| Endogenous viral elements are viral sequences of ancient extinct viruses (paleoviruses)that had been inserted in host genome. Paleovirology is the study of paleoviruses and the effects that these agents have had on the evolution of their hosts. Although there is no report about endogenous hepadnaviral elements in mammalian genomes, they have been discovered in zebra finch and related birds in Passeriformes. However, these endogenous viral elements are short and cannot be utilized to reconstruct any hepadnaviral genes or functional elements.In this study, in silico screening of potential endogenous hepadnaviral sequences in157publicly available metazoan genomes was performed using tBLASTn and polymerase protein sequences from extant orthohepadnaviruses and avihepadnavirusesas queries. Positive matches were found in budgerigar (Melopsittacus undulatus)of the order Psittaciformes. Since the extant parrot HBV (PHBV) polymerase generated the best matches in this initial screening, PHBV polymerase and PreC/C protein sequences were used as queries in a second tBLASTn search in the budgerigar genomic sequences. Atotal of61endogenous hepadnavirus sequences [collectively designated endogenous budgerigar hepatitis B viruses (eBHBV)] were identified in49contigs.Most eBHBV sequences constitute incomplete viral genome fragments. However, two eBHBV sequences containing near-or longer-than-full-length genome were identified:eBHBV1is～3.9kb in length and comprises about1.3×the genome with redundant polymerase and PreC/C sequences at the termini, whereas eBHBV2is-3.1kb in length and comprises almost exactly1.0×the genome with both termini in PreC/C. Using sequencing of cloned amplicons and southern blot, we confirm the presence of eBHBVs, including eBHBV1and eBHBV2, sequences in the budgerigar genome. According to the current model of hepadnaviral replication the terminal protein (TP) domain of polymerase, the ε packaging signal, and the direct repeat (DR) elements are all essential for viral replication. Interestingly, these features are preserved in both eBHBV1and eBHBV2.The phylogenetic trees place eBHBV1and eBHBV2in a separate branch, distinct from all the extant avihepadnaviruses, and between endogenous zebra finch HBV element J (eZHBVj) and all the extant avihepadnaviruses, suggesting the existence of a common ancestor shared by eBHBV and the common ancestor of extant avihepadnaviruses.The two copys of0.3×genome length of redundancy in eBHBV1exhibit2.01%divergence at the nucleotide level. It could be calculated that eBHBV1inserted into the budgerigar genome at least2.5to5.0million years ago. This is much longer than the age, less than6,000years, of most recent common ancestor of extant avihepadnavirus calculated by others. No eBHBVorthologous sequences were found in the genome of Black-capped Lory (Lorius lory), the most closed species to budgerigar, using sequencing of cloned amplicons and Southern blot.Therefore, the age of eBHBVs is less than14million years, which is the age of the last common ancestor of budgerigar and Black-capped Lory.The presence of near-or longer-than-full-length genome also implicates promoter activity preserved in eBHBV1and eBHBV2. Transcription activity was detected in vitrofor eBHBV1and eBHBV2, and in budgerigar primaryhepatocytes. The core gene (BHBc) is intact in eBHBV1. The Core protein was expressed as a GST-fusion protein and could be targeted by an anti-BHBc antibody raised using a BHBc peptide with high activity and specificity, which will enable the detection of endogenous expression of the Core protein.In summary, the discovery of eBHBVs, particularly the eBHBVs containing full-length genome, will improve our knowledge on the origin and evolution of hepadnaviruses, and also on the interaction between host and virus. |
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