Licorice-saponin A3 is a broad-spectrum inhibitor for COVID-19 by targeting viral spike and anti-inflammation.

Currently, human health due to corona virus disease 2019 (COVID-19) pandemic has been seriously threatened. The coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19. However, the efficacy is compromised by the SARS-CoV-2 evolvement and mutation. Here we report the SARS-CoV-2 S protein receptor-binding domain (RBD) inhibitor licorice-saponin A3 (A3) could widely inhibit RBD of SARS-CoV-2 variants, including Beta, Delta, and Omicron BA.1, XBB and BQ1.1. Furthermore, A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells, with EC50 of 1.016 µM. The mechanism was related with binding with Y453 of RBD determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis combined with quantum mechanics/molecular mechanics (QM/MM) simulations. Interestingly, phosphoproteomics analysis and multi fluorescent immunohistochemistry (mIHC) respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 MAPK pathways and rebalancing the corresponding immune dysregulation. This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.

A model to predict the risk of mortality in severely ill COVID-19 patients.

BACKGROUND: To investigate and select the useful prognostic parameters to develop and validate a model to predict the mortality risk for severely and critically ill patients with the coronavirus disease 2019 (COVID-19). METHODS: We established a retrospective cohort of patients with laboratory-confirmed COVID-19 (≥18 years old) from two tertiary hospitals: the People's Hospital of Wuhan University and Leishenshan Hospital between February 16, 2020, and April 14, 2020. The diagnosis of the cases was confirmed according to the WHO interim guidance. The data of consecutive severely and critically ill patients with COVID-19 admitted to these hospitals were analyzed. A total of 566 patients from the People's Hospital of Wuhan University were included in the training cohort and 436 patients from Leishenshan Hospital were included in the validation cohort. The least absolute shrinkage and selection operator (LASSO) and multivariable logistic regression were used to select the variables and build the mortality risk prediction model. RESULTS: The prediction model was presented as a nomograph and developed based on identified predictors, including age, chronic lung disease, C-reactive protein (CRP), D-dimer levels, neutrophil-to-lymphocyte ratio (NLR), creatinine, and total bilirubin. In the training cohort, the model displayed good discrimination with an AUC of 0.912 [95% confidence interval (CI): 0.884-0.940] and good calibration (intercept = 0; slope = 1). In the validation cohort, the model had an AUC of 0.922 [95% confidence interval (CI): 0.891-0.953] and a good calibration (intercept = 0.056; slope = 1.161). The decision curve analysis (DCA) demonstrated that the nomogram was clinically useful. CONCLUSION: A risk score for severely and critically ill COVID-19 patients' mortality was developed and externally validated. This model can help clinicians to identify individual patients at a high mortality risk.

CovidViT: a novel neural network with self-attention mechanism to detect Covid-19 through X-ray images.

Since the emergence of the novel coronavirus in December 2019, it has rapidly swept across the globe, with a huge impact on daily life, public health and the economy around the world. There is an urgent necessary for a rapid and economical detection method for the Covid-19. In this study, we used the transformers-based deep learning method to analyze the chest X-rays of normal, Covid-19 and viral pneumonia patients. Covid-Vision-Transformers (CovidViT) is proposed to detect Covid-19 cases through X-ray images. CovidViT is based on transformers block with the self-attention mechanism. In order to demonstrate its superiority, this research is also compared with other popular deep learning models, and the experimental result shows CovidViT outperforms other deep learning models and achieves 98.0% accuracy on test set, which means that the proposed model is excellent in Covid-19 detection. Besides, an online system for quick Covid-19 diagnosis is built on http://yanghang.site/covid19.

CovidViT: a novel neural network with self-attention mechanism to detect Covid-19 through X-ray images

Since the emergence of the novel coronavirus in December 2019, it has rapidly swept across the globe, with a huge impact on daily life, public health and the economy around the world. There is an urgent necessary for a rapid and economical detection method for the Covid-19. In this study, we used the transformers-based deep learning method to analyze the chest X-rays of normal, Covid-19 and viral pneumonia patients. Covid-Vision-Transformers (CovidViT) is proposed to detect Covid-19 cases through X-ray images. CovidViT is based on transformers block with the self-attention mechanism. In order to demonstrate its superiority, this research is also compared with other popular deep learning models, and the experimental result shows CovidViT outperforms other deep learning models and achieves 98.0% accuracy on test set, which means that the proposed model is excellent in Covid-19 detection. Besides, an online system for quick Covid-19 diagnosis is built on http://yanghang.site/covid19.

