Abstract
BackgroundBats are recognised as an important reservoir for a number of highly pathogenic zoonotic viruses. While many of these viruses cause severe and often fatal disease in humans, bats are able to coexist with these viruses without clinical signs of disease. The mechanism conferring this antiviral response is not fully understood. Here, we investigated the differential protein expression of immortalised Pteropus alecto kidney cells (PaKiT03) following transfection with the viral mimic, Poly I:C. Two complementary proteomic approaches, difference gel electrophoresis (DIGE) and isobaric tagging for relative and absolute quantitation (iTRAQ) were used to quantify changes in protein expression following Poly I:C stimulation at 4, 8 and 20 hr post treatment (hpt).ResultsThe expression of ISG54 gene, a known responder to virus infection and Poly I:C treatment, was significantly induced in transfected cells compared with mock-transfected cells. Through iTRAQ analysis we show that Poly I:C up-regulates key glycolytic enzymes at 4 hpt within PaKiT03 cells. In contrast, at 20 hpt PaKiT03 cells down-regulated ribosomal subunit proteins. The analysis with DIGE of Poly I:C transfected PaKiT03 cells showed over 215 individual spots differentially regulated, however only 25 spots could be unambiguously identified by LC-MS/MS. Immunoblotting confirmed the up-regulation of Eno1 and Tpi1 in PaKiT03 cells following Poly I:C transfection. A comparison with human cells (HEK293T and HeLa) and one additional bat cell line (PaLuT02), demonstrated that glycolytic pathways are also induced in these cell types, but at different intensities.ConclusionThe two techniques, DIGE and iTRAQ identified largely overlapping sets of differentially expressed proteins, however DIGE unambiguously identified significantly less proteins than iTRAQ. Poly I:C induced a rapid metabolic shift towards glycolysis within the PaKiT03 cells at 4 hpt, presumably as a consequence of increased energy requirements. On the other hand ribosomal subunit proteins were seen as down-regulated by iTRAQ, these proteins may be the limiting factors in the translational machinery available for virus replication. This study provides new insight into the antiviral response of bat cells, highlighting the importance of energy metabolism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-015-0081-6) contains supplementary material, which is available to authorized users.
Highlights
Bats are recognised as an important reservoir for a number of highly pathogenic zoonotic viruses
PaKiT03 cells are responsive to polycytidylic acid (Poly I):C transfection The viability of PaKiT03 cells was assessed at 3, 6, 22 and 46 hr following transfection with increasing concentrations of Poly I:C (0.5, 1 or 10 μg/ml) delivered with Lipofectamine
From 6 to 22 hr post transfection the viability of cells treated with 0.5 μg/ml, 1 μg/ml of Poly I:C or with Lipofectamine alone remained stable while the viability of cells treated with 10 μg/ml of Poly I:C dropped to below 70 %
Summary
Bats are recognised as an important reservoir for a number of highly pathogenic zoonotic viruses While many of these viruses cause severe and often fatal disease in humans, bats are able to coexist with these viruses without clinical signs of disease. While many bat borne pathogens cause severe and often fatal diseases in humans, bats demonstrate no clinical signs of disease when infected with these agents. Experimental infections of bats with highly pathogenic viruses such as Hendra and Nipah virus yielded no observable clinical signs. Virus isolation, seroconversion, and the excretion of virus in saliva, urine and faeces were observed [10, 11] Subclinical infections of both fruit and insectivorous bats have been reported following experimental infection with Zaire Ebola virus. High titres of Ebola virus were successfully obtained from viscera and faecal samples following experimental infection [12]
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