C. S. Choi

Immunity to PRRSV: Double-edged sword

Abstract The immune system is a double-edged sword for porcine reproductive and respiratory syndrome virus (PRRSV) infection. On one edge PRRSV has a predilection for immune cells and the disease manifestations can be linked directly to changes in the immune system. PRRSV appears to replicate extensively, if not exclusively, in cells of the immune lineage, notably macrophages; the direct replication of which may lead to immunosuppression, precipitate secondary infection and/or mediate disease. On the other edge, the virus stimulates immunity post-infection that protects an animal from re-infection. A vast array of structural and functionally distinct antibody specific to PRRSV are generated following infection or vaccination. Discrete populations of functional antibodies appear at different times and possibly reflect reactivity to different PRRSV polypeptides. Cell-mediated immune responses specific to PRRSV can be detected in various exposed pigs as well. Thus, the immune system appears to be intimately involved in both the disease process and protection from disease. It is unclear at this state of understanding what immune compartment provides protective immunity. Is it humoral (i.e. antibodies), selective functionally distinct populations of antibodies specific for selected PRRSV polypeptides or is cellular immunity essential for protection, or both. This review will attempt to summarize the current state of knowledge of the complex interaction of the immune system and PRRSV.

Structural polypeptides of the American (VR-2332) strain of porcine reproductive and respiratory syndrome virus.

SummaryThe structural polypeptides of the isolate VR-2332 of porcine reproductive and respiratory syndrome virus were analyzed in sucrose gradient-purified virions. The virus had an average density of 1.15 g/cm3 and contained, by SDS-PAGE, three major polypeptides with apparent molecular weights of 15, 19 and 26–30 kDa, which were designated as nucleocapsid (N), matrix (M) and envelope (E), respectively. The predominant structural protein was N. N-glycosidase F digestion only affected E whereas O-glycosidase or endoglycosidase H digestion had no effect, suggesting that the viral glycoproteins contain only complex N-linked carbohydrates.

Monitoring of porcine reproductive and respiratory syndrome virus infection in boars.

A major concern exists on transmission of porcine reproductive and respiratory syndrome virus (PRRSV) via semen and effect of vaccination on PRRSV shedding in semen. Recent reports suggest that the virus can be transmitted by semen from boars infected experimentally or from natural sources. Seminal shedding, viremia, and changes in semen quality in boars with or without vaccination were examined. Nine boars were divided into three groups (three boars/group). Group I boars were vaccinated with 2 ml of RespPRRS vaccine (NOBL Laboratory) intramusculary and groups II and III were non-vaccinated. At 28 post-vaccination study days, group I and group II boars were challenged with virulent PRRSV VR-2332 at 2 ml of 10(4.0) TCID50 per boar intranasally. Group III served as non-vaccinated and non-challenged control. Semen and serum samples were collected from -9 pre-vaccination study days to 85 post-challenge study days and tested for the presence of PRRSV by virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR). Prior to detection of PRRSV RNA from samples, conditions for RT-nPCR were optimized. Two primer sets, an external and an internal, were selected for RT-nPCR. The first round of PCR using an external primer set could detect 10 TCID50 of PRRSV/reaction. However, nested PCR could detect as little as 0.01 TCID50 of PRRSV/reaction. PRRS vaccine virus was not isolated from vaccinated pigs, but the vaccine virus RNA was detected from three boars, at day 6 to 15, 9 to 12, and 15 to 21 post-vaccination by RT-nPCR. Following challenge, two of non-vaccinated/challenged boars shed virus into semen up to 50 and 57 days post-challenge, respectively. The group I vaccinated boars did not shed virus into semen after challenge. The non-vaccinated/challenged group featured sperm abnormalities in the form of significantly increased incidence of proximal droplets and abnormal tails at 36-50 days post-challenge. The latter defect was observed to increase similarly in vaccinated/challenged boars as well.

Workshop studies on monoclonal antibodies reactive against porcine myeloid cells

Investigators from eight laboratories analyzed the reactivity of 22 monoclonal antibodies (mAb) against porcine myeloid cells. Based on binding data, clustering analysis and inhibition studies, workshop mAb 74-22-15 (003) and 6F3 (007) were assigned a swine workshop cluster number 3 (SWC3). These mAb recognized macrophages and neutrophils; neutrophils; a monocyte/macrophage-specific mAb was not identified by this workshop.

Pathogenicity of a skin isolate of porcine parvovirus in swine fetuses

The pathogenic properties of a skin isolate of porcine parvovirus (PPV), designated Kresse isolate, were compared with NADL-8 isolate, a prototype isolate of PPV, by in utero inoculation of mid-term and late-term gestation swine fetuses. Fetuses from pregnant sows of mid-gestation were inoculated with either NADL-8 or Kresse virus. Both isolates were highly pathogenic to mid-gestation fetuses. In contrast, dramatic differences in pathogenicity between these 2 isolates were observed in fetuses inoculated late in gestation. Such fetuses from each of 4 sows were inoculated with NADL-8 or Kresse virus isolate and sacrificed at 10, 18, 21, or 23 days postinoculation (PI). NADL-8-inoculated fetuses were grossly normal. The pathogenic effects of Kresse isolate were evident by gross pathology in fetuses collected at 18, 21, and 23 days PI, but not at 10 days PI. Hemagglutination (HA) and fluorescent antibody (FA) methods were used to identify virus in various tissues of late-gestation fetuses collected at 10 and 21 days PI. At 10 days PI, HA antigens were detected only in livers of NADL-8-inoculated fetuses, but in all tissues examined of Kresse-inoculated fetuses, including the brain. PPV specific fluorescence was demonstrated in tissues of fetuses inoculated with NADL-8 and Kresse virus. The major difference was that virus antigen was found in the brains of fetuses inoculated with Kresse virus, but not in NADL-8 infected fetuses. At 21 days PI, HA antigen was not detected in any of the tissues of fetuses inoculated with NADL-8 virus, with PPV specific fluorescence by FA being found only in the kidney. However, fetuses inoculated with Kresse virus displayed HA antigen in liver and PPV-specific fluorescence in all tissues tested including the brain. Both isolates induced similar antibody responses, 1:128 to 1:256 at 10 days and 1:512 to 1:1024 at 21 days PI. In addition, immunoglobulin G (IgG) deposits were demonstrated in kidneys and skin of fetuses inoculated with Kresse virus and IgM in brain, but not in tissues from fetuses inoculated with NADL-8 virus.

