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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.

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 × 10 6 and 2.1 × 10 4 genome copies per mL (range 3.3 × 10 0 -2.5 × 10 7 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 >10 4 genome copies per mL, i.e. above the threshold considered at risk of active hepatitis and liver damage for HBV.

Hantavirus cardiopulmonary syndrome (HPS) has gained importance in Latin America as an emerging disease, with reports of about 4000 HPS cases; however, this is probably an underestimate because of limited surveillance programs and diagnostic tools to confirm HPS. In order to address this issue and develop better serosurveillance capability, we evaluated three recombinant peptides from the Necoclí virus (NECV) nucleocapsid in antibody-capture ELISA. We cloned and expressed antigens representing the whole NECV nucleocapsid protein (NECV-rN), the immunodominant domain (NECV-rN100), and a serospecific domain (NECV-rN428), and then we compared these antigens in ELISA to detect IgG antibodies to NECV in human sera. We evaluated human sera collected during two epidemiological studies from the area where NECV was discovered. The first group included 609 sera from healthy individuals, and the second one included 89 samples from patients with undifferentiated febrile illness. In these two groups, hantavirus infection had previously been determined by the presence of IgG to Maciel virus (MCLV), a hantavirus closely related to NECV. The number of IgG-positive sera was higher using the Necoclí ELISA with the rN100 protein, which detected antibodies in a higher percentage of healthy individuals, 129/609 (21.2%), as well as in febrile patients, 11/89 (12.3%). In contrast, using MCLV ELISA, 8 of 609 (1.3%) and 4 of 89 (4.5%) samples from healthy and febrile patients, respectively, were seropositive. The agreement between the NECV and MCLV ELISA assays was ≥ 82.3%; however, the kappa indices were weak but statistically significant for rN (0.251 CI; 0.138-0.365) and rN100rN (0.153 CI; 0.084-0.223). The weak kappa indices were attributed to decreased MCLV ELISA assay sensitivity. These results suggest that NECV rN and rN100 have increased specificity and could be further validated for improved diagnosis of hantavirus infections.

A novel codrug, α-DDB-FNCG, was synthesized through coupling of α-biphenyl dimethyl dicarboxylate (α-DDB) and the nucleoside analogue FNCG, via an ester bond. The anti-HBV activity and hepatoprotective effects of this compound were investigated both in vitro and in vivo. In HBV-transfected HepG2.2.15 cell line, the secretion of HBsAg and HBeAg as well as the levels of extracellular and intracellular viral DNA were determined by ELISA and real-time fluorescent quantitative Polymerase Chain Reaction (FQ-PCR), respectively. In DHBV-infected ducks, the viral DNA levels in serum and liver were determined by FQ-PCR. In addition, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in both serum and liver were also examined. The improvement of ducks' livers was evaluated by histopathological analysis. It has been demonstrated that α-DDB-FNCG could suppress the levels of HBV antigens and viral DNA in a time- and dose-dependent manner in the HepG2.2.15 cell line. Furthermore, this codrug could also significantly inhibit the viral DNA replication and reduce the ALT and AST levels in both serum and liver of DHBV-infected ducks, with improved hepatocellular architecture in drug-treated ducks. In short, these results suggest that α-DDB-FNCG could be a promising candidate for further development of new anti-HBV agents with hepatoprotective effects.

Ethnopharmacological Relevance: Hydrocotyle sibthorpioides (Apiaceae) have been used as a folk remedy for the treatment of fever, edema, detoxication, throat pain, psoriasis and hepatitis B virus infections in China. The aim of this study is to isolate and identify an anti-HBV compound from this herb., Materials and Methods: A compound (saponin) was isolated from the active ethanol extract using bioassay-guided screening. The structure of the saponin was elucidated by spectroscopic methods and compared with published data. The anti-HBV activity of the saponin was evaluated by detecting the levels of HBV antigens, extracellular HBV DNA, nuclear covalent closed circular DNA (cccDNA) and five HBV promoters in HepG2.2.15 cells. In addition, the levels of serum HBsAg/HBeAg, DHBV DNA, ALT/AST and hepatic pathological changes were analyzed in DHBV-infected ducks., Results: The chemical analysis indicated that the saponin isolated from Hydrocotyle sibthorpioides is asiaticoside. The pharmacodynamics experimental studies showed that asiaticoside effectively suppressed the levels of HBsAg/HBeAg, extracellular HBV DNA and intracellular cccDNA in a dose-dependent manner. Furthermore, experiments demonstrated that asiaticoside markedly reduced viral DNA transcription and replication by inhibiting the activities of core, s1, s2, and X gene promoters. In addition, asiaticoside markedly reduced DHBV replication without any obvious signs of toxicity. The levels of serum DHBV DNA, HBsAg/HBeAg were increased 3 days after drug withdrawal, but the levels rebounded slightly in the asiaticoside treatment groups compared with the 3TC treatment group. Moreover, analysis of the serum ALT/AST levels and the liver pathological changes indicated that asiaticoside could alleviate liver damage., Conclusions: Our results show that asiaticoside could efficiently inhibit HBV replication both in vitro and in vivo, and asiaticoside may be a major bioactive ingredient in Hydrocotyle sibthorpioides., (© 2013 Published by Elsevier Ireland Ltd.)

