Genetic characterization and phylogenetic analysis of duck-derived waterfowl parvovirus in Anhui province, eastern China.

Recently, a novel duck-origin goose parvovirus (N-GPV) was reported to cause short beak and dwarfism syndrome in ducks. In this study, we performed complete genome sequencing and analyzed three different duck-derived parvoviruses that infected different breeds of ducks. Phylogenetic trees based on gene sequences indicated that they were classical goose parvovirus (C-GPV), Muscovy duck parvovirus (MDPV), and N-GPV. Furthermore, potential recombination events were found. These results improve our understanding of the diversity of duck-derived parvoviruses in Anhui province, eastern China, and provide a reference for the prevention of associated diseases.

POECIVIRUS IS PRESENT IN INDIVIDUALS WITH BEAK DEFORMITIES IN SEVEN SPECIES OF NORTH AMERICAN BIRDS.

Avian keratin disorder (AKD), a disease of unknown etiology characterized by debilitating beak overgrowth, has increasingly affected wild bird populations since the 1990s. A novel picornavirus, poecivirus, is closely correlated with disease status in Black-capped Chickadees (Poecile atricapillus) in Alaska, US. However, our knowledge of the relationship between poecivirus and beak deformities in other species and other geographic areas remains limited. The growing geographic scope and number of species affected by AKD-like beak deformities require a better understanding of the causative agent to evaluate the population-level impacts of this epizootic. Here, we tested eight individuals from six avian species with AKD-consistent deformities for the presence of poecivirus: Mew Gull (Larus canus), Hairy Woodpecker (Picoides villosus), Black-billed Magpie (Pica hudsonia), American Crow (Corvus brachyrhynchos), Red-breasted Nuthatch (Sitta canadensis), and Blackpoll Warbler (Setophaga striata). The birds were sampled in Alaska and Maine (1999-2016). We used targeted PCR followed by Sanger sequencing to test for the presence of poecivirus in each specimen and to obtain viral genome sequence from virus-positive host individuals. We detected poecivirus in all individuals tested, but not in negative controls (water and tissue samples). Furthermore, we used unbiased metagenomic sequencing to test for the presence of other pathogens in six of these specimens (Hairy Woodpecker, two American Crows, two Red-breasted Nuthatches, Blackpoll Warbler). This analysis yielded additional viral sequences from several specimens, including the complete coding region of poecivirus from one Red-breasted Nuthatch, which we confirmed via targeted PCR followed by Sanger sequencing. This study demonstrates that poecivirus is present in individuals with AKD-consistent deformities from six avian species other than Black-capped Chickadee. While further investigation will be required to explore whether there exists a causal link between this virus and AKD, this study demonstrates that poecivirus is not geographically restricted to Alaska, but rather occurs elsewhere in North America.

Identification and Characterization of a Distinct Strain of Beak and Feather Disease Virus in Southeast China.

Beak and feather disease virus (BFDV) is an infectious agent responsible for feather degeneration and beak deformation in birds. In March 2017, an epidemic of psittacine beak and feather disease (PBFD) struck a farm in Fuzhou in the Fujian Province of southeast China, resulting in the death of 51 parrots. In this study, the disease was diagnosed and the pathogen was identified by PCR and whole genome sequencing. A distinct BFDV strain was identified and named as the FZ strain. This BFDV strain caused severe disease symptoms and pathological changes characteristic of typical PBFD in parrots, for example, loss of feathers and deformities of the beak and claws, and severe pathological changes in multiple organs of the infected birds. Phylogenetic analysis showed that the FZ strain was more closely related to the strain circulating in New Caledonia than the strains previously reported in China. Nucleotide homology between the FZ strain and other 43 strains of BFDV ranged from 80.0% to 92.0%. Blind passage experiment showed that this strain had limited replication capability in SPF Chicken Embryos and DF-1 Cells. Furthermore, the capsid (Cap) gene of this FZ strain was cloned into the pGEX-4T-1 expression vector to prepare the polyclonal anti-Cap antibody. Western blotting analysis using the anti-Cap antibody further confirmed that the diseased parrots were infected with BFDV. In this study, a PBFD and its pathogen was identified for the first time in Fujian Province of China, suggesting that future surveillance of BFDV should be performed.

Identification and characterization of novel genotypes of psittacine beak and feather disease virus from budgerigar in China.

