Spatial-Temporal Dataset of COVID-19 Outbreak in China

Abstract: We present Coronavirus disease 2019 (COVID-19) statistics in China dataset: daily statistics of the COVID-19 outbreak in China at the city/county level. For each city/country, we include the six most important numbers for epidemic research: daily new infections, accumulated infections, daily new recoveries, accumulated recoveries, daily new deaths, and accumulated deaths. We cross validate the dataset and the estimate error rate is about 0.04%. We then give several examples to show how to trace the spreading in particular cities or provinces, and also contrast the development of COVID-19 in all cities in China at the early, middle and late stages. We hope this dataset can help researchers around the world better understand the spreading dynamics of COVID-19 at a regional level, to inform intervention and mitigation strategies for policymakers.


Can AI help in screening Viral and COVID-19 pneumonia?

Abstract: Coronavirus disease (COVID-19) is a pandemic disease, which has already caused thousands of causalities and infected several millions of people worldwide. Any technological tool enabling rapid screening of the COVID-19 infection with high accuracy can be crucially helpful to healthcare professionals. The main clinical tool currently in use for the diagnosis of COVID-19 is the Reverse transcription polymerase chain reaction (RT-PCR), which is expensive, less-sensitive and requires specialized medical personnel. X-ray imaging is an easily accessible tool that can be an excellent alternative in the COVID-19 diagnosis. This research was taken to investigate the utility of artificial intelligence (AI) in the rapid and accurate detection of COVID-19 from chest X-ray images. The aim of this paper is to propose a robust technique for automatic detection of COVID-19 pneumonia from digital chest X-ray images applying pre-trained deep-learning algorithms while maximizing the detection accuracy. A public database was created by the authors combining several public databases and also by collecting images from recently published articles. The database contains a mixture of 423 COVID-19, 1485 viral pneumonia, and 1579 normal chest X-ray images. Transfer learning technique was used with the help of image augmentation to train and validate several pre-trained deep Convolutional Neural Networks (CNNs). The networks were trained to classify two different schemes: i) normal and COVID-19 pneumonia; ii) normal, viral and COVID-19 pneumonia with and without image augmentation. The classification accuracy, precision, sensitivity, and specificity for both the schemes were 99.7%, 99.7%, 99.7% and 99.55% and 97.9%, 97.95%, 97.9%, and 98.8%, respectively.


Knowledge synthesis from 100 million biomedical documents augments the deep expression profiling of coronavirus receptors

Abstract: The COVID-19 pandemic demands assimilation of all available biomedical knowledge to decode its mechanisms of pathogenicity and transmission. Despite the recent renaissance in unsupervised neural networks for decoding unstructured natural languages, a platform for the real-time synthesis of the exponentially growing biomedical literature and its comprehensive triangulation with deep omic insights is not available. Here, we present the nferX platform for dynamic inference from over 45 quadrillion possible conceptual associations extracted from unstructured biomedical text, and their triangulation with Single Cell RNA-sequencing based insights from over 25 tissues. Using this platform, we identify intersections between the pathologic manifestations of COVID-19 and the comprehensive expression profile of the SARS-CoV-2 receptor ACE2. We find that tongue keratinocytes and olfactory epithelial cells are likely under-appreciated targets of SARS-CoV-2 infection, correlating with reported loss of sense of taste and smell as early indicators of COVID-19 infection, including in otherwise asymptomatic patients. Airway club cells, ciliated cells and type II pneumocytes in the lung, and enterocytes of the gut also express ACE2. This study demonstrates how a holistic data science platform can leverage unprecedented quantities of structured and unstructured publicly available data to accelerate the generation of impactful biological insights and hypotheses.


An updated analysis of turning point, duration and attack rate of COVID-19 outbreaks in major Western countries with data of daily new cases

Abstract: As coronavirus spreads around the world, the study of its effects is of great practical significance. We collated data on daily new cases of the COVID-19 outbreaks in the six Western countries of the Group of Seven and the dates of governments' interventions. We studied the periods before and after the dates of major governments' interventions integrally based on a segmented Poisson model. The relevant results are published in the paper of "Predicting turning point, duration and attack rate of COVID - 19 outbreaks in major Western countries" [1]. Our method can be used to update prediction daily as COVID-19 outbreaks evolve. In this article, we illustrate an updated analysis with our method to facilitate reproducibility. Both datasets used and updated are provided.


Racism is a Virus: Anti-Asian Hate and Counterhate in Social Media during the COVID-19 Crisis

Abstract: The spread of COVID-19 has sparked racism, hate, and xenophobia in social media targeted at Chinese and broader Asian communities. However, little is known about how racial hate spreads during a pandemic and the role of counterhate speech in mitigating the spread. Here we study the evolution and spread of anti-Asian hate speech through the lens of Twitter. We create COVID-HATE, the largest dataset of anti-Asian hate and counterhate spanning three months, containing over 30 million tweets, and a social network with over 87 million nodes. By creating a novel hand-labeled dataset of 2,400 tweets, we train a text classifier to identify hate and counterhate tweets that achieves an average AUROC of 0.852. We identify 891,204 hate and 200,198 counterhate tweets in COVID-HATE. Using this data to conduct longitudinal analysis, we find that while hateful users are less engaged in the COVID-19 discussions prior to their first anti-Asian tweet, they become more vocal and engaged afterwards compared to counterhate users. We find that bots comprise 10.4% of hateful users and are more vocal and hateful compared to non-bot users. Comparing bot accounts, we show that hateful bots are more successful in attracting followers compared to counterhate bots. Analysis of the social network reveals that hateful and counterhate users interact and engage extensively with one another, instead of living in isolated polarized communities. Furthermore, we find that hate is contagious and nodes are highly likely to become hateful after being exposed to hateful content. Importantly, our analysis reveals that counterhate messages can discourage users from turning hateful in the first place. Overall, this work presents a comprehensive overview of anti-Asian hate and counterhate content during a pandemic. The COVID-HATE dataset is available at http://claws.cc.gatech.edu/covid.


TweetsCOV19 -- A Knowledge Base of Semantically Annotated Tweets about the COVID-19 Pandemic

Abstract: Publicly available social media archives facilitate research in the social sciences and provide corpora for training and testing a wide range of machine learning, NLP and information retrieval methods. With respect to the recent outbreak of COVID-19, online discourse on Twitter reflects public opinion and perception related to the pandemic itself as well as mitigating measures and their societal impact. Understanding such discourse, its evolution and interdependencies with real-world events or (mis)information can foster valuable insights. On the other hand, such corpora are crucial facilitators for computational methods addressing tasks such as sentiment analysis, event detection or entity recognition. However, obtaining, archiving and semantically annotating large amounts of tweets is costly. In this paper, we describe TweetsCOV19, a publicly available knowledge base of currently more than 8 million tweets, spanning the period Oct'19-Apr'20. Metadata about the tweets as well as extracted entities, hashtags, user mentions, sentiments, and URLs are exposed using established RDF/S vocabularies, providing an unprecedented knowledge base for a range of knowledge discovery tasks. Next to a description of the dataset and its extraction and annotation process, we present an initial analysis, use cases and usage of the corpus.


The COronavirus Pandemic Epidemiology (COPE) Consortium: A Call to Action

Abstract: The rapid pace of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; COVID-19) pandemic presents challenges to the real-time collection of population-scale data to inform near-term public health needs as well as future investigations. We established the COronavirus Pandemic Epidemiology (COPE) consortium to address this unprecedented crisis on behalf of the epidemiology research community. As a central component of this initiative, we have developed a COVID Symptom Study (previously known as the COVID Symptom Tracker) mobile application as a common data collection tool for epidemiologic cohort studies with active study participants. This mobile application collects information on risk factors, daily symptoms, and outcomes through a user-friendly interface that minimizes participant burden. Combined with our efforts within the general population, data collected from nearly 3 million participants in the United States and United Kingdom are being used to address critical needs in the emergency response, including identifying potential hot spots of disease and clinically actionable risk factors. The linkage of symptom data collected in the app with information and biospecimens already collected in epidemiology cohorts will position us to address key questions related to diet, lifestyle, environmental, and socioeconomic factors on susceptibility to COVID-19, clinical outcomes related to infection, and long-term physical, mental health, and financial sequalae. We call upon additional epidemiology cohorts to join this collective effort to strengthen our impact on the current health crisis and generate a new model for a collaborative and nimble research infrastructure that will lead to more rapid translation of our work for the betterment of public health.


