Genome sequence determinations and analyses of novel circoviruses from goose and pigeon

The genomes of novel circoviruses from goose and pigeon, which were isolated using degenerate primer and inverse primer PCR methods, were cloned and sequenced. Comparative nucleotide (nt) sequence analyses showed that the goose circovirus (GCV) and pigeon circovirus (PiCV) possessed genomes which were 1821 and 2037 or 2036 nt, respectively, and which had features in common with the genomes of porcine circoviruses types 1 and 2 (PCV1, PCV2) and psittacine beak and feather disease virus (BFDV), such that they can now be assigned to the genus Circovirus of the family Circoviridae. Common features include the possession of (i) a potential stem-loop/nonanucleotide motif with which the initiation of rolling circle replication of the virus DNA is associated; (ii) two major ORFs, located on the virus (V1 ORF) and complementary (C1 ORF) strands, which encode the replication-associated protein (Rep) and capsid protein, respectively; (iii) high levels of amino acid identity (41.2--58.2%) shared with other circovirus Rep proteins; and (iv) direct/inverted repeat sequences within the putative intergenic region. On the basis of nt and amino acid sequence identities, GCV is substantially less closely related to BFDV than PiCV is to BFDV.     

Comparitive sensitivity of polymerase chain reaction diagnosis of psittacine beak and feather disease on feather samples, cloacal swabs and blood from budgerigars (Melopsittacus undulates, Shaw 18005).

A longitudinal study was performed in order to investigate virus excretion and viraemia during a clinical outbreak of the psittacine beak and feather disease in budgerigars (Melopsittacus undulatus). Viral nucleic acid was detected in feathers, cloacal swabs and blood samples. Overall, beak and feather disease virus (BFDV) DNA was detected most commonly in feather samples, followed by cloacal swabs, and least frequently from blood samples. In most cases the viraemia was short lived and correlated with clinical signs, such as feather abnormalities. Sequence analysis of the polymerase chain reaction fragment amplified from the replication-associated gene (ORF V1) indicated a close relationship with other BFDV isolates. Overall the highest level of nucleotide identity was found with the ORF V1 of another budgerigar isolate. Our results suggest that feather samples and cloacal swabs should be taken for polymerase chain reaction diagnosis to determine the presence of BFDV in an aviary, but that detection in these samples may not correlate well with psittacine beak and feather disease.     

A unique isolate of beak and feather disease virus isolated from budgerigars (Melopsittacus undulatus) in South Africa

Beak and feather disease virus (BFDV), the causative agent of psittacine beak and feather disease (PBFD) infects psittaciformes worldwide. We provide an annotated sequence record of three full-length unique genomes of BFDV isolates from budgerigars (Melopsittacus undulatus) from a breeding farm in South Africa. The isolates share >99% nucleotide sequence identity with each other and ~96% nucleotide sequence identity to two recent isolates (Melopsittacus undulatus) from Thailand but only between 91.6 and 86.6% identity with all other full-length BFDV sequences. Maximum-likelihood analysis and recombination analysis suggest that the South African budgerigar BFDV isolates are unique to budgerigars, are non-recombinant in origin, and represent a new genotype of BFDV.     

Beak and feather disease virus infection in cockatiels (Nymphicus hollandicus).

Psittacine beak and feather disease is known to occur in a wide range of psittacine species; however, there are no scientific or credible anecdotal reports of psittacine beak and feather disease occurring in the cockatiel (Nymphicus hollandicus) despite it being one of the world's most commonly kept companion bird species. Consequently, this has resulted in speculation that the species may have some innate resistance to beak and feather disease virus (BFDV) infection. To investigate this hypothesis we conducted a survey of cockatiels (n=88) at commercial aviaries to investigate whether BFDV infection occurs in cockatiels, and found that all birds were virus-free by polymerase chain reaction and haemagglutination assay and had no detectable antibody titre by haemagglutination-inhibition assay. In addition to this, we sequenced the genome of two BFDV isolates obtained from diseased cockatiel feathers and performed cross-reactivity assays 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 paper to report evidence of an antigenically distinct BFDV in psittacine birds.     

Genetic diversity of the Rep gene of beak and feather disease virus in South Africa

Summary. A study on the genetic variation of Beak and feather disease virus (BFDV) isolates in South Africa was performed by amplifying and sequencing a region within the ORF1 of the genome. Six different BFDV isolates were found in 15 psittacine species from 6 regions within South Africa, representing three unique isolates and three isolates that clustered into a budgerigar lineage (BG) previously described.     

