Molecular Architecture of the SARS-CoV-2 Virus.

SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryoelectron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were determined to average resolutions of 8.7-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diameter lumen.

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase. Both the holo-RdRp and nsp13 are essential for viral replication and are targets for treating the disease COVID-19. Here we present cryoelectron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template product in complex with two molecules of the nsp13 helicase. The Nidovirales order-specific N-terminal domains of each nsp13 interact with the N-terminal extension of each copy of nsp8. One nsp13 also contacts the nsp12 thumb. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. We also observe ADP-Mg2+ bound in the nsp12 N-terminal nidovirus RdRp-associated nucleotidyltransferase domain, detailing a new pocket for anti-viral therapy development.

Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2.

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.

Sialoglycan binding triggers spike opening in a human coronavirus.

Coronavirus spike proteins mediate receptor binding and membrane fusion, making them prime targets for neutralizing antibodies. In the cases of severe acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus 2 and Middle East respiratory syndrome coronavirus, spike proteins transition freely between open and closed conformations to balance host cell attachment and immune evasion1-5. Spike opening exposes domain S1B, allowing it to bind to proteinaceous receptors6,7, and is also thought to enable protein refolding during membrane fusion4,5. However, with a single exception, the pre-fusion spike proteins of all other coronaviruses studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, with spike proteins more commonly transitioning to open states in response to specific cues, rather than spontaneously. Here, using cryogenic electron microscopy and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes after binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state through allosteric interdomain crosstalk. Our findings provide detailed insight into coronavirus attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

Structure of replicating SARS-CoV-2 polymerase.

The new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes1-3. Here we present a cryo-electron microscopy structure of the SARS-CoV-2 RdRp in an active form that mimics the replicating enzyme. The structure comprises the viral proteins non-structural protein 12 (nsp12), nsp8 and nsp7, and more than two turns of RNA template-product duplex. The active-site cleft of nsp12 binds to the first turn of RNA and mediates RdRp activity with conserved residues. Two copies of nsp8 bind to opposite sides of the cleft and position the second turn of RNA. Long helical extensions in nsp8 protrude along exiting RNA, forming positively charged 'sliding poles'. These sliding poles can account for the known processivity of RdRp that is required for replicating the long genome of coronaviruses3. Our results enable a detailed analysis of the inhibitory mechanisms that underlie the antiviral activity of substances such as remdesivir, a drug for the treatment of coronavirus disease 2019 (COVID-19)4.

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic continues, with devasting consequences for human lives and the global economy1,2. The discovery and development of virus-neutralizing monoclonal antibodies could be one approach to treat or prevent infection by this coronavirus. Here we report the isolation of sixty-one SARS-CoV-2-neutralizing monoclonal antibodies from five patients infected with SARS-CoV-2 and admitted to hospital with severe coronavirus disease 2019 (COVID-19). Among these are nineteen antibodies that potently neutralized authentic SARS-CoV-2 in vitro, nine of which exhibited very high potency, with 50% virus-inhibitory concentrations of 0.7 to 9 ng ml-1. Epitope mapping showed that this collection of nineteen antibodies was about equally divided between those directed against the receptor-binding domain (RBD) and those directed against the N-terminal domain (NTD), indicating that both of these regions at the top of the viral spike are immunogenic. In addition, two other powerful neutralizing antibodies recognized quaternary epitopes that overlap with the domains at the top of the spike. Cryo-electron microscopy reconstructions of one antibody that targets the RBD, a second that targets the NTD, and a third that bridges two separate RBDs showed that the antibodies recognize the closed, 'all RBD-down' conformation of the spike. Several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against SARS-CoV-2.

A pneumonia outbreak associated with a new coronavirus of probable bat origin.

Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.

A Novel Coronavirus from Patients with Pneumonia in China, 2019.

In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).

Testing of Commercial Masks and Respirators and Cotton Mask Insert Materials using SARS-CoV-2 Virion-Sized Particulates: Comparison of Ideal Aerosol Filtration Efficiency versus Fitted Filtration Efficiency.

Shortages in the availability of personal protective face masks during the COVID-19 pandemic required many to fabricate masks and filter inserts from available materials. While the base filtration efficiency of a material is of primary importance when a perfect seal is possible, ideal fit is not likely to be achieved by the average person preparing to enter a public space or even a healthcare worker without fit-testing before each shift. Our findings suggest that parameters including permeability and pliability can play a strong role in the filtration efficiency of a mask fabricated with various filter media, and that the filtration efficiency of loosely fitting masks/respirators against ultrafine particulates can drop by more than 60% when worn compared to the ideal filtration efficiency of the base material. Further, a test method using SARS-CoV-2 virion-sized silica nanoaerosols is demonstrated to assess the filtration efficiency against nanoparticulates that follow air currents associated with mask leakage.

Spiky Nanostructures with Geometry-matching Topography for Virus Inhibition.

