Budgerigar Fledgling Disease Research Articles

Avian polyomavirus in wild birds: genome analysis of isolates from Falconiformes and Psittaciformes

Avian polyomavirus (APV) infections have been reported to cause fatal disease in a wide range of psittacine species. Here we demonstrate APV infections in buzzards (Buteo buteo) and in a falcon (Falco tinnunculus) found dead in Germany, and in lovebirds (Agapornis pullaria) with fatal disease, wild-caught in Moçambique. APV infection in buzzards was determined by PCR amplification of parts of the viral genome followed by Southern blot hybridisation. The genomes of the isolates obtained from the falcon and one of the lovebirds proved to be very closely related to those of Budgerigar Fledgling Disease Virus (BFDV)-1, BFDV-2 and BFDV-3, isolated from budgerigar, chicken, and parakeet, respectively. A consensus sequence was delineated from the known nucleotide sequences of APV isolates. The significance of some nucleotide changes is discussed. Infectivity of all of these isolates was neutralized by antibodies directed against BFDV-1. Data presented in this investigation show that the polyomavirus isolates obtained from different avian species so far all belong to one genotype and one serotype within the proposed subgenus Avipolyomavirus of the family Papovaviridae. The designation Budgerigar Fledgling Disease Virus (BFDV) is, therefore, misleading as this virus type infects different species of birds. The name Avian Polymavirus and the abreviation APV should be adopted to all of the isolates investigated in detail at present. The possible role of birds of passage in the epidemiology in APV infections is discussed.

A concurrent outbreak of psittacine beak and feather disease virus, and avian polyomavirus infection in budgerigars (Melopsittacus undulatus)

A dual natural infection with psittacine beak and feather disease virus and budgerigar fledgling disease virus in a breeding aviary of budgerigars (Melopsittacus undulatus) is described. One‐hundred per cent of newly‐hatched birds were affected and mortality was high (85%). Most surviving birds had diarrhoea, feather alterations and 30% mortality after a 5‐ to 10‐week period. Necropsies of 1‐week‐old and 2‐month‐old birds demonstrated non‐specific lesions, but histologically nuclear inclusions suggestive of viral infection (adenovirus, herpesvirus, polyomavirus) and also cytoplasmic inclusions suggestive of beak and feather disease were seen in integument and internal organs. Staining of tissues with viral specific DNA probes for psittacine beak and feather disease virus and for avian polyomavirus demonstrated simultaneous presence of viral DNA from both viruses in some birds. This is the first description of a concurrent outbreak of psittacine beak and feather disease virus and avian polyomavirus infection in a breeding aviary.

The Effect of Space Flight on Monoclonal Antibody Synthesis in a Hybridoma Mouse Cell Line

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [ 35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

Characterization of a calcium binding domain in the VP1 protein of the avian polyomavirus, budgerigar fledgling disease virus

Calcium ions appear to play a major role in maintaining the structural integrity and assembly of papovavirus virions and are likely involved in the process of viral uncoating. Recently it was reported that the purified recombinant VP1 protein of budgerigar fledgling disease virus (BFDV) was capable of assembling into capsid-like particles in the presence of calcium. It is now reported that the major capsid protein VP1 of BFDV binds calcium ions in an in vitro calcium binding assay. Two deletions were made in the VP1 protein to identify a calcium binding domain and to further characterize the role of calciumm ions in the capsid assembly process. Recombinant VP1 lacking a putative calcium-binding domain (Asp-237–Asp-248) failed to bind radioactive 45Ca 2+ yet associated into capsomeres. These capsomeres were similar in shape to the wild-type VP1 but were unable to assemble into capsid-like particles. Likewise, recombinant VP1 lacking ten carboxyl terminal amino acids (Glu-334–Arg-343) also formed capsomeres that were unable to assemble into capsid-like particles. In contrast to the VP1 protein with the internal deletion, the protein with the truncated carboxyl terminus bound 45Ca 2+ in the in vitro assay. These results have identified a calcium binding domain (Asp-237–Asp-248) for the BFDV VP1 protein and a crucial role for the VP1 carboxyl terminal amino acids (Glu-334–Arg-343) in capsid assembly.

