So far, only a single heron hepatitis B virus genome (HHBV-4) has been cloned and sequenced. Therefore, neither the significance of its sequence divergence from other avian hepadnaviruses nor the sequence variability of HHBV genomes in general are known. Here we have analysed the sequence heterogeneity of HHBV genome populations in several sera from naturally infected herons. A highly sensitive PCR method for full-length HHBV genome amplification was established which allowed direct sequencing of entire HHBV populations without prior cloning. Sequences of HHBV genomes from four sera were thus obtained which differed from those of HHBV-4 by up to 7%. Some of the divergent nucleotides and the corresponding amino acids of the predicted viral proteins were conserved in all four new HHBV isolates and varied only in HHBV-4. This indicates that the HHBV-4 genome is not in all aspects representative of this class of viruses. Interestingly, a highly conserved ORF upstream of the C-gene present in a position analogous to that of the mammalian hepadnavirus X-gene became apparent in all HHBV genomes. In contrast to the duck hepadnaviruses, the small (sAg-S) instead of the largest (sAg-L) envelope protein of all HHBVs has a myristylation site. These data confirm the significant sequence divergence of HHBV from other avian hepadnaviruses. Moreover, they show that HHBV has low sequence variability and indicate two new and unique features not evident in other avihepadnaviruses: an additional, highly conserved gene and potential myristylation of the sAg-S instead of the sAg-L envelope protein.


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