Karakus Umut, Crameri Michel, Lanz Caroline, Yángüez Emilio
Institute of Medical Virology, University of Zurich, Zürich, Switzerland.
Methods Mol Biol. 2018;1836:59-88. doi: 10.1007/978-1-4939-8678-1_4.
Influenza viruses are constantly circulating among humans, in which they cause seasonal epidemics of severe respiratory disease. Additionally, these zoonotic viruses infect different mammals and birds, from which new antigenic variants are occasionally transmitted to humans leading to devastating global pandemics. Surveillance programs, in which viruses from the main reservoir (waterfowl), intermediate hosts (like pigs and other farm animals), and other affected species are isolated and characterized, are crucial for the global influenza prevention strategy. This chapter gives an overview of the most commonly used methods for the propagation and titration of influenza viruses, which are key steps in surveillance procedures, as well as in vaccine development and basic research. Depending on the host and the viral strain, primary isolates are obtained from biological samples of different origin and subsequently amplified in embryonated chicken eggs or cell cultures. These propagation procedures are the focus of the first part of this chapter. Once the initial isolates have been amplified, virus titration methods based on particular characteristics of influenza viruses, such as their ability to agglutinate red blood cells (RBCs) or to induce cytopathic effects (CPE) in cell monolayers, are used to estimate the amount of viral particles. Such approaches, like the hemagglutination assay (HA assay), 50% tissue culture infectious dose (TCID50), or plaque assay, are included in the second part of this chapter. Although they are simple and cost-effective, some of these techniques have been partially replaced by faster and more sensitive methods based on the quantification of viral genomes, such as the quantitative real-time reverse transcription PCR (RT-qPCR), which is presented at the end of this section. The different protocols are explained in detail in order to facilitate the preparation and quantification of infectious virus stocks.
流感病毒一直在人类中传播，引发严重呼吸道疾病的季节性流行。此外，这些人畜共患病毒感染不同的哺乳动物和鸟类，偶尔会将新的抗原变体传播给人类，导致毁灭性的全球大流行。监测计划至关重要，该计划需从主要宿主（水禽）、中间宿主（如猪和其他农场动物）以及其他受影响物种中分离病毒并进行特征鉴定，这是全球流感预防策略的关键。本章概述了流感病毒增殖和滴定最常用的方法，这些方法是监测程序以及疫苗开发和基础研究的关键步骤。根据宿主和病毒株的不同，从不同来源的生物样本中获得原始分离株，随后在鸡胚或细胞培养物中进行扩增。这些增殖程序是本章第一部分的重点。一旦初始分离株得到扩增，就会使用基于流感病毒特定特征的病毒滴定方法，如它们凝集红细胞（RBC）的能力或在细胞单层中诱导细胞病变效应（CPE）的能力，来估计病毒颗粒的数量。此类方法，如血凝试验（HA试验）、50%组织培养感染剂量（TCID50）或空斑试验，包含在本章第二部分。尽管这些技术简单且成本效益高，但其中一些技术已部分被基于病毒基因组定量的更快、更灵敏的方法所取代，如定量实时逆转录PCR（RT-qPCR），本节末尾将对此进行介绍。为便于制备和定量感染性病毒储备液，将详细解释不同的方案。
