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Porcine circovirus 2 (PCV2) replication is characterized by high variation among infected pigs. This study investigated the role of immunologic responses in causing this variation. Twelve gnotobiotic pigs were inoculated with PCV2. Four of these pigs were treated with cyclosporin A (CysA) to monitor the effect of the adaptive immunity on the development of the PCV2 infection. Through lymph node biopsies at 10, 15, and 21 days postinoculation (DPI), PCV2 replication in lymphoid tissues was monitored. The production of total PCV2-specific and PCV2-neutralizing antibodies was followed, together with interferon-gamma (IFN-gamma) mRNA expression levels in peripheral blood monocytes as a marker for cellular immunity. In general, the CysA-treated pigs showed the highest PCV2 titers, indicating that the adaptive immunity is necessary to restrain PCV2 replication. Three different PCV2 replication patterns were observed in non-CysA-treated pigs. Pattern 1: In two pigs, PCV2 was not detected. They had the highest neutralizing antibody titers, appearing from 15 DPI. In these pigs a good cellular response was indicated by a peak in IFN-gamma mRNA at 15 DPI. Pattern 2: Five pigs contained low to moderate PCV2 titers at 15 DPI, remaining constant or decreasing towards 21 DPI. Lower neutralizing antibody titers were observed and no rise in IFN-gamma was detected. Pattern 3: In one pig, a low PCV2 titer at 15 DPI dramatically increased toward 21 DPI. Although an antibody response against PCV2 was mounted, no PCV2-neutralizing antibodies were detected. This pig also showed no rise in IFN-gamma. The study findings indicate that variation in the onset of the adaptive immunity may account for variation in PCV2 replication among pigs. Absence of PCV2-neutralizing antibodies may be an important factor in the development of an increased virus replication.

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Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal microscopy and chemical inhibition of endocytic pathways (clathrin- and caveolae-mediated endocytosis and macropinocytosis), followed by evaluation of the level of PCV2 infection. It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min post-incubation. Bound PCV2 VLPs were internalized in 47+/-5.0 % of cells. Internalization was continuous, with 70.5+/-9.7 % of bound PCV2 VLPs internalized at 360 min post-incubation. Internalizing PCV2 VLPs co-localized with clathrin. PCV2 infection was decreased significantly by chemical inhibitors that specifically blocked (i) actin-dependent processes, including cytochalasin D (75.5+/-7.0 % reduction) and latrunculin B (71.0+/-3.0 % reduction), and (ii) clathrin-mediated endocytosis, including potassium depletion combined with hypotonic shock (50.2+/-6.3 % reduction), hypertonic medium (56.4+/-5.7 % reduction), cytosol acidification (59.1+/-7.1 % reduction) and amantadine (52.6+/-6.7 % reduction). Inhibiting macropinocytosis with amiloride and caveolae-dependent endocytosis with nystatin did not decrease PCV2 infection significantly. PCV2 infection was reduced by the lysosomotropic weak bases ammonium chloride (47.0+/-7.9 % reduction) and chloroquine diphosphate (49.0+/-5.6 % reduction). Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via clathrin-mediated endocytosis and requires an acidic environment for infection.

In this in vitro study, the replication kinetics of porcine circovirus type 2 (PCV2) in porcine alveolar macrophages (PAM) and fetal cardiomyocytes (FCM), two target cells in vivo, was compared with that in PK-15 cells. Cultures were inoculated with either the postweaning multisystemic wasting syndrome (PMWS)-associated strain Stoon-1010 or the abortion-associated strain 1121. Viral proteins were visualized and virus production was determined. In PK-15 cells, the capsid protein was expressed between 6 and 12 hours post inoculation (hpi), it relocated to the nucleus between 12 and 24 hpi. At that time, Rep protein was also detected in the nucleus. This sequence of events also occurred in FCM and PAM but nuclear localized antigens appeared later (48 hpi) and in a lower percentage of cells. In PAM, clear differences in susceptibility were seen between pigs. In PAM from two out of five tested pigs, nuclear localized antigens were not detected, whereas in PAM from three other pigs they were seen in up to 20% of the antigen-positive cells. Virus production was observed in PK-15 but not in PAM or FCM cultures. In a second study, the replication kinetics of seven different PCV2 strains were compared in PK-15 cells. It was shown that the two abortion-associated strains had a different replication kinetics in comparison with PMWS or porcine dermatitis and nephropathy syndrome associated strains. With the abortion-associated strains, a higher number of infected cells was observed at 24 hpi and the percentage of infected cells with nuclear localised antigens was lower compared to that of other strains.