Quantification of infectious duck hepatitis B virus by radioimmunofocus assay

A simple method is described for the precise quantification of infectious duck hepatitis B virus (DHBV) in cell culture, using a radioimmunofocus assay (RIFA). Primary duck hepatocyte cell cultures were infected with serial dilutions of viral samples as for a plaque assay, but then maintained with liquid overlay medium. After incubation for up to 14 days, cell monolayers were fixed with acetone, then stained with a monoclonal antibody to DHBV L protein followed by secondary antibody labelled with I. Foci of infection (representing individual infectious particles in the inoculum) were detected by autoradiography. The number of foci recovered was increased by addition of dimethyl sulphoxide to culture medium, but was not appreciably altered by the use of semi-solid medium. The titre of virus suspensions determined by RIFA correlated well with titration in ducklings. The RIFA is a useful method for titration of DHBV, as it has a wide dynamic range and is well suited to parallel titration of large numbers of samples. This assay will have wide use for the analysis of DHBV growth kinetics, antiviral efficacy, and virus inactivation procedures. J. Med. Virol. 52:354–361, 1997. © 1997 Wiley-Liss, Inc.     

Alteration of infection pattern of duck hepatitis B virus by immunomodulatory drugs

The relationship between host immune state and hepatic inflammation and infection pattern of the Duck hepatitis B virus (DHBV) was investigated by experimental transmission of DHBV into 98 Japanese 7-day-old ducklings that had been pretreated with immunoregulatory drugs including cyclophosphamide, OK 432, and a steroid hormone. Immunosuppressive treatment with cyclophosphamide revealed an extension of the viremic period associated with an absence of inflammatory changes in the liver. Although immunostimulating treatment with OK 432 showed a remarkable accumulation of inflammatory cells in the liver, the viremic period was not shortened. Treatment with a steroid used as a immunosuppressant did not suppress the hepatitis; moreover, it increased viral DNA replication and extended the viremic period. This phenomenon of viral replication seemed to be caused by the direct effects of the steroid. Alteration of DHBV infection by modifying the host immune state is quite similar to that of hepatitis B virus (HBV) in humans. In DHBV infection, the host immune state seemed to have a considerable role in determining the infection pattern and degree of hepatitis activity. DHBV may be a helpful model of HBV for studying host-viral interaction and the immunological mechanism of viral hepatitis.     

Comparative sequence analysis of duck and human hepatitis B virus genomes

We have cloned and sequenced an infectious, functionally active genome of a duck hepatitis B virus (DHBV). It is 3,021 base pairs (bp) in length and shows little DNA sequence homology to the genome of human hepatitis B virus (HBV). However, the amino acid sequences of predicted viral gene products are similar between DHBV and HBV, and the genome organization present in DHBV reflects that of HBV. As in the mammalian virus the long minus strand of the DHBV genome encodes three long overlapping reading frames designated as P, S, and C. The fourth open reading frame, termed X, is absent in DHBV. A comparison with a sequence of a second DHBV isolate [Mandart et al, Journal of Virology 49:782-792, 1984] revealed a nucleotide sequence variation of 5.6% and confirmed the presented overall gene organization of DHBV.     

Expression of biomarkers of interferon type I in patients suffering from chronic diseases

Interferons (IFNs) are used widely in the treatment of viral infections, tumours and neurological disorders. The aim of this study was to evaluate the endogenous expressions of various IFN-induced compounds [specifically: neopterin (NPT), beta2microglobulin (beta2mg) and 2-5 oligoadenylate synthetase (2-5 OAS)] in patients with various chronic diseases requiring treatment with IFN type I. The results showed that patients with such chronic diseases as hepatitis C virus-associated type II mixed cryoglobulinaemia (MC), chronic hepatitis C (CHC) and relapsing-remitting multiple sclerosis (RRMS) are characterized by different activations of the IFN system. Furthermore, the interindividual variability in baseline levels of IFN-induced biomarkers was higher in patients with chronic diseases than in healthy individuals. When levels of the above biomarkers were measured 24 h after the first injection of IFN in patients with CHC or RRMS, significant increases in expression levels of IFN-induced compounds were recorded but, again, there is a broad range of variability in the degree of increase. Further, a significant inverse correlation between baseline levels of NPT, beta2mg and 2-5 OAS activity and their relative increases after IFN administration was found in patients with CHC or RRMS. Together, the results are consistent with the observation that there is considerable interindividual heterogeneity in the clinical response to IFNs, which suggests that host factors other than disease markers must be taken into account in order to manage and optimize the IFN therapy.     

