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ABSTRACT: Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent (vv) IBDV strains have emerged and induce up to 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood and the relative contribution of the two genome segments A and B in this phenomenon is not known. Isolate 94432 was previously shown as being genetically related to vvIBDVs but exhibits an atypical antigenicity and does not cause mortality. Here, the full-length genome of 94432 was determined and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as 94432 isolate. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced and their pathogenicity assessed in specific pathogen free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in an hydrophobic groove on VP1 surface, which suggests possible interactions between VP1 and another, as yet unidentified, molecule at that this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.
Journal of Virology 12/2012; · 5.40 Impact Factor
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ABSTRACT: Polyomaviruses are aetiological agents of fatal acute diseases in various bird species. Genomic analysis revealed that avian polyomavirus (APyV), crow polyomavirus (CPyV), finch polyomavirus (FPyV) and goose hemorrhagic polyomavirus (GHPyV) are closely related to each other, but nevertheless form separate viral species; however, their serological relationship was unknown so far. As only APyV can be efficiently grown in tissue culture, virus-like particles (VLPs) were generated by expression of the genomic regions encoding the major structural protein VP1 of these viruses in yeast; these were used to elicit type-specific antibodies in rabbits and as antigens in serological reactions. For increased VLP assembly, a nuclear localisation signal was introduced into APyV-VP1. VLPs derived from monkey polyomavirus SV40-VP1 served as control. APyV-, GHPyV- and CPyV-VLPs showed hemagglutinating activity with chicken and human erythrocytes. CPyV- and GHPyV-specific sera showed slight cross-reactions in immunoblotting, hemagglutination inhibition assay and indirect ELISA. The FPyV-specific serum slightly inhibited the hemagglutination activity of APyV-VLPs and showed a weak cross-neutralising activity against APyV in cell culture tests. Generally, these data indicate that the four polyomaviruses of birds are serologically distinct. However, in accordance with genetic data, a relationship between CPyV and GHPyV as well as between APyV and FPyV is evident, and grouping into two different serogroups may be suggested. The hemagglutinating activity of APyV, CPyV and GHPyV may indicate similar receptor-binding mechanisms for these viruses. Our data could be useful for the development of vaccines against the polyomavirus-induced diseases in birds and for interpretation of diagnostic test results.
Journal of General Virology 08/2012; · 3.36 Impact Factor
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ABSTRACT: Infectious bursal disease virus (IBDV) is the aetiological agent of the acute and highly contagious infectious bursal disease (IBD) or "Gumboro disease". IBD is one of the economically most important diseases that affects commercially produced chickens worldwide. Along with strict hygiene management of poultry farms, vaccination programmes with inactivated and live attenuated viruses have been used to prevent IBD. Live vaccines show a different degree of attenuation; many of them may cause bursal atrophy and thus immunosuppression with poor immune response to vaccination against other pathogens and an increase in vulnerability to various types of infections as possible consequences. Depending on their intrinsic characteristics or on the vaccination procedures, some of the vaccines may not induce full protection against the very virulent IBDV strains and antigenic variants observed in the last three decades. As chickens are most susceptible to IBDV in their first weeks of life, active immunity to the virus has to be induced early after hatching. However, maternally derived IBDV-specific antibodies may interfere with early vaccination with live vaccines. Thus new technologies and second-generation vaccines including rationally designed and subunit vaccines have been developed. Recently, live viral vector vaccines have been licensed in several countries and are reaching the market. Here, the current status of IBD vaccines is discussed.
Avian Pathology 04/2012; 41(2):133-9. · 1.71 Impact Factor
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ABSTRACT: BACKGROUND: Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. METHODOLOGY, PRINCIPAL FINDINGS: Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180. CONCLUSION, SIGNIFICANCE: The present results show that different domains of the RdRp, are essential for the in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses.
