Monoclonal antibodies to a 55-kilodalton protein present in duck liver inhibit infection of primary duck hepatocytes with duck hepatitis B virus

As an approach to identifying hepatocyte receptors for the avian hepadnavirus duck hepatitis B virus (DHBV), hybridomas were prepared from mice immunized with permissive duck hepatocytes. Monoclonal antibodies (MAbs) were screened for the ability to inhibit binding of DHBV particles to primary duck hepatocytes and to block infection. We identified two MAbs which partially blocked binding and caused marked inhibition of infection of primary duck hepatocytes with DHBV. Lack of cross-reactivity with DHBV envelope proteins suggested that inhibition of infection was due to specific interaction between the antibodies and a host cell surface molecule. Both MAbs immunoprecipitated a 55-kDa protein (p55) expressed in duck liver and several other duck tissues. p55 homologs were also identified in other birds and mammals. We predict from our data that only a small proportion of total cellular p55 molecules are expressed at the surfaces of hepatocytes and that p55 is involved in some early step in the infectious pathway.     

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One hundred and two apparently healthy Indian domestic ducks from the Poultry Research Station, Madras were screened for duck hepatitis B virus (DHBV) infection by; 1. screening for the duck hepatitis B virus surface antigen (DHBsAg) in their sera using hepatitis B virus (HBV) reagents, 2. screening for DHBsAg using specific duck hepatitis B virus (DHBV) reagents and 3. demonstration of DHBV DNA using DHBV DNA probe by dot blot hybridisation. While 5 ducks (4.9%) were consistently positive with HBV reagents, use of DHBV reagents showed a total of 4 ducks (including 3 of the above 5) to be positive for DHBsAg. DNA hybridisation showed 6 ducks to be positive for DHBV DNA. On clinical examination, 5 out of these 6 ducks did not reveal abnormalities, the other one showed hepatomegaly and ascites. Post-mortem studies showed the presence of nodules on the surface of the liver in all 5 which were positive with HBV reagents including the one with hepatomegaly. On histopathological evaluation, they were found to be hepatocellular carcinoma with or without bile duct carcinoma. The present study is a pilot report on the occurrence of DHBV infection in Indian domestic ducks and the possibility of antigenic cross reactivity between human HBV and duck hepatitis B virus antigens.     

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) was evaluated for its inhibitory effect on hepadnavirus replication in three different cell systems, i.e., human hepatoma cell lines HepG2 2.2.15 and HB611 (transfected with human hepatitis B virus (HBV)) and primary cultures of duck hepatocytes infected with duck hepatitis B virus (DHBV). PMEA inhibited HBV release from HepG2 2.2.15 cells and HB611 cells at a 50% inhibitory concentration (IC50) of 0.7 and 1.2 microM, respectively. Intracellular viral DNA synthesis was inhibited at concentrations equivalent to those required to inhibit virus release from the cells. DHBV secretion from duck hepatocytes was inhibited by PMEA at an IC50 of 0.2 microM. HBsAg secretion was inhibited by PMEA in a concentration-dependent manner in HB611 cells and DHBV-infected duck hepatocytes but not HepG2 2.2.15 cells. The 50% cytotoxic concentration, as measured by inhibition of [3H-methyl]deoxythymidine incorporation was 150 microM for the two human hepatoma cell lines and 40 microM for the duck hepatocyte cultures. In a pilot experiment PMEA was found to reduce the amounts of DHBV DNA in the serum of Pekin ducks.     

Biodistribution, stability, and antiviral efficacy of liposome-entrapped phosphorothioate antisense oligodeoxynucleotides in ducks for the treatment of chronic duck hepatitis B virus infection