Hematology of vaccinated and non-vaccinated long-billed corellas following infection with beak and feather disease virus (BFDV)

The hematological characteristics of juvenile long-billed corellas (Cacatua tenurostris), with or without prior administration of a psittacine beak and feather disease vaccine, were studied for 97 days after experimental infection with beak and feather disease virus (BFDV). It was found that the pre-challenge hematological values were similar between 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 packed cell volume and total serum protein were not significantly affected by BFDV challenge.     

Identification of a novel circovirus in Australian ravens (Corvus coronoides) with feather disease.

The complete genome of a novel Circovirus isolated from an Australian raven (Corvus coronoides) with feather lesions similar to those that occur in psittacine beak and feather disease is reported. Degenerate polymerase chain reaction primers were designed to amplify and sequence novel Circovirus DNA from affected feathers. Sequence analysis indicated that the tentatively named raven circovirus (RaCV) was 1898 nucleotides in size with two major open reading frames synonymous with other avian circoviruses, ORF C1 and ORF V1, likely to encode a putative capsid protein (Cap) and replicase-associated protein (Rep), respectively. In common with other circoviruses was the conservation of several nucleotide structures and amino acid motifs implicated in virus replication. Comparison with other members of the Circoviridae demonstrated that RaCV shares the greatest sequence homology with canary circovirus (CaCV) and pigeon circovirus (PiCV) and was more distantly related to the beak and feather disease virus, goose circovirus, duck circovirus and the two porcine circoviruses, PCV1 and PCV2. Phylogenetic analysis of the genome and the putative Cap and Rep proteins provided further evidence of the close relationship of RaCV with CaCV and PiCV.     

Molecular analysis and associated pathology of beak and feather disease virus isolated in Italy from young Congo African grey parrots (Psittacus erithacus) with an “atypical peracute form” of the disease

This study is the first report on the genetic and pathogenic characterization of beak and feather disease virus (BFDV) occurring in Italy. Twenty BFDV strains isolated in Italy from juvenile Congo African grey parrots (Psittacus erithacus) were investigated. Seventeen strains showed an “atypical peracute form” (aPF) of the disease, and three a chronic form (CF). The birds with aPF had been weaned, were independent as far as food and protection were concerned and apparently were without lesions. The gene coding for the putative coat protein was amplified in all isolates while the BFDV genome was sequenced completely in 10 samples, eight of them belonging to aPF affected birds and two from CF of the disease. All full genomes clustered into the J strain of BFDV, where two new subtypes were identified. Recombination analyses showed evidence of genetic exchanges in two BFDV genomes. In addition, a correlation between viral isolate and origin of the breeding material was shown, while an association between the genetic features of the virus and the clinical form was not observed. Histologically, apoptosis was detected frequently in aPF samples and sporadically in CF samples. Interestingly, BFDV antigens were detected in the nuclei and cytoplasm of such apoptotic cells. The data presented here support the hypothesis that, in the absence of a defined BFDV genetic variant accountable for a specific clinical form of psittacine beak and feather disease, differences in the apoptotic rate between aPF and CF are strictly host related.     

Evidence for specificity of psittacine beak and feather disease viruses among avian hosts

Beak and feather disease is a major avian disease of both captive and wild parrot and cockatoo populations. Clinical signs include beak elongation and abnormal growth, together with weight loss and in some individuals the disease is fatal. We investigated the relationship between viral genotypes and their hosts in order to test for a positive association between distinct viral genomes and avian species. Specifically, we used the polymerase chain reaction (PCR) to amplify and sequence a 605-nucleotide (nt) segment of a coding region in the Beak and Feather Disease Virus (BFDV) genome. Feather and blood samples from 25 caged birds representing 10 species were assayed and the BFDV was detected in 21 samples from New Zealand. A phylogenetic analysis of DNA sequences from 17 specimens together with previously published sequences from Australian "isolates" revealed three lineages present in New Zealand. One viral lineage was found in six cockatoos representing two species (designated CT), a second lineage was detected in a budgerigar (designated BG), and a third was found in 10 lorikeets representing seven species (designated LK). This distinctive clustering pattern of viral sequences with groups of psittacine species indicates a genotypic association between the virus and these hosts.     

Development and applications of a monoclonal antibody to a recombinant beak and feather disease virus (BFDV) capsid protein

The development of diagnostic assays for detecting beak and feather disease virus (BFDV) has traditionally been hampered by the difficulty associated with producing suitable reagents, namely purified virus and polyclonal antibodies. In an effort to develop a consistent and standardised source of antibody, a monoclonal antibody to a recombinant BFDV capsid protein has been developed and its use in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays characterised. 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 three genera of psittacine birds, including the recently described cockatiel BFDV isolate. The monoclonal antibody should have widespread application in both research and the development of diagnostic assays for BFDV.     

Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests.

