SEQUENCE HETEROGENEITY OF HERON HEPATITIS-B VIRUS GENOMES DETERMINED BY FULL-LENGTH DNA AMPLIFICATION AND DIRECT SEQUENCING REVEALS NOVEL AND UNIQUE FEATURES

So far, only a single heron hepatitis B virus genome (HHBV-4) has been cloned and sequenced. Therefore, neither the significance of its sequ ence divergence from other avian hepadnaviruses nor the sequence varia bility of HHBV genomes in general are known. Here we have analysed the sequence heterogeneity of HHBV genome populations in several sera fro m naturally infected herons. A highly sensitive PCR method for full-le ngth HHBV genome amplification was established which allowed direct se quencing of entire HHBV populations without prior cloning. Sequences o f HHBV genomes from four sera were thus obtained which differed from t hose of HHBV-4 by upto 7%. Some of the divergent nucleotides and the c orresponding amino acids of the predicted viral proteins were conserve d in all four new HHBV isolates and varied only in HHBV-4. This indica tes that the HHBV-4 genome is not in all aspects representative of thi s class of viruses. Interestingly, a highly conserved ORF upstream of the C-gene present in a position analogous to that of the mammalian he padnavirus 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 variabi lity and indicate two new and unique features not evident in other avi hepadnaviruses: an additional, highly conserved gene and potential myr istylation of the sAg-S instead of the sAg-L envelope protein.