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Phosphodiesterase 12 (PDE12) is a negative regulator of the type 1 interferon (IFN) response and here we show that PDE12 inhibitors (lead compounds 63 and 17) are associated with increased RNAseL activity, are well tolerated at the therapeutic range and inhibit, both in vitro and in vivo, the replication of several RNA viruses including hepatitis C virus (HCV), dengue virus (DENV), West Nile Virus (WNV) and SARS-CoV-2.

GB virus B (GBV‐B) is a New World monkey‐associated flavivirus used to model acute hepatitis C virus (HCV) infection. Critical for evaluation of anti‐viral or vaccine approaches is an understanding of the effect of HCV on the liver at different stages of infection. In the absence of longitudinal human tissue samples at defined time points, we have characterised changes in tamarins. As early as two weeks post infection histological changes were noticeable, and these were established in all animals by six weeks. Despite high levels of liver‐associated viral RNA there was reversal of hepatic damage on clearance of peripheral virus though fibrosis was demonstrated in four tamarins. Notably, viral RNA burden in the liver dropped to near undetectable or background levels in all animals which underwent a second viral challenge, highlighting the efficacy of the immune response in removing foci of replication in the liver. These data add to the knowledge of GBV‐B infection in New World primates which can offer attractive systems for the testing of prophylactic and therapeutic treatments and the evaluation of their utility in preventing or reversing liver pathology.

BackgroundThe SARS-CoV-2 virus causes a wide spectrum of disease severity. Initial manifestations include fever, dry cough, and constitutional symptoms, which may progress to respiratory disease. There may also be neurological and psychiatric manifestations, involving both the central and peripheral nervous system.MethodsWe performed a literature search of the databases PubMed, EMBASE, The Cochrane Library and Web of Science for observational studies reporting neurological, psychiatric, and neuropsychiatric effects of COVID-19. This was followed by a narrative synthesis to summarise the data and discuss neuropsychiatric associations, symptom severity, management, and recovery.FindingsThe most frequently reported neurological symptoms were ageusia, hyposmia/anosmia, dizziness, headache, and loss of consciousness. Statistically significant relationships were noted between Asian ethnicity and peripheral neuropathy (p=0.0001) and neuro-syndromic symptoms (p=0.001). ITU admission was found to have a statistically significant relationship with male sex (p=0.024). Depression and anxiety were also identified both during and after infection. The most frequent treatments used were intravenous immunoglobulins, followed by antibiotics, antivirals, and hydroxychloroquine; with mean treatment duration of 6 days.InterpretationVarious neuropsychiatric symptoms have been associated with COVID-19 infection. More studies are required to further our knowledge in the management of neurological and psychiatric symptoms during and after COVID-19 infection

Hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae, causing acute and chronic liver infections, which are responsible for considerable morbidity and mortality worldwide. The chronic disease burden is currently estimated at 291 million, down on estimates from 2 to 3 decades ago as a result of effective immunization campaigns against the virus. Chronically infected individuals run the risk of developing cirrhosis and hepatocellular carcinoma in later life. Antiviral treatment with interferon or nucleos(t)ide analogs offers the only means of interrupting this process. This article reviews the current level of knowledge relating to this important human pathogen and provides the latest updates on the taxonomy, structure, life cycle, replication and molecular biology of the virus, as well as current preventative approaches and therapeutic interventions.

Coronavirus disease 2019 (COVID-19) has significantly affected the well-being of individuals worldwide. We herein describe the epidemiology of COVID-19 in the Republic of Cyprus during the first epidemic wave (9 March&ndash, 3 May 2020). We analyzed surveillance data from laboratory-confirmed cases, including targeted testing and population screening. Statistical analyses included logistic regression. During the surveillance period, 64,136 tests (7322.3 per 100,000) were performed, 873 COVID-19 cases were diagnosed, and 20 deaths were reported (2.3%). Health-care workers (HCWs) represented 21.4% of cases. Overall, 19.1% of cases received hospital care and 3.7% required admission to Intensive Care Units. Male sex (adjusted Odds Ratio (aOR): 3.04, 95% Confidence Interval (CI): 1.97&ndash, 4.69), increasing age (aOR: 1.56, 95%CI: 1.36&ndash, 1.79), symptoms at diagnosis (aOR: 6.05, 95%CI: 3.18&ndash, 11.50), and underlying health conditions (aOR: 2.08, 95%CI: 1.31&ndash, 3.31) were associated with hospitalization. For recovered cases, the median time from first to last second negative test was 21 days. Overall, 119 primary cases reported 616 close contacts, yielding a pooled secondary attack rate of 12% (95%CI: 9.6&ndash, 14.8%). Three population-based screening projects, and two projects targeting employees and HCWs, involving 25,496 people, revealed 60 positive individuals (0.2%). Early implementation of interventions with targeted and expanded testing facilitated prompt outbreak control on the island.

The currently recommended treatment for chronic hepatitis B virus (HBV) infection achieves only viral suppression whilst on therapy, but rarely hepatitis B surface antigen (HBsAg) loss. The ultimate therapeutic endpoint is the combination of HBsAg loss, inhibition of new hepatocyte infection, elimination of the covalently closed circular DNA (cccDNA) pool, and restoration of immune function in order to achieve virus control. This review concentrates on new antiviral drugs that target different stages of the HBV life cycle (direct acting antivirals) and others that enhance both innate and adaptive immunity against HBV (immunotherapy). Drugs that block HBV hepatocyte entry, compounds that silence or deplete the cccDNA pool, others that affect core assembly, agents that degrade RNase-H, interfering RNA molecules, and nucleic acid polymers are likely interventions in the viral life cycle. In the immunotherapy category, molecules that activate the innate immune response such as Toll-like-receptors, Retinoic acid Inducible Gene-1 (RIG-1) and stimulator of interferon genes (STING) agonists or checkpoint inhibitors, and modulation of the adaptive immunity by therapeutic vaccines, vector-based vaccines, or adoptive transfer of genetically-engineered T cells aim towards the restoration of T cell function. Future therapeutic trends would likely be a combination of one or more of the aforementioned drugs that target the viral life cycle and at least one immunomodulator.