PLoS ONE 01/2012; 7(1):e28064. · 4.09 Impact Factor
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Ursula Heffels-Redmann,
Dirk Enderlein,
Sibylle Herzog,
Christiane Herden,
Anne Piepenbring,
Daniel Neumann, Hermann Müller,
Sara Capelli,
Heiner Müller,
Kirstin Oberhäuser,
Helga Gerlach,
Erhard F Kaleta,
Michael Lierz
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ABSTRACT: A total of 1442 live birds and 73 dead birds out of 215 bird collections in Spain, Germany, Italy, the UK and Denmark were tested for avian bornavirus (ABV) infection by four different methods. The majority of the birds were psittacines belonging to 54 different genera of the order Psittaciformes. In total, 22.8% of the birds reacted positive for ABV in at least one of the tests. Combined testing of swabs from the crop and cloaca, and serum for the diagnosis of ABV infection in live birds revealed that virus shedding and antibody production coincided in only one-fifth of the positive birds so that the examination of these three samples is recommended for reliable ABV diagnosis. By statistical analysis of this large number of samples, the ABV infection proved to be highly significant (P <0.001) associated with histopathologically confirmed proventricular dilatation disease (PDD) in dead birds as well as with clinically assumed PDD in live birds. However, ABV infection was also detected in psittacines without pathological lesions or clinical signs of PDD. Twelve non-psittacine birds belonging to the genera Aburria, Ciconia, Geopelia, Leucopsar and Pavo were tested negative for ABV infection. Within the order of Psittaciformes, birds belonging to 33 different genera reacted positive for ABV. In 16 of these psittacine genera, the ABV infection was demonstrated for the first time. The present study emphasizes the widespread occurrence of clinically variable ABV infections in Europe by analysing a large number of specimens from a broad range of bird species in several assays.
Avian Pathology 08/2011; 40(4):419-26. · 1.71 Impact Factor
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ABSTRACT: Chimpanzee polyomavirus (ChPyV) was originally detected in the faeces of a captive chimpanzee by random screening using broad-spectrum PCR. Its pathogenicity and the distribution among chimpanzees are unknown so far. Here, the major capsid protein VP1 of ChPyV was expressed in yeast cells. Virus-like particles (VLPs) with a diameter of approximately 45nm were demonstrated although the efficiency of VLP formation was low as compared to monkey polyomavirus SV40-VLPs. The ChPyV-VLP preparation did not agglutinate human erythrocytes. Low cross-reactions between ChPyV- and SV40-VLP-specific sera were detected by immunoblotting, but not by ELISA. Testing of 163 sera derived from captive and wild-caught healthy chimpanzees using an ELISA based on the ChPyV-VLPs resulted in 11.7% positive results, ranging from 0% to 56% in different groups. The VLPs may be used in future to assess the distribution of ChPyV infections among other animal species and humans.
Virus Research 02/2011; 155(2):514-9. · 2.94 Impact Factor
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ABSTRACT: Polyomaviruses of birds are aetiological agents of acute inflammatory diseases in non-immunocompromised hosts, which is in contrast to mammalian polyomaviruses. VP4, an additional structural protein encoded by the viral genomes of the known avian polyomaviruses, has been suggested to contribute to pathogenicity through loss of cells following induction of apoptosis. Four distinct bird polyomaviruses have been identified so far, which infect crows, finches, geese and parrots. Using broad-spectrum PCR, a novel polyomavirus, tentatively designated canary polyomavirus (CaPyV), was detected in diseased canary birds (Serinus canaria) that died at an age of about 40 days. Intranuclear inclusion bodies were found in the liver, spleen and kidneys. The entire viral genome was amplified from a tissue sample using rolling-circle amplification. Phylogenetic analysis of the genome sequence indicated a close relationship between CaPyV and other avian polyomaviruses. Remarkably, an ORF encoding VP4 could not be identified in the CaPyV genome. Therefore, the mechanism of pathogenicity of CaPyV may be different from that of the other avian polyomaviruses.
Journal of General Virology 12/2010; 91(Pt 12):3016-22. · 3.36 Impact Factor
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ABSTRACT: Avian polyomavirus (APV) causes a range of disease syndromes in psittacine birds, from acute fatal disease to subclinical infections, depending on age, species, and other unidentified risk factors. To determine the prevalence of APV-specific antibodies in a captive population of Spix's macaws (Cyanopsitta spixii) in Quatar, 54 birds were tested by blocking enzyme-linked immunosorbent assay. A prevalence of 48.1% for APV antibodies, which indicates viral exposure, was found. Of 36 Spix's macaws that were serially tested over a period of 4 years, 50.0% were consistently positive, 36.1% were consistently negative, 5.5% had permanently declining antibody levels, and 2.8% showed variable results. By using polymerase chain reaction testing on whole blood samples, an apparent viremia was detected in 1 of 44 birds (2.3%), although contamination provides a likely explanation for this isolated positive result in a hand-reared chick. The white blood cell count was significantly higher in antibody-positive birds compared with antibody-negative birds (P < .05). Because antibody-positive and antibody-negative birds were housed together without a change in their respective antibody status, transmission of APV within the adult breeding population appeared to be a rare event.