Tissue tropisms of porcine parvovirus in swine

SummaryLate-term gestation swine fetuses, similar to adult animals, are able to effectively mount immune response and survive porcine parvovirus (PPV) infection. An exception to this is the Kresse strain of PPV, which causes fetal death in late-term gestation swine fetuses. In an effort to understand the basis for this profound difference in pathogenicity between Kresse strain and the prototype strain of PPV, NADL-8, studies were designed to examine potential difference in sites of replication and quantity of virus produced between Kresse and NADL-8 strains. In order to define the sites of viral replication or sites of viral sequestration in situ hybridization, using digoxigenin-labeled strand-specific oligonucleotide probes, was applied to detect the presence of either double stranded viral DNA (RF) or viral single stranded DNA in tissues of infected fetuses. The presence and the state of viral DNA was confirmed by Southern blot hybridization. Relative amounts of RF-DNA synthesis in each tissue was compared between the two virus strains. Virus replication appeared to be of comparable levels in the livers of both NADL-8 and Kresse infected swine fetuses. Differences between these strains were observed in the brain and spleen; RF-DNA was detected in the brain and spleens of Kresse infected fetuses but not in NADL-8 infected ones. These findings indicate that differences in DNA replication of the PPV strains in selective sites, as well as the quantity of virus produced, may explain the distinction in pathogenesis of these viruses.

Differential infection of porcine alveolar macrophage subpopulations by nonopsonized Mycobacterium bovis involves CD14 receptors.

The resurgence in mycobacterial infection worldwide has led to renewed attention to the pathogenesis of Mycobacterium species. The purpose of this study was to characterize the infection of alveolar macrophages (AMs) by nonopsonized Mycobacterium bovis, and to elucidate the mechanism by which a differential infection of subpopulations of AM may occur. A difference in susceptiblity to Mycobacterium bovis infection of subpopulations of AMs was observed, such that the least dense cells were the least susceptible (21.4 ± 10.7%) and the most dense cells were the most readily infected (61.8 ± 5.6%). The percentage of AMs staining for CD14 receptors showed a similar differential distribution, with fewer of the least dense cells expressing CD14 and a greater percentage of the most dense cells staining for CD14 receptor expression. To investigate the role of CD14 receptors in the infection of AMs, anti‐CD14 antibody was added to the cell cultures. Infection of AM by Mycobacterium bovis was blocked by up to 60.2% by anti‐CD14 antibody but not by isotype control antibody. The results of this study suggest that Mycobacterium bovis selectively infects AM subpopulations, specifically those with the greatest expression of CD14, a putative receptor mechanism for Mycobacterium bovis infection of porcine AM. J. Leukoc. Biol. 60: 214–220; 1996.

Complete nucleotide sequence of a CDNA encoding porcine tumor necrosis factor‐alpha

Abstract Tumor necrosis factor‐alpha (TNF‐α), produced by immune cells, is a cytokine with a central role in the mediation of inflammatory responses to infection and injury. We report the nucleotide and corresponding amino acid sequence of a full length porcine TNF‐α cDNA. The complete cDNA nucleotide sequence is 1650 bp in length (not including the poly A tail) and has an open reading frame of 696 bp encoding a 232 amino acid protein. The porcine TNF cDNA sequence shows homologies of 86, 77, and 82% to human, murine, and lapine TNF cDNA sequences in the coding regions, respectively. The 5’ untranslated region of the cDNAs shows little sequence similarity. However, the 3´ untranslated region contains highly conserved sequences among all species, especially the TTATTTAT motif characteristic of cytokine messages.

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen.

SummarySwine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.

Inhibition of porcine parvovirus replication by empty virus particles

SummaryThe influence of empty porcine parvovirus (PPV) particles on viral replication was examined in cell cultures and in swine. Following extensive purification, homogenous preparations of full and empty PPV preparations were obtained and used for in vitro and in vivo analyses. In the first in vitro experiment, swine testes cells were infected with mixtures of various ratios of empty and full (E/F) particles. The production of both intracellular and extracellular virus was markedly inhibited in the presence of empty particles. This inhibition was dependent upon the concentration of empty particles present in the mixture. In the second in vitro study, various concentrations of empty particles were added prior to full virus infection. Again, marked inhibition of progeny virus production was evident and related to the concentrations of empty particles added.Based on the results of in vitro studies, the influence of empty particles on PPV infection in swine was tested by infecting mid-term and late-term gestation swine fetuses with various E/F particle ratios. Both mid-term and late-term fetuses exposed to 0:1, 1:1 and 5:1 E/F ratios displayed gross pathological evidence of PPV infection whereas fetuses exposed to E/F ratios of 30:1 or greater were grossly normal in appearance. However, fetuses infected with 30:1, 50:1 and 300:1 E/F ratios showed evidence of virus in their tissues by DNA hybridization. Regardless of the E/F ratios, late-term infected fetuses responded with high antibody titers ranging from 1024 to 4096. The results from these studies suggested that empty particles interfered with viral replication in both cell culture and in animals.