Mukherjee Swati, Pierson Theodore C, Dowd Kimberly A
Viral Pathogenesis Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2014;1138:75-97. doi: 10.1007/978-1-4939-0348-1_6.
This chapter outlines methods for the production of dengue virus (DENV) reporter virus particles (RVPs) and their use in assays that measure antibody-mediated neutralization and enhancement of DENV infection. RVPs are pseudo-infectious virions produced by complementation of a self-replicating flavivirus replicon with the DENV structural genes in trans. RVPs harvested from transfected cells are capable of only a single round of infection and encapsidate replicon RNA that encodes a reporter gene used to enumerate infected cells. RVPs may be produced using the structural genes of different DENV serotypes, genotypes, and mutants by changing plasmids used for complementation. Further modifications are possible including generating RVPs with varying levels of uncleaved prM protein, which resemble either the immature or mature form of the virus. Neutralization potency is measured by incubating RVPs with serial dilutions of antibody, followed by infection of target cells that express DENV attachment factors. Enhancement of infection is measured similarly using Fc receptor-expressing cells capable of internalizing antibody-virus complexes.
本章概述了登革病毒（DENV）报告病毒颗粒（RVP）的生产方法及其在检测抗体介导的中和作用和DENV感染增强作用的试验中的应用。RVP是通过将自我复制的黄病毒复制子与DENV结构基因反式互补而产生的假感染性病毒粒子。从转染细胞收获的RVP仅能进行一轮感染，并包裹编码用于计数感染细胞的报告基因的复制子RNA。通过改变用于互补的质粒，可使用不同DENV血清型、基因型和突变体的结构基因来生产RVP。还可以进行进一步的修饰，包括产生具有不同水平未切割prM蛋白的RVP，其类似于病毒的未成熟或成熟形式。通过将RVP与抗体的系列稀释液孵育，然后感染表达DENV附着因子的靶细胞来测量中和效力。使用能够内化抗体-病毒复合物的表达Fc受体的细胞，以类似的方式测量感染增强作用。
