Bat airway epithelial cells: a novel tool for the study of zoonotic viruses

Abstract

Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.

Figure 1. Species for which airway epithelial cells were established and their geographic distribution.

A. Seba's short-tailed bat (C. perspicillata), and B. Straw-colored fruit bat (E. helvum) (upper row), and their distribution (lower row) (map adapted from IUCN Red List of Threatened Species, http://www.iucnredlist.org).

Figure 2. Establishment of airway epithelial cell culture by outgrowth from trachea specimens.

A Trachea tissue sample in cell culture dish viewed from above (left side) with outgrowth of primary airway epithelial cells from the mucosal layer (right side, 100× magnification) (here: E. helvum) B Immortalized and subcloned airway epithelial cells from E. helvum, subclone 1 (designated EidheAEC.B-1) C Immortalized and subcloned airway epithelial cells from C. perspicillata, subclone 3 (designated CarperAEC.B-3). Black bars represent 50 µm.

Figure 3. Immunofluorescence staining for markers of epithelial origin of Tb 1 Lu and airway epithelial cells from C. perspicillata (CarperAEC.B) and E. helvum (EidheAEC.B) prior to subcloning.

The markers used to confirm epithelial origin were cytokeratin (CK, red) and zonula occludens-1 (ZO-1, green); nuclei are counterstained with DAPI (blue). Expression of both markers is present in all cell lines generated by the described methods, indicating an epithelial origin. By contrast, the commercially available Tb 1 Lu does not show expression of the respective markers.

Figure 4. Virus infection studies with vesicular stomatitis virus (VSV) (A, B) and Rift Valley fever virus (RVFV) (C, D), and at MOI of 0.1 (left side) and 0.001 (right side), in subclones of immortalized C. perspicillata airway epithelial cells (subclone 3, designated CarperAEC.B-3) and E. helvum airway epithelial cells (subclone 1, designated EidheAEC.B-1).

Viruses were detected by quantitative real-time RT-PCR. All cells were infectable and able to support replication of RVFV and VSV.