To examine the effect of Gankang Suppository on duck hepatitis B virus (DHBV), the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups: a Gankang Suppository treatment group, an acyclovir (ACV) group and a DHBV model group (control), with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medication and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values (log) of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment (P=0.0092, P=0.0070, P=0.0080, respectively). Compared with DHBV model control group, the ALT level was significantly decreased (P=0.0020, P=0.0019, respectively) on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment (P=0.0298). Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal (P=0.0016). Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group (P=0.0282, P=0.0084, P=0.0195, respectively). It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

Objective: To develop a standard duck hepatitis B virus (DHBV) animal model using a local Hubei species of duck, Ma Ya, and use it as an in vivo experimental system to study antiviral strategies against hepatitis B., Methods: Two-day-old Ma Ya ducklings were experimentally infected via intraperitoneal injection with the DHBV inocula which was collected from the transfected culture supernatant of 1.5-fold-overlength genome recombinant plasmid. Blood samples were taken twice or thrice a week during post-inoculation for 50 days. Viremia was quantified by serum real-time PCR to show the peak. Antiviral treatment of the DHBV-infected ducklings was started 3 d post-inoculation. The animals received oral administration of lamivudine (3TC) at a dose of 25 mg/kg/d for 5 d, followed by a maintenance therapy thrice weekly for 3 more weeks. Serum was quantified to show the viremia peak and liver biopsy specimens were analysed by Southern blotting and in-situ hybridization at the end of antiviral drug treatment., Results: The experimental infection rate of 2-day-old ducklings was 87.5%. Viremia started to be detectable on day 7 and reached a peak on day 11 post-inoculation, followed by a decrease and fluctuations. Four weeks of oral administration of 3TC led to a significant decrease in viremia peak during. This effect was not sustained, as a rebound in viremia was observed after drug withdrawal. Similarly, the analysis of liver biopsies at the end of 3TC treatment showed a marked decrease in DHBV DNA. However, after drug withdrawal a rebound of intrahepatic DHBV DNA was observed in duck livers., Conclusion: The Hubei duck model with experimental DHBV infection of transfected supernatant is more suitable for the hepadnavirus biologic research due to its stability and practicability.

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.

The hepatitis B virus core protein (HBcAg) is a uniquely immunogenic particulate antigen and as such has been used as a vaccine carrier platform. The use of other hepadnavirus core proteins as vaccine carriers has not been explored. To determine whether the rodent hepadnavirus core proteins derived from the woodchuck (WHcAg), ground squirrel (GScAg), and arctic squirrel (AScAg) viruses possess immunogen characteristics similar to those of HBcAg, comparative antigenicity and immunogenicity studies were performed. The results indicate that (i) the rodent core proteins are equal in immunogenicity to or more immunogenic than HBcAg at the B-cell and T-cell levels; (ii) major histocompatibility complex (MHC) genes influence the immune response to the rodent core proteins (however, nonresponder haplotypes were not identified); (iii) WHcAg can behave as a T-cell-independent antigen in athymic mice; (iv) the rodent core proteins are not significantly cross-reactive with the HBcAg at the antibody level (however, the nonparticulate "eAgs" do appear to be cross-reactive); (v) the rodent core proteins are only partially cross-reactive with HBcAg at the CD4+ T-cell level, depending on MHC haplotype; and (vi) the rodent core proteins are competent to function as vaccine carrier platforms for heterologous, B-cell epitopes. These results have implications for the selection of an optimal hepadnavirus core protein for vaccine design, especially in view of the "preexisting" immunity problem that is inherent in the use of HBcAg for human vaccine development.