Schaftoside inhibits 3CLpro and PLpro of SARS-CoV-2 virus and regulates immune response and inflammation of host cells for the treatment of COVID-19.

It is an urgent demand worldwide to control the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) are key targets to discover SARS-CoV-2 inhibitors. After screening 12 Chinese herbal medicines and 125 compounds from licorice, we found that a popular natural product schaftoside inhibited 3CLpro and PLpro with IC50 values of 1.73 ± 0.22 and 3.91 ± 0.19 µmol/L, respectively, and inhibited SARS-CoV-2 virus in Vero E6 cells with EC50 of 11.83 ± 3.23 µmol/L. Hydrogen-deuterium exchange mass spectrometry analysis, quantum mechanics/molecular mechanics calculations, together with site-directed mutagenesis indicated the antiviral activities of schaftoside were related with non-covalent interactions with H41, G143 and R188 of 3CLpro, and K157, E167 and A246 of PLpro. Moreover, proteomics analysis and cytokine assay revealed that schaftoside also regulated immune response and inflammation of the host cells. The anti-inflammatory activities of schaftoside were confirmed on lipopolysaccharide-induced acute lung injury mice. Schaftoside showed good safety and pharmacokinetic property, and could be a promising drug candidate for the prevention and treatment of COVID-19.

Rapid quantitative monitoring of SARS-CoV-2 spike protein-mediated syncytia formation using split NanoLuc.

SARS-CoV-2 infection causes syncytial pneumocyte in patients and this has been considered as a defining feature of severe COVID-19 cases. Traditional methods of syncytia quantification require the morphology characterization of fused cells either with light microscope or fluorescent microscope, which is time-consuming and not accurate. Here we developed a rapid and sensitive coculture system measuring spike-induced syncytia by using NanoLuc complementation system. We found the formation of syncytia occurred rapidly after ACE2-expressing cells exposure to spike protein. In addition, we found furin cleavage as well as the cell surface protease TMPRSS2 enhanced syncytia formation. Finally, we showed that this coculture system can be used to test the ability of different compound to inhibit syncytia formation, thus providing a useful tool to screen anti-syncytial drugs.

Risk Factors for Weak Antibody Response of SARS-CoV-2 Vaccine in Adult Solid Organ Transplant Recipients: A Systemic Review and Meta-Analysis.

Objective: This is the first systematic review and meta-analysis to determine the factors that contribute to poor antibody response in organ transplant recipients after receiving the 2-dose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Method: Data was obtained from Embase, PubMed, Web of Science, Cochrane Library, China National Knowledge Infrastructure (CNKI), and Chinese Biomedical Literature Database (CBM). Studies reporting factors associated with antibody responses to the 2-dose SARS-CoV-2 vaccine in solid organ transplant recipients were included in our study based on the inclusion and exclusion criteria. Two researchers completed the literature search, screening, and data extraction. Randomized models were used to obtain results. Egger's test was performed to determine publication bias. Sensitivity analysis was performed to determine the stability of the result. The heterogeneity was determined using the Galbraith plot and subgroup analysis. Results: A total of 29 studies were included in the present study. The factors included living donor, BNT162b2, tacrolimus, cyclosporine, antimetabolite, mycophenolic acid (MPA) or mycophenolate mofetil (MMF), azathioprine, corticosteroids, high-dose corticosteroids, belatacept, mammalian target of rapamycin (mTOR) inhibitor, tritherapy, age, estimated glomerular filtration rate (eGFR), hemoglobin, and tacrolimus level were significantly different. Multivariate analysis showed significant differences in age, diabetes mellitus, MPA or MMF, high-dose corticosteroids, tritherapy, and eGFR. Conclusion: The possible independent risk factors for negative antibody response in patients with organ transplants who received the 2-dose SARS-CoV-2 vaccine include age, diabetes mellitus, low eGFR, MPA or MMF, high-dose corticosteroids, and triple immunosuppression therapy. mTOR inhibitor can be a protective factor against weak antibody response. Systematic Review Registration: PROSPERO, identifier CRD42021257965.

One-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification.

Droplet digital polymerase chain reaction (ddPCR) is a third generation of PCR that was recently developed to overcome the limitation of direct quantification observed in real-time quantification PCR (qPCR). Recent studies have shown that ddPCR is more sensitive than the gold standard reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) samples. In combination with multiplexing, multiple RT-ddPCR assays can be developed to directly quantify different SARS-CoV-2 nucleic acid targets within a single sample, significantly saving on cost and time. Since ddPCR is tolerant to a number of inhibitors unlike qPCR, it can be used to detect and quantify samples from complex environments like wastewater. Here we present three one-step RT-ddPCR protocols on how to develop simplex (one target), duplex (two targets), and triplex probe mix (three targets) assays for SARS-CoV-2 detection and quantification. The assays can be used for diagnosis or other research-related SARS-CoV-2 applications.

