Sequence heterogeneity of heron hepatitis B virus genomes determined by full-length DNA amplification and direct sequencing reveals novel and unique features.

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.Hepadnaviruses are small, enveloped DNA viruses that replicate via reverse transcription of an RNA pregenome. The family Hepadnaviridae is divided into two genera, Orthohepadnavirus and Avihepadnavirus, each with restricted host specificity. Ortho (mammalian) hepadnaviruses have been found in humans (hepatitis B virus [HBV]) and great apes, woolly monkeys (woolly monkey HBV), woodchucks (woodchuck hepatitis virus), ground squirrels (ground squirrel hepatitis virus), arctic squirrels (arctic squirrel hepatitis virus), and Richardson ground squirrels (Richardson ground squirrel hepatitis virus). The avian hepadnaviruses include duck HBV (DHBV) (40,43,58,66), heron HBV (HHBV) (52), Ross goose HBV (RGHBV; GenBank accession no. M95589), snow goose HBV (SGHBV) (11), stork HBV (STHBV) (50), and crane HBV (CHBV) (48).Sequence similarities between the ortho-and avihepadnaviruses are minimal except in highly conserved functional domains, though the overall genome organization is similar. Proteins encoded by both groups include the nucleocapsid (core) and envelope polypeptides (L and S), the nonstructural HBeAg (a secretory protein of unknown function), and the polymerase/reverse transcriptase protein (Pol protein). Orthohepadnaviruses also encode a third envelope protein, M, and a regulatory protein, termed X, that is required for efficient replication in vivo (13,65,67). Studies with liver cell lines suggest that X stimulates signal transduction, regulates several transcription factors, and has a role in virus replicatio...

Section: Resultssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 96%

Identification and Characterization of Avihepadnaviruses Isolated from Exotic Anseriformes Maintained in Captivity

Guo

Mason

Aldrich

et al. 2005

“…The sequences of peptides X2 (ILLTAHPGTNRLIGR) and X3 (GYVELKNYTPLLRSC) correspond to DHBx amino acids 46 to 60 and 76 to 90 derived from the DHBV1 genome, respectively. The sequence of peptide p759 (AVVPCDCTFGMYHCL) is identical to the C-terminal end of the predicted X-like protein of HHBV4 (48) and almost identical to the corresponding region of DHBx proteins. All three peptides were conjugated to keyhole limpet hemocyanin and then injected into rabbits (subcutaneously and intramuscularly) to raise the ␣-X2, ␣-X3, and ␣-p759 antibodies.…”

Section: Plasmidsmentioning

confidence: 88%

“…Although X-protein-like sequences were proposed to be present in the middle of the DHBV nucleocapsid protein (DHBc) (18), functional similarities of DHBc with the mammalian hepadnavirus X proteins have never been identified. Moreover, the existence of an open reading frame (ORF) in the hepadnavirus genomes isolated from grey herons (48), snow geese (9), a Ross goose (48), and white storks (H. J. Netter, S.-F. Chang, and H. Will, unpublished observation) in a position similar to that of the X gene of orthohepadnaviruses argues that an X-like protein may be expressed from avian hepadnavirus genomes.…”

mentioning

confidence: 99%

Duck Hepatitis B Virus Expresses a Regulatory HBx-Like Protein from a Hidden Open Reading Frame