NAIST COVID: Multilingual COVID-19 Twitter and Weibo Dataset

Abstract: Since the outbreak of coronavirus disease 2019 (COVID-19) in the late 2019, it has affected over 200 countries and billions of people worldwide. This has affected the social life of people owing to enforcements, such as"social distancing"and"stay at home."This has resulted in an increasing interaction through social media. Given that social media can bring us valuable information about COVID-19 at a global scale, it is important to share the data and encourage social media studies against COVID-19 or other infectious diseases. Therefore, we have released a multilingual dataset of social media posts related to COVID-19, consisting of microblogs in English and Japanese from Twitter and those in Chinese from Weibo. The data cover microblogs from January 20, 2020, to March 24, 2020. This paper also provides a quantitative as well as qualitative analysis of these datasets by creating daily word clouds as an example of text-mining analysis. The dataset is now available on Github. This dataset can be analyzed in a multitude of ways and is expected to help in efficient communication of precautions related to COVID-19.


Rapid Structure-Based Screening Informs Potential Agents for Coronavirus Disease (COVID-19) Outbreak

Abstract: Coronavirus Disease 2019 (COVID-19), caused by the novel coronavirus, has spread rapidly across China. Consequently, there is an urgent need to sort and develop novel agents for the prevention and treatment of viral infections. A rapid structure-based virtual screening is used for the evaluation of current commercial drugs, with structures of human angiotensin converting enzyme II (ACE2), and viral main protease, spike, envelope, membrane and nucleocapsid proteins. Our results reveal that the reported drugs Arbidol, Chloroquine and Remdesivir may hinder the entry and release of virions through the bindings with ACE2, spike and envelope proteins. Due to the similar binding patterns, NHC (β-d-N4-hydroxycytidine) and Triazavirin are also in prospects for clinical use. Main protease (3CLpro) is likely to be a feasible target of drug design. The screening results to target 3CL-pro reveal that Mitoguazone, Metformin, Biguanide Hydrochloride, Gallic acid, Caffeic acid, Sulfaguanidine and Acetylcysteine seem be possible inhibitors and have potential application in the clinical therapy of COVID-19.


Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2.

Abstract: The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.


Covid-19, Coronavirus, SARS-CoV-2 and the small bowel.

Abstract: Although SARS-CoV-2 may primarily enter the cells of the lungs, the small bowel may also be an important entry or interaction site, as the enterocytes are rich in angiotensin converting enzyme (ACE)-2 receptors. The initial gastrointestinal symptoms that appear early during the course of Covid-19 support this hypothesis. Furthermore, SARS-CoV virions are preferentially released apically and not at the basement of the airway cells. Thus, in the setting of a productive infection of conducting airway epithelia, the apically released SARS-CoV may be removed by mucociliary clearance and gain access to the GI tract via a luminal exposure. In addition, post-mortem studies of mice infected by SARS-CoV have demonstrated diffuse damage to the GI tract, with the small bowel showing signs of enterocyte desquamation, edema, small vessel dilation and lymphocyte infiltration, as well as mesenteric nodes with severe hemorrhage and necrosis. Finally, the small bowel is rich in furin, a serine protease which can separate the S-spike of the coronavirus into two "pinchers" (S1 and 2). The separation of the S-spike into S1 and S2 is essential for the attachment of the virion to both the ACE receptor and the cell membrane. In this special review, we describe the interaction of SARS-CoV-2 with the cell and enterocyte and its potential clinical implications.


Structural basis of a shared antibody response to SARS-CoV-2.

Abstract: Molecular understanding of neutralizing antibody responses to SARS-CoV-2 could accelerate vaccine design and drug discovery. We analyzed 294 anti-SARS-CoV-2 antibodies and found that IGHV3-53 is the most frequently used IGHV gene for targeting the receptor-binding domain (RBD) of the spike protein. Co-crystal structures of two IGHV3-53 neutralizing antibodies with RBD, with or without Fab CR3022, at 2.33 to 3.20 Å resolution revealed that the germline-encoded residues dominate recognition of the ACE2 binding site. This binding mode limits the IGHV3-53 antibodies to short CDR H3 loops, but accommodates light-chain diversity. These IGHV3-53 antibodies show minimal affinity maturation and high potency, which is promising for vaccine design. Knowledge of these structural motifs and binding mode should facilitate design of antigens that elicit this type of neutralizing response.


Considerations around the SARS-CoV-2 Spike Protein with particular attention to COVID-19 brain infection and neurological symptoms.

Abstract: Spike protein (S protein) is the virus 'key' to infect cells being able to strongly bind to the human angiotensin-converting enzyme2 (ACE2), as it has been reported. In fact, Spike structure and function is known to be highly important for cell infection as well as entering the brain. Growing evidence indicates that different types of coronaviruses not only affect the respiratory system, but they might also invade the central nervous system (CNS). However, very few evidence have been so far reported on the presence of COVID-19 in the brain and the potential exploitation, by this virus, of lung to brain axis to reach neurons has not completely understood. In this article we assessed the SARS-CoV and SARS-CoV-2 Spike protein sequence, structure and electrostatic potential using computational approaches. Our results showed that the S proteins of SARS-CoV-2 and SARS-CoV are highly similar, sharing a sequence identity of 77%. In addition, we found that the SARS-CoV-2 S protein is slightly more positively charged than that of SARS-CoV since it contains four more positively charged residues and five less negatively charged residues which may lead to an increased affinity to bind to negatively charged regions of other molecules through non-specific and specific interactions. Analyzing of the S protein binds to the host ACE2 receptor showed a 30% higher binding energy for SARS-CoV-2 than the SARS-CoV S protein. These results might be useful for understanding the mechanism of cell entry, blood brain barrier crossing and clinical features related to the CNS infection by SARS-CoV-2.


Molecular simulation of SARS-CoV-2 spike protein binding to pangolin ACE2 or human ACE2 natural variants reveals altered susceptibility to infection.

Abstract: We constructed complex models of SARS-CoV-2 spike protein binding to pangolin or human ACE2, the receptor for virus transmission, and estimated the binding free energy changes using molecular dynamics simulation. SARS-CoV-2 can bind to both pangolin and human ACE2, but has a significantly lower binding affinity for pangolin ACE2 due to the increased binding free energy (9.5 kcal mol-1). Human ACE2 is among the most polymorphous genes, for which we identified 317 missense single-nucleotide variations (SNVs) from the dbSNP database. Three SNVs, E329G (rs143936283), M82I (rs267606406) and K26R (rs4646116), had a significant reduction in binding free energy, which indicated higher binding affinity than wild-type ACE2 and greater susceptibility to SARS-CoV-2 infection for people with them. Three other SNVs, D355N (rs961360700), E37K (rs146676783) and I21T (rs1244687367), had a significant increase in binding free energy, which indicated lower binding affinity and reduced susceptibility to SARS-CoV-2 infection.


Binding of the SARS-CoV-2 Spike Protein to Glycans

Abstract: The pandemic of SARS-CoV-2 has caused a high number of deaths in the world. To combat it, it is necessary to develop a better understanding of how the virus infects host cells. Infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate (HS) and sialic acid-containing oligosaccharides. In this study, we examined and compared the binding of the subunits and spike (S) proteins of SARS-CoV-2 and SARS-CoV, MERS-CoV to these glycans. Our results revealed that the S proteins and subunits can bind to HS in a sulfation-dependent manner, the length of HS is not a critical factor for the binding, and no binding with sialic acid residues was detected. Overall, this work suggests that HS binding may be a general mechanism for the attachment of these coronaviruses to host cells, and supports the potential importance of HS in infection and in the development of antiviral agents against these viruses.


A graph-based approach identifies dynamic H-bond communication networks in spike protein S of SARS-CoV-2

Abstract: Corona virus spike protein S is a large homo-trimeric protein embedded in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, has a relatively large H-bond cluster at the receptor binding domain, and a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.


Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization

Abstract: SARS-CoV-2, the Covid-19 causative virus, adheres to human cells through binding of its envelope Spike protein to the receptor ACE2. The Spike receptor-binding domain (S-RBD) mediates this key event and thus is a primary target for therapeutic neutralizing antibodies to mask the ACE2-interacting interface. Here, we generated 99 synthetic nanobodies (sybodies) using ribosome and phage display. The best sybody MR3 binds the RBD with KD of 1.0 nM and neutralizes SARS-CoV-2 pseudovirus with IC50 of 0.40 μg mL-1. Crystal structures of two sybody-RBD complexes reveal a common neutralizing mechanism through which the RBD-ACE2 interaction is competitively inhibited by sybodies. The structures allowed the rational design of a mutant with higher affinity and improved neutralization efficiency by ∼24-folds, lowering the IC50 from 12.32 to 0.50 μg mL-1. Further, the structures explain the selectivity of sybodies between SARS-CoV strains. Our work presents an alternative approach to generate neutralizers against newly emerged viruses. One sentence summary Structural and biochemical studies revealed the molecular basis for the neutralization mechanism of in vitro-selected and rationally designed nanobody neutralizers for SARS-CoV-2 pseudovirus.


