Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB) signaling pathway.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E). PURPOSE: The study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro. METHODS: The antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway. RESULTS: KD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells. CONCLUSIONS: KD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.

Whole Genome Analysis and Targeted Drug Discovery Using Computational Methods and High Throughput Screening Tools for Emerged Novel Coronavirus (2019-nCoV).

A novel coronavirus designated as SARS-CoV-2 in February 2020 by World Health organization (WHO) was identified as main cause of SARS like pneumonia cases in Wuhan city in Hubei Province of China at the end of 2019. This been recently declared as Global Pandemic by WHO. There is a global emergency to identify potential drugs to treat the SARS-CoV-2. Currently, there is no specific treatment against the new virus. There is a urgency to identifying potential antiviral agents to combat the disease is urgently needed. An effective and quick approach is to test existing antiviral drugs against. Whole genome analysis and alignment carried out using BLASTn, SMART BLAST and WebDSV 2.0 had shown more than 238 ORF's coding for proteins mostly origin from Bat SARS coronavirus and root genomic origin from Archaea. Molecular docking results against protein targets Furin, papain like proteases, RdRp and Spike glycoprotein had shown paritaprevir, ritonavir, entecavir and chloroquine derivatives are the best drugs to inhibit multi targets of coronavirus infection including natural compounds corosolic acid, baicalin and glycyrrhizic acid with minimal inhibitory concentrations. Thus we propose use of paritaprevir, entecavir, ritonavir and chloroquine derivatives as best drug combination along with niacinamide, folic acid and zinc supplements to treat novel coronavirus infection. We also propose use of plant protease inhibitors (PI's) and Anti-IL8, IL-6, IL-2 as future drug models against coronavirus.

ADRS due to COVID-19 in midterm pregnancy: successful management with plasma transfusion and corticosteroids.

BACKGROUND: Management of acute respiratory distress syndrome (ARDS) in pregnant women infected with new severe acute respiratory syndrome Corona virus 2 (SARS-CoV2) is a challenging clinical task. CASE: A 30- year-old woman (gravid 3, parity 2) presented at her 21 and 2/7 weeks gestation (pre pregnancy BMI: 36.1 kg/m2), with ARDS caused by SARS-CoV2 infection. She received lopinavir/ritonavir and azithromycin as well as early methyl prednisolone therapy. Given the persistent hypoxemia despite oxygen therapy via non rebreather face mask (FiO2:80%), convalescent plasma transfusion was administered that led to a mild clinical improvement as well as decrease in inflammatory markers. Growth of her fetus assessed by obstetric sonography was normal during hospital stay. CONCLUSION: Judicious corticosteroid therapy along with convalescent plasma transfusion to suppress viremia and cytokine storm can lead to favorable outcome in the pregnant women with ARDS caused by SARS-CoV2 infection without superimposed bacterial infection.

Destabilizing the structural integrity of COVID-19 by caulerpin and its derivatives along with some antiviral drugs: An in silico approaches for a combination therapy.

