Beak and feather disease virus (BFDV), a non-enveloped, icosahedral virus with a circular single stranded DNA (ssDNA) genome, is the causative agent behind psittacine beak and feather disease (PBFD), an often fatal disease affecting parrots. Symptoms include feathering abnormalities, loss of feathers, and occasionally beak and claw deformities. BFDV-induced immunosuppression results in an increased susceptibility to secondary microbial infections, which is often the cause of death in infected parrots. There is no cure, no effective treatment, and no protective vaccine for BFDV. The international trade in exotic parrots has facilitated the spread of BFDV, so that it now has a global presence. Given that over a quarter of the currently recognised 356 psittacine species are considered to be at risk of extinction in the wild, the worldwide presence of BFDV, coupled with its extreme environmental stability, poses serious concerns for the future of some of the worlds most endangered parrots. That genetic diversity exists among BFDV isolates has been established, yet in the 14 years since the genome was fully sequenced, very few full-length BFDV genome sequences have been deposited in GenBank, despite the technology to rapidly isolate and amplify entire circular ssDNA genomes being readily available. Most studies have sequenced just a portion of the genome, usually one of the open reading frames (ORFs) encoding the major viral proteins, to investigate phylogenetic relationships between isolates. However the two major BFDV ORFs, encoding the replication associated protein (Rep) and the capsid protein (CP), have been shown to evolve at different rates, with the functional Rep being generally more conserved while CP is more variable. When also considering the fact that ssDNA viruses are notoriously recombinant, it becomes clear that an analysis based on a portion of the genome is unlikely to accurately establish evolutionary relationships. Therefore the focus of the studies described in this thesis was on isolation and amplification of full-length BFDV genomes from avian blood and feather samples that first tested positive to a PCR-based BFDV screening method. Samples were collected by appropriately trained people in New Zealand, New Caledonia, and Poland, before being sent to the University of Canterbury for molecular and bioinformatic analysis. The sequences of the BFDV genomes from each region were compared to each other and to all other full BFDV genome sequences publically available in GenBank, to compare the genetic diversity among these isolates. Recombination analyses were also performed, to assess how recombination is impacting on the evolution of BFDV. New strains of BFDV and new subtypes of existing BFDV strains were discovered, indicating that the global genetic diversity may be greater than previously thought. Many strains also proved to be recombinants, in particular those from Poland. Europe has had a long history with importing and breeding exotic parrots, and the high degree of recombination among the Polish BFDV isolates coupled with the number of previously unsampled strains is an example of how maintaining populations of multiple species in captivity enables evolution through recombination, and emergence of novel viral strains. Full genome analyses can also enable tracking the source of an infection. A total of 78 full genome sequences from 487 samples tested were deposited into GenBank as a direct result of the work undertaken as part of this thesis, thereby adding to the existing knowledge base regarding BFDV. With continued global sampling and full genome analysis it may one day be possible to trace the history of BFDV to its original emergence.