Computational methods to develop potential neutralizing antibody Fab region against SARS-CoV-2 as therapeutic and diagnostic tool

Abstract: SARS-CoV-2, a global pandemic originated from Wuhan city of China in the month of December 2019. There is an urgency to identify potential antibodies to neutralize the virus and also as a diagnostic tool candidate. At present palliative treatments using existing antiviral drugs are under trails to treat SARS-CoV-2.Whole Genome sequence of Wuhan market sample of SARS-CoV-2 was obtained from NCBI Gene ID MN908947.3.Spike protein sequence PDB ID 6VSB obtained from RCSB database. Spike protein sequence had shown top V gene match with IGLV1-44*01, IGLV1-47*02 and has VL type chain. Whole Genome sequence had shown top V gene match with IGHV1-38-4*01 and has VH type chain. VD chain had shown link to allele HLA-A0206 80%, HLA-A0217 80%, HLA-A2301 75%, HLA-A0203 75%, HLA-A0202 70% and HLA-A0201 55% of binding levels. Some conserved regions of spike protein had shown strong binding affinity with HLA-A-0*201, HLA-A24, HLA-B-5701 and HLA-B-5703 alpha chains. Synthetic Fab construct BCR type antibody IgG (CR5840) had shown Polyspecific binding activity with spike glycoprotein when compared with available Anti-SARS antibody CR3022.Thus we propose CR5840 Fab constructed antibody as potential neutralizing antibody for SARS-CoV-2. Based on germline analysis we also propose cytotoxic T lymphocyte epitope peptide selective system as effective tool for the development of SARS-CoV-2 vaccine.


Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

Abstract: The recent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid international spread pose a global health emergency. The trimeric spike (S) glycoprotein interacts with its receptor human ACE2 to mediate viral entry into host-cells. Here we present cryo-EM structures of an uncharacterized tightly closed SARS-CoV-2 S-trimer and the ACE2-bound-S-trimer at 2.7-Å and 3.8-Å-resolution, respectively. The tightly closed S-trimer with inactivated fusion peptide may represent the ground prefusion state. ACE2 binding to the up receptor-binding domain (RBD) within S-trimer triggers continuous swing-motions of ACE2-RBD, resulting in conformational dynamics of S1 subunits. Noteworthy, SARS-CoV-2 S-trimer appears much more sensitive to ACE2-receptor than SARS-CoV S-trimer in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and residue Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2, and provided structural basis of the spike D614G-mutation induced enhanced infectivity. Our findings offer a thorough picture on the mechanism of ACE2-induced conformational transitions of S-trimer from ground prefusion state towards postfusion state, thereby providing important information for development of vaccines and therapeutics aimed to block receptor binding.


Extracellular vesicles containing ACE2 efficiently prevent infection by SARS-CoV-2 Spike protein-containing virus

Abstract: SARS-CoV-2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by TMPRRS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2-EVs are efficient decoys for SARS-CoV-2 S protein-containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is 500 to 1500 times more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2.


A potent neutralizing human antibody reveals the N-terminal domain of the Spike protein of SARS-CoV-2 as a site of vulnerability

Abstract: The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global public health threat. Most research on therapeutics against SARS-CoV-2 focused on the receptor binding domain (RBD) of the Spike (S) protein, whereas the vulnerable epitopes and functional mechanism of non-RBD regions are poorly understood. Here we isolated and characterized monoclonal antibodies (mAbs) derived from convalescent COVID-19 patients. An mAb targeting the N-terminal domain (NTD) of the SARS-CoV-2 S protein, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2, although it does not block the interaction between angiotensin-converting enzyme 2 (ACE2) receptor and S protein. The cryo-EM structure of the SARS-CoV-2 S protein in complex with 4A8 has been determined to an overall resolution of 3.1 Angstrom and local resolution of 3.4 Angstrom for the 4A8-NTD interface, revealing detailed interactions between the NTD and 4A8. Our functional and structural characterizations discover a new vulnerable epitope of the S protein and identify promising neutralizing mAbs as potential clinical therapy for COVID-19.


In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Abstract: Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).


Understanding the B and T cells epitopes of spike protein of severe respiratory syndrome coronavirus-2: A computational way to predict the immunogens

Abstract: The 2019 novel severe respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak has caused a large number of deaths with thousands of confirmed cases worldwide. The present study followed computational approaches to identify B- and T-cell epitopes for spike glycoprotein of SARS-CoV-2 by its interactions with the human leukocyte antigen alleles. We identified twenty-four peptide stretches on the SARS-CoV-2 spike protein that are well conserved among the reported strains. The S protein structure further validated the presence of predicted peptides on the surface. Out of which twenty are surface exposed and predicted to have reasonable epitope binding efficiency. The work could be useful for understanding the immunodominant regions in the surface protein of SARS-CoV-2 and could potentially help in designing some peptide-based diagnostics.


De novo design of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike protein

Abstract: The emergence of SARS-CoV-2 is responsible for the pandemic of respiratory disease known as COVID-19, which emerged in the city of Wuhan, Hubei province, China in late 2019. Both vaccines and targeted therapeutics for treatment of this disease are currently lacking. Viral entry requires binding of the viral spike receptor binding domain (RBD) with the human angiotensin converting enzyme (hACE2). In an earlier paper1, we report on the specific residue interactions underpinning this event. Here we report on the de novo computational design of high affinity antibody variable regions through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike protein using the software tool OptMAVEn-2.02. Subsequently, we carry out computational affinity maturation of the designed prototype variable regions through point mutations for improved binding with the target epitope. Immunogenicity was restricted by preferring designs that match sequences from a 9-mer library of “human antibodies” based on H-score (human string content, HSC)3. We generated 106 different designs and report in detail on the top five that trade-off the greatest affinity for the spike RBD epitope (quantified using the Rosetta binding energies) with low H-scores. By grafting the designed Heavy (VH) and Light (VL) chain variable regions onto a human framework (Fc), high-affinity and potentially neutralizing full-length monoclonal antibodies (mAb) can be constructed. Having a potent antibody that can recognize the viral spike protein with high affinity would be enabling for both the design of sensitive SARS-CoV-2 detection devices and for their deployment as therapeutic antibodies.


Unfractionated heparin potently inhibits the binding of SARS-CoV-2 spike protein to a human cell line

Abstract: The SARS-CoV-2 spike protein is known to bind to the receptor, ACE2, on the surface of target cells. The spike protein is processed by membrane proteases, including TMPRSS2, and is either internalised or fuses directly with the cell, leading to infection. We identified a human cell line that expresses both ACE2 and TMPRSS2, the RT4 urinary bladder transitional carcinoma, and used it to develop a proxy assay for viral interactions with host cells. A tagged recombinant form of the spike protein, containing both the S1 and S2 domains, binds strongly to RT4 cells as determined by flow cytometry. Binding is temperature dependent and increases sharply at 37°C, suggesting that processing of the spike protein is likely to be important in the interaction. As the spike protein has previously been shown to bind heparin, a soluble glycosaminoglycan, we used a flow cytometry assay to determine the effect of heparin on spike protein binding to RT4 cells. Unfractionated heparin inhibited spike protein binding with an IC50 value of <0.05U/ml whereas two low molecular weight heparins were much less effective. This suggests that heparin, particularly unfractionated forms, could be considered to reduce clinical manifestations of COVID-19 by inhibiting continuing viral infection. Despite the sensitivity to heparin, we found no evidence that host cell glycosaminoglycans such as heparan and chondroitin sulphates play a major role in spike protein attachment.


AI334 and AQ806 antibodies recognize the spike S protein from SARS-CoV-2 by ELISA

Abstract: We tested 10 recombinant antibodies directed against the spike S protein from SARS-CoV-1. Among them, antibodies AI334 and AQ806 detect by ELISA the spike S protein from SARS-CoV-2.


Structures of potent and convergent neutralizing antibodies bound to the SARS-CoV-2 spike unveil a unique epitope responsible for exceptional potency

Abstract: Understanding the mechanism of neutralizing antibodies (NAbs) against SARS-CoV-2 is critical for effective vaccines and therapeutics development. We recently reported an exceptionally potent NAb, BD-368-2, and revealed the existence of VH3-53/VH3-66 convergent NAbs in COVID-19. Here we report the 3.5-Å cryo-EM structure of BD-368-2’s Fabs in complex with a mutation-induced prefusion-state-stabilized spike trimer. Unlike VH3-53/VH3-66 NAbs, BD-368-2 fully blocks ACE2 binding by occupying all three receptor-binding domains (RBDs) simultaneously, regardless of their “up” and “down” positions. BD-368-2 also triggers fusogenic-like structural rearrangements of the spike trimer, which could impede viral entry. Moreover, BD-368-2 completely avoids the common epitope of VH3-53/VH3-66 NAbs, evidenced by multiple crystal structures of their Fabs in tripartite complexes with RBD, suggesting a new way of pairing potent NAbs to prevent neutralization escape. Together, these results rationalize a unique epitope that leads to exceptional neutralization potency, and provide guidance for NAb therapeutics and vaccine designs against SARS-CoV-2.


