Establishment, immortalisation and characterisation of pteropid bat cell lines

Abstract

Background: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions.

Methodology/findings: Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types.

Conclusions/significance: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

Figure 1. Morphological differences observed for primary cell cultures derived from P. alecto tissues.

(A) Cells derived from a brain (left) and kidney (right) after 5 days in primary cell culture. (B) Cells derived from liver (left) and kidney (right) after 12 days in primary cell culture.

Figure 2. Stable expression of SV40T proteins in transformed cells.

(A) Immunofluorescent staining of P. alecto spleen and kidney primary cells and cells transformed to detect expression of the SV40T antigens. (B) Western blot of untransformed P. alecto spleen and kidney primary cells (lanes 1 and 3, respectively) and transformed cells (lanes 2 and 4) to detect expression of the SV40 large T antigen.

Figure 3. Infection of cloned P. alecto cell lines by HeV and NiV.

(A) Comparison of infection kinetics of NiV in three different cell lines at 24 and 48 hours post infection. (B) Comparison of infection efficiency of P. alecto cloned cell lines for HeV and NiV. The images were taken 24 hours post infection. In both studies, cells were infected at high multiplicity of infection (MOI ≥100), fixed with 100% methanol and removed from the Biosafety Level-4 laboratory before being stained with HeV G protein-specific antibodies.

Figure 4. Induction of P. alecto interferon gene expression by poly I∶C.

Results shown are of fold increase in IFN-α and IFN-β transcript levels (measured by real-time PCR) after treatment of P. alecto cloned and SV40 Large T antigen immortalised brain, kidney and foetal cells with 10 µg/ml poly I∶C over the basal level of IFN gene expression in mock treated cells. Error bars represent the standard deviation of the mean derived from duplicate samples.