Aim: To establish and assess the methods for quantitative detection of serum duck hepatitis B virus (DHBV) DNA by quantitative membrane hybridization using DHBV DNA probe labeled directly with alkaline phosphatase and fluorescence quantitative PCR (qPCR)., Methods: Probes of DHBV DNA labeled directly with alkaline phosphatase and chemiluminescent substrate CDP-star were used in this assay. DHBV DNA was detected by autoradiography, and then scanned by DNA dot-blot. In addition, three primers derived from DHBV DNA S gene were designed. Semi-nested primer was labeled by AmpliSensor. Standard curve of the positive standards of DHBV DNA was established after asymmetric preamplification, semi-nested amplification and on-line detection. Results from 100 samples detected separately by alkaline phosphatase direct-labeled DHBV DNA probe with dot-blot hybridization and digoxigenin-labeled DHBV DNA probe hybridization. Seventy samples of duck serum were tested by fluorescent qPCR and digoxigenin-labeled DHBV DNA probe in dot-blot hybridization assay and the correlation of results was analysed., Results: Sensitivity of alkaline phosphatase direct-labeled DHBV DNA probe was 10 pg. The coincidence was 100% compared with digoxigenin-labeled DHBV DNA probe assay. After 30 cycles, amplification products showed two bands of about 180 bp and 70 bp by 20 g/L agarose gel electrophoresis. Concentration of amplification products was in direct proportion to the initial concentration of positive standards. The detection index was in direct proportion to the quantity of amplification products accumulated in the current cycle. The initial concentration of positive standards was in inverse proportion to the number of cycles needed for enough quantities of amplification products. Correlation coefficient of the results was (0.97, P<0.01) between fluorescent qPCR and dot-blot hybridization., Conclusion: Alkaline phosphatase direct-labeled DHBV DNA probe in dot-blot hybridization and fluorescent qPCR can be used as valuable means to quantify DHBV DNA in serum.

The recent isolation of a nonhuman primate hepadnavirus from woolly monkeys prompted an examination of other primates for potentially new hepadnaviruses. A serological analysis of 30 captive gibbons revealed that 47% were positive for at least one marker of ongoing or previous infection with a hepatitis B virus (HBV). The amino acid sequences of the core and surface genes of human and gibbon virus isolates were very similar. Phylogenetic analysis indicated that the gibbon isolates lie within the human HBV family, indicating that these HBV isolates most likely stem from infection of gibbons from a human source.

We describe the identification and functional analysis of an evolutionary distinct new avian hepadnavirus. Infection of snow geese (Anser caerulescens) with a duck hepatitis B virus (DHBV)-related virus, designated SGHBV, was demonstrated by detection of envelope proteins in sera with anti-DHBV preS and S antibodies. Comparative sequence analysis of the PCR-amplified SGHBV genomes revealed unique SGHBV sequence features compared with other avian hepadnaviruses. Unlike DHBV, SGHBV shows an open reading frame in an analogous position to orthohepadnavirus X genes. Four of five cloned genomes were competent in replication, gene expression, and virus particle secretion in chicken hepatoma cells. Primary duck hepatocytes were permissive for infection with SGHBV, suggesting a similar or identical host range. SGHBV was found to secrete a significant fraction of virion-like particles containing single-stranded viral DNA. This was observed both in cell culture medium of SGHBV DNA-transfected LMH cells and in viremic sera of several birds, suggesting that it is a stable trait of SGHBV. Taken together, SGHBV has several unique features that expand the knowledge of the functional and evolutionary diversity of hepadnaviruses and offers new experimental opportunities for studies on the life cycle of hepadnaviruses., (Copyright 1999 Academic Press.)

A high prevalence (42.6%) of hepatitis B virus (HBV) infection was suspected in 195 formerly captive orangutans due to a large number of serum samples which cross-reacted with human HBV antigens. It was assumed that such viral infections were contracted from humans during captivity. However, two wild orangutans were identified which were HBV surface antigen positive, indicating that HBV or related viruses may be occurring naturally in the orangutan populations. Sequence analyses of seven isolates revealed that orangutans were infected with hepadnaviruses but that these were clearly divergent from the six known human HBV genotypes and those of other nonhuman hepadnaviruses reported. Phylogenetic analyses revealed geographic clustering with Southeast Asian genotype C viruses and gibbon ape HBV. This implies a common origin of infection within this geographic region, with cross-species transmission of hepadnaviruses among hominoids.

The hepadnaviral DNA genome is synthesized by a viral-encoded reverse transcriptase, but the nature of this protein(s) in vivo remains obscure. We have previously described studies in which activity gel assays identified multiple DNA polymerase (DNAp) activities associated with highly purified duck hepatitis B virus (DHBV) core particles. We now report that virions isolated from viremic sera are associated with DNA-dependent DNAp activities which are nearly identical to major DNAp activities detected with highly purified DHBV core particles. These results suggest that the virion-associated polymerases are the same as those which are detected with core particles and are likely to represent DHBV pol gene products involved in replication of the genome.

