Avihepadnavirus diversity in parrots is comparable to that found amongst all other avian species.

Avihepadnaviruses have previously been isolated from various species of duck, goose, stork, heron and crane. Recently the first parrot avihepadnavirus was isolated from a Ring-necked Parakeet in Poland. In this study, 41 psittacine liver samples archived in Poland over the last nine years were tested for presence of Parrot hepatitis B virus (PHBV). We cloned and sequenced PHBV isolates from 18 birds including a Crimson Rosella, an African grey parrot and sixteen Ring-necked Parakeets. PHBV isolates display a degree of diversity (>78% genome wide pairwise identity) that is comparable to that found amongst all other avihepadnaviruses (>79% genome wide pairwise identity). The PHBV viruses can be subdivided into seven genetically distinct groups (tentatively named A-G) of which the two isolated of PHBV-G are the most divergent sharing ∼79% genome wide pairwise identity with all their PHBVs. All PHBV isolates display classical avihepadnavirus genome architecture.

Molecular characterisation of an avihepadnavirus isolated from Psittacula krameri (ring-necked parrot).

Avihepadnaviruses have been documented previously in ducks, herons, geese, storks and cranes. Here, we describe the full genome of a new avihepadnavirus isolated from Psittacula krameri (ring-necked parrot) in Poland. The parrot hepatitis B virus (PHBV) genome (3042 bp) shares <76% sequence identity with other avihepadnavirus isolates and is phylogenetically most closely related to heron and stork hepatitis B viruses isolates. PHBV has a genome organization similar to that of other hepadnaviruses and contains ORFs for a preC/C, preS/S and polyprotein. Additionally, we identified an X-like ORF in the genome of PHBV. The full-genome data will be useful in developing screening tools for avihepadnaviruses in parrots.

Heterologous replacement of the supposed host determining region of avihepadnaviruses: high in vivo infectivity despite low infectivity for hepatocytes.

Hepadnaviruses, including hepatitis B virus (HBV), a highly relevant human pathogen, are small enveloped DNA viruses that replicate via reverse transcription. All hepadnaviruses display a narrow tissue and host tropism. For HBV, this restricts efficient experimental in vivo infection to chimpanzees. While the cellular factors mediating infection are largely unknown, the large viral envelope protein (L) plays a pivotal role for infectivity. Furthermore, certain segments of the PreS domain of L from duck HBV (DHBV) enhanced infectivity for cultured duck hepatocytes of pseudotyped heron HBV (HHBV), a virus unable to infect ducks in vivo. This implied a crucial role for the PreS sequence from amino acid 22 to 90 in the duck tropism of DHBV. Reasoning that reciprocal replacements would reduce infectivity for ducks, we generated spreading-competent chimeric DHBVs with L proteins in which segments 22-90 (Du-He4) or its subsegments 22-37 and 37-90 (Du-He2, Du-He3) are derived from HHBV. Infectivity for duck hepatocytes of Du-He4 and Du-He3, though not Du-He2, was indeed clearly reduced compared to wild-type DHBV. Surprisingly, however, in ducks even Du-He4 caused high-titered, persistent, horizontally and vertically transmissable infections, with kinetics of viral spread similar to those of DHBV when inoculated at doses of 10(8) viral genome equivalents (vge) per animal. Low-dose infections down to 300 vge per duck did not reveal a significant reduction in specific infectivity of the chimera. Hence, sequence alterations in PreS that limited infectivity in vitro did not do so in vivo. These data reveal a much more complex correlation between PreS sequence and host specificity than might have been anticipated; more generally, they question the value of cultured hepatocytes for reliably predicting in vivo infectivity of avian and, by inference, mammalian hepadnaviruses, with potential implications for the risk assessment of vaccine and drug resistant HBV variants.

Identification of interspecies recombination among hepadnaviruses infecting cross-species hosts.

Members of the family Hepadnaviridae are divided into two genera, Orthohepadnavirus (from mammalian) and Avihepadnavirus (from avian). Recombination had been found to occur among human hepatitis B virus (HBV) strains of different genotypes, or between hepadnavirus strains from human and nonhuman primate. To reach a comparatively complete inspection of interspecies recombination events among hepadnavirus strains from various hosts, 837 hepadnavirus complete genome sequences from human and 112 from animals were analyzed by using fragment typing to scan for potential interspecies recombinants. Further bootscanning and phylogenetic analyses of the potential recombinants revealed six genome sequences as interspecies recombinants. Interspecies recombination events were found to occur among HBV strains from human and nonhuman primates, from gibbons of different genera, from chimpanzee and an unknown host, and between two avian hepadnavirus strains from birds of different subfamilies, which was identified for the first time. HBV interspecies recombinants were found to have recombination hot spots similar to that of human HBV intergenotype recombinants, breakpoints frequently locating near gene boundaries. Interspecies recombination found in this study may alter current views on hepadnavirus host specificity.

