Cell lines from the Egyptian fruit bat are permissive for modified vaccinia Ankara

Abstract

Bats are reservoir hosts for a spectrum of infectious diseases. Some pathogens (such as Hendra, Nipah and Marburg viruses) appear to use mainly fruit bats as reservoir. We describe designed immortalization of primary fetal cells from the Egyptian fruit bat (Rousettus aegyptiacus) to facilitate isolation and characterization of pathogens associated with these mammals. Three cell lines with different properties were recovered and successful immortalization was confirmed by continuous cultivation for over 18 months. Surprisingly, the cell lines are fully permissive for a highly attenuated poxvirus, modified vaccinia Ankara (MVA). MVA is a safe and well characterized vaccine vector that cannot replicate in most mammalian cells. High permissivity of Rousettus cell lines could justify testing bats for susceptibility to MVA as a replication competent vector with low zoonotic potential to induce herd immunity in bat colonies against viruses causing rabies or haemorrhagic fevers.

Fig. 1

Cell line generation and passage diagram. (A) GFP positive control in upper panel demonstrating transfection rate in primary Rousettus cells in the first column (scale bar is 20 μm for all images with 20× initial magnification). Foci of immortalized cells still embedded in monolayers of primary cells is shown in the second column (scale bar is 100 μm). Source primary cultures for three cell lines are shown in the third column. Within 10 passages primary cells undergo senescence and are lost leaving low passage cell lines (shown here at week 40) that at low confluency in appearance are surprisingly similar to the source. There are also only few changes in phenotype after 80 weeks of continuous culture. (B) Passage diagram demonstrating stable proliferation rates with a doubling time of approximately 50 h for R05T (after a lag phase of 10 weeks) and 120 h for R05R. R06E cell line exhibits a long lag phase for 30 weeks and thereafter sudden increase in doubling time to approximately 24 h.

Fig. 2

Spread of AdGFP in cell monolayers and transfer to HEK 293. AdGFP spreads slowly in monolayers of R06E and R05T cells without cytopathic effect. Avian CR cells can be infected but vector does not spread. AdGFP spreads rapidly with fulminant CPE in 293 cells. (B) AdGFP is lost in Rousettus cells within 5 passages. Infected R06E cells at passage 3 were mixed with an equal number of 293 sentinel cells. AdGFP infrequently is transferred from R06E (weak GFP signal) to 293 cells (bright GFP signal without CPE at day 4) by cell–cell contact but not via supernatant. Fulminant CPE in 293 cells at day 8, except for weak GFP expression no effect on Rousettus cells.

Fig. 3

High permissivity for modified vaccinia Ankara (MVA). (A) All Rousettus cell lines are permissive for highly attenuated MVA. Shown here is CPE 48 h after infection with a MOI of 0.1. (B) Yields of MVA in Rousettus lines are similar to yield obtained by infection of avian cell lines CR and CS. Vero is shown as negative control. This experiment was performed after 35 weeks of continuous cultivation.

Fig. 4

Kinetic for MVA replication declines in R06E at high passage. (A) Appearance of MVA cytopathic effect is delayed in high passage R06E (but not in R05T) and cultures of R06E have to sub-passaged until infection is visible. (B) Replication of MVA is rapid in R05T and CR, slower in BHK and clearly delayed in the high-passage R06E cell line. This experiment was performed after approximately 65 weeks of continuous cultivation. Infection was performed with a MOI of 0.1 corresponding to an input virus concentration of 2 × 10⁴ pfu/ml in all cultures at day 0.

Fig. 5

Immunofluorescence for immortalizing E1A protein and transient transfection to examine expression plasmid promoter activity. (A) All cells are positive for E1A in early passage Rousettus cell lines but signal intensity is close to the detection limit in R06E and R05R. CR cells serve as positive control, Vero as negative control. (B) In high passage cell lines signal intensity remains high in R05T but was lost in R06E. (C) Chiropteran and avian cell lines were transiently transfected with an expression plasmid where GFP reporter is under control of the promoters used in the immortalizing plasmid. hPGK, human phosphoglycerate kinase promoter; tk, herpes simplex virus thymidine kinase promoter. Note extremely low signal strength for hPGK and tk promoters in Rousettus cells but not in CR cells. Even the usually very strong hCMV promoter appears to be repressed in Rousettus cells. Images shown here were taken 24 h post-transfection.