Oral famciclovir against duck hepatitis B virus replication in hepatic and nonhepatic tissues of ducklings infected in ovo.

Detection of hepadnaviral DNA in extrahepatic tissues of human and animal models of hepatitis B virus (HBV) has raised the question of whether virus replication in organs other than the liver could be targeted for the treatment of chronic hepatitis B. Since duck hepatitis B virus (DHBV) replication is dynamic in the liver, kidney, pancreas, and spleen of newly hatched ducklings infected in ovo, we used the duck model and the new antiherpesvirus agent, famciclovir (FCV), to determine whether antiviral effect of nucleoside analogues on DHBV replication is pluripotential. Day-old ducklings hatched from eggs laid by a DHBV-carrier duck were bled and administered FCV (25 mg/kg/bd) orally for periods of 1, 2, 3, 6, 9, and 12 days. Seventeen (17) hours after the last dose of each regimen the duckling(s) was bled and postmortem samples of liver, kidney, pancreas, and spleen were snap-frozen and stored at -70 degrees C. Analysis of plasma samples of ducklings treated for 2 days and longer by dot-blot hybridisation showed that levels of DHBV DNA were reduced significantly compared to levels in samples collected before treatment begun. Southern blot hybridisation of tissue DNA corroborated these results and showed that DHBV DNA replicative intermediates in all the tissues examined were reduced to levels that reflected the amount of virus released into the blood of each treated duckling. It is concluded from these results that if antiviral agents could be transformed to active metabolites in any infected tissues including the liver, replication of hepadnaviruses would be inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical disinfection of duck hepatitis B virus: a model for inactivation of infectivity of hepatitis B virus.

The susceptibility of duck hepatitis B virus (DHBV) to the virucidal effects of sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) was compared to hepatitis B virus (HBV) with the aim of using the duck as a model for studying HBV disinfection. Using viral DNA polymerase (DNAP) as a target, inhibition of DNAP activity by chlorine disinfectants was found to be concentration-dependent but independent of contact time. Two minute exposure of minimal effective concentrations of sodium hypochlorite (domestic bleach: 3600 ppm and industrial bleach: 3180 ppm) and sodium dichloroisocyanurate (3000 ppm available chlorine) to DHBV- and HBV-rich plasma totally inhibited DNA polymerase activity. DHBV particles in DHBV-carrier duck plasma (10(4.5) ID50/mL) were treated with these concentrations and inoculated intravenously into 18 one-day old ducklings (six animals/disinfectant). Analysis of plasma (0, 7 and 14 days post-infection) and post-mortem liver (14 days post-infection) by DNA hybridization techniques showed that DHBV DNA was undetectable in samples from all animals inoculated with disinfected virus particles. However, post-inoculation plasma and liver of 18 of 18 control ducklings inoculated with untreated virions were positive for DHBV DNA. These results show for the first time that total inhibition in vitro of hepadnavirus DNA polymerase activity by chemical disinfectants is predictive of inactivation of infectivity in vivo.

Hepadnaviruses, their infections and hepatocellular carcinoma.

Ten years ago hepatitis B virus (HBV) was thought to be a unique virus, not included in any known family of viruses. Following the discovery of a number of HBV-like viruses that infect birds and mammals, the existence of a new family known as hepadnaviridae has been confirmed. Hepadnaviruses are small hepatotropic viruses that have a characteristic partially double stranded genome, exhibit a narrow host range and replicate by reverse transcription. The family currently comprises six viruses of which human hepatitis B virus is the prototype member. Other members include woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus (GSHV), tree squirrel hepatitis virus (TSHV). Peking duck hepatitis B virus (DHBV) and heron hepatitis B virus (HHBV). Candidate members of the family include kangaroo hepatitis virus (KHV) and stink snake hepatitis virus (SSHV). In humans, infection with HBV is associated with a wide spectrum of clinical conditions including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Infection with HBV is endemic throughout much of the world and the virus is maintained by the enormous reservoir of over 300 million chronic carriers. For almost 20 years experimental work on hepadnaviruses has been carried out using either natural hosts or cultured cells that were capable to support synthesis of a few viral gene products but unable to execute a complete cycle of virus replication. In this article, we have attempted to summarize the efforts made towards understanding the biology of hepadnaviruses, the nature of their infections and their association with primary liver cancer.

