Browsing by Author "Meerts, P."

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Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31
(2005) Misinzo, G.; Meerts, P.; Bublot, M.; Mast, J.; Weingartl, H. M.; Nauwynck, H. J.
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.
Cell tropism and entry of porcine circovirus 2
(Elsevier, 2011-11-11) Nauwynck, H.J.; Sanchez, R.; Meerts, P.; Lefebvre, D.J.; Saha, D.; Huang, L.; Misinzo, G.
Porcine circovirus 2 (PCV2) may induce reproductive failure (return to oestrus, embryonic death, mum- mification, weak- and stillborn piglets) and postweaning multisystemic wasting syndrome (PMWS). Furthermore, it may modulate the immunity in such a way that it aggravates the outcome of many bacterial and viral infections. In the present paper, the cellular tropism and entry of PCV2 are described and linked with the pathological and clinical consequences.
Correlation between the presence of neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease
(2006-01-30) Meerts, P.; Misinzo, G.; Lefebvre, D.; Nielsen, J.; Bøtner, A.; Kristensen, C. S.; Nauwynck, H. J.
Background: In a previous study, it was demonstrated that high replication of Porcine circovirus 2 (PCV2) in a gnotobiotic pig was correlated with the absence of PCV2-neutralizing antibodies. The aim of the present study was to investigate if this correlation could also be found in SPF pigs in which PMWS was experimentally reproduced and in naturally PMWS-affected pigs. Results: When looking at the total anti-PCV2 antibody titres, PMWS-affected and healthy animals seroconverted at the same time point, and titres in PMWS-affected animals were only slightly lower compared to those in healthy animals. In healthy animals, the evolution of PCV2-neutralizing antibodies coincided with that of total antibodies. In PMWS-affected animals, neutralizing antibodies could either not be found (sera from field studies) or were detected in low titres between 7 and 14 DPI only (sera from experimentally inoculated SPF pigs). Differences were also found in the evolution of specific antibody isotypes titres against PCV2. In healthy pigs, IgM antibodies persisted until the end of the study, whereas in PMWS-affected pigs they quickly decreased or remained present at low titres. The mean titres of other antibody isotypes (IgG1, IgG2 and IgA), were slightly lower in PMWS-affected pigs compared to their healthy group mates at the end of each study. Conclusion: This study describes important differences in the development of the humoral immune response between pigs that get subclinically infected with PCV2 and pigs that experience a high level of PCV2-replication which in 3 of 4 experiments led to the development of PMWS. These observations may contribute to a better understanding of the pathogenesis of a PCV2- infection.
Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation
(Veterinary Microbiolog, 2008-05-05) Lefebvre, D. J.; Meerts, P.; Costers, S.; Misinzo, G.; Barbe, F.; Reeth, K. V.; Nauwynck, H. J.
Previously, it was shown that modulation of the immune system enhances porcine circovirus type 2 (PCV2) replication in pigs. In the present study, the effect of the mitogen concanavalin A (ConA) on PCV2 replication was investigated. Since ConA induces T-lymphocyte activation and initiates the production of interferon-gamma (IFN-g), a cytokine that enhances PCV2 replication in porcine epithelial and monocytic cell lines in vitro, it was examined if the effects observed with ConA were mediated by IFN-g. In an in vitro study, ConA but not IFN-g enhanced PCV2 replication in peripheral blood mononuclear cells (PBMC). Up to 2.08% and 0.96% of PBMC were antigen positive for PCV2 strains 1121 and Stoon-1010, respectively, and a low virus production was observed. PCV2-infected PBMC were identified as CD4+ (40%), CD8+ (54%) and IgM+ (11%). In a subsequent in vivo study, caesarean-derived colostrum-deprived piglets were injected with ConA or IFN-g 12 h before inoculation and every 3 days for 9 days after inoculation with strain 1121. PCV2 was isolated from inguinal lymph node biopsies from 10 days post-inoculation (dpi) in ConA-treated pigs and from 15 dpi in non-treated and IFN-g-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-g-treated pigs showed a delayed humoral immune response, but this delay did not result in increased PCV2 replication in these pigs. These experiments demonstrated that ConA enhances PCV2 replication in PBMC in vitro and in lymphoid tissues in vivo. # 2008 Elsevier B.V. All rights reserved.
Replication kinetics of different porcine circovirus 2 strains in PK-15 cells, fetal cardiomyocytes and macrophages
(2005) Meerts, P.; Misinzo, G.; McNeilly, F.; Nauwynck, H. J.
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.