Molecular evolution of psittacine beak and feather disease virus (PBFDV) isolated from african grey parrots (Psittacus erithacus) reared in Italy.

The purpose of this study was to molecularly characterize BFDV strains isolated in Italy from African grey parrots affected by peracute or chronic form of BFDV disease. To our knowledge this is the first survey of BFDV infection in Italy in which, together with the classical sequence comparison analyses, we attempted to associate genetic features of the viruses with clinical forms of the illness. Mixed organs from 8 animals dead displaying peracute form of BFDV disease and blood from 2 animals harbouring chronic signs of this illness were collected. These animals were reared in 8 different breeding facilities located in North and Central Italy. The full‐length genomes of BFDV strains were analyzed using 4 primer sets that amplify overlapping DNA stretches. These fragments were sequenced and aligned with all known BFDV sequences available on GenBank using the program ClustalW (1). Bayesian methods implemented in the computer program MrBayes ver. 3.1.1 (2), were used to draw phylogenetic trees and assess statistical support for clades. All genomes contained the characteristic nonanucleotide circovirus origin of replication sequence located within a stem loop structure and the conserved motifs located within the 2 major open reading frames (ORFs). A peculiar feature of the BFDV viruses we analyzed was the presence of 5 to 6 ORFs, in contrast with published data that ascribed to BFDV genome the presence up to 7 ORFs. ORF6 was always absent. The whole nucleotide sequence identity among our isolates varied from 93.6% to 99.9% and genome sizes extended from 1,997 to 2,001 nucleotides. Full genome analysis showed that the DNA isolated from 8 animals fall into 2 subtypes of the BFDV‐J strain, namely J1 and J2, which share a 98 to 100% intra‐subtype identity, while the sequences of BFDV viruses obtained from other 2 animals, based on the classification system proposed by Varsani et al. (2011), seem to cluster into 2 new subtypes, which we defined as J4 and J5. Finally, by phylogenetic analysis, we observed a correlation among viral strains derived from the same breeding, whereas no association between phylogenetic distribution and clinical symptoms or geographical location of the breeding was noticed. Our study shows that the complete genomes of BFDV strains isolates in Italy grouped into BFDV‐J strain, according to the fact that this is the main strain infecting African grey parrots in Europe. Moreover, even if we were not able to discern an association between genetic feature of the virus and form of the illness, we identify two new circovirus subtypes, namely J4 and J5 characteristic of Italian BFDV viruses