ABSTRACT
Research activities with infectious severe acute respiratory syndrome corona-virus 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 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 (Delta S-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 [Delta S-VRP(G)] than in cells expressing other viral glycoproteins, including S. We confirmed that infection from Delta S-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with Delta S-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 Delta S-VRP(G)-infected cells with either of the anti-CoV drugs remdesivir (nucleoside analog) and 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, our findings show that 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. IMPORTANCE Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and anti-viral drugs. By deleting the essential spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment.
ABSTRACT
The Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal zoonotic pathogen that was first identified in humans in Saudi Arabia and Jordan in 2012. Intermittent sporadic cases, community clusters, and nosocomial outbreaks of MERS-CoV continue to occur. Between April 2012 and December 2019, 2499 laboratory-confirmed cases of MERS-CoV infection, including 858 deaths (34.3% mortality) were reported from 27 countries to WHO, the majority of which were reported by Saudi Arabia (2106 cases, 780 deaths). Large outbreaks of human-to-human transmission have occurred, the largest in Riyadh and Jeddah in 2014 and in South Korea in 2015. MERS-CoV remains a high-threat pathogen identified by WHO as a priority pathogen because it causes severe disease that has a high mortality rate, epidemic potential, and no medical countermeasures. This Seminar provides an update on the current knowledge and perspectives on MERS epidemiology, virology, mode of transmission, pathogenesis, diagnosis, clinical features, management, infection control, development of new therapeutics and vaccines, and highlights unanswered questions and priorities for research, improved management, and prevention.