Greg Simmons

School of Veterinary Science

University of Queensland

Retrovirus replication Reverse transcription and integration

Viral RNA cDNA(Provirus)

Host cells chromosome

Reverse transcriptase

Integrase

•RNA viruses •Copy RNA into cDNA using RT •Viral DNA(provirus) inserted into host cells chromosome using IN •If provirus integrated into germ line cells virus is “endogenous” •Provirus is then passed on to offspring Mendellian inheritencethrough

Methodology:

Blood or tissue samples collected from wild koalas DNA extraction DNA assayed with PCR, nested PCR, and/or q PCR PCR based on conserved region of pol Amplicon sequenced confirm KoRV sequence Sensitive and specific Some q PCR positive samples not able to be sequenced (low proviral copy number?)

In total 708 koalas tested 100% prevalence in Qld (277) and NSW (100) 66% in Victoria-mainland (89) 28% in Victoria-islands (80) 15% in Kangaroo island (162)

KoRV prevalence in wild populations in Australia

Location No Tested No +ve % +ve

North Queensland 27 27 100

South-East Queensland 250 250 100

Eastern New South Wales 43 43 100

Western New South Wales 57 57 100

Victorian mainland-Strezleckis 26 *18 69.2

Victorian mainland -Gippsland 20 *11 55.0

Victorian mainland - other 43 *36 81.8

Raymond Island Victoria 29 10 34.5

Snake Island Victoria 12 6 50.0

French Island Victoria 28 6 21.4

Phillip Island Victoria 11 0 0

Kangaroo Island 162 24 14.8

* An additional 18 animals (20% of 89) from Victoria mainland are “doubtful”, probably positive

Majority of koalas tested from mainland Australia KoRV positive 95% (442/466)

Proviral copy number

Gel from conventional KoRV PCR. All bands are KoRV positive. Note difference in band intensity, indicating differences in proviral copy number

Proviral copy no./genomic unit (cell) for selected groups of KoRV positive koalas. (DNA taken from ear punch biopsies) 1A – Qld koalas + ve conventional PCR 1B – Victorian koalas +ve conventional PCR 2 – Victorian koals +ve nested PCR 3 – Vicrorian koals +ve q PCR only

Significance of proviral load?

No direct evidence yet However , anecdotal evidence suggests southern populations much healthier Appear to have much lower prevalence of chlamydiosis and other disease This is true even on Kangaroo island where population is highly in bred

Transmission ?

Endogenous KoRV – inherited Exogenous KoRV? Possibly arthropod vector Can be spread in vitro by paralysis ticks using artificial feeding apparatus Other retroviruses spread by biting arthropods

KoRV infected plasma collected from a wild koala

RT q PCR to detect KoRV RNA

Tick feeding medium following feeding by KoRV fed ticks

Conclusions KoRV widespread throughout wild koalas

So far only KoRV free koalas detected are in Victoria and South Australia

Marked difference in KoRV proviral load between Qld and Victorian koalas

Suggests endogenous vs exogenous virus in different regions

This may be significant in regards to virulence of the virus

Acknowledgements Jo Meers, Paul Young, Jeff McKee

Jon Hanger and Jo Loader, Australian Wildlife hospital

Allan McKinnon and staff Moggil Koala Hospital

Steve Phillips

Bill Ellis

Kath Handasyde

Suzie Zendt

Damien Phillips