Beak and feather disease virus (BFDV) causes severe disease characterized by irreversible feather disorders and severe immunosuppression in many psittacine species. BFDV cannot be propagated in tissue or cell cultures, rendering virus propagation and thus diagnosis rather difficult. To develop reliable diagnostic methods, the region encoding the BFDV capsid protein C1 was cloned from an infected sulphur-crested cockatoo (Cacatua galerita). Phylogenetic analysis showed this gene had 76.3 to 83.2% amino acid identity to published sequences. No protein was detected after induction of full-length C1 expression in Escherichia coli. However, deletion of an amino-terminal arginine-rich sequence facilitated expression. C1(39-244)-His, a polyhistidine-tailed variant of this protein, was purified and used for immunization of chickens. The immune sera detected C1 with an apparent molecular weight of 27 kDa in western blots of organ homogenates of BFDV-infected birds. Using C1(39-244)-His as antigen, 11 psittacine sera were tested for the presence of BFDV-specific antibodies by enzyme-linked immunosorbent assay and immunoblotting. The results obtained correlated well with the BFDV-specific haemagglutination inhibition activity of the sera, suggesting C1(39-244)-His has value as a recombinant antigen for BFDV-specific serological tests.     

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.     

Baculovirus expression of beak and feather disease virus (BFDV) capsid protein capable of self-assembly and haemagglutination

Beak and feather disease virus (BFDV) is a common avian circovirus infection of wild Psittaciformes and is a recognised threat to endangered psittacine species. Currently, there is a requirement to develop BFDV antigen for diagnostic purposes and since efforts to propagate BFDV in vitro have so far been unsuccessful the entire coding region of BFDV ORF C1 was expressed in Sf9 insect cells using a baculovirus expression system. The entire coding region of BFDV ORF C1, the presumptive capsid, was expressed in Sf9 insect cells using baculovirus expression system. Electron microscopic examination of negatively stained material demonstrated that the recombinant protein self-assembled to produce virus-like particles (VLPs) thus confirming that ORF C1 is likely to be the sole determinant for capsid construction in vivo. BFDV VLPs also possessed haemagglutinating activity which provides further evidence that self-assembled BFDV VLPs retain receptor mediated biological activity and that the determinants for BFDV haemagglutination activity rely solely on the capsid protein. The recombinant protein reacted with anti-BFDV sera from naturally immune parrots and cockatoo and from chickens experimentally inoculated with native BFDV in both Western blots and haemagglutination inhibition (HI) assay. BFDV VLPs were also a suitable replacement antigen for serological detection of BFDV antibody by HI.     

A new isolate of beak and feather disease virus from endemic wild red-fronted parakeets (Cyanoramphus novaezelandiae) in New Zealand

Psittacine beak and feather disease (PBFD) is a viral disease distributed worldwide with a potentially critical impact on many rare parrots. While efforts have been made to determine its prevalence in wild and captive psittacines, only limited work has been done to document complete genomes of its causative agent, beak and feather disease virus (BFDV). Here, we describe five full genomes of BFDV isolated from wild specimens of an endemic New Zealand parrot, the red-fronted parakeet (Cyanoramphus novaezelandiae). The isolates share >99% nucleotide similarity amongst themselves and ~91–92% similarity to BFDV isolates from southern Africa, Europe and Australia. A maximum-likelihood (ML) phylogenetic tree including 42 other full-genome sequences indicated that the five isolates from red-fronted parakeets represent an undescribed genotype of BFDV. These isolates are evolutionarily most closely related to the Cacatuini isolates from Thailand and the Lorinae isolates from Australia in the rep gene ML tree; however, in the cp ML tree, the evolutionary relationship is closer to viruses found in the Psittacini.     

Characterization of a new virus from cockatoos with psittacine beak and feather disease

A novel virus isolated from the feather follicles of cockatoos diagnosed as having psittacine beak and feather disease was characterized by electron microscopy, nucleic acid content, and polypeptide composition. Purified virions displayed an icosahedral symmetry, were nonenveloped, and had a mean diameter of 14 to 16 nm negatively stained. Three major viral proteins were identified, with approximate molecular weights of 26.3, 23.7, and 15.9 kDa. The viral nucleic acid was found to be single-stranded DNA based on acridine orange staining, resistance to alkali and ribonuclease, and sensitivity to both DNAse 1 and S1 nuclease. The size of the DNA was estimated to be between 1.7 and 2.0 kb by agarose gel electrophoresis. This size and its circular conformation were confirmed by electron microscopy. A preliminary transmission study using purified virus induced pathological lesions characteristic of those observed in the natural disease. On the basis of the extremely small size of the virions and the single-stranded circular viral DNA, we propose that the etiologic agent of psittacine beak and feather disease represents a previously undescribed viral pathogen.     

