Authentication of the R06E fruit bat cell line

Abstract

Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery.

Keywords: R05T; R06E; Rousettus aegyptiacus; fruit bat cell line.

Figure 1

Species-specific PCRs. (A) Genomic DNA was isolated from different cell lines and analyzed by PCR using a primer pair designed to amplify across an intron of the conserved MACF1 gene; (B) Isolated genomic DNA was analyzed by a single-primer PCR to amplify minisatellites. Abbreviations for cell lines: BHK, Syrian baby hamster kidney; CHO-K1, Chinese hamster ovary; HEK 293, human embryonic kidney; SaOS-2, human osteosarcoma; R05R, R05T, R06E, Rousettus aegyptiacus; CR.pIX, CS, Cairina moschata retina or somites; Vero, African green monkey. NTC for non template control.

Figure 2

Comparison of ND2 sequences of R06E and GenBank AY504596 from the Egyptian rousette. The closest significant alignment is shown after query through NCBI BLAST (consensus sequence of three independent clones). All differences in nucleotide sequence to the GenBank entry are also present in the R05R and R05T sister cell lines. Capital letters: primer sequences.

Figure 3

Cocultivation of Vero.GFP with R06E. The cocultures shown differ only by frequency of splitting. (A) Bright field and fluorescence pictures were taken at different passages after spiking of Vero cells, stably expressing GFP, into R06E cell culture; (B) Species-specific MACF1 PCR of fruit bat (R05T, R06E), monkey (Vero.GFP) and spiked cell cultures with different splitting procedures (as shown in A) at various passage levels; (C) Ratio of GFP-positive Vero to R06E in different cell cultures. Cells were quantified by FACS analysis.

Figure 4

R06E at passages 50 and 90 has reduced permissivity for MVA (A) but not for VSIV (B) at 37 °C. R06E examined for VSIV was at passage 60. Cultivation and infection at 37 °C is highlighted in red with bold symbols, at 32 °C in black with open symbols. Lower passage R06E in the second panel of (A) is shown with squares and higher passage R06E is shown with triangles.