This study investigated the feasibility of using liposomes to increase the hepatic delivery and antiviral efficacy of phosphorothioate antisense oligodeoxynucleotides (PS-ODN) for the in vivo treatment of hepatitis B virus (HBV) infection. Ducks infected with duck hepatitis B virus (DHBV) were used as the model. We studied the stability of an antisense PS-ODN in duck plasma, its integrity during the process of liposome entrapment, its in vivo biodistribution, plasma clearance, and excretion. In addition, the intrahepatic distribution of a labeled free and liposome-entrapped ODN was also investigated. The results of our studies show that: 1) phosphorothioate ODN remain stable during the process of liposome entrapment; 2) are stable in duck plasma for many hours; 3) are rapidly cleared from the plasma when injected intravenously; 4) intravenous injection of antisense ODNs entrapped within liposomes enhances delivery of the ODN to the liver; and 5) inhibit DHBV replication. Serum DHBV DNA levels fell rapidly, with a corresponding decrease in intrahepatic viral replicative intermediates at the end of the 5-day study period. Although inhibition of viral replication and a fall in the target protein was observed, a marked inhibition of viral replication was also observed with high doses of a random-sequence ODN. Thus, it is not certain that inhibition of viral replication was entirely through an antisense mechanism. Therefore, liposomes may be effective vehicles to improve the delivery of antisense oligonucleotides to the liver for the therapy of hepatotropic viruses.     

Long-term therapy with the guanine nucleoside analog penciclovir controls chronic duck hepatitis B virus infection in vivo

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the antiviral guanine nucleoside analog penciclovir for 12 or 24 weeks at a dosage of 10 mg/kg of body weight per day. By the completion of both 12 and 24 weeks of therapy, molecular hybridization studies of the liver tissue revealed that the viral DNA, RNA, and protein levels were significantly reduced compared to those in the placebo-treated controls. Penciclovir treatment for 12 or 24 weeks was not associated with any toxicity, establishing the efficacy and safety of long-term penciclovir therapy in chronic DHBV infection.     

In vivo selection of duck hepatitis B virus pre-S variants which escape from neutralization

To better understand the role of specific residues within the duck hepatitis B virus (DHBV) pre-S protein in neutralization and infectivity, we have selected and identified pre-S variants which escape neutralization. A highly neutralizing monoclonal antibody (Mab 900) which recognizes an epitope 83IPQPQWTP90 localized previously on the DHBV pre-S protein, within a region suspected to mediate the virus interaction with hepatocytes, was used as immune pressure. After only two in vivo neutralization rounds with Mab 900, five different pre-S mutant genomes were identified, which harbored point mutations affecting only proline residues located at position 90 within this epitope (83IPQPQWTP90) and/or at a distance at position 5. We have shown that a single (P5L) or double proline (P5L + P90H) substitution affect neither virus replication capacity nor in vivo infectivity. However, the P5 mutation reduces mutant recognition by Mab 900 twofold, while the substitution of both prolines 5 and 90 almost completely abolishes mutant P5L + P90H reactivity with this Mab and leads to a decrease of neutralization. Therefore we describe here an experimental system which allows rapid in vivo selection and identification of DHBV pre-S variants and provide evidence that residues within and at a distance from the neutralization epitope are important in DHBV neutralization but do not affect its replication capacity and infectivity.     

Duck hepatitis B virus inactivation and 8-methoxypsoralen photoadduct formation in human platelet concentrates

Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA). To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus. A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5-6 log10 virus kill. The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium. Optimum conditions for PCI were 100 micrograms/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium. A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions. Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding. The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands. Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone. A minor subpopulation of DHBV was found to be somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.     

Dual topology of the large envelope protein of duck hepatitis B virus: determinants preventing pre-S translocation and glycosylation

The biosynthesis and topology of the large envelope protein (L protein) of hepadnaviruses was investigated using the duck hepatitis B virus (DHBV) model, which also allows the study of hepadnavirus morphogenesis in experimentally infected hepatocytes. Results from proteolysis of virus particles and from the analysis of topology and posttranslational modification of L chains synthesized in vivo or in a cell-free system both support the presence of a mixed population of L-protein molecules with their N-terminal pre-S domain located either inside or outside the virus particle. During L biosynthesis and DHBV morphogenesis, pre-S, together with the neighboring transmembrane domain (TM-I), initially remained cytoplasmically disposed and was translocated only posttranslationally. Delayed pre-S translocation into a post-endoplasmic reticulum compartment is also indicated by the absence of glycosylation at a modification-competent pre-S glycosylation site. Major features of L-protein biosynthesis and of the resulting dual topology appear to be conserved between avian and mammalian hepadnaviruses, supporting the model that pre-S domains function in part either as an internal matrix for capsid envelopment or externally as a ligand for cellular receptor binding. However, differences in the mechanisms controlling pre-S translocation were revealed by the results of mutational analyses identifying and characterizing cis-acting determinants in pre-S that delay its cotranslational translocation. Our data from DHBV demonstrate the negative influence of a cluster of positively charged amino acid residues next to TM-I, a motif that is conserved among the avian but absent from mammalian hepadnaviruses. Additional control elements, which are apparently shared between both virus groups and which may serve in chaperone binding, were mapped by deletion analysis in the central part of pre-S.     

