Related Experiment Video
Updated: Jun 5, 2025
Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting
Published on: March 1, 2019
Combined mutations in nonstructural protein 14, envelope, and membrane proteins mitigate the neuropathogenicity of
Kotou Sangare1, Shufeng Liu1, Prabhuanand Selvaraj1
1Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
Abstract:
We previously reported that mutations outside the spike protein play a role in the attenuation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1 variant in human ACE2 transgenic mice (K18-hACE2). Here, we assessed the pathogenicity of SARS-CoV-2 (WA1/2020) containing mutations from the Omicron BA.1 variant in K18-hACE2 mice. At an infection dose of 104 plaque-forming units (PFU), WA1 virus carrying Omicron BA.1 Nsp14(I42V), E(T9I), M(D3G/Q19E/A63T), but not Nsp6(Δ105-107, I189V), substitutions showed significant reduction in lethality. Interestingly, reduction of viral load is more pronounced in the brains than in the lungs. Subsequent analyses suggest that BA.1 E(T9I) and M(D3G/Q19E/A63T) substitutions result in less efficient packaging of virus-like particles. Given that Nsp14(I42V), E(T9I), M(Q19E/A63T) are well preserved in subsequent omicron subvariants, including currently circulating variants, our findings highlight the importance of understanding how non-spike mutations affect the pathogenicity of SARS-CoV-2 variants.
Importance:
Inoculation of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) with SARS-CoV-2 often leads to a fatal brain infection. Omicron BA.1 variant, however, was found to be non-lethal in this model. Here, we systematically assessed the effect of individual mutations of Omicron BA.1 on the pathogenicity of the virus in hACE2 transgenic mice and found that combination of 5 mutations of Nsp14, E, and M of BA.1 variant significantly lowered brain viral load and reduced lethality. These results provide new insights into how SARS-CoV-2 Omicron BA.1 is attenuated.
Insights
Mutations in SARS-CoV-2 Omicron BA.1, outside the spike protein, reduce lethality in mice. Specific mutations in Nsp14, E, and M proteins significantly lower brain viral load and pathogenicity.
Area of Science:
- Virology
- Immunology
- Genetics
Background:
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1 variant shows reduced pathogenicity in human ACE2 transgenic mice.
- Understanding the genetic basis of this attenuation is crucial for predicting variant evolution and impact.
Purpose of the Study:
- To systematically assess the impact of individual Omicron BA.1 mutations on SARS-CoV-2 pathogenicity in K18-hACE2 mice.
- To identify specific non-spike mutations contributing to the reduced virulence of the BA.1 variant.
Main Methods:
- Infection of K18-hACE2 mice with SARS-CoV-2 (WA1/2020) engineered with specific Omicron BA.1 mutations.
- Assessment of viral load in brain and lungs, and evaluation of lethality.
- Analysis of virus-like particle packaging efficiency for specific mutations.
Main Results:
- A combination of Nsp14(I42V), E(T9I), and M(D3G/Q19E/A63T) substitutions significantly reduced lethality in infected mice.
- Brain viral load was disproportionately reduced compared to lung viral load.
- E(T9I) and M(D3G/Q19E/A63T) mutations were associated with less efficient virus-like particle packaging.
Conclusions:
- Non-spike mutations, particularly in Nsp14, E, and M proteins, play a significant role in the attenuation of SARS-CoV-2 Omicron BA.1.
- These mutations impact viral packaging and reduce pathogenicity, especially in the brain.
- The conserved nature of these mutations in subsequent Omicron subvariants underscores their importance in SARS-CoV-2 evolution.
