RPflex: A Coarse-Grained Network Model for RNA Pocket Flexibility Study.

RNA regulates various biological processes, such as gene regulation, RNA splicing, and intracellular signal transduction. RNA's conformational dynamics play crucial roles in performing its diverse functions. Thus, it is essential to explore the flexibility characteristics of RNA, especially pocket flexibility. Here, we propose a computational approach, RPflex, to analyze pocket flexibility using the coarse-grained network model. We first clustered 3154 pockets into 297 groups by similarity calculation based on the coarse-grained lattice model. Then, we introduced the flexibility score to quantify the flexibility by global pocket features. The results show strong correlations between the flexibility scores and root-mean-square fluctuation (RMSF) values, with Pearson correlation coefficients of 0.60, 0.76, and 0.53 in Testing Sets I-III. Considering both flexibility score and network calculations, the Pearson correlation coefficient was increased to 0.71 in flexible pockets on Testing Set IV. The network calculations reveal that the long-range interaction changes contributed most to flexibility. In addition, the hydrogen bonds in the base-base interactions greatly stabilize the RNA structure, while backbone interactions determine RNA folding. The computational analysis of pocket flexibility could facilitate RNA engineering for biological or medical applications.

Histones: The critical players in innate immunity

The highly conserved histones in different species seem to represent a very ancient and universal innate host defense system against microorganisms in the biological world. Histones are the essential part of nuclear matter and act as a control switch for DNA transcription. However, histones are also found in the cytoplasm, cell membranes, and extracellular fluid, where they function as host defenses and promote inflammatory responses. In some cases, extracellular histones can act as damage-associated molecular patterns (DAMPs) and bind to pattern recognition receptors (PRRs), thereby triggering innate immune responses and causing initial organ damage. Histones and their fragments serve as antimicrobial peptides (AMPs) to directly eliminate bacteria, viruses, fungi, and parasites in vitro and in vivo. Histones are also involved in phagocytes-related innate immune response as components of neutrophil extracellular traps (NETs), neutrophil activators, and plasminogen receptors. In addition, as a considerable part of epigenetic regulation, histone modifications play a vital role in regulating the innate immune response and expression of corresponding defense genes. Here, we review the regulatory role of histones in innate immune response, which provides a new strategy for the development of antibiotics and the use of histones as therapeutic targets for inflammatory diseases, sepsis, autoimmune diseases, and COVID-19.

Real-time Speech Interruption Analysis: From Cloud to Client Deployment (preprint)

Meetings are an essential form of communication for all types of organizations, and remote collaboration systems have been much more widely used since the COVID-19 pandemic. One major issue with remote meetings is that it is challenging for remote participants to interrupt and speak. We have recently developed the first speech interruption analysis model, which detects failed speech interruptions, shows very promising performance, and is being deployed in the cloud. To deliver this feature in a more cost-efficient and environment-friendly way, we reduced the model complexity and size to ship the WavLM_SI model in client devices. In this paper, we first describe how we successfully improved the True Positive Rate (TPR) at a 1% False Positive Rate (FPR) from 50.9% to 68.3% for the failed speech interruption detection model by training on a larger dataset and fine-tuning. We then shrank the model size from 222.7 MB to 9.3 MB with an acceptable loss in accuracy and reduced the complexity from 31.2 GMACS (Giga Multiply-Accumulate Operations per Second) to 4.3 GMACS. We also estimated the environmental impact of the complexity reduction, which can be used as a general guideline for large Transformer-based models, and thus make those models more accessible with less computation overhead.

COVID-19 Vaccines and Global Stock Markets (preprint)

Global stock markets react positively when different phases of human clinical trials on COVID-19 vaccines begin. The average abnormal stock return on the first day of the trials is both statistically and economically significant at 8.08 basis points. The increase in the average abnormal stock return is threefold higher for leading vaccine candidates. The positive reaction is more pronounced upon the start of phase III trials, and it is also stronger for vaccine candidates developed by the U.S. and China. To explain the findings, we use a simple capital budgeting framework and show that stock markets convey important information about market-wide expectations on vaccine development that evolves along with the pandemic.

