Peripheral blood contains two major particular infrequent dendritic cells (DC) subsets linking the innate and specific immune system, the myeloid DC and plasmacytoid DC equivalent to the natural interferon-producing cells (NIPC). The functional characterization of these cells demands large volumes of blood, making a large animal model more appropriate and beneficial for certain studies. Here, two subsets of porcine blood mononuclear cells expressing swine workshop cluster 3 (SWC3, a SIRP family member), are described and compared to monocytes. The blood DC specialized in T-cell stimulation were major histocompatibility complex (MHC) class II+, CD80/86+, CD1+/-, CD4-, and in contrast to monocytes CD14-. A CD16- and a CD16+ subset could be discriminated. Granulocyte-macrophage colony-stimulating factor and interleukin-3 were survival factors for this DC subset, and culture induced an up-regulation of MHC class II and CD80/86. The second subset described, are porcine NIPC, typically CD4++, MHC class IIlow, CD80/86low, CD1-, CD8-/low, CD16-/low and CD45RA-/low. Porcine NIPC had high interleukin-3 binding capacity, and survived in response to this cytokine. Their unique function was strong interferon type I secretion after virus stimulation. Both subsets were endocytically active when freshly isolated, and down-regulated this activity after in vitro maturation. Taken together, the present report has delineated porcine blood DC and NIPC, permitting a more detailed understanding of innate immune defences, particularly in response to infections.

Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-α) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-α synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-α-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-α almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-α is more likely to involve a specific, multimeric structure than a definite sequence motif.

We studied the interferon-alpha (IFN-alpha) system in relation to the porcine arterivirus (PoAV), porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant porcine IFN-alpha inhibited the growth of this virus in alveolar macrophage cultures. When pigs were challenged intranasally with PoAV, their serum contained IFN-alpha in relatively low concentrations on the second day after challenge and up to 5 days at the latest. Most animals had no IFN-alpha in their lung secretions, even though PoAV replicates in the respiratory tract. In vitro, PoAV replicates in alveolar macrophages, but neither these nor peripheral blood mononuclear cells (PBMC) produced IFN-alpha in response to infection. This may be because PoAV suppresses IFN-alpha production. When macrophages treated with PoAV were superinfected with swine transmissible gastroenteritis virus (TGEV), a known good inducer of IFN, no IFN-alpha was detected. This suppressive effect was lost when the virus was inactivated by UV light. Our results suggest that downregulation of IFN-alpha production may play an important part in enabling PoAV to replicate in cell cultures and in pigs.

Epithelial cells infected with the coronavirus transmissible gastroenteritis virus (TGEV) and fixed by glutaraldehyde induced a high alpha interferon (IFN-ax) production in nonimmune porcine as well as human or bovine peripheral blood mononuclear cells (PBMC). IFN-a was detected as early as 3 h after exposure of PBMC to infected cells and at producer/inducer cell ratios as low as 1/1. Two of four monoclonal antibodies directed against the viral transmembrane glycoprotein El could block the IFN-inducing capacity of both TGEV-infected cells and viral particles. On the other hand, IFN-a induction was not markedly affected by monoclonal antibodies directed against other El epitopes, against peplomer glycoprotein E2, or against nucleocapsid protein. Thus, these findings strongly imply that IFN induction by TGEV results from interactions between an outer membrane domain of El and the PBMC membrane.

Transmissible gastroenteritis virus, an enteropathogenic coronavirus of swine, is a potent inducer of alpha interferon (IFN-a) both in vitro and in vivo. Previous studies have shown that virus-infected fixed cells or viral suspensions were able to induce an early and strong IFN-a synthesis by naive lymphocytes. Two monoclonal antibodies directed against the viral membrane glycoprotein M (29,000; formerly El) were found to markedly inhibit virus-induced IFN production, thus assigning to M protein a potential effector role in this phenomenon (B. Charley and H. Laude, J. Virol. 62:8-11, 1988). The present report describes the selection and characterization of a collection of 125 mutant viruses which escaped complement-mediated neutralization by two IFN induction-blocking anti-M protein monoclonal antibodies. Two of these mutants, designated H92 and dm49-4, were found to exhibit a markedly reduced interferogenic activity. IFN synthesis by lymphocytes incubated with purified suspensions of these mutants was 30to 300-fold lower than that of the parental virus. The transcription of IFN-a genes following induction by each mutant was decreased proportionally, as evidenced by Northern (RNA) blot analysis. The sequence of the M gene of 20 complement-mediated neutralization-resistant mutants, including the 2 defective mutants, was determined by direct sequencing of genome RNA. Thirteen distinct amino acid changes were predicted, all located at positions 6 to 22 from the N terminus of the mature M protein and within the putative ectodomain of the molecule. Two substitutions, Thr-17 to Ile and Ser-19 to Pro, were assumed to generate the defective phenotypes of mutants dm49-4 and H92, respectively. The alteration of an Asn-Ser-Thr sequence in dm49-4 virus led to the synthesis of an M protein devoid of a glycan side chain, which suggests a possible involvement of this structure in IFN induction. Overall, these data supported the view that an interferogenic determinant resides in the N-terminal, exposed part of the molecule and provided further evidence for the direct role of M protein in the induction of IFN-a by transmissible gastroenteritis virus. The acronym VIP (viral interferogenic protein) is proposed as a designation for this particular class of proteins. Alpha interferon (IFN-a) can be induced after contact of leukocytes with viruses, bacterial products, or tumor cells (8). In contrast to IFN-P, which is produced after viral