Constrained evolution of overlapping genes in viral host adaptation: Acquisition of glycosylation motifs in hepadnaviral precore/core genes.

Hepadnaviruses use extensively overlapping genes to expand their coding capacity, especially the precore/core genes encode the precore and core proteins with mostly identical sequences but distinct functions. The precore protein of the woodchuck hepatitis virus (WHV) is N-glycosylated, in contrast to the precore of the human hepatitis B virus (HBV) that lacks N-glycosylation. To explore the roles of the N-linked glycosylation sites in precore and core functions, we substituted T77 and T92 in the WHV precore/core N-glycosylation motifs (75NIT77 and 90NDT92) with the corresponding HBV residues (E77 and N92) to eliminate the sequons. Conversely, these N-glycosylation sequons were introduced into the HBV precore/core gene by E77T and N92T substitutions. We found that N-glycosylation increased the levels of secreted precore gene products from both HBV and WHV. However, the HBV core (HBc) protein carrying the E77T substitution was defective in supporting virion secretion, and during infection, the HBc E77T and N92T substitutions impaired the formation of the covalently closed circular DNA (cccDNA), the critical viral DNA molecule responsible for establishing and maintaining infection. In cross-species complementation assays, both HBc and WHV core (WHc) proteins supported all steps of intracellular replication of the heterologous virus while WHc, with or without the N-glycosylation sequons, failed to interact with HBV envelope proteins for virion secretion. Interestingly, WHc supported more efficiently intracellular cccDNA amplification than HBc in the context of either HBV or WHV. These findings reveal novel determinants of precore secretion and core functions and illustrate strong constraints during viral host adaptation resulting from their compact genome and extensive use of overlapping genes.

Conservation of the HBV RNA element epsilon in nackednaviruses reveals ancient origin of protein-primed reverse transcription.

Hepadnaviruses, with the human hepatitis B virus as prototype, are small, enveloped hepatotropic DNA viruses which replicate by reverse transcription of an RNA intermediate. Replication is initiated by a unique protein-priming mechanism whereby a hydroxy amino acid side chain of the terminal protein (TP) domain of the viral polymerase (P) is extended into a short DNA oligonucleotide, which subsequently serves as primer for first-strand synthesis. A key component in the priming of reverse transcription is the viral RNA element epsilon, which contains the replication origin and serves as a template for DNA primer synthesis. Here, we show that recently discovered non-enveloped fish viruses, termed nackednaviruses [C. Lauber et al., Cell Host Microbe 22, 387-399 (2017)], employ a fundamentally similar replication mechanism despite their huge phylogenetic distance and major differences in genome organization and viral lifestyle. In vitro cross-priming studies revealed that few strategic nucleotide substitutions in epsilon enable site-specific protein priming by heterologous P proteins, demonstrating that epsilon is functionally conserved since the two virus families diverged more than 400 Mya. In addition, other cis elements crucial for the hepadnavirus-typical replication of pregenomic RNA into relaxed circular double-stranded DNA were identified at conserved positions in the nackednavirus genomes. Hence, the replication mode of both hepadnaviruses and nackednaviruses was already established in their Paleozoic common ancestor, making it a truly ancient and evolutionary robust principle of genome replication that is more widespread than previously thought.

Comparison of the prevalence of Domestic Cat Hepadnavirus in a population of cats with uveitis and in a healthy blood donor cat population in the United Kingdom.

OBJECTIVE: Domestic Cat Hepadnavirus (DCH) is a novel virus recently identified in the domestic cat. Currently, little is known regarding its clinical significance. The hepadnaviridae family includes the Hepatitis B Virus (HBV). Co-infection of HBV and Hepatitis C in humans increases the risk of uveitis. We aimed to determine whether DCH is present in the United Kingdom (UK) and whether DCH warrants investigation as a potential cause of uveitis in cats. PROCEDURES: Clinical records from the Royal Veterinary College (RVC) and the Animal Health Trust (AHT) were reviewed for feline cases diagnosed with endogenous uveitis. A healthy control group was identified from cats presented to the RVC as blood donors. DNA was extracted from stored blood samples using commercially available kits. Polymerase chain reaction assays were performed to confirm the presence of feline DNA and to detect the presence of DCH DNA using previously described protocols. RESULTS: Blood samples were available from 65 cats with endogenous uveitis and 43 healthy control cats. Two blood samples from cats with endogenous uveitis tested positive for the presence of DCH DNA. DCH DNA was not detected in the control group. There was no statistically significant difference between the prevalence of DCH between the groups. CONCLUSIONS: Domestic Cat Hepadnavirus is present in the UK. This study failed to demonstrate a conclusive link between DCH and uveitis in cats, although further studies to investigate an association with other feline diseases are warranted.

