Never before has an electron microscope withstood the rigors and safety protocol of a BioSafety Level 3 containment environment. So it’s no wonder that the first U.S. laboratory of its kind opened its doors to a surge of virologists and infectious disease researchers eager to study Level 3 viruses and human pathogens with the magnification and resolution of a 200 keV Transmission Electron Microscope (TEM).
“We’re the only ones in the world to do this,” said Dr. Michael Sherman, who headed the construction of the new BSL3 lab, the Cryo-electron Microscopy Center for Macromolecular Systems, at the University of Texas Medical Branch (UTMB). “There was no protocol. We developed it as we worked towards completion of the lab.”
In October 2007 – two years after the initial construction began - researchers could finally examine detailed structures and surface features of Hanta, Yellow Fever,, Rift Valley Fever, Western Equine Encephalitis, SARS, and Venezuelan Equine Encephalitis viruses – all deadly and transmittable – in the effort to better understand how they replicate and how to control them through vaccines.
“There are not many places in the world to safely study these viruses,” said Dr. Stan Watowich, who has been instrumental in development of the lab with project manager Dr. Sherman. His research focuses on molecular mechanisms of virus assembly and virus-host cell attachment, with particular attention to RNA enveloped viruses such as hepatitis C (HCV), Venezuelan equine encephalitis (VEE), and encephalitic flaviviruses. Watowich is an Associate Professor, Department of Biochemistry & Molecular Biology, and is also on the faculty of the Sealy Center for Structural Biology.
Sherman, also on the faculty of UTMB’s Sealy Center for Structural Biology, uses the Cryo-EM for high-resolution structure determination of biological macromolecules and their assemblies. These include cellular organelles, viruses, and single particles with lower or without symmetry. He also plans to study Lassa Fever and SARS with the cryo-TEM.
“The microscope will be one of the first in the world to properly handle such deadly viruses,” said Watowich, “and it will also be the safest microscope in the world for operators and service people. We are handling agents that no one else should be putting in a microscope. The decontamination process is something that no one has bothered to put on an instrument of this kind before.”
A preliminary reconstruction of Western Equine Encephalitis (WEEV), the first BSL-3 agent imaged in the new UTMB facility. A member of the Alphavirus family, WEEV is an enveloped virus, meaning that there is a lipid membrane within the virion. The surface of the virion is shown in gold and the membrane can be seen through the holes. The images were obtained from the viruses grown and purified by Dr. Scott Weaver, the world expert in alphaviruses at UTMB and a PI on the Keck Grant.
Modifying the JEM-2200FS TEM for decontamination required both UTMB and JEOL to work closely together to design and produce hardware that would withstand the bakeout procedure required to decontaminate the microscope in the event of a sample becoming dislodged from the sample stage or for service. JEOL USA has made customized and unique adaptations to its microscopes over the years to suit customers’ needs, but this was a first.
From the beginning, a partnership was formed to take on the challenge, with a dedicated team to see the project through to completion. Dennis Razmus of JEOL took on a new role as Project Lead, with primary assistance coming from JEOL Regional Service Manager Craig Koht, just to manage the modification program for the instrument. “We had to design and test modifications to decontaminate the system. A normal bakeout wasn’t sufficient,” he said.
Initially the service team performed research on contamination results in their Pittsburg office, using vaporized hydrogen peroxide, which kills the virus but also “kills part of scope,” Razmus said. That’s when they turned to high temperatures and equipment modifications.
Razmus produced a regular bulletin to the service department to “inform and educate them with what I learned over time. It’s a highly regulated business that is monitored by the CDC and NIH. We invented our own protocol and wrote procedures for what we would do and with our systems. There is a sixty-page procedure. Everything has to be in writing.”
“It is a big vision but we’ve taken it one step at a time,” said Sherman. “We knew where we were going to go and we were confident we were going to get there. We are a touchstone now for other people wanting to set up a new facility. The groundwork has been laid.”
Galveston is a key center for virology research in the country, so it was a natural location for the new lab, known as the W.M. Keck Center for Virus Imaging.
“The advantage of being at UTMB,” Watowich explains, “is that we have a high concentration of virologists and infectious disease researchers down here. And we have access to pretty much every nasty virus.”
UTMB is among the world’s foremost facilities for handling viruses and studying emerging infectious diseases. The university is home to the National Institute of Health’s Center for Biodefense and Emerging Infectious Diseases and will also be the site of the nation’s first National Biocontainment Laboratory, a BSL4 facility. BSL4 is the highest biosafety rating for laboratories dealing with highly contagious organisms. BSL4 viruses currently have no vaccines or cures. Perhaps the best known such virus is Ebola.
The various researchers at UTMB have been learning the procedures necessary to submit their samples to the BSL3 lab for cryo-imaging. “They had a list of projects ready to go as soon as we were operational,” Watowich said.
Dr. Sherman determines the schedule. Virologists must have the materials of proper purity and concentrations ready to go.
The biocontainment lab can only be accessed through three levels of security and safety barriers consisting of a series of rooms with interlocks and decreasing pressure. Although only researchers with appropriate government security clearance and advanced training are allowed in the biocontainment lab, any investigator can use the remote operations capability of the facility to study highly pathogenic microbes such as SARS or avian influenza. By using remote operation to control the cryo-electron microscope, scientists from around the world can collaborate with UTMB to efficiently use the BSL3 cryo-TEM facility.
The Laboratory has three JEOL cryo-electon microscopes. The high-resolution JEM-2200FS is located in the BSL-3 facility, permitting structural imaging of pathogens. The JEM-2100 is available for imaging of non-pathogenic targets. A lower resolution JEM-1010 microscope is available for preliminary screening.
For more information on the Cryo-electron Microscopy Center for Macromolecular Systems, visit www.scsb.utmb.edu/facilities/cryoem.htm.