Brien James D, Hassert Mariah, Stone E Taylor, Geerling Elizabeth, Cruz-Orengo Lillian, Pinto Amelia K
Department of Molecular Microbiology and Immunology, Saint Louis University.
School of Veterinary Medicine University of California Davis.
J Vis Exp. 2019 Aug 15(150). doi: 10.3791/59632.
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.
所展示的方法阐述了从感染寨卡病毒的动物中分离器官以及定量病毒载量的实验室程序。该程序的目的是在感染后不同时间点或不同实验条件下，对小鼠外周和中枢神经系统区域的病毒滴度进行定量，以确定调节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可用于病灶形成试验定量和病毒滴度的定量PCR评估。快速器官分离技术旨在保存病毒滴度。通过病灶形成试验进行病毒滴度定量可实现对寨卡病毒的快速通量评估。病灶形成试验的优点是可评估感染性病毒，该试验的局限性在于存在器官毒性的可能性，从而降低检测限。病毒滴度评估与定量PCR相结合，使用重组RNA拷贝对照来评估器官内病毒基因组拷贝数，检测限较低。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，并且可应用于评估感染包括登革热病毒在内的大多数病原体的动物器官中的病毒滴度。
