Isolation and characterization of an Aves polyomavirus 1 from diseased budgerigars in China.

Aves polyomavirus 1 (APV) causes inflammatory disease in psittacine birds, especially in young budgerigar. In this study, an APV virus (SD18 strain) was isolated from a diseased psittacine birds breeding facility. The full genome (4981 bp) of SD18 was determined and analyzed. Phylogenetic analysis of full genome sequences indicated all the APV strains form two groups. The SD18 strain showed close relationship with APV isolated from Poland, however, the other Chinese strains are located in group II, which suggested different genotypes APVs are co-circulating in China. Compared with the consensus sequence of APV full genome, the SD18 strain contains 13 nucleotide mutations, and 2 unique amino acid substitutions (R179M and Q382K) located in VP2/3 and Large T proteins. To explore the pathogenicity of the virus, the SD18 strain was used to challenge 2-week-old budgerigars. All infected birds died no later than 5 days post infection, and virus was detected in multiple organs including brain, heart, ingluvies, liver, and intestine, which indicated that SD18 is fatal and causes systemic infection in young budgerigar. In vitro studies showed that SD18 replicated efficiently in CEF cells and reached the highest viral titers at 9 days post infection. Notably, replication of SD18 stimulated IFN-ß response in CEF cells and overexpression of the VP4 or VP4Delta proteins significantly inhibited IFN-ß promoter activation, which could be the strategy of APV to escape from the host innate immunity.

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.

Establishment of rapid detection method and surveillance of budgerigar fledgling disease virus using a TaqMan Real-Time PCR.

Budgerigar fledgling disease virus (BFDV) infection causes sudden death, abdominal distention, and feather abnormality in psittacine birds. In this study, we developed a TaqMan Real-time PCR assay to detect BFDV by targeting a conserved region in VP1 gene. The detection limit of the assay was 30 DNA gene copies, 1000 times more sensitive than conventional PCR. The coefficients of variation were less than 1.09% in either intra- or inter-assays, indicating high reproducibility. By using this method, the prevalence of BFDV in China was evaluated. 56 feces samples were collected from four psittacine birds breeding facilities in China. The results showed 28 out of 56 samples were positive for BFDV in Real-Time PCR assay, while only 19 samples were positive in PCR assay. Three facilities were positive for BFDV with positive rates from 60% to 87.5%. Further sequence analysis of VP1 genes from the positive samples indicated that VP1 genes fell into two different lineages in phylogenetic tree, suggesting that different genotypes BFDV are co-circulating in China.

Experimental inoculation of BFDV-positive budgerigars (Melopsittacus undulatus) with two Mycobacterium avium subsp. avium isolates.

Beak and feather disease virus- (BFDV-) positive (naturally infected) but clinically healthy budgerigars (Melopsittacus undulatus) were inoculated with two isolates of Mycobacterium avium subsp. avium isolated from naturally infected golden pheasant (Chrysolophus pictus) and peafowl (Pavo cristatus). During a period of more than two months after inoculation, samples of cloacal and crop swabs, faeces, and blood were obtained for BFDV and Mycobacterium avium testing with PCR. Birds were euthanized nine weeks after inoculation. All infected budgerigars developed signs typical of mycobacteriosis, but more advanced clinical and pathological changes were visible in the group infected with the pheasant isolate. Only a few cloacal and crop swab samples were positive for Mycobacterium avium subsp. avium despite advanced pathological changes in the internal organs. In the groups infected with mycobacterium isolates the frequency of BFDV-positive samples was higher than in the control group. In the infected groups the frequency of BFDV was substantially higher in the cloacal swabs of birds inoculated with the pheasant isolate than in the peafowl-isolate-infected group.

Endogenous hepadnaviruses in the genome of the budgerigar (Melopsittacus undulatus) and the evolution of avian hepadnaviruses.

Endogenous hepadnaviruses (hepatitis B viruses [HBVs]) were recently discovered in the genomes of passerine birds. We mined six additional avian genomes and discovered multiple copies of endogenous HBVs in the budgerigar (order Psittaciformes), designated eBHBV. A phylogenetic analysis reveals that the endogenous hepadnaviruses are more diverse than their exogenous counterparts and that the endogenous and exogenous hepadnaviruses form distinct lineages even when sampled from the same avian order, indicative of multiple genomic integration events.

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.

Molecular genetic and pathogenic characterization of psittacid herpesvirus type 1 isolated from a captive galah (Eolophus roseicapillus) in Japan.

Psittacid herpesvirus type 1 (PsHV-1) was isolated from a captive galah (Eolophus roseicapillus) in Japan that was suspected of having Pacheco's disease (PD), an acute fatal disease in psittacine birds. PsHV-1 has been classified into four genotypes based on the UL16 gene sequence. In the present study, we investigated the genetic and pathogenic characteristics of the isolated virus, FOY-1, compared with a reference strain, RSL-1. The FOY-1 strain was classified into PsHV-1 genotype 2. The FOY-1 strain was found to be less pathogenic to budgerigars than RSL-1, which was classified as genotype 4 in an in vivo study. This is the first report regarding the classification of originally isolated PsHV-1 in Japan and its characterization by animal infection experiment.

The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4.

Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting ß-hairpin observed in other polyomaviruses. We postulate that the terminal ß-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

A novel genotype of beak and feather disease virus in budgerigars (Melopsittacus undulatus).