A pandemic-induced environmental dilemma of disposable masks: solutions from the perspective of the life cycle.

The coronavirus disease 2019 (COVID-19) has swept the world and still afflicts humans. As an effective means of protection, wearing masks has been widely adopted by the general public. The massive use of disposable masks has raised some emerging environmental and bio-safety concerns: improper handling of used masks may transfer the attached pathogens to environmental media; disposable masks mainly consist of polypropylene (PP) fibers which may aggravate the global plastic pollution; and the risks of long-term wearing of masks are elusive. To maximize the utilization and minimize the risks, efforts have been made to improve the performance of masks (e.g., antivirus properties and filtration efficiency), extend their functions (e.g., respiration monitoring and acting as a sampling device), develop new disinfection methods, and recycle masks. Despite that, from the perspective of the life cycle (from production, usage, and discard to disposal), comprehensive solutions are urgently needed to solve the environmental dilemma of disposable masks in both technologies (e.g., efficient use of raw materials, prolonging the service life, and enabling biodegradation) and policies (e.g., stricter industry criteria and garbage sorting).

Digital PCR Applications in the SARS-CoV-2/COVID-19 Era: a Roadmap for Future Outbreaks.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global public health disaster. The current gold standard for the diagnosis of infected patients is real-time reverse transcription-quantitative PCR (RT-qPCR). As effective as this method may be, it is subject to false-negative and -positive results, affecting its precision, especially for the detection of low viral loads in samples. In contrast, digital PCR (dPCR), the third generation of PCR, has been shown to be more effective than the gold standard, RT-qPCR, in detecting low viral loads in samples. In this review article, we selected publications to show the broad-spectrum applications of dPCR, including the development of assays and reference standards, environmental monitoring, mutation detection, and clinical diagnosis of SARS-CoV-2, while comparing it analytically to the gold standard, RT-qPCR. In summary, it is evident that the specificity, sensitivity, reproducibility, and detection limits of RT-dPCR are generally unaffected by common factors that may affect RT-qPCR. As this is the first time that dPCR is being tested in an outbreak of such a magnitude, knowledge of its applications will help chart a course for future diagnosis and monitoring of infectious disease outbreaks.

Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells.

The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.

Long-Term Preservation of SARS-CoV-2 RNA in Silk for Downstream RT-PCR Tests.

Positive controls made of viral gene components are essential to validate the performance of diagnostic assays for pathogens like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, most of them are target-specific, limiting their application spectrum when validating assays beyond their specified targets. The use of an inactivated whole-virus RNA reference standard could be ideal, but RNA is a labile molecule that needs cold chain storage and transportation to preserve its integrity and activity. The cold chain process stretches the already dwindling storage capacities, incurs huge costs, and limits the distribution of reference materials to low-resource settings. To circumvent these issues, we developed an inactivated whole-virus SARS-CoV-2 RNA reference standard and studied its stability in silk fibroin matrices, i.e., silk solution (SS) and silk film (SF). Compared to preservation in nuclease-free water (ddH2O) and SS, SF was more stable and could preserve the SARS-CoV-2 RNA reference standard at room temperature for over 21 weeks (∼6 months) as determined by reverse transcription polymerase chain reaction (RT-PCR). The preserved RNA reference standard in SF was able to assess the limits of detection of four commercial SARS-CoV-2 RT-PCR assays. In addition, SF is compatible with RT-PCR reactions and can be used to preserve a reaction-ready primer and probe mix for RT-PCR at ambient temperatures without affecting their activity. Taken together, these results offer extensive flexibility and a simpler mechanism of preserving RNA reference materials for a long time at ambient temperatures of ≥25 °C, with the possibility of eliminating cold chains during storage and transportation.

A SARS-CoV-2 nanobody that can bind to the RBD region may be used for treatment in COVID-19 in animals.

Coronavirus disease 2019 (COVID-19) caused by an infectious virus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), poses a threat to the world. The suitable treatments must be identified for this disease in animals. Nanobody have therapeutic potential in the COVID-19. In this study, SARS-CoV-2 Spike RBD protein was used to make the nanobody. Nanobodies binding to the SARS-CoV-2 Spike RBD protein was obtained. Interestingly, the nanobody could bind to SARS-CoV-2 Spike S protein and RBD protein at the same time. Nanobodies were validated with a neutralizing antibody detection kit. The use of pseudoviruses confirmed that nanobodies could prevent pseudoviruses from infecting cells. We believe the nanobody are very valuable and could be used in the treatment of COVID-19. SARS-CoV-2 nanobodies can be rapidly mass-produced from microorganisms to block SARS-CoV-2 infection in vitro and in vivo with preventive and therapeutic effects.