Structure of the nCoV trimeric spike


Local computational methods to improve the interpretability and analysis of cryo-EM maps

Abstract: Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, ‘over-sharpening’ due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.


Prefusion spike protein stabilization through computational mutagenesis

Abstract: A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has emerged as a human pathogen, causing global pandemic and resulting in over 400,000 deaths worldwide. The surface spike protein of SARS-CoV-2 mediates the process of coronavirus entry into human cells by binding angiotensin-converting enzyme 2 (ACE2). Due to the critical role in viral-host interaction and the exposure of spike protein, it has been a focus of most vaccines’ developments. However, the structural and biochemical studies of the spike protein are challenging because it is thermodynamically metastable1. Here, we develop a new pipeline that automatically identifies mutants that thermodynamically stabilize the spike protein. Our pipeline integrates bioinformatics analysis of conserved residues, motion dynamics from molecular dynamics simulations, and other structural analysis to identify residues that significantly contribute to the thermodynamic stability of the spike protein. We then utilize our previously developed protein design tool, Eris, to predict thermodynamically stabilizing mutations in proteins. We validate the ability of our pipeline to identify protein stabilization mutants through known prefusion spike protein mutants. We finally utilize the pipeline to identify new prefusion spike protein stabilization mutants.


Genetic variants in TMPRSS2 and Structure of SARS-CoV-2 spike glycoprotein and TMPRSS2 complex

Abstract: SARS-CoV-2, a highly transmittable pathogen has infected over 3.8 million people around the globe. The spike glycoprotein of SARS-CoV-2 engages host ACE2 for adhesion, TMPRSS2 for activation and entry. With the aid of whole-exome sequencing, we report a variant rs12329760 in TMPRSS2 gene and its mutant V160M, which might impede viral entry. Furthermore, we identified TMPRSS2 cleavage sites in S2 domain of spike glycoprotein and report the structure of TMPRSS2 in complex with spike glycoprotein. We also report the structures of protease inhibitors in complex with TMPRSS2, which could hamper the interaction with spike protein. These findings advance our understanding on the role of TMPRSS2 and in the development of potential therapeutics.


Scientists have turned the structure of the coronavirus into music

Abstract: You’ve probably seen dozens of images of the novel coronavirus—now responsible for 1 million infections and tens of thousands of deaths Now, scientists have come up with a way for you to hear it: by translating the structure of its famous spike protein into music


SARS-CoV-2 Encodes a PPxY Late Domain Motif that is Known to Enhance Budding and Spread in Enveloped RNA Viruses

Abstract: Currently, the global COVID-19 (Coronavirus Disease-2019) pandemic is affecting the health and/or socioeconomic life of almost each people in the world. Finding vaccines and therapeutics is urgent but without forgetting to elucidate the molecular mechanisms that allow some viruses to become dangerous for humans. Here, analysis of all proteins of SARS-CoV-2 revealed a unique PPxY Late (L) domain motif 25PPAY28 in spike protein inside hot disordered loop predicted subject to phosphorylation and binding. It was demonstrated in enveloped RNA viruses that PPxY motif recruits Nedd4 E3 ubiquitin ligases and ultimately the ESCRT complex to enhance virus budding and release that means a high viral load, hence facilitating new infections. Note that PPxY motif is not present in proteins of SARS-CoV. This suggests that PPxY motif by its role in enhancing the viral load could explain why SARS-CoV-2 is more contagious than SARS-CoV. Of course, after the experimental verifications showing that PPxY motif plays the same role as reported for other enveloped RNA viruses, it could become an interesting target for the development of novel host-oriented antivirals therapeutics for preventing S protein to recruit Nedd4 E3 ubiquitin ligases partners.


Recombinant SARS-CoV-2 spike proteins for sero-surveillance and epitope mapping

Abstract: The newly emergent SARS-CoV-2 coronavirus is closely related to SARS-CoV which emerged in 2002. Studies on coronaviruses in general, and SARS in particular, have identified the virus spike protein (S) as being central to virus tropism, to the generation of a protective antibody response and to the unambiguous detection of past infections. As a result of this centrality SARS-CoV-2 S protein has a role in many aspects of research from vaccines to diagnostic tests. We describe a number of recombinant forms of SARS-CoV-2 S expressed in commonly available expression systems and their preliminary use in diagnostics and epitope mapping. These sources may find use in the current and future analysis of the virus and the Covid-19 disease it causes.


Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike

Abstract: Binding of the spike protein of SARS-CoV-2 to the human angiotensin converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds into a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.


Structural modeling of 2019-novel coronavirus (nCoV) spike protein reveals a proteolytically-sensitive activation loop as a distinguishing feature compared to SARS-CoV and related SARS-like coronaviruses.

Abstract: The 2019 novel coronavirus (2019-nCoV) is currently causing a widespread outbreak centered on Hubei province, China and is a major public health concern. Taxonomically 2019-nCoV is closely related to SARS-CoV and SARS-related bat coronaviruses, and it appears to share a common receptor with SARS-CoV (ACE-2). Here, we perform structural modeling of the 2019-nCoV spike glycoprotein. Our data provide support for the similar receptor utilization between 2019-nCoV and SARS-CoV, despite a relatively low amino acid similarity in the receptor binding module. Compared to SARS-CoV, we identify an extended structural loop containing basic amino acids at the interface of the receptor binding (S1) and fusion (S2) domains, which we predict to be proteolytically-sensitive. We suggest this loop confers fusion activation and entry properties more in line with MERS-CoV and other coronaviruses, and that the presence of this structural loop in 2019-nCoV may affect virus stability and transmission.


Receptor-binding domain of SARS-Cov spike protein: soluble expression in E. coli, purification and functional characterization.

Abstract: AIM Spike protein of coronavirus is responsible for virus binding, fusion and entry, and is a major inducer of neutralizing antibodies. This paper was to find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability. METHODS Three fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay. RESULTS RBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST/RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST/RBD binding to Vero E6 cells were 77.08 and 352.73 respectively. CONCLUSION In this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.


In silico design of antiviral peptides targeting the spike protein of SARS-CoV-2

Abstract: An outbreak caused by 2019 novel coronavirus (2019-nCoV) was first identified in Wuhan City, Hubei Province, China. The new virus was later named SARS-CoV-2. The virus has affected tens of thousands of patients in the world. The infection of SARS-CoV-2 causes severe pneumonia and even death. It is urgently needed to find a therapeutic method to treat patients with SARS-CoV-2 infection. Studies showed that the surface spike (S) protein is essential for the coronavirus binding and entry of host cells. The heptad repeats 1 and 2 (HR1 and HR2) in the S protein play a decisive role in the fusion of the viral membrane with the host cell membrane. We predicted the HR1 and HR2 regions in S protein by sequence alignment. We simulated a computational model of HR1/2 regions and the fusion core. The binding energy of HR1 and HR2 of the fusion core was -33.4 kcal/mol. We then designed antivirus peptides by molecular dynamics simulation of the fusion core. The binding energy of HR2-based antiviral peptide to HR1 was -43.0 kcal/mol, which was stronger than the natural stage of the fusion core, suggesting that the predicted antiviral peptide can competitively bind with HR1 to prevent forming of the fusion core. The antiviral peptides can prevent SARS-CoV-2 membrane fusion and can potentially be used for the prevention and treatment of infections.


Is SARS-CoV-2 originated from laboratory? A rebuttal to the claim of formation via laboratory recombination


A spike with which to beat COVID-19?


Role of the GTNGTKR motif in the N-terminal receptor-binding domain of the SARS-CoV-2 spike protein

Abstract: The 2019 novel coronavirus disease (COVID-19) that emerged in China has been declared as public health emergency of international concern by the World Health Organization and the causative pathogen was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this report, we analyzed the structural characteristics of the N-terminal domain of the S1 subunit (S1-NTD) of the SARS-CoV-2 spike protein in comparison to the SARS-CoV in particular, and to other viruses presenting similar characteristic in general. Given the severity and the wide and rapid spread of the SARS-CoV-2 infection, it is very likely that the virus recognizes other receptors/co-receptors besides the ACE2. The NTD of the SARS-CoV-2 contains a receptor-binding motif different from that of SARS-CoV, with some insertions that could confer to the new coronavirus new receptor binding abilities. In particular, motifs similar to the insertion 72GTNGTKR78 have been found in structural proteins of other viruses; and these motifs were located in putative regions involved in recognizing protein and sugar receptors, suggesting therefore that similar binding abilities could be displayed by the SARS-CoV-2 S1-NTD. Moreover, concerning the origin of these NTD insertions, our findings point towards an evolutionary acquisition rather than the hypothesis of an engineered virus.


