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Porcine circovirus 2 (PCV2) may induce reproductive failure (return to oestrus, embryonic death, mummification, 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.

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We examined recruitment, survival, life cycle and fecundity of two metallicolous (M, on metalliferous calamine soils) and two non-metallicolous (NM, on normal soils) populations of Thlaspi caerulescens in Belgium and Luxemburg. In each population, permanent plots were monitored over two reproductive seasons. In M populations, plots were located in two contrasting environments (grass versus grove) in order to test the influence of vegetation cover on life strategy. Our results show that the monocarpic life cycle is dominant in all populations of T. caerulescens. However the length of the pre-reproductive period varies from several months (winter annuals) to 1 year or more (perennials), and is partly related to plant origin (M versus NM). Most plants growing in metalliferous environments were annuals, whereas NM plants were mostly perennials. These differences in life cycle were related to differences in survival during summer, which was better in NM than in M populations. Within each M population, different survival conditions and life cycles were observed according to vegetation cover. Plants growing in grass areas were mostly annuals and had a low survival rate in summer whereas grove plants were mostly perennials and survived better in summer. Our results suggest the selection of stress avoiders (shortening of life cycle) in M populations of T. caerulescens but only for individuals growing in grass areas. Summer survival seems to play a key role in selection of life strategy in T. caerulescens.

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Population differentiation of alien invasive plants within their non-native range has received increasingly more attention. Common gardens are typically used to assess the levels of genotypic differentiation among populations.However, in such experiments, environmental maternal effects can influence phenotypic variation among individuals if seed sources are collected from field populations under variable environmental regimes. In the present study, we investigated the causes of an altitudinal cline in an invasive plant. Seeds were collected from Senecio inaequidens (Asteraceae) populations along an altitudinal gradient in southern France. In addition, seeds from the same populations were generated by intra-population crossings in a climatic chamber. The two seed lots were grown in a common garden in Central Belgium to identify any evidence of environmentally induced maternal effects and / or an altitudinal cline in a suite of life-history traits. Results failed to detect any environmental maternal effects. However, an altitudinal cline in plant height and aboveground biomass was found to be independent of the maternal environment.

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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.

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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.

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* The hypothesis that zinc (Zn) hyperaccumulation defends Thlaspi caerulescens against herbivores is tested with the snail Helix aspersa. We investigated the effects of leaf zinc, cadmium, glucosinolate, nitrogen and dry matter concentrations on the feeding preferences of snails. * Four T. caerulescens populations from southern France (two from metalliferous and two from normal soils) were grown on low- and high-Zn soils to obtain contrasting leaf Zn concentrations. Plants were also collected in the field, and binary feeding choices involving low- and high-Zn leaves were conducted. * Foliar Zn, Cd, N and dry matter concentrations did not affect the feeding choices of snails, whereas glucosinolate had a significant negative effect on herbivore preferences. Compared with metallicolous plants, nonmetallicolous ones appeared to be better protected against snails, whatever their Zn concentration. * These results do not support the defence hypothesis, as glucosinolates appear to decrease the degree of herbivory when Zn does not.

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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.

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Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory distress in young pigs and reproductive failure in sows. In PRRSV infected pigs, virus persists for several weeks to several months. Although IPMA antibodies are detected from 7 days post inoculation (pi), virus neutralizing (VN) antibodies are commonly detected starting from 3 weeks pi with an SN test on Marc-145 cells. Since infection of Marc-145 cells is quite different compared to infection of macrophages, the in vivo target cell, the role of these VN antibodies in in vivo protection is questionable. In our study, we demonstrated that antibodies from pigs early in infection with PRRSV Lelystad virus (14 days pi) showed no neutralization in the SN test on Marc-145 cells, but partially reduced Lelystad virus infection of porcine alveolar macrophages. At 72 days pi, VN antibodies were detected by the SN test on Marc-145 cells, and these protected macrophages completely against Lelystad virus infection. In contrast, these VN antibodies only partially reduced porcine alveolar macrophage infection of a Belgian PRRSV isolate (homologous virus), and had no effect on infection of porcine alveolar macrophages with the American type VR-2332 strain (heterologous virus). Confocal analysis of Lelystad virus attachment and internalization in macrophages showed that antibodies blocked infection through both a reduction in virus attachment, and a reduction of PRRSV internalization. Western immunoblotting analysis revealed that sera from 14 days pi, which showed no neutralization in the SN test on Marc-145 cells but partially reduced Lelystad virus infection of macrophages, predominantly recognized the Lelystad virus N protein, and reacted faintly with the M envelope protein. Sera from 72 days pi, with VN antibodies that blocked infection of Marc-145 cells and PAM, reacted with the N protein and the two major envelope proteins M and GP5. Using the Belgian PRRSV isolate 94V360 an identical but less intense reactivity profile was obtained. VN sera also recognized the VR-2332 N and M protein, but not the GP5 protein.