Presently, the SARS-CoV-2 (COVID-19) pandemic has been spreading throughout the world. Some drugs such as lopinavir, simeprevir, hydroxychloroquine, chloroquine, and amprenavir have been recommended for COVID-19 treatment by some researchers, but these drugs were not effective enough against this virus. This study based on in silico approaches was aimed to increase the anti-COVID-19 activities of these drugs by using caulerpin and its derivatives as an adjunct drug against SARS-CoV-2 receptor proteins: the SARS-CoV-2 main protease and the SARS-CoV-2 spike protein. Caulerpin exhibited antiviral activities against chikungunya virus and herpes simplex virus type 1. Caulerpin and some of its derivatives showed inhibitory activity against Alzheimer's disease. The web server ANCHOR revealed higher protein stability for the two receptors with disordered score (< 0.6). Molecular docking analysis showed that the binding energies of most of the caulerpin derivatives were higher than all the suggested drugs for the two receptors. Also, we deduced that inserting NH2, halogen, and vinyl groups can increase the binding affinity of caulerpin toward 6VYB and 6LU7, while inserting an alkyl group decreases the binding affinity of caulerpin toward 6VYB and 6LU7. So, we can modify the inhibitory effect of caulerpin against 6VYB and 6LU7 by inserting NH2, halogen, and vinyl groups. Based on the protein disordered results, the SARS-CoV-2 main protease and SARS-CoV-2 spike protein domain are highly stable proteins, so it is quite difficult to unstabilize their integrity by using individual drugs. Also, molecular dynamics (MD) simulation indicates that binding of the combination therapy of simeprevir and the candidate studied compounds to the receptors was stable and had no major effect on the flexibility of the protein throughout the simulations and provided a suitable basis for our study. So, this study suggested that caulerpin and its derivatives could be used as a combination therapy along with lopinavir, simeprevir, hydroxychloroquine, chloroquine, and amprenavir for disrupting the stability of SARS-CoV2 receptor proteins to increase the antiviral activity of these drugs.

Optimizing Safe Dental Practice During the COVID-19 Pandemic: Recommendations Based on a Guide Developed for Dental Practices in China.

The current global coronavirus disease 2019 (COVID-19) outbreak is still exerting severe global implications, and its development in various regions is complex and variable. The high risk of cross-infection poses a great challenge to the dental practice environment; it is therefore urgent to develop a set of pandemic prevention measures to ensure dental practice safety during the COVID-19 outbreak. Therefore, we combined the epidemiological characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), public emergency measures for COVID-19, characteristics of dental practice, and relevant literature reports to develop a set of dynamic practice measures for dental practices in high-, medium-, and low-risk areas affected by COVID-19. This will help dental practices to achieve standard prevention and ensure their safe and smooth operation during the pandemic. It is hoped that these measures will provide a reference basis for dental hospitals and dental clinics in their care and pandemic prevention work.

Effect of COVID-19 on Male Reproductive System - A Systematic Review.

Background: Angiotensin-converting enzyme II (ACE2), a receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to enter host cells, is widely expressed in testes and prostate tissues. The testis and prostate produce semen. At present, there are contradictory reports about whether SARS-CoV-2 can exist in the semen of infected men. Objective: To provide a comprehensive overview of the topic of whether COVID-19 can impact on male reproductive system. Methods: We reviewed the relevant publications on the possible impact of Coronavirus Disease 2019 (COVID-19) on male reproductive system and summarized the latest and most important research results so far. Literature published in English from December 2019 to January 31, 2021 regarding the existence of SARS-CoV-2 in semen, testis, and prostatic fluid and the effects of COVID-19 on male reproductive were included. Results: We identified 28 related studies, only one of which reported the presence of SARS-CoV-2 in semen. The study found that the semen quality of patients with moderate infection was lower than that of patients with mild infection and healthy controls. The impaired semen quality may be related to fever and inflammation. Pathological analysis of the testis/epididymis showed that SARS-CoV-2 viral particles were positive in 10 testicular samples, and the spermatogenic function of the testis was impaired. All 94 expressed prostatic secretion (EPS) samples were negative for SARS-CoV-2 RNA. Conclusion: The likelihood of SARS-CoV-2 in the semen of COVID-19 patients is very small, and semen should rarely be regarded as a carrier of SARS-CoV-2 genetic material. However, COVID-19 may cause testicular spermatogenic dysfunction via immune or inflammatory reactions. Long-term follow-up is needed for COVID-19 male patients and fetuses conceived during the father's infection period.

A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV-2.

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV-2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV-2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

Clinical Features of COVID-19 in Newborns, Infants, and Children: A Systematic Review and Meta-analysis.