Thermodynamics and NMR studies on Duck, Heron and Human HBV encapsidation signals.

Hepatitis B virus (HBV) replication is initiated by binding of its reverse transcriptase (P) to the apical stem-loop (AL) and primer loop (PL) of epsilon, a highly conserved RNA element at the 5'-end of the RNA pregenome. Mutation studies on duck/heron and human in vitro systems have shown similarities but also differences between their P-epsilon interaction. Here, NMR and UV thermodynamic data on AL (and PL) from these three species are presented. The stabilities of the duck and heron ALs were found to be similar, and much lower than that of human. NMR data show that this low stability stems from an 11-nt internal bulge destabilizing the stem of heron AL. In duck, although structured at low temperature, this region also forms a weak point as its imino resonances broaden to disappearance between 30 and 35 degrees C well below the overall AL melting temperature. Surprisingly, the duck- and heron ALs were both found to be capped by a stable well-structured UGUU tetraloop. All avian ALs are expected to adhere to this because of their conserved sequence. Duck PL is stable and structured and, in view of sequence similarities, the same is expected for heron - and human PL.

Hepatitis B virus taxonomy and hepatitis B virus genotypes.

Hepatitis B virus (HBV) is a member of the hepadnavirus family. Hepadnaviruses can be found in both mammals (orthohepadnaviruses) and birds (avihepadnaviruses). The genetic variability of HBV is very high. There are eight genotypes of HBV and three clades of HBV isolates from apes that appear to be additional genotypes of HBV. Most genotypes are now divided into subgenotypes with distinct virological and epidemiological properties. In addition, recombination among HBV genotypes increases the variability of HBV. This review summarises current knowledge of the epidemiology of genetic variability in hepadnaviruses and, due to rapid progress in the field, updates several recent reviews on HBV genotypes and subgenotypes.

Avian hepatitis B viruses: molecular and cellular biology, phylogenesis, and host tropism.

The human hepatitis B virus (HBV) and the duck hepatitis B virus (DHBV) share several fundamental features. Both viruses have a partially double-stranded DNA genome that is replicated via a RNA intermediate and the coding open reading frames (ORFs) overlap extensively. In addition, the genomic and structural organization, as well as replication and biological characteristics, are very similar in both viruses. Most of the key features of hepadnaviral infection were first discovered in the DHBV model system and subsequently confirmed for HBV. There are, however, several differences between human HBV and DHBV. This review will focus on the molecular and cellular biology, evolution, and host adaptation of the avian hepatitis B viruses with particular emphasis on DHBV as a model system.

Evidence from nature: interspecies spread of heron hepatitis B viruses.

Heron hepatitis B viruses (HHBVs) in three subspecies of free-living great blue herons (Ardea herodias) from Florida, USA, were identified and characterized. Eight of 13 samples were positive in all assays used, whereas sera from egrets, which are also members of the family Ardeidae, were negative in the same assays. Comparative phylogenetic analysis of viral DNA sequences from the preS/S region of previously reported and novel HHBV strains isolated from captive grey herons (Germany) and free-ranging great blue herons (USA), respectively, revealed a strong conservation (95 % sequence similarity) with two separate clusters, implying a common ancestor of all strains. Our data demonstrate for the first time that different subspecies of herons are infected by HHBV and that these infections exist in non-captive birds. Phylogenetic analysis and the fact that the different heron species are geographically isolated populations suggest that lateral transmission, virus adaptation and environmental factors all play a role in HHBV spreading and evolution.

Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity.

Five new hepadnaviruses were cloned from exotic ducks and geese, including the Chiloe wigeon, mandarin duck, puna teal, Orinoco sheldgoose, and ashy-headed sheldgoose. Sequence comparisons revealed that all but the mandarin duck viruses were closely related to existing isolates of duck hepatitis B virus (DHBV), while mandarin duck virus clones were closely related to Ross goose hepatitis B virus. Nonetheless, the S protein, core protein, and functional domains of the Pol protein were highly conserved in all of the new isolates. The Chiloe wigeon and puna teal hepatitis B viruses, the two new isolates most closely related to DHBV, also lacked an AUG start codon at the beginning of their X open reading frame (ORF). But as previously reported for the heron, Ross goose, and stork hepatitis B viruses, an AUG codon was found near the beginning of the X ORF of the mandarin duck, Orinoco, and ashy-headed sheldgoose viruses. In all of the new isolates, the X ORF ended with a stop codon at the same position. All of the cloned viruses replicated when transfected into the LMH line of chicken hepatoma cells. Significant differences between the new isolates and between these and previously reported isolates were detected in the pre-S domain of the viral envelope protein, which is believed to determine viral host range. Despite this, all of the new isolates were infectious for primary cultures of Pekin duck hepatocytes, and infectivity in young Pekin ducks was demonstrated for all but the ashy-headed sheldgoose isolate.

