Distinct pathway for evolution of enhanced receptor binding and cell entry in SARS-like bat coronaviruses.

Understanding the zoonotic risks posed by bat coronaviruses (CoVs) is critical for pandemic preparedness. Herein, we generated recombinant vesicular stomatitis viruses (rVSVs) bearing spikes from divergent bat CoVs to investigate their cell entry mechanisms. Unexpectedly, the successful recovery of rVSVs bearing the spike from SHC014, a SARS-like bat CoV, was associated with the acquisition of a novel substitution in the S2 fusion peptide-proximal region (FPPR). This substitution enhanced viral entry in both VSV and coronavirus contexts by increasing the availability of the spike receptor-binding domain to recognize its cellular receptor, ACE2. A second substitution in the spike N-terminal domain, uncovered through forward-genetic selection, interacted epistatically with the FPPR substitution to synergistically enhance spike:ACE2 interaction and viral entry. Our findings identify genetic pathways for adaptation by bat CoVs during spillover and host-to-host transmission, fitness trade-offs inherent to these pathways, and potential Achilles' heels that could be targeted with countermeasures.

Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection.

Andes virus (ANDV) and Sin Nombre virus (SNV) are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we use computational and experimental approaches to delineate the binding surface of the hantavirus glycoprotein complex on PCDH1's first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influences the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this and a neighboring residue substantially protects Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.

Genotype-specific features reduce the susceptibility of South American yellow fever virus strains to vaccine-induced antibodies.

The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acid changes at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

Efficacy and Safety of COVID-19 Convalescent Plasma in Hospitalized Patients: A Randomized Clinical Trial.

Importance: There is clinical equipoise for COVID-19 convalescent plasma (CCP) use in patients hospitalized with COVID-19. Objective: To determine the safety and efficacy of CCP compared with placebo in hospitalized patients with COVID-19 receiving noninvasive supplemental oxygen. Design, Setting, and Participants: CONTAIN COVID-19, a randomized, double-blind, placebo-controlled trial of CCP in hospitalized adults with COVID-19, was conducted at 21 US hospitals from April 17, 2020, to March 15, 2021. The trial enrolled 941 participants who were hospitalized for 3 or less days or presented 7 or less days after symptom onset and required noninvasive oxygen supplementation. Interventions: A unit of approximately 250 mL of CCP or equivalent volume of placebo (normal saline). Main Outcomes and Measures: The primary outcome was participant scores on the 11-point World Health Organization (WHO) Ordinal Scale for Clinical Improvement on day 14 after randomization; the secondary outcome was WHO scores determined on day 28. Subgroups were analyzed with respect to age, baseline WHO score, concomitant medications, symptom duration, CCP SARS-CoV-2 titer, baseline SARS-CoV-2 serostatus, and enrollment quarter. Outcomes were analyzed using a bayesian proportional cumulative odds model. Efficacy of CCP was defined as a cumulative adjusted odds ratio (cOR) less than 1 and a clinically meaningful effect as cOR less than 0.8. Results: Of 941 participants randomized (473 to placebo and 468 to CCP), 556 were men (59.1%); median age was 63 years (IQR, 52-73); 373 (39.6%) were Hispanic and 132 (14.0%) were non-Hispanic Black. The cOR for the primary outcome adjusted for site, baseline risk, WHO score, age, sex, and symptom duration was 0.94 (95% credible interval [CrI], 0.75-1.18) with posterior probability (P[cOR<1] = 72%); the cOR for the secondary adjusted outcome was 0.92 (95% CrI, 0.74-1.16; P[cOR<1] = 76%). Exploratory subgroup analyses suggested heterogeneity of treatment effect: at day 28, cORs were 0.72 (95% CrI, 0.46-1.13; P[cOR<1] = 93%) for participants enrolled in April-June 2020 and 0.65 (95% CrI, 0.41 to 1.02; P[cOR<1] = 97%) for those not receiving remdesivir and not receiving corticosteroids at randomization. Median CCP SARS-CoV-2 neutralizing titer used in April to June 2020 was 1:175 (IQR, 76-379). Any adverse events (excluding transfusion reactions) were reported for 39 (8.2%) placebo recipients and 44 (9.4%) CCP recipients (P = .57). Transfusion reactions occurred in 2 (0.4) placebo recipients and 8 (1.7) CCP recipients (P = .06). Conclusions and Relevance: In this trial, CCP did not meet the prespecified primary and secondary outcomes for CCP efficacy. However, high-titer CCP may have benefited participants early in the pandemic when remdesivir and corticosteroids were not in use. Trial Registration: ClinicalTrials.gov Identifier: NCT04364737.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Single-Dilution COVID-19 Antibody Test with Qualitative and Quantitative Readouts.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.

Computational strategies to combat COVID-19: useful tools to accelerate SARS-CoV-2 and coronavirus research.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel virus of the family Coronaviridae. The virus causes the infectious disease COVID-19. The biology of coronaviruses has been studied for many years. However, bioinformatics tools designed explicitly for SARS-CoV-2 have only recently been developed as a rapid reaction to the need for fast detection, understanding and treatment of COVID-19. To control the ongoing COVID-19 pandemic, it is of utmost importance to get insight into the evolution and pathogenesis of the virus. In this review, we cover bioinformatics workflows and tools for the routine detection of SARS-CoV-2 infection, the reliable analysis of sequencing data, the tracking of the COVID-19 pandemic and evaluation of containment measures, the study of coronavirus evolution, the discovery of potential drug targets and development of therapeutic strategies. For each tool, we briefly describe its use case and how it advances research specifically for SARS-CoV-2. All tools are free to use and available online, either through web applications or public code repositories. Contact:evbc@unj-jena.de.