Induction of an ATP-polymerizing enzyme in TMV-infected tobacco and its homology to the human 2′–5′ a synthetase

Several reports have indicated that tobacco carries an enzyme (APE) that, in the presence of poly (rI):(rC), polymerizes ATP to oligoadenylates. This paper demonstrates that the tobacco APE system comprises several proteins (estimated sizes: 32, 42, 67, and 84±10% kD). Only one of these proteins (the 67-kD form) binds to poly (rI):(rC). This APE form has been purified by affinity chromatography on a synthetic ds-RNA column. Four tobacco proteins, including the purified one, crossreact with antibodies against the human enzyme, 2–5 A synthetase. The ATP-binding capacity of some of these proteins has also been demonstrated. The amount of plant oligoadenylates obtained by polymerizing ATP with the purified APE form allows, for the first time, their direct analysis by TLC. The TLC analysis indicated that the oligomer produced by APE is not identical to the 2–5 oligoadenylate.     

Interferon vs. adenine arabinoside 5′-monophosphate in patients with anti-HBe-positive chronic hepatitis

Anti-HBe-positive patients with precore mutants may have severe, progressive liver disease. Therapy with interferon has been effective, but relapses are frequent. To evaluate and compare two antiviral treatments, lymphoblastoid interferon (ly-IFN) and adenine arabinoside 5′-monophosphate (ARA-AMP), 20 patients with anti-HBe-positive chronic hepatitis (5 cirrhosis and 15 CAH) and viral replication (HBcAg in the liver and HBV DNA in serum) were treated. Patients were randomized into two groups: 11 patients received ARA-AMP, 5 mg/kg/day during 7 weeks, and 9 received human ly-IFN, 5,000,000 units, three times per week, during 4 months. Baseline clinical, biochemical and histological features were not significantly different between the two groups. At the end of therapy, 8 (89%) patients in the interferon group and 5 (45%) in the ARA-AMP group showed normal ALT levels and no HBV DNA in serum by a liquid hybridization assay (P < 0.05). At 1 year of follow-up, a persistent response was observed in 33% of ly-IFN patients and in 27% of ARA-AMP patients, a transient response in 56% and 18%, and nonresponse in 11% and 55%, respectively. HBV DNA remained detectable by polymerase chain reaction (PCR) in 19 of the 20 patients. Among the responders, an improvement in histological lesion and the disappearance of intrahepatic HBcAg were observed; in the nonresponders, histological lesion remained stable or worsened. In conclusion, the efficacy of interferon and ARA-AMP was similar in treating anti-HBe-positive chronic hepatitis. Although interferon treatment led to initial improvement in a larger number of patients, there was a much higher rate of relapses with this drug. © 1996 Wiley-Liss, Inc.     

A sequential study of viral DNA in serum in experimental transmission of duck hepatitis B virus

To understand the relationship among the time of infection, infection patterns, and liver diseases, experimental transmission of duck hepatitis B virus (DHBV) utilizing 165 Japanese white domestic ducklings was performed. Twenty to 25 ducklings were each inoculated with DHBV-positive serum intravenously at day one, 3, 5, 7, 10, and 14 posthatch and were sacrificed during the 1st, 2nd, 3rd, and 4th (and 24th in those inoculated on day one and day 3 posthatch) week after inoculation to obtain sera and the liver. The sera served for the measurement of DHBV DNA by spot hybridization test and DNA polymerase activity, and the liver was subjected to morphological examination including immunostaining for DHBV. The ducklings inoculated with DHBV on 1 day and 3 days posthatch always revealed persistent viremia, whereas those on and after 5 days posthatch showed persistent or transient viremia. The hepatitis activity in the liver was seen in ducklings inoculated with DHBV on and after 3 days posthatch and was very weak consistent with the diagnosis of mild acute hepatitis of humans. The serum transaminase activity was not significantly elevated at the time of occurrence of histological hepatitis activity. Since host immune mechanism establish at 3 to 5 days posthatch in birds, the host immune response seemed to determine whether DHBV infection was persistent or transient and the occurrence of hepatitis activity as seen in human hepatitis B virus (HBV) infection.     

