Expression at mRNA level of cytokines and A238L gene in porcine blood-derived macrophages infected in vitro with African swine fever virus (ASFV) isolates of different virulence.

Porcine macrophage cultures were infected with two ASFV isolates of variable virulence and mRNA levels of several relevant macrophage-derived cytokines were quantified by real time PCR. At six hours post infection, a clear enhancement of mRNA expression of TNFalpha, IL6, IL12 and IL15 was observed in macrophages infected with the low virulent ASFV/NH/P68 (NHV) when compared to those infected with the highly virulent ASFV/L60 (L60). The sequence of the A238L gene homologue to the cellular IkappaB was found identical in both viral isolates and its expression at mRNA level was higher in macrophages infected with NHV when compared to macrophages infected with L60. Furthermore our results suggest a negative correlation between the mRNA expression of A238L gene and the mRNA expression of the above mentioned cytokines (with the exception of IL10) in L60 infected macrophages in opposition to the positive correlation (with exception of the IL1) suggested in NHV infection. Overall, our data strongly emphasize that virulence of ASFV isolates may depend on their capacity to regulate the expression of macrophage-derived cytokines relevant for the development of host protective responses by yet unknown mechanisms triggered by the virus at early stages of the cellular infection.

Rapid detection of porcine reproductive and respiratory syndrome viral nucleic acid in blood using a fluorimeter based PCR method.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus recognised world wide as an important cause of reproductive failure and pneumonia in pigs. American and European strains of PRRSV, differentiated antigenically and genomically, have been reported. PRRSV infections are currently diagnosed using serology, virus isolation and/or immunocytochemistry. In order to overcome various drawbacks associated with these techniques, conventional, block-based RT-PCR methods for the detection of PRRSV nucleic acid in clinical samples have been described. These methods require gel electrophoresis for analysis of PCR products and present high risk of DNA carry-over contamination between the samples tested. We describe the detection of PRRSV RNA in serum samples and in blood impregnated filter disks (FDs), obtained from experimentally inoculated pigs, using a closed-tube, fluorimeter-based PCR assay. The assay eliminates the use of gel electrophoresis, and is as sensitive and specific as the conventional block-based PCR assay, detecting positive samples as early as 1 day post-inoculation. We also report a rapid fluorimeter based PCR method for differentiating American and European strains of PRRSV.

A fluorimeter-based RT-PCR method for the detection and quantitation of porcine cytokines.

In this paper, we describe a fluorimeter-based, closed-tube, polymerase chain reaction (PCR) assay for the detection and quantification of the mRNA of porcine interleukin 1alpha (IL1alpha) and interleukin 2 (IL2) cytokines in peripheral blood leukocytes (PBLs) using melting curve analysis and compare it to a standard PCR performed in a block-based thermocycler.

Immunopathogenesis of classical swine fever: role of monocytic cells.

Virulent classical swine fever (CSF) represents an immunomodulatory viral infection that perturbs immune functions. Circulatory and immunopathological disorders include leukopenia, immunosuppression and haemorrhage. Monocytic cells - targets for CSF virus (CSFV) infection - could play critical roles in the immunopathology, owing to their production of immunomodulatory and vasoactive factors. Monocytes and macrophages (Mphi) are susceptible to virus infection, as a consequence of which prostaglandin E2 (PGE2) production is enhanced. The presence of PGE2 in serum from CSFV-infected pigs correlated with elevated PGE2 productivity by the peripheral blood mononuclear cells from these same animals. It was noted that these PGE2-containing preparations did not inhibit, but actually enhanced, lymphocyte proliferation. The proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 were not involved, although elevated IL-1 production could relate to lymphocyte activation. Nevertheless, IL-1 was not the sole element: infected Mphi produced lympho-stimulatory activity but little IL-1. This release of immunomodulatory factors, following CSFV infection of monocytic cells, was compared with other characteristics of the disease. Therein, PGE2 and IL-1 production was noted to coincide with the onset of fever and the coagulation disorders typical of CSF. Consequently, these factors are of greater relevance to the haemorrhagic disturbances, such as petechia and infarction, rather than the leukopenia found in CSF.

Modulation of monocytic cell activity and virus susceptibility during differentiation into macrophages.

