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1.
PLoS Pathog ; 18(6): e1010590, 2022 06.
Article in English | MEDLINE | ID: covidwho-1892333

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been transmitted across all over the world, in contrast to the limited epidemic of genetically- and virologically-related SARS-CoV. However, the molecular basis explaining the difference in the virological characteristics among SARS-CoV-2 and SARS-CoV has been poorly defined. Here we identified that host sialoglycans play a significant role in the efficient spread of SARS-CoV-2 infection, while this was not the case with SARS-CoV. SARS-CoV-2 infection was significantly inhibited by α2-6-linked sialic acid-containing compounds, but not by α2-3 analog, in VeroE6/TMPRSS2 cells. The α2-6-linked compound bound to SARS-CoV-2 spike S1 subunit to competitively inhibit SARS-CoV-2 attachment to cells. Enzymatic removal of cell surface sialic acids impaired the interaction between SARS-CoV-2 spike and angiotensin-converting enzyme 2 (ACE2), and suppressed the efficient spread of SARS-CoV-2 infection over time, in contrast to its least effect on SARS-CoV spread. Our study provides a novel molecular basis of SARS-CoV-2 infection which illustrates the distinctive characteristics from SARS-CoV.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Peptidyl-Dipeptidase A/metabolism , Polysaccharides/metabolism , Protein Binding , Spike Glycoprotein, Coronavirus/metabolism
2.
Influenza Other Respir Viruses ; 16(5): 837-841, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-1819905

ABSTRACT

The impact of strengthening preventive measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the prevalence of respiratory viruses in children was examined. After the SARS-CoV-2 pandemic, the rate of multiple virus detection among hospitalized children decreased. Immediately after the SARS-CoV-2 pandemic, respiratory syncytial and parainfluenza viruses were rarely detected and subsequently reemerged. Human metapneumovirus and influenza virus were not consistently detected. Non-enveloped viruses (bocavirus, rhinovirus, and adenovirus) were detected to some extent even after the pandemic. Epidemic-suppressed infectious diseases may reemerge as susceptibility accumulates in the population and should continue to be monitored.


Subject(s)
COVID-19 , Respiratory Tract Infections , COVID-19/diagnosis , COVID-19/epidemiology , Child , Child, Hospitalized , Humans , Infant , Pandemics/prevention & control , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/epidemiology , Rhinovirus , SARS-CoV-2
3.
J Infect Chemother ; 28(7): 859-865, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1739954

ABSTRACT

INTRODUCTION: Seasonal human coronavirus (HCoV)-229E, -NL63, -OC43, and -HKU1 are seasonal coronaviruses that cause colds in humans. However, the clinical characteristics of pediatric inpatients infected with HCoVs are unclear. This study aimed to compare and clarify the epidemiological and clinical features of HCoVs and respiratory syncytial virus (RSV), which commonly causes severe respiratory infections in children. METHODS: Nasopharyngeal swabs were collected from all pediatric inpatients with respiratory symptoms at two secondary medical institutions in Fukushima, Japan. Eighteen respiratory viruses, including RSV and four HCoVs, were detected via reverse transcription-polymerase chain reaction. RESULTS: Of the 1757 specimens tested, viruses were detected in 1272 specimens (72.4%), with 789 single (44.9%) and 483 multiple virus detections (27.5%). RSV was detected in 639 patients (36.4%) with no difference in clinical characteristics between RSV-A and RSV-B. HCoV was detected in 84 patients (4.7%): OC43, NL63, HKU1, and 229E in 25 (1.4%), 26 (1.5%), 23 (1.3%), and 16 patients (0.9%), respectively. Patients with HCoV monoinfection (n = 35) had a significantly shorter period from onset to hospitalization (median [interquartile range] days, 2 [1-4.5] vs. 4 [2-5]), significantly shorter hospitalization stays (4 [3-5] vs. 5 [4-6]), and more cases of upper respiratory infections (37.1% vs. 3.9%) and croup (17.1% vs. 0.3%) but less cases of lower respiratory infection (54.3% vs. 94.8%) than patients with RSV monoinfection (n = 362). CONCLUSION: Seasonal HCoV-infected patients account for approximately 5% of children hospitalized for respiratory tract infections and have fewer lower respiratory infections and shorter hospital stays than RSV-infected patients.