Os vírus são patógenos importantes na saúde das aves, podendo levar a surtos que ameaçam de forma significante a população destas. O Bornavírus aviário (ABV), o Circovírus (BFDV) e o Poliomavírus (APV) são os agentes virais mais comuns e que mais ameaçam os psitacídeos de cativeiro. O ABV é responsável pela doença da dilatação proventricular (PDD) em psitacídeos e outras aves, uma doença neurológica letal, que foi descoberta no início da década de oitenta na Europa e América do Norte. A primeira infecção por APV descrita em aves foi em periquitos australianos (Melopsittacus undulatus) jovens, sendo depois associada com elevada mortalidade e morbidade em outros psitacídeos. O BFDV é o agente causador da doença do bico e das penas de psitacídeos, que ocorre quase exclusivamente em psitacídeos, principalmente em criatórios, aves em quarentena e lojas de animais. Foram testadas 120 amostras de psitacídeos de cativeiro no Brasil, para BFDV e APV e 112 amostras para ABV, resultando em 21 (17,5%) aves positivas para APV, 41 (34,17%) para BFDV e 32 (28,57 %) para ABV. Entre os animais positivos, quatorze apresentaram infecção concomitante, sete foram positivos para BFDV e ABV, seis positivos para BFDV e APV e uma positiva para BFDV, APV e ABV. Dentre os animais positivos para BFDV, os sinais clínicos mais comuns encontrados foram apteria e apatia/anorexia, em relação às aves positivas para APV foram a apatia/anorexia, enquanto para ABV os sinais neurológicos foram os mais representados. A detecção de APV, BFDV e ABV demostra a ocorrência destes vírus testados em psitacídeos de cativeiro no Brasil, tanto em espécies exóticas como em espécies nativas. / Viruses are important pathogens in avian health and may lead to outbreaks that threaten significantly the population of birds. The Avian Bornavírus (ABV), Circovirus (BFDV) and Avian Polyomavirus (APV) are the most common viral agents that threaten parrots in captivity. The ABV is responsible for the proventricular dilation (PDD) in parrots and other birds, a lethal neurological disease, which was discovered in the early eighties in Europe and North America the disease. The first APV infection in birds has been described in young Australian budgerigars (Melopsittacus undulatus), after being associated with high mortality and morbidity in other parrots. The BFDV is the causative agent of Beak and Feathers Disease, which occurs almost exclusively in psittacines, especially in aviary, quarantine birds and pet stores. A total of 120 captivity parrots were tested in Brazil for BFDV and APV and 112 samples for ABV, resulting in 21 (17.5%) positives for APV, 41 (34.17%) for BFDV and 32 (28.57%) for ABV. Among the positive animals, fourteen had concomitant infection, six were positive for both APV and BFDV, seven for BFDV and ABV, and one sample was positive for BFDV, APV and ABV. Among BFDV positive animals, most common clinical signs were apteria and apathy/anorexia, for APV positive birds were apathy/anorexia, while for ABV were neurological signs were the most represented. The detection of APV, BFDV and ABV demostrate the occurrence of all tested viruses in captive parrots in Brazil, including exotic and native species.

Os vírus são patógenos importantes na saúde das aves, podendo levar a surtos que ameaçam de forma significante a população destas. O Bornavírus aviário (ABV), o Circovírus (BFDV) e o Poliomavírus (APV) são os agentes virais mais comuns e que mais ameaçam os psitacídeos de cativeiro. O ABV é responsável pela doença da dilatação proventricular (PDD) em psitacídeos e outras aves, uma doença neurológica letal, que foi descoberta no início da década de oitenta na Europa e América do Norte. A primeira infecção por APV descrita em aves foi em periquitos australianos (Melopsittacus undulatus) jovens, sendo depois associada com elevada mortalidade e morbidade em outros psitacídeos. O BFDV é o agente causador da doença do bico e das penas de psitacídeos, que ocorre quase exclusivamente em psitacídeos, principalmente em criatórios, aves em quarentena e lojas de animais. Foram testadas 120 amostras de psitacídeos de cativeiro no Brasil, para BFDV e APV e 112 amostras para ABV, resultando em 21 (17,5%) aves positivas para APV, 41 (34,17%) para BFDV e 32 (28,57 %) para ABV. Entre os animais positivos, quatorze apresentaram infecção concomitante, sete foram positivos para BFDV e ABV, seis positivos para BFDV e APV e uma positiva para BFDV, APV e ABV. Dentre os animais positivos para BFDV, os sinais clínicos mais comuns encontrados foram apteria e apatia/anorexia, em relação às aves positivas para APV foram a apatia/anorexia, enquanto para ABV os sinais neurológicos foram os mais representados. A detecção de APV, BFDV e ABV demostra a ocorrência destes vírus testados em psitacídeos de cativeiro no Brasil, tanto em espécies exóticas como em espécies nativas. / Viruses are important pathogens in avian health and may lead to outbreaks that threaten significantly the population of birds. The Avian Bornavírus (ABV), Circovirus (BFDV) and Avian Polyomavirus (APV) are the most common viral agents that threaten parrots in captivity. The ABV is responsible for the proventricular dilation (PDD) in parrots and other birds, a lethal neurological disease, which was discovered in the early eighties in Europe and North America the disease. The first APV infection in birds has been described in young Australian budgerigars (Melopsittacus undulatus), after being associated with high mortality and morbidity in other parrots. The BFDV is the causative agent of Beak and Feathers Disease, which occurs almost exclusively in psittacines, especially in aviary, quarantine birds and pet stores. A total of 120 captivity parrots were tested in Brazil for BFDV and APV and 112 samples for ABV, resulting in 21 (17.5%) positives for APV, 41 (34.17%) for BFDV and 32 (28.57%) for ABV. Among the positive animals, fourteen had concomitant infection, six were positive for both APV and BFDV, seven for BFDV and ABV, and one sample was positive for BFDV, APV and ABV. Among BFDV positive animals, most common clinical signs were apteria and apathy/anorexia, for APV positive birds were apathy/anorexia, while for ABV were neurological signs were the most represented. The detection of APV, BFDV and ABV demostrate the occurrence of all tested viruses in captive parrots in Brazil, including exotic and native species.