The complete nucleotide sequence of the genome of a hepatitis B virus isolated from a naturally infected chimpanzee.

The complete nucleotide sequence of a strain of hepatitis B virus, originally isolated from a naturally infected chimpanzee, has been determined. Interesting features of the sequence include the presence of an in-phase stop codon in the 'pre-core' region of the core antigen open reading frame. The sequence shows approximately 10% nucleotide divergence from all of the other hepatitis B virus sequences previously published and the possibility that this divergence is the result of passage through chimpanzees is discussed.

Restriction of sensitivity of HIV-1-antigen ELISA by serum anti-core antibodies.

Addition of varying concentrations of HIV-1-seropositive plasma to purified virus particles and soluble viral antigen preparation inhibited the detection of HIV-1-antigen by ELISA. The degree of inhibition on p24 antigen ELISA depended on the relative concentrations of viral antigen and anti-p24 antibodies in the mixtures. The relevance of these observations to clinical specimens was demonstrated when serial plasma samples from nine AIDS-related complex (ARC) patients in a clinical trial of foscarnet therapy were assayed for p24 antigen. Six (66.6%) out of nine patients were negative on screening. However, when their plasma was centrifuged through a sucrose solution, virus particles were pelleted that were depleted of anti-p24 and virus-specific p24 antigen was detected in resuspended pellets obtained from samples from all six patients. Eight serial samples collected from a male homosexual over 50 weeks following seroconversion were also subjected to the separation procedure. HIV-1-antigen was detected in all eight samples. In the light of these observations, the application of the separation technique in monitoring the efficacy of zidovudine or other anti-retroviral therapy should reveal more precise antigen levels in patients who otherwise appear to be antigen-negative in HIV-antigen assays. We propose that the natural history of HIV infections follows a pattern of chronic viral infection with continuous shedding of virus particles circulating as immune complexes.

Suramin treatment for chronic active hepatitis B--toxic and ineffective.

Suramin has recently been shown to inhibit the activity of the duck hepatitis B virus DNA polymerase (DHBV DNAp) in vitro. However, we found no demonstrable in vivo suppression of human hepatitis B virus DNA polymerase (HBV DNAp) activity in three male patients with severe chronic active hepatitis. Suramin treatment resulted in prolongation of the prothrombin time in all cases and a rise in bilirubin in two and it may have led to haemorrhage from oesophageal varices in one patient and to hepatic encephalopathy in another. Its use in chronic hepatitis is not recommended.

Antiviral activity of the polybasic anion, suramin and acyclovir in Hepadna virus infection.

Acyclovir and suramin were examined for their efficacy alone and in combination against duck hepatitis B virus (DHBV) in persistently infected Pekin ducks. In ducks the peak plasma concentration of acyclovir was reached thirty minutes after oral administration. Oral acyclovir and suramin administered intravenously suppressed the replication and production of infectious virions as measured by marked reduction of DNA polymerase activity during treatment. However, rebound of enzyme activity was observed soon after cessation of drug therapy. In contrast, sustained reduction of polymerase activity was attained by combined therapy of acyclovir followed by suramin, demonstrating a significant enhancement of anti-DHBV activity which requires confirmation in a larger experimental study. This report establishes that the duck model is ideal for screening antiviral compounds in treatment of infection with Hepadna viruses.

Maternal transmission of duck hepatitis B virus in pedigree Pekin ducks.

