The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Å resolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

中文翻译：

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一种新的灭活方法，可促进需要高度遏制的带包裹RNA病毒的冷冻EM：使用委内瑞拉马脑炎病毒（VEEV）的案例研究。

在生物安全级别3和4的收容设施中使用电子显微镜（EM）所面临的挑战已经减慢了对高危病毒的冷冻EM研究的进展。我们在委内瑞拉马脑炎病毒（VEEV）株TC-83的案例研究中解决了这一空白。病毒的化学失活可能会物理扭曲结构，因此，为了验证天然结构的保留，我们选择VEEV TC-83菌株来开发此方法，因为该病毒具有4.8Å分辨率的cryo-EM结构。在我们的方法中，扩增的VEEV TC-83通过30％的蔗糖垫直接从上清液中浓缩，重新悬浮，并用1％的戊二醛化学灭活。再用30％的蔗糖缓冲垫去除可能干扰单颗粒分析的任何过量戊二醛。固定的低温电磁图 灭活VEEV的分辨率确定为7.9。该图保留了天然病毒的结构特征，如二十面体对称性，衣壳核心和三聚体尖峰的组织。我们的结果表明，我们的策略可轻松适用于灭活其他需要BSL-3或BSL-4进行冷冻EM的包膜RNA病毒。但是，灭活的验证需要每个机构的生物安全委员会的监督。