Working with bacteria and viruses that cause life-threatening diseases with no currently available treatment options takes guts. Most scientists are familiar with the routine requirements of good aseptic technique, are highly aware of laboratory safety requirements, and are more than familiar with autoclaves and sterilization issues, but if we make a mistake the consequences are usually only lost time or a spoiled experiment—not a lost life.
Scientists working with highly virulent organisms deal with a whole other level of risk that requires adherence to the strictest of safety regulations, and these containment regulations can sometimes place constraints on the type of experiment that can be performed with dangerous pathogens. A paper published in the April 2014 issue of Assay and Drug Development Technologies brought this to my attention and reminded me of the serious issues some scientists face on a daily basis as they research ways to combat infectious diseases.
Regulations for Infectious agents are classified into various biological safety levels (BS1-BS4 or P1-P4) according to the danger posed; from level 1 (minimal risk) to level 4 (highest risk). Examples of level 1-category risk include such innocuous organisms as laboratory strains of E. coli, for which no extra precautions are required beyond the routine practices used in all microbiology labs. In contrast, level 4 precautions are reserved for biological agents such as Ebola and Marburg viruses, which require maximum containment procedures including sealed laboratories, positive pressure suits for researchers and decontamination of air exiting the facility.
The Assay and Drug Development Technologies paper (Tigabu, B., et al. (2014) Assay Drug Dev. Technol. 3, 155-162), describes development and validation of a high-throughput screen for inhibitors of Nipah Virus (NiV) cytopathic effect that could be conducted under biosafety level 4 (BSL-4) laboratory conditions. Nipah virus can cause encephalitis, may be fatal, and there is no vaccine or effective drug treatment available. Traditional methods of screening for cytopathic effect, such as visual examination and plaque assays, are not amenable to large-scale screening operations. The authors state that up until their study, high-throughput screening of level 4 viral pathogens typically required attenuation of the virus so that it could be removed to a lower containment level for screening purposes.
The paper came to our attention because the method described uses the Promega ViralTox-Glo™ Assay. The assay measures cellular ATP as an indication of host cell viability, and cytopathic effect due to viral infection is indicated by ATP depletion. The assay reagent is added directly to cell cultures, and the ATP is quantitated by a luminescent signal.
The authors developed and validated a high-throughput screen for inhibitors of Nipah Virus (NiV) cytopathic effect using the ViralTox™ Glo Assay under biosafety level 4 (BSL-4) laboratory conditions. The ability to perform screens under containment conditions allowed them to use wildtype viruses and to evaluate compounds against all stages in the viral life cycle. They screened a library of 10,000 compounds in 384-well plates in a pilot study, and confirmed “hits” in dose response and viral titer reduction assays.
The minimal number of manipulations required and the ability to detect cytopathic effect with a luminescent method were some of the key factors that made it possible to perform this screen inside the containment laboratory.
Here’s the Paper
Tigabu B, Rasmussen L, White EL, Tower N, Saeed M, Bukreyev A, Rockx B, LeDuc JW, & Noah JW (2014). A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus. Assay and drug development technologies, 12 (3), 155-61 PMID: 24735442
Isobel Maciver
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