Cloning, expression and antiviral activity of IFNγ from the Australian fruit bat, Pteropus alecto

Abstract

Bats are natural reservoir hosts to a variety of viruses, many of which cause morbidity and mortality in other mammals. Currently there is a paucity of information regarding the nature of the immune response to viral infections in bats, partly due to a lack of appropriate bat specific reagents. IFNγ plays a key role in controlling viral replication and coordinating a response for long term control of viral infection. Here we describe the cloning and expression of IFNγ from the Australian flying fox, Pteropus alecto and the generation of mouse monoclonal and chicken egg yolk antibodies specific to bat IFNγ. Our results demonstrate that P. alecto IFNγ is conserved with IFNγ from other species and is induced in bat splenocytes following stimulation with T cell mitogens. P. alecto IFNγ has antiviral activity on Semliki forest virus in cell lines from P. alecto and the microbat, Tadarida brasiliensis. Additionally recombinant bat IFNγ was able to mitigate Hendra virus infection in P. alecto cells. These results provide the first evidence for an antiviral role for bat IFNγin vitro in addition to the application of important immunological reagents for further studies of bat antiviral immunity.

Fig. 1

Pteropus alecto IFNγ is closely related to other mammalian IFNγ genes. (A) Alignment of the deduced amino acid sequence of bat IFNγ genes with IFNγ genes from other species. The six α helixes corresponding to the conserved secondary structure of IFNγ are indicated. Potential N-linked glycosylation sites and the conserved NLS (KRKR) are shown in bold. Dashes indicated similarity and dots indicate gaps. (B) Phylogenetic analysis based on amino acid alignments of bat IFNγ with representative vertebrate species. Branch support is indicated as the percentage out of 1000 bootstrap replicates and is shown where support is greater than 60%. The P. vampyrus and M. lucifugus sequences were obtained from the publicly available whole genome sequences available in Ensembl. The accession numbers of all other IFNγ sequences can be found in Section 2.

Fig. 2

Detection of bat IFNγ in Western blot and ELISA using mouse monoclonal and IgY antibodies to recombinant bat IFNγ. (A) SDS–PAGE and coomassie blue staining of the purified rbIFNγ (lane 2) indicates its approximate molecular weight as 17 kDa. (B) Western blot of rbIFNγ was probed with anti-bat mAb 2G6 (lane 2) and yolk derived polyclonal anti-bat IgY (lane 4) to rbIFNγ and were visualised with anti-mouse HRP and anti-chicken HRP, respectively, in a chemiluminescence system. Negative controls run on lanes 1 and 3 was an unrelated E. coli expressed and purified recombinant bat protein of similar size to rbIFNγ. (C) Capture ELISA developed using the anti-bat IgY (for coating the plate), anti-bat mAb 2G6 (for detection) and anti-mouse-HRP (secondary Ab) has the sensitivity to detect 10 ng/ml of rbIFNγ. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Pteropus alecto spenocytes produce IFNγ in response to mitogen stimulation. (A) IFNγ mRNA was measured in RNA from cultured splenocytes from two individual P. alecto by qRT-PCR and normalised against 18s rRNA. (B) Native IFNγ protein was measured in supernatant from cultured cells by capture ELISA using IFNγ specific anti-bat IgY as the coating antibody and IFNγ specific anti-bat mAb as the detection antibody. Data are mean values of triplicates, and the bars represent ± standard error of the means.

Fig. 4

Recombinant bat IFNγ displays antiviral activity against Semliki forest virus in P. alecto and T. brasiliensis cell lines. Cells were pre-treated overnight with serial dilutions of supernatants from CHO cells transfected with pCI plasmid containing bat IFNγ (rbIFNγ^CHO) or pCI vector alone (CHO mock). Cells were then infected with SFV for 48 h. Assay controls included replicates pre-treated with media alone and cultured for a further 48 h either infected (media + SFV) or uninfected (media). Cell death due to viral infection was determined by a colorimetric assay using the viral dye neutral red. Pre-treatment of (A) P. alecto PaKiT02 cells or (B) T. brasiliensis Tb1-Lu cells with rbIFNγ^CHO protects from SFV infection in a dose dependent manner. Data are mean values of triplicates, and the error bars represent SEs.

Fig. 5

Recombinant bat IFNγ displays antiviral activity against Hendra virus. P. alecto PaKiT02 cells were pre-treated overnight with serial dilutions of supernatant from CHO cells transfected with pCI plasmid containing bat IFNγ (rbIFNγ^CHO) or pCI vector alone (CHO mock) and then infected with HeV for a further 48 h. HeV positive cells were enumerated by immunofluorescent labelling with anti HeV glycoprotein G antibody. Data are mean values of five replicates, and the error bars represent SEs. Asterisks indicate statistical significance with p ⩽ 0.05.