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2.
Sci China Life Sci ; 2021 Apr 28.
Article in English | MEDLINE | ID: covidwho-1212915

ABSTRACT

Prolonged viral RNA shedding and recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in coronavirus disease 2019 (COVID-19) patients have been reported. However, the clinical outcome and pathogenesis remain unclear. In this study, we recruited 43 laboratory-confirmed COVID-19 patients. We found that prolonged viral RNA shedding or recurrence mainly occurred in severe/critical patients (P<0.05). The average viral shedding time in severe/critical patients was more than 50 days, and up to 100 days in some patients, after symptom onset. However, chest computed tomography gradually improved and complete absorption occurred when SARS-CoV-2 RT-PCR was still positive, but specific antibodies appeared. Furthermore, the viral shedding time significantly decreased when the A1,430G or C12,473T mutation occurred (P<0.01 and FDR<0.01) and increased when G227A occurred (P<0.05 and FDR<0.05). High IL1R1, IL1R2, and TNFRSF21 expression in the host positively correlated with viral shedding time (P<0.05 and false discovery rate <0.05). Prolonged viral RNA shedding often occurs but may not increase disease damage. Prolonged viral RNA shedding is associated with viral mutations and host factors.

3.
Signal Transduct Target Ther ; 6(1): 167, 2021 04 24.
Article in English | MEDLINE | ID: covidwho-1203416

ABSTRACT

The ongoing 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 has posed a worldwide pandemic and a major global public health threat. The severity and mortality of COVID-19 are associated with virus-induced dysfunctional inflammatory responses and cytokine storms. However, the interplay between host inflammatory responses and SARS-CoV-2 infection remains largely unknown. Here, we demonstrate that SARS-CoV-2 nucleocapsid (N) protein, the major structural protein of the virion, promotes the virus-triggered activation of NF-κB signaling. After binding to viral RNA, N protein robustly undergoes liquid-liquid phase separation (LLPS), which recruits TAK1 and IKK complex, the key kinases of NF-κB signaling, to enhance NF-κB activation. Moreover, 1,6-hexanediol, the inhibitor of LLPS, can attenuate the phase separation of N protein and restrict its regulatory functions in NF-κB activation. These results suggest that LLPS of N protein provides a platform to induce NF-κB hyper-activation, which could be a potential therapeutic target against COVID-19 severe pneumonia.


Subject(s)
/metabolism , NF-kappa B/metabolism , RNA, Viral/metabolism , Signal Transduction , A549 Cells , Acrylates/pharmacology , Animals , /pathology , Chlorocebus aethiops , HEK293 Cells , HeLa Cells , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Phosphoproteins/metabolism , Vero Cells
4.
Emerg Infect Dis ; 27(5)2021 05.
Article in English | MEDLINE | ID: covidwho-1200875

ABSTRACT

Understanding the immune response to Middle East respiratory syndrome coronavirus (MERS-CoV) is crucial for disease prevention and vaccine development. We studied the antibody responses in 48 human MERS-CoV infection survivors who had variable disease severity in Saudi Arabia. MERS-CoV-specific neutralizing antibodies were detected for 6 years postinfection.

5.
Signal Transduct Target Ther ; 6(1): 155, 2021 04 15.
Article in English | MEDLINE | ID: covidwho-1189204

ABSTRACT

Disease progression prediction and therapeutic drug target discovery for Coronavirus disease 2019 (COVID-19) are particularly important, as there is still no effective strategy for severe COVID-19 patient treatment. Herein, we performed multi-platform omics analysis of serial plasma and urine samples collected from patients during the course of COVID-19. Integrative analyses of these omics data revealed several potential therapeutic targets, such as ANXA1 and CLEC3B. Molecular changes in plasma indicated dysregulation of macrophage and suppression of T cell functions in severe patients compared to those in non-severe patients. Further, we chose 25 important molecular signatures as potential biomarkers for the prediction of disease severity. The prediction power was validated using corresponding urine samples and plasma samples from new COVID-19 patient cohort, with AUC reached to 0.904 and 0.988, respectively. In conclusion, our omics data proposed not only potential therapeutic targets, but also biomarkers for understanding the pathogenesis of severe COVID-19.