A 14-month old female Pekin duck experimentally infected as an embryo with duck hepatitis B virus via the amniotic route has been a chronic carrier of duck hepatitis B virus with very high (P/N) values of DNA polymerase activity since hatching. All the progeny were, on evaluation for congenital infection, found to be duck hepatitis B virus positive by endogenous DNA polymerase reaction and electron microscopy. These offspring remained persistently viremic throughout the study. Maternal transmission therefore bred true to a total of 49 offspring--24 ducklings (less than 24 hr old) and 25 ducks--studied. Six of these 25 ducks matched for age and sex and bled weekly for 6 weeks exhibited fluctuating plasma levels of DNA polymerase activity. Higher DNA polymerase activity was detected in newly hatched ducklings than in older viremic ducks. This observation was corroborated with the results of electron microscopic examination of thin sections of liver. Duck hepatitis B virus particles, located within vesicles of rough endoplasmic reticulum in the cytoplasm of hepatocytes, were more abundant, and therefore more readily observed, in ducklings than in older ducks.

Studies by electron microscopy on the assembly of duck hepatitis B virus in the liver.

Liver specimens from 1-day-old ducklings infected in ovo with maternally transmitted duck hepatitis B virus (DHBV) were examined by electron microscopy. Complete and incomplete DHBV particles were located within hypertrophied cisternae of the endoplasmic reticulum of the hepatocytes. The complete viral particles found intracellularly have inner cores with a diameter ranging from 35 to 37.5 nm and an outer coat or envelope. The whole particle measures approximately 45-65 nm in diameter. Naked core particles were located in the nuclei, free in the cytoplasm, and also near or on the cisternal membrane of the endoplasmic reticulum on the cytoplasmic face. Duck hepatitis B virions appear to share morphological characteristics with the viral coat and core of human hepatitis B virus (HBV). Electron microscopy suggested that the core particles of DHBV migrate from the nucleus into the cytoplasm through the nuclear pores. The complete viral particles are probably formed by protrusion of the core particles through the endoplasmic reticulum and by simultaneous encapsulation with a coat derived from the endoplasmic reticulum.

Experimental in ovo transmission of duck hepatitis B virus.

Inoculation of fertile Pekin duck eggs with diluted serum containing DHBV into eggs incubated for 24 h and into the extra-embryonic cavities of 14-day-old embryos resulted in a high proportion of viraemic ducklings irrespective of the route of inoculation. Long-term observation of som of the ducks established that the viraemia induced experimentally is long-lasting and has persisted for periods up to 16 mth post-hatch. Separation of DHBV from the plasma of carrier ducks by rate zonal centrifugation was examined by DNA polymerase (DNAP) activity. Particles in the fraction with peak DNAP activity had a buoyant density of 1.16 g X cm-3 in sucrose and an estimated sedimentation coefficient, S20.w of 77. DHBV particles, the morphology of which could be resolved under the electron microscope, consisted of a coat (about 10 nm in thickness) surrounding a core with a diameter measuring 40 nm but not 27 nm as previously reported. Spike-like projections were found on the surface of the core as described previously by W.S. Mason, G. Seal and J. Summers, 1980, J. Virol. 36, 829-836.

Acute hepatitis A infection in hepatitis B chimpanzee carriers.

Two hepatitis B virus carrier chimpanzees which were superinfected with hepatitis A virus developed acute hepatitis followed by the production of antibodies to hepatitis A virus. The Southern blot technique employed to monitor liver hepatitis B virus DNA revealed that the amount of viral DNA in both animals was significantly reduced during the acute phase of hepatitis A infection. The levels of plasma hepatitis B DNA polymerase activity were also reduced in one chimpanzee. The high titers of HBsAg in the circulation remained unchanged throughout the study, and antibodies to the surface antigen and to e antigen were not detected. The morphological lesions in the liver were severe in one chimpanzee from whom one specimen showed both periportal focal necrosis and zonal parenchymal necrosis.

The mechanism of replication of hepatitis B virus: evidence of asymmetric replication of the two DNA strands.

