Generation of self-replicating airway organoids from the cave nectar bat Eonycteris spelaea as a model system for studying host–pathogen interactions in the bat airway epithelium

ABSTRACT

Bats are reservoir hosts for various zoonotic viruses with pandemdic potential in humans and livestock. In vitro systems for studying bat host–pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air–liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species-specific virus susceptibility and the reservoir likelihood for emerging infectious agents.

KEYWORDS:

• Bat
• Chiroptera
• Eonycteris spelaea
• airway organoids
• airway epithelial cells

Acknowledgement

We thank the NUS Medicine BSL-3 Core Facility team for their support in BSL-3 procedures. We thank the staffs in NUS Otolaryngology Department for their help in tissue culture procedures. We thank National Centre of Infectious Diseases, Singapore for the SARS-CoV-2 Omicron isolate used for the study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Conflict of interest statement

The authors declare no conflict of interest.

Additional information

Funding

This research was funded in part by Singapore National Research Foundation (NRF2012NRF-CRP001-056, NRF2016NRF-NSFC002-013), the Singapore National Medical Research Council of Singapore (MOH-COVID19RF2-0001, MOH-COVID19RF-003, MOH-OFIRG19MAY-0011, MOH-OFIRG10NOV-0050) and the Ministry of Education Singapore (MOE2019-T2-2-130); Singapore National Medical Research Council Centre Grant Program – Diabetes, Tuberculosis and Neuroscience No. CGAug16M009; the NUS Reimagine Research Grant, Singapore; and the Nanyang Technological University Lee Kong Chian School of Medicine LEARN grant (021914-00001). LLYC is supported by the Lee Kong Chian School of Medicine, Nanyang Technological University under the Dean’s Postdoctoral Fellowship and the Wong Peng Onn Fellowship (002823-00001).