Subject(s)
/blood , Drug Discovery , Lipidomics , Proteomics , /metabolism , Biomarkers/blood , Female , Humans , Male
6.
Preprint | bioRxiv | ID: ppbiorxiv-439793

ABSTRACT

SARS-CoV-2 infection in human can cause medical complications across various tissues and organs. Despite of the advances to understanding the pathogenesis of SARS-CoV-2, its tissue tropism and interactions with host cells have not been fully understood. Existing clinical data have suggested possible SARS-CoV-2 infection in human skeleton system. In the present study, we found that authentic SARS-CoV-2 could efficiently infect human and mouse bone marrow-derived macrophages (BMMs) and alter the expression of macrophage chemotaxis and osteoclast-related genes. Importantly, in a mouse SARS-CoV-2 infection model that was enabled by the intranasal adenoviral (AdV) delivery of human angiotensin converting enzyme 2 (hACE2), SARS-CoV-2 was found to be present in femoral BMMs as determined by in situ immunofluorescence analysis. Using single-cell RNA sequencing (scRNA-Seq), we characterized SARS-CoV-2 infection in BMMs. Importantly, SARS-CoV-2 entry on BMMs appeared to be dependent on the expression of neuropilin-1 (NRP1) rather than the widely recognized receptor ACE2. It was also noted that unlike brain macrophages which displayed aging-dependent NRP1 expression, BMMs from neonatal and aged mice had constant NRP1 expression, making BMMs constantly vulnerable target cells for SARS-CoV-2. Furthermore, it was found that the abolished SARS-CoV-2 entry in BMM-derived osteoclasts was associated with the loss of NRP1 expression during BMM-to-osteoclast differentiation. Collectively, our study has suggested that NRP1 can mediate SARS-CoV-2 infection in BMMs, which precautions the potential impact of SARS-CoV-2 infection on human skeleton system.

7.
Lancet Infect Dis ; 2020 Oct 06.
Article in English | MEDLINE | ID: covidwho-1162009

ABSTRACT

BACKGROUND: Middle East respiratory syndrome (MERS) remains of global public health concern. Dromedary camels are the source of zoonotic infection. Over 70% of MERS coronavirus (MERS-CoV)-infected dromedaries are found in Africa but no zoonotic disease has been reported in Africa. We aimed to understand whether individuals with exposure to dromedaries in Africa had been infected by MERS-CoV. METHODS: Workers slaughtering dromedaries in an abattoir in Kano, Nigeria, were compared with abattoir workers without direct dromedary contact, non-abattoir workers from Kano, and controls from Guangzhou, China. Exposure to dromedaries was ascertained using a questionnaire. Serum and peripheral blood mononuclear cells (PBMCs) were tested for MERS-CoV specific neutralising antibody and T-cell responses. FINDINGS: None of the participants from Nigeria or Guangdong were MERS-CoV seropositive. 18 (30%) of 61 abattoir workers with exposure to dromedaries, but none of 20 abattoir workers without exposure (p=0·0042), ten non-abattoir workers or 24 controls from Guangzhou (p=0·0002) had evidence of MERS-CoV-specific CD4+ or CD8+ T cells in PBMC. T-cell responses to other endemic human coronaviruses (229E, OC43, HKU-1, and NL-63) were observed in all groups with no association with dromedary exposure. Drinking both unpasteurised camel milk and camel urine was significantly and negatively associated with T-cell positivity (odds ratio 0·07, 95% CI 0·01-0·54). INTERPRETATION: Zoonotic infection of dromedary-exposed individuals is taking place in Nigeria and suggests that the extent of MERS-CoV infections in Africa is underestimated. MERS-CoV could therefore adapt to human transmission in Africa rather than the Arabian Peninsula, where attention is currently focused. FUNDING: The National Science and Technology Major Project, National Institutes of Health.