During the first quarter of 2020 the world is experiencing a pandemic of Severe Acute Respiratory Syndrome‑Coronavirus‑2 (SARS‑CoV‑2), a novel beta coronavirus that is responsible for the 2019 novel coronavirus disease (COVID‐19). The COVID‐19 pandemic revealed that healthcare systems around the world were not prepared to deal with either the direct effects of the pandemic or with the indirect effects that are imposed on the health of patients with chronic disorders such as cancer patients. Some challenges and dilemmas currently faced during the pandemic include the management of cancer patients during the treatment and follow‐up phases, the assessment of the safety of treatments currently used for the management of SARS‑CoV‑2 for use in cancer patients, the development of psychoeducation and emotional support for cancer patients and the safe conduct of clinical trials involving participation of cancer patients. Evidence from the literature supports the need for the urgent development of a holistic contingency plan which will include clear guidelines for the protection and comprehensive care of cancer patients. The implementation of such a plan is expected to have many beneficial effects by mainly minimizing the increased morbidity and mortality of cancer patients that could result as an adverse consequence of the COVID‐19 or future pandemics., The COVID‐19 pandemic has revealed that health care systems around the world were not prepared to deal with either the direct effects of the pandemic or with the indirect effects that are imposed on the health of cancer patients. The evidence from the literature supports that the development of a contingency plan is as urgent as ever to minimise the increased morbidity and mortality of cancer patients that could result as a consequence of the current COVID‐19 pandemic or any other future pandemics.

Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.

Chronic hepatitis B virus (HBV) infection may progress to cirrhosis, hepatocellular carcinoma (HCC) and end-stage liver failure with time. Interruption of this process can only be achieved through effective antiviral treatment. This approach has so far involved the use of immunomodulators such as pegylated interferon alpha (Peg-IFNα) for a finite period of up to a year and nucleos-(t)ide analogues (NUCs) for treatment over much longer periods of time. The latter act by suppressing HBV replication at the level of DNA synthesis by inhibiting the viral reverse transcriptase/DNA polymerase and causing premature termination of DNA synthesis. The ideal treatment end point is loss of HBsAg in both HBeAg+ve and HBeAg-ve patients following monotherapy. This, however, is only achievable in a minority of patients. Secondary outcomes are durable HBeAg loss and seroconversion to anti-HBe, which occur in about 18-30% of HBeAg+ve patients depending on the antiviral used, and sustained suppression of HBV-DNA accompanied by biochemical normalization and histological improvement in non-HBeAg+ve seroconverting and HBeAg-ve patients. There is therefore a need for additional direct-acting antivirals (DAAs) targeting different stages of the life cycle of the virus, as well as immunotherapeutic approaches. Such developments may pave the way for their use either alone or more likely in combination in the fight against chronic HBV infection. Such drugs or approaches, which are currently undergoing preclinical or clinical testing, are the subject of this review.

Translation initiation of the Hepatitis C virus (HCV) genome is driven by an internal ribosome entry site (IRES), located within the 5' non-coding region. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. However, the role of the viral proteins on HCV translation remains controversial. In this report, we confirmed previous studies showing that NS5A down-regulates IRES activity in HepG2 but not in Huh7 cells suggesting that the NS5A effect on HCV IRES is cell-type dependent. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. Furthermore, we present data indicating that the NS5A-mediated inhibitory effect on IRES-dependent translation could be linked with the PKR inactivation. Finally, we show that NS5A from GBV-C but not from GBV-B down-regulates HCV IRES activity in the absence or the presence of PKR over expression. Notably, HCV and GBV-C but not GBV-B NS5A contains a previously identified PKR interacting protein domain.

In chronic hepatitis B virus (HBV) infection, variants with mutations in the basal core promoter (BCP) and precore region predominate and associate with more severe disease forms. Studies on their effect on viral replication remain controversial. Increasing evidence shows that epigenetic modifications of cccDNA regulate HBV replication and disease outcome. Here we determined the transcription and viral replication efficiency of well-defined BCP and precore mutations and their effect on cccDNA epigenetic control. HBV monomers bearing BCP mutations A1762T/G1764A and A1762T/G1764A/C1766T, and precore mutations G1896A, G1899A and G1896A/G1899A, were transfected into HepG2 cells using a plasmid-free approach. Viral RNA transcripts were detected by Northern blot hybridization and RT PCR, DNA replicative intermediates by Southern blotting and RT PCR, and viral release was measured by ELISA. Acetylation of cccDNA-bound histones was assessed by Chromatin ImmunoPrecipitation (ChIP) assay and methylation of cccDNA by bisulfite sequencing. BCP mutations resulted in low viral release, mRNA transcription and pgRNA/cccDNA ratios that paralleled the acetylation of cccDNA-bound H4 histone and inversely correlated with the HDAC1 recruitment onto cccDNA. Independently of the mutations, cccDNA was a target for methylation, accompanied by the upregulation of DNMT1 expression and DNMT1 recruitment onto cccDNA. Our results suggest that BCP mutations decrease viral replication capacity possibly by modulating the acetylation and deacetylation of cccDNA-bound histones while precore mutations do not have a significant effect on viral replication. These data provide evidence that epigenetic factors contribute to the regulation of HBV viral replication.

Epigenetic modifications are stable alterations in gene expression that do not involve mutations of the genetic sequence itself. It has become increasingly clear that epigenetic factors contribute to the outcome of chronic hepatitis B virus (HBV) infection by affecting cellular and virion gene expression, viral replication and the development of hepatocellular carcinoma. HBV persists in the nucleus of infected hepatocytes as a stable non-integrated covalently closed circular DNA (cccDNA) which functions as a minichromosome. There are two major forms of HBV epigenetic regulation: posttranslational modification of histone proteins associated with the cccDNA minichromosome and DNA methylation of viral and host genomes. This review explores how HBV can interphase with host epigenetic regulation in order to evade host defences and to promote its own survival and persistence. We focus on the effect of cccDNA bound-histone modifications and the methylation status of HBV DNA in regulating viral replication. Investigation of HBV epigenetic control has important clinical correlates with regards to the development of potential therapeutic regimens that will successfully eradicate HBV infection and deal with HBV reactivation in those undergoing treatment with demethylating agents.

