A novel hepadnavirus in domestic dogs.

Hepadnaviruses have been identified in several animal species. The hepadnavirus prototype, human hepatitis B virus (HBV), is a major public health problem associated with chronic liver diseases and hepatocellular carcinoma. Recently, a novel hepadnavirus, similar to HBV, was identified in domestic cats. Since several pathogens can be shared between cats and dogs, we hypothesized that dogs could also harbor hepadnaviruses and we tested a collection of canine sera with multiple molecular strategies. Overall, hepadnavirus DNA was identified in 6.3% (40/635) of canine serum samples, although the viral load in positive sera was low (geometric mean of 2.70 × 102 genome copies per mL, range min 1.36 × 102-max 4.03 × 104 genome copies per mL). On genome sequencing, the canine hepadnaviruses revealed high nucleotide identity (about 98%) and similar organization to the domestic cat hepadnavirus. Altered hepatic markers were found in hepadnavirus-positive dogs, although the role of hepadnavirus in canine health remains to be elucidated.

A longitudinal observational study in two cats naturally-infected with hepadnavirus.

Hepatitis B virus (HBV) is a major cause of liver disease in humans including chronic hepatitis and hepatocellular carcinoma. Domestic cat hepadnavirus (DCH), a novel HBV-like hepadnavirus, was identified in domestic cats in 2018. From 6.5 %-10.8 % of pet cats are viremic for DCH and altered serological markers suggestive of liver damage have been identified in 50 % of DCH-infected cats. DCH DNA has been detected in association with characteristic lesions of chronic hepatitis and with hepatocellular carcinoma in cats, suggesting a possible association. In this study longitudinal molecular screening of cats infected with DCH was performed to determine if DCH can cause chronic infections in cats. Upon re-testing of sera from five DCH-positive animals, 2-10 months after the initial diagnosis, three cats tested negative for DCH on two consecutive occasions using quantitative PCR. Two other cats remained DCH-positive, including an 8-month-old female cat re-tested four months after the initial positive result, and a 9-year-old male cat, which tested positive for DCH on six occasions over an 11-month period. The latter had a history of chronic hepatopathy with jaundice, lethargy and elevated serum alanine transaminase levels (ALT). During the period of observation, DCH titers ranged between 1.64 × 105 and 2.09 × 106 DNA copies/mL and ALT was persistently elevated, suggesting chronic infection. DCH DNA was not detected in oral, conjunctival, preputial and rectal swabs from the two animals collected at several time points. Long-term (chronic) infection would be consistent with the relatively high number of viremic cats identified in epidemiological investigations, with the possible association of DCH with chronic hepatic pathologies and with what described with HBV in human patients.

Molecular detection and characterisation of Domestic Cat Hepadnavirus (DCH) from blood and liver tissues of cats in Malaysia.

BACKGROUND: A new domestic cat hepadnavirus (DCH, family Hepadnaviridae) was first reported from whole blood samples of domestic cats in Australia in 2018, and from cat serum samples in Italy in 2019. The pathogenesis of DCH is unknown, but it was reported in cats with viraemia (6.5-10.8%), chronic hepatitis (43%) and hepatocellular carcinoma (28%). Recent reports suggest that DCH resembles the human hepatitis B virus (HBV) and its related hepatopathies. This study aims to detect and characterize DCH among domestic cats in Malaysia. A cross-sectional study was performed on 253 cats, of which 87 had paired blood and liver samples, entailing whole-genome sequencing and phylogenetic analysis of DCH from a liver tissue sample. RESULTS: Among the 253 cats included in this study, 12.3% of the whole blood samples tested positive for DCH. The detection rate was significantly higher in pet cats (16.6%, n = 24/145) compared to shelter cats (6.5%, n = 7/108). Liver tissues showed higher a DCH detection rate (14.9%, n = 13/87) compared to blood; 5 out of these 13 cats tested positive for DCH in their paired liver and blood samples. Serum alanine transaminase (ALT) was elevated (> 95 units/L) in 12 out of the 23 DCH-positive cats (52.2%, p = 0.012). Whole-genome sequence analysis revealed that the Malaysian DCH strain, with a genome size of 3184 bp, had 98.3% and 97.5% nucleotide identities to the Australian and Italian strains, respectively. The phylogenetic analysis demonstrated that the Malaysian DCH genome was clustered closely to the Australian strain, suggesting that they belong to the same geographically-determined genetic pool (Australasia). CONCLUSIONS: This study provided insights into a Malaysian DCH strain that was detected from a liver tissue. Interestingly, pet cats or cats with elevated ALT were significantly more likely to be DCH positive. Cats with positive DCH detection from liver tissues may not necessarily have viraemia. The impact of this virus on inducing liver diseases in felines warrants further investigation.

Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats.

Hepatitis B virus (HBV) is a human pathogen of global concern, while a high diversity of viruses related to HBV have been discovered in other animals during the last decade. Recently, the novel mammalian hepadnavirus, tentatively named domestic cat hepadnavirus (DCH), was detected in an immunocompromised cat. Herein, a collection of 209 cat sera and 15 hepato-diseased cats were screened for DCH using PCR, resulting in 12.4% and 20% positivity in the tested sera and necropsied cats, respectively. Among the DCH-positive sera, a significantly high level of co-detection with retroviral infection was found, with the highest proportion being co-detection with feline immunodeficiency virus (FIV). Full-length genome characterization of DCH revealed the genetic diversity between the nine Thai DCH sequences obtained, and that they phylogenetically formed three distinct monophyletic clades. A putative DCH recombinant strain was found, suggesting a possible role of recombination in DCH evolution. Additionally, quantitative PCR was used to determine the viral copy number in various organs of the DCH-moribund cats, while the pathological findings were compared to the viral localization in hepatocytes, adjacent to areas of hepatic fibrosis, by immunohistochemical (IHC) and western blot analysis. In addition to the liver, positive-DCH immunoreactivity was found in various other organs, including kidneys, lung, heart, intestine, brain, and lymph nodes, providing evidence of systemic infection. Ultrastructure of infected cells revealed electron-dense particles in the nucleus and cytoplasm of hepatocytes, bronchial epithelial cells, and fibroblasts. We propose the intracellular development mechanism of this virus. Although the definitive roles of pathogenicity of DCH remains undetermined, a contributory role of the virus associated with systemic diseases is possible.

Identification of hepadnavirus in the sera of cats.

Hepadnaviruses infect several animal species. The prototype species, human hepatitis B virus (HBV), increases the risk of liver diseases and may cause cirrhosis and hepatocellular carcinoma. Recently a novel hepadnavirus, similar to HBV, has been identified through transcriptomics studies in a domestic cat with large cell lymphoma in Australia. Herewith, a collection of 390 feline serum samples was screened for hepadnavirus. Overall, the virus was identified in 10.8% of the sera with a significantly higher prevalence (17.8%) in the sera of animals with a clinical suspect of infectious disease. Upon genome sequencing, the virus was closely related (97.0% nt identity) to the prototype Australian feline virus Sydney 2016. The mean and median values of hepadnavirus in the feline sera were 1.3 × 106 and 2.1 × 104 genome copies per mL (range 3.3 × 100-2.5 × 107 genome copies per mL). For a subset of hepadnavirus-positive samples, information on the hemato-chemical parameters was available and in 10/20 animals a profile suggestive of liver damage was present. Also, in 7/10 animals with suspected hepatic disease, virus load was >104 genome copies per mL, i.e. above the threshold considered at risk of active hepatitis and liver damage for HBV.

Discovery of a highly divergent hepadnavirus in shrews from China.

Limited sampling means that relatively little is known about the diversity and evolutionary history of mammalian members of the Hepadnaviridae (genus Orthohepadnavirus). An important case in point are shrews, the fourth largest group of mammals, but for which there is limited knowledge on the role they play in viral evolution and emergence. Here, we report the discovery of a novel shrew hepadnavirus. The newly discovered virus, denoted shrew hepatitis B virus (SHBV), is divergent to be considered a new species of Orthohepadnavirus. Phylogenetic analysis revealed that these viruses were usually most closely related to TBHBV (tent-making bat hepatitis B virus), known to be able to infect human hepatocytes, and had a similar genome structure, although SHBV fell in a more basal position in the surface protein phylogeny. In sum, these data suggest that shrews are natural hosts for hepadnaviruses and may have played an important role in their long-term evolution.