Replication of the hepadnavirus DNA genome is accomplished via reverse transcription of an intermediate, pregenomic RNA molecule. This process is likely to be carried out by a virally encoded, multifunctional polymerase which possesses DNA- and RNA-dependent DNA polymerase and RNase H activities. However, the nature of the product(s) of the polymerase gene predicted to mediate these functions is unclear. Biochemical studies of the polymerase protein(s) have been limited by its apparent low abundance in virus particles and, until recently, the inability to express active polymerase protein(s) heterologously. We have used activity gel assays to detect DNA- and RNA-dependent DNA polymerase activities associated with highly purified duck hepatitis B virus (DHBV) core particles (S. M. Oberhaus and J. E. Newbold, J. Virol. 67:6558-6566, 1993). Now we report that the same approach identifies a 35-kDa RNase H activity in association with highly purified DHBV core particles and crude preparations of virions from DHBV-infected ducks and woodchuck hepatitis virus-infected woodchucks. This is the first report of the detection of an hepadnavirus-associated RNase H activity. Its apparent size is smaller than any of the DNA polymerase activities that we detected previously and significantly smaller than the full-length protein predicted from the polymerase open reading frame (p85 for DHBV). These data suggest that the viral polymerase and RNase H activities are separable and that these enzymes may coordinate their activities in vivo by forming a complex.

In order to define, in more detail, the virological events which occur after completion of antiviral chemotherapy, ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated for 4 weeks with the nucleoside analogue ganciclovir and followed up over a 7-day period. Specimens of serum and liver were collected daily during follow-up for virological analysis. Treatment resulted in a substantial reduction in both viraemia and liver DHBV DNA replicative intermediates. However, after cessation of treatment, viraemia returned to detectable levels within 4 days. In the liver, the viral supercoiled DNA (SC DNA) was the form least affected by therapy and returned to near control levels by day 2 post-treatment. The other hepatic replicative intermediates reached pretreatment levels within 4 days of cessation of therapy. This study has defined the kinetics of relapse of viral replication after completion of antiviral therapy in the duck hepatitis B model. Of all viral replicative forms, the SC DNA appears to be the one which is most resistant to nucleoside analogue therapy and is presumably responsible for the relapse phenomenon observed post-treatment.

The antiviral activity and ability of mismatched double-stranded RNA (m-dsRNA), r(I)n.r(C12-U)n, to induce interferon (IFN) were evaluated in ducks chronically infected with duck hepatitis B virus (DHBV). When m-dsRNA was administered intravenously at a single dose of 5 mg/kg, serum DHBV DNA concentrations decreased significantly for 3 days (P < 0.002). However, the DHBV DNA concentrations returned to the pretreatment levels 4 days after treatment. Inhibition of DHBV DNA replication in the liver was also observed 2 days after treatment. Serum IFN activity peaked 3 hours after administration of m-dsRNA, then rapidly declined. 2'-5' Oligo-adenylate synthetase (2'-5'AS) activity increased gradually after treatment and remained elevated for at least 48 hours. In ducks receiving m-dsRNA once daily for 7 consecutive days, serum DHBV DNA concentrations on the last day of treatment were decreased by 76 +/- 12% (P < 0.05) in ducks that received 0.2 mg of m-dsRNA per kg and by 65 +/- 12% (P < 0.05) in ducks that received 1 mg of m-dsRNA per kg. This decrease persisted for at least 2 weeks after the cessation of treatment in all ducks. These results suggest that m-dsRNA effectively inhibits DHBV replication in vivo, and that IFN induction and stimulation of 2'-5'AS activity contribute to the inhibition of DHBV replication by m-dsRNA.

We investigated effects of various DNAs on duck hepatitis B virus replication in vivo. One-day-old ducks were infected intravenously with DHBV. Various DNAs were then injected intravenously, and duck hepatitis B virus levels were followed for up to 20 days after the inoculation. When M13 bacteriophage DNA (M13 DNA), heat-denatured Escherichia coli DNA or phi X 174 phage DNA was injected intravenously at a dose of 2.45 mg/kg body wt daily for 10 days, a significant decrease of serum duck hepatitis B virus DNA was detected within 10 days. The efficacy was twice that reported with antisense DNA on a weight basis and far more than that reported on a molar basis. M13 DNA was superior, on the basis of effective dose, to acyclovir as an anti-duck hepatitis B virus agent. On treatment with M13 DNA, serum 2-5 A synthetase level was increased five to six times, suggesting that the antiviral effect of M13 DNA is at least partly due to induction of endogenous interferon, which in turn induces 2-5 A synthetase. No significant inhibitory effect on replication of duck hepatitis B virus was demonstrated by DNAs obtained from herring testes, herring sperm, salmon testes, human placenta or calf thymus. On discontinuation of M13 DNA injection on day 10, duck hepatitis B virus reappeared in the serum at later time points. Digestion of M13 DNA with S1 nuclease resulted in marked reduction of antiviral activity. These results show that M13 DNA, not its digested product, has potent antiviral activity.