Effects of simeprevir on the replication of SARS-CoV-2 in vitro and in transgenic hACE2 mice.

In a bid to contain the current COVID-19 (coronavirus disease 2019) pandemic, various countermeasures have been applied. To date, however, there is a lack of an effective drug for the treatment of COVID-19. Through molecular modelling studies, simeprevir, a protease inhibitor approved for the management of hepatitis C virus infection, has been predicted as a potential antiviral against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causative agent of COVID-19. Here we assessed the efficacy of simeprevir against SARS-CoV-2 both in vitro in Vero E6 cells and in vivo in a human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model. The results showed that simeprevir could inhibit SARS-CoV-2 replication in Vero E6 cells with a half-maximal effective concentration (EC50) of 1.41 ± 0.12 µM. In a transgenic hACE2 mouse model of SARS-CoV-2 infection, intraperitoneal administration of simeprevir at 10 mg/kg/day for 3 consecutive days failed to suppress viral replication. These findings collectively imply that simeprevir does not inhibit SARS-CoV-2 in vivo and therefore do not support its application as a treatment against COVID-19 at a dosage of 10 mg/kg/day.

New Insights into Unexpected Severe PM2.5 Pollution during the SARS and COVID-19 Pandemic Periods in Beijing.

During the SARS period in 2003 and COVID-19 pandemic period in 2020, unexpected severe particulate matter pollution occurred in northern China, although the anthropogenic activities and associated emissions have assumed to be reduced dramatically. This anomalistic increase in PM2.5 pollution raises a question about how source emissions impact the air quality during these pandemic periods. In this study, we investigated the stable Cu and Si isotopic compositions and typical source-specific fingerprints of PM2.5 and its sources. We show that the primary PM2.5 emissions (PM2.5 emitted directly from sources) actually had no reduction but redistribution during these pandemic periods, rather than the previous thought of being greatly reduced. This finding provided critical evidence to interpret the anomalistic PM2.5 increase during the pandemic periods in north China. Our results also suggested that both the energy structure adjustment and stringent regulations on primary emissions should be synergistically implemented in a regional scale for clean air actions in China.

Natural triterpenoids from licorice potently inhibit SARS-CoV-2 infection.

Introduction: The COVID-19 global epidemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a great public health emergency. Discovering antiviral drug candidates is urgent for the prevention and treatment of COVID-19. Objectives: This work aims to discover natural SARS-CoV-2 inhibitors from the traditional Chinese herbal medicine licorice. Methods: We screened 125 small molecules from Glycyrrhiza uralensis Fisch. (licorice, Gan-Cao) by virtual ligand screening targeting the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. Potential hit compounds were further evaluated by ELISA, SPR, luciferase assay, antiviral assay and pharmacokinetic study. Results: The triterpenoids licorice-saponin A3 (A3) and glycyrrhetinic acid (GA) could potently inhibit SARS-CoV-2 infection, with EC50 of 75 nM and 3.17 µM, respectively. Moreover, we reveal that A3 mainly targets the nsp7 protein, and GA binds to the spike protein RBD of SARS-CoV-2. Conclusion: In this work, we found GA and A3 from licorice potently inhibit SARS-CoV-2 infection by affecting entry and replication of the virus. Our findings indicate that these triterpenoids may contribute to the clinical efficacy of licorice for COVID-19 and could be promising candidates for antiviral drug development.

Association between glucocorticoids treatment and viral clearance delay in patients with COVID-19: a systematic review and meta-analysis.