Host Biology and Anthropogenic Factors Affect Hepadnavirus Infection in a Neotropical Bat.

The tent-making bat hepatitis B virus (TBHBV) is a hepadnavirus closely related to human hepatitis B virus. The ecology of TBHBV is unclear. We show that it is widespread and highly diversified in Peters' tent-making bats (Uroderma bilobatum) within Panama, while local prevalence varied significantly between sample sites, ranging from 0 to 14.3%. Females showed significantly higher prevalence than males, and pregnant females were more often acutely infected than non-reproductive ones. The distribution of TBHBV in bats was significantly affected by forest cover, with higher infection rates in areas with lower forest cover. Our data indicate that loss of natural habitat may lead to positive feedback on the biotic factors driving infection possibility. These results underline the necessity of multidisciplinary studies for a better understanding of mechanisms in pathogen-host relationships and for predictions in disease ecology.

A Novel Hepadnavirus Identified in an Immunocompromised Domestic Cat in Australia.

High-throughput transcriptome sequencing allows for the unbiased detection of viruses in host tissues. The application of this technique to immunosuppressed animals facilitates the detection of viruses that might otherwise be excluded or contained in immunocompetent individuals. To identify potential viral pathogens infecting domestic cats we performed high-throughput transcriptome sequencing of tissues from cats infected with feline immunodeficiency virus (FIV). A novel member of the Hepadnaviridae, tentatively named domestic cat hepadnavirus, was discovered in a lymphoma sample and its complete 3187 bp genome characterized. Phylogenetic analysis placed the domestic cat hepadnavirus as a divergent member of mammalian orthohepadnaviruses that exhibits no close relationship to any other virus. DNA extracted from whole blood from pet cats was positive for the novel hepadnavirus by PCR in 6 of 60 (10%) FIV-infected cats and 2 of 63 (3.2%) FIV-uninfected cats. The higher prevalence of hepadnavirus viraemia detected in FIV-infected cats mirrors that seen in human immunodeficiency virus-infected humans coinfected with hepatitis B virus. In summary, we report the first hepadnavirus infection in a carnivore and the first in a companion animal. The natural history, epidemiology and pathogenic potential of domestic cat hepadnavirus merits additional investigation.

Extensive diversity and evolution of hepadnaviruses in bats in China.

To better understand the evolution of hepadnaviruses, we sampled bats from Guizhou, Henan and Zhejiang provinces, China, and rodents from Zhejiang province. Genetically diverse hepadnaviruses were identified in a broad range of bat species, with an overall prevalence of 13.3%. In contrast, no rodent hepadnaviruses were identified. The newly discovered bat hepadnaviruses fell into two distinct phylogenetic groups. The viruses within the first group exhibited high diversity, with some closely related to viruses previously identified in Yunnan province. Strikingly, the newly discovered viruses sampled from Jiyuan city in the second phylogenetic group were most closely related to those found in bats from West Africa, suggestive of a long-term association between bats and hepadnaviruses. A co-phylogenetic analysis revealed frequent cross-species transmission among bats from different species, genera, and families. Overall, these data suggest that there are likely few barriers to the cross-species transmission of bat hepadnaviruses.

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.

Deciphering the Origin and Evolution of Hepatitis B Viruses by Means of a Family of Non-enveloped Fish Viruses.

Hepatitis B viruses (HBVs), which are enveloped viruses with reverse-transcribed DNA genomes, constitute the family Hepadnaviridae. An outstanding feature of HBVs is their streamlined genome organization with extensive gene overlap. Remarkably, the ∼1,100 bp open reading frame (ORF) encoding the envelope proteins is fully nested within the ORF of the viral replicase P. Here, we report the discovery of a diversified family of fish viruses, designated nackednaviruses, which lack the envelope protein gene, but otherwise exhibit key characteristics of HBVs including genome replication via protein-primed reverse-transcription and utilization of structurally related capsids. Phylogenetic reconstruction indicates that these two virus families separated more than 400 million years ago before the rise of tetrapods. We show that HBVs are of ancient origin, descending from non-enveloped progenitors in fishes. Their envelope protein gene emerged de novo, leading to a major transition in viral lifestyle, followed by co-evolution with their hosts over geologic eras.