Background: hepatitis B virus (HBV) is accounting for over one million deaths annually due to immune-mediated chronic liver damage. Natural killer (NK) cells are abundant in the liver and contribute in HBV persistence. NK cytotoxic effects are controlled by signals from activating and inhibitory receptors. HBV may circumvent host antiviral immunity via the regulation of NK receptors and their ligands. We investigated the effect of viral replication and HBeAg mutations on NK mediators expression in the livers of chronic HBV (CHB) patients and in cell cultures.Methods: HBV monomers bearing hotspot mutations in the basal core promoter and precore region were transfected into HepG2 cells using a plasmid-free assay. Serum viremia and liver HBV RNA were measured in 19 CHB patients. The expression of HBV RNA and of NKG2D ligands, B7H6, DNAX accessory molecule-1, lectin-like transcript 1 (LLT1), LFA-1 and TRAIL was measured in the livers of CHB patients and transfected cells.Results: in general, high HBV replication in CHB patients and cell lines upregulated the mRNA of all NK cell ligands and particularly the inhibitory NK cell ligand, LLT1. The exception was the NKG2D ligand, MICA, that was significantly decreased in patients with high serum viremia and intrahepatic HBV RNA levels.Conclusions: HBV replication has differential effects on NK cell ligands suggesting a potential escape mechanisms through up-regulation of LLT1 and down-regulation of MICA. A general trend towards upregulating NK cell ligands can be counteracted by decreasing MICA and hence weakening NK surveillance

Hepatitis A virus (HAV) and hepatitis E virus (HEV) are enteric hepatotropic viruses and their prevalence is related to the sanitary conditions of the region under investigation. There are only a few studies on the seroepidemiology of these two viruses in the general Iranian population. The purpose of this investigation was to measure the prevalence of hepatitis A and E infections in the general population. Between 2006 and 2007, a cross sectional study was performed in Tehran, Iran. Blood specimens were collected and questionnaires were filled in for 551 persons. Patient sera were tested by ELISA for anti-HEV and anti-HAV IgGs. The χ(2) test and independent t-test were used for statistical analysis and p0.05 was considered significant. The overall seroprevalence rates of anti-HEV IgG and anti-HAV IgG were 9.3% and 90%, respectively. The prevalence of antibodies to HAV and HEV was greater among men than women and increased with age. However, there was no significant relationship between age and gender with the existence of anti- HAV and HEV IgG antibodies. Our results show the seroprevalence of HAV and HEV antibodies are high and both viruses are endemic in this region. These findings are in accordance with results obtained from previous studies. We recommend that foreign travelers to Iran are vaccinated against HAV.

Background & Aims Translation of HBsAg depends on transcription of the appropriate mRNAs from cccDNA, but its relation to other hepatitis B virus (HBV) replication parameters is not known, inasmuch as integrated sequences of HBV-DNA may also contribute to its serum levels, especially in HBeAg-negative chronic hepatitis B (CHB) patients. Methods We investigated HBsAg serum levels, its hepatocellular expression, and their relationship to HBV replicative- and host-response parameters before treatment in 54 HBeAg-negative CHB patients and in 15 of them after 40.1±33.3months of virological response on oral antiviral (NUC) therapy also. Liver cccDNA and HBV-DNA quantitation, HBsAg- and HBcAg-immunostaining were performed in the same needle biopsy material, while serum HBsAg and HBV-DNA levels were measured in samples drawn on the day of liver biopsy. Results In untreated patients, serum HBsAg correlated positively with HBsAg-positive hepatocytes/mm 2 ( p =0.003) and weakly with serum HBV-DNA, but not with cccDNA, liver HBV-DNA, HBcAg-positive hepatocytes/mm 2 , or ALT. cccDNA correlated significantly with liver HBV-DNA ( p 0.00001), ALT ( p =0.001), and serum HBV-DNA levels ( p =0.012) but not with liver HBsAg or HBcAg. Antiviral therapy decreased serum HBsAg levels by 79.6% ( p =0.012) and liver HBV-DNA by 84.4% ( p =0.026) in paired comparisons and, as expected, significantly decreased serum HBV-DNA and ALT levels, but not cccDNA. Conclusions In untreated HBeAg-negative CHB, serum HBsAg levels reflect liver HBsAg, but not cccDNA or liver HBV-DNA, suggesting that they are not solely dependent on the replicative cycle of HBV. Effective NUC therapy for 3.34years significantly lowers serum HBsAg and liver HBV-DNA, but not cccDNA.

Introduction. Acute hepatitis C virus (HCV) infection is rarely studied, but virus sequence evolution and host-virus dynamics during this early stage may influence the outcome of infection. Hypervariable region 1 (HRV1) is genetically diverse and under selective pressure from the host immune response. We analyzed HRV1 evolution by frequent sampling of an acutely infected HCV cohort.Methods. Three or more pretreatment samples were obtained from each of 10 acutely infected subjects. Polymerase chain reaction amplification was performed with multiple primer combinations to identify the full range of sequences present. Positive samples were cloned and sequenced. Phylogenetic analyses were used to assess viral diversity.Results. Eight of the 10 subjects were coinfected with at least 2 HCV subtypes. Multiple subtypes were detected in individual samples, and their relative proportions changed through actute infection. The subjects with the most complex subtype structure also had a dynamic viral load; however, changes in viral load were not directly linked to changes in subtype.Conclusions. This well-sampled cohort with acute HCV infection was characterized by dynamic coinfection with multiple viral subtypes, representing a highly complex virologic landscape extremely early in infection.

