Bioluminescent and Fluorescent Reporter-Expressing Recombinant SARS-CoV-2

Vasquez, Desarey Morales; Chiem, Kevin; Ye, Chengjin; Martínez-Sobrido, Luis

doi:10.1007/978-1-0716-2453-1_18

Cited by 3 publications

(1 citation statement)

References 53 publications

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“…For validation of heat-inactivation, the SARS-CoV-2 expressing Venus fluorescent reporter gene ( Morales Vasquez et al., 2022 ) was subjected to heat treatment in a screw-capped cryovial at 95°C water bath for 10 minutes. To validate inactivation, 80% Confluent monolayers of A549-ACE2 or Vero E6-TMPRSS2-T2A-ACE2 cells, grown in a black 24-well clear-bottom plate (ibidi # 82426), were incubated with treated or untreated virus (MOI of 0.16) for only one hour at 37°C, 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories

Chaki,

Kahl-McDonagh,

Neuman

et al. 2024

Front. Cell. Infect. Microbiol.

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IntroductionPathogen leak from a high-containment laboratory seriously threatens human safety, animal welfare, and environmental security. Transportation of pathogens from a higher (BSL4 or BSL3) to a lower (BSL2) containment laboratory for downstream experimentation requires complete pathogen inactivation. Validation of pathogen inactivation is necessary to ensure safety during transportation. This study established a validation strategy for virus inactivation. MethodsSARS-CoV-2 wild type, delta, and omicron variants underwent heat treatment at 95°C for 10 minutes using either a hot water bath or a thermocycler. To validate the inactivation process, heat-treated viruses, and untreated control samples were incubated with A549-hACE2 and Vero E6-TMPRSS2-T2A-ACE2 cells. The cells were monitored for up to 72 hours for any cytopathic effects, visually and under a microscope, and for virus genome replication via RT-qPCR. The quality of post-treated samples was assessed for suitability in downstream molecular testing applications. ResultsHeat treatment at 95°C for 10 minutes effectively inactivated SARS-CoV-2 variants. The absence of cytopathic effects, coupled with the inability of virus genome replication, validated the efficacy of the inactivation process. Furthermore, the heat-treated samples proved to be qualified for COVID-19 antigen testing, RT-qPCR, and whole-genome sequencing. DiscussionBy ensuring the safety of sample transportation for downstream experimentation, this validation approach enhances biosecurity measures. Considerations for potential limitations, comparisons with existing inactivation methods, and broader implications of the findings are discussed.

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Section: Methodsmentioning

confidence: 99%

Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories

Chaki,

Kahl-McDonagh,

Neuman

et al. 2024

Front. Cell. Infect. Microbiol.

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Bacterial Artificial Chromosome Reverse Genetics Approaches for SARS-CoV-2

Chiem,

Nogales,

Almazán

et al. 2023

Methods in Molecular Biology

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Fluorescent and Bioluminescent Reporter Mouse-Adapted Ebola Viruses Maintain Pathogenicity and Can Be Visualized in Vivo

Davies

Welch

Jain

et al. 2023

The Journal of Infectious Diseases

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Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA)-EBOVs, including one based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 80% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using hand-held blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models.

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Bioluminescent and Fluorescent Reporter-Expressing Recombinant SARS-CoV-2

Cited by 3 publications

References 53 publications

Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories

Validating the inactivation of viral pathogens with a focus on SARS-CoV-2 to safely transfer samples from high-containment laboratories

Bacterial Artificial Chromosome Reverse Genetics Approaches for SARS-CoV-2

Fluorescent and Bioluminescent Reporter Mouse-Adapted Ebola Viruses Maintain Pathogenicity and Can Be Visualized in Vivo

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