Host cell-virus cross talk: phosphorylation of a hepatitis B virus envelope protein mediates intracellular signaling

Phosphorylation of cytosolic pre-S domains of the duck hepatitis B virus (DHBV) large envelope protein (L) was identified as a regulatory modification involved in intracellular signaling. By using biochemical and mass spectrometric analyses of phosphopeptides obtained from metabolically radiolabeled L protein, a single phosphorylation site was identified at serine 118 as part of a PX(S/T)P motif, which is strongly preferred by ERK-type mitogen-activated protein kinases (MAP kinases). ERK2 specifically phosphorylated L at serine 118 in vitro, and L phosphorylation was inhibited by a coexpressed MAP kinase-specific phosphatase. Furthermore, L phosphorylation and ERK activation were shown to be induced in parallel by various stimuli. Functional analysis with transfected cells showed that DHBV L possesses the ability to activate gene expression in trans and, by using mutations eliminating (S-->A) or mimicking (S-->D) serine phosphorylation, that this function correlates with L phosphorylation. These mutations had, however, no major effects on virus production in cell culture and in vivo, indicating that L phosphorylation and transactivation are not essential for hepadnavirus replication and morphogenesis. Together, these data suggest a role of the L protein in intracellular host-virus cross talk by varying the levels of pre-S phosphorylation in response to the state of the cell.     

Kinetics of duck hepatitis B virus infection following low dose virus inoculation: one virus DNA genome is infectious in neonatal ducks

Using pooled serum from congenitally duck hepatitis B virus (DHBV)-infected ducks as inoculum, we examined the effect of virus dose on the incubation period of infection and on the patterns of spread of virus infection in the liver. The pooled serum inoculum contained 9.5 x 10(9) DHBV genomes per milliliter and had an infectivity titre (ID50) in newly hatched ducks of 1.5 x 10(10) per milliliter with a 95% confidence interval of 3.0 x 10(9) to 6.3 x 10(10) ID50/ml, indicating the equivalence between one DHBV genome and one infectious unit within the limits of the assays. The incubation period of infection was inversely related to the dose of inoculum and the onset of viraemia ranged from Day 6 with the highest dose to Day 14 or 29 with the lowest dose inoculum. To study the spread of virus infection from a low percentage of initially infected cells we inoculated newly hatched ducks intravenously with sufficient DHBV (1.5 x 10(3) ID50) to infect only approximately 0.0001% of total liver cells. DHBV infection first reached detectable levels on Day 4 postinoculation (p.i.) and was detected in approximately 0.035% of hepatocytes, most of which occurred as single cells or pairs of cells, indicating that a number of rounds of infection had occurred with the spread of virus both to adjoining cells, i.e., by cell-to-cell spread, and to cells located in other parts of the liver lobule. Despite some bird-to-bird variation in timing, the percentage of infected hepatocytes increased exponentially with a mean doubling time of 16 hr from Day 4 to Day 14 p.i., by which time replication was seen in > 95% of hepatocytes. This rapid dissemination from a small number of infected hepatocytes suggests that, in neonatal ducks, there are no major delays in virus replication within the liver, that any innate and adaptive defence mechanisms operating during the first 10 to 14 days of infection are insufficient to contain virus spread, and that even a small number of infected hepatocytes produce enough progeny to rapidly infect the remaining hepatocytes.     

The GDPAL region of the pre-S1 envelope protein is important for morphogenesis of woodchuck hepatitis virus

The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein.     

Are there alternative avian influenza viruses for generation of stable attenuated avian-human influenza A reassortant viruses?

