Andrew Peters

Mutability Dynamics of an Emergent Single Stranded DNA Virus in a Naïve Host

Quasispecies variants and recombination were studied longitudinally in an emergent outbreak of beak and feather disease virus (BFDV) infection in the orange-bellied parrot (Neophema chrysogaster). Detailed health monitoring and the small population size (<300 individuals) of this critically endangered bird provided an opportunity to longitudinally track viral replication and mutation events occurring in a circular, single-stranded DNA virus over a period of four years within a novel bottleneck population. Optimized PCR was used with different combinations of primers, primer walking, direct amplicon sequencing and sequencing of cloned amplicons to analyze BFDV genome variants. Analysis of complete viral genomes (n = 16) and Rep gene sequences (n = 35) revealed that the outbreak was associated with mutations in functionally important regions of the normally conserved Rep gene and immunogenic capsid (Cap) gene with a high evolutionary rate (3.41×10−3 subs/site/year) approaching that for RNA viruses; simultaneously we observed significant evidence of recombination hotspots between two distinct progenitor genotypes within orange-bellied parrots indicating early cross-transmission of BFDV in the population. Multiple quasispecies variants were also demonstrated with at least 13 genotypic variants identified in four different individual birds, with one containing up to seven genetic variants. Preferential PCR amplification of variants was also detected. Our findings suggest that the high degree of genetic variation within the BFDV species as a whole is reflected in evolutionary dynamics within individually infected birds as quasispecies variation, particularly when BFDV jumps from one host species to another.

EVIDENCE OF PSITTACINE BEAK AND FEATHER DISEASE VIRUS SPILLOVER INTO WILD CRITICALLY ENDANGERED ORANGE-BELLIED PARROTS (NEOPHEMA CHRYSOGASTER)

Abstract We report the recent emergence of a novel beak and feather disease virus (BFDV) genotype in the last remaining wild population of the critically endangered Orange-bellied Parrot (Neophema chrysogaster). This virus poses a significant threat to the recovery of the species and potentially its survival in the wild. We used PCR to detect BFDV in the blood of three psittacine beak and feather disease (PBFD)–affected wild Orange-bellied Parrot fledglings captured as founders for an existing captive breeding recovery program. Complete BFDV genome sequence data from one of these birds demonstrating a 1,993-nucleotide-long read encompass the entire circular genome. Maximum-likelihood (ML) and neighbor-joining (NJ) phylogenetic analysis supported the solitary position of this viral isolate in a genetically isolated branch of BFDV. On Rep gene sequencing, a homologous genotype was present in a second wild orange-bellied parrot and the third bird was infected with a distantly related genotype. These viruses have newly appeared in a population that has been intensively monitored for BFDV for the last 13 yr. The detection of two distinct lineages of BFDV in the remnant wild population of Orange-bellied Parrots, consisting of fewer than 50 birds, suggests a role for other parrot species as a reservoir for infection by spillover into this critically endangered species. The potential for such a scenario to contribute to the extinction of a remnant wild animal population is supported by epidemiologic theory.

Phylogeny of beak and feather disease virus in cockatoos demonstrates host generalism and multiple-variant infections within Psittaciformes

Phylogenetic analyses of the highly genetically diverse but antigenically conserved, single-stranded circular, DNA genome of the avian circovirus, beak and feather disease virus (BFDV) from cockatoo species throughout Australia demonstrated a high mutation rate for BFDV (orders of magnitude fall in the range of 10(-4) substitutions/site/year) along with strong support for recombination indicating active cross-species transmission in various subpopulations. Multiple variants of BFDV were demonstrated with at least 30 genotypic variants identified within nine individual birds, with one containing up to 7 variants. Single genetic variants were detected in feathers from 2 birds but splenic tissue provided further variants. The rich BFDV genetic diversity points to Australasia as the most likely geographical origin of this virus and supports flexible host switching. We propose this as evidence of Order-wide host generalism in the Psittaciformes characterised by high mutability that is buffered by frequent recombination and slow replication strategy.

Evidence of a deep viral host switch event with beak and feather disease virus infection in rainbow bee-eaters ( Merops ornatus )

Since the characterization of psittacine beak and feather disease (PBFD) in 1984, a wide range of avian circoviruses have been discovered with varying pathogenic effects amongst a diverse range of avian hosts. Until recently these circovirus species were thought to be restricted to within avian Orders such as the Psittaciformes for beak and feather disease virus (BFDV) and Columbiformes for pigeon circovirus with little evidence of cross-family transmission or replication. We report evidence of a naturally occurring novel host switch event with self-limiting BFDV infection in a group of rainbow bee-eaters (Merops ornatus) a species of Coraciiformes unrelated to parrots and not previously known to be susceptible to any avian circovirus. The outbreak highlights important and unexpected aspects of disease emergence and host-switching pertinent to other situations when viruses might cross species boundaries as well as the potential of avian circoviruses to infect disparate host species.

