Establishing a Disease‐in‐a‐Dish Model to Study SARS‐CoV‐2 Infection During Prenatal Development

Song, Ann; Ona, Jack; Osuna, Claudia; Phandthong, Rattapol; Talbot, Prue

doi:10.1002/cpz1.759

Cited by 8 publications

(11 citation statements)

References 29 publications

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“…Human SARS-CoV-2 pseudoparticles were produced using previously published procedures ( Crawford et al, 2020 ; Song et al, 2023 ). Generated pseudoparticles were first titered with HEK293T-ACE2 cells using flow cytometry to detect the ZsGreen viral backbone ( Figures 5A–C ).…”

Section: Resultsmentioning

confidence: 99%

“…SARS-CoV-2 pseudoparticles, which cannot replicate, can be generated in a Biosafety Lab 2 cabinet and provide a safer alternative than using live virus. This infection system has been adapted to other cell types and species ( Song et al, 2023 ). Pseudotyping has been used previously to study virus-host receptor binding ( Wang et al, 2008 ; Lu et al, 2014 ; Phandthong et al, 2022 , 2023 ) and can be applied to Mv1Lu cells, which are a robust in vitro model for studying coronavirus infection in mink.…”

Section: Discussionmentioning

confidence: 99%

“…Our study provides a framework for future work on viral entry mechanisms and development of therapeutics for SARS-CoV-2 infections in zoonotic species. While our study focused on the wild-type spike strain (Wuhan-Hu-1), our protocol could be adapted for use with other variants and tested using other zoonotic species ( Song et al, 2023 ). Finally, our approach could be used to explore an alternative endocytic entry pathway facilitated by soluble ACE2 ( Yeung et al, 2021 ), highlighting the versatility of this infection system for drug screening.…”

Section: Discussionmentioning

confidence: 99%

“…Virus aliquots were stored at −80 • C. Prior to the experiments, the transduction efficiency of each batch of viruses was tested via flow cytometry of infected HEK 293T-ACE2. Viral titers were calculated using the Poisson formula (Crawford et al, 2020;Song et al, 2023). The flow cytometry conditions were the same as those for the Mv1Lu cell protocol.…”

Section: Lentiviral Production To Generate Sars-cov-2 Pseudoparticlesmentioning

confidence: 99%

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Endocytosis inhibitors block SARS-CoV-2 pseudoparticle infection of mink lung epithelium

Song,

Phandthong,

Talbot

2023

Front. Microbiol.

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IntroductionBoth spill over and spill back of SARS-CoV-2 virus have been reported on mink farms in Europe and the United States. Zoonosis is a public health concern as dangerous mutated forms of the virus could be introduced into the human population through spillback.MethodsThe purpose of our study was to determine the SARS-CoV-2 entry mechanism using the mink lung epithelial cell line (Mv1Lu) and to block entry with drug inhibitors.ResultsMv1Lu cells were susceptible to SARS-CoV-2 viral pseudoparticle infection, validating them as a suitable disease model for COVID-19. Inhibitors of TMPRSS2 and of endocytosis, two pathways of viral entry, were tested to identify those that blocked infection. TMPRSS2 inhibitors had minimal impact, which can be explained by the apparent lack of activity of this enzyme in the mink and its localization within the cell, not on the cell surface.DiscussionDyngo4a, a small molecule endocytosis inhibitor, significantly reduced infection, supporting the conclusion that the entry of the SARS-CoV-2 virus into Mv1Lu cells occurs primarily through endocytosis. The small molecule inhibitors that were effective in this study could potentially be used therapeutically to prevent SARS-CoV-2 infection in mink populations. This study will facilitate the development of therapeutics to prevent zoonotic transmission of SARS-CoV-2 variants to other animals, including humans.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Lentiviral Production To Generate Sars-cov-2 Pseudoparticlesmentioning

confidence: 99%

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Endocytosis inhibitors block SARS-CoV-2 pseudoparticle infection of mink lung epithelium

Song,

Phandthong,

Talbot

2023

Front. Microbiol.