Identification of in vivo interaction between Hepatitis C Virus core protein and 5′ and 3′ UTR RNA

Here, we investigated the ability of the Hepatitis C Virus (HCV) core protein to interact specifically with the 5′ and 3′ untranslated regions (UTRs) of HCV using an in vivo cell-based translation inhibition assay. HCV core protein interacts weakly but specifically with the SLIII stem loop in the 5′ UTR in which the SLIIIb subdomain is the major determinant and the SL2 loop in the X region of the 3′ UTR. These results revealed for the first time in vivo interaction of the core protein with 5′ and 3′ UTRs involved in the viral life cycle. This system provides a useful tool for further investigating interactions between the HCV core protein and 5′ and 3′ UTRs.     

Effects of 2'-fluorinated arabinosyl-pyrimidine nucleosides on duck hepatitis B virus DNA level in serum and liver of chronically infected ducks.

The 2'-fluorinated arabinosyl-pyrimidine nucleosides, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), are new antiviral compounds with in vitro inhibitory activity against the DNA polymerase of hepadnaviruses. Those compounds also induced permanent inhibition of viral replication in woodchucks chronically infected by woodchuck hepatitis virus. The effects of these antiviral compounds were assessed in ducks chronically infected by duck hepatitis B virus (DHBV). Following intraperitoneal administration for 5 days, FMAU (2 mg/kg/day) and FIAC (10 mg/kg/day) induced a transient decrease in DHBV replication, as shown by the decrease in both the serum and liver DHBV DNA level. After stopping therapy, DHBV replication rebounded immediately to the pretreatment level. The supercoiled form of liver viral DNA was found to be less affected by the therapy. By contrast, no obvious antiviral effect was observed with vidarabine monophosphate (ara-AMP) (80 mg/kg/day) therapy. No sign of toxicity was observed during the course of the treatment. These preliminary results confirmed in the DHBV model the higher efficacy of FIAC and FMAU as compared to ara-AMP. Pharmacokinetic studies are needed to explain the differences observed in viral replication in these 2 models of HBV infection.     

Evaluation of Phyllanthus amarus and Phyllanthus maderaspatensis as agents for postexposure prophylaxis in neonatal duck hepatitis B virus infection

The therapeutic potential of plant extracts of Phyllanthus amarus and Phyllanthus maderaspatensis for postexposure prophylaxis against infection by Hepadnaviruses was studied in ducklings infected by the duck hepatitis B virus (DHBV). Forty-four Pekin ducklings were inoculated intraperitoneally with DHBV at 24 hr posthatch. They were treated by intraperitoneal injection of Phyllanthus amarus (aqueous extract) (100 mg/kg body weight) or Phyllanthus mad eraspatensis (alcoholic extract) (100 mg/kg body weight) for a period of 4 weeks. Infected ducklings treated with saline served as controls. Weekly serum samples obtained before, during, and after treatment were analysed for the presence of DHBV DNA in serum by dot blot hybridisation using α ³²P-labelled probes. Liver tissue was collected after killing the ducks at various time intervals and was studied for replicative status of the viral DNA and liver histopathology; 17 of 21 ducks were viraemic on completion of treatment with Phyllanthus amarus. At 16 week posttreatment follow-up four of seven animals remained viraemic. Similar results were obtained with Phyllanthus maderaspatensis. There was no alteration in DHBV replication in the liver. No toxicity was observed with this treatment. These observations suggest that Phyllanthus amarus and Phyllanthus maderaspatensis are not useful as therapeutic agents for postexposure prophylaxis against DHBV infection. © 1993 Wiley-Liss, Inc.     

