Cell Cultures for Virology: Usability, Advantages, and Prospects

Dolskiy, Alexander A.; Grishchenko, I. V.; Yudkin, Dmitry V.

doi:10.3390/ijms21217978

Cited by 40 publications

(32 citation statements)

References 105 publications

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“…Because of that, lack of viable viruses in air samples does not necessarily correlate to the absence of viable virus in RNA positive samples, and the presence of RNA should be interpreted as the probable presence of a viable virus, especially considering that viral culture is very difficult as it requires a week or more for completion and specialized laboratory equipment and skills, therefore being much less sensitive than detection by molecular methods 89,90 . Nonetheless, alternative methods for assessing viral infectivity, such as sample pre‐treatment before nucleic acid extraction with propidium monoazide (PMA) should be explored and validated 91‐93 . This method is based on the assumption that virus inactivation is associated with the loss of integrity of viral outer structures such as the envelope and capsid 94 .…”

Section: Discussionmentioning

confidence: 99%

“…Regarding viral infectivity in air, Tang et al 41 [91][92][93] This method is based on the assumption that virus inactivation is associated with the loss of integrity of viral outer structures such as the envelope and capsid. 94 As PMA is a photoreactive intercalating dye with a high affinity for DNA and RNA, it forms a covalent linkage upon exposure to intense visible light.…”

Section: Referencementioning

confidence: 99%

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SARS‐CoV ‐2 air sampling: A systematic review on the methodologies for detection and infectivity

et al. 2022

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This systematic review aims to present an overview of the current aerosol sampling methods (and equipment) being used to investigate the presence of SARS-CoV-2 in the air, along with the main parameters reported in the studies that are essential to analyze the advantages and disadvantages of each method and perspectives for future research regarding this mode of transmission. A systematic literature review was performed on PubMed/MEDLINE, Web of Science, and Scopus to assess the current air sampling methodologies being applied to SARS-CoV-2. Most of the studies took place in indoor environments and healthcare settings and included air and environmental sampling. The collection mechanisms used were impinger, cyclone, impactor, filters, water-based condensation, and passive sampling. Most of the reviewed studies used RT-PCR to test the presence of SARS-CoV-2 RNA in the collected samples. SARS-CoV-2 RNA was detected with all collection mechanisms. From the studies detecting the presence of SARS-CoV-2 RNA, fourteen assessed infectivity. Five studies detected viable viruses using impactor, water-based condensation, and cyclone collection mechanisms. There is a need for a standardized protocol for sampling SARS-CoV-2 in air, which should also account for other influencing parameters, including air exchange ratio in the room sampled, relative humidity, temperature, and lighting conditions.

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Section: Discussionmentioning

confidence: 99%

Section: Referencementioning

confidence: 99%

SARS‐CoV ‐2 air sampling: A systematic review on the methodologies for detection and infectivity

et al. 2022

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“…First, the tropism of a virus for a particular cell line can be enhanced by adapting the virus to the cell line. 33 Differences in the number of passages of different virus strains may cause the variations compared to the SARS-CoV-2 USA-WA1/2020 strain that we used in this study. Another possibility is that the NP produced by different VoC may have mutations that lead to different binding affinities of labeled MAbs used in this assay, causing changes in the FRET between the donor and acceptor.…”

Section: Discussionmentioning

confidence: 99%

SARS-CoV-2 Nucleocapsid Protein TR-FRET Assay Amenable to High Throughput Screening

Gorshkov

Vasquez

Chiem

et al. 2022

ACS Pharmacol. Transl. Sci.

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Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have been put forth, only a few compounds are in late-stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here, we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies conjugated to donor and acceptor fluorophores that produce a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC 50 ) for remdesivir of 9.3 μM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, β), Brazilian/Japanese P.1 (Gamma, γ), and Californian (Epsilon, ε) variants of concern (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

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“…The differences we observed in the detection of NP from the different VoC are interesting and could be due to several factors. First, the tropism of a virus for a particular cell line can be enhanced by adapting the virus to the cell line 33 . Differences in the number of passages of different virus strains may cause the variations compared to the SARS-CoV-2 USA-WA1/2020 strain that we used in this study.…”

Section: Discussionmentioning

confidence: 99%

A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

Gorshkov

Vasquez

Chiem

et al. 2021

Preprint

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Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 μM against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, β), Brazilian/Japanese variant P.1 (Gamma, γ), and Californian (Epsilon, ε), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

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Cell Cultures for Virology: Usability, Advantages, and Prospects

Cited by 40 publications

References 105 publications

SARS‐CoV ‐2 air sampling: A systematic review on the methodologies for detection and infectivity

SARS‐CoV ‐2 air sampling: A systematic review on the methodologies for detection and infectivity

SARS-CoV-2 Nucleocapsid Protein TR-FRET Assay Amenable to High Throughput Screening

A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

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