Chang

Netter

Hildt

et al. 2001

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho-and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.Since the identification of hepatitis B virus (HBV) in humans, several related viruses have been isolated from mammalian and avian species (9, 53, 68). These viruses, known as hepadnaviruses, display high liver tropism, have a narrow host range, and cause acute and chronic hepatitis. Chronic infection with mammalian hepadnaviruses (orthohepadnaviruses) is associated with the development of liver cancer.The hepadnaviruses are small enveloped DNA viruses with a unique virion ultrastructure. The viral genome is a partially duplexed, relaxed circular DNA molecule (rcDNA) with a size of about 3 kb which upon entry of the cell is converted into a covalently closed circular episome. This covalently closed circular DNA is then transcribed by the host RNA polymerase II, synthesizing subgenomic mRNAs and a greater-than-genomelength RNA known as pregenomic RNA or C-mRNA. Distinctive for all hepadnaviruses is the method of replication by reverse transcription of the pregenomic RNA into rcDNA. Transcripts encoding the envelope proteins, the nucleocapsid protein, the polymerase protein (P protein), and, as exclusively described so far in orthohepadnaviruses, the X protein have been identified. The organization of the viral genome is very compact, with overlapping reading frames and promoters and enhancer elements located within coding regions (20).The X proteins of orthohepadnaviruses affect signal transduction pathways, transcription, cell transformation, and proliferation (1,8,46,70). The HBV-specific X protein (HBx) is expressed in vivo, as shown by immunohistology and indirectly by identification of an HBx-specific immune response in infected individuals (64). In vivo expressio...

“…viruses (Fig. 5a), i.e., isolates 26 and 3 of DHBV, clones 1 to 5 of snow goose HBV (11), Ross goose HBV (J. Newbold, personal communication), isolate 4 of heron HBV (51,58), and clone 21 of stork HBV (53). CT preferentially cleaves after aromatic and large hydrophobic side chains of phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W).…”

Section: Ct-treated Svps Inhibit Infection Of Ct-treated Dhbvmentioning

confidence: 99%

Entry of Duck Hepatitis B Virus into Primary Duck Liver and Kidney Cells after Discovery of a Fusogenic Region within the Large Surface Protein

Maenz

Chang

Iwanski

et al. 2007

Hepatitis B viruses exhibit a narrow host range specificity that is believed to be mediated by a domain of the large surface protein, designated L. For duck hepatitis B virus, it has been shown that the pre-S domain of L binds to carboxypeptidase D, a cellular receptor present in many species on a wide variety of cell types. Nonetheless, only hepatocytes become infected. It has remained vague which viral features determine host range specificity and organotropicity. By using chymotrypsin to treat duck hepatitis B virus, we addressed the question of whether a putative fusogenic region within the amino-terminal end of the small surface protein may participate in viral entry and possibly constitute one of the determinants of the host range of the virus. Addition of the enzyme to virions resulted in increased infectivity. Remarkably, even remnants of enzyme-treated subviral particles proved to be inhibitory to infection. A noninfectious deletion mutant devoid of the binding region for carboxypeptidase D could be rendered infectious for primary duck hepatocytes by treatment with chymotrypsin. Although because of the protease treatment mutant and wild-type viruses may have become infectious in an unspecific and receptor-independent manner, their host range specificity was not affected, as shown by the inability of the virus to replicate in different hepatoma cell lines, as well as primary chicken hepatocytes. Instead, the organotropicity of the virus could be reduced, which was demonstrated by infection of primary duck kidney cells.The family Hepadnaviridae, with the human hepatitis B virus (HBV) as the prototype member, consists of small, DNAcontaining, enveloped viruses which cause liver diseases in humans, rodents, and birds (22). A characteristic feature of HBV and other hepadnaviruses is a strong species specificity where, e.g., HBV infects humans and chimpanzees (1) and duck HBV (DHBV) infects only certain duck and goose species (49). Moreover, all of the hepadnaviruses show relatively strict organotropicity, infecting mainly hepatocytes.Facilitated by the identification of neutralizing epitopes (12) and the allocation of host range-specific determinants to the pre-S region of L (29), it was possible to demonstrate the binding of DHBV to a putative hepatocellular receptor molecule via a certain domain on pre-S (7,8,30,39,63). This receptor molecule has been identified as gp180, a transmembrane protein of the carboxypeptidase D family that, however, has also been found in nonliver duck tissues that are not susceptible to DHBV infection (16,38,62). Furthermore, it has been shown that LMH cells, a chicken hepatoma cell line which resists infection but is capable of virus production after transfection of full-length DHBV DNA, also express gp180 (38). In addition, it was demonstrated that this molecule is concentrated in the Golgi apparatus, to cycle to and from the plasma membrane, where it colocalizes with pre-S (6, 7). Expression of duck gp180 in HuH7 cells, a human hepatoma cell line, led merely to internalizatio...