key: cord-0979717-f2bwtz7k authors: Zhang, Xianwen; Liu, Yang; Liu, Jianying; Bailey, Adam L.; Plante, Kenneth S.; Plante, Jessica A.; Zou, Jing; Xia, Hongjie; Bopp, Nathen E.; Aguilar, Patricia V.; Ren, Ping; Menachery, Vineet D.; Diamond, Michael S.; Weaver, Scott C.; Xie, Xuping; Shi, Pei-Yong title: A trans-complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence date: 2021-02-23 journal: Cell DOI: 10.1016/j.cell.2021.02.044 sha: 4a585919c839f9e1dc7a302c7dde559f526e9fd7 doc_id: 979717 cord_uid: f2bwtz7k The biosafety level-3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round, but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development. After intranasal inoculation with 5×10 3 TCID 50 of S-IV-P5-Vero-P2 virion (the highest possible 170 dose), hamsters did not lose weight or develop detectable disease ( Fig. S5A-B) . Collectively, the results indicate that both ∆ORF3-E mNG virion and S-IV-P5-Vero-P2 virion are highly 172 attenuated and do not disseminate or cause disease in hamsters. To corroborate the hamster results, we tested ∆ORF3-E mNG virion in more susceptible intracranial route with 500, 50, 5, and 1 TCID 50 of WT SARS-CoV-2 developed 100%, 25%, 181 25%, and 0% mortality, respectively (Fig. 3K) . Similar to the ∆ORF3-E mNG virion, no morbidity 182 or mortality was observed after mice were inoculated by the intranasal or intracranial route with 183 2.5×10 3 or 5×10 2 TCID 50 of S-IV-P5-Vero-P2 virion, respectively ( Fig. S5C-F) . Together, the 184 results demonstrate that both single-round ∆ORF3-E mNG virion and multiple-round S-IV-P5- Vero-P2 virion lack virulence in K18-hACE2 mice. patients were measured by two assays for comparison: the ∆ORF3-E mNG virion assay and the gold standard plaque-reduction neutralization test (PRNT). The two assays produced 191 comparable 50% neutralization titers (NT 50 ) for all specimens (Table 1 and Fig. 4B-C) . In 192 addition, the ∆ORF3-E mNG virion assay could also be used to measure the 50% effective The EC 50 discrepancy between the two cell types is likely due to different efficiencies in 198 converting Remdesivir to its triphosphate form, as previously reported (Pruijssers et al., 2020; 199 Xie et al., 2020b) . Collectively, the results demonstrate that the ∆ORF3-E virion assay can be 200 used for high-throughput neutralization testing and antiviral drug discovery. We generated and characterized a trans-complementation system for SARS-CoV-2. The 204 system produced a high yield of single-round infectious ∆ORF3-E virion that could be used for 205 neutralization and antiviral testing. An mNG reporter was introduced into the ∆ORF3-E virion to 206 indicate viral replication. Depending on research needs, other reporter genes, such as luciferase 207 or GFP, could be engineered into the system. A reliable high-throughput neutralization assay is 1 <20 <20 2 <20 <20 3 59 80 4 81 80 5 169 160 6 225 200 7 274 320 8 353 320 9 370 320 10 392 320 11 394 400 12 568 320 13 585 800 14 666 400 15 677 640 16 744 320 17 909 800 18 925 640 19 1196 800 20 1789 1600 To assess viral RNA levels, a quantitative RT-PCR assay was conducted using an iTaq Viroporins E, 3a, and 8a in Replication and Pathogenesis Simulation of the clinical and pathological manifestations of 686 COVID-19) in golden Syrian hamster model: implications for 687 disease pathogenesis and transmissibility Derivation of a novel SARS-coronavirus 689 replicon cell line and its application for anti-SARS drug screening Evaluation of a 691 recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine 692 platform Rapid identification of coronavirus replicase inhibitors using a selectable replicon RNA SARS-CoV-2 Reverse Genetics 698 Reveals a Variable Infection Gradient in the Respiratory Tract Characteristics of SARS-CoV-2 and COVID-19 Furin Cleavage Site Is Key to SARS Subgenomic replicons of the flavivirus Kunjin: 705 construction and applications SARS-CoV-2 growth, furin-cleavage-site 708 adaptation and neutralization using serum from acutely infected hospitalized COVID-19 709 patients A PCR amplicon-based SARS-CoV-2 711 replicon for antiviral evaluation Molecular determinants and mechanism for antibody cocktail 714 preventing SARS-CoV-2 escape Fast-spreading U.K. virus variant raises alarms A high-throughput neutralizing antibody assay for COVID-19 diagnosis 740 and vaccine evaluation Generation of a replication-742 competent, propagation-deficient virus vector based on the transmissible gastroenteritis 743 coronavirus genome Spike mutation D614G alters SARS-CoV-2 746 fitness Remdesivir Inhibits SARS-CoV-749 2 in Human Lung Cells and Chimeric SARS-CoV Expressing the SARS-CoV-2 RNA 750 Polymerase in Mice A simple method of estimating fifty percent endpoints NIH Image to ImageJ: 25 years of 754 image analysis A live-attenuated Zika virus vaccine candidate 757 induces sterilizing immunity in mouse models The severe acute respiratory syndrome coronavirus 3a is a novel structural protein Rapid reconstruction of SARS-CoV-2 using 763 a synthetic genomics platform The Structure of the Membrane Protein of SARS-CoV-2 Resembles the 765 Sugar Transporter SemiSWEET SARS-CoV-2 infection of human ACE2-transgenic 774 mice causes severe lung inflammation and impaired function Evasion of Type I Interferon by SARS-CoV-2 Neutralization of SARS-CoV-2 779 spike 69/70 deletion, E484K, and N501Y variants by BNT162b2 vaccine-elicited sera Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and 783 N501Y variants by BNT162b2 vaccine-elicited sera An Infectious cDNA Clone of SARS-CoV-2 A nanoluciferase SARS-CoV-2 for rapid 789 neutralization testing and screening of anti-infective drugs for COVID-19 Molecular Architecture of the SARS-CoV-2 Virus Rewiring the severe acute 794 respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a 795 recombination-resistant genome Neutralizing antibody against SARS-CoV-2 In brief develop a trans-complementation system to produce single-round infectious SARS-CoV-2 that recapitulates authentic viral infection and replication. They then show the system can be safely used at biosafety level-2 for high-throughput antiviral testing