Related Experiment Video
Updated: Jan 2, 2026
In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus
Published on: December 2, 2012
A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study
Amar D Parvate1, Frank Vago1, S Saif Hasan1
1Department of Biological Sciences, Purdue University, West Lafayette, IN, USA.
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.
Insights
We developed a cryo-electron microscopy (cryo-EM) method to study high-risk viruses like Venezuelan Equine Encephalitis Virus (VEEV) in containment. This technique preserves native virus structure after chemical inactivation, enabling structural analysis of dangerous pathogens.
Area of Science:
- Structural Biology
- Virology
- Biophysics
Background:
- Electron microscopy (EM) in high-containment facilities (BSL-3/4) is challenging for studying dangerous viruses.
- Previous methods for inactivating viruses for EM studies risked structural distortion.
- Venezuelan Equine Encephalitis Virus (VEEV) has a known high-resolution cryo-EM structure, making it a suitable model.
Purpose of the Study:
- To develop and validate a cryo-EM methodology for studying chemically inactivated, high-consequence viruses.
- To assess the structural integrity of VEEV after inactivation using cryo-EM.
- To establish a protocol adaptable for other enveloped RNA viruses requiring high biosafety levels.
Main Methods:
- Concentration of VEEV TC-83 from cell culture supernatant using sucrose cushions.
- Chemical inactivation of concentrated VEEV using glutaraldehyde.
- Purification of inactivated VEEV with a second sucrose cushion to remove excess fixative.
- Cryo-electron microscopy data collection and image processing to generate a 3D map.
Main Results:
- A cryo-EM map of inactivated VEEV TC-83 was obtained at 7.9 Å resolution.
- The determined structure retained key features of the native virus, including icosahedral symmetry and the arrangement of capsid proteins and spikes.
- The methodology demonstrated successful preservation of structural integrity post-inactivation.
Conclusions:
- The developed method allows for cryo-EM structural analysis of chemically inactivated enveloped RNA viruses.
- This approach facilitates structural studies of pathogens in BSL-3 and BSL-4 laboratories.
- Institutional Biosafety Committee approval is necessary for validating inactivation efficacy for each specific virus.
