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1.
PLoS One ; 17(4): e0265670, 2022.
Article in English | MEDLINE | ID: covidwho-1775445

ABSTRACT

Host biomarkers are increasingly being considered as tools for improved COVID-19 detection and prognosis. We recently profiled circulating host-encoded microRNA (miRNAs) during SARS-CoV-2 infection, revealing a signature that classified COVID-19 cases with 99.9% accuracy. Here we sought to develop a signature suited for clinical application by analyzing specimens collected using minimally invasive procedures. Eight miRNAs displayed altered expression in anterior nasal tissues from COVID-19 patients, with miR-142-3p, a negative regulator of interleukin-6 (IL-6) production, the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-30c-2-3p, miR-628-3p and miR-93-5p) independently classifies COVID-19 cases with 100% accuracy. This study further defines the host miRNA response to SARS-CoV-2 infection and identifies candidate biomarkers for improved COVID-19 detection.


Subject(s)
COVID-19 , MicroRNAs , Biomarkers , COVID-19/diagnosis , Gene Expression Profiling , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Respiratory System/metabolism , SARS-CoV-2/genetics
2.
J Virol ; 95(14): e0011121, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1358015

ABSTRACT

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.


Subject(s)
Ferrets , Influenza A Virus, H1N1 Subtype , Interferon-alpha/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Orthomyxoviridae Infections/immunology , Animals , Cell Line , Conserved Sequence , Disease Models, Animal , Ferrets/immunology , Ferrets/metabolism , Ferrets/virology , Humans , Models, Molecular , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/metabolism , Polymerase Chain Reaction , Sequence Analysis, Protein , Up-Regulation
3.
PLoS Pathog ; 17(7): e1009759, 2021 07.
Article in English | MEDLINE | ID: covidwho-1329138

ABSTRACT

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , COVID-19/genetics , MicroRNAs/genetics , SARS-CoV-2 , Adult , Aged , Animals , COVID-19/blood , Case-Control Studies , Diagnosis, Differential , Disease Models, Animal , Female , Ferrets , Gene Expression , Host Microbial Interactions/genetics , Humans , Influenza A Virus, H1N1 Subtype , Longitudinal Studies , Male , MicroRNAs/blood , Middle Aged , Orthomyxoviridae Infections/diagnosis , Orthomyxoviridae Infections/genetics , Pandemics , Supervised Machine Learning
4.
J Virol ; 95(15): e0032721, 2021 07 12.
Article in English | MEDLINE | ID: covidwho-1305507

ABSTRACT

The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains; previously C6orf106) was identified as a proviral factor for Hendra virus infection and was recently characterized to function as an inhibitor of type I interferon expression. Here, we have utilized transcriptome sequencing (RNA-seq) to define cellular pathways regulated by ILRUN in the context of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of Caco-2 cells. We find that inhibition of ILRUN expression by RNA interference alters transcription profiles of numerous cellular pathways, including upregulation of the SARS-CoV-2 entry receptor ACE2 and several other members of the renin-angiotensin aldosterone system. In addition, transcripts of the SARS-CoV-2 coreceptors TMPRSS2 and CTSL were also upregulated. Inhibition of ILRUN also resulted in increased SARS-CoV-2 replication, while overexpression of ILRUN had the opposite effect, identifying ILRUN as a novel antiviral factor for SARS-CoV-2 replication. This represents, to our knowledge, the first report of ILRUN as a regulator of the renin-angiotensin-aldosterone system (RAAS). IMPORTANCE There is no doubt that the current rapid global spread of COVID-19 has had significant and far-reaching impacts on our health and economy and will continue to do so. Research in emerging infectious diseases, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is growing rapidly, with new breakthroughs in the understanding of host-virus interactions to assist with the development of innovative and exciting therapeutic strategies. Here, we present the first evidence that modulation of the human protein-coding gene ILRUN functions as an antiviral factor for SARS-CoV-2 infection, likely through its newly identified role in regulating the expression of SARS-CoV-2 entry receptors ACE2, TMPRSS2, and CTSL. These data improve our understanding of biological pathways that regulate host factors critical to SARS-CoV-2 infection, contributing to the development of antiviral strategies to deal with the current SARS-CoV-2 pandemic.


Subject(s)
Angiotensin-Converting Enzyme 2/biosynthesis , COVID-19/metabolism , Down-Regulation , Gene Expression Regulation, Enzymologic , Neoplasm Proteins/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , Caco-2 Cells , Cathepsin L/biosynthesis , Cathepsin L/genetics , Chlorocebus aethiops , Humans , Neoplasm Proteins/genetics , Renin-Angiotensin System , SARS-CoV-2/genetics , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Vero Cells
5.
J Virol ; 95(14): e0011121, 2021 06 24.
Article in English | MEDLINE | ID: covidwho-1287245

ABSTRACT

The current fears of a future influenza pandemic have resulted in an increased emphasis on the development and testing of novel therapeutic strategies against the virus. Fundamental to this is the ferret model of influenza infection, which is critical in examining pathogenesis and treatment. Nevertheless, a precise evaluation of the efficacy of any treatment strategy in ferrets is reliant on understanding the immune response in this model. Interferon-inducible transmembrane proteins (IFITMs) are interferon-stimulated proteins shown to be critically important in the host immune response against viral infections. These proteins confer intrinsic innate immunity to pH-dependent viruses such as influenza viruses and can inhibit cytosolic entry of such viruses to limit the severity of infection following interferon upregulation. Mutations in IFITM genes in humans have been identified as key risk factors for worsened disease progression, particularly in the case of avian influenza viruses such as H7N9. While the IFITM genes of humans and mice have been well characterized, no studies have been conducted to classify the IFITM locus and interferon-driven upregulation of IFITMs in ferrets. Here, we show the architecture of the ferret IFITM locus and its synteny to the IFITM locus of other mammalian and avian species. Furthermore, we show that ferret IFITM1, -2, and -3 are functionally responsive to both interferon-α (IFN-α) and influenza virus stimulation. Thus, we show that ferret IFITMs exhibit interferon-stimulated properties similar to those shown in other species, furthering our knowledge of the innate immune response in the ferret model of human influenza virus infections. IMPORTANCE IFITM proteins can prevent the entry of several pH-dependent viruses, including high-consequence viruses such as HIV, influenza viruses, and SARS-coronaviruses. Mutations in these genes have been associated with worsened disease outcomes with mutations in their IFITM genes, highlighting these genes as potential disease risk factors. Ferrets provide a valuable tool to model infectious diseases; however, there is a critical shortage of information regarding their interferon-stimulated genes. We identified the putative ferret IFITM genes and mapped their complete gene locus. Thus, our study fills a critical gap in knowledge and supports the further use of the ferret model to explore the importance of IFITMs in these important diseases.


Subject(s)
Ferrets , Influenza A Virus, H1N1 Subtype , Interferon-alpha/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Orthomyxoviridae Infections/immunology , Animals , Cell Line , Conserved Sequence , Disease Models, Animal , Ferrets/immunology , Ferrets/metabolism , Ferrets/virology , Humans , Models, Molecular , Orthomyxoviridae Infections/genetics , Orthomyxoviridae Infections/metabolism , Polymerase Chain Reaction , Sequence Analysis, Protein , Up-Regulation
6.
Int J Mol Sci ; 22(7)2021 Mar 25.
Article in English | MEDLINE | ID: covidwho-1154425

ABSTRACT

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions; however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here, we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.


Subject(s)
Cytokines/genetics , RNA Processing, Post-Transcriptional , Ribosomes/metabolism , Ribosomes/virology , SARS-CoV-2/immunology , Transcription Factors/genetics , Animals , Antiviral Agents/antagonists & inhibitors , Cell Line, Tumor , Chlorocebus aethiops , Computational Biology , Cytokines/metabolism , Epithelial Cells/immunology , Epithelial Cells/virology , Gene Expression Profiling , Host Microbial Interactions , Humans , Immunity, Innate/genetics , Lung/immunology , Lung/virology , RNA, Messenger/metabolism , RNA-Seq , Ribosomes/genetics , SARS-CoV-2/metabolism , Transcription Factors/metabolism , Transcriptome , Vero Cells
7.
J Virol Methods ; 286: 113977, 2020 12.
Article in English | MEDLINE | ID: covidwho-800505

ABSTRACT

The development of medical countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires robust viral assays. Here we have adapted a protocol for polyethylene glycol (PEG)-mediated precipitation of SARS-CoV-2 stocks without the need for ultracentrifugation. Virus precipitation resulted in a ∼1.5 log10 increase in SARS-CoV-2 titres of virus prepared in VeroE6 cells and enabled the infection of several immortalized human cell lines (Caco-2 and Calu-3) at a high multiplicity of infection not practically achievable without virus concentration. This protocol underscores the utility of PEG-mediated precipitation for SARS-CoV-2 and provides a resource for a range of coronavirus research areas.


Subject(s)
Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Pneumonia, Viral/virology , Polyethylene Glycols/chemistry , Animals , COVID-19 , COVID-19 Testing , Caco-2 Cells , Chlorocebus aethiops , Coronavirus Infections/diagnosis , Humans , Pandemics , Pneumonia, Viral/diagnosis , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Ultracentrifugation/methods , Vero Cells
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