An in-depth investigation of the safety and immunogenicity of an inactivated SARS-CoV-2 vaccine (preprint)

BACKGROUND In-depth investigations of the safety and immunogenicity of inactivated SARS-CoV-2 vaccines are needed. METHOD In a phase I randomized, double-blinded, and placebo-controlled trial involving 192 healthy adults 18-59 years of age, two injections of three different doses (50 EU, 100 EU and 150 EU) of an inactivated SARS-CoV-2 vaccine or the placebo were administered intramuscularly with a 2- or 4-week interval between the injections. The safety and immunogenicity of the vaccine were evaluated within 28 days. FINDING In this study, 191 subjects assigned to three doses groups or the placebo group completed the 28-day trial. There were 44 adverse reactions within the 28 days, most commonly mild pain and redness at the injection site or slight fatigue, and no abnormal variations were observed in 48 cytokines in the serum samples of immunized subjects. The serum samples diluted from 1:32 to 1:4096 and incubated with the virus did not show antibody-dependent enhancement effects (ADEs) with regard to human natural killer cells, macrophages or dendritic cells. At day 14, the seroconversion rates had reached 92%, 100% and 96% with geometric mean titers (GMTs) of 18.0, 54.5 and 37.1, and at day 28, the seroconversion rates had reached 80%, 96% and 92% with GMTs of 10.6, 15.4 and 19.6in 0, 14 and 0, 28 procedures, respectively. Seroconversion was associated with the synchronous upregulation of ELISA antibodies against the S protein, N protein and virion and a cytotoxic T lymphocyte (CTL) response. Transcriptome analysis shaped the genetic diversity of immune response induced by the vaccine. INTERPRETATION In a population aged 18-59 years, this inactivated SARS-CoV-2 vaccine was safe and immunogenic.

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.

Autoproteolytic Products of the SARS-CoV-2 Nucleocapsid Protein are Primed for Antibody Evasion and Virus Proliferation (preprint)

The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes autoproteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We used mass spectrometry to show that N binds to a monoclonal antibody raised against full-length N. No antibody interactions were detected for N proteoforms without C-terminal residues, therefore locating antigenic regions towards the C-terminus. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. We propose that the proteoforms generated via autoproteolysis evade antibody detection through removal of the antigenic C-terminus and facilitate interactions with structured RNA or cyclophilin thereby enabling the virus to proliferate.

The strength of a NES motif in the nucleocapsid protein of human coronaviruses is related to genus, but not to pathogenic capacity (preprint)

Seven members of the Coronaviridae family infect humans, but only three (SARS-CoV, SARS-CoV-2 and MERS-CoV) cause severe disease with a high case fatality rate. Using in silico analyses (machine learning techniques and comparative genomics), several features associated to coronavirus pathogenicity have been recently proposed, including a potential increase in the strength of a predicted novel nuclear export signal (NES) motif in the nucleocapsid protein. Here, we have used a well-established nuclear export assay to experimentally establish whether the recently proposed nucleocapsid NESs are capable of mediating nuclear export, and to evaluate if their activity correlates with coronavirus pathogenicity. The six NES motifs tested were functional in our assay, but displayed wide differences in export activity. Importantly, these differences in NES strength were not related to strain pathogenicity. Rather, we found that the NESs of the strains belonging to the genus Alphacoronavirus were markedly stronger than the NESs of the strains belonging to the genus Betacoronavirus. We conclude that, while some of the genomic features recently identified in silico could be crucial contributors to coronavirus pathogenicity, this does not appear to be the case of nucleocapsid NES activity, as it is more closely related to coronavirus genus than to pathogenic capacity.