To enable assessment of recombinant BFDV capsid protein (recBFDVcap) for vaccination to protect against PBFD, commercially available lovebirds (Agapornis sp.) were tested for evidence of past and current BFDV infection using PCR, HI and HA to identify suitable BFDV-free birds in which to test the vaccine. During this attempt, it was found that lovebirds from commercial aviaries were endemically infected with BFDV with evidence of up to 100% prevalence of BFDV DNA in blood samples from individual birds over time. Such an approach was abandoned as unlikely to yield suitable numbers of naïve birds to conduct a BFDV vaccination trial. As commercially available lovebirds were considered to be a poor source of BFDV-free birds, wild caught cockatoo nestlings and eggs (long-billed corella; Cacatua tenuirostris and galah; Eolophus roseicapillus) were used to assess the efficacy of BFDV vaccination using baculovirus recombinant BFDV capsid. Eggs were artificially incubated and 3 eggs successfully hatched and 1 was successfully hand-reared. All nestlings were screened for BFDV DNA in blood using PCR upon arrival then on days 11, 18 and 25 and tested for anti-BFDV antibody on the day of arrival. All hatched birds were determined to be free of BFDV DNA and BFDV HI antibody in the peripheral blood throughout the hand rearing period and the flock was considered to be suitable for a BFDV vaccination trial. Corellas (n=13) were injected with 1 mL of vaccine containing 10 μg recBFDVcap on day 0 and 0.4 mL vaccine containing 66.8 μg recBFDVcap on day 11. All vaccinated corellas and 5 non-vaccinated control corellas were given 0.4 mL BFDV suspension (titre = log2 12 HAU/50 μL) intramuscularly and 0.1 mL orally 16 days after booster vaccination. Blood was collected periodically during the vaccination period and blood and feathers collected before and after BFDV administration. Testing included BFDV DNA detection by PCR and qRT PCR (on blood) as well as serum antibody detection by haemagglutination inhibition (HI) and BFDV DNA and antigen was detected by qRT PCR and haemagglutination (HA) (on feathers), respectively. Four of 97 blood samples collected from vaccinated birds post BFDV challenge tested positive by PCR, whereas 17 of 35 samples taken from non-vaccinated control corellas tested positive. Vaccinated birds did not develop feather lesions, had only transient PCR detectable viraemia and had no evidence of persistent infection 270 days post-challenge using PCR, histopathology and immunohistochemistry (IHC). Non-vaccinated control corellas developed transient feather lesions and PCR, HI and HA test results consistent with PBFD. They were BFDV PCR positive for up to 41 days post-challenge and qRT PCR demonstrated reduced virus replication in vaccinated birds compared to non-vaccinated control birds. Thus, administration of recBFDVcap vaccine alone was found to incite an adaptive immune response in BFDV-free corellas that subsequently conferred protection against inoculation with BFDV. A commonly utilized method for excising blood dried on filter paper was proven to be of high risk of carryover contamination facilitated by a hole punch used for processing several samples. Therefore a practical method of avoiding carryover contamination was developed and used in the DNA testing procedures of the vaccination trial. Finally, the haematological characteristics of the above mentioned cockatoos were studied before and for 97 days after experimental infection with BFDV. It was found that the pre-challenge haematological values were similar between the vaccinated and non-vaccinated corellas. Most pre-challenge parameters were comparable to previously reported values of other cockatoos and psittacine birds. Significant differences were seen in both groups when comparing pre-challenge values with post challenge values for total and differential leukocyte concentrations, but PCV and TSP were not significantly affected by BFDV challenge.

Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss and immunosuppression leading to secondary infections and eventually death in psittacine birds. The development of standardised reagents for the detection and characterisation of BFDV infections and for the production of protective vaccines has been difficult as no cell culture system has yet been found to grow the virus successfully in vitro. However, the development of consistent and effective diagnostic tests and vaccines is now more practical through the application of nucleic acid-based detection methods and recombinant technology. A quantitative real-time PCR assay for the detection of BFDV DNA was developed, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye, with assays run on a Corbett RotorGene 3000. A synthetic oligonucleotide was used to establish standard curves for the quantitation of viral load in both blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/ìL. The assay was developed using BFDV-positive DNA extracts from the feathers of 10 different species of birds and validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was reliably detected in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with the feather dander of BFDV-infected birds meant that HA feather preparations were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples in future investigations. A recombinant BFDV capsid protein was also produced and a specific monoclonal antibody developed against it. The behaviour of the protein in haemagglutination (HA) assays and the behaviour of the monoclonal antibody in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays were characterised. The protein had the ability to agglutinate galah erythrocytes as per the wild-type virus and this agglutination was successfully inhibited by antibodies to wild-type BFDV from naturally immune psittacine birds. Furthermore, the protein self-assembled into virus-like particles as determined by electron microscopy. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from 3 genera of psittacine birds, including the recently described cockatiel BFDV isolate. A novel “blocking” (or “competitive”) ELISA (bELISA) for the detection of anti- BFDV antibodies in psittacine sera (Ab-bELISA) was also developed and validated with 166 samples from eastern long-billed corellas vaccinated with the recombinant capsid protein and challenged with live virus. The bELISA was found to be both sensitive and specific and correlated strongly with the HI test, thus it should have wide application for the serodiagnosis of BFDV. A survey of cockatiels (n=88) housed at commercial aviaries was conducted to investigate whether BFDV infection occurs in cockatiels. All birds were diagnosed as being virus-free by PCR and HA and had no detectable antibody titre by HI assay. In addition to this, the genomes of two BFDV isolates obtained from diseased cockatiel feathers were sequenced and cross-reactivity assays performed using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first report of an antigenically distinct BFDV in psittacine birds. Since the Ab-bELISA has a lower limit of detection than the HI assay, it was used to repeat the cockatiel sero-survey. No antibodies were detectable in any of the cockatiels tested and thus questions about the real prevalence of BFDV infection in cockatiels and the possible existence of a novel BFDV serotype adapted to cockatiels remain unanswered. The successful control of PBFD in both pet and wild birds depends on the development of vaccines that incite a strong specific immune response and can be efficiently produced in large quantities. Recombinant BFDV capsid proteins have recently been considered as candidate vaccines against BFDV and recombinant techniques allow the development of other candidate vaccines, including DNA vaccines. In order to examine the potential of DNA vaccination as a strategy for the prevention and control of BFDV, two DNA vaccines, based on the nucleotide sequence encoding the capsid protein of BFDV, were developed using the mammalian expression vector pVAX1. The vaccine constructs encoding both the full length and NLS-truncated capsid protein resulted in protein expression both in vitro and in vivo. Protein was detected in COS-7 cells transfected with the constructs with an indirect immunocytochemistry assay using the monoclonal antibody described in Chapter 5. Protein was present in the nucleus of cells transfected with the vaccine encoding the full-length nucleotide sequence and in the cytoplasm of cells transfected with the vaccine encoding the NLS-truncated sequence as expected. Both DNA vaccine constructs induced detectable levels of anti-BFDV antibodies in vaccinated birds, determined using the Ab-bELISA described in Chapter 5. Thus, DNA vaccines similar to those presented here may have application in the prevention and control of BFDV and some options for the further development of these vaccines into effective methods for the control of BFDV are discussed.