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1.
Virol J ; 19(1): 76, 2022 Apr 26.
Article in English | MEDLINE | ID: covidwho-1817229

ABSTRACT

BACKGROUND: During the ongoing Covid-19 pandemic caused by the emerging virus SARS-CoV-2, research in the field of coronaviruses has expanded tremendously. The genome of SARS-CoV-2 has rapidly acquired numerous mutations, giving rise to several Variants of Concern (VOCs) with altered epidemiological, immunological, and pathogenic properties. METHODS: As cell culture models are important tools to study viruses, we investigated replication kinetics and infectivity of SARS-CoV-2 in the African Green Monkey-derived Vero E6 kidney cell line and the two human cell lines Caco-2, a colon epithelial carcinoma cell line, and the airway epithelial carcinoma cell line Calu-3. We assessed viral RNA copy numbers and infectivity of viral particles in cell culture supernatants at different time points ranging from 2 to 96 h post-infection. RESULTS: We here describe a systematic comparison of growth kinetics of the five SARS-CoV-2 VOCs Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, and Omicron/B.1.1.529 and a non-VOC/B.1.1 strain on three different cell lines to provide profound information on the differential behaviour of VOCs in different cell lines for researchers worldwide. We show distinct differences in viral replication kinetics of the SARS-CoV-2 non-VOC and five VOCs on the three cell culture models Vero E6, Caco-2, and Calu-3. CONCLUSION: This is the first systematic comparison of all SARS-CoV-2 VOCs on three different cell culture models. This data provides support for researchers worldwide in their experimental design for work on SARS-CoV-2. It is recommended to perform virus isolation and propagation on Vero E6 while infection studies or drug screening and antibody-based assays should rather be conducted on the human cell lines Caco-2 and Calu-3.


Subject(s)
COVID-19 , Carcinoma , Caco-2 Cells , Cell Culture Techniques , Chlorocebus aethiops , Humans , Kinetics , Pandemics , SARS-CoV-2/genetics
2.
MAbs ; 14(1): 2060724, 2022.
Article in English | MEDLINE | ID: covidwho-1774258

ABSTRACT

As of early 2022, the coronavirus disease 2019 (COVID-19) pandemic remains a substantial global health concern. Different treatments for COVID-19, such as anti-COVID-19 neutralizing monoclonal antibodies (mAbs), have been developed under tight timelines. Not only mAb product and clinical development but also chemistry, manufacturing, and controls (CMC) process development at pandemic speed are required to address this highly unmet patient need. CMC development consists of early- and late-stage process development to ensure sufficient mAb manufacturing yield and consistent product quality for patient safety and efficacy. Here, we report a case study of late-stage cell culture process development at pandemic speed for mAb1 and mAb2 production as a combination therapy for a highly unmet patient treatment. We completed late-stage cell culture process characterization (PC) within approximately 4 months from the cell culture process definition to the initiation of the manufacturing process performance qualification (PPQ) campaign for mAb1 and mAb2, in comparison to a standard one-year PC timeline. Different strategies were presented in detail at different PC steps, i.e., pre-PC risk assessment, scale-down model development and qualification, formal PC experiments, and in-process control strategy development for a successful PPQ campaign that did not sacrifice quality. The strategies we present may be applied to accelerate late-stage process development for other biologics to reduce timelines.


Subject(s)
COVID-19 , Pandemics , Animals , CHO Cells , COVID-19/prevention & control , Cell Culture Techniques , Cricetinae , Cricetulus , Humans
3.
Int J Mol Sci ; 23(7)2022 Mar 22.
Article in English | MEDLINE | ID: covidwho-1753510

ABSTRACT

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is a common clinical problem, leading to significant morbidity and mortality, and no effective pharmacotherapy exists. The problem of ARDS causing mortality became more apparent during the COVID-19 pandemic. Biotherapeutic products containing multipotent mesenchymal stromal cell (MMSC) secretome may provide a new therapeutic paradigm for human healthcare due to their immunomodulating and regenerative abilities. The content and regenerative capacity of the secretome depends on cell origin and type of cultivation (two- or three-dimensional (2D/3D)). In this study, we investigated the proteomic profile of the secretome from 2D- and 3D-cultured placental MMSC and lung fibroblasts (LFBs) and the effect of inhalation of freeze-dried secretome on survival, lung inflammation, lung tissue regeneration, fibrin deposition in a lethal ALI model in mice. We found that three inhaled administrations of freeze-dried secretome from 2D- and 3D-cultured placental MMSC and LFB protected mice from death, restored the histological structure of damaged lungs, and decreased fibrin deposition. At the same time, 3D MMSC secretome exhibited a more pronounced trend in lung recovery than 2D MMSC and LFB-derived secretome in some measures. Taking together, these studies show that inhalation of cell secretome may also be considered as a potential therapy for the management of ARDS in patients suffering from severe pneumonia, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, their effectiveness requires further investigation.