Structural basis for repurposing a 100-years-old drug suramin for treating COVID-19 (preprint)

The COVID-19 pandemic by non-stop infections of SARS-CoV-2 has continued to ravage many countries worldwide. Here we report the discovery of suramin, a 100-year-old drug, as a potent inhibitor of the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) through blocking the binding of RNA to the enzyme. In biochemical assays, suramin and its derivatives are at least 20-fold more potent than remdesivir, the currently approved nucleotide drug for COVID-19. The 2.6 [A] cryo-EM structure of the viral RdRp bound to suramin reveals two binding sites of suramin, with one site directly blocking the binding of the RNA template strand and the other site clash with the RNA primer strand near the RdRp catalytic active site, therefore inhibiting the viral RNA replication. Furthermore, suramin potently inhibits SARS-CoV-2 duplication in Vero E6 cells. These results provide a structural mechanism for the first non-nucleotide inhibitor of the SARS-CoV-2 RdRp and a rationale for repurposing suramin for treating COVID-19.

SARS-CoV-2 infection damages airway motile cilia and impairs mucociliary clearance (preprint)

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. We examined the functional and structural consequences of SARS-CoV-2 infection in a reconstituted human bronchial epithelium model. SARS-CoV-2 replication caused a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remained limited. Rather, SARS-CoV-2 replication led to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. The motile cilia function was compromised, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramped up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrated the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Sub-second heat inactivation of coronavirus (preprint)

Heat treatment denatures viral proteins that comprise the virion, making virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and 4 proteins, 3 of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature of as low as 75 oC and treatment duration of 15 min can effectively inactivate CoV. The applicability of a CoV heat inactivation method greatly depends on the length of time of a heat treatment and the temperature needed to inactivate the virus. With the goal of finding conditions where sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple system that can measure sub-second heat inactivation of CoV. The system is composed of capillary stainless-steel tubing immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can be flowed at various speeds. Flowing virus solution at different speeds, along with a real-time temperature monitoring system, allows the virus to be accurately exposed to a desired temperature for various durations of time. Using mouse hepatitis virus (MHV), a beta-coronavirus, as a model system, we identified that 85.2 oC for 0.48 s exposure is sufficient to obtain > 5 Log10 reduction in viral titer (starting titer: 5 x 107 PFU/mL), and that when exposed to 83.4 oC for 0.95 s, the virus was completely inactivated (zero titer, > 6 Log10 reduction).

A genetic variant protective for COVID-19 is inherited from Neanderthals (preprint)

It was recently shown that the major genetic risk factor associated with becoming severely ill with COVID-19 when infected by SARS-CoV-2 is inherited from Neandertals. Thanks to new genetic association studies additional risk factors are now being discovered. Using data from a recent genome-wide associations from the Genetics of Mortality in Critical Care (GenOMICC) consortium, we show that a haplotype at a region associated with requiring intensive care is inherited from Neandertals. It encodes proteins that activate enzymes that are important during infections with RNA viruses. As compared to the previously described Neandertal risk haplotype, this Neandertal haplotype is protective against severe COVID-19, is of more moderate effect, and is found at substantial frequencies in all regions of the world outside Africa.

GNS561 exhibits potent in vitro antiviral activity against SARS-CoV-2 through autophagy inhibition (preprint)

Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/2019-nCoV) has spread quickly worldwide, with more than 29 million cases and 920,000 deaths. Interestingly, coronaviruses were found to subvert and hijack the autophagic process to allow their viral replication. One of the spotlights had been focused on the autophagy inhibitors as a target mechanism effective in the inhibition of SARS-CoV-2 infection. Consequently, chloroquine (CQ) and hydroxychloroquine (HCQ), a derivative of CQ, was suggested as the first potentially be therapeutic strategies as they are known to be autophagy inhibitors. Then, they were used as therapeutics in SARS-CoV-2 infection along with remdesivir, for which the FDA approved emergency use authorization. Here, we investigated the antiviral activity and associated mechanism of GNS561, a small basic lipophilic molecule inhibitor of late-stage autophagy, against SARS-CoV-2. Our data indicated that GNS561 showed the highest antiviral effect for two SARS-CoV-2 strains compared to CQ and remdesivir. Focusing on the autophagy mechanism, we showed that GNS561, located in LAMP2-positive lysosomes, together with SARS-CoV-2, blocked autophagy by increasing the size of LC3-II spots and the accumulation of autophagic vacuoles in the cytoplasm with the presence of multilamellar bodies characteristic of a complexed autophagy. Finally, our study revealed that the combination of GNS561 and remdesivir was associated with a strong synergistic antiviral effect against SARS-CoV-2. Overall, our study highlights GNS561 as a powerful drug in SARS-CoV-2 infection and supports that the hypothesis that autophagy inhibitors could be an alternative strategy for SARS-CoV-2 infection.

