FLOW CYTOMETRIC ANALYSIS OF INVITRO BLUETONGUE VIRUS-INFECTION OF BOVINE BLOOD MONONUCLEAR-CELLS
FLOW CYTOMETRIC ANALYSIS OF INVITRO BLUETONGUE VIRUS-INFECTION OF BOVINE BLOOD MONONUCLEAR-CELLS
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DOI：
10.1099/0022-1317-73-8-1953
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发表时间：
1992-08-01
影响因子：
3.8
通讯作者：
MACLACHLAN, NJ
中科院分区：
文献类型：
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作者：
BARRATTBOYES, SM;ROSSITTO, PV;MACLACHLAN, NJ
用蓝舌病毒(BTV)10型感染牛外周血单个核细胞(PBM)贴壁和非贴壁细胞，部分贴壁细胞在接种前用白介素2(IL-2)和刀豆蛋白A(ConA)刺激24 h。以不同的时间间隔收集细胞，直到接种后72h。将一组针对牛CD2、CD4或CD8、单核细胞和粒细胞、B细胞、γ-βT细胞或IL-2受体(IL-2R)的白细胞分化抗原特异性单抗(MAb)直接与异硫氰酸荧光素偶联，将针对BTV主要核心蛋白VP7的单抗直接与藻红蛋白偶联。在单标记和双标记免疫荧光研究中，用结合的单抗标记细胞，以特异性地识别接种培养物中感染BTV的细胞。用碘化丙啶排除法测定细胞存活率，并用流式细胞仪进行分析。病毒滴定证实PBM细胞培养物有生产性感染。这些数据显示，牛PBM细胞亚群在体外对BTV感染的易感性存在明显差异。单核细胞容易感染BTV，刺激的CD4+细胞也易感染，感染对单核细胞和刺激的淋巴细胞具有细胞病变作用。感染细胞比例在培养24小时后下降，病毒滴度在培养72小时后明显下降。未受刺激的非贴壁细胞接种BTV后，培养的CD4+细胞IL-2R表达增加。这些发现可能与牛BTV感染的发病机制有关。
Cultures of adherent and non-adherent bovine peripheral blood mononuclear (PBM) cells were inoculated with bluetongue virus (BTV) serotype 10. Some cultures of non-adherent cells were stimulated with interleukin 2 (IL-2) and concanavalin A for 24 h prior to virus inoculation. Cells were harvested at various intervals up to 72 h after inoculation. A panel of leukocyte differentiation antigen-specific monoclonal antibodies (MAbs), specific for bovine CD2, CD4 or CD8, monocytes and granulocytes, B cells, gamma-delta-T cells or the IL-2 receptor (IL-2r), was directly conjugated to fluorescein isothiocyanate, and a MAb specific for the BTV major core protein VP7 was directly conjugated to phycoerythrin. Cells were labelled with conjugated MAbs in single- and double-label immunofluorescence studies to identify specifically the BTV-infected cells in inoculated cultures. The viability of cells was determined by propidium iodide exclusion, and all analyses were done using flow cytometry. Productive infection of cultures of PBM cells was confirmed by virus titration. The data revealed a clear difference between subsets of bovine PBM cells in susceptibility to infection with BTV in vitro. Monocytes were readily infected with BTV, as were stimulated CD4+ cells, and infection was cytopathic to monocytes and stimulated lymphocytes. The proportion of infected cells decreased after 24 h and virus titres dropped markedly by 72 h in all cultures. CD4+ cells in cultures of unstimulated non-adherent cells inoculated with BTV showed increased expression of IL-2r. The possible relevance of these findings to the pathogenesis of BTV infection of cattle is discussed.