In vitro use of autologous dendritic cells improves detection of T cell responses to hepatitis B virus (HBV) antigens

T lymphocyte responses to hepatitis B virus (HBV) core antigen (HBcAg) are vigorous and easily detectable in vitro during recovery from acute hepatitis B but significantly weaker in patients with chronic HBV infection. In contrast, T cell responses to hepatitis B surface antigen (HBsAg) are almost undetectable during infection and even in a substantial fraction of subjects receiving vaccination with HBsAg. The aim of this study was to investigate whether the use of dendritic cells (DCs) in an in vitro assay could increase the detection of HBV‐specific T cells in these conditions. Autologous monocyte‐derived DCs, compared to direct HBsAg addition to the cultures, increased the stimulation of HBs‐ specific T cells. These were detected in 73% of healthy subjects who had recently received hepatitis B vaccine and in 43% of patients recovering from acute hepatitis B. Likewise, proliferation in response to DC‐presented HBcAg was detected in both CD4⁺ and CD8⁺ T cells from the majority of chronic hepatitis B patients. A longitudinal evaluation of HBc‐specific T cell responses during and after a 1‐year treatment with pegylated interferon (IFN)‐α showed that HBc‐specific CD4⁺ T cell responses had no correlation with sustained virus suppression whereas CD8⁺ T cell responses were more frequently detected in patients able to control HBV replication after therapy interruption. The use of autologous DCs as antigen‐presenting cells appears applicable to clinically relevant in vitro evaluation of T cell responses, particularly in those conditions characterized by low frequency of circulating antigen‐specific cells and suboptimal in vivo activation. J. Med. Virol. 81:332–339, 2009. © 2008 Wiley‐Liss, Inc.     

Amplification of GB virus-C/hepatitis G virus RNA with primers from different regions of the viral genome

GB virus-C/hepatitis G virus (GBV-C/HGV) is a newly identified RNA virus. The aim of the study was to compare three primer pairs from the 5′ untranslated region (5′UTR), envelope region 2 (E 2) and nonstructural region 3 (NS 3) of GBV-C/HGV genome for their ability to detect GBV-C/HGV RNA by polymerase chain reaction (PCR) assays. By using PCR with primers from different regions of the viral genome, serum GBV-C/HGV RNA was assayed in 200 at-risk individuals. The sensitivity of this assay was assessed by a titration experiment, and nucleotide sequences of the amplified products were determined directly. Of 200 serum samples, 43 (21.5%) were positive for GBV-C/HGV RNA with at least one of the primer pairs. The positive rates by 5′UTR, NS 3, and E 2 primers were 100%, 98%, and 84%, respectively, and the sensitivity of PCR assays using 5′UTR primers was 10 to 100 times more likely to detect GBV-C/HGV RNA than that of NS 3 and E 2 primers. The average homology of amplified targets to the prototype HGV genome was 89%, 80%, and 85% and the similarity between each amplified target was up to 100%, 90%, and 92% in the 5′UTR, E 2, and NS 3 regions, respectively. Therefore, the 5′UTR of GBV-C/HGV genome is highly conserved and primers deduced from this region can provide a sensitive and specific PCR assay for GBV-C/HGV RNA. J. Med. Virol. 51:284–289, 1997. © 1997 Wiley-Liss, Inc.     

Effect of Phyllanthus amarus on duck hepatitis B virus replication in vivo

Nine ducks congenitally infected with the duck hepatitis B virus (DHBV) were treated either orally (four ducks for 10 weeks) or intraperitoneally (five ducks for 12 weeks) with the Indian traditional herbal remedy Phyllanthus amarus. Compared to placebo-treated control ducks, these treatments did not result in a reduction of circulating viral DNA in the serum or in the level of viral DNA replication in the liver. In two of the five intraperitoneal-treated ducks, a reduction in the levels of duck hepatitis B surface antigenaemia (DHBsAg) was observed. The data strongly suggest that Phyllanthus amarus has no significant inhibitory effect on DHBV DNA replication and only a minor effect on DHBsAg production.     