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors (preprint)

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

Assessing and Addressing the Coronavirus-Induced Economic Crisis: Evidence From 1.5 Billion Sales Invoices (preprint)

We probe the effects of the COVID-19 pandemic and the subsequent containment policies on business activities in China by exploiting big data on 1.5 billion sales invoices. Using a difference-in-differences approach, we estimate that the average drop in sales is between 23% and 35%, depending on firm size, for the 12-week period after Wuhan's lockdown. The unprecedented plunge in countrywide economic activities is more evident in the first eight weeks, and firm sales gradually resume to 85% of the normal level afterward. Firms in industries requiring more intensive face-to-face interactions suffer more during the public health measures. Also, cities relying on investment-driven economic growth are more resilient. Lastly, local governments' economic stimulus policies, aimed at alleviating financial losses for small and micro firms, are actually more effective for medium-sized and large firms. Our results provide implications for other economies seeking to develop strategies to contain the disease and reopen the economy.

To procrastinate or not to procrastinate: the optimal timing of containing the global COVID-19 pandemic spread (preprint)

As global public health is under threat by the 2019-nCoV, an urgent question to ask is what the optimal strategy of epidemic prevention and control (P&C) measures would be, especially in terms of the timing of enforcing aggressive policy response so as to maximise health efficacy and to contain pandemic spread. Here, we developed a logistic probability function configured SEIR model to analyse the COVID-19 outbreak and estimate its transmission pattern under different “anticipate- or delay-to-activate” policy response scenarios in containing the pandemic. We wound that the potential positive effects of stringent P&C measures would be cancelled out in case of significantly delayed action, whereas a partially procastinatory wait-and-see control policy may still be able to contribute to containing the degree of epidemic spread although its effectiveness may be significantly compromised compared to a scenario of early intervention coupled with stringent P&C measures. A laissez faire policy adopted by the government and health authority to tackling the uncertainly of COVID19-type pandemic development during the early stage of the outbreak turns out to be a very bad strategy from optimal control perspective, as significant damages would be produced in that case. Authors Jun Li and Yimin Zhou contributed equally to this work.

Analysis and Prediction of False Negative Results for SARS-CoV-2 Detection with Pharyngeal Swab Specimen in COVID-19 Patients: A Retrospective Study (preprint)

