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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-332289

ABSTRACT

The gastrointestinal tract is constitutively exposed to proteases including trypsin, a serine protease originating from the pancreas1. Elevated trypsin levels in the large intestine have been implicated in pathological conditions including infectious and inflammatory bowel disease2-4. Here we show that trypsin is regulated via degradation by members of the gut microbiota. After passing through the small intestine, trypsin activity is markedly reduced in the caecum of specific pathogen-free (SPF) mice, whereas germ-free (GF) mice have high luminal trypsin levels. We have successfully identified and isolated Paraprevotella strains from the faecal microbiome of healthy human donors as potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins and promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium . Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus, a mouse coronavirus dependent on trypsin and trypsin-like proteases for entry into host cells5,6. Congruently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced diarrhoea severity in patients with SARS-CoV2 infection. Therefore, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

2.
Jpn J Infect Dis ; 2022 Feb 28.
Article in English | MEDLINE | ID: covidwho-1698801

ABSTRACT

Prominent genomic recombination has been observed between the Delta and Alpha variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from clinical specimens in Japan. Interestingly, the recombination variant detected in this study carries a spike protein identical to the one in the domestic Delta variant, thereby suggesting that further risks would not be concerned with infectivity and immune escape. The recombinant has been classified as XC lineage in the PANGOLIN database. It is necessary to intensively study such marked genetic variations and characterize the emerging variants after careful verification of their lineage and clade assignment.

3.
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
4.
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
7.
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.….

8.
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
9.
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 , COVID-19/drug therapy , Chlorocebus aethiops , Dogs , HeLa Cells , Humans , Madin Darby Canine Kidney Cells , Vero Cells
10.
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
11.
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
12.
Jpn J Infect Dis ; 73(3): 181-186, 2020 May 22.
Article in English | MEDLINE | ID: covidwho-628700

ABSTRACT

Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) is usually diagnosed through highly sensitive and specific genetic tests such as real-time reverse transcription polymerase chain reaction (RT-PCR). Currently, two real-time RT-PCR assays targeting the upE and ORF1a regions of the MERS-CoV genome are widely used, and these are the standard assays recommended by the World Health Organization (WHO). The MERS outbreaks to date suggest that rapid diagnosis and subsequent isolation of infected patients, particularly superspreaders, are critical for containment. However, conventional real-time RT-PCR assays require large laboratory instruments, and amplification takes approximately 2 h. These disadvantages limit rapid diagnosis. Here, an ultra-rapid real-time RT-PCR test was established comprising a multiplex assay for upE and ORF1a running on a mobile PCR1100 device. As few as five copies of the MERS-CoV RNA can be detected within 20 min using the standard WHO assays in the mobile PCR device, with the sensitivity and specificity being similar to those of a conventional real-time PCR instrument such as the LightCyler, thereby enabling timely intervention to control MERS-CoV infection.


Subject(s)
Coronavirus Infections/virology , Middle East Respiratory Syndrome Coronavirus/isolation & purification , Point-of-Care Systems , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Coronavirus Infections/diagnosis , Disease Outbreaks , Sensitivity and Specificity , Time Factors
13.
Microbiol Immunol ; 64(9): 635-639, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-613452

ABSTRACT

In this study, the anti-severe acute respiratory syndrome coronavirus-2 (anti-SARS-CoV-2) activity of mycophenolic acid (MPA) and IMD-0354 was analyzed. These compounds were chosen based on their antiviral activities against other coronaviruses. Because they also inhibit dengue virus (DENV) infection, other anti-DENV compounds/drugs were also assessed. On SARS-CoV-2-infected VeroE6/TMPRSS2 monolayers, both MPA and IMD-0354, but not other anti-DENV compounds/drugs, showed significant anti-SARS-CoV-2 activity. Although MPA reduced the viral RNA level by only approximately 100-fold, its half maximal effective concentration was as low as 0.87 µ m, which is easily achievable at therapeutic doses of mycophenolate mofetil. MPA targets the coronaviral papain-like protease and an in-depth study on its mechanism of action would be useful in the development of novel anti-SARS-CoV-2 drugs.


Subject(s)
Antiviral Agents/pharmacology , Benzamides/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Mycophenolic Acid/pharmacology , Pneumonia, Viral/drug therapy , Animals , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/virology , Dengue Virus/drug effects , Humans , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Vero Cells , Virus Replication/drug effects
14.
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
15.
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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