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1.
Mol Cell ; 2021 Mar 03.
Article in English | MEDLINE | ID: covidwho-1117323

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.

2.
Nat Ecol Evol ; 5(5): 600-608, 2021 05.
Article in English | MEDLINE | ID: covidwho-1111986

ABSTRACT

Bats are the suggested natural hosts for severe acute respiratory syndrome coronavirus (SARS-CoV) and the causal agent of the coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2. The interaction of viral spike proteins with their host receptor angiotensin-converting enzyme 2 (ACE2) is a critical determinant of potential hosts and cross-species transmission. Here we use virus-host receptor binding and infection assays to examine 46 ACE2 orthologues from phylogenetically diverse bat species, including those in close and distant contact with humans. We found that 24, 21 and 16 of them failed to support infection by SARS-CoV, SARS-CoV-2 or both viruses, respectively. Furthermore, we confirmed that infection assays in human cells were consistent with those in two bat cell lines. Additionally, we used genetic and functional analyses to identify critical residues in bat ACE2 receptors associated with viral entry restrictions. Our results suggest that many bat species may not be the potential hosts of one or both viruses and that no correlation was identified between proximity to humans and probability of being natural hosts of SARS-CoV or SARS-CoV-2. This study demonstrates dramatic variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species and adds knowledge towards a better understanding of coronavirus-bat interaction.


Subject(s)
Chiroptera , Animals , Humans , Peptidyl-Dipeptidase A/genetics , Receptors, Virus/genetics , Spike Glycoprotein, Coronavirus/genetics
3.
Cell Res ; 31(4): 395-403, 2021 04.
Article in English | MEDLINE | ID: covidwho-1091494

ABSTRACT

The upcoming flu season in the Northern Hemisphere merging with the current COVID-19 pandemic raises a potentially severe threat to public health. Through experimental coinfection with influenza A virus (IAV) and either pseudotyped or live SARS-CoV-2 virus, we found that IAV preinfection significantly promoted the infectivity of SARS-CoV-2 in a broad range of cell types. Remarkably, in vivo, increased SARS-CoV-2 viral load and more severe lung damage were observed in mice coinfected with IAV. Moreover, such enhancement of SARS-CoV-2 infectivity was not observed with several other respiratory viruses, likely due to a unique feature of IAV to elevate ACE2 expression. This study illustrates that IAV has a unique ability to aggravate SARS-CoV-2 infection, and thus, prevention of IAV infection is of great significance during the COVID-19 pandemic.


Subject(s)
/pathology , Coinfection/pathology , Influenza A virus/physiology , Orthomyxoviridae Infections/pathology , /physiology , /deficiency , /metabolism , Animals , Cathepsin L/genetics , Cathepsin L/metabolism , Cell Line , Coinfection/virology , Humans , Influenza A virus/isolation & purification , Lung/pathology , Mice , Mice, Transgenic , Orthomyxoviridae Infections/virology , RNA, Guide/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Severity of Illness Index , Viral Load , Virus Internalization
4.
Protein Cell ; 2020 Nov 09.
Article in English | MEDLINE | ID: covidwho-915250

ABSTRACT

In the original publication the author's name 'Dimitri Lavillete' is published incorrectly. The correct author name should be spelt as 'Dimitri Lavillette' is provided in this correction.

6.
Preprint | SSRN | ID: ppcovidwho-587

ABSTRACT

Circulating in China and 75 other countries and territories, the ongoing COVID-19 outbreak has caused devastating mortality and posed a great threat to public h

8.
Protein Cell ; 11(10): 723-739, 2020 10.
Article in English | MEDLINE | ID: covidwho-697126

ABSTRACT

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus Infections/drug therapy , Oxidoreductases/antagonists & inhibitors , Pandemics , Pneumonia, Viral/drug therapy , RNA Viruses/drug effects , Thiazoles/pharmacology , Animals , Antiviral Agents/therapeutic use , Betacoronavirus/drug effects , Betacoronavirus/physiology , Binding Sites/drug effects , Cell Line , Coronavirus Infections/virology , Crotonates/pharmacology , Cytokine Release Syndrome/drug therapy , Drug Evaluation, Preclinical , Gene Knockout Techniques , Humans , Influenza A virus/drug effects , Leflunomide/pharmacology , Mice , Mice, Inbred BALB C , Orthomyxoviridae Infections/drug therapy , Oseltamivir/therapeutic use , Oxidoreductases/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Pneumonia, Viral/virology , Protein Binding/drug effects , Pyrimidines/biosynthesis , RNA Viruses/physiology , Structure-Activity Relationship , Thiazoles/therapeutic use , Toluidines/pharmacology , Ubiquinone/metabolism , Virus Replication/drug effects
9.
Virol Sin ; 2020 Jul 21.
Article in English | MEDLINE | ID: covidwho-659402

