将病毒滴定到易感细胞系上是用于定量感染性病毒滴度的重要病毒学技术。它是流行病出血性疾病病毒(EHDV)研究的组成部分,包括估计传染性,计算感染的多重性,并确认病毒在细胞培养物中的繁殖。然而,量化传染性EHDV的能力对于疾病控制也至关重要,特别是在爆发的情况下。常规EHD诊断不能准确量化传染性病毒,这将允许准确预测向前传播的风险,但相反,通常在性质上更定性(例如,病毒分离)或仅定量病毒基因组拷贝(例如,实时PCR),通常在感染性病毒从宿主中清除后很长时间内仍可检测。通常通过检测易感哺乳动物细胞培养物单层中可见的细胞病变效应(CPE)来定量感染性EHDV滴度。然而,并非所有易感细胞系在EHDV感染时都表现出可见的CPE,包括KC细胞等细胞系,它们来自EHDV生物昆虫媒介,库仑。本章介绍了将EHDV阳性样品滴定到相关样品上的综合方法,易感哺乳动物(Vero)和昆虫(KC)细胞系,并描述了可用于可视化EHDV感染的替代方法,通过CPE或病毒蛋白的免疫荧光标记,以实现感染性EHDV滴度的计算。
The titration of viruses onto susceptible cell lines is an important virological technique used to quantify infectious viral titers. It forms an integral component of epizootic hemorrhagic disease virus (EHDV) research, including estimating infectivity, calculating multiplicity of infection, and confirming virus propagation in cell culture. However, the ability to quantify infectious EHDV is also critical for disease control, particularly in the event of an outbreak. Routine EHD diagnostics do not accurately quantify infectious virus, which would allow accurate prediction of the onward transmission risk, but instead are typically more qualitative in nature (e.g., virus isolation) or only quantify viral genome copies (e.g., real-time PCR) which often remain detectable long after infectious virus is cleared from the host.Infectious EHDV titers are typically quantified through the detection of visible cytopathic effect (CPE) in the monolayer of susceptible mammalian cell cultures. However, not all susceptible cell lines demonstrate visible CPE upon EHDV infection, including cell lines such as KC cells, which are derived from the EHDV biological insect vector, Culicoides sonorensis. This chapter presents a comprehensive method for the titration of EHDV-positive samples onto relevant, susceptible mammalian (Vero) and insect (KC) cell lines and describes alternative methods that can be used to visualize EHDV infection, by CPE or immunofluorescent labeling of viral proteins, to enable the calculation of infectious EHDV titers.