Low density blood granulocytic cells induced during classical swine fever are targets for virus infection

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Abstract

Classical swine fever virus infection of pigs causes a severe leukopenia and immunosuppression. In the present study, the kinetics of virus infection, and identification of target cells for the virus in peripheral blood were analysed. Virus infection was often not detectable before 5–7 days p.i. A minority of animals yielded detectable infected cells at 3 days p.i., but <5% PBMC. It was not until 10 days p.i. that this figure increased-to 35–70% PBMC depending on the animal. Detailed analysis of Ficoll–Hypaque-purified PBMC identified the major population to be SWC3+SWC8+CD14+MHCII granulocytic cells. Microscopic observations determined that these low density granulocytic cells in the PBMC from CSFV infected animals were indeed immature cells. Both the low density granulocytic cells and monocytes were major targets for CSFV infection in the peripheral blood. This is the first demonstration that low density granulocytic cells dominate the blood leukocyte population during CSF, and that such cells are targets for virus infection. The present work also demonstrates that the leukocyte population changes, such as B lymphocyte depletion and the relative dominance of myeloid cells in the blood during CSF, occur before virus infection of the affected cells. Thus, the pathological mechanism therein is not a direct consequence of virus infection.

Introduction

Classical swine fever (CSF) virus, a member of the family Flaviviridae, genus Pestivirus, is a small enveloped RNA virus causing an economically important and fatal disease of pigs. Previous studies showed that classical swine fever affects the immune system, a main characteristic being generalized leukopenia (Trautwein, 1988; Thiel et al., 1996). Lymphoid organs are also affected by the infection. Thymus and bone marrow atrophy (Trautwein, 1988; Cheville and Mengeling, 1969), as well as a generalized depletion of lymphocytes (Ressang, 1973), wherein a B lymphocyte deficiency was prominent (Susa et al., 1992), have been recorded. A general dysfunction of T lymphocyte activity has also been observed (Pauly et al., 1998; Summerfield et al., 1998). CSF virus (CSFV) has a particular affinity for cells of the reticuloendothelial system, being detectable in macrophages, endothelial cells and reticular cells (Trautwein, 1988; Thiel et al., 1996), although in terminal stages of the disease, cells of other mononuclear leukocyte populations can be infected (Trautwein, 1988; Pauly et al., 1998).

Previous studies not only described a leukopenia, but also a B lymphopenia and an increase of neutrophilic bands in the peripheral blood compartment (Susa et al., 1992). Detailed information on the target cells therein, and the role of virus infection in the induced haematological changes is lacking. This, together with the diagnostic importance of analyses on the peripheral blood, formed the rationale behind the present study: a kinetic analysis of haematological changes during CSF, and how these relate to virus infection of peripheral blood mononuclear cells (PBMC). The analysis of virus infection was effected using a co-culture with `indicator' cells, the polymerase chain reaction (PCR) technique for viral RNA detection, and the flow cytometric detection of cells expressing viral glycoprotein E2, providing a broad analytical spectrum for the identification of target cells for CSFV infection. The results demonstrated that early B cell lymphopenia and the appearance of low density granulocytic cells were characteristic changes during CSF, which were apparent before virus infection in PBMC was detectable. Furthermore, the latter cell population was identified to be immature granulocytic cells, and to be targets for CSFV infection.

Section snippets

Infection of pigs with CSFV

All experimental procedures and animal management protocols were undertaken in accordance with the requirements of the competent local Animal Care and Ethics Committee. For the various experiments, a total of 12 animals were used; representative examples are shown in the figures for reasons of clarity. Two virulent strains of CSFV were used to infect four month old pigs, in groups of 2–4, which had been bred at the institute under SPF conditions: the moderate virulent CSFV Alfort/187 strain

Infection of PBMC by CSFV

All infected animals in the present study showed clinical symptoms of CSF with an incubation time of 3–5 days before the onset of fever. In order to relate the analysis of immunopathological events in CSF to the virus infection, it was necessary to identify leukocytes infected by CSFV. Initial experiments employed PBMC from blood samples taken daily during the acute phase of the disease. The cells were analysed (i) for virus infection by co-culture with CSFV-susceptible PK-15 cells, (ii) for

Discussion

CSF is characterized in immunopathological terms by a leukopenia, wherein a B lymphopenia is particular evident (Susa et al., 1992, Summerfield et al., 1998). In the present study, the B lymphocyte deficiency was noted to be a particular early and characteristic haematological change detectable within the PBMC compartment. In contrast to the lymphocytes, the absolute numbers of myeloid cells in the peripheral blood of CSFV-infected animals were more stable. Consequently, due to the decrease in

Acknowledgements

This work was supported by the Swiss Federal Veterinary Office. We thank H. Gerber for hybridoma and mAb preparation, R. Tschudin, R. Schaffner and S. Knötig for technical assistance with the cell preparations, S. Bossy for technical assistance with the PCR, and P. Zulliger, M. Mader and A. Michel for animal care.

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