ABSTRACT

We recently reported that inhibitors against human dihydroorotate dehydrogenase (DHODH) have broad-spectrum antiviral activities including their inhibitory efficacies on SARS-CoV-2 replication in infected cells. However, there are limited data from clinical studies to prove the application of DHODH inhibitors in Coronavirus disease 2019 (COVID-19) patients. In the present study, we evaluated Leflunomide, an approved DHODH inhibitor widely used as a modest immune regulator to treat autoimmune diseases, in treating COVID-19 disease with a small-scale of patients. Cases of 10 laboratory-confirmed COVID-19 patients of moderate type with obvious opacity in the lung were included. Five of the patients were treated with Leflunomide, and another five were treated as blank controls without a placebo. All the patients accepted standard supportive treatment for COVID-19. The patients given Leflunomide had a shorter viral shedding time (median of 5 days) than the controls (median of 11 days, P = 0.046). The patients given Leflunomide also showed a significant reduction in C-reactive protein levels, indicating that immunopathological inflammation was well controlled. No obvious adverse effects were observed in Leflunomide-treated patients, and they all discharged from the hospital faster than controls. This preliminary study on a small-scale compassionate use of Leflunomide provides clues for further understanding of Leflunomide as a potential antiviral drug against COVID-19.

10.
Emerg Microbes Infect ; 9(1): 1175-1179, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-361278

ABSTRACT

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10-4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Multiplex Polymerase Chain Reaction , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction , DNA Primers , DNA Probes , Humans , Multiplex Polymerase Chain Reaction/methods , Pandemics , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Sensitivity and Specificity , Viral Load
11.
Emerg Microbes Infect ; 9(1): 1259-1268, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-342833

ABSTRACT

Quantitative real time PCR (RT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. However, due to the low viral load specimens and the limitations of RT-PCR, significant numbers of false negative reports are inevitable, which results in failure to timely diagnose, cut off transmission, and assess discharge criteria. To improve this situation, an optimized droplet digital PCR (ddPCR) was used for detection of SARS-CoV-2, which showed that the limit of detection of ddPCR is significantly lower than that of RT-PCR. We further explored the feasibility of ddPCR to detect SARS-CoV-2 RNA from 77 patients, and compared with RT-PCR in terms of the diagnostic accuracy based on the results of follow-up survey. 26 patients of COVID-19 with negative RT-PCR reports were reported as positive by ddPCR. The sensitivity, specificity, PPV, NPV, negative likelihood ratio (NLR) and accuracy were improved from 40% (95% CI: 27-55%), 100% (95% CI: 54-100%), 100%, 16% (95% CI: 13-19%), 0.6 (95% CI: 0.48-0.75) and 47% (95% CI: 33-60%) for RT-PCR to 94% (95% CI: 83-99%), 100% (95% CI: 48-100%), 100%, 63% (95% CI: 36-83%), 0.06 (95% CI: 0.02-0.18), and 95% (95% CI: 84-99%) for ddPCR, respectively. Moreover, 6/14 (42.9%) convalescents were detected as positive by ddPCR at 5-12 days post discharge. Overall, ddPCR shows superiority for clinical diagnosis of SARS-CoV-2 to reduce the false negative reports, which could be a powerful complement to the RT-PCR.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Real-Time Polymerase Chain Reaction/methods , False Negative Reactions , Humans , Limit of Detection , Pandemics , RNA, Viral/genetics , Viral Load/methods
12.
Nature ; 582(7813): 557-560, 2020 06.
Article in English | MEDLINE | ID: covidwho-137432

ABSTRACT

The ongoing outbreak of coronavirus disease 2019 (COVID-19) has spread rapidly on a global scale. Although it is clear that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted through human respiratory droplets and direct contact, the potential for aerosol transmission is poorly understood1-3. Here we investigated the aerodynamic nature of SARS-CoV-2 by measuring viral RNA in aerosols in different areas of two Wuhan hospitals during the outbreak of COVID-19 in February and March 2020. The concentration of SARS-CoV-2 RNA in aerosols that was detected in isolation wards and ventilated patient rooms was very low, but it was higher in the toilet areas used by the patients. Levels of airborne SARS-CoV-2 RNA in the most public areas was undetectable, except in two areas that were prone to crowding; this increase was possibly due to individuals infected with SARS-CoV-2 in the crowd. We found that some medical staff areas initially had high concentrations of viral RNA with aerosol size distributions that showed peaks in the submicrometre and/or supermicrometre regions; however, these levels were reduced to undetectable levels after implementation of rigorous sanitization procedures. Although we have not established the infectivity of the virus detected in these hospital areas, we propose that SARS-CoV-2 may have the potential to be transmitted through aerosols. Our results indicate that room ventilation, open space, sanitization of protective apparel, and proper use and disinfection of toilet areas can effectively limit the concentration of SARS-CoV-2 RNA in aerosols. Future work should explore the infectivity of aerosolized virus.


