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evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic infecting more than 111 million people and causing 2.4 million deaths. Clinical disease in humans ranges from asymptomatic infection to pneumonia, severe respiratory compromise, multi-organ failure and systemic inflammatory syndromes. The rapid expansion and prolonged nature of the COVID-19 pandemic and its accompanying morbidity, mortality and destabilizing socioeconomic effects have made the development of SARS-CoV-2 therapeutics and vaccines an urgent global health priority 1. Indeed, the emergency use authorization and rapid deployment of antibody-based countermeasures, including mAbs, immune plasma therapy and messenger RNA, and inactivated and viral-vectored vaccines has provided hope for curtailing disease and ending the pandemic. The spike protein of the SARS-CoV-2 virion binds the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) to promote entry into human cells 2. Because the spike protein is critical for viral entry, it has been targeted for vaccine development and therapeutic antibody interventions. SARS-CoV-2 S proteins are cleaved to yield S1 and S2 fragments. The S1 protein includes the N-terminal (NTD) and receptor-binding (RBD) domains, whereas the S2 protein promotes membrane fusion. The RBD is recognized by many potently neutralizing monoclonal antibodies 3-7 , protein-based inhibitors 8 and serum antibodies 9. The current suite of antibody therapeutics and vaccines was designed with a spike protein based on strains circulating during the early phases of the pandemic in 2020. More recently, variants with enhanced transmissibility have emerged in the United Kingdom (B.1.1.7), South Africa (B.1.351), Brazil (B.1.1.248) and elsewhere with multiple substitutions in the spike protein, including in the NTD and the receptor-binding motif (RBM) of the RBD. Preliminary studies with pseudoviruses suggest that neutralization by some antibodies and immune sera may be diminished against variants expressing mutations in the spike gene 10-13. Given these

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.

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We engineered three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion + N501Y + D614G from UK; and E484K + N501Y + D614G from SA. Neutralization geometric mean titers (GMTs) of 20 BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81-to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses. We previously reported that BNT162b2, a nucleoside-modified RNA vaccine that encodes the SARS-CoV-2 full-length, prefusion-stabilized spike glycoprotein, elicited dose-dependent SARS-CoV-2-neutralizing GMTs that were similar to or higher than the GMT of a panel of SARS-CoV-2 convalescent human serum samples 1. We subsequently reported that, in a randomized, placebo-controlled trial of approximately 44,000 participants 16 years of age or older, a two-dose regimen of BNT162b2 conferred 95% protection against Coronavirus Disease 2019 (COVID-19) 2. Since the previously reported studies were conducted, rapidly spreading variants of SARS-CoV-2 have arisen in the UK, SA and other regions 3,4. These variants have multiple mutations in their spike glycoproteins, which are key targets of virus-neutralizing antibodies. The emerged spike mutations have raised concerns of vaccine efficacy against these new strains. The goal of this study was to examine the effect of several key spike mutations from the UK and SA strains on BNT162b2 vaccine-elicited neutralization. Using an infectious complementary DNA (cDNA) clone of SARS-CoV-2 (ref. 5), we engineered three spike mutant viruses on the genetic background of clinical strain USA-WA1/2020 (Supplementary Fig. 1). 1) Mutant N501Y virus contains the N501Y mutation that is shared by both the UK and SA variants. This mutation is located in the viral receptor-binding domain (RBD) for cell entry, increases binding to the angiotensin-converting enzyme 2 receptor and enables the virus to expand its host range to infect mice 5,6. 2) Mutant Δ69/70 + N501Y + D614G virus contains two additional changes present in the UK variants: amino acid 69 and 70 deletion (Δ69/70) and D614G substitution. Amino acids 69 and 70