东部马脑炎病毒(EEEV)是一种新兴的公共卫生威胁,随着近年来美国报告的病例数增加。EEEV是一种BSL3病原体,和北美菌株是美国联邦选择代理(SA)。这些限制使得EEEV的实验难以进行,由于BSL3空间中通常无法使用高科技设备,并且担心在操作过程中会产生气溶胶。因此,一系列适用于不同下游分析方法的灭活方法对于推进EEEV的研究至关重要。我们使用了热量,化学,和基于紫外线(UV)的方法,用于灭活被非选择剂Madariaga病毒(MADV)感染的细胞和上清液。尽管MADV和EEEV菌株在遗传上是不同的,在氨基酸水平上相差8-11%,预计它们同样容易受到各种灭活方法的影响。我们确定以下是有效的灭活方法:加热,TRIzolLS,4%PFA,10%福尔马林,和紫外线辐射感染的上清液;TRIzol,2.5%SDS与BME,0.2%NP40,4%PFA,和10%福尔马林用于感染细胞。我们的结果有可能扩展EEEV研究人员进行的实验和分析的类型和复杂性。
The Eastern Equine Encephalitis Virus (EEEV) is an emerging public health threat, with the number of reported cases in the US increasing in recent years. EEEV is a BSL3 pathogen, and the North American strain is a US Federal Select Agent (SA). These restrictions make experiments with EEEV difficult to perform, as high-tech equipment is often unavailable in BSL3 spaces and due to concerns about generating aerosols during manipulations. Therefore, a range of inactivation methods suitable for different downstream analysis methods are essential for advancing research on EEEV. We used heat, chemical, and ultraviolet (UV)-based methods for the inactivation of infected cells and supernatants infected with the non-select agent Madariaga virus (MADV). Although the MADV and EEEV strains are genetically distinct, differing by 8-11% at the amino acid level, they are expected to be similarly susceptible to various inactivation methods. We determined the following to be effective methods of inactivation: heat, TRIzol LS, 4% PFA, 10% formalin, and UV radiation for infected supernatants; TRIzol, 2.5% SDS with BME, 0.2% NP40, 4% PFA, and 10% formalin for infected cells. Our results have the potential to expand the types and complexity of experiments and analyses performed by EEEV researchers.