Review of psittacine beak and feather disease and its effect on Australian endangered species

BACKGROUND Since it was first described in the early 1980s, psittacine beak and feather disease (PBFD) has become recognised as the dominant viral pathogen of psittacine birds in Australia. Our aim was to evaluate and review the effect of PBFD and its position as a key threatening process to Australian psittacine bird species. We review the origin/evolutionary pathways and potential threat of PBFD to endangered psittacine bird populations and captive-breeding flocks. CONCLUSIONS The most recent beak and feather disease virus (BFDV) phylogenetic analyses indicate that all endangered Australian psittacine bird species are susceptible to, and equally likely to be infected by, BFDV genotypes from a range of host psittacine species. Management of the disease in captive-breeding programs has relied on testing and culling, which has proven costly. The risk of PBFD should be considered very carefully by management teams contemplating the establishment of captive-breeding flocks for endangered species. Alternative disease prevention tools, including vaccination, which are increasingly being used in wildlife health, should be considered more seriously for managing and preventing PBFD in captive flocks of critically endangered species.

Evolution of circoviruses in lorikeets lags behind its hosts.

The presence of endogenous viral elements in host genomes hints towards much older host-virus relationships than predicted by exogenous phylogenies, with highly mutable single-stranded DNA (ssDNA) viruses and RNA viruses often occupying entangled multispecies ecological niches. The difficulty lies in unravelling the long-term evolutionary history of vertebrate virus-host relationships and determining the age of a potentially ancient tree based only fresh shoots at the tips. Resolving such lineages, and the sometimes great discrepancy amongst evolutionary timescales, is problematic, especially when purifying selection or recombination can significantly alter the accuracy of phylogenetic reconstruction methods. Pathogens which occupy entangled multispecies ecological niches add a further layer of complexity but we show that multi-host scenarios may also provide opportunities to identify allopatric or sympatric paleobiological signals that can unlock longer term phylogenies. We identified host-based, cryptic, sympatric differentiation in beak and feather disease virus in the Psittaciformes tribe Loriini along with endogenous circovirus motifs in Kea (Nestor notabilis) and Gondwanan vicariance estimates to infer the evolutionary timescale of the circoviruses. This demonstrated a chronology of psittacine circovirus speciation aligned to conservative Zealandic divergences for relic circovirus motifs in Kea and a 10million year divergence coinciding with the Papuan central range orogeny that triggered the radiation of Loriini and segregation of an antecedent viral clade in Australian lorikeets. Estimates of circovirus speciation in birds highlighted a Gondwanan dominant group in Neoaves with passerine, columbid and larid circoviruses deeply separated from those in waterfowl, consistent with a Triassic divergence of Galloanserae. The circovirus tree had a deep ancestry in invertebrates with a Palaeozoic expansion in fish and mammals. We show that longer term evolutionary relationships in viruses which have a high rate of mutation and admixture can be disentangled, highlighting that contemporary virus host-switching can be explained by deep intra-lineage host phylogeny.

Beak and feather disease virus genotypes in Australian parrots reveal flexible host-switching

OBJECTIVE To discover beak and feather disease virus (BFDV) genotypes in Australian parrots that might threaten vulnerable and endangered psittacine bird species. METHODS Phylogenetic analyses of new DNA sequence data from Australian birds including the Rep gene (n = 55) and nine whole genomes, were compared with all available published BFDV genomes to assess host- and geographically-based divergence as well as probable host-switch events. RESULTS Strong support for flexible host-switching and recombination was detected, indicating active cross-species transmission in various subpopulations. CONCLUSION The data suggested that all endangered Australian psittacine bird species are equally likely to be infected by BFDV genotypes from any other close or distantly related host reservoir species.

Haemolytic anaemia associated with Theileria sp. in an orphaned platypus.

CASE REPORT The clinical and laboratory findings in an orphaned juvenile female platypus (Ornithorhynchus anatinus) that presented with a severe anaemia and tick infestation are reported. The animal developed a terminal septicaemia and died. Antemortem clinical pathology, postmortem histopathology and 18S rDNA sequencing supported a diagnosis of extravascular haemolytic anaemia secondary to Theileria ornithorhynchi infection. CONCLUSION Although T. ornithorhynchi infection is common in the platypus, this is the first case in which it has been shown to cause a haemolytic anaemia in this species and molecular characterisation of the organism has been described. A review of the previous literature concerning T. ornithorhynchi and possible treatment options for future cases are discussed.

Haemochromatosis in a Brazilian tapir (Tapirus terrestris) in an Australian zoo

A 23-year-old Brazilian, or lowland, tapir with a 6-month history of loss of body condition developed clinical signs and laboratory findings consistent with liver failure. The animal was euthanased and a diagnosis of hepatic haemochromatosis was made based on histopathology. Two other healthy tapirs in the same collection had chronically elevated serum and tissue iron concentrations. The excessive accumulation of iron in tissues with resultant tissue damage (i.e. haemochromatosis) has been reported in a range of captive species. This and other reported cases of haemochromatosis in the Brazilian tapir would suggest that this condition is an important consideration in the management of this species in zoos. Further research into the endogenous regulation of iron metabolism, especially the role of hepcidin, in tapirs and other species at risk of iron storage disorders may be helpful in the prevention of this condition.

Whole-Genome Sequence Characterization of a Beak and Feather Disease Virus in a Wild Regent Parrot (Polytelis anthopeplus monarchoides)

ABSTRACT The whole-genome sequence of beak and feather disease virus (BFDV) from a wild Australian regent parrot (Polytelis anthopeplus monarchoides) was characterized. The genome consists of 1,993 bp and has a typical stem-loop structure between open reading frame 1 (ORF1) and ORF2. This is the first evidence of BFDV infection as well as the complete genome sequence for this host species, globally.