Interferon Alpha Induces Multiple Cellular Proteins That Coordinately Suppress Hepadnaviral Covalently Closed Circular DNA Transcription.

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.

ICTV Virus Taxonomy Profile: Hepadnaviridae.

The family Hepadnaviridae comprises small enveloped viruses with a partially double-stranded DNA genome of 3.0-3.4 kb. All family members express three sets of proteins (preC/C, polymerase and preS/S) and replication involves reverse transcription within nucleocapsids in the cytoplasm of hepatocytes. Hepadnaviruses are hepatotropic and infections may be transient or persistent. There are five genera: Parahepadnavirus, Metahepadnavirus, Herpetohepadnavirus, Avihepadnavirus and Orthohepadnavirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hepadnaviridae, which is available at ictv.global/report/hepadnaviridae.

Human genetic basis of fulminant viral hepatitis.

In rare cases, hepatitis A virus (HAV) and hepatitis B virus (HBV) can cause fulminant viral hepatitis (FVH), characterized by massive hepatocyte necrosis and an inflammatory infiltrate. Other viral etiologies of FVH are rarer. FVH is life-threatening, but the patients are typically otherwise healthy, and normally resistant to other microbes. Only a small minority of infected individuals develop FVH, and this is the key issue to be addressed for this disease. In mice, mouse hepatitis virus 3 (MHV3) infection is the main model for dissecting FVH pathogenesis. Susceptibility to MHV3 differs between genetic backgrounds, with high and low mortality in C57BL6 and A/J mice, respectively. FVH pathogenesis in mice is related to uncontrolled inflammation and fibrinogen deposition. In humans, FVH is typically sporadic, but rare familial forms also exist, suggesting that there may be causal monogenic inborn errors. A recent study reported a single-gene inborn error of human immunity underlying FVH. A patient with autosomal recessive complete IL-18BP deficiency was shown to have FVH following HAV infection. The mechanism probably involves enhanced IL-18- and IFN-γ-dependent killing of hepatocytes by NK and CD8 T cytotoxic cells. Proof-of-principle that FVH can be genetic is important clinically, for the affected patients and their families, and immunologically, for the study of immunity to viruses in the liver. Moreover, the FVH-causing IL18BP genotype suggests that excessive IL-18 immunity may be a general mechanism underlying FVH, perhaps through the enhancement of IFN-γ immunity.

Genetic and molecular features for hepadnavirus and plague infections in the Himalayan marmot.

The Himalayan marmot (Marmota himalayana), a natural host and transmitter of plague, is also susceptible to the hepadnavirus infection. To reveal the genetic basis of the hepadnavirus susceptibility and the immune response to plague, we systematically characterized the features of immune genes in Himalayan marmot with those of human and mouse. We found that the entire major histocompatibility complex region and the hepatitis B virus pathway genes of the Himalayan marmot were conserved with those of humans. A Trim (tripartite motif) gene cluster involved in immune response and antiviral activity displays dynamic evolution, which is reflected by the duplication of Trim5 and the absence of Trim22 and Trim34. Three key regions of Ntcp, which is critical for hepatitis B virus entry, had high identity among seven species of Marmota. Moreover, we observed a severe alveolar hemorrhage, inflammatory infiltrate in the infected lungs and livers from Himalayan marmots after infection of EV76, a live attenuated Yersinia pestis strain. Lots of immune genes were remarkably up-regulated, which several hub genes Il2rγ, Tra29, and Nlrp7 are placed at the center of the gene network. These findings suggest that Himalayan marmot is a potential animal model for study on the hepadnavirus and plague infection.

Codon usage pattern and its influencing factors in different genomes of hepadnaviruses.