Chimeras of duck and heron hepatitis B viruses provide evidence for functional interactions between viral components of pregenomic RNA encapsidation.

Packaging of hepadnavirus pregenomic RNA (pgRNA) into capsids, or encapsidation, requires several viral components. The viral polymerase (P) and the capsid subunit (C) are necessary for pgRNA encapsidation. Previous studies of duck hepatitis B virus (DHBV) indicated that two cis-acting sequences on pgRNA are required for encapsidation: epsilon, which is near the 5' end of pgRNA, and region II, located near the middle of pgRNA. Later studies suggested that the intervening sequence between these two elements may also make a contribution. It has been demonstrated for DHBV that epsilon interacts with P to facilitate encapsidation, but it is not known how other cis-acting sequences contribute to encapsidation. We analyzed chimeras of DHBV and a related virus, heron hepatitis B virus (HHBV), to gain insight into the interactions between the various viral components during pgRNA encapsidation. We learned that having epsilon and P derived from the same virus was not sufficient for high levels of encapsidation, implying that other viral interactions contribute to encapsidation. Chimeric analysis showed that a large sequence containing region II may interact with P and/or C for efficient encapsidation. Further analysis demonstrated that possibly an RNA-RNA interaction between the intervening sequence and region II facilitates pgRNA encapsidation. Together, these results identify functional interactions among various viral components that contribute to pgRNA encapsidation.

Characterization of the cis-acting contributions to avian hepadnavirus RNA encapsidation.

Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two cis-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), epsilon and region II. epsilon, an RNA stem-loop near the 5' end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, epsilon and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between epsilon and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between epsilon and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.

Identification and characterization of a novel replicative intermediate of heron hepatitis B virus.

We have identified and characterized a novel intracellular DNA replicative intermediate that is synthesized by heron hepatitis B virus (HHBV) and not by other avian hepadnaviruses. The new DNA form is synthesized in all host cells tested. The HHBV nucleic acid template, and not HHBV proteins, is responsible for the formation of the new form. The new form is comprised of a full-length minus-strand DNA and an incomplete plus-strand DNA whose 5' ends are mapped to DR2, predominantly. The 3' ends of its plus-strand are located between nucleotides 946 and 1046. Genetic analysis indicates that the sequences responsible for the formation of the new form lie between nucleotides 910 and 1364. The endogenous polymerase activity of capsids isolated from cells converted the new form into RC DNA. Intracellular capsids containing the new form are secreted inefficiently as virions, in comparison to RC- and DL DNA-containing capsids. Our analysis suggests that the new form is an incomplete RC DNA molecule that is due to a specific block or pause in the synthesis of plus-strand DNA. Our analysis also suggests that capsids become competent for efficient secretion sometime after the synthesis of 1500 nucleotides of plus-strand DNA.

cis-Acting sequences 5E, M, and 3E interact to contribute to primer translocation and circularization during reverse transcription of avian hepadnavirus DNA.

Hepadnaviral reverse transcription requires template switches for the genesis of relaxed circular (RC) DNA, the major genomic form in virions. Two template switches, primer translocation and circularization, are required during the synthesis of the second, or plus, strand of DNA. Studies of duck hepatitis B virus (DHBV) indicate that in addition to the requirement for repeated sequences at the donor and acceptor sites, template switching requires at least three other cis-acting sequences, 5E, M, and 3E. In this study we analyzed a series of variant heron hepatitis B viruses (HHBV) in which the regions of the genome that would be expected to contain 5E, M, and 3E were replaced with DHBV sequence. We found that all single and double chimeras were partially defective in the synthesis of RC DNA. In contrast, the triple chimera was able to synthesize RC DNA at a level comparable to that of unchanged HHBV. These results indicate that the three cis-acting sequences, 5E, M, and 3E, need to be compatible to contribute to RC DNA synthesis, suggesting that these sequences interact during plus-strand synthesis. Second, we found that the defect in RC DNA synthesis for several of the single and double chimeric viruses resulted from a partial defect in primer translocation/utilization and a partial defect in circularization. These findings indicate that the processes of primer translocation and circularization share a mechanism during which 5E, M, and 3E interact.