Development and Application of an Immunohistochemical Staining Technique to Detect Avian Polyomaviral Antigen in Tissue Sections

An immunohistochemical staining technique was developed to detect polyomaviral antigens of budgerigar fledgling disease in formalin-fixed tissue sections. This technique used an indirect avidin-biotin, alkaline phosphatase labeling system with a mixture of monoclonal antibodies developed against the virus major capsid protein. The staining technique was applied retrospectively to 24 avian accessions which were originally diagnosed as budgerigar fledgling disease or avian polyomavirus infection based on microscopic findings including typical intranuclear inclusions. Immunohistochemical staining resulted in positive reactions in some tissues from 17 of 24 cases. The tissues most frequently containing typical intranuclear inclusions or positive immunohistochemical staining were the spleen, liver, and kidney. Neither of the 2 nonpsittacine cases was positive immunohistochemically. This technique may be used wither as a rapid test on routinely processed diagnostic samples to confirm the presence of avian polyomavirus or for pathogenesis research studies.

Early and late pre-mRNA processing of budgerigar fledgling disease virus 1: identification of viral RNA 5' and 3' ends and internal splice junctions.

Budgerigar fledgling disease virus 1 (BFDV-1) is the first avian polyomavirus to be identified, and it possesses uncommon structural and biological properties. Here we present an analysis of the processed viral RNAs in infected chicken embryo fibroblast cells. Two early and 18 late BFDV-1 mRNAs were defined according to their 5' ends and internal splice patterns. In the early region of the genome an incomplete splice reaction covering 195 nt is responsible for creating two mRNAs that could encode small t and large T antigens, which would be initiated from a hypothetical early promoter, PE. The late mRNA 5' ends define two putative promoter regions (PL1 and PL2), 111 nt apart in the BFDV-1 genome non-coding region. The overall splicing pattern of the late mRNAs is further complicated by an alternative splice reaction of intron 2 (deletion of either 64 nt in intron 2a or of 256 nt in intron 2b) and a splice removing intron 3 (870 nt), resulting in deletion of most of the VP2-VP3 coding region. The positions of the late mRNA 5' ends and the splicing pattern indicate the existence of two open reading frames, putatively encoding two pairs of agnoproteins, in the 5' region of several late mRNAs. These mRNAs appear to be bicistronic and to encode one of the agnoproteins together with one of the viral coat proteins.

Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

Infection with the avian polyomavirus, BFDV, selectively affects myofibril structure in embryonic chick ventricle cardiomyocytes.

Embryonic cardiomyocytes can both beat and divide. They assemble cardiac muscle-specific proteins into sarcomeric myofibrils and contract. In addition, they periodically synthesize DNA, complete mitosis, disassemble sarcomeric myofibrils in the area of the mitotic spindle, assemble cytoplasmic isoform-specific proteins into a cleavage furrow contractile ring, undergo cytokinesis, and then reform sarcomeric myofibrils in daughter cells. Little is known about how embryonic cardiomyocytes disassemble their myofibrils as they traverse the cell cycle and divide. In the present study, beating embryonic avian ventricular cardiomyocytes in primary culture were stimulated to initiate DNA synthesis without subsequent mitosis or cytokinesis by infection with the lytic avian polyomavirus, Budgerigar Fledgling Disease Virus (BFDV). Within 48 hours, infected, adherent cardiomyocytes disassemble most of their sarcomeric myofibrils, retaining cardiac myosin only in thin myofibrils with disrupted sarcomeric periodicity and in amorphous nonfibrillar pools. By 72 hours, infected cardiomyocytes contain no myofibrils and no longer react with antibodies to cardiac myosin. In contrast, infected cardiomyocytes continue to display cytoplasmic myosin localized in stress-fiber-like-structures in adherent cells, or in disrupted fibers and dispersed pools in detaching cells. Infected cardiomyocytes also continue to display interphase-like arrays of polymerized microtubules, even when rounded-up just prior to lysis. These results suggest that polyomavirus infection may provide a useful model system for further study of the regulation of myofibrils disassembly in embryonic cardiomyocytes.