Codon usage bias (CUB) arises from the preference for a codon over codons for the same amino acid. The major factors contributing to CUB are evolutionary forces, compositional properties, gene expression, and protein properties. The present analysis was performed to investigate the compositional properties and the extent of CUB across the genomes of members of the family Hepadnaviridae, as previously no work using bioinformatic tools has been reported. The viral genes were found to be AT rich with low CUB. Analysis of relative synonymous codon usage (RSCU) was used to identify overrepresented and underrepresented codons for each amino acid. Correlation analysis of overall nucleotide composition and its composition at the third codon position suggested that mutation pressure might influence the CUB. A highly significant correlation was observed between GC12 and GC3 (r = 0.910, p < 0.01), indicating that directional mutation affected all three codon positions across the genome. Translational selection (P2) and mutational responsive index (MRI) values of genes suggested that mutation plays a more important role than translational selection in members of the family Hepadnaviridae.

The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation.

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.

New Avian Hepadnavirus in Palaeognathous Bird, Germany.

In 2015, we identified an avian hepatitis B virus associated with hepatitis in a group of captive elegant-crested tinamous (Eudromia elegans) in Germany. The full-length genome of this virus shares <76% sequence identity with other avihepadnaviruses. The virus may therefore be considered a new extant avian hepadnavirus.

Distinct Viral Lineages from Fish and Amphibians Reveal the Complex Evolutionary History of Hepadnaviruses.

UNLABELLED: Hepadnaviruses (hepatitis B viruses [HBVs]) are the only animal viruses that replicate their DNA by reverse transcription of an RNA intermediate. Until recently, the known host range of hepadnaviruses was limited to mammals and birds. We obtained and analyzed the first amphibian HBV genome, as well as several prototype fish HBVs, which allow the first comprehensive comparative genomic analysis of hepadnaviruses from four classes of vertebrates. Bluegill hepadnavirus (BGHBV) was characterized from in-house viral metagenomic sequencing. The African cichlid hepadnavirus (ACHBV) and the Tibetan frog hepadnavirus (TFHBV) were discovered using in silico analyses of the whole-genome shotgun and transcriptome shotgun assembly databases. Residues in the hydrophobic base of the capsid (core) proteins, designated motifs I, II, and III, are highly conserved, suggesting that structural constraints for proper capsid folding are key to capsid protein evolution. Surface proteins in all vertebrate HBVs contain similar predicted membrane topologies, characterized by three transmembrane domains. Most striking was the fact that BGHBV, ACHBV, and the previously described white sucker hepadnavirus did not form a fish-specific monophyletic group in the phylogenetic analysis of all three hepadnaviral genes. Notably, BGHBV was more closely related to the mammalian hepadnaviruses, indicating that cross-species transmission events have played a major role in viral evolution. Evidence of cross-species transmission was also observed with TFHBV. Hence, these data indicate that the evolutionary history of the hepadnaviruses is more complex than previously realized and combines both virus-host codivergence over millions of years and host species jumping. IMPORTANCE: Hepadnaviruses are responsible for significant disease in humans (hepatitis B virus) and have been reported from a diverse range of vertebrates as both exogenous and endogenous viruses. We report the full-length genome of a novel hepadnavirus from a fish and the first hepadnavirus genome from an amphibian. The novel fish hepadnavirus, sampled from bluegills, was more closely related to mammalian hepadnaviruses than to other fish viruses. This phylogenetic pattern reveals that, although hepadnaviruses have likely been associated with vertebrates for hundreds of millions of years, they have also been characterized by species jumping across wide phylogenetic distances.

Detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in China.

