Highlights d The receptor-binding motif (RBM) is a highly variable region of SARS-CoV-2 spike d RBM mutation N439K has emerged independently in multiple lineages d N439K increases spike affinity for hACE2; viral fitness and disease are unchanged d N439K confers resistance to several mAbs and escapes some polyclonal responses

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to £10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

SARS-CoV-2 can mutate to evade immunity, with consequences for the efficacy of emerging vaccines and antibody therapeutics. Herein we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is the most divergent region of S, and provide epidemiological, clinical, and molecular characterization of a prevalent RBM variant, N439K. We demonstrate that N439K S protein has enhanced binding affinity to the hACE2 receptor, and that N439K virus has similar clinical outcomes and in vitro replication fitness as compared to wild-type. We observed that the N439K mutation resulted in immune escape from a panel of neutralizing monoclonal antibodies, including one in clinical trials, as well as from polyclonal sera from a sizeable fraction of persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

The bat connection The heterogeneity of COVID-19 makes it challenging to predict the course of infection in an individual. Upon virus infection, interferons (IFNs) generate the initial signals for cellular defenses. Knowing that defects in IFN signaling are associated with more severe COVID-19, Wickenhagen et al . used IFN-stimulated gene expression screening on human lung cells from which they identified a gene for 2′-5′-oligoadenylate synthetase 1 (OAS1) (see the Perspective by Schoggins). OAS1 stimulates RNase L to inhibit the virus with a surprising degree of specificity, targeting the membranous organelles in which it replicates. In most mammals, OAS1 is attached to membranes by a prenyl group. However, billions of humans do not have the prenylated OAS1 haplotype, including many experiencing severe COVID-19. The same is true for horseshoe bats, prolific sources of betacoronaviruses, because of an ancient retrotransposition event. —CA