acidification inhibitors increased PCV2 infection. Treatment of cells with a combination of IFN-g and ELS acidification inhibitors had an additional increase on PCV2 infection (Fig.1.) and combined treatment increased PCV2 yield by up to 50 times. This augmented virus replication in PK-15 cells may be helpful in the production of PCV2 vaccines.

October 2008

Further Reading

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