OBJECTIVE: An epidemic of acute hepatitis C virus (HCV) infection in HIV-positive men-who-have-sex-with-men (MSM) is emerging in Europe, Australia and the USA. The aim of this study was to characterise the natural history of primary HCV in this setting and to assess host and viral factors which predict spontaneous clearance. METHODS: This prospective longitudinal cohort study was carried out in 112 HIV-positive patients who were followed in a single centre (the St Mary's Acute HCV Cohort). Plasma and peripheral blood mononuclear cells (PBMCs) were obtained at monthly intervals for 3 months and at 3-monthly intervals thereafter for a median of 45 months (IQR = 29-69 months). The primary end point was spontaneous clearance of HCV. Cox regression was used to assess the impact of clinical and virological variables on outcome, including liver function, CD4 count, rate of HCV RNA decline, T cell response and clonal sequence evolution within the HCV E2 envelope gene. RESULTS: 15% of patients cleared HCV spontaneously, while 85% progressed towards chronicity. The latter group included a significant proportion of 'fluctuating' progressors (37.5%), in whom a fall followed by a rise (>1 log₁₀) in viraemia was observed. This was associated with superinfection with new HCV strains and partially effective T cell responses. Spontaneous clearance was strongly associated with a 2.2 log₁₀ viral load drop within 100 days of infection (HR = 1.78; p < 0.0001), elevated bilirubin (≥ 40 μmol/l; HR = 5.04; p = 0.006), elevated alanine aminotransferase (ALT; ≥ 1000 IU/ml; HR = 2.62; p = 0.048) and baseline CD4 count ≥ 650 × 10⁶/l (HR = 2.66; p = 0.045), and only occurred in patients with genotype 1 infection. Evolution to spontaneous clearance occurred in patients with low viral diversity in the presence of an early multispecific T cell response. CONCLUSIONS: Spontaneous clearance of acute HCV in HIV-positive men can be predicted by a rapid decline in viral load, high CD4 count, elevated bilirubin and ALT, and is associated with low viral diversity and strong T cell responses.

Co-infection with HBV and HCV seems to be associated with more severe liver disease in retrospective and cross-sectional studies in adults, but no data are available when co-infection is acquired in youth. The long-term outcome of infection acquired in youth was assessed in patients co-infected with HBV and HCV and in patients with HBV infection only. Twenty-seven patients with HBV and HCV co-infection and 27 patients infected with HBV only were enrolled. Seventy-six per cent of the patients were treated with alpha-interferon for 1 year. After a median follow-up of 23 years, the annual progression rate of fibrosis was 0.07 in patients co-infected with HBV and HCV, and in those infected with HBV it was 0.07 and 0.11 (P < 0.004) for HBe and anti-HBe-positive patients, respectively. In co-infected patients, the development of cirrhosis was observed in 2 (7.4%) and of hepatocellular carcinoma (HCC) in 1 (3.7%), while in those with HBV, cirrhosis appeared in one patient (3.7%). Alcohol intake (OR = 9.5 +/- 1.2; 95% CI = 6.6-13.9; P < 0.0001) was independently associated with cirrhosis and HCC. alpha-interferon showed no efficacy during treatment, but the treated group showed higher HCV RNA clearance during post-treatment follow-up. Co-infection with HBV and HCV and single HBV infection acquired in youth showed a low rate of progression to liver fibrosis, no liver failure, and low development of HCC during a median follow-up of 23 years (range 17-40).

An epidemic of acute hepatitis C virus (HCV) infection among HIV-positive men who have sex with men is occurring in urban centers in Western Europe and the United States. Early diagnosis and treatment of HCV results in improved sustained virological response rates. This study compared the sensitivity of reverse transcriptase PCR (RT-PCR) versus antibody screening for the diagnosis of early HCV infection in HIV-positive patients and estimated the length of time from HCV infection to the development of anti-HCV antibodies.Patients from the St Mary's Acute Hepatitis C Cohort (SMACC) were recruited retrospectively and prospectively between 2004 and 2008.Archived plasma samples, obtained at 1-3 monthly intervals for routine monitoring of HIV viral load were assayed retrospectively for HCV in order to assess the sensitivity of RT-PCR and enzyme-linked immunosorbent assay (ELISA).: Forty-three HIV-positive patients with early HCV infection were identified. The median CD4 cell count was 570 cells/microl. The median alanine transaminase at the time of the first positive HCV PCR was 65 IU/ml. At this time, 75% of patients had a negative HCV antibody test. Three months later, 37% of patients still had a negative result. After 9 months, 10% of patients had a negative test and 5% remained negative after 1 year.Delayed seroconversion in HIV-positive individuals with acute HCV may result in delayed diagnosis and treatment. Where there is a clinical suspicion of recent HCV infection, for example, elevated alanine transaminase levels, HIV-infected patients should be screened for HCV RNA by RT-PCR.

— Six patients with chronic hepatitis B virus (HBV)-induced liver disease were treated with human lymphoblastoid interferon. Daily, alternate day or thrice weekly regimens were employed in two, one and three patients respectively. Thrice weekly intra-muscular injections of 7.5 to 10 megaunits of interferon/m2 appeared to be as effective as daily injections in producing inhibition of HBV replication, were associated with fewer side effects and could be continued for up to 3 months. The effectiveness of this regimen in producing long-term inhibition of viral replication must now be determined.

Acute exacerbations in HBeAg negative patients with chronic hepatitis B virus (HBV) infection are invariably associated with concurrent increases in the index of IgM class antibodies against the core protein (anti-HBc) of the virus. This study aimed to investigate whether this was related to the clearance of variants from the quasispecies pool and the appearance of new ones, with aminoacid substitutions in well recognized B-cell epitopes. In this study, 5 HBeAg negative patients (A to E) with 13 sequential serum samples (A1-A2, B1-B2-B3, C1-C2, D1-D2-D3, E1-E2-E3) were investigated after amplification of the entire core encoding region followed by cloning/sequencing studies. The sequences at different time points were compared with those from a single HBeAg positive patient with no apparent acute exacerbations. The results from sequence comparison showed that virus variants emerged in all (A2, B3, C2, D3, E2, and E3) but two (B2 and D2) subsequent sera with amino-acid substitutions affecting B-cell epitopes. It is concluded that the rise in the values of IgM anti-HBc may be attributed to the alteration of the antigenic epitopes leading to new antibody production in the majority of the cases. However, it appears that increases in IgM anti-HBc indexes in a few cases may relate to other possible mechanisms which are discussed.