BACKGROUND: In recent years, novel hepadnaviruses, hepeviruses, hepatoviruses, and hepaciviruses have been discovered in various species of bat around the world, indicating that bats may act as natural reservoirs for these hepatitis viruses. In order to further assess the distribution of hepatitis viruses in bat populations in China, we tested the presence of these hepatitis viruses in our archived bat liver samples that originated from several bat species and various geographical regions in China. METHODS: A total of 78 bat liver samples (involving two families, five genera, and 17 species of bat) were examined using nested or heminested reverse transcription PCR (RT-PCR) with degenerate primers. Full-length genomic sequences of two virus strains were sequenced followed by phylogenetic analyses. RESULTS: Four samples were positive for hepadnavirus, only one was positive for hepevirus, and none of the samples were positive for hepatovirus or hepacivirus. The hepadnaviruses were discovered in the horseshoe bats, Rhinolophus sinicus and Rhinolophus affinis, and the hepevirus was found in the whiskered bat Myotis davidii. The full-length genomic sequences were determined for one of the two hepadnaviruses identified in R. sinicus (designated BtHBVRs3364) and the hepevirus (designated BtHEVMd2350). A sequence identity analysis indicated that BtHBVRs3364 had the highest degree of identity with a previously reported hepadnavirus from the roundleaf bat, Hipposideros pomona, from China, and BtHEVMd2350 had the highest degree of identity with a hepevirus found in the serotine bat, Eptesicus serotinus, from Germany, but it exhibited high levels of divergence at both the nucleotide and the amino acid levels. CONCLUSIONS: This is the first study to report that the Chinese horseshoe bat and the Chinese whiskered bat have been found to carry novel hepadnaviruses and a novel hepevirus, respectively. The discovery of BtHBVRs3364 further supports the significance of host switches evolution while opposing the co-evolutionary theory associated with hepadnaviruses. According to the latest criterion of the International Committee on Taxonomy of Viruses (ICTV), we hypothesize that BtHEVMd2350 represents an independent genotype within the species Orthohepevirus D of the family Hepeviridae.

Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity.

Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt -85 to -11 in eBHBV1 Cp was critical for the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt -64 to -50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt -58 to -54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt -21 to -17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses.

Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes.

The hepatitis B virus (HBV), family Hepadnaviridae, is one of most relevant human pathogens. HBV origins are enigmatic, and no zoonotic reservoirs are known. Here, we screened 3,080 specimens from 54 bat species representing 11 bat families for hepadnaviral DNA. Ten specimens (0.3%) from Panama and Gabon yielded unique hepadnaviruses in coancestral relation to HBV. Full genome sequencing allowed classification as three putative orthohepadnavirus species based on genome lengths (3,149-3,377 nt), presence of middle HBV surface and X-protein genes, and sequence distance criteria. Hepatic tropism in bats was shown by quantitative PCR and in situ hybridization. Infected livers showed histopathologic changes compatible with hepatitis. Human hepatocytes transfected with all three bat viruses cross-reacted with sera against the HBV core protein, concordant with the phylogenetic relatedness of these hepadnaviruses and HBV. One virus from Uroderma bilobatum, the tent-making bat, cross-reacted with monoclonal antibodies against the HBV antigenicity determining S domain. Up to 18.4% of bat sera contained antibodies against bat hepadnaviruses. Infectious clones were generated to study all three viruses in detail. Hepatitis D virus particles pseudotyped with surface proteins of U. bilobatum HBV, but neither of the other two viruses could infect primary human and Tupaia belangeri hepatocytes. Hepatocyte infection occurred through the human HBV receptor sodium taurocholate cotransporting polypeptide but could not be neutralized by sera from vaccinated humans. Antihepadnaviral treatment using an approved reverse transcriptase inhibitor blocked replication of all bat hepadnaviruses. Our data suggest that bats may have been ancestral sources of primate hepadnaviruses. The observed zoonotic potential might affect concepts aimed at eradicating HBV.

Liver-directed gene therapy of chronic hepadnavirus infection using interferon alpha tethered to apolipoprotein A-I.

BACKGROUND & AIMS: Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. METHODS: Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. RESULTS: In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. CONCLUSIONS: Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.

Snow goose hepatitis B virus (SGHBV) envelope and capsid proteins independently contribute to the ability of SGHBV to package capsids containing single-stranded DNA in virions.

