Single-particle quantification of SARS-CoV-2 virus-like particles using flow virometry.

Area of Science:

• Virology
• Biotechnology
• Molecular Biology

Background:

• SARS-CoV-2 virus-like particles (VLPs) are crucial tools for studying viral mechanisms.
• Traditional VLP production often involves complex multi-plasmid systems.
• Efficient and quantifiable VLP generation is essential for research and diagnostics.

Purpose of the Study:

• To develop a simplified and more efficient method for producing fluorescent SARS-CoV-2 VLPs.
• To enable fluorescent quantification and single-particle analysis of VLPs.
• To compare the efficiency of a novel two-plasmid system against a traditional four-plasmid system.

Main Methods:

• Generation of SARS-CoV-2 VLPs using HEK 293T cells with four plasmids encoding S, N, M, and E proteins.
• Fusion of enhanced green fluorescent protein (EGFP) to S and N proteins for fluorescent labeling.
• Development of a two-plasmid system incorporating internal ribosomal entry site elements.
• Analysis of VLP production, protein concentration (ELISA), size, and concentration (flow virometry).

Main Results:

• Fluorescent VLPs were successfully produced using both four- and two-plasmid systems.
• The two-plasmid system yielded a fourfold higher N protein concentration (800 ng/mL vs. 200 ng/mL).
• Flow virometry confirmed VLP production with an average diameter of 80 nm and a concentration of 1.68 × 10^8 particles/mL.

Conclusions:

• The two-plasmid system significantly enhances the efficiency of fluorescent SARS-CoV-2 VLP production.
• Flow virometry is a reliable technique for characterizing VLP size and concentration.
• This optimized VLP production method facilitates further research into SARS-CoV-2.