Localization of avian bornavirus RNA by in situ hybridization in tissues of psittacine birds with proventricular dilatation disease

Proventricular dilatation disease (PDD) of psittacine birds is caused by a number of different genotypes of a novel viral species, avian bornavirus (ABV). Here we present an in situ hybridization (ISH) procedure using digoxigenin-labeled RNA probes for localizing viral genomic and mRNA of ABV-2 and ABV-4 in tissues of affected birds. Out of eleven immunohistochemically positive birds ISH signals were only found in seven. Partial sequencing of the viral genome had shown that four of them were infected with ABV-2, two with ABV-4 and one had a mixed infection with ABV-2 and ABV-4. ISH signals were present in the brain, in the vegetative nerve system, glandular epithelia and smooth muscle cells of the intestinal tract and in cardiomyocytes. Hybridization signals for viral genome were more abundant than signals for mRNA. As the probes were not strictly genotype-specific, four of the birds had hybridization signals with both, the ABV-2 and ABV-4 probes. The signals achieved with the homologous probes were more intense and more abundant than those resulting from heterologous probes. Taken together, the results of this study show that ISH can be used as a tool for localizing ABV sequences in tissues of birds with PDD and confirm the causative role of ABVs by showing viral replication in affected tissues.