Nominated Principal Applicant
Joyce Wilson, University of Saskatchewan
Co-Applicant
Darryl Falzarano, Vaccine and Infectious Disease Organization (VIDO) and CoVaRR-Net Pillar 3 Deputy
Collaborators
Anil Kumar, University of Saskatchewan
Objectives
This research aimed to develop reverse-genetic tools to study virus-host interactions and variants of concern.
The research had two main objectives:
- Generate SARS-CoV-2 replication tools to rapidly assess virus replication fitness in biosafety levels 2 and 3.
- Develop reverse genetic clones of SARS-CoV-2 variants to study the roles of specific sequence changes that evolved in SARS-CoV-2
Major Successes
For objective 1: Generation of SARS-CoV-2 replication tools to rapidly assess virus replication fitness in biosafety levels 3 and 2
The team’s research focused on creating tools to study how SARS-CoV-2 interacts with human cells and how different virus variants behave. They made molecular copies of the Wuhan-1, Delta, and Omicron variants of the virus, some with a glowing marker called NanoLuc (Nluc), which helps them track the virus. Using these copies, they produced live versions of the virus in lab-grown cells.
The modified versions of Wuhan, Delta, and Omicron variants behaved similarly to the original virus in terms of how they replicate. Importantly, the special versions of the virus that contain the NanoLuc marker let the research team easily measure virus activity and they used them to test antiviral drugs like Remdesivir, Molnupiravir, and Nirmatrelvir. They found that these drugs reduced the virus’ activity, showing similar effectiveness to other testing methods.
Finally, the team created a simple tool to study how SARS-CoV-2 replicates its RNA inside cells. This tool is a piece of DNA that can be copied in bacteria and then introduced into other cells to start the virus’s RNA replication and transcription processes. Importantly, this DNA doesn’t have the parts needed to form new virus particles, so it can’t produce infectious viruses. Again, the researchers included the marker Nluc that makes the cells glow, helping them measure how much the virus’s RNA is replicating. They again tested antiviral drugs like Remdesivir, Molnupiravir, and Nirmatrelvir and found that these drugs effectively stopped the RNA replication in one’s system. The big advantage of their system was safety. Studying the actual SARS-CoV-2 virus requires high-level biosafety labs (BSL-3), but their non-infectious system can be used in lower-level biosafety labs (BSL-2), making research more accessible.
Thus, this research team successfully developed several Nluc reporter systems to study SARS-CoV-2 interactions with human cells and assess antiviral drug efficacy, including one that allows for safe and effective testing of antiviral compounds in biosafety level 2 laboratories, as it does not involve handling infectious virus particles.
More specific, scientific, list of key major successes:
- Creation of Omicron wild-type and Omicron Nluc molecular clones and viruses;
- Development of Delta wild type and Delta Nluc molecular clones and viruses;
- Showed proof of principle antiviral assays using their reporter viruses to confirm that Nluc activity levels are a good proxy for virus replication and antiviral activity. Their assay generates similar IC50 values as those cited in the literature for virus inhibition by Remdesivir, Molnupiravir, and Nirmatrelvir;
- Confirmed that a SARS-CoV-2 subgenomic replicon DNA clone produces RNA that replicates authentically in cells, expresses Nluc and can be inhibited by antiviral drugs, but does not produce infectious particles so can be used in biosafety level 2 labs.
For objective 2 – generation and evaluation of adaptive mutations that evolved in the SARS-CoV-2 non-structural protein 6 (NSP6), non-structural protein 4 (NSP4), and nucleocapsid (N) genes of Delta and Omicron variants:
The team’s research aimed to understand how SARS-CoV-2 adapts to humans and how these changes affect its behavior. They focused on mutations outside the spike protein, particularly in the NSP6, NSP4, and N proteins. By creating mutant versions of the Delta and Omicron variants with these specific mutations reverted to their original forms, the researchers observed that certain changes in the N and NSP4 proteins enhanced the virus’s ability to replicate. Surprisingly, a mutation in NSP6 found in Omicron variants actually reduced replication efficiency. These findings help understand the pressures driving virus evolution during a pandemic.
More specific, scientific, description of major success here:
Synthetic molecular clone plasmids can be used to generate de-evolved versions of SARS-CoV-2 variants. SARS-CoV-2 viruses derived from clones that have been de-evolved to remove adaptive mutations in NSP6, NSP4, and N have modified replication capacity in cell culture versus wild type versions. This team will continue this work to identify the mechanism of action of the mutations and how they affect virus pathogenesis.
Budget
CoVaRR-Net is funding this research, which was first proposed to the Canadian Institutes of Health Research’s (CIHR) Emerging COVID-19 Research Gaps and Priorities – Variants funding call, with a $305,775 cash contribution.