Greg Simmons
School of Veterinary Science
University of Queensland
Retrovirus replication
Reverse transcription and integration
•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
Viral RNA
cDNA
(Provirus)
Reverse
transcriptase
Integrase
Host cells
chromosome
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?)
KoRV prevalence in wild
populations in Australia
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)
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
KoRV infected plasma collected
from a wild koala
Tick feeding medium
following feeding by
KoRV fed ticks
Exogenous KoRV?
Possibly arthropod vector
Can be spread in vitro
by paralysis ticks using
artificial feeding apparatus
Other retroviruses spread
by biting arthropods
RT q PCR to detect KoRV RNA
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