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1.
Viruses ; 13(8)2021 08 23.
Article in English | MEDLINE | ID: covidwho-1524167

ABSTRACT

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , SARS-CoV-2/drug effects , Tretinoin/pharmacology , Animals , Cell Line, Tumor , Chlorocebus aethiops , Cysteine Proteinase Inhibitors/pharmacology , DEAD Box Protein 58/metabolism , High-Throughput Screening Assays , Humans , Receptors, Immunologic/metabolism , SARS-CoV-2/enzymology , SARS-CoV-2/physiology , Vero Cells , Virus Replication/drug effects
2.
Sci Rep ; 11(1): 20638, 2021 10 19.
Article in English | MEDLINE | ID: covidwho-1475483

ABSTRACT

The COVID-19 pandemic is an unprecedented threat to humanity that has provoked global health concerns. Since the etiopathogenesis of this illness is not fully characterized, the prognostic factors enabling treatment decisions have not been well documented. Accurately predicting the progression of the disease would aid in appropriate patient categorization and thus help determine the best treatment option. Here, we have introduced a proteomic approach utilizing data-independent acquisition mass spectrometry (DIA-MS) to identify the serum proteins that are closely associated with COVID-19 prognosis. Twenty-seven proteins were differentially expressed between severely ill COVID-19 patients with an adverse or favorable prognosis. Ingenuity Pathway Analysis revealed that 15 of the 27 proteins might be regulated by cytokine signaling relevant to interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF), and their differential expression was implicated in the systemic inflammatory response and in cardiovascular disorders. We further evaluated practical predictors of the clinical prognosis of severe COVID-19 patients. Subsequent ELISA assays revealed that CHI3L1 and IGFALS may serve as highly sensitive prognostic markers. Our findings can help formulate a diagnostic approach for accurately identifying COVID-19 patients with severe disease and for providing appropriate treatment based on their predicted prognosis.


Subject(s)
Biomarkers/blood , COVID-19 Serological Testing/methods , COVID-19/blood , Gene Expression Profiling , Proteomics/methods , Chitinase-3-Like Protein 1/metabolism , Enzyme-Linked Immunosorbent Assay , Gas Chromatography-Mass Spectrometry , Gene Expression Regulation , Humans , Inflammation , Interleukin-1beta/biosynthesis , Interleukin-6/biosynthesis , Prognosis , SARS-CoV-2 , Tumor Necrosis Factor-alpha/biosynthesis , Virus Diseases
5.
J Mol Cell Biol ; 2021 Aug 27.
Article in English | MEDLINE | ID: covidwho-1376314

ABSTRACT

The uncontrolled spread of the COVID-19 pandemic has led to the emergence of different SARS-CoV-2 variants across the globe. The ongoing global vaccination strategy to curtail the COVID-19 juggernaut, is threatened by the rapidly spreading Variants of Concern (VOC) and other regional mutants, which are less responsive to neutralization by infection or vaccine derived antibodies. We have previously developed the hiVNT system which detects SARS-CoV-2 neutralizing antibodies in sera in less than three hours. In this study, we modify the hiVNT for rapid qualitative screening of neutralizing antibodies (nAb) to multiple VOC of SARS-CoV-2, and assess the neutralizing efficacy of the BNT162b2 mRNA vaccine on seven epidemiologically relevant SARS-CoV-2 variants. Here we show that the BNT162b2 mRNA vaccine can activate humoral immunity against the major SARS-CoV-2 mutants that are currently in circulation. Albeit a small sample size, we observed that one dose of vaccine was sufficient to elicit a protective humoral response in previously infected people. Using a panel of seven SARS-CoV-2 variants and a single prototype virus, our modified hiVNT would be useful for large-scale community wide testing to detect protective immunity that may confer vaccine/immune passport in the ongoing COVID-19 pandemic.

