Identification of mixed infections with different genotypes of avian bornaviruses in psittacine birds with proventricular dilatation disease

Proventricular dilatation disease (PDD) is a fatal, progressive neurological disorder of psittacine birds, which is caused by a single-stranded RNA virus, the avian bornavirus (ABV). The disease pattern includes lymphoplasmacytic inflammation of the central, peripheral and autonomic nervous system. Seven avian bornavirus genotypes have been identified during the last years. So far only monoinfections with a single genotype of ABV have been attributed to PDD cases. However, after a recent survey discovered a case of a double infection with two different ABV genotypes, this seemed to indicate the need for a more systematic search for mixed infections. Brain specimens from 21 psittacine birds affected with PDD were examined. Aim of the investigation was to generate partial ABV sequences of a part of the matrix protein (M) gene and to evaluate whether sequences of more than one ABV genotype were present. RNA was extracted, and subjected to reverse transcriptase PCR with primer pairs generating a partial sequence of the matrix protein (M) gene, followed by a cloning procedure. Ten clones per case were sequenced in order to elucidate whether sequences characteristic for one or more than one genotype were present. In 19 of 21 cases clear M gene sequences could be generated; in two cases nucleic acid amplification failed. Seven birds were infected with ABV 2 and nine with ABV 4, representing the predominant genotypes in Europe. Two cases showed a mixed infection with ABV 2 and ABV 4, and one case a mixed infection with ABV 2 and ABV 6. These results suggest that the molecular cloning method is a useful tool for distinguishing between single and multiple infection events by different ABV genotypes.