Subject(s)
Aerosols/analysis , Aerosols/chemistry , Bathroom Equipment , Betacoronavirus/isolation & purification , Coronavirus Infections/virology , Hospitals , Pneumonia, Viral/virology , Workplace , Betacoronavirus/genetics , China/epidemiology , Coronavirus Infections/epidemiology , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Crowding , Disinfection , Humans , Intensive Care Units , Masks , Medical Staff , Pandemics/prevention & control , Patients/statistics & numerical data , Pneumonia, Viral/epidemiology , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , RNA, Viral/analysis , Social Isolation , Ventilation
13.
Emerg Microbes Infect ; 9(1): 761-770, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-29222

ABSTRACT

Circulating in China and 158 other countries and areas, the ongoing COVID-19 outbreak has caused devastating mortality and posed a great threat to public health. However, efforts to identify effectively supportive therapeutic drugs and treatments has been hampered by our limited understanding of host immune response for this fatal disease. To characterize the transcriptional signatures of host inflammatory response to SARS-CoV-2 (HCoV-19) infection, we carried out transcriptome sequencing of the RNAs isolated from the bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) specimens of COVID-19 patients. Our results reveal distinct host inflammatory cytokine profiles to SARS-CoV-2 infection in patients, and highlight the association between COVID-19 pathogenesis and excessive cytokine release such as CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B. Furthermore, SARS-CoV-2 induced activation of apoptosis and P53 signalling pathway in lymphocytes may be the cause of patients' lymphopenia. The transcriptome dataset of COVID-19 patients would be a valuable resource for clinical guidance on anti-inflammatory medication and understanding the molecular mechansims of host response.


Subject(s)
Bronchoalveolar Lavage Fluid , Chemokines/analysis , Coronavirus Infections/genetics , Cytokines/analysis , Leukocytes, Mononuclear , Pneumonia, Viral/genetics , Transcriptome , Apoptosis , Betacoronavirus , Coronavirus Infections/blood , Coronavirus Infections/immunology , Humans , Lymphopenia , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/immunology , RNA-Seq , Signal Transduction , Tumor Suppressor Protein p53
15.
Emerg Microbes Infect ; 9(1): 313-319, 2020.
Article in English | MEDLINE | ID: covidwho-337

ABSTRACT

From December 2019, an outbreak of unusual pneumonia was reported in Wuhan with many cases linked to Huanan Seafood Market that sells seafood as well as live exotic animals. We investigated two patients who developed acute respiratory syndromes after independent contact history with this market. The two patients shared common clinical features including fever, cough, and multiple ground-glass opacities in the bilateral lung field with patchy infiltration. Here, we highlight the use of a low-input metagenomic next-generation sequencing (mNGS) approach on RNA extracted from bronchoalveolar lavage fluid (BALF). It rapidly identified a novel coronavirus (named 2019-nCoV according to World Health Organization announcement) which was the sole pathogens in the sample with very high abundance level (1.5% and 0.62% of total RNA sequenced). The entire viral genome is 29,881 nt in length (GenBank MN988668 and MN988669, Sequence Read Archive database Bioproject accession PRJNA601736) and is classified into ß-coronavirus genus. Phylogenetic analysis indicates that 2019-nCoV is close to coronaviruses (CoVs) circulating in Rhinolophus (Horseshoe bats), such as 98.7% nucleotide identity to partial RdRp gene of bat coronavirus strain BtCoV/4991 (GenBank KP876546, 370 nt sequence of RdRp and lack of other genome sequence) and 87.9% nucleotide identity to bat coronavirus strain bat-SL-CoVZC45 and bat-SL-CoVZXC21. Evolutionary analysis based on ORF1a/1b, S, and N genes also suggests 2019-nCoV is more likely a novel CoV independently introduced from animals to humans.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Disease Outbreaks , Pneumonia, Viral/epidemiology , Adult , China , Female , Genome, Viral , Humans , Male , Phylogeny , RNA, Viral/genetics , Young Adult
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