6.
J Infect Public Health ; 14(9): 1212-1217, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1364265

ABSTRACT

BACKGROUND: Many health care workers around the world tackled with COVID-19, however sadly, the infection of many medical care workers were reported. To reduce the risk of infection, we launched selected team (Team COVID) of non-specialists and brought in active telemedicine method and computed tomography (CT)-first protocol. We describe our actual practice and the health status of medical doctors dealing with COVID-19 patients. METHODS: Between April 17, 2020 and May 24, 2020, 10 doctors worked with COVID-19 patients as part of Team COVID. The Team COVID doctors used a CT-first triage protocol for outpatients and telemedicine for inpatients and outpatients. We evaluated paired serum-specific antibodies for SARS-CoV-2 at the initial and end of the study duration and PCR results for SARS-CoV-2 at the end of the study duration. Furthermore, 36-item short-form of the Medical Outcome Study Questionnaire (SF-36) at the beginning and end of the study period were evaluated. RESULTS: Ten doctors worked as Team COVID: seven internal medicine doctors and three surgeons. During the study period, Team COVID treated 165 individuals in the outpatient clinic and isolated hospitalized patients for 315 person-days. There were no positive results of serum-specific antibody testing and PCR testing for SARS-CoV-2 in Team COVID doctors. Furthermore, the SF-36 showed no deterioration in physical and mental QOL status. No in-hospital infection occurred during the study period. CONCLUSIONS: The Team COVID fulfilled the treatment using the active telemedicine and CT-first triage protocol without in hospital infection and excess stress. The combination strategy seems acceptable for both the protection and stress relief among the medical staff.


Subject(s)
COVID-19 , Telemedicine , Humans , Quality of Life , SARS-CoV-2 , Tomography, X-Ray Computed , Triage
7.
PLoS One ; 16(8): e0256022, 2021.
Article in English | MEDLINE | ID: covidwho-1352710

ABSTRACT

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic rapidly increases the use of mechanical ventilation (MV). Such cases further require extracorporeal membrane oxygenation (ECMO) and have a high mortality. OBJECTIVE: We aimed to identify prognostic biomarkers pathophysiologically reflecting future deterioration of COVID-19. METHODS: Clinical, laboratory, and outcome data were collected from 102 patients with moderate to severe COVID-19. Interleukin (IL)-6 level and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copy number in plasma were assessed with ELISA kit and quantitative PCR. RESULTS: Twelve patients died or required ECMO owing to acute respiratory distress syndrome despite the use of MV. Among various variables, a ratio of oxygen saturation to fraction of inspired oxygen (SpO2/FiO2), IL-6, and SARS-CoV-2 RNA on admission before intubation were strongly predictive of fatal outcomes after the MV use. Moreover, among these variables, combining SpO2/FiO2, IL-6, and SARS-CoV-2 RNA showed the highest accuracy (area under the curve: 0.934). In patients with low SpO2/FiO2 (< 261), fatal event-rate after the MV use at the 30-day was significantly higher in patients with high IL-6 (> 49 pg/mL) and SARS-CoV-2 RNAaemia (> 1.5 copies/µL) compared to those with high IL-6 or RNAaemia or without high IL-6 and RNAaemia (88% vs. 22% or 8%, log-rank test P = 0.0097 or P < 0.0001, respectively). CONCLUSIONS: Combining SpO2/FiO2 with high IL-6 and SARS-CoV-2 RNAaemia which reflect hyperinflammation and viral overload allows accurately and before intubation identifying COVID-19 patients at high risk for ECMO use or in-hospital death despite the use of MV.


Subject(s)
COVID-19/mortality , Interleukin-6/blood , RNA, Viral/metabolism , SARS-CoV-2/genetics , Adult , Aged , Aged, 80 and over , Area Under Curve , COVID-19/pathology , COVID-19/virology , Female , Hospital Mortality , Humans , Male , Middle Aged , Oxygen Consumption , Prognosis , Prospective Studies , ROC Curve , Respiration, Artificial , SARS-CoV-2/isolation & purification , Viral Load
9.
Front Microbiol ; 12: 661187, 2021.
Article in English | MEDLINE | ID: covidwho-1241181

ABSTRACT

Objective: There is scarce evidence regarding the long-term persistence of neutralizing antibodies among coronavirus disease 2019 (COVID-19) survivors. This study determined neutralizing antibody titers (NT50) and antibodies against spike protein (SP) or nucleocapsid protein (NP) antigens approximately 6 months after the diagnosis of COVID-19. Methods: COVID-19 survivors in Japan were recruited. Serum samples and data related to patients' characteristics and COVID-19 history were collected. NT50 and titers of antibodies against NP and SP antigens were measured at 20-32 weeks after the first positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test results. Factors associated with NT50 were identified using the multivariable linear regression and the correlations among NT50 and titers of immunoglobulin G (IgG) and total immunoglobulins (Igs) against NP and SP were assessed by Spearman's correlation. Results: Among 376 participants (median [range] days after testing positive for SARS-CoV-2, 180 (147-224); median [range] years of age, 50 (20-78); 188 [50%] male), most tested positive for NT50 (n = 367, 98%), SP-IgG (n = 344, 91%), SP-total Ig (n = 369, 98%), NP-IgG (n = 314, 84%), and NP-total Ig (n = 365, 97%). Regression analysis indicated that higher BMI, fever, and the requirement of mechanical ventilation or extracorporeal membrane oxygenation were significantly associated with higher NT50. Anti-SP antibodies correlated moderately with NT50 (Spearman's correlation: 0.63 for SP IgG; 0.57 for SP-total Ig), while the correlation was weak for anti-NP antibodies (0.37 for NP IgG; 0.32 for NP-total Ig). Conclusions: Most COVID-19 survivors had sustained neutralizing antibodies and tested positive for SP-total Ig and NP-total Ig approximately 6 months after infection.