Subject(s)
COVID-19 , Respiratory Syncytial Virus, Human , Respiratory Tract Infections , COVID-19/epidemiology , Child , Child, Hospitalized , Humans , Infant , Pandemics , Respiratory Tract Infections/epidemiology , Seasons
5.
Pathogens ; 11(3)2022 Feb 27.
Article in English | MEDLINE | ID: covidwho-1715599

ABSTRACT

In the ongoing coronavirus diseases 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), real-time RT-PCR based diagnostic assays have been used for the detection of infection, but the positive signal of real-time RT-PCR does not necessarily indicate the infectivity of the patient. Due to the unique replication system of the coronavirus, primer/probe sets targeted nucleocapsid (N) and spike (S) protein detect the abundantly synthesized subgenomic RNAs as well as the virus genome, possibly making the assay unsuitable for estimation of the infectivity of the specimen, although it has an advantage for the diagnostic tests. In this study, the primer/probe set targeting the open reading frame 1a (ORF1a) gene was developed to specifically detect viral genomic RNA. Then the relation between the ORF1a signal and infectivity of the clinical specimens was validated by virus isolation using VeroE6 cells, which constitutively express transmembrane protease, serine 2, (VeroE6/TMPRSS2). The analytical sensitivity of developed ORF1a set was similar to that of previously developed N and S sets. Nevertheless, in the assay of the clinical specimen, detection rate of the ORF1a gene was lower than that of the N and S genes. These data indicated that clinical specimens contain a significant amount of subgenomic RNAs. However, as expected, the isolation-succeeded specimen always showed an RT-PCR-positive signal for the ORF1a gene, suggesting ORF1a detection in combination with N and S sets could be a more rational indicator for the possible infectivity of the clinical specimens.

7.
Jpn J Infect Dis ; 75(1): 96-101, 2022 Jan 24.
Article in English | MEDLINE | ID: covidwho-1637698

ABSTRACT

Various variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began emerging worldwide from the end of 2020 to the beginning of 2021. The variants GRY/VOC202012/01 (B1.1.7), GH/N501Y.V2 (B1.351), and GR/N501Y.V3 (P1) are characterized by N to Y amino acid substitution at position 501 in the S protein. The variant containing L to R substitution at position 452 in the S protein G/L452R.V3 (B1.617) was endemic to India. The heightened concern regarding these variants is related to their increased viral infectivity. Information about nucleotide mismatch(es) on the primer/probe sequence is important for maintaining good performance of real-time PCR assays. In this study, real-time RT-PCR assays developed by the National Institute of Infectious Diseases, Japan (NIID-N2 and NIID-S2 assays), were reviewed to analyze nucleotide mismatches of variants in primer/probe sequences. The frequency of mismatched sequences in three variants (GRY/VOC202012/01, GH/N501Y.V2, and GR/N501Y.V3) was lower than that in all SARS-CoV-2 sequences. The mismatch, that G to C substitution at nucleotide 8 in reverse primer of S2 set, elevated to about 16.3% in G/L452R.V3, however the substitution did not affect the analytical sensitivity of assay. Therefore, the study indicates that the NIID-N2 and NIID-S2 sets detect VOCs of SARS-CoV-2 with reliable efficiency.


