Development of a Single-Cycle Infectious SARS-CoV-2 Virus Replicon Particle System for Use in Biosafety Level 2 Laboratories

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.

Middle East respiratory syndrome coronavirus vaccine based on a propagation-defective RNA replicon elicited sterilizing immunity in mice.

Self-amplifying RNA replicons are promising platforms for vaccine generation. Their defects in one or more essential functions for viral replication, particle assembly, or dissemination make them highly safe as vaccines. We previously showed that the deletion of the envelope (E) gene from the Middle East respiratory syndrome coronavirus (MERS-CoV) produces a replication-competent propagation-defective RNA replicon (MERS-CoV-ΔE). Evaluation of this replicon in mice expressing human dipeptidyl peptidase 4, the virus receptor, showed that the single deletion of the E gene generated an attenuated mutant. The combined deletion of the E gene with accessory open reading frames (ORFs) 3, 4a, 4b, and 5 resulted in a highly attenuated propagation-defective RNA replicon (MERS-CoV-Δ[3,4a,4b,5,E]). This RNA replicon induced sterilizing immunity in mice after challenge with a lethal dose of a virulent MERS-CoV, as no histopathological damage or infectious virus was detected in the lungs of challenged mice. The four mutants lacking the E gene were genetically stable, did not recombine with the E gene provided in trans during their passage in cell culture, and showed a propagation-defective phenotype in vivo. In addition, immunization with MERS-CoV-Δ[3,4a,4b,5,E] induced significant levels of neutralizing antibodies, indicating that MERS-CoV RNA replicons are highly safe and promising vaccine candidates.

Minimum determinants of transmissible gastroenteritis virus enteric tropism are located in the n-terminus of spike protein

Transmissible gastroenteritis virus (TGEV) is an enteric coronavirus causing high morbidity and mortality in porcine herds worldwide, that possesses both enteric and respiratory tropism. The ability to replicate in the enteric tract directly correlates with virulence, as TGEVs with an exclusive respiratory tropism are attenuated. The tissue tropism is determined by spike (S) protein, although the molecular bases for enteric tropism remain to be fully characterized. Both pAPN and sialic acid binding domains (aa 506-655 and 145-155, respectively) are necessary but not sufficient for enteric tract infection. Using a TGEV infectious cDNA and enteric (TGEV-SC11) or respiratory (TGEV-SPTV) isolates, encoding a full-length S protein, a set of chimeric recombinant viruses, with a sequential modification in S protein amino terminus, was engineered. In vivo tropism, either enteric, respiratory or both, was studied by inoculating three-day-old piglets and analyzing viral titers in lung and gut. The data indicated that U655 G change in S gene (S219A in S protein) was required to confer enteric tropism to a respiratory virus that already contains the pAPN and sialic acid binding domains in its S protein. Moreover, an engineered virus containing U655 G and a 6 nt insertion at position 1124 (Y374-T375insND in S protein) was genetically stable after passage in cell cultures, and increased virus titers in gut by 1000-fold. We postulated that the effect of these residues in enteric tropism may be mediated by the modification of both glycosaminoglycan binding and S protein structure. Copyright © 2019 by the authors. Licensee MDPI, Basel, Switzerland.