Targeting the SARS-CoV-2 spike glycoprotein prefusion conformation: virtual screening and molecular dynamics simulations applied to the identification of potential fusion inhibitors

Abstract: The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a renewed interest in studying the role of the spike S glycoprotein in regulating coronavirus infections in the natural host. Taking advantage of the cryo-electron microscopy structure of SARS-CoV-2 S trimer in the prefusion conformation, we performed a virtual screening simulation with the aim to identify novel molecules that could be used as fusion inhibitors. The spike glycoprotein structure has been completed using modeling techniques and its inner cavity, needful for the postfusion transition of the trimer, has been scanned for the identification of strongly interacting available drugs. Finally, the stability of the protein-drug top complexes has been tested using classical molecular dynamics simulations. The free energy of interaction of the molecules to the spike protein has been evaluated through the MM/GBSA method and per-residue decomposition analysis. Results have been critically discussed considering previous scientific knowledge concerning the selected compounds and sequence alignments have been carried out to evaluate the spike glycoprotein similarity among the betacoronavirus family members. Finally, a cocktail of drugs that may be used as SARS-CoV-2 fusion inhibitors has been suggested.


Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses

Abstract: Over the past 20 years, several coronaviruses have crossed the species barrier into humans, causing outbreaks of severe, and often fatal, respiratory illness. Since SARS-CoV was first identified in animal markets, global viromics projects have discovered thousands of coronavirus sequences in diverse animals and geographic regions. Unfortunately, there are few tools available to functionally test these viruses for their ability to infect humans, which has severely hampered efforts to predict the next zoonotic viral outbreak. Here, we developed an approach to rapidly screen lineage B betacoronaviruses, such as SARS-CoV and the recent SARS-CoV-2, for receptor usage and their ability to infect cell types from different species. We show that host protease processing during viral entry is a significant barrier for several lineage B viruses and that bypassing this barrier allows several lineage B viruses to enter human cells through an unknown receptor. We also demonstrate how different lineage B viruses can recombine to gain entry into human cells, and confirm that human ACE2 is the receptor for the recently emerging SARS-CoV-2.


Understanding the B and T cell epitopes of spike protein of severe acute respiratory syndrome coronavirus-2: A computational way to predict the immunogens

Abstract: The 2019 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak has caused a large number of deaths, with thousands of confirmed cases worldwide. The present study followed computational approaches to identify B- and T-cell epitopes for the spike (S) glycoprotein of SARS-CoV-2 by its interactions with the human leukocyte antigen alleles. We identified 24 peptide stretches on the SARS-CoV-2 S protein that are well conserved among the reported strains. The S protein structure further validated the presence of predicted peptides on the surface, of which 20 are surface exposed and predicted to have reasonable epitope binding efficiency. The work could be useful for understanding the immunodominant regions in the surface protein of SARS-CoV-2 and could potentially help in designing some peptide-based diagnostics. Also, identified T-cell epitopes might be considered for incorporation in vaccine designs.


Developing a Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein Model in a Viral Membrane

Abstract: This technical study describes all-atom modeling and simulation of a fully glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB: 6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19) so that researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.


Developing a Fully-glycosylated Full-length SARS-CoV-2 Spike Protein Model in a Viral Membrane

Abstract: This technical study describes all-atom modeling and simulation of a fully-glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB:6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently-determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully-glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19), so researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.


Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Abstract: Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome-coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resolution of 2.9 angstroms, with a local resolution of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the molecular basis for coronavirus recognition and infection.


Identification of 22 N-glycosites on spike glycoprotein of SARS-CoV-2 and accessible surface glycopeptide motifs: implications for vaccination and antibody therapeutics

Abstract: Coronaviruses hijack human enzymes to assemble the sugar coat on their spike glycoproteins. The mechanisms by which human antibodies may recognize the antigenic viral peptide epitopes hidden by the sugar coat are unknown. Glycosylation by insect cells differs from the native form produced in human cells, but insect cell-derived influenza vaccines have been approved by the US Food and Drug Administration. In this study, we analyzed recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein secreted from BTI-Tn-5B1-4 insect cells, by trypsin and chymotrypsin digestion followed by mass spectrometry analysis. We acquired tandem mass spectrometry (MS/MS) spectrums for glycopeptides of all 22 predicted N-glycosylated sites. We further analyzed the surface accessibility of spike proteins according to cryogenic electron microscopy and homolog-modeled structures, and available antibodies that bind to SARS-CoV-1. All 22 N-glycosylated sites of SARS-CoV-2 are modified by high-mannose N-glycans. MS/MS fragmentation clearly established the glycopeptide identities. Electron densities of glycans cover most of the spike receptor-binding domain of SARS-CoV-2, except YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, similar to a region FSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQ in SARS-CoV-1. Other surface-exposed domains include those located on central helix, connecting region, heptad repeats, and N-terminal domain. Because the majority of antibody paratopes bind to the peptide portion with or without sugar modification, we propose a snake-catching model for predicted paratopes: a minimal length of peptide is first clamped by a paratope, and sugar modifications close to the peptide either strengthen or do not hinder the binding.


Antibody signature induced by SARS-CoV-2 spike protein immunogens in rabbits

Abstract: Multiple vaccine candidates against SARS-CoV-2 based on viral spike protein are under development. However, there is limited information on the quality of antibody responses generated with these vaccine modalities. To better understand antibody responses induced by spike protein-based vaccines, we performed a qualitative study by immunizing rabbits with various SARS-CoV-2 spike protein antigens: S ectodomain (S1+S2; amino acids 16 to 1213), which lacks the cytoplasmic and transmembrane domains (CT-TM), the S1 domain (amino acids 16 to 685), the receptor binding domain (RBD) (amino acids 319 to 541), and the S2 domain (amino acids 686 to 1213, lacking the RBD, as control). Resulting antibody quality and function were analyzed by enzyme-linked immunosorbent assay (ELISA), RBD competition assay, surface plasmon resonance (SPR) against different spike proteins in native conformation, and neutralization assays. All three antigens (S1+S2 ectodomain, S1 domain, and RBD), but not S2, generated strong neutralizing antibodies against SARS-CoV-2. Vaccination-induced antibody repertoire was analyzed by SARS-CoV-2 spike genome fragment phage display libraries (SARS-CoV-2 GFPDL), which identified immunodominant epitopes in the S1, S1-RBD, and S2 domains. Furthermore, these analyses demonstrated that the RBD immunogen elicited a higher antibody titer with five-fold higher affinity antibodies to native spike antigens compared with other spike antigens, and antibody affinity correlated strongly with neutralization titers. These findings may help guide rational vaccine design and facilitate development and evaluation of effective therapeutics and vaccines against COVID-19 disease.


Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Abstract: The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.


Structural and simulation analysis of hotspot residues interactions of SARS-CoV 2 with human ACE2 receptor

Abstract: The novel corona virus disease 2019 (SARS-CoV 2) pandemic outbreak was alarming. The binding of SARS-CoV (CoV) spike protein (S-Protein) Receptor Binding Domain (RBD) to Angiotensin converting enzyme 2 (ACE2) receptor initiates the entry of corona virus into the host cells leading to the infection. However, considering the mutations reported in the SARS-CoV 2 (nCoV), the structural changes and the binding interactions of the S-protein RBD of nCoV were not clear. The present study was designed to elucidate the structural changes, hot spot binding residues and their interactions between the nCoV S-protein RBD and ACE2 receptor through computational approaches. Based on the sequence alignment, a total of 58 residues were found mutated in nCoV S-protein RBD. These mutations led to the structural changes in the nCoV S-protein RBD 3d structure with 4 helices, 10 sheets and intermittent loops. The nCoV RBD was found binding to ACE2 receptor with 11 hydrogen bonds and 1 salt bridge. The major hot spot amino acids involved in the binding identified by interaction analysis after simulations includes Glu 35, Tyr 83, Asp 38, Lys 31, Glu 37, His 34 amino acid residues of ACE2 receptor and Gln 493, Gln 498, Asn 487, Tyr 505 and Lys 417 residues in nCoV S-protein RBD. Based on the hydrogen bonding, RMSD and RMSF, total and potential energies, the nCoV was found binding to ACE2 receptor with higher stability and rigidity. Concluding, the hotspots information will be useful in designing blockers for the nCoV spike protein RBD. [Formula: see text]Communicated by Ramaswamy H. Sarma.