10.
Cell Rep Med ; 2(6): 100311, 2021 Jun 15.
Article in English | MEDLINE | ID: covidwho-1230816

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a major global public health concern. Although rapid point-of-care testing for detecting viral antigen is important for management of the outbreak, the current antigen tests are less sensitive than nucleic acid testing. In our current study, we produce monoclonal antibodies (mAbs) that exclusively react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and exhibit no cross-reactivity with other human coronaviruses, including SARS-CoV. Molecular modeling suggests that the mAbs bind to epitopes present on the exterior surface of the nucleocapsid, making them suitable for detecting SARS-CoV-2 in clinical samples. We further select the optimal pair of anti-SARS-CoV-2 nucleocapsid protein (NP) mAbs using ELISA and then use this mAb pair to develop immunochromatographic assay augmented with silver amplification technology. Our mAbs recognize the variants of concern (501Y.V1-V3) that are currently in circulation. Because of their high performance, the mAbs of this study can serve as good candidates for developing antigen detection kits for COVID-19.

11.
Dig Endosc ; 2021 Feb 06.
Article in English | MEDLINE | ID: covidwho-1132881

ABSTRACT

OBJECTIVES: Gastrointestinal endoscopy (GIE) is useful for the early detection and treatment of many diseases; however, GIE is considered a high-risk procedure in the coronavirus disease 2019 (COVID-19) pandemic era. This study aimed to explore the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity in saliva and gastrointestinal fluids to which endoscopy medical staff are exposed. METHODS: The study was a single-center cross-sectional study. From June 1 to July 31, 2020, all patients who underwent GIE at Yokohama City University Hospital were registered. All patients provided 3 mL of saliva. For upper GIE, 10 mL of gastric fluid was collected through the endoscope. For lower GIE, 10 mL of intestinal fluid was collected through the endoscope. The primary outcome was the positive rate of SARS-CoV-2 in saliva and gastrointestinal fluids. We also analyzed serum-specific antibodies for SARS-CoV-2 and patients' background information. RESULTS: A total of 783 samples (560 upper GIE and 223 lower GIE samples) were analyzed. Polymerase chain reaction (PCR) on saliva samples did not show any positive results in either upper or lower GIE samples. However, 2.0% (16/783) of gastrointestinal fluid samples tested positive for SARS-CoV-2. No significant differences in age, sex, purpose of endoscopy, medication, or rate of antibody test positivity were found between PCR positive and PCR negative cases. CONCLUSIONS: Asymptomatic patients, even those with no detectable virus in their saliva, had SARS-CoV-2 in their gastrointestinal tract. Endoscopy medical staff should be aware of infection when performing procedures. The study was registered as UMIN000040587.

12.
Front Microbiol ; 11: 628281, 2020.
Article in English | MEDLINE | ID: covidwho-1058428

ABSTRACT

Objectives: Serological tests for COVID-19 have been instrumental in studying the epidemiology of the disease. However, the performance of the currently available tests is plagued by the problem of variability. We have developed a high-throughput serological test capable of simultaneously detecting total immunoglobulins (Ig) and immunoglobulin G (IgG) against nucleocapsid protein (NP) and spike protein (SP) and report its performance in detecting COVID-19 in clinical samples. Methods: We designed and prepared reagents for measuring NP-IgG, NP-Total Ig, SP-IgG, and SP-Total Ig (using N-terminally truncated NP (ΔN-NP) or receptor-binding domain (RBD) antigen) dedicated automated chemiluminescent enzyme immunoassay analyzer AIA-CL1200. After determining the basal thresholds based on 17 sera obtained from confirmed COVID-19 patients and 600 negative sera, the clinical validity of the assay was evaluated using independent 202 positive samples and 1,000 negative samples from healthy donors. Results: All of the four test parameters showed 100% specificity individually (1,000/1,000; 95%CI, 99.63-100). The sensitivity of the assay increased proportionally to the elapsed time from symptoms onset, and all the tests achieved 100% sensitivity (153/153; 95%CI, 97.63-100) after 13 days from symptoms onset. NP-Total Ig was the earliest to attain maximal sensitivity among the other antibodies tested. Conclusion: Our newly developed serological testing exhibited 100% sensitivity and specificity after 13 days from symptoms onset. Hence, it could be used as a reliable method for accurate detection of COVID-19 patients and to evaluate seroprevalence and possibly for surrogate assessment of herd immunity.