The response to interferon-alpha treatment of patients with chronic hepatitis B under the current protocol is not satisfactory. The aim of this study was to try an alternative approach to improve treatment outcome. Of 374 HBeAg-positive patients, 127 of them received 5 million units of interferon-alpha thrice weekly for 6 months and constituted the control group, while 247 in the study group received the same dosage but the duration of treatment was tailored. The study protocol provided for continuation of treatment if HBV DNA levels were continuously decreasing. The treatment ended when viral, antigenic and biochemical endpoints were reached or when HBV DNA levels were no longer decreasing. The median length of tailored treatment was 10 (range 6-24) months. The end-of-treatment response rates were 39.3% and 23.6% (P = 0.002), and after 12-month, follow-up, the sustained response rates were 40.5% and 28.3% (P = 0.013) in the study and control groups, respectively. Excluding the patients who dropped out, 228 and 115 completed a median of 40- and 44-month-long follow-up; the long-term response was thus 45.3% and 33.1% (P = 0.014) in the respective groups. Interferon-alpha treatment tailored in length demonstrated significantly increased efficacy in patients with chronic hepatitis B.

There are two hypotheses explaining a fulminant outcome after hepatitis B virus (HBV) infection, both of which may be applicable at the same time: (i) basal core promoter (BCP) mutations increase viral replication, allowing rapid spread of the virus through the liver, and (ii) pre-core (pre-C) mutations abrogating hepatitis B e antigen (HBeAg) synthesis remove its tolerogenic effect, leading to a vigorous immune response. This study investigated the effect of these mutations on virus replication efficiency and HBeAg production. Substitutions A1762T/G1764A and T1753C, C1766T and T1768A in the BCP region, and G1896A and G1899A in the pre-C region, were examined either alone or in combination, using a common genetic background. Huh7 cells were transfected with these constructs and real-time PCR was used to quantify released virion-associated and intracellular HBV DNA, pregenomic RNA and pre-C mRNA. In addition, culture supernatants were tested for hepatitis B surface antigen (HBsAg) and HBeAg. The double BCP mutation (A1762T/G1764A) and the pre-C mutations (G1896A, G1899A), either alone or in combination, had no appreciable effect on the replication capacity of the virus. In contrast, clones with mutations at positions 1766/1768, 1762/1764/1766 and 1753/1762/1764 exhibited increased-replication phenotypes. HBeAg was undetectable in all cultures transfected with constructs bearing the G1896A stop-codon mutation, as expected. In contrast, constructs with additional mutations in the BCP region had appreciably lower levels of HBeAg expression than the wild type. Thus, core promoter mutations other than those at 1762/1764 appear to upregulate viral DNA replication and, at the same time, greatly reduce HBeAg production.

The natural history of chronic hepatitis B is dependent on the age of acquiring the hepatitis B infection. Those who are infected at adolescence or adulthood (including most of the Caucasians) tend to have stable disease after hepatitis B e antigen seroconversion with normal serum alanine aminotransaminase (ALT) and hepatitis B virus (HBV) DNA levels

Variants of hepatitis B surface antigen have been described in different clinical settings, but their replicative capacity in vitro has remained unexplored. Point mutations leading to sT131I, sK141E, and sG145R amino-acid substitutions were engineered by site-directed mutagenesis into an infectious plasmid clone of the virus. The mutated constructs were transfected into Huh7 cells, and their replication capacity was documented by LightCycler (Roche Diagnostics) measurements of virion-associated hepatitis B virus (HBV) DNA, intracellular relaxed circular double-stranded DNA, and pregenomic RNA. The sT131I and sG145R variants replicated with efficiency equal to that of the wild type, whereas the sK141E variant was replication impaired. Hepatitis B virus (HBV) particles have an outer lipid bilayer envelope into which are inserted the large, middle, and small hepatitis B surface antigen (HBsAg) proteins. All 3 proteins share the group-specific “a” antigenic determinant, a hydrophilic region with a conformational structure that constitutes the main neutralization epitope of the virus [1]. Variants of the virus that have amino acid substitutions in this region have been described in different epidemiological and clinical settings. These variants exhibit altered antigenicity, which causes assays either to fail to detect them or to have greatly reduced sensitivity. Such variants have been described in vaccinees, livertransplant patients who have received monoclonal or polyclonal anti-HBs treatment after transplant, cases of occult infection

To determine the long-term response to interferon-alpha therapy in patients with hepatitis B e antigen-negative chronic hepatitis B, and the factors independently associated with response and survival.Sixty-three patients with documented hepatitis B e antigen-negative chronic hepatitis B treated with interferon-alpha for a year were followed-up for a period of 6 years.Sustained biochemical and virological response was seen in 34.91% and 33.33% of patients at 6 and 12 months of follow-up, respectively, and histological improvement in 54.5% of sustained responders compared with non-responders (7.1%, P = 0.004, chi-squared test), at 6 months of follow-up. Multivariate analysis showed that patients with hepatitis B virus-DNA levels at 6 months of treatment10,000 copies/mL had a low probability of relapse, compared with those with levels10 000 copies/mL (P = 0.032). Age (65 years) and hepatitis B virus-DNA level at 6 months of treatment (10,000 copies/mL) were the independent factors for disease progression and survival (P = 0.041 and P = 0.044 respectively). At 6 years, a sustained response was still present in 19.04% of patients and 4.8% of them had developed anti-HBs.Hepatitis B virus-DNA monitoring by quantitative polymerase chain reaction at 6 months of treatment may allow for early prediction of response to interferon-alpha, and may serve as an indicator of disease progression in the future.

Patients who are chronically infected with either hepatitis B or C viruses run the risk of developing cirrhosis and hepatocellular carcinoma in later life. Antiviral treatment offers the only means of interrupting this progression. To date, recombinant interferon alpha and the nucleos(t)ide analogues lamivudine and adefovir dipivoxil are the only licensed drugs for the treatment of chronic hepatitis B, whilst recombinant or pegylated interferons in combination with ribavirin are the ones used for chronic hepatitis C virus infections. The efficacy of these treatments, reasons for treatment failure, drug resistance and future options are discussed in the present review.