Molecular and biological characteristics of avian polyomaviruses: isolates from different species of birds indicate that avian polyomaviruses form a distinct subgenus within the polyomavirus genus

The isolation and characterization of two avian polyomaviruses, from chicken (BFDV-2) and a parrot (BFDV-3), is reported. Both isolates are closely related to the non-mammalian polyomavirus budgerigar fledgling disease virus (BFDV) isolated from budgerigars (now called BFDV-1), and all three viral genomes are shown to have the same basic size of 4981 bp. A 151 bp insertion was, however, observed in the non-coding region of BFDV-2 which represented an exact duplication of the left half of the non-coding region, including the putative early promoter and amino terminus of the large T antigen. With a further 15 base pairs exchanged elsewhere throughout the three genomes, these viruses have distinct degrees of tropism for various avian species. The production of antibodies directed against a beta-galactosidase-large T antigen fusion protein of BFDV-1 is described. These antibodies detected the large T antigen, with an M(r) of approximately 80K, and the small t antigen, with an M(r) of approximately 24K, in cells infected with BFDV isolates. Whereas these antibodies bind with low affinity to the large T antigen of simian virus 40 (SV40), SV40- or mouse polyomavirus-specific antibodies will not bind to the BFDV large T antigen. Antibodies directed against BFDV structural polypeptides exhibit broad, reciprocal cross-reactivities with all three structural proteins of mammalian polyomaviruses. The significance of polyomavirus infections in various avian species is discussed. Based on unique structural and biological properties we propose that these viruses should be placed in a distinct subgenus (Avipolyomavirus) within the polyomaviruses.

Production and Characterization of Monoclonal Antibodies to Budgerigar Fledgling Disease Virus Major Capsid Protein VP 1

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

Nuclear localization of budgerigar fledgling disease virus capsid protein VP2 is conferred by residues 308–317

The capsid protein VP2 of budgerigar fledgling disease virus (BFDV) contains two sequences (residues 309–315 and 334–340) which are homologous to the prototypic nuclear localization sequence (NLS) of the simian virus 40 T-antigen. Using recombinant potential NLS-β-galactosidase fusion proteins we identified amino acid residues 308–317 (VPKRKRKLPT) to be the NLS of BFDV capsid proteins VP2 and VP3. Microfluorometry studies show that the BFDV-VP2 signal is considerably more efficient in nuclear transport kinetics, than the NLS of SV40-VP2, corresponding to amino acid residues 317–326 (PNKKKRKLSR).

Polymerase chain reaction assay for avian polyomavirus.

A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

Polyomavirus Infection in Budgerigars (Melopsittacus undulatus): Clinical and Aetiological Studies

In order to get insight into the aetiology of French Moult (FM) and Budgerigar Fledgling Disease (BFD), and to determine relationships between the two diseases, 298 budgerigars from 49 different breeding flocks were examined. From all birds with BFD and from several birds with FM, viruses were isolated which produced characteristic cytopathic changes in chicken embryo fibroblasts. They were insensitive to chloroform treatment, and their replication was inhibited in the presence of 5-iododeoxyuridine. One of these isolates, from a bird exhibiting clinical signs of BFD, was determined by biochemical and biophysical methods to be a polyomavirus (BFDV). Nestling budgerigars 3 to 10 days of age, were inoculated with this BFDV isolate. They developed an acute systemic disease with high mortality rates, similar to naturally occurring infections. In this regard, BFDV differs markedly from all the other polyomaviruses known until now which only cause clinically asymptomatic, persistent infections in their natural hosts. Most of the birds examined with clinical signs of BFD or FM exhibited low titers of antibodies neutralizing the BFDV isolate, whereas in clinically healthy birds from flocks that never had any problems with FM or BFD, no antibodies against BFDV could be detected. On account of the results of our experiments described here we suspect that FM might be a milder, more protracted form of a BFDV infection.

Myristoylation of budgerigar fledgling disease virus capsid protein VP2

We present evidence that the structural protein VP2 of budgerigar fledgling disease virus, an avian polyomavirus, is specifically modified by covalent attachment of myristic acid. The fatty acid linkage is insensitive to hydroxylamine treatment and thus represents the amide type of fatty acylation of proteins.