Although it was thought that children were not susceptible to 2019-nCoV in the early days of the COVID-19 infection outbreak, there are currently reports of children and even one-day-old newborns being infected by the virus and hospitalized around the world. Recognizing the symptoms of the infection in children is of great value since a large number of children are asymptomatic or have mild symptoms, which may act as facilitators of the virus transmission. This review aimed to identify and summarize the existing evidence on clinical characteristics of COVID-19 in pediatric patients. A systematic search was conducted in the PubMed, Web of Science, Scopus, Google Scholar, and WHO database for eligible publications. The review proposal was registered with the PROSPERO. The quality assessment was done based on JBI Critical appraisal tools. The random-effects model was used to pool clinical features in the meta-analysis. From the identified 256 potentially relevant studies, 32 articles met the predetermined inclusion and exclusion criteria. Twenty-one studies fulfilled the criteria for this meta-analysis. Fever (58%) and cough (48%) were reported as the most common symptoms of infected children. Disease severity was mild in 51% and moderate in 39% of cases. A total of 63% of cases had respiratory and 25% of children had gastrointestinal symptoms, particularly diarrhea and nausea/vomiting. Approximately 24% of patients were asymptomatic. Results demonstrated that fever and cough were the most common symptoms of COVID-19-infected children and the majority of cases had mild-to-moderate disease severity.

Immunogenic potential of DNA vaccine candidate, ZyCoV-D against SARS-CoV-2 in animal models.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500 µg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also elicited Th-1 response as evidenced by elevated IFN-γ levels.

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at ≥21 days from symptom onset but had higher sensitivity with samples collected at ≤5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

Combined use of the hepatitis C drugs and amentoflavone could interfere with binding of the spike glycoprotein of SARS-CoV-2 to ACE2: the results of a molecular simulation study.

The worldwide rapid spread of the COVID-19 disease necessitates the search for fast and effective treatments. The repurposing of existing drugs seems to be the best solution in this situation. In this study, the molecular docking method was used to test 248 drugs against the receptor-binding domain (RBD) of spike glycoprotein of SARS-CoV-2, which is responsible for viral entry into the host cell. Among the top-ranked ligands are drugs that are used for hepatitis C virus (HCV) treatments (paritaprevir, ledipasvir, simeprevir) and a natural biflavonoid amentoflavone. The binding sites of the HCV drugs and amentoflavone are different. Therefore, the ternary complexes of the HCV drug, amentoflavone, and RBD can be created. For the 5 top-ranked ligands, the validating molecular dynamics simulations of binary and ternary complexes with RBD were performed. According to the MMPBSA-binding free energies, the HCV drugs ledipasvir and paritaprevir (in a neutral form) are the most efficient binders of the RBD when used in combination with amentoflavone.Communicated by Ramaswamy H. Sarma.

The envelope protein of SARS-CoV-2 increases intra-Golgi pH and forms a cation channel that is regulated by pH.

KEY POINTS: We report a novel method for the transient expression of SARS-CoV-2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes. Intracellular expression of SARS-CoV-2 E protein increases intra-Golgi pH. By targeting the SARS-CoV-2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH. This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID-19. ABSTRACT: The envelope (E) protein of coronaviruses such as SARS-CoV-1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS-CoV-2 in mammalian cells by directed targeting using a carboxyl-terminal fluorescent protein tag, mKate2. The wild-type SARS-CoV-2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum-Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS-CoV-2 E to the plasma membrane, which enabled biophysical analysis using whole-cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two-electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+ , Cs+ and K+ . The E current is nearly time- and voltage-independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time- and voltage-dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS-CoV-2 in vitro and possible efficacy in treating COVID-19.