Mining of epitopes on spike protein of SARS-CoV-2 from COVID-19 patients


Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients' B Cells

Abstract: The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.


Is the Rigidity of SARS-CoV-2 Spike Receptor-Binding Motif the Hallmark for Its Enhanced Infectivity? Insights from All-Atom Simulations

Abstract: The severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic is setting the global health crisis of our time, causing a devastating societal and economic burden. An idiosyncratic trait of coronaviruses is the presence of spike glycoproteins on the viral envelope, which mediate the virus binding to specific host receptor, enabling its entry into the human cells. In spite of the high sequence identity of SARS-CoV-2 with its closely related SARS-CoV emerged in 2002, the atomic-level determinants underlining the molecular recognition of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor and, thus, the rapid virus spread into human body, remain unresolved. Here, multi-microsecond-long molecular dynamics simulations enabled us to unprecedentedly dissect the key molecular traits liable of the higher affinity/specificity of SARS-CoV-2 toward ACE2 as compared to SARS-CoV. This supplies a minute per-residue contact map underlining its stunningly high infectivity. Harnessing this knowledge is pivotal for urgently developing effective medical countermeasures to face the ongoing global health crisis.


Comparing the Binding Interactions in the Receptor Binding Domains of SARS-CoV-2 and SARS-CoV

Abstract: SARS-CoV-2, since emerging in Wuhan, China, has been a major concern because of its high infection rate and has left more than six million infected people around the world. Many studies endeavored to reveal the structure of the SARS-CoV-2 compared to the SARS-CoV, in order to find solutions to suppress this high infection rate. Some of these studies showed that the mutations in the SARS-CoV spike (S) protein might be responsible for its higher affinity to the ACE2 human cell receptor. In this work, we used molecular dynamics simulations and Monte Carlo sampling to compare the binding affinities of the S proteins of SARS-CoV and SARS-CoV-2 to the ACE2. Our results show that the protein surface of the ACE2 at the receptor binding domain (RBD) exhibits negative electrostatic potential, while a positive potential is observed for the S proteins of SARS-CoV/SARS-CoV-2. In addition, the binding energies at the interface are slightly higher for SARS-CoV-2 because of enhanced electrostatic interactions. The major contributions to the electrostatic binding energies result from the salt bridges forming between R426 and ACE-2-E329 in the case of SARS-CoV and K417 and ACE2-D30 in the SARS-CoV-2. In addition, our results indicate that the enhancement in the binding energy is not due to a single mutant but rather because of the sophisticated structural changes induced by all these mutations together. This finding suggests that it is implausible for the SARS-CoV-2 to be a lab-engineered virus.


The MERS-CoV Receptor DPP4 as a Candidate Binding Target of the SARS-CoV-2 Spike

Abstract: The ongoing outbreak of the novel coronavirus pneumonia COVID-19 has caused great number of cases and deaths, but our understanding about the pathogen SARS-CoV-2 remains largely unclear. The attachment of the virus with the cell-surface receptor and a cofactor is the first step for the infection. Here, bioinformatics approaches combining human-virus protein interaction prediction and protein docking based on crystal structures have revealed the high affinity between human dipeptidylpeptidase 4 (DPP4) and the spike (S) receptor-binding domain of SARS-CoV-2. Intriguingly, the crucial binding residues of DPP4 are identical to those that are bound to the MERS-CoV-S. Moreover, E484 insertion and adjacent substitutions should be most essential for this DPP4-binding ability acquirement of SARS-CoV-2-S compared with SARS-CoV-S. This potential utilization of DPP4 as a binding target for SARS-CoV-2 may offer novel insight into the viral pathogenesis and help the surveillance and therapeutics strategy for meeting the challenge of COVID-19.


Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody

Abstract: The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 11,900 laboratory-confirmed human infections, including 259 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. These results suggest that CR3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a critical impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.


Design of engineered surfaces for prospective detection of Sars-CoV-2 using quartz crystal microbalance based techniques

Abstract: INTRODUCTION: Rapid transmission of the severe acute respiratory syndrome coronavirus 2 has affected the whole world and forced it to a halt (lockdown). A fast and label free detection method for the novel coronavirus needs to be developed along with the existing enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) based methods. AREAS COVERED: In this report, biophysical aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein are outlined based on its recent reported electron microscopy structure. Protein binding sites are analyzed theoretically, which consisted of hydrophobic and positive charged amino acid residues. Different strategies to form mixed self-assembled monolayers (SAMs) of hydrophobic (CH3) and negatively charged (COOH) groups are discussed to be used for the specific and strong interactions with spike protein. Bio-interfacial interactions between the spike protein and device (sensor) surface and its implications towards designing suitable engineered surfaces are summarized. EXPERT OPINION: Implementation of the engineered surfaces in quartz crystal microbalance (QCM) based detection techniques for the diagnosis of the novel coronavirus from oral swab samples is highlighted. The proposed strategy can be explored for the label free and real time detection with sensitivity up to ng level. These engineered surfaces can be reused after desorption.


Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) in Henan Province, China: Discovery of a New Bunyavirus

Abstract: Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (>99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS.


Main Routes of Entry and Genomic Diversity of SARS-CoV-2, Uganda.

Abstract: We established rapid local viral sequencing to document the genomic diversity of severe acute respiratory syndrome coronavirus 2 entering Uganda. Virus lineages closely followed the travel origins of infected persons. Our sequence data provide an important baseline for tracking any further transmission of the virus throughout the country and region.


About the origin of the first two Sars-CoV-2 infections in Italy: inference not supported by appropriate sequence analysis.

Abstract: In the 5th February 2020 issue of Journal of Medical Virology a paper was published by Giovannetti et al., entitled "The first two cases of 2019-nCoV in Italy: where they come from?"1 . In this paper a phylogenetic and evolutionary analysis was applied to the virus identified in the first two subjects diagnosed in Italy with 2019-nCoV infection, recently renamed SARS-CoV-22 , two Chinese spouses arrived in Italy for tourism. The diagnosis was performed by the virology team under direction of Maria R. Capobianchi, at the National Institute of Infectious Diseases (INMI) in Rome, Italy, where the patients are currently hospitalized. This article is protected by copyright. All rights reserved.


SARS-CoV-2 Phylogenetic Analysis, Lazio Region, Italy, February-March 2020.

Abstract: We report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus 2 virus strains from the Lazio region of Italy and provide information about the dynamics of virus spread. Data suggest effective containment of clade V strains, but subsequently, multiple waves of clade G strains were circulating widely in Europe.


Detection of coronaviruses in Pteropus & Rousettus species of bats from different States of India.

Abstract: Background & objectives : Bats are considered to be the natural reservoir for many viruses, of which some are potential human pathogens. In India, an association of Pteropus medius bats with the Nipah virus was reported in the past. It is suspected that the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also has its association with bats. To assess the presence of CoVs in bats, we performed identification and characterization of bat CoV (BtCoV) in P. medius and Rousettus species from representative States in India, collected during 2018 and 2019. Methods : Representative rectal swab (RS) and throat swab specimens of Pteropus and Rousettus spp. bats were screened for CoVs using a pan-CoV reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. A single-step RT-PCR was performed on the RNA extracted from the bat specimens. Next-generation sequencing (NGS) was performed on a few representative bat specimens that were tested positive. Phylogenetic analysis was carried out on the partial sequences of RdRp gene sequences retrieved from both the bat species and complete viral genomes recovered from Rousettus spp. Results : Bat samples from the seven States were screened, and the RS specimens of eight Rousettus spp. and 21 Pteropus spp. were found positive for CoV RdRp gene. Among these, by Sanger sequencing, partial RdRp sequences could be retrieved from three Rousettus and eight Pteropus bat specimens. Phylogenetic analysis of the partial RdRp region demonstrated distinct subclustering of the BtCoV sequences retrieved from these Rousettus and Pteropus spp. bats. NGS led to the recovery of four sequences covering approximately 94.3 per cent of the whole genome of the BtCoVs from Rousettus bats. Three BtCoV sequences had 93.69 per cent identity to CoV BtRt-BetaCoV/GX2018. The fourth BtCoV sequence was 96.8 per cent identical to BtCoV HKU9-1. Interpretation & conclusions : This study was a step towards understanding the CoV circulation in Indian bats. Detection of potentially pathogenic CoVs in Indian bats stresses the need for enhanced screening for novel viruses in them. One Health approach with collaborative activities by the animal health and human health sectors in these surveillance activities shall be of use to public health. This would help in the development of diagnostic assays for novel viruses with outbreak potential and be useful in disease interventions. Proactive surveillance remains crucial for identifying the emerging novel viruses with epidemic potential and measures for risk mitigation.


