Validation of the Filovirus Plaque Assay for Use in Preclinical Studies.
Validation of the Filovirus Plaque Assay for Use in Preclinical Studies.
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DOI：
10.3390/v8040113
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发表时间：
2016-04-21
期刊：
影响因子：
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通讯作者：
Bavari S
中科院分区：
文献类型：
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作者：
Shurtleff AC;Bloomfield HA;Mort S;Orr SA;Audet B;Whitaker T;Richards MJ;Bavari S
用于定量病毒储备液、制备的病毒攻毒接种物和研究动物样品中丝状病毒的空斑试验最近已得到充分表征和标准化，可在多个开展生物安全4级（BSL-4）研究的机构中使用。标准化研究完成后，在美国陆军传染病医学研究所（USAMRIID）开始了符合药物非临床研究质量管理规范（GLP）的空斑试验方法验证研究，以证明马尔堡病毒安哥拉（MARV）变体和埃博拉病毒基奎特（EBOV）变体的可靠和可重现测量的适用性。检测的验证参数包括准确度、精密度、线性、耐用性、病毒贮备液稳定性和系统适用性。经确认，MARV和EBOV检测试剂盒的准确度为±0.5 log 10 PFU/mL。重复性精密度、中间精密度和重现性精密度足以返回变异系数（%CV）≤ 30%的病毒滴度，认为变异对于基于细胞的生物测定是可接受的。当由两名分析员对相同噬斑进行定量时，用于评价试验精密度的组内相关统计技术返回的值符合验收标准，表明分析员之间的一致性较高。当在空斑试验培养基中稀释的非人灵长类动物（NHP）血清和血浆样品上运行时，该试验显示出准确度和特异性，基质效应可忽略不计。病毒贮备液在试验复检期间正常使用的典型冻融循环中表现出稳定性。结果表明，EBOV和MARV空斑试验对于与GLP动物模型研究性能相关的样品中丝状病毒滴定具有准确度、精密度和耐用性。
A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing Biosafety Level 4 (BSL-4) studies. After standardization studies were completed, Good Laboratory Practices (GLP)-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of the Marburg Virus Angola (MARV) variant and Ebola Virus Kikwit (EBOV) variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to ±0.5 log10 PFU/mL. Repeatability precision, intermediate precision and reproducibility precision were sufficient to return viral titers with a coefficient of variation (%CV) of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay’s precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (NHP) serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.