Psittacine beak and feather disease (PBFD) is a common disease in psittacine bird that caused by beak and feather disease virus (BFDV). BFDV is widely spread and threatening psittacine birds worldwide. However, the BFDV infection in China remains largely unknown. In this study, a surveillance study of BFDV was conducted in three budgerigar breeding facilities, which showed that 66.6% of collected faeces samples were positive for BFDV. Full genomes of nine BFDV circulating in the three budgerigar breeding facilities (three for each facility) were determined and analysed. The full genomes shared 75.9% to 87.5% identity with the known genotype BFDV. Phylogenetic analysis of the full genome indicated that the BFDV circulating in China formed a separated group, and the nine isolates fell into three subgroups, suggesting that different unique BFDV genotypes are circulating in China. Notably, the Cap genes of three strains (SD3, SD5 and SD9) showed low identity (67.9% to 70%) to all the known genotypes of BFDV. Phylogenetic analysis showed that these three Cap genes formed a unique lineage that is different from all known genotypes, which suggested that the SD3, SD5 and SD9 strains identified in this study belong to a novel genotype that has not been reported. However, the origin of this genotype remains unclear. All the data indicated that the different unique genotypes of BFDV are co-circulating in China, and active surveillance of BFDV is warranted.

Novel goose parvovirus in domestic Linwu sheldrakes with short beak and dwarfism syndrome, China.

Recently, short beak and dwarfism syndrome (SBDS) had a sudden outbreak in Cherry Valley duck flocks, followed by Pekin ducks and mule ducks in various regions of mainland China. This widely spreading infectious disease was characterized by growth retardation, smaller beak and tarsus with high morbidity and low mortality rate. In this study, we identified and characterized virus from domestic Linwu sheldrakes (namely as HuN18) with SBDS. HuN18 isolates shared high nucleotide identity with novel goose parvovirus (N-GPV). A 5110-nucleotide full-length genome sequence of HuN18 was found with no deletion in ITR region. Alignment studies of HuN18 showed 96.8%-99.0% identity with other N-GPVs and 92.9%-96.3% identity with classic GPV. According to the recombination analysis, HuN18 showed the potential major parent was the N-GPV sdlc01 strain, the potential minor parent was the classical GPV Y strain, and the secondary potential minor parent was the SYG61v strain. To the best of our knowledge, this is the first report of N-GPV in domestic Linwu sheldrakes with SBDS; these data provide evidence that attenuated live viruses are involved in genetic recombination with prevailing wild parvoviruses, which contributes to the novel emerging variants of waterfowl parvoviruses.

Avian keratin disorder of Alaska black-capped chickadees is associated with Poecivirus infection.

BACKGROUND: Avian keratin disorder (AKD) is an epizootic of debilitating beak deformities, first documented in black-capped chickadees (Poecile atricapillus) in Alaska during the late 1990s. Similar deformities have now been recorded in dozens of species of birds across multiple continents. Despite this, the etiology of AKD has remained elusive, making it difficult to assess the impacts of this disease on wild populations. We previously identified an association between infection with a novel picornavirus, Poecivirus, and AKD in a small cohort of black-capped chickadees. METHODS: To test if the association between Poecivirus and AKD holds in a larger study population, we used targeted PCR followed by Sanger sequencing to screen 124 symptomatic and asymptomatic black-capped chickadees for Poecivirus infection. We further compared the efficacy of multiple non-terminal field sampling methods (buccal swabs, cloacal swabs, fecal samples, and blood samples) for Poecivirus screening. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to determine if virus is actively replicating in beak tissue. RESULTS: Poecivirus was detected in 28/28 (100%) individuals with AKD, but only 9/96 (9.4%) asymptomatic individuals with apparently normal beaks (p < 0.0001). We found that cloacal swabs are the most sensitive of these sample types for detecting Poecivirus in birds with AKD, but that buccal swabs should be combined with cloacal swabs in evaluating the infection status of asymptomatic birds. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to provide evidence of active viral replication. CONCLUSION: The data presented here show a strong, statistically significant relationship between Poecivirus infection and AKD, and provide evidence that Poecivirus is indeed an avian virus, infecting and actively replicating in beak tissue of AKD-affected BCCH. Taken together, these data corroborate and extend the evidence for a potential causal association between Poecivirus and AKD in the black-capped chickadee. Poecivirus continues to warrant further investigation as a candidate agent of AKD.

Pathogenicity of a variant goose parvovirus, from short beak and dwarfism syndrome of Pekin ducks, in goose embryos and goslings.