Genomic analysis of SARS-CoV-2 strains among Indians returning from Italy, Iran & China, & Italian tourists in India.


Dominant and rare SARS-Cov2 variants responsible for the COVID-19 pandemic in Athens, Greece.

Abstract: SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel Coronavirus responsible for the Coronavirus Disease-2019 (COVID-19) pandemic. Since the beginning of the pandemic, the virus has spread in almost the entire world. Tracing and tracking virus international and local transmission has been an enormous challenge. Chains of infections starting from various countries worldwide seeded the outbreak of COVID-19 in Athens, capital city of Greece. Full-genome analysis of isolates from Athens' Hospitals and other healthcare providers revealed the variety of SARS-CoV-2 that initiated the pandemic before lock-down and passenger flight restrictions. The present work may serve as reference for resolving future lines of infection in the area and Europe especially after resumption of passenger flight connections to Athens and Greece during summer of 2020.


A genome epidemiological study of SARS-CoV-2 introduction into Japan

Abstract: Background: After the first case of COVID-19 in Japan on 15 January 2020, multiple nationwide COVID-19 clusters were identified by the end of February. The Japanese government focused on mitigating emerging COVID-19 clusters by conducting active nationwide epidemiological surveillance. However, an increasing number of cases appeared until early April, many with unclear infection routes exhibiting no recent history of travel outside Japan. We aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from COVID-19 cases until early April and characterise the genealogical networks to demonstrate possible routes of spread in Japan. Methods: Nasopharyngeal specimens were collected from patients and a quantitative reverse transcription polymerase chain reaction testing for SARS-CoV-2 was performed. Positive RNA samples were subjected whole genome sequencing and a haplotype network analysis was performed. Findings: Some of the primary clusters identified during January and February in Japan directly descended from Wuhan-Hu-1-related isolates in China and other distinct clusters. Clusters were almost contained until mid-March; the haplotype network analysis demonstrated that COVID-19 cases from late March through early April may have caused an additional large cluster related to the outbreak in Europe, leading to additional spread within Japan. National self-restraint during February was effective in mitigating the COVID-19 spread, but late action on stopping immigration and declaring national emergency in Japan might be involved in the later increase in cases. Interpretation: Genome surveillance suggested that at least two distinct SARS-CoV-2 introductions from China and other countries occurred. Funding: Japan Agency for Medical Research and Development.


Phylogenomics and phylodynamics of SARS-CoV-2 retrieved genomes from India

Abstract: The ongoing SARS-CoV-2 pandemic is one of the biggest outbreaks after the Spanish flu of 1918. Understanding the epidemiology of viral outbreaks is the first step towards vaccine development programs. This is the first phylodynamics study attempted on of SARS-CoV-2 genomes from India to infer its current evolution in the context of an ongoing pandemic. Out of 286 retrieved SARS-CoV-2 whole genomes from India, 138 haplotypes were generated and analyzed. Median-joining network was built to investigate the relatedness of SARS-CoV-2 haplotypes in India. The BDSIR package of BEAST2 was used to calculate the reproduction number (R0) and the infectious rate of the virus. Past and current population trend was investigated using the stamp date method in coalescent Bayesian skyline plot, implemented in BEAST2 and by exponential growth prior in BEAST 1.10.4. Median-joining network reveals two distinct ancestral clusters A and B showing genetic affinities with Wuhan outbreak sample. The network also illustrates the autochthonous development of isolates in a few instances. High basic reproduction number of SARS-nCoV-2 in India points towards the phase of active community transmission. The Bayesian skyline plot revel exponential rise in the effective population size (Ne) of Indian isolates from the first week of January to the first week of April 2020. More genome sequencing and analyses of the virus will be required in coming days to monitor COVID19 after the upliftment of lock down in India.


Distribution of COVID-19 and phylogenetic tree construction of sars-CoV-2 in Indonesia

Abstract: Coronavirus disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has spread quickly across the world and has been declared a pandemic. Indonesia has many COVID-19 cases, with a high mortality rate. This study aimed to describe the distribution of COVID-19 in Indonesia and constructed the SARS-CoV-2 phylogenetic tree from Indonesian isolates and those from other countries, including other CoVs to determine their relationship. The distribution data of COVID-19 in Indonesia were obtained from the COVID-19 Management Handling Unit and descriptively analyzed. SARS-CoV-2 isolates were retrieved from the GenBank® (National Center of Biotechnology Information, USA) and GISAID EpiCoV™ databases and were used to construct phylogenetic trees using MEGA X software. Of the 37 provinces in Indonesia, five provinces with the highest case fatality rates were DKI Jakarta, Jawa Barat, Jawa Timur, and Banten, and the five provinces with the highest cure rate were Kepulauan Riau, Bali, Aceh, Gorontalo, and DI Yogyakarta. SARS-CoV-2 Indonesian isolates were closely related to SARS-CoV-2 isolates from other countries. The rapid and widespread distribution of SARS-CoV-2 in Indonesia was caused by the lack of compliance with territorial restrictions and dishonesty with medical personnel. These data revealed that mutations can occur during the transmission process, which can be caused by a history of travel and increased patient immunity.


Multiple SARS-CoV-2 introductions shaped the early outbreak in Central Eastern Europe: comparing Hungarian data to a worldwide sequence data-matrix

Abstract: Severe Acute Respiratory Syndrome Coronavirus 2 is the third highly pathogenic human coronavirus in history. Since the emergence in Hubei province, China, during late 2019 the situation evolved to pandemic level. Following China, Europe was the second epicenter of the pandemic. To better comprehend the detailed founder mechanisms of the epidemic evolution in Central-Eastern Europe, particularly in Hungary, we determined the full-length SARS-CoV-2 genomes from 32 clinical samples collected from laboratory confirmed COVID-19 patients over the first month of disease in Hungary. We applied a haplotype network analysis on all available complete genomic sequences of SARS-CoV-2 from GISAID database as of the 21th of April, 2020. We performed additional phylogenetic and phylogeographic analyses to achieve the recognition of multiple and parallel introductory events into our region. Here we present a publicly available network imaging of the worldwide haplotype relations of SARS-CoV-2 sequences and conclude the founder mechanisms of the outbreak in Central-Eastern Europe.


Phylogenetic pattern of SARS-CoV-2 from COVID-19 patients from Bosnia and Herzegovina: lessons learned to optimize future molecular and epidemiological approaches

Abstract: Whole Genome Sequence of four samples from COVID-19 outbreaks was done in two laboratories in Bosnia and Herzegovina (Veterinary Faculty Sarajevo and Alea Genetic Center). All four BiH sequences cluster mainly with European ones (Italy, Austria, France, Sweden, Cyprus, England). The constructed phylogenetic tree indicates probable multiple independent introduction events. The success of future containment measures concernig new introductions will be highly challenging for country due to the significant proportion of BH population living abroad.


Diagnostics and spread of SARS-CoV-2 in Western Africa: An observational laboratory-based study from Benin

Abstract: Information on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread in Africa is limited by fragile 2 surveillance systems and insufficient diagnostic capacity. 3 We assessed the coronavirus disease-19 (COVID-19)-related diagnostic workload in Benin, Western Africa, 4 characterized SARS-CoV-2 genomes from 12 acute cases of COVID-19, used those together with public data to 5 estimate SARS-CoV-2 transmission dynamics in a Bayesian framework, validated a widely used diagnostic dual target 6 RT-PCR kit donated to African countries, and conducted serological analyses in 68 sera from confirmed COVID-19 7 cases and from febrile patients sampled before the predicted SARS-CoV-2 introduction. 8 We found a 15-fold increase in the monthly laboratory workload due to COVID-19. Genomic surveillance showed 9 introductions of three distinct SARS-CoV-2 lineages. SARS-CoV-2 genome-based analyses yielded an R0 estimate of 10 4.4 (95% confidence interval: 2.0-7.7), suggesting intense spread of SARS-CoV-2 in Africa. RT-PCR-based tests 11 were highly sensitive but showed variation of internal controls and between diagnostic targets. Commercially available 12 SARS-CoV-2 ELISAs showed up to 25% false-positive results depending on antigen and antibody types, likely due 13 to unspecific antibody responses elicited by acute malaria according to lack of SARS-CoV-2-specific neutralizing 14 antibody responses and relatively higher parasitemia in those sera. 15 We confirm an overload of the diagnostic capacity in Benin and provide baseline information on the usability of 16 genome-based surveillance in resource-limited settings. Sero-epidemiological studies needed to assess SARS-CoV-2 17 spread may be put at stake by low specificity of tests in tropical settings globally. The increasing diagnostic challenges 18 demand continuous support of national and supranational African stakeholders.