The present study evaluated gull influenza A viruses as donors of attenuating genes for the production of live, attenuated influenza A H1N1 and H3N2 avian-human (ah) reassortant viruses for use as vaccines to prevent disease due to influenza A viruses in humans. The previously evaluated duck influenza A viruses were abandoned as donors of attenuating avian influenza virus genes because clinical evaluation of H1N1 and H3N2 ah reassortant virus vaccines derived from duck viruses documented residual virulence of H1N1 reassortants for seronegative infants and young children. Gull influenza A viruses occupy an independent ecologic niche and are rarely isolated from species other than gulls. The possibility of using gull influenza A viruses as donors of internal gene segments in ah reassortant viruses was evaluated in the present study using three different gull viruses and three human influenza A viruses. Gull-human H3N2 reassortant influenza A viruses with the desired 6-2 genotype (six internal avian influenza virus genes and the two human influenza virus surface glycoprotein genes) were readily generated and were found to be attenuated for squirrel monkeys and chimpanzees. However, ah reassortant viruses with gull and human influenza A H1N1 genes were difficult to generate, and reassortants that had the desired genotype of six gull virus genes with human influenza A H1 and N1 genes were not isolated despite repeated attempts. The gull PB2, NP and NS genes were not present in any of the gull-human H1N1 reassortants generated. The under-representation of these three gene segments suggests that reassortants bearing one or more of these three gene segments might have reduced viability indicative of a functional incompatibility in their gene products. The difficulties encountered in the generation of a 6-2 gull-human H1N1 reassortant virus are sufficient to conclude that the gull influenza A viruses tested would not be useful as donors of sets of six internal genes to attenuate human influenza A viruses. This study also identifies influenza virus gene segments that appear to be incompatible for generation of reassortants. Elucidation of the molecular basis of this restriction may provide information on intergenic interactions involved in virion assembly or packaging.     

Primary structure and strand specificity of BstF5I-1 DNA methyltransferase which recognizes 5''-GGATG-3''

1. Ovomucoids were purified from Muscovy duck, domestic duck and domestic goose. 2. Peptide maps of cyanogen bromide-cleaved ovomucoids from Muscovy duck and domestic duck were very similar to one another, but differed from that of goose. 3. Muscovy duck ovomucoid showed the same protease inhibitory pattern as ovomucoid from domestic duck, inhibiting trypsin in the molar ratio of 1:2 and chymotrypsin 1:1. 4. Inhibitory complexes could be detected between chymotrypsin and ovomucoid from both Muscovy and domestic duck, but not from goose, by using non-denaturing gels. 5. No complexes could be detected between DFP-inactivated chymotrypsin and any of the ovomucoids. 6. The results show that of ovomucoid from Muscovy duck more closely resembles that from domestic duck than goose.     

Photochemical inactivation of duck hepatitis B virus in human platelet concentrates: a model of surrogate human hepatitis B virus infectivity

BACKGROUND: Photochemical decontamination of platelet concentrates (PCs) has been demonstrated by the use of 8-methoxypsoralen and ultraviolet A light. Systems for studying the inactivation of blood-borne viruses facilitate the evaluation of photochemical decontamination protocols. STUDY DESIGN AND METHODS: Duck hepatitis B virus (HBV), a model for human HBV, was adapted for the study of hepadnavirus inactivation. A highly specific in vitro infectivity assay used primary duck hepatocyte cultures and was followed by the detection of replicated duck HBV sequences. RESULTS: Duck HBV-infected primary duck hepatocyte cultures produced authentic infectious virus. High-titer (> 10(9) virus genome equivalents/mL) duck HBV-infected sera were completely inactivated in serum or PCs by the use of 100 micrograms per mL of 8-methoxypsoralen and 70 J per cm2 of ultraviolet A light. Intracellular duck HBV (> 4.2 log10) in PCs was also inactivated. Culture results were confirmed by a sensitive duckling infectivity assay that indicated that 6.3 log10 of infectious duck HBV had been inactivated by photochemical decontamination. CONCLUSION: The sensitivity of the culture assay was comparable to that of the duckling assay using polymerase chain reaction gene amplification to detect duck HBV. Duck HBV inactivation in PCs was dependent on the dose of ultraviolet A light and independent of 8-methoxypsoralen concentrations of 100 to 300 micrograms per mL: 100 micrograms per mL 8-methoxypsoralen inactivated 4 to 5 log10 of virus in conjunction with 20 to 40 J per cm2 of ultraviolet A light. The polymerase chain reaction-enhanced duck HBV culture system has utility in optimizing photochemical decontamination protocols.