RESUMO

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 Šresolution 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.

Assuntos

Microscopia Crioeletrônica/métodos , Vírus da Encefalite Equina Venezuelana/fisiologia , Vírus de RNA/fisiologia , Inativação de Vírus , Animais , Capsídeo/química , Proteínas do Capsídeo , Linhagem Celular , Chlorocebus aethiops , Contenção de Riscos Biológicos/métodos , Vírus da Encefalite Equina Venezuelana/genética , Glutaral/química , Glutaral/metabolismo , Cavalos , Células Vero , Virologia/métodos , Replicação Viral