Identification and analysis of a new hepadnavirus in white storks.

We identified, cloned, and functionally characterized a new avian hepadnavirus infecting storks (STHBV). STHBV has the largest DNA genome of all avian hepadnaviruses and, based on sequence and phylogenetic analysis, is most closely related to, but distinct from, heron hepatitis B virus (HHBV). Unique for STHBV among the other avian hepadnaviruses is a potential HNF1 binding site in the preS promoter. In common only with HHBV, STHBV has a myristylation signal on the S and not the preS protein, two C terminally located glycosylation sites on the precore/core proteins and lacks the phosphorylation site essential for the transcriptional transactivation activity of duck-HBV preS protein. The cloned STHBV genomes were competent in gene expression, replication, and viral particle secretion. STHBV infected primary duck hepatocytes very inefficiently suggesting a restricted host range, similar to other hepadnaviruses. This discovery of stork infections unravels novel evolutionary aspects of hepadnaviruses and provides new opportunities for hepadnavirus research.

Sequence- and structure-specific determinants in the interaction between the RNA encapsidation signal and reverse transcriptase of avian hepatitis B viruses.

Hepatitis B viruses (HBVs) replicate by reverse transcription of an RNA intermediate. Packaging of this RNA pregenome into nucleocapsids and replication initiation depend crucially on the interaction of the reverse transcriptase, P protein, with the cis-acting, 5' end-proximal encapsidation signal epsilon. The overall secondary structure is similar in all of the hepadnaviral epsilon signals, with a lower and an upper stem, separated by a bulge, and an apical loop. However, while epsilon is almost perfectly conserved in all mammalian viruses, the epsilon signals of duck HBV (DHBV) and heron HBV (D epsilon and H epsilon, respectively) differ substantially in their upper stem regions, both in primary sequence and in secondary structure; nonetheless, H epsilon interacts productively with DHBV P protein, as shown by its ability to stimulate priming, i.e., the covalent attachment of a deoxynucleoside monophosphate to the protein. In this study, we extensively mutated the variable and the conserved positions in the upper stem of D epsilon and correlated the functional activities of the variant RNAs in a priming assay with secondary structure and physical P protein binding. These data revealed a proper overall structure, with the bulge and certain key residues, e.g., in the loop, being important constraints in protein binding. Many mutations at the evolutionarily variable positions complied with these criteria and yielded priming-competent RNAs. However, most mutants at the conserved positions outside the loop were defective in priming even though they had epsilon-like structures and bound to P protein; conversely, one point mutant in the loop with an apical structure different from those of D epsilon and H epsilon was priming competent. These results suggest that P protein binding can induce differently structured epsilon RNAs to adopt a new, common conformation, and they support an induced-fit model of the epsilon-P interaction in which both components undergo extensive structural alterations during formation of a priming-competent ribonucleoprotein complex.

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 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.

Previously unsuspected cis-acting sequences for DNA replication revealed by characterization of a chimeric heron/duck hepatitis B virus.

Heron hepatitis B virus (HHBV) is an avian hepadnavirus that is closely related to duck hepatitis B virus (DHBV). To learn more about the mechanism of hepadnavirus replication, we characterized a clone of HHBV that contains a substitution of DHBV sequence from nucleotide coordinates 403 to 1364. This clone, named HDE1, expresses a chimeric pregenomic RNA, a chimeric polymerase (P) protein, and a core (C) protein with a one-amino-acid substitution at its carboxy terminus. We have shown that HDE1 is defective for minus-strand DNA synthesis, resulting in an overall reduction of viral DNA. HDE1 was also defective for plus-strand DNA synthesis, resulting in aberrant ratios of replication intermediates. Genetic complementation assays indicated that HDE1 replication proteins, C and P, are functional for replication and wild-type HHBV proteins do not rescue either defect. These findings indicate that the HDE1 substitution mutation acts primarily in cis. By restoring nucleotides 403 to 902 to the HHBV sequence, we showed that cis-acting sequences for plus-strand DNA synthesis are located in the 5' half of the HDE1 chimeric region. These data indicate the presence of one or more formerly unrecognized cis-acting sequences for DNA synthesis within the chimeric region (nucleotides 403 to 1364). These cis-acting sequences in the middle of the genome might interact directly or indirectly with known cis elements that are located near the ends of the genome. Our findings suggest that a specific higher-order template structure is involved in the mechanism of hepadnavirus DNA replication.