A version of SARS-CoV-2, the virus that causes COVID-19 disease, has been successfully modified to shine brightly in animal cells and tissues, providing a real-time way to track the spread and intensity of viral infection as it occurs in animal models, researchers at the Texas Biomedical Research Institute (Texas Biomed) report in the journal Proceedings of the National Academy of Sciences (PNAS).
Now we can track where the virus is going in animal models for COVID-19. Being able to see how the virus is progressing, and which organs and cell types it specifically targets, will be of great help in understanding the virus and optimizing antiviral drugs and vaccines. “
Luis Martinez-Sobrido, Ph.D., professor, Texas Biomed, virologist and senior author of articles
In addition to tracking the virus, Martinez-Sobrido and colleagues have already started using reporter viruses to assess the effectiveness of neutralizing antibodies against different variants of concern, as was recently reported in the Journal of Virology.
Turn on the lights
To make the reporter virus, Martinez-Sobrido and his team combined several advanced molecular biology tools to add the genetic sequence of fluorescent or bioluminescent “reporter” proteins to the virus’s genetic code. As the virus code is replicated and transcribed, so is the code of glowing proteins.
In an earlier study, the team replaced one of the virus’s genes with the glowing protein gene, but this resulted in a very weak signal – the gene was not expressed enough to be easily detected in animals. To increase the brightness, the researchers had to figure out how to get the virus to produce greater amounts of reporter proteins.
Their solution: They inserted the reporter gene next to a different gene in SARS-CoV-2, specifically the gene encoding the core protein. “It is the most expressed protein in SARS-CoV-2,” said molecular biologist Chengjin Ye, Ph.D., a member of Martinez-Sobrido’s lab. This time the signal was so bright, “it almost blinded me when I looked through the fluorescence microscope,” he said.
Reporter proteins work in cells and living animal models, in combination with imaging systems that detect the wavelengths of light emitted by proteins. Being able to visually observe viral load and localization offers many advantages over other methods. It is much simpler and faster, which saves time and materials.
“Instead of needing a large team to screen 2,000 compounds to see if they work against the virus, one person could do it with a reporter virus in a matter of hours,” Ye said.
It also allows the virus to be followed in the same animal throughout infection and treatment, reducing the number of animals needed to obtain similar information.
The team adapted the reporter viruses to express different stained proteins attached to the variants of concern of SARS-CoV-2, which they described in a separate article in the Journal of Virology. Critically, this approach allowed them to test the effectiveness of a neutralizing antibody against two variants in a single assay, at the same time.
“This is a significant advantage in saving time and resources, especially with so many basic materials like plastics and reagents in such high demand and limited supply due to the pandemic,” said Kevin Chiem, Ph.D. candidate and member of the Martinez-Sobrido laboratory. “As new variants emerge, we can easily adapt the system and quickly research how well the antibodies are working against them. “
Powerful and precise
Importantly, the group demonstrated that reporter viruses behave similarly to a wild-type version of the virus. This is because they did not delete any viral genes and designed the reporter protein to immediately separate from the virus core protein so that it functions normally. Their research shows that the brightness of the reporter protein correlates well with viral load, although protein build-up can occur over several days, leading to a slightly stronger signal in some cases.
The advancement relies on several powerful techniques, including reverse genetics techniques to generate recombinant SARS-CoV-2, which link pieces of genetic code together to produce the complete virus.
Martinez-Sobrido and his team have shared their recombinant SARS-CoV-2 and non-infectious precursor materials, called plasmids, with more than 100 labs around the world. They can now share reporter viruses with qualified laboratories with access to Biocontainment Security Level (BSL) -3, which is necessary to work with SARS-CoV-2, to help fight the COVID-pandemic. 19 still in progress.
“We believe it is our responsibility to share these new tools and technologies with other researchers around the world to help end the pandemic as quickly as possible,” Martinez-Sobrido said.
Texas Biomedical Research Institute
Yes c., et al. (2021) Analysis of the dynamics of SARS-CoV-2 infection in vivo using reporter viruses. PNAS. doi.org/10.1073/pnas.2111593118.