Subject(s)
Acute Lung Injury , COVID-19 , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Pneumonia , Respiratory Distress Syndrome , Acute Lung Injury/therapy , Animals , COVID-19/therapy , Cell Culture Techniques , Female , Fibrin , Humans , Mesenchymal Stem Cell Transplantation/methods , Mice , Pandemics , Placenta , Pregnancy , Proteomics , Respiratory Distress Syndrome/therapy , SARS-CoV-2
4.
Viruses ; 12(6)2020 06 08.
Article in English | MEDLINE | ID: covidwho-1726020

ABSTRACT

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.


Subject(s)
Betacoronavirus/drug effects , Coronavirus Infections/virology , Pneumonia, Viral/virology , Virus Inactivation/drug effects , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , COVID-19 , Cell Culture Techniques/methods , Chlorocebus aethiops , Containment of Biohazards/methods , Containment of Biohazards/standards , Humans , Nasopharynx/virology , Pandemics , RNA, Viral/isolation & purification , SARS-CoV-2 , Specimen Handling/methods , Vero Cells , Viral Load/methods
5.
Viruses ; 14(3)2022 02 28.
Article in English | MEDLINE | ID: covidwho-1715784

ABSTRACT

Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Cell Culture Techniques , Humans , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , SARS-CoV-2/genetics
6.
Int J Med Mushrooms ; 24(2): 23-30, 2022.
Article in English | MEDLINE | ID: covidwho-1690631

ABSTRACT

The antiviral properties of water extracts from pharmaceutical raw materials of the chaga mushroom, Inonotus obliquus, were studied against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). All studies with infectious materials were carried out in an isolated virological laboratory of the State Research Center of Virology and Biotechnology Vector of Rospotrebnadzor, which has a sanitary and epidemiological conclusion for the right to work with pathogenic biological agents of I-II pathogenicity groups. Antiviral activity was determined by the ability of I. obliquus water extracts to inhibit the replication of SARS-CoV-2 (nCoV/Victoria /1/2020 strain) in Vero E6 and Vero cell cultures. The results of these studies showed that water extracts of I. obliquus are characterized by low toxicity in Vero and Vero E6 cell cultures and have antiviral activity against SARS-CoV-2. The 50% inhibitory concentration ranged from 0.75 to 11.6 µg/mL. A patent for the invention was received (Patent RU, 2741714 C 1, 2021).


Subject(s)
Agaricales , Basidiomycota , COVID-19 , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Cell Culture Techniques , Chlorocebus aethiops , Inonotus , SARS-CoV-2 , Vero Cells , Water
7.
J Virol ; 95(24): e0117421, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1691429

ABSTRACT

Defective interfering particles (DIPs) of influenza A virus (IAV) are naturally occurring mutants that have an internal deletion in one of their eight viral RNA (vRNA) segments, rendering them propagation-incompetent. Upon coinfection with infectious standard virus (STV), DIPs interfere with STV replication through competitive inhibition. Thus, DIPs are proposed as potent antivirals for treatment of the influenza disease. To select corresponding candidates, we studied de novo generation of DIPs and propagation competition between different defective interfering (DI) vRNAs in an STV coinfection scenario in cell culture. A small-scale two-stage cultivation system that allows long-term semi-continuous propagation of IAV and its DIPs was used. Strong periodic oscillations in virus titers were observed due to the dynamic interaction of DIPs and STVs. Using next-generation sequencing, we detected a predominant formation and accumulation of DI vRNAs on the polymerase-encoding segments. Short DI vRNAs accumulated to higher fractions than longer ones, indicating a replication advantage, yet an optimum fragment length was observed. Some DI vRNAs showed breaking points in a specific part of their bundling signal (belonging to the packaging signal), suggesting its dispensability for DI vRNA propagation. Over a total cultivation time of 21 days, several individual DI vRNAs accumulated to high fractions, while others decreased. Using reverse genetics for IAV, purely clonal DIPs derived from highly replicating DI vRNAs were generated. We confirm that these DIPs exhibit a superior in vitro interfering efficacy compared to DIPs derived from lowly accumulated DI vRNAs and suggest promising candidates for efficacious antiviral treatment. IMPORTANCE Defective interfering particles (DIPs) emerge naturally during viral infection and typically show an internal deletion in the viral genome. Thus, DIPs are propagation-incompetent. Previous research suggests DIPs as potent antiviral compounds for many different virus families due to their ability to interfere with virus replication by competitive inhibition. For instance, the administration of influenza A virus (IAV) DIPs resulted in a rescue of mice from an otherwise lethal IAV dose. Moreover, no apparent toxic effects were observed when only DIPs were administered to mice and ferrets. IAV DIPs show antiviral activity against many different IAV strains, including pandemic and highly pathogenic avian strains, and even against nonhomologous viruses, such as SARS-CoV-2, by stimulation of innate immunity. Here, we used a cultivation/infection system, which exerted selection pressure toward accumulation of highly competitive IAV DIPs. These DIPs showed a superior interfering efficacy in vitro, and we suggest them for effective antiviral therapy.


