Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse
Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse
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DOI：
10.3791/59632
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发表时间：
2019-08-01
影响因子：
1.2
通讯作者：
Pinto, Amelia K.
中科院分区：
文献类型：
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作者：
Brien, James D.;Hassert, Mariah;Pinto, Amelia K.
正在介绍的方法展示了从感染寨卡病毒的动物身上分离器官和量化病毒载量的实验室程序。该程序的目的是量化感染后不同时间点或不同实验条件下小鼠周围和中枢神经系统区域的病毒滴度，以确定调控寨卡病毒感染的病毒学和免疫学因素。所展示的器官分离程序允许对病毒滴度进行病灶形成分析和定量聚合酶链式反应评估。快速器官分离技术是为保存病毒滴度而设计的。通过病灶形成试验进行病毒滴度定量，可以快速评估寨卡病毒的吞吐量。病灶形成试验的好处是对感染性病毒的评估，该试验的局限性是有可能降低器官毒性，降低检测限。病毒滴度评估与定量聚合酶链式反应相结合，并使用重组RNA复制控制器官内的病毒基因组拷贝数以较低的检测下限进行评估。总体而言，这些技术为分析寨卡病毒感染动物外周和中枢神经系统中的寨卡病毒滴度提供了一种准确、快速、高通量的方法，并可用于评估包括登革热病毒在内的大多数病原体感染的动物器官中的病毒滴度。
The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.