8.
Fundamental Research ; 2021.
Article | WHO COVID | ID: covidwho-1116731

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a major public health threat worldwide Insight into protective and pathogenic aspects of SARS-CoV-2 immune responses is critical to work out effective therapeutics and develop vaccines for controlling the disease Here, we review the present literature describing the innate and adaptive immune responses including innate immune cells, cytokine responses, antibody responses and T cell responses against SARS-CoV-2 in human infection, as well as in AEC2-humanized mouse infection We also summarize the now known and unknown about the role of the SARS-CoV-2 immune responses By better understanding the mechanisms that drive the immune responses, we can tailor treatment strategies at specific disease stages and improve our response to this worldwide public health threat

9.
Genome Med ; 13(1): 30, 2021 02 22.
Article in English | MEDLINE | ID: covidwho-1097198

ABSTRACT

BACKGROUND: Since early February 2021, the causative agent of COVID-19, SARS-CoV-2, has infected over 104 million people with more than 2 million deaths according to official reports. The key to understanding the biology and virus-host interactions of SARS-CoV-2 requires the knowledge of mutation and evolution of this virus at both inter- and intra-host levels. However, despite quite a few polymorphic sites identified among SARS-CoV-2 populations, intra-host variant spectra and their evolutionary dynamics remain mostly unknown. METHODS: Using high-throughput sequencing of metatranscriptomic and hybrid captured libraries, we characterized consensus genomes and intra-host single nucleotide variations (iSNVs) of serial samples collected from eight patients with COVID-19. The distribution of iSNVs along the SARS-CoV-2 genome was analyzed and co-occurring iSNVs among COVID-19 patients were identified. We also compared the evolutionary dynamics of SARS-CoV-2 population in the respiratory tract (RT) and gastrointestinal tract (GIT). RESULTS: The 32 consensus genomes revealed the co-existence of different genotypes within the same patient. We further identified 40 intra-host single nucleotide variants (iSNVs). Most (30/40) iSNVs presented in a single patient, while ten iSNVs were found in at least two patients or identical to consensus variants. Comparing allele frequencies of the iSNVs revealed a clear genetic differentiation between intra-host populations from the respiratory tract (RT) and gastrointestinal tract (GIT), mostly driven by bottleneck events during intra-host migrations. Compared to RT populations, the GIT populations showed a better maintenance and rapid development of viral genetic diversity following the suspected intra-host bottlenecks. CONCLUSIONS: Our findings here illustrate the intra-host bottlenecks and evolutionary dynamics of SARS-CoV-2 in different anatomic sites and may provide new insights to understand the virus-host interactions of coronaviruses and other RNA viruses.


Subject(s)
/prevention & control , Genome, Viral/genetics , High-Throughput Nucleotide Sequencing/methods , Polymorphism, Single Nucleotide , /genetics , /virology , Gene Frequency , Genotype , Haplotypes , Host-Pathogen Interactions , Humans , Phylogeny , /physiology
11.
J Biol Chem ; : 100435, 2021 Feb 18.
Article in English | MEDLINE | ID: covidwho-1087033

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and Angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat) and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex.

12.
ACS Chem Biol ; 16(3): 491-500, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1084488

ABSTRACT

The outbreak of novel coronavirus SARS-CoV-2 has caused a worldwide threat to public health. COVID-19 patients with SARS-CoV-2 infection can develop clinical symptoms that are often confused with the infections of other respiratory pathogens. Sensitive and specific detection of SARS-CoV-2 with the ability to discriminate from other viruses is urgently needed for COVID-19 diagnosis. Herein, we streamlined a highly efficient CRISPR-Cas12a-based nucleic acid detection platform, termed Cas12a-linked beam unlocking reaction (CALIBURN). We show that CALIBURN could detect SARS-CoV-2 and other coronaviruses and influenza viruses with little cross-reactivity. Importantly, CALIBURN allowed accurate diagnosis of clinical samples with extremely low viral loads, which is a major obstacle for the clinical applications of existing CRISPR diagnostic platforms. When tested on the specimens from SARS-CoV-2-positive and negative donors, CALIBURN exhibited 73.0% positive and 19.0% presumptive positive rates and 100% specificity. Moreover, unlike existing CRISPR detection methods that were mainly restricted to respiratory specimens, CALIBURN displayed consistent performance across both respiratory and nonrespiratory specimens, suggesting its broad specimen compatibility. Finally, using a mouse model of SARS-CoV-2 infection, we demonstrated that CALIBURN allowed detection of coexisting pathogens without cross-reactivity from a single tissue specimen. Our results suggest that CALIBURN can serve as a versatile platform for the diagnosis of COVID-19 and other respiratory infectious diseases.


