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

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)

BFDV is highly genetically

diverse

Cockatoo clade ?

Parrot clade ?

Lorikeet clade ?

Cross-species infection?

2009

Diagnosis

HI titre = antibody (blood, serum, plasma)

HA titre = virus excretion (feather)

PCR = infection ≠ diseaseDNA sequencing

Preferred samples FeatherBlood 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.

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)

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

200829% PCR positive35% HI positive

Infected

Recovere

d

Susceptible

Total = 54% infected

N=71

6

3

Adelaide Zoo:2 of 20 birds tested

PCR positive

Brief Methodology

95°C

3 min

95°C

30 sec57°C

45 sec

68°C

2 min68°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

Results

Initial genome sequence data 2 main genotypes (red & blue)

Unclear ancestry

poly

tom

y

Codon-based Bayesian

analysis in whole

genome

Wild birds

Why is this one

different?

Rep geneWild birds

RecombinationUsing 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.

Evolutionary rates of BFDV

Mean evolutionary rate

DNA sequencing amplicons vs clones

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

Acknowledgements

Australian Research Council

CSU-PRS

EH Graham Centre