A Sweep of Earth's Virome Reveals Host-Guided Viral Protein Structural Mimicry and Points to Determinants of Human Disease.

Viruses deploy genetically encoded strategies to coopt host machinery and support viral replicative cycles. Here, we use protein structure similarity to scan for molecular mimicry, manifested by structural similarity between viral and endogenous host proteins, across thousands of cataloged viruses and hosts spanning broad ecological niches and taxonomic range, including bacteria, plants and fungi, invertebrates, and vertebrates. This survey identified over 6,000,000 instances of structural mimicry; more than 70% of viral mimics cannot be discerned through protein sequence alone. We demonstrate that the manner and degree to which viruses exploit molecular mimicry varies by genome size and nucleic acid type and identify 158 human proteins that are mimicked by coronaviruses, providing clues about cellular processes driving pathogenesis. Our observations point to molecular mimicry as a pervasive strategy employed by viruses and indicate that the protein structure space used by a given virus is dictated by the host proteome. A record of this paper's transparent peer review process is included in the Supplemental Information.

Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts.

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

A Virion-Based Assay for Glycoprotein Thermostability Reveals Key Determinants of Filovirus Entry and Its Inhibition.

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

A Structure-Informed Atlas of Human-Virus Interactions.

While knowledge of protein-protein interactions (PPIs) is critical for understanding virus-host relationships, limitations on the scalability of high-throughput methods have hampered their identification beyond a number of well-studied viruses. Here, we implement an in silico computational framework (pathogen host interactome prediction using structure similarity [P-HIPSTer]) that employs structural information to predict ∼282,000 pan viral-human PPIs with an experimental validation rate of ∼76%. In addition to rediscovering known biology, P-HIPSTer has yielded a series of new findings: the discovery of shared and unique machinery employed across human-infecting viruses, a likely role for ZIKV-ESR1 interactions in modulating viral replication, the identification of PPIs that discriminate between human papilloma viruses (HPVs) with high and low oncogenic potential, and a structure-enabled history of evolutionary selective pressure imposed on the human proteome. Further, P-HIPSTer enables discovery of previously unappreciated cellular circuits that act on human-infecting viruses and provides insight into experimentally intractable viruses.

Author Correction: The discovery of Bombali virus adds further support for bats as hosts of ebolaviruses.

In the version of this Article originally published, the bat species for 12 individuals were incorrectly identified in Supplementary Table 1 and 2. After resequencing the MT-CytB and MT-CO1 segments and reviewing the data, the authors have corrected the errors for these 12 animals. In the amended version of the Supplementary Information, Supplementary Tables 1 and 2 have been replaced to include the corrected host species information. None of the 12 bats affected were positive for the Bombali virus, and the conclusions of the study are therefore unchanged.

The discovery of Bombali virus adds further support for bats as hosts of ebolaviruses.

Here we describe the complete genome of a new ebolavirus, Bombali virus (BOMV) detected in free-tailed bats in Sierra Leone (little free-tailed (Chaerephon pumilus) and Angolan free-tailed (Mops condylurus)). The bats were found roosting inside houses, indicating the potential for human transmission. We show that the viral glycoprotein can mediate entry into human cells. However, further studies are required to investigate whether exposure has actually occurred or if BOMV is pathogenic in humans.

Substrate translocation involves specific lysine residues of the central channel of the conjugative coupling protein TrwB.

Conjugative transfer of plasmid R388 requires the coupling protein TrwB for protein and DNA transport, but their molecular role in transport has not been deciphered. We investigated the role of residues protruding into the central channel of the TrwB hexamer by a mutational analysis. Mutations affecting lysine residues K275, K398, and K421, and residue S441, all facing the internal channel, affected transport of both DNA and the relaxase protein in vivo. The ATPase activity of the purified soluble variants was affected significantly in the presence of accessory protein TrwA or DNA, correlating with their behaviour in vivo. Alteration of residues located at the cytoplasmic or the inner membrane interface resulted in lower activity in vivo and in vitro, while variants affecting residues in the central region of the channel showed increased DNA and protein transfer efficiency and higher ATPase activity, especially in the absence of TrwA. In fact, these variants could catalyze DNA transfer in the absence of TrwA under conditions in which the wild-type system was transfer deficient. Our results suggest that protein and DNA molecules have the same molecular requirements for translocation by Type IV secretion systems, with residues at both ends of the TrwB channel controlling the opening-closing mechanism, while residues embedded in the channel would set the pace for substrate translocation (both protein and DNA) in concert with TrwA.

A High-Throughput Strategy for Dissecting Mammalian Genetic Interactions.

Comprehensive delineation of complex cellular networks requires high-throughput interrogation of genetic interactions. To address this challenge, we describe the development of a multiplex combinatorial strategy to assess pairwise genetic interactions using CRISPR-Cas9 genome editing and next-generation sequencing. We characterize the performance of combinatorial genome editing and analysis using different promoter and gRNA designs and identified regions of the chimeric RNA that are compatible with next-generation sequencing preparation and quantification. This approach is an important step towards elucidating genetic networks relevant to human diseases and the development of more efficient Cas9-based therapeutics.