Castillo-Olivares J, Tearle J P, Montesso F, Westcott D, Kydd J H, Davis-Poynter N J, Hannant D
Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK.
Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK.
J Gen Virol. 2003 Oct;84(Pt 10):2745-2753. doi: 10.1099/vir.0.19144-0.
Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a (51)Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain (51)Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8(+)/CD4(+) ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8(+) T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.
马动脉炎病毒（EAV）可引起马科动物的全身性感染，其结果各异，从亚临床感染到严重疾病，还能导致怀孕母马流产以及种马持续性感染。感染动物体内必然会产生的血清病毒中和抗体反应会持续数月或数年，据信在病毒清除过程中发挥着重要作用。然而，由于缺乏评估这些免疫反应的方法，人们对针对EAV的细胞免疫了解甚少。在本研究中，我们描述了使用（51）Cr释放细胞溶解试验检测EAV康复小马外周血中细胞毒性T淋巴细胞（CTL）前体的方法。用作CTL靶标的原代马皮肤细胞显示表达MHC I但不表达MHC II，并且能有效保留（51）Cr并支持EAV复制。从经活EAV孵育或未经活EAV孵育的EAV康复小马采集的外周血单核细胞（PBMC）用作效应细胞。EAV诱导的PBMC培养物显示有淋巴细胞母细胞扩增和激活的迹象，与模拟诱导的PBMC相比，CD8（+）/CD4（+）比值增加。EAV刺激的PBMC诱导的细胞毒性具有病毒特异性，表现出基因限制性，由CD8（+）T淋巴细胞介导，并且在感染后4个月至1年多的时间内均可检测到。这些发现和方法有望有助于理解马体内病毒与宿主的相互作用，特别是EAV感染期间发生的病毒清除机制。