Fludarabine is used widely for the treatment of chronic lymphocytic leukaemia, but not as yet implicated in the emergence of hepatitis B surface antigen (HBsAg) variants following hepatitis B virus (HBV) reactivation. Such a variant was detected in a 78-year-old female who was HBsAg(-)/anti-HBc(+)/anti-HBs(+)/anti-HBe(+), and with normal ALT levels, who developed HBV reactivation after fludarabine treatment. She had high HBV-DNA levels, and became positive for HBeAg, in the absence of detectable HBsAg. HBV-DNA was extracted from serum and the HBsAg encoding region of the genome was amplified by PCR, followed by cloning and sequencing. The HBV strain appeared to be subtype adw, but had higher nucleotide homology with ayw than adw isolates, supported further by phylogenetic tree analysis. Amino-acid sequence comparisons over the alpha determinant region revealed the following substitutions: C124N, G130R, and N146S. There were also unique substitutions outside the alpha determinant. All these mutations appeared to have a profound effect on the antigenicity of this region, which resulted in failure to detect HBsAg by commercially available diagnostic assays. It is concluded that a surface variant emerged in an HBsAg(-)/anti-HBs(+) patient with chronic lymphocytic leukaemia following fludarabine treatment, with an unprecedented number of amino-acid substitutions in the alpha determinant region of HBsAg, including a subtype switch.

A patient with agammaglobulinemia developed acute hepatitis that progressed to chronic liver disease with high levels of hepatitis B virus (HBV) DNA in the absence of detectable HBsAg. Sequencing of the a determinant region of HBsAg revealed multiple amino acid substitutions that, unusually, also included a substitution at position 122 that defines subtype specificity. All of these mutations had a profound effect on the antigenicity of this region, which led to the complete failure of variant detection by commercially available routine diagnostic assays or laboratory-based monoclonal antibody assays.

To determine the pattern and significance of the HCV genetic heterogeneity before and during treatment with recombinant-2b or lymphoblastoid α-interferon, hypervariable region 1 (HVR-1) and NS5A quasispecies were characterised by cloning and sequencing in 12 HCV-1b-infected subjects. Patients were either responder-relapsers or non-responders to treatment. Extensive amino acid sequence analysis was applied to reveal the significance of HCV variation at key sites within HVR-1 and NS5A regions. Genetic complexity, genetic diversity, and the non-synonymous to synonymous substitution ratios of HVR-1 quasispecies decreased during treatment in responder-relapser patients only, and more markedly so following lymphoblastoid α-interferon. In non-responders, the HVR-1 quasispecies broadened. Amino acids G406 and Q409, which represent a major viral epitope, were highly conserved throughout treatment. Responder-relapser patients had a higher mutation frequency in NS5A than non-responders. Lymphoblastoid α-interferon promoted the selection of intermediate Interferon Sensitivity Determining Region (ISDR) sequences, whereas recombinant-2b α-interferon favoured maintenance or selection of conserved ISDR sequences. Variability upstream of the ISDR was associated with treatment response, but the amino acid substitutions conferring higher replicative ability to in vitro HCV replicons were absent in in vivo isolates. In conclusion, the pattern of HVR-1 quasispecies evolution correlates with the clinical response, and the conservation of specific amino acids may be useful for immune targeting in vivo. In responder-relapser patients, the initial HVR-1 evolution resembles that found in sustained responders. Variability within the entire NS5A, as opposed to a single region (ISDR), may have a role in influencing α-interferon treatment outcome. A differential effect of different α-interferon preparations on HCV quasispecies kinetics may exist. J. Med. Virol. 70:62–73, 2003. © 2003 Wiley-Liss, Inc.

The N-terminal end of the hepatitis C virus (HCV) envelope glycoprotein E2 contains a stretch of 27 amino acids that exhibit increased variability. This hypervariable region 1 (HVR-1), as it is normally referred to, is thought to contain epitopes that come under humoral immune attack. In the present study, 10 patients (5 children and 5 adults) with humoral immune defects and chronic HCV infection were investigated, to see how HVR-1 sequences behave over time in these patients who are unable to produce antibodies. Amplicons of this region showed little or no variation at all over time, indicating that quasispecies variation in this region is driven by the host's humoral immune response.

Hospital-acquired hepatitis B (HBV) and C virus (HCV) infections continue to occur despite increased awareness of this problem among the medical community. One hundred six patients were infected in a haematology oncology ward for children, over the time period 1996 to 2000. Serum samples from 45 such patients and 3 from infected medical personnel were used for nucleic acid amplification. HBV core, as well as HCV core and hypervariable region 1 (HVR1) nucleotide sequences, were analysed by phylogenetic tree analysis, in order to characterise the epidemiological pattern of viral transmission on the ward. Samples from 32 patients were positive for HBV-DNA or HCV-RNA by PCR. Ten patients were positive for both markers. Seventeen out of twenty-three HCV core gene sequences were found to be evolutionarily related and clustered separately from other local sequences in the phylogenetic tree, indicating nosocomial transmission. This was confirmed by analysis of HVR1 gene sequences. One nurse and one physician from the ward were HCV RNA positive, but their HCV sequences were not related evolutionarily to those of the patient cluster. Fifteen out of nineteen HBV core gene sequences were also clustered together and were positioned separately in the relevant tree. Epidemiological investigation excluded a common source infection and indicated that spread of infection was most likely due to inappropriate infection control measures on the ward. No obvious risk factors for transmission were identified during the retrospective survey in patients with related sequences, except use of multidose vials for saline and poor staff compliance with routine hand hygiene procedures. The preventive measures that were introduced reduced the incidence of infection significantly. No new cases of HBV infection and only three anti-HCV seroconversions occurred over a period of 19 months. The introduction and maintenance of strict prevention measures over a 2 year period, combined with HBV vaccination, reduced significantly the incidence of new HCV and HBV infections.