Structure, Dynamics, Receptor Binding, and Antibody Binding of the Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein in a Viral Membrane.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell entry by binding to angiotensin-converting enzyme 2 (ACE2) and is considered the major target for drug and vaccine development. We previously built fully glycosylated full-length SARS-CoV-2 S protein models in a viral membrane including both open and closed conformations of the receptor-binding domain (RBD) and different templates for the stalk region. In this work, multiple µs-long all-atom molecular dynamics simulations were performed to provide deeper insights into the structure and dynamics of S protein and glycan functions. Our simulations reveal that the highly flexible stalk is composed of two independent joints and most probable S protein orientations are competent for ACE2 binding. We identify multiple glycans stabilizing the open and/or closed states of the RBD and demonstrate that the exposure of antibody epitopes can be captured by detailed antibody-glycan clash analysis instead of commonly used accessible surface area analysis that tends to overestimate the impact of glycan shielding and neglect possible detailed interactions between glycan and antibodies. Overall, our observations offer structural and dynamic insights into the SARS-CoV-2 S protein and potentialize for guiding the design of effective antiviral therapeutics.

Immune Responses to SARS-CoV-2 in Solid Organ Transplant Recipients.

PURPOSE OF REVIEW: Coronavirus disease 2019 (COVID-19) is caused by a complex interplay between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dynamics and host immune responses. Hosts with altered immunity, including solid organ transplant recipients, may be at increased risk of complications and death due to COVID-19. A synthesis of the available data on immune responses to SARS-CoV-2 infection is needed to inform therapeutic and preventative strategies in this special population. RECENT FINDINGS: Few studies have directly compared immune responses to SARS-CoV-2 between transplant recipients and the general population. Like non-transplant patients, transplant recipients mount an exuberant inflammatory response following initial SARS-CoV2 infection, with IL-6 levels correlating with disease severity in some, but not all studies. Transplant recipients display anti-SARS-CoV-2 antibodies and activated B cells in a time frame and magnitude similar to non-transplant patients-limited data suggest these antibodies can be detected within 15 days of symptom onset and may be durable for several months. CD4+ and CD8+ T lymphopenia, a hallmark of COVID-19, is more profound in transplant recipients, but SARS-CoV-2-reactive T cells can be detected among patients with both mild and severe disease. SUMMARY: The limited available data indicate that immune responses to SARS-CoV-2 are similar between transplant recipients and the general population, but no studies have been sufficiently comprehensive to understand nuances between organ types or level of immunosuppression to meaningfully inform individualized therapeutic decisions. The ongoing pandemic provides an opportunity to generate higher-quality data to support rational treatment and vaccination strategies in this population.

Association of Age With Likelihood of Developing Symptoms and Critical Disease Among Close Contacts Exposed to Patients With Confirmed SARS-CoV-2 Infection in Italy.

Importance: Solid estimates of the risk of developing symptoms and of progressing to critical disease in individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are key to interpreting coronavirus disease 2019 (COVID-19) dynamics, identifying the settings and the segments of the population where transmission is more likely to remain undetected, and defining effective control strategies. Objective: To estimate the association of age with the likelihood of developing symptoms and the association of age with the likelihood of progressing to critical illness after SARS-CoV-2 infection. Design, Setting, and Participants: This cohort study analyzed quarantined case contacts, identified between February 20 and April 16, 2020, in the Lombardy region of Italy. Contacts were monitored daily for symptoms and tested for SARS-CoV-2 infection, by either real-time reverse transcriptase-polymerase chain reaction using nasopharyngeal swabs or retrospectively via IgG serological assays. Close contacts of individuals with laboratory-confirmed COVID-19 were selected as those belonging to clusters (ie, groups of contacts associated with an index case) where all individuals were followed up for symptoms and tested for SARS-CoV-2 infection. Data were analyzed from February to June 2020. Exposure: Close contact with individuals with confirmed COVID-19 cases as identified by contact tracing operations. Main Outcomes and Measures: Age-specific estimates of the risk of developing respiratory symptoms or fever greater than or equal to 37.5 °C and of experiencing critical disease (defined as requiring intensive care or resulting in death) in SARS-CoV-2-infected case contacts. Results: In total, 5484 case contacts (median [interquartile range] age, 50 [30-61] years; 3086 female contacts [56.3%]) were analyzed, 2824 of whom (51.5%) tested positive for SARS-CoV-2 (median [interquartile range] age, 53 [34-64] years; 1604 female contacts [56.8%]). The proportion of infected persons who developed symptoms ranged from 18.1% (95% CI, 13.9%-22.9%) among participants younger than 20 years to 64.6% (95% CI, 56.6%-72.0%) for those aged 80 years or older. Most infected contacts (1948 of 2824 individuals [69.0%]) did not develop respiratory symptoms or fever greater than or equal to 37.5 °C. Only 26.1% (95% CI, 24.1%-28.2%) of infected individuals younger than 60 years developed respiratory symptoms or fever greater than or equal to 37.5 °C; among infected participants older than 60 years, 6.6% (95% CI, 5.1%-8.3%) developed critical disease. Female patients were 52.7% (95% CI, 24.4%-70.7%) less likely than male patients to develop critical disease after SARS-CoV-2 infection. Conclusions and Relevance: In this Italian cohort study of close contacts of patients with confirmed SARS-CoV-2 infection, more than one-half of individuals tested positive for the virus. However, most infected individuals did not develop respiratory symptoms or fever. The low proportion of children and young adults who developed symptoms highlights the possible challenges in readily identifying SARS-CoV-2 infections.

