Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
1.
Methods Mol Biol ; 2452: 111-129, 2022.
Article in English | MEDLINE | ID: covidwho-1844263

ABSTRACT

In late 2019, the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. Since its emergence, SARS-CoV-2 has been responsible for a world-wide pandemic resulting in over 80 million infections and over 1.8 million deaths. The severity of the pandemic has prompted widespread research efforts to more fully understand SARS-CoV-2 and the disease it causes, COVID-19. Research into this novel virus will be facilitated by the availability of clearly described and effective protocols that enable the propagation and quantification of infectious virus. Here, we describe protocols for the propagation of SARS-CoV-2 in Vero E6 cells as well as two human cells lines, the intestinal epithelial Caco-2 cell line and the respiratory epithelial Calu-3 cell line. Additionally, we provide protocols for the quantification of SARS-CoV-2 by plaque assays and immunofocus forming assays in Vero E6 cells utilizing liquid overlays. These protocols provide a foundation for laboratories acquiring the ability to study SARS-CoV-2 to address this ongoing pandemic.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Caco-2 Cells , Chlorocebus aethiops , Humans , Pandemics , Vero Cells
2.
Eur J Med Chem ; 226: 113862, 2021 Dec 15.
Article in English | MEDLINE | ID: covidwho-1433178

ABSTRACT

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 µM; IC50(Calu3) = 0.24 µM), 3'-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 µM; IC50(Calu3) = 0.18 µM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 µM; IC50(Calu3) = 0.25 µM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 µM; IC50(Calu3) = 0.06 µM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , COVID-19/virology , Ebolavirus/drug effects , Fatty Acids/chemistry , SARS-CoV-2/drug effects , Adenosine Monophosphate/chemical synthesis , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/chemical synthesis , Alanine/chemistry , Alanine/pharmacology , Antiviral Agents/chemistry , Humans , SARS-CoV-2/isolation & purification
3.
Cell Rep ; 36(5): 109479, 2021 08 03.
Article in English | MEDLINE | ID: covidwho-1328702

ABSTRACT

Coronaviruses rely on host membranes for entry, establishment of replication centers, and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we test small molecule inhibitors that target the PI3 kinase VPS34 or fatty acid metabolism for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity. Our studies determine that compounds targeting VPS34 are potent SARS-CoV-2 inhibitors. Mechanistic studies with compounds targeting multiple steps up- and downstream of fatty acid synthase (FASN) identify the importance of triacylglycerol production and protein palmitoylation as requirements for efficient viral RNA synthesis and infectious virus production. Further, FASN knockout results in significantly impaired SARS-CoV-2 replication that can be rescued with fatty acid supplementation. Together, these studies clarify roles for VPS34 and fatty acid metabolism in SARS-CoV-2 replication and identify promising avenues for the development of countermeasures against SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/virology , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Lipid Metabolism/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects , Aminopyridines/pharmacology , Animals , Caco-2 Cells , Cell Line , Chlorocebus aethiops , Class III Phosphatidylinositol 3-Kinases/metabolism , Fatty Acid Synthases/drug effects , Fatty Acid Synthases/genetics , Gene Knockout Techniques , Humans , Lipoylation/drug effects , Pyrimidines/pharmacology , RNA, Viral/metabolism , Triglycerides/metabolism , Vero Cells
4.
Viruses ; 13(3)2021 03 11.
Article in English | MEDLINE | ID: covidwho-1181628

ABSTRACT

The ongoing SARS-CoV-2 pandemic has resulted in an increased need for technologies capable of efficiently disinfecting public spaces as well as personal protective equipment. UV light disinfection is a well-established method for inactivating respiratory viruses. Here, we have determined that broad-spectrum, pulsed UV light is effective at inactivating SARS-CoV-2 on multiple surfaces in vitro. For hard, non-porous surfaces, we observed that SARS-CoV-2 was inactivated to undetectable levels on plastic and glass with a UV dose of 34.9 mJ/cm2 and stainless steel with a dose of 52.5 mJ/cm2. We also observed that broad-spectrum, pulsed UV light is effective at reducing SARS-CoV-2 on N95 respirator material to undetectable levels with a dose of 103 mJ/cm2. We included UV dosimeter cards that provide a colorimetric readout of UV dose and demonstrated their utility as a means to confirm desired levels of exposure were reached. Together, the results presented here demonstrate that broad-spectrum, pulsed UV light is an effective technology for the in vitro inactivation of SARS-CoV-2 on multiple surfaces.


Subject(s)
COVID-19/virology , Disinfection/methods , Masks/virology , SARS-CoV-2/radiation effects , Virus Inactivation/radiation effects , COVID-19/prevention & control , Disinfection/instrumentation , Humans , SARS-CoV-2/physiology , Ultraviolet Rays
5.
Viruses ; 13(3):460, 2021.
Article in English | MDPI | ID: covidwho-1126017

ABSTRACT

The ongoing SARS-CoV-2 pandemic has resulted in an increased need for technologies capable of efficiently disinfecting public spaces as well as personal protective equipment. UV light disinfection is a well-established method for inactivating respiratory viruses. Here, we have determined that broad-spectrum, pulsed UV light is effective at inactivating SARS-CoV-2 on multiple surfaces in vitro. For hard, non-porous surfaces, we observed that SARS-CoV-2 was inactivated to undetectable levels on plastic and glass with a UV dose of 34.9 mJ/cm2 and stainless steel with a dose of 52.5 mJ/cm2. We also observed that broad-spectrum, pulsed UV light is effective at reducing SARS-CoV-2 on N95 respirator material to undetectable levels with a dose of 103 mJ/cm2. We included UV dosimeter cards that provide a colorimetric readout of UV dose and demonstrated their utility as a means to confirm desired levels of exposure were reached. Together, the results presented here demonstrate that broad-spectrum, pulsed UV light is an effective technology for the in vitro inactivation of SARS-CoV-2 on multiple surfaces.

6.
Science ; 370(6521)2020 12 04.
Article in English | MEDLINE | ID: covidwho-873450

ABSTRACT

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.


Subject(s)
COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/metabolism , Host Microbial Interactions , Mitochondrial Membrane Transport Proteins/metabolism , Protein Interaction Maps , SARS Virus/metabolism , SARS-CoV-2/metabolism , Severe Acute Respiratory Syndrome/metabolism , Conserved Sequence , Coronavirus Nucleocapsid Proteins/genetics , Cryoelectron Microscopy , Humans , Mitochondrial Membrane Transport Proteins/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Conformation
7.
Viruses ; 12(6)2020 06 06.
Article in English | MEDLINE | ID: covidwho-548042

ABSTRACT

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Viral Plaque Assay/methods , Virus Inactivation , Animals , Betacoronavirus/drug effects , Betacoronavirus/growth & development , Betacoronavirus/pathogenicity , COVID-19 , Cellulose , Chlorocebus aethiops , Culture Media/chemistry , Formaldehyde , Guanidines/pharmacology , HEK293 Cells , Humans , Pandemics , Phenols/pharmacology , Propiolactone/pharmacology , SARS-CoV-2 , Sepharose , Vero Cells
SELECTION OF CITATIONS
SEARCH DETAIL