Serological Evaluation of Some Psittaciformes for Budgerigar Fledgling Disease Virus

Breeding psittaciform birds (psittacines) from three geographically separated aviaries experiencing fledgling mortality were monitored during 1983 and 1984 for specific serum antibody to budgerigar fledgling disease virus (BFDV) using a fluorescent-antibody virus-neutralization test. Neither the time nor the extent of exposure to the virus was known. Serological titers were positive in 45% of birds sampled from Aviary 1, 25% from Aviary 2, and 11% from Aviary 3. Several species of psittacine birds within each aviary were serologically positive for BFDV. The results indicated that a papovavirus similar to BFDV appears to infect a wide range of captive adult psittacine birds. Macaws (Ara sp. and Anodorhynchus sp.) were evaluated for distribution of infection. Each species within these two genera showed positive serological titers to BFDV. Three groups of birds showed a decrease in serum antibody titer to BFDV at 1 and 2.5 months after the first sampling. Positive titers decreased from 66 to 20% for one group and from 60 to 50% for a second group in 1 month, and they decreased from 42 to 17% for a third group in 2.5 months.

Papovavirus‐like infection in budgerigars (Melopsittacus Undulatus)

Outbreaks of a disease in two budgerigar aviaries caused 20% and 53% mortality respectively in recently hatched chicks during the 1985 breeding season. Clinical signs were first observed at 5 to 7 days of age. In most birds darkening of the normal skin colour was followed by death in 2 to 10 days. In individual nests all or part of a hatch could be affected. Occasional survivors had abnormal plumage. Few gross lesions were found at post-mortem examination. Histologically, many large basophilic intranuclear inclusions were present in multifocal degenerative lesions throughout the epidermis, kidney, heart, brain and other tissues. These inclusions were shown by transmission electron microscopy to contain numerous virus particles which, on direct examination, had a .diameter of 46 to 56 nm and a papovavirus-like morphology. These outbreaks were considered to be cases of budgerigar fledgling disease, a syndrome that has been reported in other countries but not previously described in Great Britain.

Cloning and characterization of budgerigar fledgling disease virus, an avian polyomavirus

The DNA of a virus isolated from fledgling budgerigars, designated BFDV, was cloned and analyzed with regard to its relationship to the polyomavirus subgroup of the papovavirus family. Under relaxed conditions, the DNA of BFDV cross-hybridized with the DNAs of members of the polyomavirus subgroup, such as the mouse polyomavirus, the monkey viruses simian virus 40, stump-tailed macaque virus, and lymphotropic papovavirus, and the human viruses JCV and BKV. Under stringent conditions, however, no homologies could be detected. Furthermore, BFDV propagated in chicken embryo cells was antigenically related relthe capsid antigen(s) of the other polyomaviruses. The virus was able to transform hamster embryo cells in vitro which is a typical feature of polyomaviruses. These data clearly indicate that BFDV is a new distinct member of the polyomavirus genus representing the first nonmammalian polyomavirus.

A light and electron microscopic examination of budgerigar fledgling disease virus in tissue and in cell culture

Morphological changes induced by a newly described avian virus in budgerigar (Melopsittacus undulatus) tissues were examined with light and electron microscopes. Infected cells viewed with the light microscope were found to have enlarged nuclei containing marginated chromatin. Cytoplasmic contents were frequently clear in appearance. Tissues affected included skin, feather, follicle, kidney, uropygial gland, crop, lung, liver, heart, bone marrow, spleen and brain. Serum from infected birds contained viral particles. No tumours were found in affected birds. Electron microscopy demonstrated viral particles that were naked, predominantly icosehedral, and 42-49 nm in diameter. Occasional elongate forms of the virions were seen in kidney tissue. Small groups of virions were occasionally enclosed within nuclear and cytoplasmic membranes. Viral particles were observed in chicken embryo fibroblast cultures 18 hours post-inoculation. The particles first appeared in swollen nuclei and subsequently were found in the cytoplasm of more senescent cells. Cytoplasmic disruption and swollen rough endoplasmic reticulum were also observed in infected cells. Negatively stained preparations of the fluid from infected chicken embryo fibroblast cultures contained typical cubic viral particles as well as elongate forms similar to those seen in excised tissue preparations.