Subject(s)
Bacterial Proteins/genetics , /diagnosis , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Endodeoxyribonucleases/genetics , RNA, Viral/analysis , /chemistry , Adenoviridae/chemistry , Animals , Fluorescent Dyes/chemistry , Humans , Limit of Detection , Mice, Inbred BALB C , Nucleic Acid Amplification Techniques , RNA Probes/genetics , RNA, Viral/genetics , Specimen Handling , Spectrometry, Fluorescence
13.
J Med Chem ; 2021 Feb 01.
Article in English | MEDLINE | ID: covidwho-1057678

ABSTRACT

The outbreak of coronavirus disease 2019 (COVID-19) has resulted in a global pandemic due to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the time of this manuscript's publication, remdesivir is the only COVID-19 treatment approved by the United States Food and Drug Administration. However, its effectiveness is still under question due to the results of the large Solidarity Trial conducted by the World Health Organization. Herein, we report that the parent nucleoside of remdesivir, GS-441524, potently inhibits the replication of SARS-CoV-2 in Vero E6 and other cell lines. Challenge studies in both an AAV-hACE2 mouse model of SARS-CoV-2 and in mice infected with murine hepatitis virus, a closely related coronavirus, showed that GS-441524 was highly efficacious in reducing the viral titers in CoV-infected organs without notable toxicity. Our results support that GS-441524 is a promising and inexpensive drug candidate for treating of COVID-19 and other CoV diseases.

14.
J Exp Med ; 218(4)2021 04 05.
Article in English | MEDLINE | ID: covidwho-1035695

ABSTRACT

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2-specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2-infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2-specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2-infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.


Subject(s)
/immunology , Epitopes, T-Lymphocyte/immunology , Host-Pathogen Interactions/physiology , T-Lymphocytes/immunology , T-Lymphocytes/virology , /genetics , Animals , Antibodies, Neutralizing/blood , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/virology , Chlorocebus aethiops , Cross Reactions , Epitope Mapping , Interferon Type I/immunology , Interferon Type I/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Middle East Respiratory Syndrome Coronavirus/immunology , SARS Virus/immunology , /pathogenicity , Vero Cells
15.
Life Sci ; 269: 119046, 2021 Mar 15.
Article in English | MEDLINE | ID: covidwho-1030918

ABSTRACT

BACKGROUND: The pandemic of the coronavirus disease 2019 (COVID-19) has brought a global public health crisis. However, the pathogenesis underlying COVID-19 are barely understood. METHODS: In this study, we performed proteomic analyses of airway mucus obtained by bronchoscopy from severe COVID-19 patients. In total, 2351 and 2073 proteins were identified and quantified in COVID-19 patients and healthy controls, respectively. RESULTS: Among them, 92 differentiated expressed proteins (DEPs) (46 up-regulated and 46 down-regulated) were found with a fold change >1.5 or <0.67 and a p-value <0.05, and 375 proteins were uniquely present in airway mucus from COVID-19 patients. Pathway and network enrichment analyses revealed that the 92 DEPs were mostly associated with metabolic, complement and coagulation cascades, lysosome, and cholesterol metabolism pathways, and the 375 COVID-19 only proteins were mainly enriched in amino acid degradation (Valine, Leucine and Isoleucine degradation), amino acid metabolism (beta-Alanine, Tryptophan, Cysteine and Methionine metabolism), oxidative phosphorylation, phagosome, and cholesterol metabolism pathways. CONCLUSIONS: This study aims to provide fundamental data for elucidating proteomic changes of COVID-19, which may implicate further investigation of molecular targets directing at specific therapy.


