Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

作者： Jean K. Millet1,2, Tiffany Tang3, Lakshmi Nathan3, Javier A. Jaimes4, Hung-Lun Hsu3,5, Susan Daniel3, Gary R. Whittaker1 1Department of Microbiology and Immunology, College of Veterinary Medicine,Cornell University, 2INRA, Virologie et Immunologie Moléculaires, 3Robert Frederick Smith School of Chemical and Biomolecular Engineering,Cornell University, 4Department of Microbiology, College of Agricultural and Life Sciences,Cornell University, 5Horae Gene Therapy Center,University of Massachusetts Medical School

简介：

Overview

This protocol outlines the generation of pseudotyped particles incorporating spike proteins from MERS and SARS coronaviruses in a BSL-2 environment. These particles enable the study of viral entry into host cells using a luciferase reporter gene for quantification.

Key Study Components

Area of Science

• Virology
• Cell Biology
• Infectious Diseases

Background

• Pseudotyped viruses are safer models for studying pathogenic viruses.
• The technique allows for the examination of viral entry mechanisms.
• HEK293T cells are commonly used for transfection and virus production.
• Monitoring cell health is crucial for successful transfection and infection.

Purpose of Study

• To develop a safe method for studying viral entry of MERS and SARS.
• To quantify the infectivity of pseudotyped particles.
• To assess receptor usage in pseudovirions.

Methods Used

• Transfection of HEK293T cells with plasmids encoding viral proteins.
• Collection and purification of pseudotyped particles.
• Infection of target cells with pseudotyped particles.
• Measurement of luciferase activity to quantify infectivity.

Main Results

• Pseudotyped particles exhibited strong infectivity in target cells.
• Luciferase assays demonstrated a concentration-dependent response.
• Receptor usage was confirmed to be similar to that of native viruses.
• Western blot assays indicated successful incorporation of spike proteins.

Conclusions

• The developed protocol provides a reliable method for studying viral entry.
• Pseudotyped particles serve as effective surrogates for pathogenic viruses.
• This approach can be adapted for other viruses beyond coronaviruses.

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