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

Susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of sialoadhesin and CD163

<p>Abstract</p> <p>Background</p> <p>Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. <it>In vivo</it>, the virus infects a subpopulation of tissue macrophages. <it>In vitro</it>, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, sialoadhesin (Sn) and CD163 (CHO^Sn-CD163and PK15^Sn-CD163) and evaluated their potential for production of PRRSV.</p> <p>Results</p> <p>Detailed analysis of PRRSV infection revealed that LV and VR-2332 virus particles could attach to and internalize into the CHO^Sn-CD163and PK15^Sn-CD163cells. Initially, this occurred less efficiently for macrophage grown virus than for Marc-145 grown virus. Upon internalization, disassembly of the virus particles was observed. The two cell lines could be infected with PRRSV strains LV and VR-2332. However, it was observed that Marc-145 grown virus infected the cells more efficiently than macrophage grown virus. If the cells were treated with neuraminidase to remove cis-acting sialic acids that hinder the interaction of the virus with Sn, the amount of infected cells with macrophage grown virus increased. Comparison of both cell lines showed that the PK15^Sn-CD163cell line gave in general better results than the CHO^Sn-CD163cell line. Only 2 out of 5 PRRSV strains replicated well in CHO^Sn-CD163cells. Furthermore, the virus titer of all 5 PRRSV strains produced after passaging in PK15^Sn-CD163cells was similar to the virus titer of those strains produced in Marc-145 cells. Analysis of the sequence of the structural proteins of original virus and virus grown for 5 passages on PK15^Sn-CD163cells showed either no amino acid (aa) changes (VR-2332 and 07V063), one aa (LV), two aa (08V194) or three aa (08V204) changes. None of these changes are situated in known neutralizing epitopes.</p> <p>Conclusions</p> <p>A PRRSV susceptible cell line was constructed that can grow virus to similar levels compared to currently available cell lines. Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes. Together, the results show that this cell line can be used to produce vaccine virus and for PRRSV virus isolation.</p

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP3, GP4 and GP5 envelope glycoproteins, only the M/GP5 glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP5. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines

Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages

Genome-wide transcriptional response of primary alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is a major cause of economic loss for the swine industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) triggers weak and atypical innate immune responses, but key genes and mechanisms by which the virus interferes with the host innate immunity have not yet been elucidated. In this study, genes that control the response of the main target of PRRSV, porcine alveolar macrophages (PAMs), were profiled in vitro with a time-course experiment spanning the first round of virus replication. PAMs were obtained from six piglets and challenged with the Lelystad PRRSV strain, and gene expression was investigated using Affymetrix microarrays and real-time PCR. Of the 1409 differentially expressed transcripts identified by analysis of variance, two, five, 25, 16 and 100 differed from controls by a minimum of 1.5-fold at 1, 3, 6, 9 and 12 h post-infection (p.i.), respectively. A PRRSV infection effect was detectable between 3 and 6 h p.i., and was characterized by a consistent downregulation of gene expression, followed by the start of the host innate immune response at 9 h p.i. The expression of beta interferon 1 (IFN-β), but not of IFN-α, was strongly upregulated, whilst few genes commonly expressed in response to viral infections and/or induced by interferons were found to be differentially expressed. A predominance of anti-apoptotic transcripts (e.g. interleukin-10), a shift towards a T-helper cell type 2 response and a weak upregulation of tumour necrosis factor-α expression were observed within 12 h p.i., reinforcing the hypotheses that PRRSV has developed sophisticated mechanisms to escape the host defence

Assessing the functionality of viral entry-associated domains of porcine reproductive and respiratory syndrome virus during inactivation procedures, a potential tool to optimize inactivated vaccines

Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses in the pig industry worldwide. Currently, vaccines based on inactivated PRRSV provide limited protection of pigs against infection, most likely because viral epitopes associated with the induction of neutralizing antibodies are not or poorly conserved during inactivation. To analyze the effect of inactivation procedures on the interaction of PRRSV with receptors involved in virus entry, a new assay was set up in this study. Viral entry-associated domains are most likely important for the induction of neutralizing antibodies, since neutralizing antibodies block interaction of PRRSV with cellular receptors. To investigate the interaction of PRRSV with the cellular receptors upon different inactivation procedures, attachment to and internalization of inactivated PRRSV into macrophages were monitored. AT-2 could not inactivate PRRSV completely and is therefore not useful for vaccine development. PRRSV inactivated with ultraviolet light, binary ethyleneimine and gamma irradiation, which all mainly have an effect at the genomic level, showed no difference compared to control live virus at all levels of virus entry, whereas PRRSV treated with formaldehyde, glutaraldehyde and pH changes, which all have a modifying effect on proteins, was not able to internalize into macrophages anymore. These results suggest that inactivation with methods with a main effect on the viral genome preserve PRRSV entry-associated domains and are useful for future development of an effective inactivated vaccine against PRRSV. Although PRRSV incubation at 37 $^{\circ}$C can completely inactivate PRRSV with preservation of entry-associated domains, this method is not recommended for vaccine development, since the mechanism is yet unknown

Inhibition of Endosome-Lysosome System Acidification Enhances Porcine Circovirus 2 Infection of Porcine Epithelial Cells▿

Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH4Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH4Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% ± 110%, 1,212% ± 34%, and 1,100% ± 179%, respectively, in porcine kidney (PK-15) cells; by 128% ± 7%, 158% ± 3%, and 142% ± 11% in swine kidney cells; by 160% ± 28%, 446% ± 50%, and 162% ± 56% in swine testicle (ST) cells; and by 313% ± 25%, 611% ± 86%, and 352% ± 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibition of endosome-lysosome system acidification