Beak and feather disease virus (BFDV) is a causative agent for psittacine beak and feather disease (PBFD), which shows a characteristic feather disorder in psittacine birds. Nineteen budgerigars, which were clinically suspected to have PBFD, were examined by two polymerase chain reactions (PCR), which target each of open reading frames (ORFs) V1 and C1. All of the 19 samples were detected BFDV by the PCR targeting ORF C1, whereas only two of them were detected by the PCR targeting ORF V1. It was assumed that BFDV derived from budgerigar (budgerigar BFDV) has two genotypes, which are tentatively classified as budgerigar BFDV genotype 1 and genotype 2 by the PCR amplification patterns. Whole genome sequences of six budgerigar BFDVs were determined to reveal the existence of two genotypes. In the phylogenic analysis, six budgerigar BFDV sequences formed a unique group branched from the other 23 published BFDV sequences. The budgerigar BFDV genotype 1 and genotype 2 were also segregated each other, and budgerigar BFDV genotype 2 was particularly distantly related with the other BFDVs. These results suggest budgerigar BFDV is a unique in the known BFDVs and is divided into two genotypes.

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 approximately 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.

Molecular characterization of avian polyomavirus isolated from psittacine birds based on the whole genome sequence analysis.

Seven avian polyomaviruses (APVs) were isolated from seven psittacine birds of four species. Their whole genome sequences were genetically analyzed. Comparing with the sequence of BFDV1 strain, nucleotide substitutions in the sequences of seven APV isolates were found at 63 loci and a high level of conservation of amino acid sequence in each viral protein (VP1, VP2, VP3, VP4, and t/T antigen) was predicted. An A-to-T nucleotide substitution was observed in non-control region of all seven APV sequences in comparison with BFDV1 strain. Two C-to-T nucleotide substitutions were also detected in non-coding regions of one isolate. A phylogenetic analysis of the whole genome sequences indicated that the sequences from the same species of bird were closely related. APV has been reported to have distinct tropism for cell cultures of various avian species. The present study indicated that a single amino acid substitution at position 221 in VP2 was essential for propagating in chicken embryonic fibroblast culture and this substitution was promoted by propagation on budgerigar embryonic fibroblast culture. For two isolates, three serial amino acids appeared to be deleted in VP4. However, this deletion had little effect on virus propagation.

A novel budgerigar-adenovirus belonging to group II avian adenovirus of Siadenovirus.

Five budgerigars in the same breeding facility died or showed ruffled feathers. To determine the cause, five dead or euthanized budgerigars were examined. Splenomegaly was observed at necropsy in all birds examined. Histopathology of the spleen revealed a slight-to-moderate deletion of lymphocytes and increase of macrophages. Concurrent congestions in several tissues such as liver, lung, kidney, and/or brain and basophilic intranuclear inclusion bodies in the epithelial cells of renal tubules were found in all the birds examined. Psittacine adenoviral DNA was detected in the kidney of one of the five budgerigars by PCR. Sequencing and phylogenetic analysis of the hexon gene revealed that the adenovirus gene detected in the budgerigar was derived from an unknown adenovirus belonging to the genus Siadenovirus. Using a new pair of primers based on the obtained sequence, we confirmed the presence of the newly found adenovirus in all five birds. The newly found unknown adenovirus is designated as Budgerigar Adenovirus 1.

Molecular characterizations of avian polyomavirus isolated from budgerigar in China.

Budgerigar fledgling disease is an acute viral infectious disease caused by avian polyomavirus (APV). In this study, 34 liver tissue samples of young, dead budgerigar with typical symptoms were collected in 2004. All the samples had positive polymerase chain reaction (PCR) test based on the VP1 specific primers. VP1 genes of these samples were sequenced and had high similarities to each other (99%-100%). A strain (HBYM02) was isolated and sequenced. As shown in the phylogenetic tree, there are two branches. One branch was composed by strains isolated from Passeriformes, and the other was composed only by one strain isolated from Falconiformes. The genome similarities between our isolate and other reported isolates were very high (> 99%), and the evolution distances in the phylogenetic tree were very short (< 0.005), which suggests that APV in China has the same genotype as those in other regions. The results will be useful for the diagnoses of, and vaccine development for, APV.

An outbreak of the polyomavirus infection in budgerigars and cockatiels in Slovakia, including a genome analysis of an avian polyomavirus isolate.

In winter 2003-04, large numbers of budgerigars (Mellopsitacus undulatus) and cockatiels (Nymphicus hollandicus) fell ill and died in a large parrot-breeding aviary in Slovakia. In budgerigars, the disease outbreak occurred at the age of 2-3 weeks; cockatiels died within their first 7 days of life. In budgerigars, symptoms of the disease included delayed growth, tremor, darkish discoloration of skin, quill bleeding, and feathering defects. cockatiels often died without any symptoms and with a full crop; feathering defects occurred sporadically. Electron microscopy with negative staining of aqueous lysates of the affected skin and of bleeding quills showed isolated or clustered polyomavirus particles 45-50 nm in size. Long filamentous forms of the virus were also found in virion clusters of skin lysates from the budgerigars. In ultrathin sections through the pathologically altered skin tissue of budgerigars, virus particles were present in both nuclei and cytoplasm of epidermal cells, often in crystalline form. In infected cells, enlarged nuclei showed an extensive chromatin margination. On the DNA level, presence of a polyomavirus infection was conclusively proved by the polymerase chain reaction using avian polyomavirus (APV)-specific primers. A sequence analysis of the gene encoding viral protein (VP)1 and of the combined region for VP2 and VP3 proteins revealed a previously undescribed synonymous mutation in this isolate. This report extended the knowledge of the area of APV occurrence and of the spectrum of hosts in the context of genomic and morphologic variability of APV isolates.