Background: False negative results of SARS-CoV-2 nucleic acid detection pose threats to COVID-19 patients and medical workers alike. Objective: To develop multivariate models to determine clinical characteristics that contribute to false negative results of SARS-CoV-2 nucleic acid detection, and use them to predict false negative results as well as time windows for testing positive. Design: Retrospective Cohort Study (Ethics number of Tongji Hospital: No. IRBID: TJ-20200320) Setting: A database of outpatients in Tongji Hospital (University Hospital) from 15 January 2020 to 19 February 2020. Patients: 1,324 outpatients with COVID-19 Measurements: Clinical information on CT imaging reports, blood routine tests, and clinic symptoms were collected. A multivariate logistic regression was used to explain and predict false negative testing results of SARS-CoV-2 detection. A multivariate accelerated failure model was used to analyze and predict delayed time windows for testing positive. Results: Of the 1,324 outpatients who diagnosed of COVID-19, 633 patients tested positive in their first SARS-CoV-2 nucleic acid test (47.8%), with a mean age of 51 years (SD=14.9); the rest, which had a mean age of 47 years (SD=15.4), tested negative in the first test. Ground glass opacity in a CT imaging report was associated with a lower chance of false negatives (aOR, 0.56), and reduced the length of time window for testing positive by 26%. Consolidation was associated with a higher chance of false negatives (aOR, 1.57), and extended the length of time window for testing positive by 44%. In blood routine tests, basophils (aOR, 1.28) and eosinophils (aOR, 1.29) were associated with a higher chance of false negatives, and were found to extend the time window for testing positive by 23% and 41%, respectively. Age and gender also affected the significantly. Limitation: Data were generated in a large single-center study. Conclusion: Testing outcome and positive window of SARS-CoV-2 detection for COVID-19 patients were associated with CT imaging results, blood routine tests, and clinical symptoms. Taking into account relevant information in CT imaging reports, blood routine tests, and clinical symptoms helped reduce a false negative testing outcome. The predictive AFT model, what we believe to be one of the first statistical models for predicting time window of SARS-CoV-2 detection, could help clinicians improve the accuracy and efficiency of the diagnosis, and hence, optimizes the timing of nucleic acid detection and alleviates the shortage of nucleic acid detection kits around the world. Primary Funding Source: None.

The role of institutional trust in preventive and treatment-seeking behaviors during the 2019 novel coronavirus (2019-nCoV) outbreak among residents in Hubei, China (preprint)

Background Since December 2019, pneumonia associated with the 2019 novel coronavirus (2019-nCoV) has emerged in Wuhan, China. The exponential increase of the confirmed number of cases of 2019n-CoV is of great concern to the global community. The fears and panic among residents in the epicenters have prompted diverse responses, which are understudied. During such a crisis, community trust and support for the government and health authorities are important to contain the outbreak. We aimed to investigate the influence of institutional trust on public responses to the 2019-nCoV outbreak. Methods An anonymous Internet-based, cross-sectional survey was administered on January 29, 2020. The study population comprised all residents currently residing or working in the province of Hubei, where Wuhan is the capital city. The level of trust in information provision and preventive instructions, individual preventive behaviors and treatment-seeking behaviors were queried. Findings The majority of the participants expressed a great extent of trust in the information and preventive instructions provided by the central government than by the local government. A high uptake of 2019-nCoV preventive measures was found, particularly among people who had been placed under quarantine. Being under quarantine (adjusted odds ratio [OR] = 2.35, 95% confidence interval [CI] 1.80 to 3.08) and having a high institutional trust score (OR = 2.23, 95% CI 1.96 to 2.53) were both strong and significant determinants of higher preventive behavior scores. The majority of study participants (85.7%, n = 3,640) reported that they would seek hospital treatment if they suspected themselves to have been infected with 2019 n-CoV. Few of the participants from Wuhan (16.6%, n = 475) and those participants who were under quarantine (13.8%, n = 550) expressed an unwillingness to seek hospital treatment. Similarly, being under quarantine (OR = 2.36, 95% CI 1.80 to 3.09) and having a high institutional trust score (OR = 2.20, 95% CI 1.96 to 2.49) were two strong significant determinants of hospital treatment-seeking. Interpretation The results of this study suggest that institutional trust is an important factor influencing adequate preventive behavior and seeking formal medical care during an outbreak. In view of the 2019-nCoV being highly pathogenic and extremely contagious, our findings also underscore the importance of public health intervention to reach individuals with poor adherence to preventive measures and who are reluctant to seek treatment at formal health services. Funding National Key R&D Program of China, Ningbo Health Branding Subject Fund, Sanming Project of Medicine in Shenzhen, K.C. Wong Magna Fund in Ningbo University, National Natural Science Foundation of China, Fundamental Research Funds for the Central Universities, China Postdoctoral Science Foundation, and Natural Science Basic Research Program of Shanxi Province. Keywords: 2019-nCoV; institutional trust; preventive behaviors