The pathogenicity of a variant goose parvovirus (GPV), isolated from short beak and dwarfism syndrome of Pekin ducks (strain Cherry Valley), was investigated in embryonating goose eggs and goslings. The virus was easily grown in GPV antibody-free goose embryos and caused high mortality and severe lesions of goose embryos, indicating that the variant GPV has good adaptation and high pathogenicity to embryonated goose eggs similar to the classical GPV. Like the third egg-passage virus (strain H) of a classical GPV, the third egg-passage virus (strain JS1) of the variant GPV caused Derzsy's disease in 2-day-old goslings with high mortality. The findings suggest that the variant GPV strain, which had specifically adapted to Pekin ducks, still retained high pathogenicity for its original host. The mortality (73.3-80%) caused by the first and third egg-passages of the variant GPV was somewhat lower than that (93.3%) caused by the third passage virus of the classical GPV, reflecting the higher pathogenicity of the classical GPV for its original host. These findings are likely to reinforce the importance of surveillance for parvoviruses in different waterfowl species and stimulate further study to elucidate the impact of mutations in the GPV genome on its pathogenicity to goslings and ducks.

Beak and feather disease virus carriage by Knemidocoptes pilae in a sulphur-crested cockatoo (Cacatua galerita).

BACKGROUND: This paper describes the pathology associated with psittacine beak and feather disease in a wild sulphur-crested cockatoo with concurrent knemidocoptic mange, cestodiasis and mycotic encephalitis. METHODS & RESULTS: Large numbers of Knemidocoptes pilae Lavoipierre and Griffiths, 1951 (Acari: Epidermoptidae, Knemidokoptinae) were identified in affected skin associated with enhanced expression of beak and feather disease virus (BFDV) determined by immunohistochemistry. Also, BFDV antigen was demonstrated in high concentration in the gut and faecal sacs of mites, raising the possibility of ectoparasites as fomites and vectors of BFDV transmission. Large numbers of Raillietina spp. cestodes were present in the intestines. Within the brain there was a focally extensive region of necrosis and inflammation associated with branching, septate, pigmented hyphae consistent with zygomycete fungal infection. CONCLUSION: This case highlights the potential immunosuppressive effects of BFDV infection and its potential as a keystone pathogen in the Australian environment.

Pathogenicity of Pekin duck- and goose-origin parvoviruses in Pekin ducklings.

Goose parvovirus (GPV) usually affects goslings and Muscovy ducks but not Pekin ducks. Earlier works showed that a variant GPV can cause short beak and dwarfism syndrome (SBDS) in Pekin ducks. Here, we investigated the pathogenicity of a variant GPV of Pekin duck-origin (JS1) and a classical GPV of goose-origin (H) in Pekin ducklings. Following intramuscular infection at two days of age, both JS1 and H strains influenced weight gain and development of beaks and bones of wings and legs, and caused microscopic lesions of internal organs of ducks. However, the clinical signs typical of SBDS could only be replicated with the JS1 isolate. The findings suggest that both variant and classical GPVs are pathogenic for Pekin ducklings, while the former is more virulent than the latter. Using a quantitative real-time PCR assay, high levels of viral load were detected from bloods, internal organs, leg muscles, and ileac contents in JS1- and H-infected ducks from 6h to 35days postinfection (DPI). Using a GPV VP3-based ELISA, antibodies in sera of JS1- and H-infected ducks were detectable at 1 DPI and then persistently rose during the subsequent five weeks. These results suggest that both variant and classical GPVs can infect Pekin ducklings. The present work contributes to the understanding of pathogenicity of GPV to Pekin ducks and may provide clues to pathogenesis of GPV-related SBDS.

Development of a taqman-based real-time PCR assay for the rapid and specific detection of novel duck- origin goose parvovirus.

A real-time PCR assay was developed for specific detection of novel duck-origin goose parvovirus (N-GPV), the etiological agent of duck beak atrophy and dwarfism syndrome (BADS). The detection limit of the assay was 102 copies. The assay was useful in the prevention and control of BADS.

Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China.

