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Abstract：
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.
摘要：
与在生物安全3级和4级防护设施中操作电子显微镜（EM）相关的挑战减缓了高后果病毒的低温EM研究的进展。我们在委内瑞拉马脑炎病毒（VEEV）毒株TC-83的案例研究中解决了这一差距。病毒的化学灭活可能会物理上扭曲结构，因此为了验证天然结构的保留，我们选择了VEEV毒株TC-83来开发这种方法，因为该病毒具有4.8µ分辨率的低温EM结构。在我们的方法中，扩增的VEEVTC-83通过30％蔗糖垫直接从上清液中浓缩，重新暂停,并用1%戊二醛化学灭活。第二个30%蔗糖垫去除了任何可能干扰单颗粒分析的过量戊二醛。固定的低温电磁成像图，灭活的VEEV的分辨率为7.9µ。该图谱保留了天然病毒的结构特征，如二十面体对称，衣壳核心和三聚体尖峰的组织。我们的结果表明，我们的策略可以很容易地适应其他被包裹的失活，需要BSL-3或BSL-4用于冷冻EM的RNA病毒。然而,灭活的验证需要每个机构的生物安全委员会的监督。
