Establishment of a LacZ marker rescue assay to detect infectious RD114 virus
Establishment of a LacZ marker rescue assay to detect infectious RD114 virus
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DOI：
10.1292/jvms.70.785
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发表时间：
2008-08-01
影响因子：
1.2
通讯作者：
Miyazawa, Takayuki
中科院分区：
文献类型：
--
作者：
Sakaguchi, Shoichi;Okada, Masaya;Miyazawa, Takayuki
猫具有传染性内源性逆转录病毒，称为RD 114病毒，并且存在RD 114病毒污染减毒活疫苗的可能性，其中猫细胞用作底物。为了监测猫用疫苗中的传染性RD 114病毒，我们开发了一种LacZ标记拯救试验来检测传染性RD 114病毒。在检查的四种人细胞系中，TE 671细胞（人横纹肌肉瘤）对RD 114病毒最敏感，并有效支持RD 114复制。在病毒吸附过程中，通过在培养基中加入浓度为2至8 μ g/m2的聚凝胺，感染增强约5倍。4小时病毒吸附期足以获得最大滴度。通过将样品接种到用lacZ标记基因转导的TE 671细胞中，将有限稀释的样品（即，小于10个感染单位）在接种后12小时通过LacZ标记拯救测定检测到。基于本研究中获得的结果，我们提出了一个标准的LacZ标志物拯救试验检测传染性RD 114病毒的协议。
Cats have an infectious endogenous retrovials, named RD114 virus, and there is a possibility that RD114 virus has contaminated live attenuated vaccines, for which feline cells are used as a substrate. To monitor infectious RD114 virus in vaccines for cats, we developed a LacZ marker rescue assay to detect infectious RD 114 virus. Among four human cell lines examined, TE671 cells (human rhabdomyosarcoma) were most susceptible to RD 114 virus and supported RD 114 replication efficiently. Infection was enhanced approximately 5 times by the addition of polybrene at concentrations of 2 to 8 mu g/m/ in the medium during viral adsorption. A 4-hr viral adsorption period was sufficient to obtain the maximum titer. By inoculating samples into TE671 cells transduced with the lacZ marker gene, the limiting diluted sample (i.e., less than 10 infectious units) was detected at 12 clays post-inoculation by the LacZ marker rescue assay. Based on the results obtained in this study, we propose a standard protocol of the LacZ marker rescue assay to detect infectious RD114 virus.