In this study we have characterised the DNA replicative intermediates of hepatitis B virus and have shown that HBV-DNA replication is asymmetric. This pattern of HBV replication is similar to that reported for the related duck hepatitis virus (DHBV) and suggests the involvement of a similar reverse transcription process in HBV-DNA replication.

A new method for detecting hepatitis B surface antigen in liver by silver staining.

Staining of tubular and circular structures within the cisternae of the endoplasmic reticulum of the cytoplasm of liver cells infected with hepatitis B virus was enhanced by the use of 1% aqueous silver proteinate.

Assay of HBV DNA in the plasma of HBV-carrier chimpanzees superinfected with non-A, non-B hepatitis.

A dot hybridisation technique was used to monitor the levels of hepatitis B virus (HBV) DNA in the plasma of two HBV-carrier chimpanzees which had been inocula ed with documented infectious non-A, non-B hepatitis agents. A marked decrease in the quantity of HBV DNA in the plasma during the acute phase of the non-A, non-B hepatitis was observed in both carriers. The possible role of interferon or a similar antiviral agent in modulation of the HBV-carrier state is discussed. Hybridisation may become, in due course, the method of choice for examining blood samples for infectious hepatitis B virus.

Non-A, non-B hepatitis in persistent carriers of hepatitis B virus.

There are reports in the literature that infection with hepatitis A virus in hepatitis B carriers can result in resolution of the carrier state. In an attempt to induce clearance of the carrier state of hepatitis B virus in two persistently infected chimpanzees, the chimpanzees were infused with documented non-A, non-B infectious material. Biochemical and histopathological evidence of hepatitis was accompanied by the unique abnormalities of endoplasmic reticulum associated with non-A, non-B hepatitis in the chimpanzees. Elevation of alanine aminotransferase was accompanied by fourfold reduction in one chimpanzee and sixfold reduction in the other in the plasma levels of HBV-associated DNA polymerase activity and simultaneously by twofold reduction in the concentration of hepatitis B surface antigen in both chimpanzees. A mediator may account for these changes in markers of hepatitis B virus infection, and this mechanism may also explain the occurrence of spontaneous regression in some persistently infected carriers. The significance of transient red cell anaemia in non-A, non-B hepatitis, which was observed in one of the chimpanzees, is yet to be established.

A study of ultrastructural alterations in experimental non-A, non-B hepatitis by electron-beam analysis.

An electron-beam X-ray microanalysis was carried out on sections of liver biopsy specimens obtained from chimpanzees infected with non-A, non-B hepatitis. The microanalysis was concentrated over areas where typical derangement of the endoplasmic reticulum, with the formation of tubular forms possessing walls with electron-dense central membrane, was visualized. These tubular structures are regarded as the most notable pathological alteration in affected hepatocytes. However, the electron-probe microanalysis showed no deviation of the energy spectrum when compared with the background or control analysis.

Application of electron microscopy to the study of structural changes in the liver in non-A, non-B hepatitis.

Ultrastructural studies employing techniques such as alternative electron metal stain, high-angle tilting and high-voltage electron microscopy were carried out on liver biopsies obtained from chimpanzees infected with non-A, non-B hepatitis. Typical derangement of the endoplasmic reticulum leading to the formation of tubular structures in hepatocytes was observed. The use of potassium permanganate as an alternative stain revealed two features which have not been previously described. The first of these shows the wall of the tubular structures to be composed of a well-defined fibrillar-like meshwork with a periodicity of approximately 15 nm. The second feature is the demonstration of clusters of fibrin-like inclusions consisting of striated fibrils in the neighborhood of the tubular structures. The presence of intracytoplasmic fibrin may indicate non-specific morphological evidence of cell injury. Crystalline structures containing arrays of particles with an average size of 24 nm were also observed in the endoplasmic reticulum of endothelial cells of the hepatic sinusoids. Morphological differences between the crystalline lattice and the reticular arrangement, demonstrated with the use of high-angle tilting of the specimen in the electron microscopy suggest that the arrays may not be viral particules but a reflection of pathological response of the host cell.