David Lefebvre

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

BackgroundIn 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.ResultsWhen 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.ConclusionThis 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.

Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells

ABSTRACT Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% ± 8% for HS-deficient and 12% ± 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.

Porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages

Different viruses have evolved strategies that inhibit apoptosis of the host cell early in infection and/or induce apoptosis in the host cell late in infection. In this study, it was investigated if and when porcine reproductive and respiratory syndrome virus (PRRSV) modulates apoptosis in PRRSV-infected macrophages. The PRRSV replication cycle in macrophages was completed within 12 h post-inoculation (hpi). PRRSV-infected macrophages, treated with staurosporine at 4, 5, 6 and 8 hpi, were significantly protected against staurosporine-induced apoptosis, but PRRSV-infected macrophages, treated with staurosporine at 12 hpi, were not. In contrast, starting from 12 hpi, all PRRSV-infected macrophages died by caspase-dependent apoptosis, which culminated in secondary necrosis. Treatment of PRRSV-infected macrophages with Z-Val-DL-Asp-fluoromethylketone indicated that apoptosis late in infection was not essential for efficient virus release. Anti- and pro-apoptotic activities were also observed in PRRSV-infected Marc-145 cells. In conclusion, this study shows that PRRSV stimulates anti-apoptotic pathways in macrophages early in infection and that PRRSV-infected macrophages die by apoptosis late in infection.

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells.

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3+CD8high cells starting from 14 dpi. Although CD3+CD8high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3+CD8high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.

GP4 of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro

Glycoprotein 4 (GP4) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a highly variable neutralizing epitope. The present study aimed to investigate whether this epitope is susceptible to immunoselection by antibodies in vitro. Cultivation of PRRSV in vitro in the continuous presence of neutralizing monoclonal antibodies (mAbs) directed against this epitope resulted in the selection of mAb-resistant PRRSV strains within five passages. Comparison of the GP4 amino acid (aa) sequence of the original PRRSV strain with the GP4 aa sequences of the mAb-resistant PRRSV strains revealed aa substitutions within this epitope. In conclusion, this study shows that the neutralizing epitope on GP4 is susceptible to immunoselection by antibodies in vitro.

Porcine circovirus 2 infection of epithelial cells is clathrin-, caveolae- and dynamin-independent, actin and Rho-GTPase-mediated, and enhanced by cholesterol depletion

Epithelial cells are the major in vivo target cells for porcine circovirus type 2 (PCV2). Although these cells are used for most studies of PCV2 gene expression and, little is known on PCV2 entry, attachment and internalization, in epithelial cells. PCV2 attachment to epithelial cells occurred rapidly and in a time-dependent manner. In contrast to attachment, internalization was slow. Immunofluorescent stainings revealed that during internalization, PCV2 co-localized with clathrin, but not caveolin. Blocking clathrin-mediated endocytosis increased instead of decreased the number of PCV2-infected cells by threefold, suggesting that it does not represent the main internalization pathway leading to a full replication. Further analysis with different inhibitors revealed that also macropinocytosis, dynamin-dependent internalization and membrane cholesterol play no role in PCV2 entry that leads to infection. Inhibition of small GTPases with Clostridium difficile toxin B reduced the number of PCV2-infected PK-15, SK and STs to 63+/-25%, 47+/-21% and 14+/-6%, respectively. Finally, inhibiting actin polymerization also blocked PCV2 infection, showing the need for actin during PCV2 infection. Together, these data indicate that a dynamin- and cholesterol-independent, but actin- and small GTPase-dependent pathway, allows PCV2 internalization in epithelial cells that leads to infection and that clathrin-mediated PCV2 internalization in epithelial cells is not followed by a full replication.

Recombination of two porcine circovirus type 2 strains

Pigs can be concurrently infected with different PCV2 strains. In this study, a cell-culture-adapted PCV2 strain, originating from a PMWS-affected pig, was purified by limiting dilution. Three different strains were obtained, and one of them was a perfect mosaic of the other two, with recombination breakpoints in ORF1 and ORF2. Incongruence was observed between phylogenetic trees constructed with the whole genome, ORF1 and ORF2. Amplification of ORF1 and ORF2 from original material, followed by cloning and sequencing, resulted in sequences corresponding to the parental strains, but not with the mosaic strain. These results demonstrate that PCV2 can undergo recombination.

Increased porcine circovirus type 2 replication in porcine leukocytes in vitro and in vivo by concanavalin A stimulation

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-gamma), 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-gamma. In an in vitro study, ConA but not IFN-gamma 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-gamma 12h 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 15dpi in non-treated and IFN-gamma-treated pigs. ConA increased PCV2 replication levels, but disease was not observed. Half of the ConA-treated and IFN-gamma-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.

Single amino acid mutations in the capsid switch the neutralization phenotype of porcine circovirus 2

Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases in pigs. Previously, it was demonstrated that mAbs 16G12, 38C1, 63H3 and 94H8 directed against the PCV2 capsid protein recognize PCV2 strains Stoon-1010 (PCV2a), 48285 (PCV2b), 1121 (PCV2a), 1147 (PCV2b) and II9F (PCV2b), but only neutralize Stoon-1010 and 48285. This points to the existence of two distinct PCV2 neutralization phenotypes: phenotype α (mAb recognition with neutralization; Stoon-1010 and 48285) and phenotype β (mAb recognition without neutralization; 1121, 1147 and II9F). In the present study, amino acids that are important in determining the neutralization phenotype were identified in the capsid. Mutation of T at position 190 to A in strain 48285 (phenotype α) resulted in a capsid resembling that of strain 1147 (phenotype β) and caused a loss of neutralization (switch from α to β). Mutations of P at position 151 to T and A at position 190 to T in strain II9F (phenotype β) resulted in a capsid resembling that of strain 48285 (phenotype α) and gave a gain of neutralization (switch from β to α). Mutations of T at position 131 to P and of E at position 191 to R in Stoon-1010 (phenotype α) changed the capsid into that of 1121 (phenotype β) and reduced neutralization (switch from α to β). This study demonstrated that single amino acid changes in the capsid result in a phenotypic switch from α to β or β to α.

Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification.

SummaryTreatment of porcine kidney (PK-15) cells with either interferon-gamma (IFN-γ) or endosomal- lysosomal system acidification inhibitors increases replication of porcine circovirus type 2 (PCV2). In the present study, the effect of a combination of these treatments on the number of infected cells and virus yield was tested. The number of PCV2 (Stoon-1010)-infected PK-15 cells increased in cells treated with ammonium chloride (445 ± 39% increase), IFN-γ (446 ± 8%), ammonium chloride + IFN-γ (1721 ± 283%), chloroquine diphosphate (1037 ± 121%), chloroquine diphosphate + IFN-γ (2199 ± 255%), monensin (950 ± 178%) and monensin + IFN-γ (1948 ± 60%). Combined IFN-γ and endosomal-lysosomal system acidification inhibitors increased PCV2 yield by up to 50 times compared to untreated PK-15. This augmented virus replication in PK-15 cells may be helpful in the production of PCV2 vaccines.