Subject(s)
COVID-19 , Communicable Diseases , Humans , Japan , Mutation , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2
8.
Microbiol Immunol ; 66(1): 15-23, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1437961

ABSTRACT

Spike (S) protein cleavage is a crucial step in coronavirus infection. In this review, this process is discussed, with particular focus on the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared with influenza virus and paramyxovirus membrane fusion proteins, the cleavage activation mechanism of coronavirus S protein is much more complex. The S protein has two cleavage sites (S1/S2 and S2'), and the cleavage motif for furin protease at the S1/S2 site that results from a unique four-amino acid insertion is one of the distinguishing features of SARS-CoV-2. The viral particle incorporates the S protein, which has already undergone S1/S2 cleavage by furin, and then undergoes further cleavage at the S2' site, mediated by the type II transmembrane serine protease transmembrane protease serine 2 (TMPRSS2), after binding to the receptor angiotensin-converting enzyme 2 (ACE2) to facilitate membrane fusion at the plasma membrane. In addition, SARS-CoV-2 can enter the cell by endocytosis and be proteolytically activated by cathepsin L, although this is not a major mode of SARS-CoV-2 infection. SARS-CoV-2 variants with enhanced infectivity have been emerging throughout the ongoing pandemic, and there is a close relationship between enhanced infectivity and changes in S protein cleavability. All four variants of concern carry the D614G mutation, which indirectly enhances S1/S2 cleavability by furin. The P681R mutation of the delta variant directly increases S1/S2 cleavability, enhancing membrane fusion and SARS-CoV-2 virulence. Changes in S protein cleavability can significantly impact viral infectivity, tissue tropism, and virulence. Understanding these mechanisms is critical to counteracting the coronavirus pandemic.


Subject(s)
Proteolysis , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/metabolism , COVID-19 , Humans , Serine Endopeptidases , Virus Internalization
9.
Jpn J Infect Dis ; 74(5): 465-472, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1436361

ABSTRACT

Soon after the 2019 outbreak of coronavirus disease 2019 in Wuhan, China, a protocol for real-time RT-PCR assay detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was established by the National Institute of Infectious Diseases (NIID) in Japan. The protocol used Charité's nucleocapsid (Sarbeco-N) and NIID nucleocapsid (NIID-N2) assays. During the following months, SARS-CoV-2 spread and caused a global pandemic, and various SARS-CoV-2 sequences were registered in public databases, such as the Global Initiative on Sharing All Influenza Data (GISAID). In this study, we evaluated the S2 assay (NIID-S2) that was newly developed to replace the Sarbeco-N assay and the performance of the NIID-N2 and NIID-S2 assays, referring to mismatches in the primer/probe targeted region. We found that the analytical sensitivity and specificity of the NIID-S2 set were comparable to those of the NIID-N2 assay, and the detection rate for clinical specimens was identical to that of the NIID-N2 assay. Furthermore, among the available sequences (approximately 192,000), the NIID-N2 and NIID-S2 sets had 2.6% and 1.2% mismatched sequences, respectively, although most of these mismatches did not affect the amplification efficiency, except the 3' end of the NIID-N2 forward primer. These findings indicate that the previously developed NIID-N2 assay is suitable for the detection of SARS-CoV-2 with support from the newly developed NIID-S2 set.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , DNA Primers/genetics , Humans , Japan , Phosphoproteins/genetics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics
12.
Front Microbiol ; 12: 651403, 2021.
Article in English | MEDLINE | ID: covidwho-1231355

ABSTRACT

Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 µM, IC90 = 2.31 µM, and IC99 = 4.39 µM in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.

13.
iScience ; 24(4): 102367, 2021 Apr 23.
Article in English | MEDLINE | ID: covidwho-1157438

ABSTRACT

Antiviral treatments targeting the coronavirus disease 2019 are urgently required. We screened a panel of already approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as remdesivir and chroloquine in VeroE6/TMPRSS2 cells: the anti-inflammatory drug cepharanthine and human immunodeficiency virus protease inhibitor nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry through the blocking of viral binding to target cells, while nelfinavir suppressed viral replication partly by protease inhibition. Consistent with their different modes of action, synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation was highlighted. Mathematical modeling in vitro antiviral activity coupled with the calculated total drug concentrations in the lung predicts that nelfinavir will shorten the period until viral clearance by 4.9 days and the combining cepharanthine/nelfinavir enhanced their predicted efficacy. These results warrant further evaluation of the potential anti-SARS-CoV-2 activity of cepharanthine and nelfinavir.