Subject(s)
Antiviral Agents/pharmacology , Drug Design/methods , Influenza A virus , Influenza, Human/virology , RNA, Viral , Animals , Cell Culture Techniques , Cell Line , Defective Viruses/genetics , Dogs , Gene Deletion , Genome, Viral , Humans , Immunity, Innate/drug effects , Madin Darby Canine Kidney Cells , Oscillometry , Real-Time Polymerase Chain Reaction , Viral Load/drug effects , Virus Replication/drug effects
8.
Int J Mol Sci ; 23(3)2022 Feb 07.
Article in English | MEDLINE | ID: covidwho-1686815

ABSTRACT

Quantitative and functional analysis of mononuclear leukocyte populations is an invaluable tool to understand the role of the immune system in the pathogenesis of a disease. Cryopreservation of mononuclear cells (MNCs) is routinely used to guarantee similar experimental conditions. Immune cells react differently to cryopreservation, and populations and functions of immune cells change during the process of freeze-thawing. To allow for a setup that preserves cell number and function optimally, we tested four different cryopreservation media. MNCs from 15 human individuals were analyzed. Before freezing and after thawing, the distribution of leukocytes was quantified by flow cytometry. Cultured cells were stimulated using lipopolysaccharide, and their immune response was quantified by flow cytometry, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). Ultimately, the performance of the cryopreservation media was ranked. Cell recovery and viability were different between the media. Cryopreservation led to changes in the relative number of monocytes, T cells, B cells, and their subsets. The inflammatory response of MNCs was altered by cryopreservation, enhancing the basal production of inflammatory cytokines. Different cryopreservation media induce biases, which needs to be considered when designing a study relying on cryopreservation. Here, we provide an overview of four different cryopreservation media for choosing the optimal medium for a specific task.


Subject(s)
Cell Culture Techniques/methods , Cryopreservation/methods , Leukocytes, Mononuclear/cytology , Cell Survival , Cells, Cultured , Female , Flow Cytometry , Humans , Leukocyte Count , Leukocytes, Mononuclear/metabolism , Male
9.
J Hazard Mater ; 428: 128251, 2022 04 15.
Article in English | MEDLINE | ID: covidwho-1683301

ABSTRACT

This study evaluated the inactivation of SARS-CoV-2, the virus responsible for COVID-19, by ozone using virus grown in cell culture media either dried on surfaces (plastic, glass, stainless steel, copper, and coupons of ambulance seat and floor) or suspended in liquid. Treatment in liquid reduced SARS-CoV-2 at a rate of 0.92 ± 0.11 log10-reduction per ozone CT dose(mg min/L); where CT is ozone concentration times exposure time. On surface, the synergistic effect of CT and relative humidity (RH) was key to virus inactivation; the rate varied from 0.01 to 0.27 log10-reduction per ozone CT value(g min/m3) as RH varied from 17% to 70%. Depletion of ozone by competitive reactions with the medium constituents, mass transfer limiting the penetration of ozone to the bulk of the medium, and occlusion of the virus in dried matrix were postulated as potential mechanisms that reduce ozone efficacy. RH70% was found plausible since it provided the highest disinfection rate while being below the critical RH that promotes mould growth in buildings. In conclusion, through careful choice of (CT, RH), gaseous ozone is effective against SARS-CoV-2 and our results are of significance to a growing field where ozone is applied to control the spread of COVID-19.


