Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand.

In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified.

Avian Polyomavirus Genome Sequences Recovered from Parrots in Captive Breeding Facilities in Poland.

Eight genomes of avian polyomaviruses (APVs) were recovered and sequenced from deceased Psittacula eupatria, Psittacula krameri, and Melopsittacus undulatus from various breeding facilities in Poland. Of these APV-positive samples, six had previously tested positive for beak and feather disease virus (BFDV) and/or parrot hepatitis B virus (PHBV).

Genome Sequences of Beak and Feather Disease Virus in Urban Rainbow Lorikeets (Trichoglossus haematodus).

Beak and feather disease viral genomes were recovered from two deceased juvenile urban rainbow lorikeets (Trichoglossus haematodus) that lacked tail feathers. These genomes share ~95% pairwise identity with two beak and feather disease virus (BFDV) genomes identified in wild and captive Australian T. haematodus birds and ~92% identity to those in wild New Caledonian T. haematodus deplanchii birds.

Identification of an avian polyomavirus associated with Adélie penguins (Pygoscelis adeliae).

Little is known about viruses associated with Antarctic animals, although they are probably widespread. We recovered a novel polyomavirus from Adélie penguin (Pygoscelis adeliae) faecal matter sampled in a subcolony at Cape Royds, Ross Island, Antarctica. The 4988 nt Adélie penguin polyomavirus (AdPyV) has a typical polyomavirus genome organization with three ORFs that encoded capsid proteins on the one strand and two non-structural protein-coding ORFs on the complementary strand. The genome of AdPyV shared ~60 % pairwise identity with all avipolyomaviruses. Maximum-likelihood phylogenetic analysis of the large T-antigen (T-Ag) amino acid sequences showed that the T-Ag of AdPyV clustered with those of avipolyomaviruses, sharing between 48 and 52 % identities. Only three viruses associated with Adélie penguins have been identified at a genomic level, avian influenza virus subtype H11N2 from the Antarctic Peninsula and, respectively, Pygoscelis adeliae papillomavirus and AdPyV from capes Crozier and Royds on Ross Island.

Diverse small circular single-stranded DNA viruses identified in a freshwater pond on the McMurdo Ice Shelf (Antarctica).

Antarctica has some of the harshest environmental conditions for existence of life on Earth. In this pilot study we recovered eight diverse circular single-stranded DNA (ssDNA) viral genome sequences (1904-3120 nts) from benthic mats dominated by filamentous cyanobacteria in a freshwater pond on the McMurdo Ice Shelf sampled in 1988. All genomes contain two to three major open reading frames (ORFs) that are uni- or bi-directionally transcribed and all have an ORF encoding a replication-associated protein (Rep). In one genome, the second ORF has similarity to a capsid protein (CP) of Nepavirus which is most closely related to geminiviruses. Additionally, all genomes have two intergenic regions that contain putative stem loop structures, six genomes have NANTATTAC as the nonanucleotide motif, while one has CCTTATTAC, and another has a non-canonical stem loop. In the large intergenic region, we identified iterative sequences flanking the putative stem-loop elements which are a hallmark of most circular ssDNA viruses encoding rolling circle replication (RCR) initiators of the HUH endonuclease superfamily. The Reps encoded by ssDNA viral genomes recovered in this study shared <38% pairwise identity to all other Reps of known ssDNA viruses. A previous study on Lake Limnopolar (Livingston Island, South Shetland Islands), using next-generation sequencing identified circular ssDNA viruses and their putative Reps share <35% pairwise identity to those from the viral genomes removed in this study. It is evident from our pilot study that the global diversity of ssDNA viruses is grossly underestimated and there is limited knowledge on ssDNA viruses in Antarctica.

A novel papillomavirus in Adélie penguin (Pygoscelis adeliae) faeces sampled at the Cape Crozier colony, Antarctica.

Papillomaviruses are epitheliotropic viruses that have circular dsDNA genomes encapsidated in non-enveloped virions. They have been found to infect a variety of mammals, reptiles and birds, but so far they have not been found in amphibians. Using a next-generation sequencing de novo assembly contig-informed recovery, we cloned and Sanger sequenced the complete genome of a novel papillomavirus from the faecal matter of Adélie penguins (Pygoscelis adeliae) nesting on Ross Island, Antarctica. The genome had all the usual features of a papillomavirus and an E9 ORF encoding a protein of unknown function that is found in all avian papillomaviruses to date. This novel papillomavirus genome shared ~60 % pairwise identity with the genomes of the other three known avian papillomaviruses: Fringilla coelebs papillomavirus 1 (FcPV1), Francolinus leucoscepus papillomavirus 1 (FlPV1) and Psittacus erithacus papillomavirus 1. Pairwise identity analysis and phylogenetic analysis of the major capsid protein gene clearly indicated that it represents a novel species, which we named Pygoscelis adeliae papillomavirus 1 (PaCV1). No evidence of recombination was detected in the genome of PaCV1, but we did detect a recombinant region (119 nt) in the E6 gene of FlPV1 with the recombinant region being derived from ancestral FcPV1-like sequences. Previously only paramyxoviruses, orthomyxoviruses and avian pox viruses have been genetically identified in penguins; however, the majority of penguin viral identifications have been based on serology or histology. This is the first report, to our knowledge, of a papillomavirus associated with a penguin species.

Pigeon circoviruses display patterns of recombination, genomic secondary structure and selection similar to those of beak and feather disease viruses.