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“…In vitro model systems have proven to be very useful tools for deciphering molecular mechanisms related to normal development and homeostasis, or disruptions of those processes predisposing onset of disease. Examples include cell culture systems used to study developmental processes such as X-chromosome inactivation in mammals (Almeida et al, 2017; Patrat et al, 2009) or early embryogenesis (Bao et al, 2022; Lau et al, 2022), or to elucidate the cellular and molecular etiology of many different diseases based on the now popular “disease-in-a-dish” approach (Davaapil et al, 2020; Song et al, 2023). We assembled an in vitro system in which we combined the three cell types normally involved in i) the initial exposure to disruptive environmental effects (somatic cells), ii) the first phase of generational epigenetic reprogramming in the preimplantation embryo (pluripotent cells), and iii) the second phase of generational reprogramming in the developing germ line (germ cells).…”

Section: Discussionmentioning

confidence: 99%

Endocrine disruptor-induced epimutagenesisin vitro: Insight into molecular mechanisms

Lehle,

Lin,

Gomez

et al. 2024

Preprint

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Endocrine disrupting chemicals (EDCs) such as bisphenol S (BPS) are xenobiotic compounds that can disrupt endocrine signaling following exposure due to steric similarities to endogenous hormones within the body. EDCs have been shown to induce disruptions in normal epigenetic programming (epimutations) that accompany dysregulation of normal gene expression patterns that appear to predispose disease states. Most interestingly, the prevalence of epimutations following exposure to many different EDCs often persists over multiple subsequent generations, even with no further exposure to the causative EDC. Many previous studies have described both the direct and prolonged effects of EDC exposure in animal models, but many questions remain about molecular mechanisms by which EDCs initially induce epimutations or contribute to the propagation of EDC-induced epimutations either within the exposed generation or to subsequent generations. Additional questions remain regarding the extent to which there may be differences in cell type-specific susceptibilities to various EDCs, and whether this susceptibility is correlative with expression of relevant hormone receptors and/or the location of relevant hormone response elements (HREs) in the genome. To address these questions, we exposed cultured mouse pluripotent (induced pluripotent stem [iPS]), somatic (Sertoli and granulosa), and germ (primordial germ cell like [PGCLCs]) cells to BPS and measured changes in DNA methylation levels at the epigenomic level and gene expression at the transcriptomic level. We found that there was indeed a difference in cell type-specific susceptibility to EDC-induced epimutagenesis and that this susceptibility correlated with differential expression of relevant hormone receptors and, in many cases, tended to generate epimutations near relevant HREs within the genome. Additionally, however, we also found that BPS can induce epimutations in a cell type that does not express relevant receptors and in genomic regions that do not contain relevant HREs, suggesting that both canonical and non-canonical signaling mechanisms can be disrupted by BPS exposure. Most interestingly, we found that when iPS cells were exposed to BPS and then induced to differentiate into PGCLCs, the prevalence of epimutations and differentially expressed genes (DEGs) initially induced in the iPSCs was largely retained in the resulting PGCLCs, however, >90% of the specific epimutations and DEGs were not conserved but were rather replaced by novel epimutations and DEGs following the iPSC to PGCLC transition. These results suggest a unique mechanism by which an EDC-induced epimutated state may be propagated transgenerationally following a single exposure to the causative EDC.

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Endocytosis Inhibitors Block SARS-CoV-2 Pseudoparticle Infection of Mink Lung Epithelium

Song

Phandthong

Talbot

2023

Preprint

Self Cite

View full text Add to dashboard Cite

Both spill over and spill back of SARS-CoV-2 virus have been reported on mink farms in Europe and the United States. Zoonosis is a public health concern as dangerous mutated forms of the virus could be introduced into the human population through spillback. The purpose of our study was to determine the SARS-CoV-2 entry mechanism using mink lung epithelial cell line (Mv1Lu) and to block entry with drug inhibitors. Mv1Lu cells were susceptible to SARS-CoV-2 viral pseudoparticle infection, validating them as a suitable disease model for COVID-19. Inhibitors of TMPRSS2 and of endocytosis, two pathways of viral entry, were tested to identify those that blocked infection. Dyngo4a, a small molecule endocytosis inhibitor, significantly reduced infection, while TMPRSS2 inhibitors had minimal impact, supporting the conclusion that the entry of the SARS-CoV-2 virus into Mv1Lu cells occurs primarily through endocytosis. The small molecule inhibitors that were effective in this study could potentially be used therapeutically to prevent SARS-CoV-2 infection in mink populations. This study will facilitate the development of therapeutics to prevent zoonotic transmission of SARS-CoV-2 variants to other animals, including humans.

show abstract

Establishing a Disease‐in‐a‐Dish Model to Study SARS‐CoV‐2 Infection During Prenatal Development

Abstract: Establishing a disease-in-a-dish model to study SARS-CoV-2 infection during prenatal development. Current Protocols, 3, e759.

Cited by 8 publications

References 29 publications

Endocytosis inhibitors block SARS-CoV-2 pseudoparticle infection of mink lung epithelium

Endocytosis inhibitors block SARS-CoV-2 pseudoparticle infection of mink lung epithelium

Endocrine disruptor-induced epimutagenesisin vitro: Insight into molecular mechanisms

Endocytosis Inhibitors Block SARS-CoV-2 Pseudoparticle Infection of Mink Lung Epithelium

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