- Korean researchers engineered three mutant SARS-CoV-2 viruses using a laboratory technique called a SARS-CoV-2 Bacmid reverse genetic (RG) system.
- The mutant viruses were engineered using site-directed mutagenesis and contained already known mutations P323L and G671S, combined with genetic material from fireflies and the hepatitis D virus, among other components.
- The researchers found that the mutant viruses exhibited more potent infection than the naturally occurring wild-type coronavirus, enhancing its replication, and transmission ability.
- The mutant viruses were tested on female ferrets in an enhanced biosafety level 3 (BSL3) containment laboratory approved by the Korea Centers for Disease Control and Prevention.
- BSL-3 laboratories have strict engineering and containment requirements and are used to study infectious agents or toxins that can be transmitted through the air and cause potentially lethal infections.
- The study, titled “SARS-CoV-2 variants show temperature-dependent enhanced polymerase activity in the upper respiratory tract and high transmissibility,” is archived in the U.S. National Library of Medicine (NLM), which publishes papers that result from research funded by the U.S. National Institutes of Health.
A team of Korean researchers published a pre-print version of their study in BioRxiv, which is archived in the U.S. National Library of Medicine (NLM), titled “SARS-CoV-2 variants show temperature-dependent enhanced polymerase activity in the upper respiratory tract and high transmissibility.” NLM publishes preprints that result from research funded by the U.S. National Institutes of Health (NIH).
The Sep 2022 study collected SARS-CoV-2 viruses from COVID-19 patients at a hospital between February 2020 and March 2021.
In the study, researchers used a laboratory technique called a “SARS-CoV-2 Bacmid reverse genetic (RG) system” to engineer three “mutant viruses.” The researchers call these mutant viruses RG-P323L, RG-G671S, and RG-P323L/G671S.
“To evaluate how the P323L and G671S mutations affected SARS-CoV-2 fitness and transmission, we used the SARS-CoV-2 Bacmid reverse genetic (RG) system to generate three isogenic SARS-CoV-2 variants (Spike D614G) containing the NSP12 WT, P323L, G671S, or P323L/G617S mutations 24,” the study authors write.
The designators “P323L” and “G671S” signify already well-known mutations within the coronavirus. The “RG” prefix signifies the study authors’ lab-engineered forms.
The RG-P323L, RG-G671S, and RG-P323L/G671S mutant viruses “consistently exhibited”
more potent infection than wild-type coronavirus, which occur naturally in the wild, according to the authors.
The researchers found that the mutations enhanced the virus’ replication, its ability to make copies of itself, and enhances the virus’ transmission, its ability to move from one person to another.
The mutant viruses were created when researchers combined genetic material from the SARS-CoV-2 virus with genetic material from fireflies and the hepatitis D virus, among other components.
The genetic material from the COVID virus included proteins (NSP12, NSP7, and NSP8) essential for viral replication and genomic transcription, the process of copying genetic information from DNA to RNA.
The researchers combined this mixture with human cells using “site-directed mutagenesis,” the goal being the “construction” of coronavirus protein “mutants.” The authors write:
The cell-based reporter system included the bicistronic reporter construct containing firefly luciferase in the sense orientation, and Nano-glo® luciferase in the anti-sense orientation, which was flanked by the antisense 3ʹ- and 5ʹ-UTR of SARS-CoV-2 and the hepatitis D virus ribozyme (HDV) self-cleavage sequence. The reporter plasmid contained the sense firefly luciferase gene (GenBank®: U47295), hepatitis D virus ribozyme sequence, antisense 3ʹuntranslated region (UTR) of SARS-CoV-2, anti-sense Nano-glo® luciferase gene (NLuc) (GenBank®: KM359770), and HDV ribozyme sequence, which were synthesized in a row by GENEWIZ, and cloned into the HindIII and XhoI sites of the pcDNA3.1(+) plasmid (Invitrogen, Carlsbad, CA, USA). The C-terminal 10× His-tagged NSP12 gene, and Nterminal Flag-tagged NSP7 and NSP8 gene (GenBank®: NC_045512) were cloned into the BamHI and XhoI, and NheI and XhoI sites, respectively, of the pcDNA3.1(+) plasmid. For construction of NSP12 mutants (P323L, P323L/G617S, and G617S), site-directed mutagenesis was performed using the following primer sets.
The researchers then separated the three SARS-CoV-2 proteins (NSP12, NSP7, NSP8) before purifying and storing them. They combined these three “mutant” proteins with another piece of RNA from the coronavirus.
With the product, the study authors infected female ferrets that were 20-24 months old “through the nose” and even “sacrificed” some of the ferrets to test if the virus was present in the animals’ organs.
The researchers’ experiments required “approval” from the Medical Research Institute, a member of the Laboratory Animal Research Center of Chungbuk National University (LARC). The approval number is CBNUA-1731-22-01.
The handling of these mutant viruses was performed in an “enhanced biosafety level 3 (BSL3) containment laboratory” that was approved by the Korea Centers for Disease Control and Prevention (protocol KCDC-14-3-07), according to the study.
BSL-3 laboratories are used to study infectious agents or toxins that can be transmitted through the air and cause potentially lethal infections. They have strict engineering and containment requirements, including controlled airflow, two self-closing, interlocked doors, sealed windows, and filtered ventilation systems. Researchers must wear appropriate personal protective equipment and all work with infectious agents or toxins must be performed within an appropriate biosafety cabinet. Access to the laboratory is restricted and medical surveillance of laboratory workers is required. The laboratory must have a hands-free sink and eyewash near the exit, and exhaust air cannot be recirculated.
Read the full study below:
