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1.
Life Sci Alliance ; 5(4)2022 04.
Article in English | MEDLINE | ID: covidwho-1614505

ABSTRACT

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.


Subject(s)
COVID-19/diagnosis , In Situ Hybridization, Fluorescence/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/virology , Caco-2 Cells , Cell Line, Tumor , Chlorocebus aethiops , Humans , In Situ Hybridization/methods , Microscopy, Electron/methods , RNA, Viral/ultrastructure , Reproducibility of Results , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sensitivity and Specificity , Vero Cells , Virus Release/drug effects , Virus Release/genetics , Virus Release/physiology , Virus Replication/drug effects , Virus Replication/physiology
2.
Nature ; 595(7868): 596-599, 2021 07.
Article in English | MEDLINE | ID: covidwho-1322487

ABSTRACT

Biomolecular condensates have emerged as an important subcellular organizing principle1. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm2,3. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation4,5. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers6.


Subject(s)
/virology , Respiratory Syncytial Virus, Human/drug effects , Veratrum Alkaloids/pharmacology , Virus Replication/drug effects , Animals , Antiviral Agents/pharmacology , Cell Line , Female , Humans , Inclusion Bodies , Lung/virology , Mice , Mice, Inbred BALB C , Respiratory Syncytial Virus, Human/physiology , Transcription Factors , Viral Proteins
3.
Front Immunol ; 12: 683902, 2021.
Article in English | MEDLINE | ID: covidwho-1282386

ABSTRACT

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases. IMPORTANCE: Respiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.


Subject(s)
Antibodies, Viral/administration & dosage , Immunization, Passive , Immunoglobulin G/administration & dosage , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus, Human/immunology , Administration, Intranasal , Animals , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , Disease Models, Animal , Humans , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Lung/drug effects , Lung/virology , Neutralization Tests , Respiratory Syncytial Virus Infections/virology , Turbinates/drug effects , Turbinates/virology , Viral Fusion Proteins/immunology , Virus Replication/drug effects
4.
Eur J Clin Microbiol Infect Dis ; 40(10): 2235-2241, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1156953

ABSTRACT

We report evaluation of 30 assays' (17 rapid tests (RDTs) and 13 automated/manual ELISA/CLIA assay (IAs)) clinical performances with 2594 sera collected from symptomatic patients with positive SARS-CoV-2 rRT-PCR on a respiratory sample, and 1996 pre-epidemic serum samples expected to be negative. Only 4 RDT and 3 IAs fitted both specificity (> 98%) and sensitivity (> 90%) criteria according to French recommendations. Serology may offer valuable information during COVID-19 pandemic, but inconsistent performances observed among the 30 commercial assays evaluated, which underlines the importance of independent evaluation before clinical implementation.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/blood , Immunoassay/methods , SARS-CoV-2/immunology , COVID-19/virology , Humans , Immunoassay/economics , Immunoglobulin M/blood , Reagent Kits, Diagnostic , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
5.
BMC Geriatr ; 21(1): 120, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1090696

ABSTRACT

BACKGROUND: Acquired infections in hospitalized elderly people are a growing concern. In long-term care facilities with multiple staff and visitor contacts, virus outbreaks are a common challenge for infection prevention teams. Although several studies have reported nosocomial RSV outbreaks in long term care facilities, molecular epidemiology data are scarce. METHODS: RSV RNA was detected in respiratory samples from 19 patients in a long-term care hospital for elderly in Paris in March 2019 over a 3 weeks period. Genotyping was performed using nucleotide sequencing. Sociodemographic and clinical characteristics of cases part of a unique cluster, were retrospectively reviewed. RESULTS: Molecular investigation of theses RSV cases, revealed a unique cluster of 12 nosocomial cases in 2 adjacent wards. Mean age of these outbreak's cases was 89. All patients had underlying medical conditions. Seven exhibited lower respiratory symptoms and three experienced decompensation of underlying chronic heart condition. Two patients died. CONCLUSIONS: This case report highlights the importance of RSV in causing substantial disease in elderly in case of nosocomial outbreak and the contributions of molecular epidemiology in investigation and management of such outbreak.


Subject(s)
Cross Infection , Respiratory Syncytial Virus Infections , Aged , Cross Infection/diagnosis , Cross Infection/epidemiology , Disease Outbreaks , Hospitals , Humans , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Infections/epidemiology , Retrospective Studies
6.
Open Forum Infect Dis ; 7(11): ofaa394, 2020 Nov.
Article in English | MEDLINE | ID: covidwho-873048

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a current pandemic worldwide. This virus can reach all organs and disturbs the immune system, leading to a cytokine storm in severe forms. We aimed to report cutaneous features among coronavirus disease 2019 (COVID-19) hospitalized patients. METHODS: We performed a cross-sectional study on 1 given day among all patients hospitalized in acute care for COVID-19 and included all patients with cutaneous features. Follow-up 48 hours later was obtained. RESULTS: Among 59 adult patients hospitalized on the day of the study in an infectious diseases ward for SARS-CoV-2 infection who were confirmed by molecular assay and/or radiological findings (computed tomography scan), 40 were included. Several cutaneous manifestations were found: macular exanthema (80%), face edema (32%), livedo (13%), urticarial rash (8%), purpura (5%), oral lichenoid lesions (33%), and conjunctivitis (18%). Cutaneous biopsy was performed in 17 patients. Histological findings showed mast cell hyperplasia (100%), superficial perivascular infiltrate of lymphocytes (94%), and superficial edema (47%) consistent with capillary leak. CONCLUSIONS: Various dermatological signs can be encountered during COVID-19. A macular rash was the most frequent. All cutaneous features could be related to a vascular leak process.

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