Pigeon circovirus (PiCV) has a ~2 kb genome circular ssDNA genome. All but one of the known PiCV isolates have been found infecting pigeons in various parts of the world. In this study, we screened 324 swab and tissue samples from Polish pigeons and recovered 30 complete genomes, 16 of which came from birds displaying no obvious pathology. Together with 17 other publicly available PiCV complete genomes sampled throughout the Northern Hemisphere and Australia, we find that PiCV displays a similar degree of genetic diversity to that of the related psittacine-infecting circovirus species, beak and feather disease virus (BFDV). We show that, as is the case with its pathology and epidemiology, PiCV also displays patterns of recombination, genomic secondary structure and natural selection that are generally very similar to those of BFDV. It is likely that breeding facilities play a significant role in the emergence of new recombinant PiCV variants and given that ~50 % of the domestic pigeon population is infected subclinically, all pigeon breeding stocks should be screened routinely for this virus.

Avihepadnavirus diversity in parrots is comparable to that found amongst all other avian species.

Avihepadnaviruses have previously been isolated from various species of duck, goose, stork, heron and crane. Recently the first parrot avihepadnavirus was isolated from a Ring-necked Parakeet in Poland. In this study, 41 psittacine liver samples archived in Poland over the last nine years were tested for presence of Parrot hepatitis B virus (PHBV). We cloned and sequenced PHBV isolates from 18 birds including a Crimson Rosella, an African grey parrot and sixteen Ring-necked Parakeets. PHBV isolates display a degree of diversity (>78% genome wide pairwise identity) that is comparable to that found amongst all other avihepadnaviruses (>79% genome wide pairwise identity). The PHBV viruses can be subdivided into seven genetically distinct groups (tentatively named A-G) of which the two isolated of PHBV-G are the most divergent sharing ∼79% genome wide pairwise identity with all their PHBVs. All PHBV isolates display classical avihepadnavirus genome architecture.

Extensive recombination detected among beak and feather disease virus isolates from breeding facilities in Poland.

Beak and feather disease virus (BFDV) causes the highly contagious, in some cases fatal, psittacine beak and feather disease in parrots. The European continent has no native parrots, yet in the past has been one of the world's biggest importers of wild-caught exotic parrot species. Following the banning of this practice in 2007, the demand for exotic pet parrots has largely been met by established European breeding facilities, which can also supply buyers outside Europe. However, the years of unregulated importation have provided numerous opportunities for BFDV to enter Europe, meaning the likelihood of birds within captive breeding facilities being BFDV positive is high. This study examined the BFDV status of such facilities in Poland, a country previously shown to have BFDV among captive birds. A total of 209 birds from over 50 captive breeding facilities across Poland were tested, and 43 birds from 18 different facilities tested positive for BFDV. The full BFDV genomes from these 43 positive birds were determined, and phylogenetic analysis revealed that these samples harboured a relatively high degree of diversity and that they were highly recombinant. It is evident that there have been multiple introductions of BFDV into Poland over a long period of time, and the close association of different species of birds in the captive environment has probably facilitated the evolution of new BFDV strains through recombination.

Evidence of multiple introductions of beak and feather disease virus into the Pacific islands of Nouvelle-Caledonie (New Caledonia).

Beak and feather disease virus (BFDV) is a circular ssDNA virus that causes psittacine beak and feather disease and has almost global presence. Here, we report for the first time the presence of in Nouvelle-Calédonie (New Caledonia). One hundred and sixty-eight exotic and 79 endemic birds were sampled in Nouvelle-Calédonie, 26 were found to be positive for BFDV. We characterized the full genomes of 26 isolates and phylogenetic analysis placed nine of the isolates into the BFDV-J strain, with the remaining 17 isolates from Deplanche's Rainbow Lorikeet (Trichoglossus haematodus deplanchii) forming a novel strain, BFDV-P. Of more concern was the discovery of an infected bird from the vulnerable and endemic New Caledonian Parakeet (Cyanoramphus saisseti). Our results reveal that there have been at least two introductions of BFDV into Nouvelle-Calédonie.

Molecular characterisation of beak and feather disease virus (BFDV) in New Zealand and its implications for managing an infectious disease.

Beak and feather disease virus (BFDV) infections are often fatal to both captive and wild parrot populations. Its recent discovery in a wild population of native red-fronted parakeets has raised concerns for the conservation of native parrots, all of which are threatened or endangered. The question of a recent introduction versus a native genotype of the virus poses different conservation-management challenges, and thus, a clear understanding of the molecular phylogeny of BDFV is a crucial step towards integrated management planning. This study represents the first comprehensive attempt to screen New Zealand's endangered and threatened psittacines systematically for BFDV. We sampled and screened kakapos (Strigops habroptilus), kakas (Nestor meridionalis), keas (N. notabilis), Chatham parakeets (Cyanoramphus forbesi), Malherbe's parakeets (Cyanoramphus malherbi), yellow-crowned parakeets (C. auriceps) and red-fronted parakeets (Cyanoramphus novaezelandiae), as well as eastern rosellas (Platycercus eximius), an introduced species that is now common throughout the North Island, for BFDV. Out of all species and populations sampled (786 individuals), we found 16 BFDV-positive red-fronted parakeets from Little Barrier Island/Hauturu, seven eastern rosellas from the Auckland region, and eight yellow-crowned parakeets from the Eglinton Valley in the South Island. The full genomes of the viral isolates from the red-fronted parakeets share 95-97 % sequence identity to those from the invasive eastern rosellas and 92.7-93.4 % to those isolates from the South Island yellow-crowned parakeets. The yellow-crowned parakeet BFDV isolates share 92-94 % sequence identity with those from eastern rosellas. The low level of diversity among all BFDV isolates from red-fronted parakeets could suggest a more recent infection among these birds compared to the yellow-crowned parakeets, whereas the diversity in the eastern rosellas indicates a much more established infection. Pro-active screening and monitoring of BFDV infection rates in aviaries as well as in wild populations are necessary to limit the risk of transmission among threatened and endangered parrot populations in New Zealand.