Journal of Avian Medicine and Surgery 09/2010; 24(3):192-8. · 0.63 Impact Factor
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ABSTRACT: Different avian bornavirus (ABV) genotypes have recently been detected in psittacine birds with proventricular dilatation disease (PDD), an inflammatory fatal central and peripheral nervous system disorder. An indirect immunofluorescence assay (IIFA) for intra vitam demonstration of ABV-specific serum antibodies was established since reverse transcription-PCR (RT-PCR) assays may not detect all ABV variants.
Journal of clinical microbiology 06/2010; 48(6):2282-4. · 4.16 Impact Factor
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ABSTRACT: Techniques for the single-step amplification of whole genomes have been developed into powerful tools for phylogenetic analyses, epidemiological studies and studies on genome organization. Recently, the bacteriophage phi29 DNA polymerase has been used for the efficient amplification of circular DNA viral genomes without the need of specific primers by a rolling-circle amplification (RCA) mechanism. Various protocols have been applied for detection of novel viruses, for differentiation between circular and linear forms of viral genomes and for generation of infectious genomic clones directly from specimens. Here, we summarize the broad application of the RCA technique to DNA viruses infecting humans, animals and plants.
Trends in Microbiology 05/2009; 17(5):205-11. · 7.91 Impact Factor
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ABSTRACT: Pigeon circovirus (PiCV) infection and young pigeon disease syndrome (YPDS), associated with high morbidity and mortality, have been recognized in young racing pigeons from large portions of Central Europe. There exist a number of data indicating that YPDS is a consequence of PiCV infection and subsequent immunosuppression. In order to prove PiCV to be one of the crucial factors of YPDS, an experimental infection with PiCV was performed under controlled conditions. Twenty-four domestic pigeons (Columba livia forma domestica) were divided into two groups with 12 pigeons each; an infection group and a control group. All birds were between their fourth to eighth week of life. Pigeons in the infection group were infected both intramuscularly and orally with PiCV purified from naturally infected birds, while pigeons in the control group received a placebo. To test a possible influence of the PiCV infection on the immune system, the animals in both groups were vaccinated simultaneously, on the same day, against PMV-1 (Lasovac plus, IDT, Dessau-Tornau, Germany). Weekly virologic testing showed a viraemic period, and excretion of the infection virus, in pigeons in the infection group. Replication of PiCV could be proved on the basis of histologic findings of multiglobular inclusion bodies, mainly observed in macrophages of the bursa of Fabricius. A PiCV, genetically distinct from the experimental virus, was detected in the control group by polymerase chain reaction (PCR) testing, but any histologic findings comparable to the infection group were absent. None of the pigeons revealed clinical signs of illness, or hints that immunosuppression had occurred, regardless of their group. The absence of stressful conditions, considered as a trigger for the development of YPDS, may be responsible for the failure of disease reproduction in our infection model.
Avian Diseases 10/2008; 52(3):380-6. · 1.46 Impact Factor
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ABSTRACT: Infections of young racing pigeons with pigeon herpesvirus (PiHV), fowl adenovirus (FAdV) and pigeon circovirus (PiCV) are reported frequently. The role of these viruses in the pathogenesis of a disease complex called young pigeon disease syndrome (YPDS) is generally accepted. All of these viruses cause inclusion bodies in the liver so liver samples are particularly useful for the detection of infection. Consequently a multiplex polymerase chain reaction (PCR) was developed for the detection of PiHV, FAdV and PiCV in liver samples from racing pigeons. The detection limits were 10(1) genome equivalents for the detection of PiHV and PiCV and 10(3) genome equivalents for FAdV. The absence of PCR inhibitors was shown by the detection of cytochrome B gene as an internal control. No PCR products were amplified from related herpes and circoviruses or negative controls, demonstrating the specificity of the multiplex PCR. The addition of cellular DNA from liver samples or Q-solution to the reaction mix had no influence on its sensitivity. The usefulness of the multiplex PCR was demonstrated by re-investigation of liver samples from young racing pigeons previously tested positive by uniplex PCRs.
