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Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response.
mBio. 2022 Jan 18 [Online ahead of print]MBIO

Abstract

The dynamics of SARS-CoV-2 infection in COVID-19 patients are highly variable, with a subset of patients demonstrating prolonged virus shedding, which poses a significant challenge for disease management and transmission control. In this study, the long-term dynamics of SARS-CoV-2 infection were investigated using a human well-differentiated nasal epithelial cell (NEC) model of infection. NECs were observed to release SARS-CoV-2 virus onto the apical surface for up to 28 days postinfection (dpi), further corroborated by viral antigen staining. Single-cell transcriptome sequencing (sc-seq) was utilized to explore the host response from infected NECs after short-term (3-dpi) and long-term (28-dpi) infection. We identified a unique population of cells harboring high viral loads present at both 3 and 28 dpi, characterized by expression of cell stress-related genes DDIT3 and ATF3 and enriched for genes involved in tumor necrosis factor alpha (TNF-α) signaling and apoptosis. Remarkably, this sc-seq analysis revealed an antiviral gene signature within all NEC cell types even at 28 dpi. We demonstrate increased replication of basal cells, absence of widespread cell death within the epithelial monolayer, and the ability of SARS-CoV-2 to replicate despite a continuous interferon response as factors likely contributing to SARS-CoV-2 persistence. This study provides a model system for development of therapeutics aimed at improving viral clearance in immunocompromised patients and implies a crucial role for immune cells in mediating viral clearance from infected epithelia. IMPORTANCE Increasing medical attention has been drawn to the persistence of symptoms (long-COVID syndrome) or live virus shedding from subsets of COVID-19 patients weeks to months after the initial onset of symptoms. In vitro approaches to model viral or symptom persistence are needed to fully dissect the complex and likely varied mechanisms underlying these clinical observations. We show that in vitro differentiated human NECs are persistently infected with SARS-CoV-2 for up to 28 dpi. This viral replication occurred despite the presence of an antiviral gene signature across all NEC cell types even at 28 dpi. This indicates that epithelial cell intrinsic antiviral responses are insufficient for the clearance of SARS-CoV-2, implying an essential role for tissue-resident and infiltrating immune cells for eventual viral clearance from infected airway tissue in COVID-19 patients.

Authors+Show Affiliations

Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore.Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Biosafety Level 3 Core Facility, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore.Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore.Laboratory of Systems Biology and Data Analytics, Genome Institute of Singaporegrid.418377.e, Singapore.Laboratory of Systems Biology and Data Analytics, Genome Institute of Singaporegrid.418377.e, Singapore.Laboratory of Systems Biology and Data Analytics, Genome Institute of Singaporegrid.418377.e, Singapore.Laboratory of Systems Biology and Data Analytics, Genome Institute of Singaporegrid.418377.e, Singapore.Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Department of Otolaryngology-Head & Neck Surgery, National University Health System, Singapore.Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Department of Otolaryngology-Head & Neck Surgery, National University Health System, Singapore.Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. National Public Health Laboratory, National Centre for Infectious Diseases, Singapore.Laboratory of Systems Biology and Data Analytics, Genome Institute of Singaporegrid.418377.e, Singapore.Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore. SingHealth Duke-NUS Global Health Institute, Singapore.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

35038898

Citation

Gamage, Akshamal M., et al. "Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection in Vitro, Despite Eliciting a Prolonged Antiviral Response." MBio, 2022, pp. e0343621.
Gamage AM, Tan KS, Chan WOY, et al. Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response. mBio. 2022.
Gamage, A. M., Tan, K. S., Chan, W. O. Y., Lew, Z. Z. R., Liu, J., Tan, C. W., Rajagopalan, D., Lin, Q. X. X., Tan, L. M., Venkatesh, P. N., Ong, Y. K., Thong, M., Lin, R. T. P., Prabhakar, S., Wang, Y., & Wang, L. F. (2022). Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response. MBio, e0343621. https://doi.org/10.1128/mbio.03436-21
Gamage AM, et al. Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection in Vitro, Despite Eliciting a Prolonged Antiviral Response. mBio. 2022 Jan 18;e0343621. PubMed PMID: 35038898.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response. AU - Gamage,Akshamal M, AU - Tan,Kai Sen, AU - Chan,Wharton O Y, AU - Lew,Zhe Zhang Ryan, AU - Liu,Jing, AU - Tan,Chee Wah, AU - Rajagopalan,Deepa, AU - Lin,Quy Xiao Xuan, AU - Tan,Le Min, AU - Venkatesh,Prasanna Nori, AU - Ong,Yew Kwang, AU - Thong,Mark, AU - Lin,Raymond Tzer Pin, AU - Prabhakar,Shyam, AU - Wang,De Yun, AU - Wang,Lin-Fa, Y1 - 2022/01/18/ PY - 2022/1/18/entrez PY - 2022/1/19/pubmed PY - 2022/1/19/medline KW - COVID-19 KW - SARS-CoV-2 KW - nasal epithelial cells KW - single-cell sequencing KW - viral persistence SP - e0343621 EP - e0343621 JF - mBio JO - mBio N2 - The dynamics of SARS-CoV-2 infection in COVID-19 patients are highly variable, with a subset of patients demonstrating prolonged virus shedding, which poses a significant challenge for disease management and transmission control. In this study, the long-term dynamics of SARS-CoV-2 infection were investigated using a human well-differentiated nasal epithelial cell (NEC) model of infection. NECs were observed to release SARS-CoV-2 virus onto the apical surface for up to 28 days postinfection (dpi), further corroborated by viral antigen staining. Single-cell transcriptome sequencing (sc-seq) was utilized to explore the host response from infected NECs after short-term (3-dpi) and long-term (28-dpi) infection. We identified a unique population of cells harboring high viral loads present at both 3 and 28 dpi, characterized by expression of cell stress-related genes DDIT3 and ATF3 and enriched for genes involved in tumor necrosis factor alpha (TNF-α) signaling and apoptosis. Remarkably, this sc-seq analysis revealed an antiviral gene signature within all NEC cell types even at 28 dpi. We demonstrate increased replication of basal cells, absence of widespread cell death within the epithelial monolayer, and the ability of SARS-CoV-2 to replicate despite a continuous interferon response as factors likely contributing to SARS-CoV-2 persistence. This study provides a model system for development of therapeutics aimed at improving viral clearance in immunocompromised patients and implies a crucial role for immune cells in mediating viral clearance from infected epithelia. IMPORTANCE Increasing medical attention has been drawn to the persistence of symptoms (long-COVID syndrome) or live virus shedding from subsets of COVID-19 patients weeks to months after the initial onset of symptoms. In vitro approaches to model viral or symptom persistence are needed to fully dissect the complex and likely varied mechanisms underlying these clinical observations. We show that in vitro differentiated human NECs are persistently infected with SARS-CoV-2 for up to 28 dpi. This viral replication occurred despite the presence of an antiviral gene signature across all NEC cell types even at 28 dpi. This indicates that epithelial cell intrinsic antiviral responses are insufficient for the clearance of SARS-CoV-2, implying an essential role for tissue-resident and infiltrating immune cells for eventual viral clearance from infected airway tissue in COVID-19 patients. SN - 2150-7511 UR - https://www.unboundmedicine.com/medline/citation/35038898/Human_Nasal_Epithelial_Cells_Sustain_Persistent_SARS_CoV_2_Infection_In_Vitro_despite_Eliciting_a_Prolonged_Antiviral_Response_ DB - PRIME DP - Unbound Medicine ER -