13.
Microorganisms ; 8(10)2020 Oct 20.
Article in English | MEDLINE | ID: covidwho-890391

ABSTRACT

Favipiravir was initially developed as an antiviral drug against influenza and is currently used in clinical trials against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (COVID-19). This agent is presumably involved in RNA chain termination during influenza virus replication, although the molecular interactions underlying its potential impact on the coronaviruses including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) remain unclear. We performed in silico studies to elucidate detailed molecular interactions between favipiravir and the SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza virus RNA-dependent RNA polymerases (RdRp). As a result, no interactions between favipiravir ribofuranosyl-5'-triphosphate (F-RTP), the active form of favipiravir, and the active sites of RdRps (PB1 proteins) from influenza A (H1N1)pdm09 virus were found, yet the agent bound to the tunnel of the replication genome of PB1 protein leading to the inhibition of replicated RNA passage. In contrast, F-RTP bound to the active sites of coronavirus RdRp in the presence of the agent and RdRp. Further, the agent bound to the replicated RNA terminus in the presence of agent, magnesium ions, nucleotide triphosphate, and RdRp proteins. These results suggest that favipiravir exhibits distinct mechanisms of action against influenza virus and various coronaviruses.

14.
J Antimicrob Chemother ; 76(2): 283-285, 2021 01 19.
Article in English | MEDLINE | ID: covidwho-889571

ABSTRACT

Severe COVID-19 is a biphasic illness, with an initial viral replication phase, followed by a cascade of inflammatory events. Progression to severe disease is predominantly a function of the inflammatory cascade, rather than viral replication per se. This understanding can be effectively translated to changing our approach in managing the disease. The natural course of disease offers us separate windows of specific time intervals to administer either antiviral or immunomodulatory therapy. Instituting the right attack at the right time would maximize the benefit of treatment. This concept must also be factored into studies that assess the efficacy of antivirals and immunomodulatory agents against COVID-19.


Subject(s)
Antiviral Agents/administration & dosage , COVID-19/drug therapy , Immunomodulation/drug effects , Immunosuppressive Agents/administration & dosage , Time-to-Treatment , Antiviral Agents/therapeutic use , COVID-19/immunology , COVID-19/virology , Cytokines/blood , Disease Progression , Humans , Immunomodulation/immunology , Immunosuppressive Agents/therapeutic use , SARS-CoV-2/drug effects , Virus Replication/drug effects
15.
Biochem Biophys Res Commun ; 533(1): 195-200, 2020 11 26.
Article in English | MEDLINE | ID: covidwho-753910

ABSTRACT

The pandemic of COVID-19 is spreading unchecked due to the lack of effective antiviral measures. Silver nanoparticles (AgNP) have been studied to possess antiviral properties and are presumed to inhibit SARS-CoV-2. Due to the need for an effective agent against SARS-CoV-2, we evaluated the antiviral effect of AgNPs. We evaluated a plethora of AgNPs of different sizes and concentration and observed that particles of diameter around 10 nm were effective in inhibiting extracellular SARS-CoV-2 at concentrations ranging between 1 and 10 ppm while cytotoxic effect was observed at concentrations of 20 ppm and above. Luciferase-based pseudovirus entry assay revealed that AgNPs potently inhibited viral entry step via disrupting viral integrity. These results indicate that AgNPs are highly potent microbicides against SARS-CoV-2 but should be used with caution due to their cytotoxic effects and their potential to derange environmental ecosystems when improperly disposed.


Subject(s)
Antiviral Agents/administration & dosage , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Metal Nanoparticles/administration & dosage , Pneumonia, Viral/drug therapy , Silver/administration & dosage , Animals , Antiviral Agents/toxicity , Betacoronavirus/physiology , COVID-19 , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus Infections/epidemiology , Coronavirus Infections/virology , Dose-Response Relationship, Drug , Humans , Metal Nanoparticles/toxicity , Metal Nanoparticles/ultrastructure , Pandemics , Particle Size , Pneumonia, Viral/epidemiology , Pneumonia, Viral/virology , SARS-CoV-2 , Silver/toxicity , Vero Cells , Virus Internalization/drug effects
16.
J Allergy Clin Immunol ; 146(2): 330-331, 2020 08.
Article in English | MEDLINE | ID: covidwho-597639
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