BACKGROUND: Evidence of glucocorticoids on viral clearance delay of COVID-19 patients is not clear. METHODS: In this systematic review and meta-analysis, we searched for studies on Medline, Embase, EBSCO, ScienceDirect, Web of Science, Cochrane Library, and ClinicalTrials.gov from 2019 to April 20, 2021. We mainly pooled the risk ratios (RRs) and mean difference (MD) for viral clearance delay and did subgroup analyses by the severity of illness and doses of glucocorticoids. RESULTS: 38 studies with a total of 9572 patients were identified. Glucocorticoids treatment was associated with delayed viral clearance in COVID-19 patients (adjusted RR 1.52, 95% CI 1.29 to 1.80, I2 = 52%), based on moderate-quality evidence. In subgroup analyses, risk of viral clearance delay was significant both for COVID-19 patients being mild or moderate ill (adjusted RR 1.86, 95% CI 1.35 to 2.57, I2 = 48%), and for patients of being severe or critical ill (adjusted RR 1.59, 95% CI 1.23 to 2.07, I2 = 0%); however, this risk significantly increased for patients taking high doses (unadjusted RR 1.85, 95% CI 1.08 to 3.18; MD 7.19, 95% CI 2.78 to 11.61) or medium doses (adjusted RR 1.86, 95% CI 0.96 to 3.62, I2 = 45%; MD 3.98, 95% CI 3.07 to 4.88, I2 = 4%), rather those taking low doses (adjusted RR 1.38, 95% CI 0.94 to 2.02, I2 = 59%; MD 1.46, 95% CI -0.79 to 3.70, I2 = 82%). CONCLUSIONS: Glucocorticoids treatment delayed viral clearance in COVID-19 patients of taking high doses or medium doses, rather in those of taking low doses of glucocorticoids.

Comparison of Associations Between Glucocorticoids Treatment and Mortality in COVID-19 Patients and SARS Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: The response to glucocorticoids treatment may be different between coronavirus disease 2019 (Covid-19) and severe acute respiratory syndrome (SARS). METHODS: In this systematic review and meta-analysis, we searched studies on Medline, Embase, EBSCO, ScienceDirect, Web of Science, Cochrane Library, ClinicalTrials.gov, International Clinical Trials Registry Platform from 2002 to October 7, 2020. We used fixed-effects and random-effects models to compute the risk ratio of death in the group receiving glucocorticoids treatment and the control group for COVID-19 and SARS, respectively. RESULTS: Ten trials and 71 observational studies, with a total of 45,935 patients, were identified. Glucocorticoids treatment was associated with decreased all-cause mortality both in COVID-19 (risk ratio, 0.88; 95% confidence interval, 0.82-0.94; I2 = 26%) and SARS (0.48; 0.29-0.79; 10%), based on high-quality evidence, as well as decreased all-cause mortality-including composite outcome of COVID-19 (0.89; 0.82-0.98; 0%). In subgroup analyses, all-cause mortality was significantly lower among COVID-19 patients being accompanied by severe ARDS but not mild ARDS, taking low-dose or pulse glucocorticoids, being critically severe but not only severe, being of critical severity and old but not young, being of critical severity and men but not women, non-early taking glucocorticoids, taking dexamethasone or methylprednisolone, and with the increased inflammatory state; but for SARS, lower mortality was observed among those who were taking medium-high dose glucocorticoids, being severe or critically severe, early taking glucocorticoids, and taking methylprednisolone or prednisolone. CONCLUSIONS: Glucocorticoids treatment reduced mortality in COVID-19 and SARS patients of critical severity; however, different curative effects existed between the two diseases among subpopulations, mainly regarding sex- and age-specific effects, optimal doses, and use timing of glucocorticoids.

Rapid determination of infectious SARS-CoV-2 in PCR-positive samples by SDS-PMA assisted RT-qPCR.

The ongoing COVID-19 pandemic has generated a global health crisis that needs well management of not only patients but also environments to reduce SARS-CoV-2 transmission. The gold standard RT-qPCR method is sensitive and rapid to detect SARS-CoV-2 nucleic acid, but does not answer if PCR-positive samples contain infectious virions. To circumvent this problem, we report an SDS-propidium monoazide (PMA) assisted RT-qPCR method that enables rapid discrimination of live and dead SARS-CoV-2 within 3 h. PMA, a photo-reactive dye, can react with viral RNA released or inside inactivated SARS-CoV-2 virions under assistance of 0.005% SDS, but not viral RNA inside live virions. Formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, RT-qPCR detection of heat-inactivated SARS-CoV-2 resulted in larger than 9 Ct value differences between PMA-treated and PMA-free groups, while less than 0.5 Ct differences were observed in the detection of infectious SARS-CoV-2 ranging from 20 to 5148 viral particles. Using a cutoff Ct difference of 8.6, this method could differentiate as low as 8 PFU live viruses in the mixtures of live and heat-inactivated virions. Further experiments showed that this method could successfully monitor the natural inactivation process of SARS-CoV-2 on plastic surfaces during storage with comparable results to the gold standard plaque assay. We believe that the culture-free method established here could be used for rapid and convenient determination of infectious SARS-CoV-2 virions in PCR-positive samples, which will facilitate better control of SARS-CoV-2 transmission.