Subject(s)
Amino Acids/metabolism , Mucus/virology , Proteins/metabolism , Aged , Bronchoscopy , Case-Control Studies , Cholesterol/metabolism , Critical Illness , Female , Humans , Male , Middle Aged , Proteomics , Severity of Illness Index
16.
Nat Commun ; 12(1): 250, 2021 01 11.
Article in English | MEDLINE | ID: covidwho-1019817

ABSTRACT

Understanding the mechanism for antibody neutralization of SARS-CoV-2 is critical for the development of effective therapeutics and vaccines. We recently isolated a large number of monoclonal antibodies from SARS-CoV-2 infected individuals. Here we select the top three most potent yet variable neutralizing antibodies for in-depth structural and functional analyses. Crystal structural comparisons reveal differences in the angles of approach to the receptor binding domain (RBD), the size of the buried surface areas, and the key binding residues on the RBD of the viral spike glycoprotein. One antibody, P2C-1F11, most closely mimics binding of receptor ACE2, displays the most potent neutralizing activity in vitro and conferred strong protection against SARS-CoV-2 infection in Ad5-hACE2-sensitized mice. It also occupies the largest binding surface and demonstrates the highest binding affinity to RBD. More interestingly, P2C-1F11 triggers rapid and extensive shedding of S1 from the cell-surface expressed spike glycoprotein, with only minimal such effect by the remaining two antibodies. These results offer a structural and functional basis for potent neutralization via disruption of the very first and critical steps for SARS-CoV-2 cell entry.


Subject(s)
/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/immunology , /immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Viral/immunology , Binding Sites , /virology , Disease Models, Animal , Epitopes , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Virus/immunology , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
17.
J Clin Invest ; 130(10): 5235-5244, 2020 10 01.
Article in English | MEDLINE | ID: covidwho-969923

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus 2019 (COVID-19) pneumonia. Little is known about the kinetics, tissue distribution, cross-reactivity, and neutralization antibody response in patients with COVID-19. Two groups of patients with RT-PCR-confirmed COVID-19 were enrolled in this study: 12 severely ill patients in intensive care units who needed mechanical ventilation and 11 mildly ill patients in isolation wards. Serial clinical samples were collected for laboratory detection. Results showed that most of the severely ill patients had viral shedding in a variety of tissues for 20-40 days after onset of disease (8/12, 66.7%), while the majority of mildly ill patients had viral shedding restricted to the respiratory tract and had no detectable virus RNA 10 days after onset (9/11, 81.8%). Mildly ill patients showed significantly lower IgM response compared with that of the severe group. IgG responses were detected in most patients in both the severe and mild groups at 9 days after onset, and remained at a high level throughout the study. Antibodies cross-reactive to SARS-CoV and SARS-CoV-2 were detected in patients with COVID-19 but not in patients with MERS. High levels of neutralizing antibodies were induced after about 10 days after onset in both severely and mildly ill patients which were higher in the severe group. SARS-CoV-2 pseudotype neutralization test and focus reduction neutralization test with authentic virus showed consistent results. Sera from patients with COVID-19 inhibited SARS-CoV-2 entry. Sera from convalescent patients with SARS or Middle East respiratory syndrome (MERS) did not. Anti-SARS-CoV-2 S and N IgG levels exhibited a moderate correlation with neutralization titers in patients' plasma. This study improves our understanding of immune response in humans after SARS-CoV-2 infection.


Subject(s)
Antibodies, Viral/blood , Betacoronavirus/metabolism , Coronavirus Infections/blood , Pneumonia, Viral/blood , Viral Load , Virus Shedding , Adult , Aged , Antibody Specificity , Cross Reactions , Female , Humans , Kinetics , Male , Middle Aged , Pandemics , Severity of Illness Index
18.
STAR Protocols ; : 100169, 2020.
Article in English | WHO COVID | ID: covidwho-917458

ABSTRACT

Summary Common laboratory mice such as BALB/c and C57BL/6 mice are not permissive to SARS-CoV2 infection Sensitization of laboratory mice with Adenovirus expressing human ACE2 (Ad5-hACE2) provides a rapid model for testing viral intervention in vivo Despite the lack of lethal outcome, Ad5-hACE2-sensitized mice show 20% weight loss on average upon viral challenge with infectious virus being detected at the site of sensitization This protocol describes the sensitization and subsequent infection of common laboratory mice for use in testing anti-viral interventions For complete details on the use and execution of this protocol, please refer to Sun et al (2020)

20.
Microbiol Resour Announc ; 9(8)2020 Feb 20.
Article in English | MEDLINE | ID: covidwho-833422

ABSTRACT

We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China.

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