The study of antiviral drugs targeting SARS-CoV-2 nucleocapsid and spike proteins through large-scale compound repurposing.

Contributing to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clinical treatment, a drug library encompassing approximately 3,142 clinical-stage or FDA-approved small molecules is profiled to identify the candidate therapeutic inhibitors targeting nucleocapsid protein (N) and spike protein (S) of SARS-CoV-2. 16 screened candidates with higher binding affinity are evaluated via virtual screening. Comparing to those under trial/temporarily used antivirus drugs (i.e., umifenovir, lopinavir), ceftriaxone, cefotaxime, and cefuroxime show higher binding affinities to the N-terminal domain of N protein (N-NTD), C-terminal domain of N protein (N-CTD), and receptor-binding domain of S protein (S-RBD). Cefotaxime and cefuroxime have high binding affinities towards S-RBD with angiotensin-converting enzyme 2 (ACE2) complex via influence the critical interface sites at the interface of S-RBD (Arg403, Tyr453, Trp495, Gly496, Phe497, Asn501and Tyr505) and ACE2 (Asn33, His34, Glu37, Asp38, Lys353, Ala386, Ala387, Gln388, Pro389, Phe390 and Arg393) complex.

Ginkgolic acid and anacardic acid are specific covalent inhibitors of SARS-CoV-2 cysteine proteases.

BACKGROUND: In the urgent campaign to develop therapeutics against SARS-CoV-2, natural products have been an important source of new lead compounds. RESULTS: We herein identified two natural products, ginkgolic acid and anacardic acid, as inhibitors using a high-throughput screen targeting the SARS-CoV-2 papain-like protease (PLpro). Moreover, our study demonstrated that the two hit compounds are dual inhibitors targeting the SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) in addition to PLpro. A mechanism of action study using enzyme kinetics further characterized the two compounds as irreversible inhibitors against both 3CLpro and PLpro. Significantly, both identified compounds inhibit SARS-CoV-2 replication in vitro at nontoxic concentrations. CONCLUSIONS: Our finding provides two novel natural products as promising SARS-CoV-2 antivirals.

Platelet-activating immune complexes identified in critically ill COVID-19 patients suspected of heparin-induced thrombocytopenia.