The hepatitis B surface antigen (HBsAg), first named as Australia antigen, was identified and linked to hepatitis in the late 1960s by Blumberg and colleagues. It is now well established that this protein forms the outer envelope of the hepatitis B virus (HBV) and constitutes the main diagnostic marker of both acute and chronic infection. This is facilitated by the fact that the protein is produced in large amount, not all of which is virus associated. Apart from the mature infectious virions known as Dane particles, two types of subviral particles devoid of nucleic acid are also produced; the 22nm spheres and filaments of longer length. The latter two forms outnumber virions by as much as 105. The high levels of circulating HBsAg have been linked to potential T-cell anergy during chronic infection, thus constituting a mechanism of immune evasion. There are 3 forms of the protein known as the small (S), medium (M) and Large (L), the latter two of which contain the S domain at their carboxyl end which is preceded by the Pre-S2 domain in the case of the M protein and Pre-S1+Pre-S2 domains in the L protein. These proteins are translated from the relevant mRNAs which are transcribed from the intrahepatic covalently closed circular DNA (cccDNA), and their level may thus reflect the replicative capacity of the virus. However, HBsAg may also be encoded by HBV-DNA sequences integrated into the hepatocyte DNA [1]. The efficacy of antiviral drugs such as pegylated interferon alpha (Peg-IFNa) or nucleos(t)ide analogues (NUCs) which are used in the treatment of chronic HBV carriers is monitored by measurement of HBV-DNA levels by sensitive real time polymerase chain reaction techniques. In recent years, quantitative measurement of HBsAg levels expressed in IU/ml has become possible through the use of two commercially available tests, namely the Architect QT and the Elecsys HBsAg II Quant assays. A number of studies have attempted to correlate HBsAg levels with the various phases of the natural course of chronic HBV infection, as well as determine whether they can be used as a predictive factor for a favourable outcome following antiviral treatment. In the former case, HBsAg levels during the immune tolerant phase appear to have a mean of 4.75 log10 IU/ml, as opposed to 4.2, 2.7 and 3.5 log10 IU/ml in the immune clearance, immune control and reactivation phases respectively. HBV DNA levels appear to mirror the HBsAg changes during the various phases, except for the immune control phase where HBV-DNA may be detectable at very low levels or not at all [2][3]. There is evidence to suggest that HBsAg levels may differ depending on genotype, these being higher in patients infected with genotype A and lowest in genotype D, with those for genotypes B and C being intermediate in a transfection system [4]. However, using patient sera in the immune clearance phase, Sonneveld and colleagues reported higher levels of HBsAg for genotypes A and D than B and C [5]. Clearly, this issue warrants further thorough investigation with clear definition of the patients’ stage of chronic liver disease. Various investigators have attempted to use HBsAg and HBV-DNA levels as a guide in defining a cut-off value for separating inactive carriers from those with active disease. HBsAg and HBV-DNA levels of < 1000 and ≤ 2000 IU/ml in HBeAg-ve patients have been shown to identify patients with inactive disease with a positive predictive value of 87.9% [3][5][6]. What is more, it has been suggested that HBsAg and HBV DNA levels of < 100 and < 200 IU/ml respectively, can predict HBsAg loss within 6 years with a diagnostic accuracy of 91.5%, sensitivity of 83.3%, specificity of 92.1%, positive predictive value of 45.5%, and negative predictive value of 98.6% [7]. HBsAg levels decline with Peg-IFN treatment in both HBeAg+ and HBeAg- patients. In HBeAg+ patients, this decline from baseline to week 48 averages 0.75 log10 IU/ml and there appears to be an additive effect when used in combination with a NUC (Lamivudine, Telbivudine) leading to a drop of 1.3 log10 IU/ml [8][9]. In the case of HBeAg- patients the drops are still substantial, but there is no difference when Peg-IFN is used in combination with a NUC (0.65 log10 IU/ml) [10]. When NUCs are used alone a more modest drop in HBsAg level is recorded ranging from 0.1-0.4 log10 IU/ml [9][10][11]. Quantitative measurements of HBsAg levels at 12 and 24 weeks of treatment with Peg-IFN in HBeAg+ patients have shown that a no decline in HBsAg or levels > 20000 IU/ml are associated with poor sustained virological response (SVR) ranging from 3-18% at both time points [8], whilst a decline to 1500 IU/ml is associated with SVR in 54-58% of patients [12][13], rising to 62-75% if the levels drop to 1 log10 [15][16]. The utility of measuring HBsAg levels during NUC treatment is less clear as can be deduced from the small number of studies published, which appear to have included in the analysis heterogeneous groups of patients. The decline in HBsAg levels is slower and does not correlate with HBV-DNA levels. However, a rapid decline in HBsAg by >1 log10 IU/ml during therapy with Entecavir was associated with a higher loss of HBeAg after 1 year of treatment [11]. HBsAg seroclearance in HBeAg+ patients treated either with Tenofovir, Entecavir or Lamivudine ranged between 2-8% after 1-3 years of treatment, whilst in the case of HBeAg- patients, HBsAg loss occured less frequently and estimates suggest that this may require periods longer than 10 years [17][18]. In conclusion, a reduction in HBsAg levels during treatment, particularly at 12 and 24 weeks, may be predictive of a favourable outcome in the case of Peg-IFN treatment in HBeAg+ positive patients. However, suitable cut-off levels need to be more accurately defined. In the case of HBeAg- patients, both HBsAg and HBV-DNA decline levels at 12 weeks can be used as a guide to applying stopping rules. The picture in patients treated with NUCs is far from clear at the moment and clarification of the issues involved must await further detailed studies.

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.

One hundred forty-two precore/core sequences were obtained from Gambian chronic hepatitis B virus (HBV) carriers and the predominant variants defined. The two point mutations, from A to T and G to A at nt positions 1762 and 1764 in the basic core promoter region, were found in only 7/99 (7%) of the samples where this region was sequenced. These mutations were found in both HBeAg-positive and -negative patients. The precore stop-codon mutation at nt position 1896 was found in 14/51 (27%) of HBeAg-negative samples, which is a lower prevalence rate in comparison with other parts of the world with high carrier rates. In HBeAg-positive patients the core amino acid sequences were conserved, but after seroconversion to anti-HBe significantly more changes were apparent. Several of the amino acid substitutions found have been described previously been in wild-type viruses of other genotypes.