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.

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.

Early mesozoic coexistence of amniotes and hepadnaviridae.

Hepadnaviridae are double-stranded DNA viruses that infect some species of birds and mammals. This includes humans, where hepatitis B viruses (HBVs) are prevalent pathogens in considerable parts of the global population. Recently, endogenized sequences of HBVs (eHBVs) have been discovered in bird genomes where they constitute direct evidence for the coexistence of these viruses and their hosts from the late Mesozoic until present. Nevertheless, virtually nothing is known about the ancient host range of this virus family in other animals. Here we report the first eHBVs from crocodilian, snake, and turtle genomes, including a turtle eHBV that endogenized >207 million years ago. This genomic "fossil" is >125 million years older than the oldest avian eHBV and provides the first direct evidence that Hepadnaviridae already existed during the Early Mesozoic. This implies that the Mesozoic fossil record of HBV infection spans three of the five major groups of land vertebrates, namely birds, crocodilians, and turtles. We show that the deep phylogenetic relationships of HBVs are largely congruent with the deep phylogeny of their amniote hosts, which suggests an ancient amniote-HBV coexistence and codivergence, at least since the Early Mesozoic. Notably, the organization of overlapping genes as well as the structure of elements involved in viral replication has remained highly conserved among HBVs along that time span, except for the presence of the X gene. We provide multiple lines of evidence that the tumor-promoting X protein of mammalian HBVs lacks a homolog in all other hepadnaviruses and propose a novel scenario for the emergence of X via segmental duplication and overprinting of pre-existing reading frames in the ancestor of mammalian HBVs. Our study reveals an unforeseen host range of prehistoric HBVs and provides novel insights into the genome evolution of hepadnaviruses throughout their long-lasting association with amniote hosts.

Genetic characterization of hepadnaviruses associated with histopathological changes in the liver of duck and goose embryos.

Avian hepadnaviruses are etiological agents of hepatitis B, that has been identified primarily in ducks, and more recently in various avian species. In this paper, 16 hepadnaviruses were detected by polymerase chain reaction (PCR) in the field samples from dead embryos of commercially reared domestic duck and goose. Based on the molecular analysis of the S-protein gene sequences and phylogenetic Neighbor-joining tree, identified viruses were clustered in the same genetic group, indicating no host-related diversity. Both duck and goose-origin hepadnaviruses were grouped within the cluster consisting of "Western-country" and "Chinese" duck hepatitis B (DHBV) isolates, showing more evolutionary distances with other known avian hepadnaviruses. Histopathologically, the lesions observed in the liver tissue from hepadnavirus positive duck and goose embryos varied from low to mild degree of perivascular mononuclear cells and mixed cell infiltrations, followed by mild vacuolar changes. Small focal necrotic changes in the liver parenchyma, and bile ductular proliferation were also found in examined liver samples. Generally, the microscopic findings resemble those described in experimentally infected ducks, while this was the first description of hepadnavirus associated lesions in domestic goose. Although hepadnaviruses are considered to have a very narrow host range, this study showed that domestic ducks and geese are susceptible to infection with genetically almost identical hepadnaviruses, that were likely to produce similar microscopic changes in the liver of both duck and goose embryos. The impact of naturally occurred hepadnavirus infection and possible synergistic interactions with other infectious or non-infectious agents on embryo viability needs further investigation.

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.

The first full-length endogenous hepadnaviruses: identification and analysis.

In silico screening of metazoan genome data identified multiple endogenous hepadnaviral elements in the budgerigar (Melopsittacus undulatus) genome, most notably two elements comprising about 1.3 × and 1.0 × the full-length genome. Phylogenetic and molecular dating analyses show that endogenous budgerigar hepatitis B viruses (eBHBV) share an ancestor with extant avihepadnaviruses and infiltrated the budgerigar genome millions of years ago. Identification of full-length genomes with preserved key features like ε signals could enable resurrection of ancient BHBV.

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.