14.
Int J Mol Sci ; 22(6)2021 Mar 19.
Article in English | MEDLINE | ID: covidwho-1143519

ABSTRACT

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 µM and 99% at 15 µM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 µM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Oxysterols/chemistry , Oxysterols/pharmacology , SARS-CoV-2/drug effects , Administration, Oral , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/pharmacokinetics , COVID-19/drug therapy , Cell Survival/drug effects , Chlorocebus aethiops , Mice , Nucleocapsid Proteins/drug effects , Oxysterols/administration & dosage , Oxysterols/pharmacokinetics , SARS-CoV-2/genetics , Vero Cells , Viral Replication Compartments/drug effects , Virus Replication/drug effects
15.
Viruses ; 12(6)2020 06 10.
Article in English | MEDLINE | ID: covidwho-1120057

ABSTRACT

Although infection by SARS-CoV-2, the causative agent of coronavirus pneumonia disease (COVID-19), is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked Middle East respiratory syndrome coronavirus (MERS-CoV) S protein-mediated cell fusion by targeting transmembrane serine protease 2 (TMPRSS2), and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, angiotensin I converting enzyme 2 (ACE2) and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an effective concentration (EC)50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. On the other hand, a significantly higher dose (EC50 around 30 mM) was required for VeroE6/TMPRSS2 cells, where the TMPRSS2-independent but cathepsin-dependent endosomal infection pathway likely predominates. Together, our study shows that nafamostat mesylate potently inhibits SARS-CoV-2 S protein-mediated fusion in a cell fusion assay system and also inhibits SARS-CoV-2 infection in vitro in a cell-type-dependent manner. These findings, together with accumulated clinical data regarding nafamostat's safety, make it a likely candidate drug to treat COVID-19.


Subject(s)
Anticoagulants/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Guanidines/pharmacology , Pneumonia, Viral/drug therapy , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2 , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Benzamidines , Betacoronavirus/metabolism , COVID-19 , Cell Line , Chlorocebus aethiops , Coronavirus Infections/virology , Esters , Gabexate/analogs & derivatives , Gabexate/pharmacology , HEK293 Cells , Humans , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , SARS-CoV-2 , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
16.
Antimicrob Agents Chemother ; 2021 Mar 01.
Article in English | MEDLINE | ID: covidwho-1112313

ABSTRACT

Favipiravir (T-705, commercial name Avigan), a drug developed to treat influenza virus infection, has been used in some countries as an oral treatment for COVID-19; however, its clinical efficacy in this context is controversial.….

17.
Jpn J Infect Dis ; 74(1): 29-34, 2021 Jan 22.
Article in English | MEDLINE | ID: covidwho-1049199

ABSTRACT

The disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Wuhan, China, in December 2019, has rapidly spread worldwide. SARS-CoV-2 is usually detected via real-time reverse-transcription polymerase chain reaction (RT-PCR). However, the increase in specimen load in institutions/hospitals necessitates a simpler detection system. Here, we present an ultra-rapid, real-time RT-PCR assay for SARS-CoV-2 detection using PCR1100 device. Although PCR1100 tests only one specimen at a time, the amplification period is less than 20 min and the sensitivity and specificity match those of conventional real-time RT-PCR performed on large instruments. The method is potentially helpful when daily multiple SARS-CoV-2 testing is needed, for example to confirm virus-free status prior to patient discharge.


Subject(s)
COVID-19 Testing/instrumentation , COVID-19/virology , Real-Time Polymerase Chain Reaction/instrumentation , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , SARS-CoV-2/isolation & purification , COVID-19 Testing/methods , Humans , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity
20.
Jpn J Infect Dis ; 73(4): 304-307, 2020 07 22.
Article in English | MEDLINE | ID: covidwho-678395

ABSTRACT

During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Humans , Japan , Pandemics , Polyproteins , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
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