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Emerging viral infections are difficult to control as heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. SARS-CoV and MERS-CoV successively emerged causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. Here we show that a nucleotide prodrug GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat-CoVs, prepandemic bat-CoVs and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory functions. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and possibly most importantly, emerging CoV of the future.

The coronavirus disease 2019 (COVID-19) is determined by SARS-CoV-2 replication and host immune response, but studies evaluating viral evasion of immune response are lacking. Here we employed unbiased screening to identify SARS-CoV-2 proteins that antagonize type-I interferon (IFN-I) response. Three proteins were found to antagonize IFN-I production via distinct mechanisms: nsp6 binds TBK1 to suppress IRF3 phosphorylation; nsp13 binds and blocks TBK1 phosphorylation; and ORF6 binds importin KPNA2 to inhibit IRF3 nuclear translocation. Two sets of viral proteins were identified to antagonize IFN-I signaling through blocking STAT1/STAT2 phosphorylation or nuclear translocation. Remarkably, SARS-CoV-2 nsp1 and nsp6 suppressed IFN-I signaling more efficiently than SARS-CoV and MERS-CoV. Thus, when treated with IFN-I, a SARS-CoV2 replicon replicated to a higher level than chimeric replicons containing nsp1 or nsp6 from SARS-CoV or MERS-CoV. Altogether, the study has provided insights on SARS-CoV-2 evasion of IFN-I response and its potential impact on viral transmission and pathogenesis.

The emergence of a novel coronavirus (2019-nCoV) has awakened the echoes of SARS-CoV from nearly two decades ago. Yet, with technological advances and important lessons gained from previous outbreaks, perhaps the world is better equipped to deal with the most recent emergent group 2B coronavirus.

Graphical Abstract Highlights d A reverse genetic system has been established for SARS-CoV-2 d Recombinant SARS-CoV-2 replicates as efficiently as the original clinical isolate d A stable mNeonGreen reporter SARS-CoV-2 has been developed d The mNeonGreen SARS-CoV-2 can be used to screen antiviral inhibitors SUMMARYThe ongoing pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the urgency to develop experimental systems for studying this virus and identifying countermeasures. We report a reverse genetic system for SARS-CoV-2. Seven complimentary DNA (cDNA) fragments spanning the SARS-CoV-2 genome were assembled into a full-genome cDNA. RNA transcribed from the full-genome cDNA was highly infectious after electroporation into cells, producing 2.9 3 10 6 plaque-forming unit (PFU)/mL of virus. Compared with a clinical isolate, the infectiousclone-derived SARS-CoV-2 (icSARS-CoV-2) exhibited similar plaque morphology, viral RNA profile, and replication kinetics. Additionally, icSARS-CoV-2 retained engineered molecular markers and did not acquire other mutations. We generated a stable mNeonGreen SARS-CoV-2 (icSARS-CoV-2-mNG) by introducing this reporter gene into ORF7 of the viral genome. icSARS-CoV-2-mNG was successfully used to evaluate the antiviral activities of interferon (IFN). Collectively, the reverse genetic system and reporter virus provide key reagents to study SARS-CoV-2 and develop countermeasures. -Y.S. have filed a provisional patent on the reverse genetic system of SARS-CoV-2. Other authors have no conflicts of interest to declare.