Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

<div><p>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, <i>Carollia perspicillata</i> (Seba's short-tailed bat) and <i>Eidolon helvum</i> (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.</p></div

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

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

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

<p><b>A</b> 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: <i>E. helvum</i>) <b>B</b> Immortalized and subcloned airway epithelial cells from <i>E. helvum</i>, subclone 1 (designated EidheAEC.B-1) <b>C</b> Immortalized and subcloned airway epithelial cells from <i>C. perspicillata</i>, subclone 3 (designated CarperAEC.B-3). Black bars represent 50 µm.</p

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

<p>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.</p

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 <i>C. perspicillata</i> airway epithelial cells (subclone 3, designated CarperAEC.B-3) and <i>E. helvum</i> airway epithelial cells (subclone 1, designated EidheAEC.B-1).

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

Characteristics of bat species chosen for the establishment of bat airway epithelial cells.

<p>Characteristics of bat species chosen for the establishment of bat airway epithelial cells.</p

Viruses associated with the bat species <i>C. perspicillata</i> and <i>E. helvum</i>.

<p>Viruses were detected either as virus isolates, on the basis of genetic sequence, or indirectly by detection of antibodies. Table adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084679#pone.0084679-Luis1" target="_blank">[32]</a>; modified and supplemented. Viruses that have been reported to infect humans are marked with *.</p

Alterations detected in the K22 resistant variants of HCoV-229E.

a<p>Detected by comparison of the nucleotide sequences of HCoV-229E subjected to 10–13 passages in the presence of K22 including its plaque purified variants A-R with those of initial virus or mock-passaged virus (accession number KF293665).</p>b<p>Plaque purified HCoV-229E that served as initial material for the virus passages.</p>c<p>IC50 (µM).</p>d<p>Fold resistance to K22 as related to initial virus is shown in parentheses.</p>e<p>Virus preparation and its plaque purified variants M-R obtained in separate K22 selection experiment.</p>f<p>The virus used for preparation of recombinant nsp6 mutants.</p>g<p>K22 resistant recombinant viruses.</p

K22 structure, antiviral activity, and cytotoxicity.

<p>(<b>A</b>) K22 structure. (<b>B</b>) Anti-HCoV-229E activity of K22 in MRC-5 cells. K22 and HCoV-229E were added to MRC-5 cells, and the number of viral plaques developed after 48 h were assessed. Data shown are means (±SD) of duplicate determinations from three independent experiments. PFU, plaque forming unit. (<b>C</b>) Viability and proliferation of MRC-5 cells in the presence of K22. MRC-5 cells were incubated with K22 or DMSO solvent for 48 h at 37°C and the cell viability determined using tetrazolium-based reagent while cell proliferation was assayed by counting of cells. Data shown are means (±SD) of duplicate determinations from two independent experiments. (<b>D</b>) K22 affects the post-entry phase of viral life cycle. K22 (4 µM) or DMSO solvent were incubated with cells for a period of 2 h either before (−2 h), during (0 h) or after a 2 h period of cell inoculation with HCoV-229E, and the number of viral plaques developed after 48 h were assessed. Data shown are means of duplicate determinations from three independent experiments.*<i>P</i><0.05; <i>n</i> = 3. ***<i>P</i><0.005; <i>n</i> = 3. (<b>E-F</b>) K22 exhibits potent antiviral activity when added up to 6 h after infection of cells. MRC-5 cells were inoculated with HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) added at specific time points relative to the end of inoculation period. The culture medium and cells were harvested after 24 h of incubation at 37°C, and the viral RNA (<b>E</b>) and infectivity (<b>F</b>) determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. EX, extracellular medium; CA, cell-associated sample.</p

Analysis of recombinant HCoV-229E nsp6 mutants.

<p>(<b>A</b>) Predicted topological structure of HCoV-229E nsp6 indicating the location of K22 resistance mutations. Concerning transmembrane domains VI and VII two proposed topologies are shown. (<b>B-C</b>) Recombinant nsp6 mutant viruses are resistant to K22. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) was added at specific time points relative to the end of inoculation period. The infectious cell culture medium and cells were harvested after 24 h of incubation at 37°C, and copy numbers of cell-associated (CA) or extracellular (EX) viral RNA was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. (<b>D-F</b>) Replication kinetics of recombinant nsp6 mutant viruses. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at an moi of 0.05 for 1 h at 4°C. The infectious cell culture medium and cells were harvested at specific time points relative to the end of inoculation period, and copy numbers of cell-associated (CA; <b>D</b>) or extracellular (EX; <b>E</b>) viral RNA and infectivity (<b>F</b>) was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments.</p