R. Raue

Assessment of genetic, antigenic and pathotypic criteria for the characterization of IBDV strains

The aim of this work was the selection and comparison of representative infectious bursal disease virus (IBDV) strains. Nine strains of IBDV, isolated at different times and from different geographic regions of Europe and China, were characterized. Batches of all strains were prepared following standardized protocols and checked for the absence of contaminating viruses. Criteria used for their characterization were: (i) the nucleotide sequence of the VP2 variable region, (ii) binding to a panel of neutralizing monoclonal antibodies in antigen capture enzyme-linked immunosorbent assays, and (iii) virulence in specific pathogen free chickens after infection with a standardized number of median embryo infective doses. Based on the first two criteria, two of nine strains were classified as classical virulent (cv) IBDV (F52/70, Cu-1wt), and five as very virulent (vv) IBDV (849VB, 96108, HK46, GX, Harbin). Remarkably, although a clear-cut difference was demonstrable between European cvIBDV (F52/70 and Cu-1wt) and vvIBDV (849VB and 96108) strains, there was a continuum in the pathogenicity of Chinese vvIBDVs. Our results indicate the probable existence of differences in virulence within IBDV lineages determined on the basis of antigenic typing using monoclonal antibodies and the alignment of the VP2 sequences. This indicates limitations in the analysis of IBDV pathotypes based on the VP2 variable region and emphasizes that these criteria may not be sufficient for the classification of IBDV strains.

Rapid identification of "very virulent" strains of infectious bursal disease virus by reverse transcription-polymerase chain reaction combined with restriction enzyme analysis

A reverse transcription-polymerase chain reaction (RT-PCR) combined with restriction enzyme analysis (REA) was developed for differentiation of classical virulent (cv) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The VP2 gene was used for primer annealing to amplify the hypervariable region. An amplification product was obtained with serotype 1 and serotype 2 strains of IBDV.The restriction enzymes SacI and BspMI were used to identify and to differentiate the serotype 1 strains: SacI only cleaved cvIBDV RT-PCR products, whereas products obtained with vvIBDV strains were only cleaved with BspMI; serotype 2 strain products were not cleaved by either of these restriction enzymes. RT-PCR combined with REA as described in this study is thus suitable to detect IBDV of both serotypes and to distinguish rapidly between cvIBDV,vvIBDV and serotype 2 IBDV.Our investigation of a total of 11 different IBDV isolates and available nucleotide sequence data of other isolates indicate that the application of this protocol might be a useful tool for the rapid identification of vvIBDV isolates, a prerequisite for the effective control of this economically important virus infection of commercial poultry.

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.

Phylogenetic analysis of the hexon loop 1 region of an adenovirus from psittacine birds supports the existence of a new psittacine adenovirus (PsAdV).

Summary.Adenovirus infections in psittacine birds have been well known. Most of these infections were caused by fowl adenoviruses (FAdV). In this study, liver samples showing typical histological signs of an adenovirus infection were collected from Poicephalus spp. with acute disease. A PCR amplifying the variable loop 1 region of the hexon gene was developed using primers located in two conserved pedestal regions. A PCR product of approximately 590 bp in size was amplified and sequenced. The sequence obtained grouped outside of the FAdV reference strains of the 12 serotypes as well as egg drop syndrome virus and turkey adenovirus 3 indicating that a new avian adenovirus was detected. In comparison to the FAdV reference strains, the percentage of identical nucleotides ranged between 60.3 and 67.0 and that of identical amino acids (aa) between 51.3 and 61.0. Furthermore, 37 unique aa exchanges were observed; out of these, 27 are located in the 4 hypervariable regions of loop 1, which encode the serotype-specific epitops. The g/c content, the isoelectric point and the charge of the amplified fragment, however, are in the range as those of group I avian adenoviruses. It was proposed, therefore, to designate this new adenovirus as psittacine adenovirus (PsAdV).

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. C139–244-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 C139–244-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 C139–244-His has value as a recombinant antigen for BFDV-specific serological tests.

Different Domains of the RNA Polymerase of Infectious Bursal Disease Virus Contribute to Virulence

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.

Investigations on the aetiology of pinching off syndrome in four white-tailed sea eagles (Haliaeetus albicilla) from Germany

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.

Molecular Cloning of a Bangladeshi Strain of very Virulent Infectious Bursal Disease Virus of Chickens and its Adaptation in Tissue Culture by Site-Directed Mutagenesis

Full-length cDNA of both genome segments of a Bangladeshi strain of very virulent infectious bursal disease virus (BD 3/99) were cloned in plasmid vectors along with the T7 promoter tagged to the 5′-ends. Mutations were introduced in the cloned cDNA to bring about two amino acid exchanges (Q253H and A284T) in the capsid protein VP2. Transfection of primary chicken embryo fibroblast cells with RNA transcribed in vitro from the full-length cDNA resulted in the formation of mutant infectious virus particles that grow in tissue culture. The pathogenicity of this molecularly-cloned, tissue-culture-adapted virus (BD-3tc) was tested in commercial chickens. The parental wild-type strain, BD 3/99, was included for comparison. The subclinical course of the disease and delayed bursal atrophy in BD-3tc-inoculated birds suggested that these amino acid substitutions made BD-3tc partially attenuated.

Circovirus-Infektionen bei Ziervögeln und Tauben: eine Übersicht

Bei Vogeln sind Circovirus-Infektionen seit langem bekannt. Neben dem Virus der infektiosen Anamie der Kuken (chicken anaemia virus, CAV) ist hier vor allem das Virus der Schnabel- und Federkrankheit der Papageien (psitta-cine beak and feather disease virus, PBFDV) zu nennen. Die meisten Spezies der Ordnung Psittaciformes sowie einige andere Arten sind fur das PBFDV empfanglich. Die Erkrankung manifestiert sich nicht nur bei den Nestlingen der Papageien, sondern auch bei adulten Vogeln. Bei diesen kommt es zu irreversiblen Schaden im Federkleid, bis hin zur vollstandigen Federlosigkeit. Zudem erfahren infizierte Tiere eine starke Immunsuppression, die sie sehr anfallig fur Sekundarinfektionen macht. Neben dem PBFDV wurden kurzlich weitere Circoviren beschrieben, die bei Tauben oder Kanarienvogeln vorkommen. In der vorliegen-den Arbeit soll eine Ubersicht uber die Circovirus-Infektio-nen bei Ziervogeln und Tauben gegeben werden. Neben der klinischen Symptomatik sind die zur Zeit gebrauchlichen diagnostischen Nachweisverfahren und die Moglichkeiten zur Prophylaxe weitere Schwerpunkte.