Subject(s)
COVID-19 , Ozone , Cell Culture Techniques , Humans , SARS-CoV-2 , Virus Inactivation
10.
Chemistry ; 28(4): e202200039, 2022 Jan 19.
Article in English | MEDLINE | ID: covidwho-1633218

ABSTRACT

Invited for the cover of this issue are Manfred Marschall, Svetlana B. Tsogoeva and co-workers at Friedrich-Alexander University of Erlangen-Nürnberg. The image depicts a new anti-SARS-CoV-2 compound in front of SARS-CoV-2 viruses. Read the full text of the article at 10.1002/chem.202103861.


Subject(s)
COVID-19 , Quinolines , Cell Culture Techniques , Humans , SARS-CoV-2
11.
Int J Mol Sci ; 23(2)2022 Jan 13.
Article in English | MEDLINE | ID: covidwho-1625839

ABSTRACT

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.


Subject(s)
COVID-19/virology , Nasal Mucosa/cytology , Nasal Mucosa/virology , Tissue Culture Techniques/methods , Adolescent , Adult , Angiotensin-Converting Enzyme 2/metabolism , Cell Culture Techniques , Cell Differentiation , Epithelial Cells/cytology , Epithelial Cells/virology , Female , Humans , Male , Middle Aged , Models, Biological , SARS-CoV-2 , Virus Internalization
12.
Sci Total Environ ; 815: 152914, 2022 Apr 01.
Article in English | MEDLINE | ID: covidwho-1612004

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been extensively detected in raw wastewater in studies exploring wastewater-based epidemiology (WBE) for early warning purposes. Nonetheless, only a few limited studies investigated the presence of SARS-CoV-2 in treated wastewaters to determine the potential health risks across the water cycle. The detection of SARS-CoV-2 has been done mostly by RT-qPCR and ddPCR, which only provides information on the presence of nucleic acids rather than information on potential infectivity. In this study, we set to develop and evaluate the use of viability RT-qPCR for the selective discrimination and surveillance of infectious SARS-CoV-2 in secondary-treated wastewater. Enzymatic (nuclease) and viability dye (Reagent D) pretreatments were applied to infer infectivity through RT-qPCR using porcine epidemic diarrhea virus (PEDV) as a CoV surrogate. Infectivity tests were first performed on PEDV purified RNA, then on infectious and heat-inactivated PEDV, and finally on heat inactivated PEDV spiked in concentrated secondary-treated wastewater. The two viability RT-qPCR methods were then applied to 27 secondary-treated wastewater samples positive for SARS-CoV-2 RNA at the outlet of five large urban wastewater treatment plants in Portugal. Reagent D pretreatment showed similar behavior to cell culture for heat-inactivated PEDV and both viability RT-qPCR methods performed comparably to VERO E6 cell culture for SARS-CoV-2 present in secondary-treated wastewater, eliminating completely the RT-qPCR signal. Our study demonstrated the lack of infectious SARS-CoV-2 viral particles on secondary-treated wastewater through the application of two pretreatment methods for the rapid inference of infectivity through RT-qPCR, showing their potential application in environmental screening. This study addressed a knowledge gap on the public health risks of SARS-CoV-2 across the water cycle.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Cell Culture Techniques , Humans , RNA, Viral , Swine , Waste Water
13.
Cell Rep ; 37(13): 110167, 2021 12 28.
Article in English | MEDLINE | ID: covidwho-1596401

ABSTRACT

Cross-reactivity and direct killing of target cells remain underexplored for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific CD8+ T cells. Isolation of T cell receptors (TCRs) and overexpression in allogeneic cells allows for extensive T cell reactivity profiling. We identify SARS-CoV-2 RNA-dependent RNA polymerase (RdRp/NSP12) as highly conserved, likely due to its critical role in the virus life cycle. We perform single-cell TCRαß sequencing in human leukocyte antigen (HLA)-A∗02:01-restricted, RdRp-specific T cells from SARS-CoV-2-unexposed individuals. Human T cells expressing these TCRαß constructs kill target cell lines engineered to express full-length RdRp. Three TCR constructs recognize homologous epitopes from common cold coronaviruses, indicating CD8+ T cells can recognize evolutionarily diverse coronaviruses. Analysis of individual TCR clones may help define vaccine epitopes that can induce long-term immunity against SARS-CoV-2 and other coronaviruses.


Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/immunology , HLA-A2 Antigen/immunology , SARS-CoV-2/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , COVID-19/therapy , Cell Culture Techniques , Cross Reactions/immunology , Epitopes, T-Lymphocyte/immunology , HLA-A Antigens/immunology , HLA-A2 Antigen/genetics , Humans , Immunodominant Epitopes/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , RNA, Viral/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology
14.
Vet Microbiol ; 264: 109299, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1559479

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteric coronavirus that causes severe diarrhea in piglets and results in serious economic losses. There are no effective vaccines and antiviral drugs to prevent and treat PDCoV infection currently. Griffithsin (GRFT) is a lectin with potent antiviral activity against enveloped viruses because of its ability to specifically bind N-linked high-mannose oligosaccharides. GRFT has been reported to possess antiviral activity against severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and porcine epidemic diarrhea virus (PEDV). Here, we first confirmed the antiviral activity of GRFT against PDCoV in vitro. The infected cells (%) and virus titers were significantly decreased at concentration 1 µg/mL or above of GRFT. Time-course experiments revealed that GRFT inhibits PDCoV infection at the adsorption and penetration step. GRFT binding to PDCoV spike (S) protein on the surface wraps the virus and blocks its entry. The outstanding antiviral potency indicates that GRFT has the potential value as a candidate drug for the prevention and treatment of PDCoV infection.


Subject(s)
Deltacoronavirus , Plant Lectins , Animals , Antiviral Agents/pharmacology , Cell Culture Techniques/veterinary , Coronavirus Infections/drug therapy , Coronavirus Infections/veterinary , Deltacoronavirus/drug effects , Plant Lectins/pharmacology , Swine , Swine Diseases/drug therapy
15.
Sci Rep ; 11(1): 23465, 2021 12 06.
Article in English | MEDLINE | ID: covidwho-1556248

ABSTRACT

Human coronavirus NL63 (HCoV-NL63) mainly affects young children and immunocompromised patients, causing morbidity and mortality in a subset of patients. Since no specific treatment is available, this study aims to explore the anti-SARS-CoV-2 agents including favipiravir and remdesivir for treating HCoV-NL63 infection. We first successfully modelled the 3D structure of HCoV-NL63 RNA-dependent RNA polymerase (RdRp) based on the experimentally solved SARS-CoV-2 RdRp structure. Molecular docking indicated that favipiravir has similar binding affinities to SARS-CoV-2 and HCoV-NL63 RdRp with LibDock scores of 75 and 74, respectively. The LibDock scores of remdesivir to SARS-CoV-2 and HCoV-NL63 were 135 and 151, suggesting that remdesivir may have a higher affinity to HCoV-NL63 compared to SARS-CoV-2 RdRp. In cell culture models infected with HCoV-NL63, both favipiravir and remdesivir significantly inhibited viral replication and production of infectious viruses. Overall, remdesivir compared to favipiravir is more potent in inhibiting HCoV-NL63 in cell culture. Importantly, there is no evidence of resistance development upon long-term exposure to remdesivir. Furthermore, combining favipiravir or remdesivir with the clinically used antiviral cytokine interferon-alpha resulted in synergistic effects. These findings provided a proof-of-concept that anti-SARS-CoV-2 drugs, in particular remdesivir, have the potential to be repurposed for treating HCoV-NL63 infection.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Amides/chemistry , Antiviral Agents/chemistry , Coronavirus NL63, Human/enzymology , Pyrazines/chemistry , RNA-Dependent RNA Polymerase/chemistry , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Adenosine Monophosphate/pharmacology , Alanine/chemistry , Alanine/metabolism , Alanine/pharmacology , Amides/metabolism , Amides/pharmacology , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Binding Sites , Cell Culture Techniques , Cell Line , Coronavirus NL63, Human/physiology , Haplorhini , Humans , Molecular Docking Simulation , Pyrazines/metabolism , Pyrazines/pharmacology , RNA-Dependent RNA Polymerase/metabolism , Virus Replication/drug effects
16.
J Cell Mol Med ; 26(1): 235-238, 2022 01.
Article in English | MEDLINE | ID: covidwho-1555067

ABSTRACT

Due to the restrictions in accessing research laboratories and the challenges in providing proper storage and transportation of cells during the COVID-19 pandemic, having an effective and feasible mean to solve these challenges would be of immense help. Therefore, we developed a 3D culture setting of cancer cells using alginate beads and tested its effectiveness in different storage and transportation conditions. The viability and proliferation of cancer cells were assessed using trypan blue staining and quantitative CCK-8 kit, respectively. The developed beads allowed cancer cells survival up to 4 weeks with less frequent maintenance measures such as change of the culture media or subculture of cells. In addition, the recovery of cancer cells and proliferation pattern were significantly faster with better outcomes in the developed 3D alginate beads compared to the standard cryopreservation of cells or the 2D culture conditions. The 3D alginate beads also supported the viability of cells while the shipment at room temperature for a duration of up to 5 days with no humidity or CO2  support. Therefore, 3D culture in alginate beads can be used to store or ship biological cells with ease at room temperature with minimal preparations.