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The aim of the present study was to determine if porcine circovirus type 2 (PCV2) is able to infect embryonic cells of in vivo produced porcine embryos with and without zona pellucida (ZP). ZP-intact and ZP-free morulae (6-day post-insemination) and early blastocysts (7-day post-insemination), and hatched blastocysts (8-day post-insemination) were exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15). At 48 h post-incubation, the percentage of infected embryos and the percentage of viral antigen-positive cells per embryo were determined by indirect immunofluorescence (IF). Significantly different percentages of infected embryos were detected: 15% for ZP-free morulae, 50% for ZP-free early blastocysts and 100% for hatched blastocysts. The percentage of cells that expressed viral antigens was similar for the three stages of development. PCV2 exposure did not affect the in vitro development of the embryos during the 48 h study period. All ZP-intact embryos remained negative for viral antigens. In an additional experiment the diameter of the channels in the porcine ZP was determined. After incubation of early blastocysts with fluorescent microspheres of three different sizes, beads with a diameter of 20 nm and beads with a diameter of 26 nm crossed the zona whereas beads with a diameter of 200 nm did not. In conclusion, it can be stated that PCV2 is able to replicate in in vivo produced ZP-free morulae and blastocysts and that the susceptibility increases during development. The ZP forms a barrier to PCV2 infection, but based on the size of the channels in the ZP the possibility that PCV2 particles cross the ZP cannot be excluded.

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• Genetic variation structure and breeding system were investigated in metallicolous (MET) and nonmetallicolous (NONMET) populations of the heavy-metal hyperaccumulator Thlaspi caerulescens from Belgium, Luxembourg and the French Mediterranean region. • Allozyme variation showed a clear differentiation between the two ecotypes in Belgium and Luxembourg but not in southern France, in line with the lower degree of geographical isolation between the two ecotypes in this region. • In both regions inbreeding coefficient (FIS ) of NONMET populations was significantly higher compared to MET populations. Pollen/Ovule ratios were significantly higher in MET compared with NONMET populations. • These results suggest that NONMET populations of T. caerulescens are more self-fertile than their MET counterparts. This contrasts with earlier studies on other metal-tolerant species in which selfing rates were higher in MET populations. This pattern may be explained by the fact that both ecotypes are not in sympatry in the populations studied, and therefore reproductive isolation has not been selected to maintain the adaptations to metal-contaminated soils. In addition, higher selfing rates in NONMET populations may have evolved as a mechanism of reproductive assurance, because these populations generally are at low densities.

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In eastern Belgium, diploid and tetraploid knapweeds (Centaurea jacea L. s. l.) show a parapatric distribution with a contact zone. To compare the success of inter- and intra-cytotype crosses, and to investigate the pattern of allozyme segregation, controlled crosses involving plants from this contact zone were performed. Polysomic inheritance at two loci was observed in tetraploids, suggesting an autopolyploid origin. Two crosses allowed the detection of double reduction events in tetraploids at one locus, but no such event was detected among the 217 progenies. Null alleles were detected in tetraploids at two loci. Both cytotypes were highly self-incompatible. Inter-cytotype crosses were much less successful than intra-cytotype crosses in terms of seed set (five-fold reduction) and germination rates (three-fold reduction), suggesting a 'triploid block' effect. Progenies from inter-cytotype crosses most often had the maternal cytotype and resulted from selfing or pollen contamination. However, six triploids were detected. The significance of these results for the understanding of the geographical distribution of the cytotypes and inter-cytotype gene flow is discussed.

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In the northeastern part of Belgium, the Centaurea jacea complex shows extensive morphological variation and is represented by a diploid (2n = 22) and a tetraploid (2n = 44) cytotype. Polysomic inheritance of allozyme markers in the tetraploids, suggesting autopolyploidy, is here demonstrated for the first time. In order to test whether the two cytotypes occupy distinct habitats and possess different gene pools, patterns of allozymic and morphological variation were investigated in relation to ploidy level and site characteristics in 26 populations from the Belgian Ardennes. The two cytotypes showed a parapatric distribution, the diploids occurring at higher elevations (mostly above 500 m) than the tetraploids (mostly below 500 m). Three mixed populations were found near the contact zone of the two cytotypes. Within the mixed populations no triploid plant and no evidence for gene flow between cytotypes were found, despite widely overlapping flowering periods. The two cytotypes can be distinguished on the basis of morphological traits and enzymatic gene pools. The congruence of morphological and allozymic variation with chromosome numbers suggests a secondary contact between the two cytotypes with limited gene flow between them. The origin and persistence of the parapatric distribution are discussed.

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Polygonum aviculare is an annual weedy species showing extensive genetic variation in seed and leaf size and colonizing various types of man-disturbed habitats. A growth analysis was conducted on 12 genotypes representative of three regimes of disturbance of natural habitat (trampling, weeding, and no disturbance in the course of the growing season), grown under productive conditions in order to test whether relative growth rate (RGR) varies at the intraspecific level and, if so, which growth parameters may explain its variation. RGR showed significant genotypic variation (0.355-0.452 g g-1 day-1), positively correlated with specific leaf area (SLA) and leaf mass ratio (LMR) and negatively correlated with unit leaf rate per unit leaf area (ULRA). Thus, the paramount importance of leaf area ratio (LAR=SLA×LMR) in determining growth rate variation between different herbaceous species is confirmed at the intraspecific level in this species. Genotypes originating from trampled habitats had smaller seeds and smaller leaves than genotypes from habitats subject to other disturbance regimes. Additionally, they showed a lower LAR, not entirely compensated for by a higher ULRA, which resulted in a positive allometric relationship between seed size and RGR. It is hypothesized that their lower SLA, correlated with a higher leaf dry matter content (possibly a consequence of a higher cell wall content per unit leaf area) and their lower LMR have been co-selected with small leaf size as adaptations promoting resistance to trampling stress. It is suggested that variation in cell size and/or gibberellin content might be the mediators of the correlation found between seed size, leaf size and growth parameters within this species.

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