BACKGROUND: Thrombocytopenia and thrombosis are prominent in coronavirus disease 2019 (COVID-19), particularly among critically ill patients; however, the mechanism is unclear. Such critically ill COVID-19 patients may be suspected of heparin-induced thrombocytopenia (HIT), given similar clinical features. OBJECTIVES: We investigated the presence of platelet-activating anti-platelet-factor 4 (PF4)/heparin antibodies in critically ill COVID-19 patients suspected of HIT. PATIENTS/METHODS: We tested 10 critically ill COVID-19 patients suspected of HIT for anti-PF4/heparin antibodies and functional platelet activation in the serotonin release assay (SRA). Anti-human CD32 antibody (IV.3) was added to the SRA to confirm FcγRIIA involvement. Additionally, SARS-CoV-2 antibodies were measured using an in-house ELISA. Finally, von Willebrand factor (VWF) antigen and activity were measured along with A Disintegrin And Metalloprotease with ThromboSpondin-13 Domain (ADAMTS13) activity and the presence of anti-ADAMTS13 antibodies. RESULTS: Heparin-induced thrombocytopenia was excluded in all samples based on anti-PF4/heparin antibody and SRA results. Notably, six COVID-19 patients demonstrated platelet activation by the SRA that was inhibited by FcγRIIA receptor blockade, confirming an immune complex (IC)-mediated reaction. Platelet activation was independent of heparin but inhibited by both therapeutic and high dose heparin. All six samples were positive for antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS-CoV-2 virus. These samples also featured significantly increased VWF antigen and activity, which was not statistically different from the four COVID-19 samples without platelet activation. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, thus ruling out a primary thrombotic microangiopathy. CONCLUSIONS: Our study identifies platelet-activating ICs as a novel mechanism that contributes to critically ill COVID-19.

Rapid SARS-CoV-2 Spike Protein Detection by Carbon Nanotube-Based Near-Infrared Nanosensors.

To effectively track and eliminate COVID-19, it is critical to develop tools for rapid and accessible diagnosis of actively infected individuals. Here, we introduce a single-walled carbon nanotube (SWCNT)-based optical sensing approach toward this end. We construct a nanosensor based on SWCNTs noncovalently functionalized with ACE2, a host protein with high binding affinity for the SARS-CoV-2 spike protein. The presence of the SARS-CoV-2 spike protein elicits a robust, 2-fold nanosensor fluorescence increase within 90 min of spike protein exposure. We characterize the nanosensor stability and sensing mechanism and passivate the nanosensor to preserve sensing response in saliva and viral transport medium. We further demonstrate that these ACE2-SWCNT nanosensors retain sensing capacity in a surface-immobilized format, exhibiting a 73% fluorescence turn-on response within 5 s of exposure to 35 mg/L SARS-CoV-2 virus-like particles. Our data demonstrate that ACE2-SWCNT nanosensors can be developed into an optical tool for rapid SARS-CoV-2 detection.

MicroRNA Mimics or Inhibitors as Antiviral Therapeutic Approaches Against COVID-19.

Coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease 2019 (COVID-19) pandemic, present a significant threat to human health by inflicting a wide variety of health complications and even death. While conventional therapeutics often involve administering small molecules to fight viral infections, small non-coding RNA sequences, known as microRNAs (miRNAs/miR-), may present a novel antiviral strategy. We can take advantage of their ability to modulate host-virus interactions through mediating RNA degradation or translational inhibition. Investigations into miRNA and SARS-CoV-2 interactions can reveal novel therapeutic approaches against this virus. The viral genomes of SARS-CoV-2, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) were searched using the Nucleotide Basic Local Alignment Search Tool (BLASTn) for highly similar sequences, to identify potential binding sites for miRNAs hypothesized to play a role in SARS-CoV-2 infection. miRNAs that target angiotensin-converting enzyme 2 (ACE2), the receptor used by SARS-CoV-2 and SARS-CoV for host cell entry, were also predicted. Several relevant miRNAs were identified, and their potential roles in regulating SARS-CoV-2 infections were further assessed. Current treatment options for SARS-CoV-2 are limited and have not generated sufficient evidence on safety and efficacy for treating COVID-19. Therefore, by investigating the interactions between miRNAs and SARS-CoV-2, miRNA-based antiviral therapies, including miRNA mimics and inhibitors, may be developed as an alternative strategy to fight COVID-19.