Sequence and structural analysis of the 5′ noncoding region of hepatitis C virus in patients with chronic infection

Hepatitis C virus (HCV), exhibits considerable genetic diversity, but presents a relatively well conserved 5′ noncoding region (5′ NCR) among all genotypes. In this study, the structural features and translational efficiency of the HCV 5′ NCR sequences were analyzed using the programs RNAfold, RNAshapes and RNApdist and with a bicistronic dual luciferase expression system, respectively. RNA structure prediction software indicated that base substitutions will alter potentially the 5′ NCR structure. The heterogeneous sequence observed on 5′ NCR led to important changes in their translation efficiency in different cell culture lines. Interactions of the viral RNA with cellular transacting factors may vary according to the cell type and viral genome polymorphisms that may result in the translational efficiency observed. J. Med. Virol. 81:1212–1219, 2009. © 2009 Wiley‐Liss, Inc.     

Cyclic AMP-dependent and independent effects of prostaglandins on the contraction-relaxation cycle of spontaneously beating isolated rat atria

The effects of prostaglandin (PG) F_2α, E₂, E₁ and I₂ on the amplitude, duration of the contraction-relaxation cycle (CRC), the second derivative of developed tension and the cyclic adenosine-3′,5′-monophosphate (cAMP) level and on ⁴⁵Ca uptake were studied in isolated spontaneously beating rat atria. The order of capacity to generate positive inotropic effects was PGF_2α>PGE₂>PGE₁?PGI₂. Only PGI₂ and PGE₁ decreased the duration of CRC. PGF_2α produced an increase during the first 2.5min, whereafter the duration returned to the initial level. PGE₂ had no significant effect on the shape of the CRC. The ratios of the maximum and minimum of the second derivative of the developed tension were reduced by PGI₂ and PGF_2α 2.5 and 5 min after administration, respectively. The ⁴⁵Ca uptake was stimulated equally by all of the tested PGs, but only PGI₂ and PGE₁ could significantly increase the cAMP level. The results do not support the conception that cAMP could mediate the positive inotropic effect of PGs. Rather the contrary, cAMP, increased by PGE₁ or PGI₂, could be responsible for increased relaxation, which might prevent the full development of tension.     

Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays

Methods for identifying the genotype of hepatitis C virus (HCV) in clinical specimens are frequently based upon the direct characterisation of viral RNA sequences by polymerase chain reaction (PCR) amplification, or by serologically based methods, in which the infecting genotype is inferred from the pattern of antibody reactivity to type-specific peptides or recombinant proteins used as antigens in an Enzyme Linked Immunosorbent Assay (ELISA). Although genotyping by direct, PCR-based methods show generally highly concordant results with the genotype inferred from serological typing assays (>95% agreement), there exist a small number of samples that produce discrepant results. To investigate the underlying reasons for the discrepancies, we obtained eleven samples from haemophiliacs and four samples from patients with chronic hepatitis C that produced discordant results between a PCR based assay (InnoLipa I and II) and a serotyping assay (Murex HC02). Nucleotide sequences in the 5′noncoding region (5′NCR), core, and NS4 region were used to identify the genotype of the circulating virus and to identify amino acid changes in NS4 that might alter antigenicity. In 14 samples, sequence analysis of all three regions was concordant with the results of the InnoLipa assay. There were few if any amino acid substitutions in NS4 that might have accounted for the discrepant serotyping results, which were found predominantly in samples from individuals with a history of multiple exposure to HCV. It remains unclear whether the detection of antibody in such discrepant samples corresponds to previous expression of a different genotype than detected by PCR, or whether the virus population in plasma is more restricted in genotype diversity than the population in the liver or at other sites of viral replication. J. Med. Virol. 53:237–244, 1997. © 1997 Wiley-Liss, Inc.