A major component of innate immune responses relies on monocytes and macrophages, virus infection of which will pose a particular problem for immunological defense. Consequently, the monocytic cell differentiation pathway was analyzed in terms of cellular modulations therein and their relation to monocytotropic virus infection. Differentiation was characterized by down-regulation of CD14, MHC Ags, the monocytic SWC1 marker, and p53; concomitant up-regulation of the SWC9 macrophage marker, a putative porcine CD80 (detected with anti-human CD80 Ab), and acid phosphatase secretion were also characteristic. Elevated phagocytic and endocytic activities as well as endosomal/lysosomal acidification were identified as being important to the macrophage. In contrast, monocytes possessed high accessory activity. This was multifactorial, concomitantly requiring 1) high MHC Ag expression; 2) enzyme activity of esterase, peroxidase, myeloperoxidase, and 5' nucleotidase in preference to glucosidase, galactosidase, and glucuronidase; and 3) elevated capacity for spontaneous IL-1 production. Only with all parameters was efficient stimulation of Ag-specific lymphocytes possible. These results point to a continuous process during differentiation, involving inter-related characteristics linking the more accessory monocyte to the scavenger macrophage, both in vitro and in vivo. Of particular interest was how these characteristics related to monocytotropic virus infection, and how a particular virus could show a clear preference for the differentiating macrophages. Such results not only further our understanding of porcine immunology, but also provide evidence and a potential model for the determination and characterization of monocytotropic virus-host cell interactions.

The reverse transcription polymerase chain reaction for the diagnosis of porcine reproductive and respiratory syndrome: comparison with virus isolation and serology.

A single-tube reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of porcine reproductive and respiratory syndrome (PRRS) virus in blood samples from infected pigs was developed. This test was assessed for sensitivity and application as a rapid diagnostic tool by comparison with virus isolation and detection of PRRS virus antibody in blood. The RT-PCR test was slightly more sensitive than virus isolation for detection of virus in serum and markedly more sensitive than virus isolation from plasma from experimentally infected pigs. The RT-PCR test was also applicable when using whole blood-impregnated filter paper discs, with 94% of the specimens taken by this procedure being positive when compared to RT-PCR performed on serum. PRRS viral nucleic acid was detected in blood samples as early as 24 h after infection and persisted for some time, whereas circulating antibody to PRRS virus was not detected in the same animals until 9 days after infection. These results indicate that the RT-PCR may be an useful technique for the early identification of PRRS viral nucleic acid in blood samples of infected pigs.

Distribution of cytopathic and noncytopathic bovine viral diarrhea virus antigens in tissues of calves following acute experimental infection.

The distribution of cytopathic and noncytopathic biotypes of bovine viral diarrhea virus (BVDV) in the tissues of colostrum-fed and colostrum-deprived calves was investigated. Colostrum-fed (group A) and colostrum-deprived (group B) calves were experimentally infected with the BVDV isolate 80/1, which contains both BVDV biotypes. Colostrum-deprived calves were also experimentally infected with a noncytopathic BVDV (group C) or with a cytopathic BVDV (group D) cloned from the 80/1 isolate. All calves were sequentially euthanized, and a wide range of tissue samples were processed for immunofluorescent and virus isolation studies. In group A, consistent immunofluorescent staining for BVDV was detected in vascular smooth muscle of numerous blood vessels in the tissues examined, mainly at 11 and 13 days postinoculation. A predominance of samples containing cytopathic BVDV was observed in the calves of this group, following virus isolation studies. Both cytopathic and noncytopathic BVDV were detected/recovered from a larger range of specimens in the calves in group B than from the calves in group A. In the calves in all the experimental groups, large amounts of BVDV antigen were detected mainly in tissue samples from the lymphoid and gastrointestinal systems, whereas only minimal amounts of BVDV were detected in the respiratory tract. Abundant noncytopathic BVDV antigen was also detected in pituitary gland and in Langerhans islets in pancreases of colostrum-deprived calves infected with the cloned noncytopathic BVDV. Noncytopathic BVDV was isolated from a wider range of tissues from calves in group C than in the colostrum-deprived calves infected with both BVDV biotypes. A cytopathic BVDV was isolated/detected in retropharyngeal, mesenteric, and abomasal lymph nodes and in thymus of 2 calves in group C. Cytopathic BVDV was detected/isolated mainly from mesenteric lymph nodes and Peyer's patches of the calves in group D.