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Researchers at Montana State University have validated and compared two methods of inactivating the causative agent of coronavirus disease 2019 (COVID-19) that would allow studying the virus at a lower biosafety level (BSL) than is currently required.
The risk posed to researchers by this highly infectious agent – severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – currently dictates that live virus research be performed under BSL3 conditions.
However, this requirement slows the speed of response to this global pandemic, which continues to pose an unprecedented threat to public health and the world economy.
Working with SARS-CoV-2 at a more accessible and lower biosecurity level could accelerate research. However, this requires that the virus be completely inactivated, without destroying the viral components necessary for the research to be effective.
Researcher at the US Centers for Disease Control, Atlanta, Georgia, working with the influenza virus under biosafety level 3 conditions, with a respirator inside a biosafety cabinet (BSC). Photo Credit: James Gathany Content Provider: CDC
Now, Matthew Taylor and colleagues have validated and compared two protocols for SARS-CoV-2 inactivation – heat inactivation and ultraviolet (UV) irradiation – and described the effects each technique had on virion components.
Both techniques effectively neutralized SARS-CoV-2, each with different effects on important viral characteristics.
“We describe the strengths and weaknesses of each method so that investigators can choose the one that best meets their research objectives,” the team writes.
A pre-printed version of the document is available on bioRxiv * server, while the article is peer reviewed.
Electron microscopic analysis of virion morphology Semi-purified virionic preparations were identified on a grid and imaged to evaluate virion morphology. The top row of images was taken from UV inactivated samples. The bottom row of images was taken from heat-inactivated samples. The relative size is indicated by the scale bar in the lower corner of each image.
The challenges posed by BLS3 restrictions
While confining SARS-CoV-2 research to BSL3 facilities only is highly effective in keeping researchers and the wider community safe, it limits the ability to provide the timely response needed during this unprecedented pandemic.
“Viral inactivation can mitigate the risk of working with SARS-CoV-2 at a lower biosecurity level, while accelerating scientific goals,” the researchers write.
However, many of the SARS-CoV-2 inactivation methods that have been developed destroy the structural and genetic components that must be kept intact for the research to be effective.
‘The most useful inactivation methods are effective and reliable without being overly destructive to the virion components,’ the team says.
What did the researchers do?
The team validated two SARS-CoV-2 neutralization techniques: heat inactivation and UV irradiation. Each method was optimized to ensure that the virus was completely non-infectious and to limit any destructive effects on the viral components.
For example, UV exposure was reduced by removing plastic lids from Petri dishes which would otherwise absorb much of the incoming UV-C radiation, and heat inactivation was performed in low volume water bath tubes to ensuring that the heating and inactivation of the samples were uniform.
What did the study find?
Complete inactivation of the high titer stocks of SARS-CoV-2 was achieved after only 15 minutes of incubation at 65 ° C.
Complete inactivation was also achieved using a hundred times lower UV power than that used in comparable studies, the team says.
Although both techniques completely inactivated SARS-CoV-2, each method had unique effects on important viral characteristics which then influenced downstream applications.
According to Taylor and colleagues, thermal inactivation is ideal for preserving genetic material, while UV irradiation allows for the purification of high-quality virions.
The thermal inactivation protocol left the virions mostly intact, which the researchers say is an encouraging observation for protocols that enrich virions based on the biophysical properties of intact structures. The heat treatment also left the viral genomes mostly intact.
On the other hand, UV irradiation resulted in a 2 log reduction of detectable genomes, compared to heat inactivation.
‘This made thermal inactivation the preferred method for assessments using genome-based assays such as PCR,’ the team writes.
However, UV irradiation was particularly effective in maintaining virion morphology and antigenicity. High-resolution imaging of inactivated virions showed that UV-irradiated samples retain much of their native viral structure.
These samples were also significantly more detectable by enzyme-linked immunosorbent assay (ELISA) than the heat-treated samples.
The two inactivation protocols produced nearly equivalent amounts of viral proteins, although protein quality varied when assessed by Western blot. This is important to note when considering downstream applications for antigen detection or vaccine development, say the researchers.
Both methods are suitable for use in BSL2 environments
Taylor and colleagues state that the results show that both heat inactivation and UV irradiation represent viable SARS-CoV-2 inactivation methods for use in BSL2 facilities.
“Both methods left the virion mostly intact while the effects on other viral properties differed,” they write.
‘From this study, it is clear that both the extent and method of inactivation have important ramifications on SARS-CoV-2 virions that should be considered when planning experiments or downstream applications,’ the team concludes.
*Important Notice
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as consolidated information.
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