Laboratory based surveillance of SARS-CoV-2 in Pakistan

Abstract: COVID-19 cases are alarmingly increasing in Pakistan since May 2020. Laboratory based surveillance system has been in place since the start of the pandemic. The genomic surveillance of SARS-CoV-2 strains isolated locally has been conducted based on partial ORF1b. The sequences were classified to show the phylogenetic correlation and showed 100% homology with those detected in neighboring countries India and China. The rapid increase in cases has led to development of robust strategies to enhance the laboratory testing capacity. We are currently meeting the country requirement to diagnose the virus in the community. Nonetheless, factors like recent ease in lockdown measures has led to massive rise in number of cases in few weeks time.


CoronaHiT: large scale multiplexing of SARS-CoV-2 genomes using Nanopore sequencing

Abstract: The COVID-19 pandemic has spread to almost every country in the world since it started in China in late 2019. Controlling the pandemic requires a multifaceted approach including whole genome sequencing to support public health interventions at local and national levels. One of the most widely used methods for sequencing is the ARTIC protocol, a tiling PCR approach followed by Oxford Nanopore sequencing (ONT) of up to 24 samples at a time. There is a need for a higher throughput method to reduce cost per genome. Here we present CoronaHiT, a method capable of multiplexing up to 95 small genomes on a single Nanopore flowcell, which uses transposase mediated addition of adapters and PCR based addition of symmetric barcodes. We demonstrate the method using 48 and 94 SARS-CoV-2 genomes per flowcell, amplified using the ARTIC protocol, and compare performance with Illumina and ARTIC ONT sequencing. Results demonstrate that all sequencing methods produce inaccurate genomes when the RNA extract from SARS-CoV-2 positive clinical sample has a cycle threshold (Ct) >= 32. Results from set same set of 23 samples with a broad range of Cts show that the consensus genomes have >90% coverage (GISAID criteria) for 78.2% of samples for CoronaHiT-48, 73.9% for CoronaHiT-94, 78.2% for Illumina and 73.9% for ARTIC ONT, and all have the same clustering on a maximum likelihood tree. In conclusion, we demonstrate that CoronaHiT can multiplex up to 94 SARS-CoV-2 genomes per nanopore flowcell without compromising the quality of the resulting genomes while reducing library preparation complexity and significantly reducing cost. This protocol will aid the rapid expansion of SARS-CoV-2 genome sequencing globally, to help control the pandemic.


Early phylodynamics analysis of the COVID-19 epidemics in France

Abstract: France was one of the first countries to be reached by the COVID-19 pandemics. Here, we analyse 196 SARS-Cov-2 genomes collected between Jan 24 and Mar 24 2020, and perform a phylodynamics analysis. In particular, we analyse the doubling time, reproduction number (Rt) and infection duration associated with the epidemic wave that was detected in incidence data starting from Feb 27. We show that a slowing down of the epidemic spread can be detected in Mar, which is consistent with the implementation of the national lock-down on Mar 17. The inferred distributions for the infection duration and Rt are in line with those estimated from contact tracing data. Overall, this analysis shows the potential to use sequence genomic data to inform public health decisions in an epidemic crisis context.


Introductions and early spread of SARS-CoV-2 in France

Abstract: Following the emergence of coronavirus disease (COVID-19) in Wuhan, China in December 2019, specific COVID-19 surveillance was launched in France on January 10, 2020. Two weeks later, the first three imported cases of COVID-19 into Europe were diagnosed in France. We sequenced 97 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from samples collected between January 24 and March 24, 2020 from infected patients in France. Phylogenetic analysis identified several early independent SARS-CoV-2 introductions without local transmission, highlighting the efficacy of the measures taken to prevent virus spread from symptomatic cases. In parallel, our genomic data reveals the later predominant circulation of a major clade in many French regions, and implies local circulation of the virus in undocumented infections prior to the wave of COVID-19 cases. This study emphasizes the importance of continuous and geographically broad genomic sequencing and calls for further efforts with inclusion of asymptomatic infections.


Comprehensive genome analysis of 6,000 USA SARS-CoV-2 isolates reveals haplotype signatures and localized transmission patterns by state and by country

Abstract: Genomic analysis of SARS-CoV-2 sequences is crucial in determining the effectiveness of prudent safer at home measures in the United States (US). By haplotype analysis of 6,356 US isolates, we identified a pattern of strongly localized outbreaks at the city-, state-, and country-levels, and temporal transmissions. This points to the effectiveness of existing travel restriction policies and public health measures in controlling the transmission of SARS-CoV-2.


Computational search of hybrid human/ SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from other coronavirus strains

Abstract: The role of the RNAi/Dicer/Ago system to degrade RNA viruses has been elusive in mammals, which prompted authors to think that interferon (IFN) synthesis is essential in this clade relegating the RNAi defense strategy against viral infection as accessory function. We explore the theoretical possibilities that RNAi triggered by SARS-CoV-2 might degrade some host transcripts in the opposite direction although this hypothesis seems counter intuitive. SARS-CoV-2 genome was therefore computational searched for exact intra pairing within the viral RNA and also hybrid exact pairing with human transcriptome over a minimum 20 bases length. Minimal segments of 20 bases length of SARS-CoV-2 RNA were found based on the theoretical matching with existing complementary strands in the human host transcriptome. Few human genes potentially annealing with SARS-CoV-2 RNA, among them mitochondrial deubiquitinase USP30, a subunit of ubiquitin protein ligase complex FBXO21 along with two long coding RNAs were retrieved. The hypothesis that viral originated RNAi might mediate degradation of messengers of the host transcriptome was corroborated by clinical observation and phylogenetic comparative analysis indicating a strong specificity of these hybrid pairing sequences for both SARS-CoV-2 and human genomes.


Analyzing hCov genome sequences: Applying Machine Intelligence and beyond

Abstract: Covid-19 pandemic, caused by the sars-cov-2 strain of coronavirus, has affected millions of people all over the world and taken thousands of lives. It is of utmost importance that the character of this deadly virus be studied and its nature be analysed. We present here an analysis pipeline comprising phylogenetic analysis on strains of this novel virus to track its evolutionary history among the countries uncovering several interesting relationships, followed by a classification exercise to identify the virulence of the strains and extraction of important features from its genetic material that are used subsequently to predict mutation at those interesting sites using deep learning techniques. In a nutshell, we have prepared an analysis pipeline for hCov genome sequences leveraging the power of machine intelligence and uncovered what remained apparently shrouded by raw data.


Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses

Abstract: An initial outbreak of coronavirus disease 2019 (COVID-19) in China has resulted in a massive global pandemic causing well over 10,000,000 cases and 500,000 deaths worldwide. The virus responsible, SARS-CoV-2, has been found to possess a very close association with Bat-CoV RaTG13 and Pangolin-CoV MP789. The nucleocapsid protein can serve as a decent model for determining phylogenetic, evolutionary, and structural relationships between coronaviruses. Therefore, this study uses the nucleocapsid gene and protein to further investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses. Sequence and phylogenetic analyses have revealed the nucleocapsid gene and protein in SARS-CoV-2 are both closely related to those found in Bat-CoV RaTG13 and Pangolin-CoV MP789. Evidence of recombination was detected within the N gene, along with the presence of a double amino acid insertion found in the N-terminal region. Homology modeling for the N-Terminal Domain revealed similar structures but distinct electrostatic surfaces and topological variations in the β-hairpin that likely reflect specific adaptive functions. In respect to SARS-CoV-2, two amino acids (37S and 267A) were found to exist only in its N protein, along with an extended β-hairpin in the N-Terminal Domain. Collectively, this study strengthens the relationship among SARS-CoV-2, Bat-CoV RaTG13, and Pangolin-CoV MP789, providing additional insights into the structure and adaptive nature of the nucleocapsid protein found in these coronaviruses. Furthermore, this data will help enhance our understanding of the complete history behind SARS-CoV-2 and help assist in antiviral and vaccine development.


An Analysis of SARS-CoV-2 Using ViReport

Abstract: The ongoing outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in millions of cases and hundreds of thousands of deaths. Given the current lack of treatments or vaccines available, it may be useful to trace the evolu-tion and spread of the virus to better develop methods of preventative intervention. In this study, we analyzed over 4,000 full genome sequences of human SARS-CoV-2 using novel tool ViReport [13], an automated workflow for performing phylogenetic analyses on viral sequences and generating comprehensive molecular epidemiologi-cal reports. The complete ViReport output can be found at https://github.com/mirandajsong/ViReport-SARS-CoV-2.