Journal of Virological Methods 04/2008; 148(1-2):226-31. · 2.01 Impact Factor
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ABSTRACT: Borna disease virus (BDV) is a neurotropic agent infecting distinct neuronal subpopulations in the central nervous system of various mammalian species possibly including humans. Horses, a major natural host for BDV, show gastrointestinal dysfunctions besides characteristic neurological symptoms. Therefore, we hypothesized that enteric neurons may be targets of BDV replication. The presence of BDV-specific antigen in subpopulations of the ENS was investigated. Four-week-old Lewis rats were infected intracerebrally and sacrificed 4-14 weeks post infection (p.i.). BDV-immunoreactive neurons were found in submucous and myenteric neurons of the proximal colon. Fourteen weeks p.i., the proportion of BDV-positive neurons was 44+/-17 and 24+/-7% in the submucous and myenteric plexus, respectively. The majority of BDV-positive myenteric neurons showed immunoreactivity for choline acetyltransferase. Expression of Calbindin D-28k (CALB) was found in 96% of submucous and 67% of myenteric BDV-immunoreactive neurons. Additionally, the number of CALB-immunoreactive neurons was significantly higher in the myenteric plexus of infected rats compared to controls. These data indicate that BDV infects specific subpopulations of enteric neurons. Therefore, the ENS might serve as a site for BDV replication and as an immunoprivileged reservoir for BDV. In addition, upregulation of CALB in neurons of the myenteric plexus is probably induced during BDV-infection.
Veterinary Microbiology 04/2008; 127(3-4):275-85. · 3.33 Impact Factor
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Journal of Virology 12/2007; 81(21):11554-9. · 5.40 Impact Factor
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Kerstin Müller,
Elvira Schettler,
Helga Gerlach,
Leo Brunnberg,
Hafez Mohamed Hafez,
Kim Hattermann,
Reimar Johne,
Rainer Kollmann,
Oliver Krone,
Michael Lierz,
Sonja Linke,
Dörte Lueschow,
Annette Mankertz, Hermann Müller,
Christina Prusas,
Rüdiger Raue,
Dirk Soike,
Stephanie Speck,
Petra Wolf,
Kai Frölich
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ABSTRACT: The purpose of this study was to investigate the aetiology of the pinching off syndrome (POS), a generalized feather abnormality affecting free-living nestling of the white-tailed sea eagle (Haliaeetus albicilla) in Europe. For the first time, extensive clinical, haematological, biochemical, virological, bacteriological, nutritional, histopathological, parasitological and electron microscopical examinations were performed on three females and one male suffering from POS. Early and increased cytokeratin formation at the base of regenerating feathers and their follicle was observed in affected birds. Ultrathin sections of the feather papillae revealed an extended stratum transitivum and a compact, thickened keratinized stratum corneum. The transitional cells in POS feathers contained vacuoles often associated with the nucleus. Lipofuscin accumulations in neurons, glial cells and islet cells of the pancreas were found in all examined birds. It was not clear whether there is an association between the occurrence of lipofuscin and POS. No evidence was found to suggest that infectious agents (parasites, bacteria, fungi or viruses), malnutrition or hormonal imbalances are involved in the aetiology of POS in white-tailed sea eagles. It remains unclear whether there is a genetic background of POS.
Avian Pathology 07/2007; 36(3):235-43. · 1.71 Impact Factor
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ABSTRACT: Avian polyomavirus (APV) is the causative agent of an acute fatal disease in psittacine and some non-psittacine birds. In contrast to mammalian polyomaviruses, the APV genome encodes the additional capsid protein VP4 and its variant VP4Delta, truncated by an internal deletion. Both proteins induce apoptosis. Mutation of their common initiation codon prevents virus replication. Here, the generation of replication competent deletion mutants expressing either VP4 or VP4Delta is reported. In contrast to infection with wild-type virus, chicken embryo cells showed no cytopathic changes after infection with the mutants, and induction of apoptosis as well as virus release from the infected cells were delayed. Electron microscopy revealed the presence of a high proportion of small particles and tubules in preparations of the VP4 deletion mutant, indicating a scaffolding function for VP4. Wild-type and mutant viruses elicited neutralizing antibodies against APV after intramuscular and intraperitoneal infection of chicken; however, VP4-specific antibodies were only detected after infection with wild-type virus. Using the oculonasal route of infection, seroconversion was only observed in chickens infected with the wild-type virus, indicating a strongly reduced infectivity of the mutants. Based on the biological properties of the deletion mutants, they could be considered as candidates for APV marker vaccines.