Subject(s)
Alginates/pharmacology , COVID-19/epidemiology , Cell Culture Techniques , Hydrogels/pharmacology , Osteoblasts/drug effects , A549 Cells , Alginates/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Hep G2 Cells , Humans , Hydrogels/chemistry , Osteoblasts/cytology , SARS-CoV-2/pathogenicity , Time Factors
17.
Chemistry ; 28(4): e202103861, 2022 Jan 19.
Article in English | MEDLINE | ID: covidwho-1549185

ABSTRACT

The presently ongoing pandemic of human SARS-CoV-2 infections (COVID-19) presents an enormous challenge in surveillance, vaccine and antiviral drug development. Here we report the synthesis of new bioactive quinoline-morpholine hybrid compounds and their virological evaluation, which proves pronounced cell culture-based inhibitory profile against SARS-CoV-2. Thus, selected quinoline compounds may suggest specific hit-to-lead development.


Subject(s)
COVID-19 , Quinolines , Antiviral Agents/pharmacology , Cell Culture Techniques , Humans , Pandemics , Quinolines/pharmacology , SARS-CoV-2
18.
J Virol ; 96(3): e0183721, 2022 02 09.
Article in English | MEDLINE | ID: covidwho-1546443

ABSTRACT

Research activities with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are currently permitted only under biosafety level 3 (BSL3) containment. Here, we report the development of a single-cycle infectious SARS-CoV-2 virus replicon particle (VRP) system with a luciferase and green fluorescent protein (GFP) dual reporter that can be safely handled in BSL2 laboratories to study SARS-CoV-2 biology. The spike (S) gene of SARS-CoV-2 encodes the envelope glycoprotein, which is essential for mediating infection of new host cells. Through deletion and replacement of this essential S gene with a luciferase and GFP dual reporter, we have generated a conditional SARS-CoV-2 mutant (ΔS-VRP) that produces infectious particles only in cells expressing a viral envelope glycoprotein of choice. Interestingly, we observed more efficient production of infectious particles in cells expressing vesicular stomatitis virus (VSV) glycoprotein G [ΔS-VRP(G)] than in cells expressing other viral glycoproteins, including S. We confirmed that infection from ΔS-VRP(G) is limited to a single round and can be neutralized by anti-VSV serum. In our studies with ΔS-VRP(G), we observed robust expression of both luciferase and GFP reporters in various human and murine cell types, demonstrating that a broad variety of cells can support intracellular replication of SARS-CoV-2. In addition, treatment of ΔS-VRP(G)-infected cells with either of the anti-CoV drugs remdesivir (nucleoside analog) and GC376 (CoV 3CL protease inhibitor) resulted in a robust decrease in both luciferase and GFP expression in a drug dose- and cell-type-dependent manner. Taken together, our findings show that we have developed a single-cycle infectious SARS-CoV-2 VRP system that serves as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment. IMPORTANCE Due to the highly contagious nature of SARS-CoV-2 and the lack of immunity in the human population, research on SARS-CoV-2 has been restricted to biosafety level 3 laboratories. This has greatly limited participation of the broader scientific community in SARS-CoV-2 research and thus has hindered the development of vaccines and antiviral drugs. By deleting the essential spike gene in the viral genome, we have developed a conditional mutant of SARS-CoV-2 with luciferase and fluorescent reporters, which can be safely used under biosafety level 2 conditions. Our single-cycle infectious SARS-CoV-2 virus replicon system can serve as a versatile platform to study SARS-CoV-2 intracellular biology and to perform high-throughput screening of antiviral drugs under BSL2 containment.


Subject(s)
Genetic Engineering , Recombination, Genetic , Replicon , SARS-CoV-2/genetics , COVID-19/virology , Cell Culture Techniques , Cell Line , Containment of Biohazards/standards , Genes, Reporter , Humans , Laboratories/standards , Viral Proteins/genetics , Virus Replication
20.
Nat Protoc ; 16(11): 5171-5192, 2021 11.
Article in English | MEDLINE | ID: covidwho-1500485

ABSTRACT

Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research.


Subject(s)
Cell Differentiation , Gastrointestinal Tract , Organoids , Animals , Cell Culture Techniques , Epithelial Cells/cytology , Mice
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