Perler L, Pfister H, Schweizer M, Peterhans E, Jungi T W
Institute of Veterinary Virology, University of Bern, Switzerland.
J Immunol Methods. 1999 Jan 1;222(1-2):189-96. doi: 10.1016/s0022-1759(98)00198-7.
The expression of type I interferons (IFNs) in eukaryotic cells represents a first line of defense against viral infection. Cells pretreated by IFNs do not support viral replication and are protected from virus-induced cell destruction. A challenge of IFN-pretreated cells with vesicular stomatitis virus (VSV) is frequently used to quantitate this cytokine because, on the one hand, the replication of VSV is highly sensitive to IFNs and, on the other hand, in unprotected cells this virus induces a rapid cytopathic effect that can readily be quantified. However, as VSV may infect humans and is known to cause severe disease in a variety of animal species, this virus must be considered a biohazard. In this paper, we describe a bioassay for bovine IFN using Sendai virus, a paramyxovirus that grows readily in MDBK cells yet is released from these cells in a non-infectious form. The sensitivity and dynamic range of this assay are similar to those of the popular VSV-based IFN assay. We demonstrate that the Sendai-virus-based IFN assay permits rapid quantitation of recombinant bovine type I IFN, and also of native type I IFNs which are present in the supernatants of monocyte-derived macrophages infected with various pathogens. In view of the possible artifacts induced by viruses in samples to be assayed for IFN activity, we evaluated several methods of virus inactivation. Treatment with beta-propiolactone led to virus inactivation without affecting the bioactivity of IFNs as detected in the Sendai-virus-based assay.
I型干扰素(IFN)在真核细胞中的表达是抵御病毒感染的第一道防线。经IFN预处理的细胞不支持病毒复制,并受到保护免受病毒诱导的细胞破坏。用水泡性口炎病毒(VSV)攻击经IFN预处理的细胞常用于定量这种细胞因子,一方面,VSV的复制对IFN高度敏感,另一方面,在未受保护的细胞中,这种病毒会诱导一种可轻易定量的快速细胞病变效应。然而,由于VSV可能感染人类且已知会在多种动物物种中引起严重疾病,这种病毒必须被视为生物危害物。在本文中,我们描述了一种使用仙台病毒的牛IFN生物测定法,仙台病毒是一种副粘病毒,在MDBK细胞中易于生长,但以非感染性形式从这些细胞中释放。该测定法的灵敏度和动态范围与流行的基于VSV的IFN测定法相似。我们证明基于仙台病毒的IFN测定法能够快速定量重组牛I型干扰素,以及存在于感染各种病原体的单核细胞衍生巨噬细胞上清液中的天然I型干扰素。鉴于待检测IFN活性的样品中病毒可能诱导的假象,我们评估了几种病毒灭活方法。用β-丙内酯处理导致病毒灭活,而不影响基于仙台病毒测定法检测到的IFN生物活性。