An in vivo duck hepatitis B virus model recapitulates key aspects of nucleic acid polymer treatment outcomes in chronic hepatitis B patients.

Nucleic acid polymers (NAPs) are an attractive treatment modality for chronic hepatitis B (CHB), with REP2139 and REP2165 having shown efficacy in CHB patients. A subset of patients achieve functional cure, whereas the others exhibit a moderate response or are non-responders. NAP efficacy has been difficult to recapitulate in animal models, with the duck hepatitis B virus (DHBV) model showing some promise but remaining underexplored for NAP efficacy testing. Here we report on an optimized in vivo DHBV duck model and explore several characteristics of NAP treatment. REP2139 was efficacious in reducing DHBV DNA and DHBsAg levels in approximately half of the treated ducks, whether administered intraperitoneally or subcutaneously. Intrahepatic or serum NAP concentrations did not correlate with efficacy, nor did the appearance of anti-DHBsAg antibodies. Furthermore, NAP efficacy was only observed in experimentally infected ducks, not in endogenously infected ducks (vertical transmission). REP2139 add-on to entecavir treatment induced a deeper and more sustained virological response compared to entecavir monotherapy. Destabilized REP2165 showed a different activity profile with a more homogenous antiviral response followed by a faster rebound. In conclusion, subcutaneous administration of NAPs in the DHBV duck model provides a useful tool for in vivo evaluation of NAPs. It recapitulates many aspects of this class of compound's efficacy in CHB patients, most notably the clear division between responders and non-responders.

Identification of domestic cat hepadnavirus from a cat blood sample in Japan.

The hepatitis B virus (Hepadnaviridae) induces chronic hepatitis and hepatic cancer in humans. A novel domestic cat hepadnavirus (DCH) was recently identified in several countries, however, the DCH infection status of cats in Japan is unknown. Therefore, we investigated the DCH infection rate of 139 cat samples collected in Japan. We identified one positive blood sample (0.78%) from a 17-year-old female cat with chronically elevated alanine aminotransferase. Phylogenetic analysis demonstrated that the DCH strain identified in this study is genetically different from strains in other countries. Further investigations are required to elucidate the evolution of DCH and the impact of DCH infection on hepatic diseases in domestic cats.

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.

Mammalian deltavirus without hepadnavirus coinfection in the neotropical rodent Proechimys semispinosus.

Hepatitis delta virus (HDV) is a human hepatitis-causing RNA virus, unrelated to any other taxonomic group of RNA viruses. Its occurrence as a satellite virus of hepatitis B virus (HBV) is a singular case in animal virology for which no consensus evolutionary explanation exists. Here we present a mammalian deltavirus that does not occur in humans, identified in the neotropical rodent species Proechimys semispinosus The rodent deltavirus is highly distinct, showing a common ancestor with a recently described deltavirus in snakes. Reverse genetics based on a tandem minus-strand complementary DNA genome copy under the control of a cytomegalovirus (CMV) promoter confirms autonomous genome replication in transfected cells, with initiation of replication from the upstream genome copy. In contrast to HDV, a large delta antigen is not expressed and the farnesylation motif critical for HBV interaction is absent from a genome region that might correspond to a hypothetical rodent large delta antigen. Correspondingly, there is no evidence for coinfection with an HBV-related hepadnavirus based on virus detection and serology in any deltavirus-positive animal. No other coinfecting viruses were detected by RNA sequencing studies of 120 wild-caught animals that could serve as a potential helper virus. The presence of virus in blood and pronounced detection in reproductively active males suggest horizontal transmission linked to competitive behavior. Our study establishes a nonhuman, mammalian deltavirus that occurs as a horizontally transmitted infection, is potentially cleared by immune response, is not focused in the liver, and possibly does not require helper virus coinfection.

Orthohantavirus Isolated in Reservoir Host Cells Displays Minimal Genetic Changes and Retains Wild-Type Infection Properties.

