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1.
Nat Commun ; 13(1): 6100, 2022 Oct 15.
Article in English | MEDLINE | ID: covidwho-2077054

ABSTRACT

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Humans , Mice , Cathepsins , Furin/genetics , Furin/metabolism , Mice, Knockout , Peptide Hydrolases , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
2.
Pathogens ; 11(8)2022 Aug 03.
Article in English | MEDLINE | ID: covidwho-1969405

ABSTRACT

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

3.
Influenza Other Respir Viruses ; 16(5): 837-841, 2022 09.
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
4.
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.

6.
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
7.
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
8.
Uirusu ; 70(2): 155-166, 2020.
Article in Japanese | MEDLINE | ID: covidwho-1413729
9.
Jpn J Infect Dis ; 74(4): 285-292, 2021 Jul 21.
Article in English | MEDLINE | ID: covidwho-1323436

ABSTRACT

Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.


Subject(s)
Coronavirus/isolation & purification , Epithelium/virology , Respiratory System/virology , Respiratory Tract Infections/virology , Coronavirus/genetics , Genome, Viral/genetics , Humans , Japan
11.
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
12.
Jpn J Infect Dis ; 73(6): 465-468, 2020 Nov 24.
Article in English | MEDLINE | ID: covidwho-976563

ABSTRACT

Human orthopneumovirus, also known as the respiratory syncytial virus (RSV), is a leading cause of respiratory tract infections in children worldwide. The World Health Organization has taken steps toward establishing a global surveillance system for RSV, based on the global influenza surveillance and response system initiated in 2015. The US Centers for Disease Control and Prevention (CDC) has developed a genetic detection method based on real-time reverse transcription polymerase chain reaction (RT-PCR), which is used in global RSV surveillance. In Japan, immunoassay-based rapid antigen detection kits are widely used for the detection of RSV. In this study, an ultra-rapid real-time RT-PCR method for the rapid detection of RSV was developed using the PCR1100 device based on the US CDC assay in order to detect RSV in comparable time to rapid test kits. The ultra-rapid real-time RT-PCR could detect RSV viral RNA in less than 20 min while maintaining sensitivity and specificity comparable to conventional real-time RT-PCR using large installed instruments. Furthermore, combining ultra-rapid real-time RT-PCR with the M1 Sample Prep kit reduced the total working time for the detection of RSV from clinical specimen to less than 25 min, suggesting this method could be used for point-of-care RSV testing.


Subject(s)
RNA, Viral/isolation & purification , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus, Human/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , Child , Humans , Japan , Nasopharynx/virology , Point-of-Care Testing , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Virus, Human/genetics , Respiratory Tract Infections/diagnosis , Respiratory Tract Infections/virology , Sensitivity and Specificity
13.
J Virol ; 95(1)2020 12 09.
Article in English | MEDLINE | ID: covidwho-968111

ABSTRACT

Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and Middle East respiratory syndrome coronavirus (MERS-CoV) replication. Ciclesonide, an inhaled corticosteroid, suppressed the replication of MERS-CoV and other coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), in cultured cells. The 90% effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 µM. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in nonstructural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed the replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under a microscope, the viral RNA replication-transcription complex in cells, which is thought to be detectable using antibodies specific for nsp3 and double-stranded RNA, was observed to fall in the presence of ciclesonide in a concentration-dependent manner. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients.IMPORTANCE The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. New and effective antiviral agents that combat the disease are needed urgently. Here, we found that an inhaled corticosteroid, ciclesonide, suppresses the replication of coronaviruses, including betacoronaviruses (murine hepatitis virus type 2 [MHV-2], MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alphacoronavirus (human coronavirus 229E [HCoV-229E]), in cultured cells. Ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS and COVID-19.


Subject(s)
COVID-19/metabolism , Pregnenediones/pharmacology , RNA, Double-Stranded/biosynthesis , RNA, Viral/biosynthesis , SARS-CoV-2/physiology , Virus Replication/drug effects , Animals , Chlorocebus aethiops , Dogs , HeLa Cells , Humans , Madin Darby Canine Kidney Cells , Vero Cells
14.
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
15.
J Infect Dis ; 222(7): 1098-1102, 2020 09 01.
Article in English | MEDLINE | ID: covidwho-661147

ABSTRACT

During a COVID-19 outbreak on the Diamond Princess cruise ship we sampled environmental surfaces after passengers and crew vacated cabins. SARS-CoV-2 RNA was detected in 58 of 601 samples (10%) from case cabins 1-17 days after cabins were vacated but not from noncase cabins. There was no difference in detection proportion between cabins of symptomatic (15%, 28/189; cycle quantification [Cq], 29.79-38.86) and asymptomatic cases (21%, 28/131; Cq, 26.21-38.99). No SARS-CoV-2 virus was isolated from any of the samples. Transmission risk of SARS-CoV-2 from symptomatic and asymptomatic patients may be similar and surfaces could be involved in transmission.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/epidemiology , Disease Outbreaks , Environmental Monitoring , Pneumonia, Viral/epidemiology , RNA, Viral/isolation & purification , Betacoronavirus/genetics , COVID-19 , Coronavirus Infections/transmission , Coronavirus Infections/virology , Humans , Pandemics , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , SARS-CoV-2 , Sampling Studies , Ships , Specimen Handling
16.
Jpn J Infect Dis ; 73(5): 366-368, 2020 Sep 24.
Article in English | MEDLINE | ID: covidwho-141681

ABSTRACT

The coronavirus induced disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan (China) in December 2019 is currently spreading rapidly worldwide. We recently reported a laboratory protocol for the diagnosis of SARS-CoV-2 based on real-time reverse transcriptase PCR (RT-PCR) assays using two primer sets, N and N2. On January 30-31, 2020, the protocol and the reagents for these assays were distributed to the local public health institutes and quarantine depots in Japan nationwide, and thereafter SARS-CoV-2 diagnostic testing was initiated. For further validation, the assays were compared with the commercially available kits using the SARS-CoV-2 viral RNA and clinical specimens obtained from COVID19-suspected individuals. The LightMix Modular SARS and Wuhan CoV E-gene (LM S&W-E) assay was highly sensitive for the SARS-CoV-2, as was the N2 set, as both the assays showed consistent results for the clinical specimens. While the LM S&W-E set targets the highly conserved region of E gene in the SARS-CoV and SARS-CoV-2, the N2 set was designed to target specifically the unique region in the SARS-CoV-2 N gene. Therefore, the N2 set exhibits high specificity and sensitivity for SARS-CoV-2 detection. These results indicate that the protocol using the N and N2 sets is comparable to the commercially available kits, and thus is reliable for laboratory diagnosis of COVID-19.


Subject(s)
Betacoronavirus/isolation & purification , Molecular Diagnostic Techniques/methods , Betacoronavirus/genetics , COVID-19 Testing , COVID-19 Vaccines , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Humans , Japan/epidemiology , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Sensitivity and Specificity , Viral Proteins/genetics
17.
Proc Natl Acad Sci U S A ; 117(13): 7001-7003, 2020 03 31.
Article in English | MEDLINE | ID: covidwho-8451

ABSTRACT

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.


Subject(s)
Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Pneumonia, Viral/virology , Serine Endopeptidases/metabolism , Animals , COVID-19 , Cell Line , Chlorocebus aethiops , Disease Outbreaks , Humans , Pandemics , RNA, Viral/metabolism , SARS-CoV-2 , Vero Cells , Virus Cultivation
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