UNLABELLED: Hepadnaviruses selectively package capsids containing mature double-stranded DNA (dsDNA) genomes in virions. Snow goose hepatitis B virus (SGHBV) is the only known hepadnavirus that packages capsids containing single-stranded DNA (ssDNA) in virions. We found that cells replicating SGHBV produce virions containing ssDNA as efficiently as virions containing mature dsDNA. We determined that SGHBV capsid and envelope proteins independently contribute to the production of virions containing ssDNA, with the capsid protein (Cp) making a larger contribution. We identified that amino acid residues 74 and 107 of SGHBV Cp contribute to this feature of SGHBV. When we changed these residues in duck hepatitis B virus (DHBV) Cp, capsids containing immature ssDNA were packaged in virions. This result suggests that residues 74 and 107 contribute to the appearance of the "capsid packaging signal" on the surface of capsids and interact with the envelope proteins during virion formation. We also found that cells replicating SGHBV package a larger fraction of the total dsDNA they synthesize into virions than do those replicating DHBV. We determined that the SGHBV envelope proteins are responsible for this property of SGHBV. Determining if the ability of SGHBV envelope proteins to cause the formation of virions containing ssDNA is related to its ability to support high levels of virion production or if these two properties are mechanistically distinct will provide insights into virion morphogenesis. IMPORTANCE: Cells replicating hepadnaviruses contain cytoplasmic capsids that contain mature and immature genomes. However, only capsids containing mature dsDNA genomes are packaged in virions. A mechanistic understanding of this phenomenon, which is currently lacking, is critical to understanding the process of hepadnaviral virion morphogenesis. In this study, we determined that the envelope proteins contribute to the ability of hepadnaviruses to selectively produce virions containing mature dsDNA genomes. Our finding sheds new light on the mechanisms underlying virion morphogenesis and challenges the dogma that "capsid maturation," and therefore the capsid protein (Cp), is solely responsible for the selective production of virions containing mature dsDNA genomes. Further, we identified amino acid residues of Cp that contribute to its ability to cause the selective production of virions containing mature dsDNA genomes. Future studies on the role of these residues in selective secretion will broaden our understanding of this poorly understood aspect of virus replication.

Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes.

We report the discovery of endogenous viral elements (EVEs) from Hepadnaviridae, Bornaviridae and Circoviridae in the speckled rattlesnake, Crotalus mitchellii, the first viperid snake for which a draft whole genome sequence assembly is available. Analysis of the draft assembly reveals genome fragments from the three virus families were inserted into the genome of this snake over the past 50 Myr. Cross-species PCR screening of orthologous loci and computational scanning of the python and king cobra genomes reveals that circoviruses integrated most recently (within the last approx. 10 Myr), whereas bornaviruses and hepadnaviruses integrated at least approximately 13 and approximately 50 Ma, respectively. This is, to our knowledge, the first report of circo-, borna- and hepadnaviruses in snakes and the first characterization of non-retroviral EVEs in non-avian reptiles. Our study provides a window into the historical dynamics of viruses in these host lineages and shows that their evolution involved multiple host-switches between mammals and reptiles.

Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes.

Primary Tupaia hepatocytes (PTHs) are susceptible to woolly monkey hepatitis B virus (WMHBV) infection, but the identity of the cellular receptor(s) mediating WMHBV infection of PTHs remains unclear. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for human hepatitis B virus (HBV) infection of primary human and Tupaia hepatocytes. In this study, a synthetic pre-S1 peptide from WMHBV was found to bind specifically to cells expressing Tupaia NTCP (tsNTCP) and it efficiently blocked WMHBV entry into PTHs; silencing of tsNTCP in PTHs significantly inhibited WMHBV infection. Ectopic expression of tsNTCP rendered HepG2 cells susceptible to WMHBV infection. These data demonstrate that tsNTCP is a functional receptor for WMHBV infection of PTHs. The result also indicates that NTCP's orthologs likely act as a common cellular receptor for all known primate hepadnaviruses.

Hepadnavirus detected in bile and liver samples from domestic pigs of commercial abattoirs.

BACKGROUND: Preliminary studies showed the prevalence of a virus similar to human hepatitis B virus (HBV-like) in swine from farms in China and the molecular evidence of Hepadnavirus infection in domestic pigs herds in Brazil. In this study, we genetically characterize the swine Hepadnavirus strains in swine from slaughterhouses located in certified abattoirs from Rio de Janeiro State, Brazil and evaluate its hepatotropic potential. RESULTS: Bile and liver samples from swine were positive for partial genome amplification (ORF S and ORF C), direct sequencing and viral load quantification. Sequencing of the gene encoding the surface antigen allowed classification of Hepadnavirus into genotypes, similar to HBV genotype classification. Indirect immunofluorescence confirmed the presence of HBsAg antigen in liver tissue sections. CONCLUSIONS: So far our data suggest that commercial swine house an HBV-like virus and this relevant finding should be considered in studies on the origin and viral evolution.

Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism.

The human APOBEC3 (A3A-A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome.