1. Tracking of viral evolution during an outbreak of beak and feather disease virus (BFDV) infection in the critically endangered orange- bellied parrot (Neophema chrysogaster) Subir Sarker, Ali Ghorashi, Jade Forwood, Andrew Peters & Shane Raidal Charles Sturt University

2. PBFD Widespread in wild birds throughout Australasia New Zealand parrots susceptible Nominated as a key threatening process in 1995 mainly following outbreaks in orange-bellied parrots (since 1985) and Norfolk I green parrot Listed as KTP in 2001 by the Australian Government Environment Protection and Biodiversity Conservation Act (1999) Threat Abatement Plan developed in 2005 Standard diagnostic tests (Raidal et al 2008) Quarantine and Hygiene protocols (Cross, 2006)

3. BFDV is highly genetically diverse Cockatoo clade ? Parrot clade ? Lorikeet clade ? Cross-species infection? 2009

4. Diagnosis HI titre = antibody (blood, serum, plasma) HA titre = virus excretion (feather) PCR = infection ≠ disease DNA sequencing Preferred samples Feather Blood collected onto filter paper In combination these tests act as internal controls Khalesi (2005). A comparison of HA, HI & PCR for the detection of BFDV infection. J Gen Virol. 86:3039-46.

5. Historic Distribution of OBP Currently 3 captive flocks in VIC, TAS, SA - releasing birds each year Less than 50 birds remaining in the wild (IUCN, 2013)

6. Epidemiology  Most wild Australian flocks are infected  Wild SCC have a disease prevalence of 5-20% & seroprevalence of 60-80%  Spread horizontally  Virus persists in nests for many months ~ decades  Clinically normal carriers - lorikeets Recent outbreak in Tasmanian OBP

7. 2008 29% PCR positive 35% HI positive Infected Recovered Susceptible Total = 54% infected

8. N=71 6 3 Adelaide Zoo: 2 of 20 birds tested PCR positive

9. Brief Methodology

10. 95°C 3 min 95°C 30 sec 57°C 45 sec 68°C 2 min 68°C 5 min 40 cycles Optimized PCR conditions for amplification of BFDV genome Cloning of gel purified amplicon and insert were sequenced in AGRF, Australia

11. Results

12. Initial genome sequence data 2 main genotypes (red & blue) Unclear ancestry polytomy

13. Codon-based Bayesian analysis in whole genome Wild birds Why is this one different?

14. Rep gene Wild birds

15. Recombination Using SBP, GARD, DualBrothers and RDP4 program First recombination Between captive bird (11-1361) from Victoria and Tasmanian bird (12-0827-20213) originally sourced from the wild. Second recombination Between wild-caught isolate (12-0827-20214) and a captive-bred (08-423) isolate found in the Tasmanian flock.

16. Evolutionary rates of BFDV Mean evolutionary rate Full genome sequences (n=15) 3.41×10 -3 subs/site/year (95% HPD: 3.43×10 -4 to 8.08×10 -3 ) Partial Rep gene data set (n=35) 3.10×10 -5 subs/site/year (95% HPD: 2.38×10 -7 to 1.55×10 -5 )

17. DNA sequencing amplicons vs clones

19. Conclusions OBP was infected with unique BFDV genotypes Evidence of BFDV quasispecies within individual birds Recombination and high mutation rate The establishment and physical separation of three insurance flocks in TAS, VIC & SA did not prevent the re-emergence & spread of PBFD amongst OBP flocks

20. Acknowledgements Australian Research Council CSU-PRS EH Graham Centre