A newly emerged duck parvovirus, which causes beak atrophy and dwarfism syndrome (BADS) in Cherry Valley ducks, has appeared in Northern China since March 2015. To explore the genetic diversity among waterfowl parvovirus isolates, the complete genome of an identified isolate designated SDLC01 was sequenced and analyzed in the present study. Genomic sequence analysis showed that SDLC01 shared 90.8%-94.6% of nucleotide identity with goose parvovirus (GPV) isolates and 78.6%-81.6% of nucleotide identity with classical Muscovy duck parvovirus (MDPV) isolates. Phylogenetic analysis of 443 nucleotides (nt) of the fragment A showed that SDLC01 was highly similar to a mule duck isolate (strain D146/02) and close to European GPV isolates but separate from Asian GPV isolates. Analysis of the left inverted terminal repeat regions revealed that SDLC01 had two major segments deleted between positions 160-176 and 306-322 nt compared with field GPV and MDPV isolates. Phylogenetic analysis of Rep and VP1 encoded by two major open reading frames of parvoviruses revealed that SDLC01 was distinct from all GPV and MDPV isolates. The viral pathogenicity and genome characterization of SDLC01 suggest that the novel GPV (N-GPV) is the causative agent of BADS and belongs to a distinct GPV-related subgroup. Furthermore, N-GPV sequences were detected in diseased ducks by polymerase chain reaction and viral proliferation was demonstrated in duck embryos and duck embryo fibroblast cells.

Psittacine beak and feather disease-like illness in Gouldian finches (Chloebia gouldiae).

Beak and feather disease virus (BFDV) is a member of the genus Circovirus and causes psittacine beak and feather disease (PBFD) in Psittaciformes. PBFD is a severe disease generally characterized by immunodeficiency and beak and feather disorders. Although Circovirus spp. have been detected in several nonpsittacine species, little is known about the symptoms and the disease associated with this infection in birds other than Psittaciformes. In this study, we report the identification of Circovirus infection in a flock of Gouldian finches showing beak and feather disorders. Sequence analyses on the rep gene of the virus highlighted a strong similarity at nucleotide and amino acid levels with the corresponding regions of BFDV from psittacine species. By contrast, it was more distant to circoviruses identified in finch and canary.

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.

Beak necrosis in Hungarian partridges (Perdix perdix) associated with beak-bits and avian poxvirus infection.

Proliferative growth, consistent with poxvirus infection, encapsulated plastic beak-bits and covered the dorsal portion of the upper beak and nares of adult male and female captive-raised Hungarian partridges. Three representative birds were submitted to the Wisconsin Veterinary Diagnostic Laboratory for necropsy. Lesions in the necropsied birds extended through the nares, where the plastic bit ends are designed to rest. The lesions also variably extended caudally into the oropharynx and cranially within the beak epithelium, and included palate deformity and beak necrosis. Poxvirus was diagnosed in all of the birds examined based on histopathology, electron microscopy, and polymerase chain reaction amplification and sequencing. This report is the first to describe avian pox lesions associated with the application of beak-bits and the resulting beak and oral pathology.

A review of DNA viral infections in psittacine birds.

To date, several DNA viral infections have been reported in psittacine birds. Psittacine beak and feather disease (PBFD) is characterized by symmetric feather dystrophy and loss and development of beak deformities. PBFD is caused by beak and feather virus, which belongs to the Circoviridae, and is the most important infection in psittacine birds worldwide. Avian polyomavirus infection causes acute death, abdominal distention, and feather abnormalities. Pacheco's disease (PD), which is caused by psittacid herpesvirus type 1, is an acute lethal disease without a prodrome. Psittacine adenovirus infections are described as having a clinical progression similar to PD. The clinical changes in psittacine poxvirus-infected birds include serious ocular discharge, rhinitis, and conjunctivitis, followed by the appearance of ulcerations on the medial canthi of the eyes. Internal papillomatosis of parrots (IPP) is a tumor disease characterized by progressive development of papillomas in the oral and cloacal mucosa. IPP has been suggested to caused by papillomavirus or herpesvirus. However, information about these diseases is limited. Here we review the etiology, clinical features, pathology, epidemiology, and diagnosis of these DNA viruses.

Short beak and dwarfism syndrome of mule duck is caused by a distinct lineage of goose parvovirus.

From the early 1970s to the present, numerous cases of short beak and dwarfism syndrome (SBDS) have been reported in mule ducks from France. The animals showed strong growth retardation with smaller beak and tarsus. It was suggested that the syndrome was caused by goose parvovirus on the basis of serological investigation, but the causative agent has not been isolated and the disease has not so far been reproduced by experimental infection. The aim of the present study was to characterize the virus strains isolated from field cases of SBDS, and to reproduce the disease experimentally. Phylogenetic analysis proved that the parvovirus isolates obtained from SBDS of mule duck belonged to a distinct lineage of goose parvovirus-related group of waterfowl parvoviruses. The authors carried out experimental infections of 1-day-old, 2-week-old and 3-week-old mule ducks by the oral route with three different parvovirus strains: strain D17/99 of goose parvovirus from Derzsy's disease, strain FM of Muscovy duck parvovirus from the parvovirus disease of Muscovy ducks, and strain D176/02 isolated from SBDS of mule duck. The symptoms of SBDS of the mule duck could only be reproduced with the mule duck isolate (strain D176/02) following 1-day-old inoculation. Infection with a genetically different strain of goose parvovirus isolated from classical Derzsy's disease (D17/99) or with the Muscovy duck parvovirus strain (FM) did not cause any clinical symptoms or pathological lesions in mule ducks.

