Virus Propagation and Plaque Assay for Dengue Virus

登革热病毒 病毒定量 病毒 病毒学 抗体依赖性增强 细胞培养 效价 登革热 生物 中和 滴定法 化学 遗传学 无机化学
作者
Pedro Henrique Carneiro,Lucas Mendes-Monteiro,Ronaldo Mohana‐Borges
出处
期刊:Methods in molecular biology 卷期号:: 3-9 被引量:5
标识
DOI:10.1007/978-1-0716-1879-0_1
摘要

This chapter describes the methods of propagation and titration for DENV-1 to -4, which are required for most of the experiments using live viruses. DENV does not reach titers as high as those of other viruses or as high as desired for their use in biological assays. Although DENV grows in many different cell lines derived from both vertebrate and invertebrate cells, the most common cell lines used for virus isolation and propagation are mosquito cells C6/36 from Aedes albopictus. Amongst several methods for virus quantification, plaque assay stands out as being very practical and inexpensive. This technique is carried out essentially to estimate virion density in a particular material, being extremely important when evaluating the effect of an antiviral treatment or antibody neutralization capacity, for example. In this assay, viral particles are serially diluted and added onto confluent cell monolayers immersed in a semisolid medium, which is responsible for limiting virus spread throughout the culture. Therefore, regarding the medium consistency, once a virion successfully infects a cell, the newly produced particles can only infect neighboring cells, eventually leading them to death. This phenomenon can be observed as round gaps or plaques in the culture after staining live cells with a crystal violet solution. Then, plaques are counted and used to determine plaque-forming units per milliliter. Here, we describe a protocol established by our group for dengue virus (DENV) titration with porcine kidney (PS) cells.
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