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Recent Publication

Development of a Single-cycle Infectious SARS-CoV-2 Virus Replicon Particle System for use in BSL2 Laboratories


Research activities with infectious severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The Spike (S) gene of SARS-CoV-2 encodes for the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (ΔS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G (ΔS-VRP(G)) as compared to cells expressing other viral glycoproteins including S. We confirmed that infection from ΔS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with ΔS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of ΔS-VRP(G) infected cells with anti-CoV drugs remdesivir (nucleoside analog) or GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug-dose and cell-type dependent manner. Taken together, we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high throughput screening of antiviral drugs under BSL2 containment.

  • Johnny Malicoat, Senthamizharasi Manivasagam, Sonia Zuñiga, Isabel Sola, Dianne McCabe, Lijun Rong, Stanley Perlman, Luis Enjuanes, Balaji Manicassamy