Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen

Abstract

It is speculated that bats are important reservoir hosts for numerous viruses, with 27 viral families reportedly detected in bats. Majority of these viruses have not been isolated and there is little information regarding their biology in bats. Establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. Currently, few bat cell lines have been developed and only Tb1-Lu, derived from Tadarida brasiliensis is commercially available. Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). To our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. The cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture.

Keywords: Big brown bat; Cell-line; HSV; Kidney; MERS-CoV; PED-CoV; VSV.

Fig. 1

Transfection with SV40 and MyPV T-antigens increases DNA content in cells. Vero cells were transfected with plasmids expressing either SV40Tag, MyPVTag or empty vector (pcDNA). Twenty-four hr after transfection, cells were immune-stained for cytoplasmic T-antigen and with propidium iodide to quantify DNA. The DNA content of T-antigen and pcDNA transfected cells was determined by flow cytometry and expressed as the fold increase in DNA content relative to pcDNA transfected cells. The ratio for cells transfected with pcDNA was taken as ‘1′. Experiments were done in triplicate and mean values plotted. Error bars represent standard deviation. Statistical difference was calculated using Mann-Whitney U test for two independent samples. * < 0.05.

Fig. 2

Efk3 and Efk clones multiplication curve. The cell division curve for the three clones and Efk3 parental cell line was determined by counting viable cells at the indicated time-points.

Fig. 3

CPE observed in Efk cells. CPE observed following infection with MERS-CoV, VSV and PED-CoV is indicated by arrows.

Fig. 4

Efk clones support replication of viruses. The amount of virus (or viral nucleic acid) produced by Efk cell clones was measured by determining log₁₀ TCID₅₀ (MERS-CoV Fig. 4A), WST₅₀ (VSV, Fig. 4B), plaque assay (HSV, Fig. 4C) and qRT-PCR (PED-CoV, Fig. 4D). The data confirm that the viruses replicated in the Efk cell lines as well as the relevant positive control cell lines (Vero, MRC5).