Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

Abstract

BackgroundBats 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/FindingsBlack 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/SignificanceThe 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.

Novel bat adenoviruses with an extremely large E3 gene

“…The collection and storage of the bat samples were performed as described previously (Li et al, 2010). (Crameri et al, 2009). Four primary bat cell lines were successfully immortalized with SV40 large T-antigen.…”

“…Four primary bat cell lines were successfully immortalized with SV40 large T-antigen. PaKi E9 cells were kindly provided by Dr Lin-Fa Wang (Crameri et al, 2009), and MdKi cells were prepared as described previously (Li et al, 2010;Yang et al, 2015). Bat cells were cultured in DMEM/F12-Ham (Sigma-Aldrich) supplemented with 10 % FBS and 1 % antibiotics.…”

Characterization of the Antigen Processing Machinery and Endogenous Peptide Presentation of a Bat MHC Class I Molecule

“…PaKiT03 (immortalized P. alecto kidney-derived cell line) (26) and 721.221 (class I-deficient human B cell line) (27) cells were transfected with pIRES2-ZsGreen1 Ptal-N*01:01 using Lipofectamine 2000 (Life Technologies) or electroporation (250 V, 975 mF Bio-Rad Gene Pulser Xcell), respectively. Transfectants were selected using Geneticin (Life Technologies), and the bicistronic expression of a GFP reporter protein (a feature of pIRES2-ZsGreen1) allowed for transfection efficiency to be determined by flow cytometry.…”

Isolation of multiple novel paramyxoviruses from pteropid bat urine

“…At the present time, isolation of live virus from bats remains a challenge. In recent years, our group has focused on developing optimized procedures for isolation of live viruses; from urine collection protocols, sample transportation medium and storage conditions to the development of specialized bat primary cell lines (Crameri et al, 2009). In previous reports, we have described the isolation of Hendra virus (HeV) (Smith et al, 2011), Menangle virus (MenPV) and the novel henipavirus, Cedar virus (CedPV) , from beneath colonies of pteropid bats (commonly known as flying foxes) in Queensland (QLD), Australia, utilizing these optimized procedures.…”

“…Briefly, the samples were thawed at room temperature and centrifuged to pellet debris. The urine was diluted 1 : 10 in cell culture medium (Dulbecco's modified Eagle's medium nutrient mixture F-12 Ham supplemented with double-strength antibiotic/antimycotic and 10 % FCS), centrifuged again to clarify it and then dispersed across both Vero and primary Pteropus alecto kidney (PaKi) (Crameri et al, 2009) cell monolayers. The flasks were rocked for 30 min at 37 u C, cell culture medium was added and the cells were incubated for 7 days at 37 u C. Supernatant and cells were passaged two more times if no CPE was observed.…”