A comparison of haemagglutination, haemagglutination inhibition and PCR for the detection of psittacine beak and feather disease virus infection and a comparison of isolates obtained from loriids.

Psittacine beak and feather disease (PBFD) is recognized as a threat for endangered psittacine birds in Australia, New Zealand and South Africa. Several diagnostic methods for the detection of beak and feather disease virus (BFDV) infection have been developed but there are few studies comparing the relative merits or sensitivity and specificity of each diagnostic test. In this report, the results of PCR, haemagglutination (HA) and haemagglutination inhibition (HI) testing of diagnostic samples collected from 679 samples from a range of psittacine bird species suspected of being infected with BFDV are summarized and compared. There was a strong agreement (kappa = 0.757; P<0.0001) between PCR and HA testing of feather samples and PCR-negative birds were 12.7 times more likely to have HI antibody than PCR-positive birds. False-positive HA results with titres up to 1:320 were identified in six feather samples that were PCR negative; the haemagglutination detected in these samples was not inhibited by anti-BFDV antisera and was removed by filtration through a 0.22 microm filter. Similarly, one false-negative PCR result was detected in a feather sample that had a high HA titre (>1:40,960) and four false-positive PCR results were detected in a batch of four feather samples. Of 143 birds that were feather PCR positive, only two had detectable HI antibody, and these birds were also feather HA negative, suggesting that they were developing immunity to recent infection. All birds with HI antibody were negative on feather HA testing. The assays confirmed BFDV infection in two endangered swift parrots (Lathamus discolor) and phylogenetic analysis of the sequence data generated from ORF V1 of these isolates provide further evidence of BFDV genotypes clustering in parallel with the Loriidae, Cacatuidae and Psittacidae.

Nucleotide sequence analysis of a C1 gene fragment of psittacine beak and feather disease virus amplified by real-time polymerase chain reaction indicates a possible existence of genotypes.

To investigate sequence diversity of psittacine beak and feather disease virus, samples collected from 31 psittacine species with or without clinical signs were tested for the presence of the viral genome. A real-time polymerase chain reaction was developed amplifying a 202 base pair fragment of the region encoding the capsid protein C1 and detecting 100 to 1000 genome equivalents. The nucleotide sequences of the polymerase chain reaction products showed 84.1 to 100% identity with no consistent pattern with regard to the infected bird species. Amino acid exchanges were concentrated mainly in five of the 42 deduced positions. Sequences obtained from an outbreak of acute beak and feather disease in lories clustered in a separate branch of a phylogenetic tree. Sequences in samples from African grey parrots with feather disorders grouped together, whereas those from the same species with immunosuppression clustered in other branches. These results indicate the possible existence of beak and feather disease virus genotypes.

A universal polymerase chain reaction for the detection of psittacine beak and feather disease virus.

A universal PCR assay was designed that consistently detected psittacine beak and feather disease virus (BFDV) in psittacine birds affected with psittacine beak and feather disease (PBFD) from different geographic regions across Australia. Primers within open reading frame 1 (ORF1) of the BFDV genome consistently amplified a 717 bp product from blood and/or feathers of 32 birds with PBFD lesions. The PCR did not amplify a product from the feathers or blood from 7 clinically normal psittacine birds. Primers based on regions outside of ORF1 did not consistently produce a PCR product, suggesting there was some genomic variation outside ORF1. The amplified ORF1 PCR products of 10 BFDV isolates, from different psittacine species and from various regions around Australia, were cloned and comparative DNA sequence analysis demonstrated 88-99% of the ORF1 fragments. The derived amino acid sequences of the amplified ORF1 fragments demonstrated similar identity between all 10 isolates. Within ORF1, there was complete conservation of the putative nucleotide binding site and marked conservation of 2 other motifs previously identified as essential components of the replication-associated proteins of other circoviruses and geminiviruses.