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.

Antigenic differences among porcine circovirus type 2 strains, as demonstrated by the use of monoclonal antibodies.

This study examined whether antigenic differences among porcine circovirus type 2 (PCV-2) strains could be detected using monoclonal antibodies (mAbs). A subtractive immunization protocol was used for the genotype 2 post-weaning multisystemic wasting syndrome (PMWS)-associated PCV-2 strain Stoon-1,010. Sixteen stable hybridomas that produced mAbs with an immunoperoxidase monolayer assay (IPMA) titre of 1,000 or more to Stoon-1,010 were obtained. Staining of recombinant PCV-2 virus-like particles demonstrated that all mAbs were directed against the PCV-2 capsid protein. Cross-reactivity of mAbs was tested by IPMA and neutralization assay for genotype 1 strains 48,285, 1,206, VC2,002 and 1,147, and genotype 2 strains 1,121 and 1,103. Eleven mAbs (9C3, 16G12, 21C12, 38C1, 43E10, 55B1, 63H3, 70A7, 94H8, 103H7 and 114C8) recognized all strains in the IPMA and demonstrated neutralization of Stoon-1,010, 48,285, 1,206 and 1,103, but not VC2,002, 1,147 and 1,121. mAbs 31D5, 48B5, 59C6 and 108E8 did not react with genotype 1 strains or had a reduced affinity compared with genotype 2 strains in the IPMA and neutralization assay. mAb 13H4 reacted in the IPMA with PMWS-associated strains Stoon-1,010, 48,285, 1,206 and VC2,002, and the porcine dermatitis and nephropathy syndrome-associated strain 1,147, but not with reproductive failure-associated strains 1,121 and 1,103. mAb 13H4 did not neutralize any of the tested strains. It was concluded that, despite the high amino acid identity of the capsid protein (>or=91 %), antigenic differences at the capsid protein level are present among PCV-2 strains with a different genetic and clinical background.

Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin.

Heparan sulphate and sialoadhesin were previously identified on porcine macrophages as receptors for porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the exact role and cooperation of heparan sulphate and sialoadhesin during PRRSV attachment and internalization was analysed. It was observed that both heparan sulphate and sialoadhesin mediate PRRSV attachment and that only these two receptors are involved in attachment. Analysis of attachment kinetics of PRRSV to macrophages revealed that early attachment is mediated mainly via an interaction with heparan sulphate, followed by a gradual increase in interaction with sialoadhesin. By using wild-type CHO and CHO deficient in heparan sulphate expression, it was shown that heparan sulphate alone is sufficient to mediate PRRSV attachment, but not entry, and that heparan sulphate is not necessary for sialoadhesin to function as a PRRSV internalization receptor, but enhances the interaction of the virus with sialoadhesin.

Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages.

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.