: Orthohantaviruses are globally emerging zoonotic pathogens. While the reservoir host role of several rodent species is well-established, detailed research on the mechanisms of host-othohantavirus interactions has been constrained by the lack of an experimental system that is able to effectively replicate natural infections in controlled settings. Here we report the isolation, and genetic and phenotypic characterization of a novel Puumala orthohantavirus (PUUV) in cells derived from its reservoir host, the bank vole. The isolation process resulted in cell culture infection that evaded antiviral responses, persisted cell passaging, and had minor viral genome alterations. Critically, experimental infections of bank voles with the new isolate resembled natural infections in terms of viral load and host cell distribution. When compared to an attenuated Vero E6 cell-adapted PUUV Kazan strain, the novel isolate demonstrated delayed virus-specific humoral responses. A lack of virus-specific antibodies was also observed during experimental infections with wild-type PUUV, suggesting that delayed seroconversion could be a general phenomenon during orthohantavirus infection in reservoir hosts. Our results demonstrate that orthohantavirus isolation on cells derived from a vole reservoir host retains wild-type infection properties and should be considered the method of choice for experimental infection models to replicate natural processes.

Simple and visible detection of duck hepatitis B virus in ducks and geese using loop-mediated isothermal amplification.

In this study, loop-mediated isothermal amplification (LAMP) was used to establish a rapid, specific, and visual detection method for duck hepatitis B virus (DHBV). The design and synthesis of 4 specific LAMP primers were based on the conserved gene region of the DHBV genome, and the optimum temperature and time of the LAMP reaction were 63°C and 50 min, respectively. The LAMP assay was confirmed to be specific for DHBV detection and had the same sensitivity as the quantitative PCR assay. A visual detection method for rapid determination of results was developed using a color indicator containing phenol red and cresol red. A color change was produced based on a pH change in the reaction system, indicating a positive reaction. For the detection of samples from ducks and geese, the LAMP method has the advantages of simplicity, high sensitivity and specificity, good visibility, and low cost. Moreover, it is more practical and convenient than PCR-related assays for the clinical detection of DHBV.

A Novel Hepadnavirus is Associated with Chronic Hepatitis and Hepatocellular Carcinoma in Cats.

In 2015, over 850,000 people died from chronic hepatitis and hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV). A novel hepatitis B-like virus has recently been identified in domestic cats. The pathogenic potential of domestic cat hepadnavirus (DCH), for which 6.5% to 10.8% of pet cats are viremic, is unknown. We evaluated stored formalin-fixed, paraffin-embedded biopsies of diseased and normal feline liver for the presence of DCH using PCR and in situ hybridization (ISH). DCH was detected in 43% (6/14) of chronic hepatitis cases and 28% (8/29) of HCCs, whereas cholangitis (n = 6), biliary carcinoma (n = 18) and normal liver (n = 15) all tested negative for DCH. Furthermore, in DCH-associated cases, the histologic features of inflammation and neoplasia, and the viral distribution on ISH were strikingly similar to those seen with HBV-associated disease. Several histological features common in human HBV-associated hepatitis, including piecemeal necrosis and apoptotic bodies, were identified in DCH-positive cases of chronic hepatitis. In two cases of HCC examined, the proliferation index in regions that were ISH-positive was higher than in ISH-negative regions. The intracellular distribution of virus in both hepatitis and HCC demonstrated that viral nucleic acid is present in both nuclear and cytoplasmic forms. Collectively, these findings demonstrate a compelling association between DCH and some cases of chronic hepatitis and hepatocellular carcinoma in the cat that mirrors features of HBV-associated hepatopathies. Future investigations of viral epidemiology and natural history are needed to establish the impact of DCH on feline health.

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.

Genetic diversity of bat orthohepadnaviruses in China and a proposed new nomenclature.