Journal of General Virology 04/2007; 88(Pt 3):823-30. · 3.36 Impact Factor
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ABSTRACT: Goose hemorrhagic polyomavirus (GHPV) is the causative agent of hemorrhagic nephritis and enteritis of geese (HNEG), a fatal disease of young geese with high mortality rates. GHPV cannot be efficiently propagated in tissue culture. To provide antigens for diagnostic tests and vaccines, its major structural protein VP1 was recombinantly expressed in Sf9 insect cells and in the yeast Saccharomyces cerevisiae. As demonstrated by density gradient centrifugation and electron microscopy, GHPV-VP1 expressed in insect cells formed virus-like particles (VLPs) with a diameter of 45 nm indistinguishable from infectious polyomavirus particles. However, efficiency of VLP formation was low as compared to the monkey polyomavirus SV-40-VP1. In yeast cells, GHPV-VP1 alone formed smaller VLPs, 20 nm in diameter. Remarkably, co-expression of GHPV-VP2 resulted in VLPs with a diameter of 45 nm. All three types of GHPV-VLPs were shown to hemagglutinate chicken erythrocytes. ELISA and hemagglutination inhibition tests using the VLPs as antigen detected GHPV-specific antibodies in up to 85.7% of sera derived from flocks with HNEG but in none of the sera of a clinically healthy flock. However, GHPV-specific antibodies were also detected in sera from two other flocks without HNEG indicating a broad distribution of GHPV due to subclinical or unrecognised infections.
Virus Research 10/2006; 120(1-2):128-37. · 2.94 Impact Factor
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ABSTRACT: The genus Circovirus comprises small non-enveloped viruses with a circular single-stranded DNA genome. By using PCR with degenerate primers, a novel circovirus (starling circovirus, StCV) was detected in spleen samples of wild starlings (Sturnus vulgaris and Sturnus unicolor) found dead during an epidemic outbreak of septicaemic salmonellosis in northeastern Spain. Using a specific PCR, StCV was also detected in apparently healthy birds from the same population. The genome was amplified using multiply primed rolling-circle amplification and cloned. Open reading frames (ORFs) with similarities to the replication-associated protein and the capsid protein of circoviruses as well as an additional ORF encoding a protein of 106 aa were evident from the sequence. Phylogenetic analysis of circovirus genomes revealed the highest degree of similarity (67.1 %) between StCV and canary circovirus. A similar analysis of the evolutionarily conserved cytochrome b gene of the circovirus host species revealed a strict co-evolution of circoviruses with their hosts; however, the circoviruses showed about a threefold higher genetic divergence than their hosts.
Journal of General Virology 06/2006; 87(Pt 5):1189-95. · 3.36 Impact Factor
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ABSTRACT: Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds--avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)--are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5' region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses.
Journal of Virology 05/2006; 80(7):3523-31. · 5.40 Impact Factor
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ABSTRACT: In order to collect more convincing data on the aetiological agent of young pigeon disease syndrome (YPDS), a comprehensive study was performed on pigeons in German lofts with or without outbreaks of YPDS. The investigations included examination of histories, clinical signs and pathology, as well as parasitological and microbiological analysis. Pigeons in their 4th to 12th week of life exhibited clinical signs at higher frequency and with greater severity than pigeons of other ages. Greenish liquid in the crop, proventriculus and ventriculus, and yellow fluid in the small intestine were seen more often in YPDS-affected pigeons. Escherichia coli and Klebsiella pneumoniae were isolated more frequently from these birds. Depletion of splenic and bursal lymphocytes was only seen in pigeons with YPDS. Inclusion bodies were present in various organs, especially the bursa of Fabricius. The genome of pigeon circovirus was detected in lymphoid tissues from all pigeons with YPDS. The results of this study indicate that YPDS is a multifactorial disease in which pigeon circovirus might be a crucial factor, possibly by inducing immunosuppression in infected birds.
Avian Pathology 11/2005; 34(5):418-25. · 1.71 Impact Factor
