Investigations into the effect of koala retrovirus infection on the immune system of koalas

Abstract

The aim of this thesis is to investigate the effect of infection with Koala retrovirus (KoRV) on the immune system of koalas. This required the development of real-time RT-PCR assays to measure key cytokines including interleukin 4, interleukin 6, interleukin 10 and interferon gamma and CD4 and CD8β. The response of koala cells to three common mitogen stimulation protocols was assessed and appropriate reference genes (GAPDH and 28s) were validated. These qPCR methods were then used to examine the resting cytokine and CD4:CD8 mRNA expression in Victorian koalas that had either negative, positive or mixed KoRV and Chlamydia infection status. KoRV positive koalas had significantly lower levels of IL17A and IFNγ expression along with a decreased CD4:CD8 compared to negative koalas. qPCR was also used to measure gene expression by mitogen stimulated lymphocytes of koalas infected with a newly discovered variant of KoRV (KoRV B) compared to those without; this was measured four times to control for seasonal variation. KoRV B positive koalas showed significantly increased upregulation of IL17A and IL10 in three out of four sampling periods and IFNγ, IL6, IL4 and TNFα in two out of four. There was also seasonal variation in up-regulation for most cytokines and the CD4:CD8. qPCR was used to detect KoRV A and B from DNA samples from koalas in eight geographically separate populations in NSW. KoRV A was detected in 217/217 of koalas, thus it is likely endogenous in NSW. KoRV B was detected in 21/217 individuals and in all but one population examined in NSW. A pilot study was performed to establish a protocol for incubation of the synthetic peptide CKS-17 (immunosuppressive domain of gamma retroviruses), with mitogen stimulated PBMC’s from KoRV positive and KoRV negative koalas.