The orthohepadnaviruses, which include the major human pathogen hepatitis B virus, exist in a wide range of hosts. Since 2013, a large group of orthohepadnaviruses has been identified in bats worldwide and classified as 4 species within the genus Orthohepadnavirus. To further investigate orthohepadnaviruses in the Chinese bat population, 554 archived bat samples from 20 colonies covering 3 southern provinces were screened with results showing that 9 (1.6%) were positive. A systematic phylogenetic analysis has indicated the need for a new nomenclature for bat hepatitis B virus-like viruses: BtHBV, with the addition of 3 new species, one being divided into 6 genotypes. Viruses identified here shared 9.0-19.2% full genome divergence and classified into 3 different genotypes. This study illustrates the genetic diversity of orthohepadnaviruses in the Chinese bat population, and emphasizes need for further investigation of their public health significance.

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.

Suspected Hepadnavirus Association with a Hepatocellular Carcinoma in a Black-Tailed Prairie Dog (Cynomys ludovicianus).

Hepatocellular carcinomas are the most commonly reported neoplasm of black-tailed prairie dogs (Cynomys ludovicianus). In several other closely related Sciuridae species, infection with species-specific hepadnaviruses is associated with the development of these tumours, but such a hepadnavirus has not yet been identified in any prairie dog species, although its presence has been hypothesized previously. An adult prairie dog was humanely destroyed due to progressive illness and the identification of a cranial abdominal mass that was determined on histopathology to be a hepatocellular carcinoma. Deep sequencing of the tumour tissue identified the presence of a hepadnavirus, similar in its genetic structure to woodchuck hepatitis virus. Electron microscopy showed the presence of viral particles similar in structure to other hepadnaviral particles. This report suggests that a hepadnavirus may be associated with the development of hepatocellular carcinomas in the prairie dog.

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.

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.

Comparative Transcriptomic Analysis of Primary Duck Hepatocytes Provides Insight into Differential Susceptibility to DHBV Infection.

Primary duck hepatocytes (PDH) displays differential susceptibility to duck hepatitis B virus when maintained in the media supplemented with fetal bovine serum or dimethyl sulfoxide (DMSO) which has been widely used for the maintenance of hepatocytes, and prolonging susceptibility to hepadnavirus. However the mechanism underlying maintenance of susceptibility to hepadnavirus by DMSO treatment remains unclear. In this study, a global transcriptome analysis of PDHs under different culture conditions was conducted for investigating the effects of DMSO on maintenance of susceptibility of PDH to DHBV in vitro. The 384 differential expressed genes (DEGs) were identified by comparisons between each library pair (PDHs cultured with or without DMSO or fresh isolated PDH). We analyzed canonical pathways in which the DEGs were enriched in Hepatic Fibrosis / Hepatic Stellate Cell Activation, Bile Acid Biosynthesis and Tight Junction signaling. After re-annotation against human genome data, the 384 DEGs were pooled together with proteins belonging to hepatitis B pathway to construct a protein-protein interaction network. The combination of decreased expression of liver-specific genes (CYP3A4, CYP1E1, CFI, RELN and GSTA1 et al) with increased expression of hepatocyte-dedifferentiation-associated genes (PLA2G4A and PLCG1) suggested that in vitro culture conditions results in the fading of hepatocyte phenotype in PDHs. The expression of seven DEGs associated with tight junction formation (JAM3, PPP2R2B, PRKAR1B, PPP2R2C, MAGI2, ACTA2 and ACTG2) was up-regulated after short-term culture in vitro, which was attenuated in the presence of DMSO. Those results could shed light on DHBV infection associated molecular events affected by DMSO.

Identification of a novel Orthohepadnavirus in pomona roundleaf bats in China.

Bats in Myanmar, Gabon, and Panama have been found to harbor diverse hepadnaviruses. Here, we report a novel hepadnavirus in 4 of 20 pomona roundleaf bats from Yunnan province, China. This virus contains 3,278 nucleotides (nt) in the full circularized genome, with four predicted open frames (ORFs) reading in the same direction. Full genomic sequence comparisons and evolutionary analysis indicate that this virus is a member of a new species within the genus Orthohepadnavirus.

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.