Geometry-matching has been known to benefit the formation of stable biological interactions in natural systems. Herein, we report that the spiky nanostructures with matched topography to the influenza A virus (IAV) virions could be used to design next-generation advanced virus inhibitors. We demonstrated that nanostructures with spikes between 5 and 10 nm bind significantly better to virions than smooth nanoparticles, due to the short spikes inserting into the gaps of glycoproteins of the IAV virion. Furthermore, an erythrocyte membrane (EM) was coated to target the IAV, and the obtained EM-coated nanostructures could efficiently prevent IAV virion binding to the cells and inhibit subsequent infection. In a postinfection study, the EM-coated nanostructures reduced >99.9% virus replication at the cellular nontoxic dosage. We predict that such a combination of geometry-matching topography and cellular membrane coating will also push forward the development of nanoinhibitors for other virus strains, including SARS-CoV-2.

Lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (SARS-CoV-2).

PURPOSE: Lianhuaqingwen (LH) as traditional Chinese medicine (TCM) formula has been used to treat influenza and exerted broad-spectrum antiviral effects on a series of influenza viruses and immune regulatory effects Ding et al. (2017). The goal of this study is to demonstrate the antiviral activity of LH against the novel SARS-CoV-2 virus and its potential effect in regulating host immune response. METHODS: The antiviral activity of LH against SARS-CoV-2 was assessed in Vero E6 cells using CPE and plaque reduction assay. The effect of LH on virion morphology was visualized under transmission electron microscope. Pro-inflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. RESULTS: LH significantly inhibited SARS-CoV-2 replication in Vero E6 cells and markedly reduced pro-inflammatory cytokines (TNF-α, IL-6, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Furthermore, LH treatment resulted in abnormal particle morphology of virion in cells. CONCLUSIONS: LH significantly inhibits the SARS-COV-2 replication, affects virus morphology and exerts anti-inflammatory activity in vitro. These findings indicate that LH protects against the virus attack, making its use a novel strategy for controlling the COVID-19 disease.

Cardiovascular Risks in Patients with COVID-19: Potential Mechanisms and Areas of Uncertainty.

PURPOSE OF REVIEW: COronaVirus Disease 2019 (COVID-19) has spread at unprecedented speed and scale into a global pandemic with cardiovascular risk factors and complications emerging as important disease modifiers. We aim to review available clinical and biomedical literature on cardiovascular risks of COVID-19. RECENT FINDINGS: SARS-CoV2, the virus responsible for COVID-19, enters the cell via ACE2 expressed in select organs. Emerging epidemiological evidence suggest cardiovascular risk factors are associated with increased disease severity and mortality in COVID-19 patients. Patients with a more severe form of COVID-19 are also more likely to develop cardiac complications such as myocardial injury and arrhythmia. The true incidence of and mechanism underlying these events remain elusive. Cardiovascular diseases appear intricately linked with COVID-19, with cardiac complications contributing to the elevated morbidity/mortality of COVID-19. Robust epidemiologic and biologic studies are urgently needed to better understand the mechanism underlying these associations to develop better therapies.

Ultrastructural analysis of SARS-CoV-2 interactions with the host cell via high resolution scanning electron microscopy.

SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 h after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2.

Immune Responses During Human Coronavirus Infection: Suggestions for Future Studies.

Severe Acute Respiratory human Coronavirus 2 (SARS-hCOV 2) infection which began in December 2019 has rapidly disseminated worldwide due to non-availability of anti-viral treatment or vaccine, no knowledge of virus-human interaction, lack of prognostic factors for stages of illness and ability of hCoV 2 to rapidly mutate and infect multiple cell types. Host inflammation and evasion of host immune responses by viruses are believed to play major roles in disease severity of human Corona viruses (hCoVs), thus uses of anti-inflammatory and immune-boosting agents apart from complete multi-disciplinary approach are suggested to combat the ranvaging SAR-hCOV 2 infection. This paper related the structural proteins and life cycle of CoV with host immune responses to CoV. This is to bring out gaps in knowledge for possible future researches.

Flexible Nanoporous Template for the Design and Development of Reusable Anti-COVID-19 Hydrophobic Face Masks.

Since the outbreak of the severe respiratory disease caused by the novel coronavirus (COVID-19), the use of face masks has become ubiquitous worldwide to control the rapid spread of this pandemic. As a result, the world is currently facing a face mask shortage, and some countries have placed limits on the number of masks that can be bought by each person. Although the surgical grade N95 mask provides the highest level of protection currently available, its filtration efficiency for sub-300 nm particles is around 85% due to its wider pore size (∼300 nm). Because the COVID-19 virus shows a diameter of around 65-125 nm, there is a need for developing more efficient masks. To overcome these issues, we demonstrate the development of a flexible, nanoporous membrane to achieve a reusable N95 mask with a replaceable membrane and enhanced filtration efficiency. We first developed a flexible nanoporous Si-based template on a silicon-on-insulator wafer using KOH etching and then used the template as a hard mask during a reactive ion etching process to transfer the patterns onto a flexible and lightweight (<0.12 g) polymeric membrane. Pores with sizes down to 5 nm were achieved with a narrow distribution. Theoretical calculations show that airflow rates above 85 L/min are possible through the mask, which confirms its breathability over a wide range of pore sizes, densities, membrane thicknesses, and pressure drops. Finally, the membrane is intrinsically hydrophobic, which contributes to antifouling and self-cleaning as a result of droplets rolling and sliding on the inclined mask area.