Prevalence and genetic characterization of avian polyomavirus and psittacine beak and feather disease virus isolated from budgerigars in Mainland China

Budgerigar fledgling disease (BFD) and psittacine beak and feather disease (PBFD) are caused by avian polyomavirus (APV) and psittacine beak and feather disease virus (PBFDV), respectively. These diseases frequently infect psittacine birds and result in similar clinical manifestations. In this study, we observed the prevalence of PBFDV infection and a dual infection of APV and PBFDV in a budgerigar (Melopsittacus undulatus) in Mainland China for the first time. One PBFDV isolate and two APV isolates were harvested using chicken embryos. Genetic characterization and phylogenetic analysis of the complete genome of the two APV isolates revealed nucleotide similarity ranging from 99.0% to 99.6% to other sequences in GenBank, and a 14-bp insertion was observed in the genome of one APV isolate. The results of complete genome analysis of the PBFDV isolate showed nucleotide similarity ranging from 83.0% to 95.0% with other PBFDV sequences in GenBank. Genetic characterization and phylogenetic analysis of the APV and PBFDV strains isolated in this study indicated that the isolates from China were closely related to their Japanese counterparts. The results of this study will help to identify molecular determinants and will aid further research on the prevention and control of APV and PBFD infection.     

Genome characterization and genetic diversity of beet curly top Iran virus: a geminivirus with a novel nonanucleotide

Beet curly top Iran virus (BCTIV) was previously reported as a distinct curtovirus in Iran. Complete nucleotide sequences of three BCTIV isolates, one each from central, southern, and south eastern Iran were determined to be 2844, 2844, and 2845 nt long, respectively. BCTIV shared highest nucleotide sequence identity (52.3%) with Spinach curly top virus (SpCTV) and lowest identity (46.6%) with Horseradish curly top virus (HrCTV). The BCTIV genome comprises three virion-sense (V1, V2, and V3) and two complementary-sense (C1 and C2) ORFs. ORFs C3 and C4 were not found in BCTIV genome. Based on a comparison of nucleotide sequence identity of individual genes, the three virion-sense ORFs were 72.7–79.9% related to the corresponding ORFs of curtoviruses, whereas no significant relationship was found between the C1 and C2 ORFs of BCTIV and curtoviruses. These two ORFs, however, were only distantly related with those of mastreviruses. Similar to the latter viruses, the BCTIV genome comprises two intergenic regions. The BCTIV large intergenic region included a sequence capable of forming a stem loop structure and a novel nonanucleotide (TAAGATT/CC) with a unique nick site. Phylogenetic analysis using deduced amino acid sequence of individual ORFs revealed that the V2 and V3 ORFs are monophyletic and the V1 ORF is classified with the related ORF of curtoviruses. Whereas the two complementary-sense ORFs are grouped with those of mastreviruses. Computer-based prediction suggested that BCTIV has a chimeric genome which may have arisen by a recombination event involving curto- and mastrevirus ancestors. Percent nucleotide sequence identities of the coat protein gene of ten isolates of BCTIV, collected from a wide range of geographical regions in Iran, varied from 87.1 to 99.9, with the isolates being distributed between two subgroups. Based on biological and molecular properties, BCTIV is proposed as a new member of the genus Curtovirus.     

Development of novel real-time PCR assays for detecting DNA virus infections in psittaciform birds

Viral diseases of psittacine birds are detected presently by PCR. However, conventional PCR methods are not quantitative and the products can sometimes include non-specific products of the same size. To avoid these problems, real-time PCR assays based on the SYBR Green assay system were developed for the detection and quantitation of four virus diseases of psittacine birds: psittacine beak and feather disease, avian polyomavirus infection, psittacid herpesvirus infection, and psittacine adenovirus infection. Up to 1x10(2) copies of virus DNA were detected, indicating that these assays are as sensitive as conventional PCR assays. The assays are specific because they did not amplify any other pathogens including other viruses, bacteria, and fungi in psittacine birds. The assays measured successfully virus loads in clinical samples (blood, feathers, and tissues), showing that these specimens were suitable targets for the detection and quantitation of viral DNA in psittacine birds.     

Comparison of three animal viruses with circular single-stranded DNA genomes

Summary No common antigenic determinants and no DNA sequence homologies were detected when three animal viruses, chicken anaemia agent (CAA), porcine circovirus (PCV), and psittacine beak and feather disease virus (PBFDV), all of which possess circular single-stranded DNA genomes, were compared. Negative contrast electron microscopy showed that PCV and PBFDV particles were 30% smaller than CAA particles and lacked the surface structure of CAA.