Background Early diagnosis of hepatitis C virus (HCV) infection in HIV-infected patients has significant implications for patient management. However, the currently recommended serological screening strategy for identifying such patients could be improved. Little is known about the performance of routine antibody-only tests, compared with newer serological antigen–antibody detection assays and nucleic acid testing in this patient group. Objectives To compare the performance of antibody and antigen–antibody detecting methods with nucleic acid testing in the diagnosis of acute HCV in HIV-infected individuals. Study design 123 samples from 25 HIV-infected patients with acute HCV infection were tested retrospectively. The time of infection was estimated. The performance of antibody, antigen–antibody and nucleic acid detecting methods in diagnosing acute HCV infection was assessed and the sensitivity of the assays compared. Results Only 20% of samples that were positive for HCV RNA were simultaneously positive for HCV antibody. In contrast, 68% of the total number of samples were positive and 32% negative by the antigen–antibody assay. Patients became antibody-positive on average 7 months after HCV RNA was detected. Conclusion In HIV-infected patients nucleic acid testing is the most sensitive means of diagnosing acute HCV C infection. A serological assay offering combined detection of antibody and antigen enhances sensitivity of detection, compared to antibody-only assays.

Since its characterization in 1995, there has been increasing interest in the significance of GB virus B (GBV-B) due to its close phylogenetic relationship to hepatitis C virus (HCV). The genome of GBV-B is similar in length and organization to that of HCV and the two viruses share sequence similarity in their 5′ untranslated regions (5′UTR). A secondary structure model of the GBV-B 5′UTR has been proposed by comparative sequence analysis with HCV. The highly conserved secondary structure, present in HCV and the pestiviruses, is also present in the 5′UTR of GBV-B. Translation of the HCV polyprotein initiates via an internal ribosome entry site (IRES) and it is proposed that the GBV-B UTR may function in a similar manner. Dicistronic reporter constructs were made to investigate the function of the GBV-B 5′UTR. Mutational analysis and in vitro translation experiments demonstrate that GBV-B initiates translation via an IRES.

Persistent infection with hepatitis G virus (HGV) or GB virus-C (GBV-C) is common and may last for years. In addition, the principal site of virus replication remains undefined. Sequencing studies of E2 in four patients showed that a hypervariable region equivalent to that of hepatitis C virus (HCV) was absent and that viral quasispecies were less frequent than in HCV infection, particularly with respect to amino acid variation. Recurrence of viraemia following interferon treatment did not result in the emergence of new quasispecies. Virus persistence therefore does not appear to be related to immune escape by strains bearing a hypervariable E2 region. We also investigated whether virus replication occurred in peripheral blood mononuclear cells. The positive-RNA strand of the virus, but no negative strand, was detected in both serum and lymphocytes. The lymphocytes harbouring the virus were CD4 and CD19 positive. Direct sequencing and cloning of amplicons from the region of the non-structural 3 (NS3) protein showed that the nucleotide sequences in lymphocytes were different from those in serum and did not represent any of the minor serum quasispecies. Although evidence of replication in lymphocytes has not been forthcoming, the differences in sequences between serum and lymphocytes suggest that circulating virus originates from a non-hepatic site, other than lymphocytes.

The aim of this study was to determine the prevalence of infection with the newly described hepatitis G virus (HGV) in a liver transplant cohort, and to establish the frequency and nature of hepatitis in those with and without HGV infection. A reverse transcriptase-polymerase chain reaction technique was employed to determine viraemia in the patients, and liver biopsies taken at different times after transplantation were assessed histologically. Hepatitis G virus RNA was detected in 47% of the liver transplant recipients investigated. Those positive for HGV had received significantly more blood or blood products than the HGV-negative patients. The frequency of abnormal liver function tests was similar in HGV-positive and HGV-negative recipients. Bile duct epithelial cell damage was more frequently seen in those with HGV viraemia. This study indicates that almost half of the liver transplant recipients in Northern England are positive for HGV, and that infection is associated with exposure to blood and blood products. It appears that, in the immunosuppressed patient, HGV does not cause clinically significant liver disease, at least up to 2 years after transplantation. If HGV infection is associated with hepatitis outside this clinical setting, it is likely that the liver damage is immunopathologically mediated rather than as a result of direct viral cytotoxicity.

The hepatitis D virus (HDV) relies on the helper hepatitis B virus (HBV) for the provision of its envelope, which consists of hepatitis B surface antigen (HBsAg). The RNA genome of HDV is a circular rod-like structure due to its extensive intramolecular base-pairing. HDV-RNA has ribozyme activity which includes autocatalytic cleavage and self-ligation properties, essential in virus replication via the rolling circle mechanism. Replication of the RNA is thought to be effected by cellular RNA polymerase II. Hepatitis D antigen (HDAg) is the only protein encoded by HDV-RNA and its long and short forms have a regulatory role in the replication and morphogenesis of the virus. Superinfected HBV carriers who become chronically infected with HDV are at increased risk of developing cirrhosis. Attempts to treat such carriers with interferon have not been particularly successful. In recent years the epidemiology of HDV has changed primarily due to the impact of HBV vaccination in preventing an increase in the pool of susceptible individuals. © 1998 John Wiley & Sons, Ltd.

The cellular localization of the precore/core and core proteins was studied by immunofluorescence following transfection of 143 thymidine kinase-negative (TK ) and Hep-G2 cells with expression constructs containing wild-type (hepatitis B e antigen [HBeAg]-positive) and precore mutant (HBeAg-negative) sequences. Precore/core constructs with the wild-type phenotype result in strong nuclear staining, while, in contrast, constructs expressing core antigen alone have strong cytoplasmic staining. These differences in the pattern of immunofluorescence staining may be caused by expression of the precore/core protein, some of which may be translocated into the nucleus, following removal of the signal peptide. In vitro translation experiments showed that the main protein products obtained in the presence of microsomal membranes were the precore/core protein and a truncated product representing the same protein without its signal peptide. Core protein expression from the precore mutant constructs was very much reduced, indicating that translational re-initiation was not very efficient. The significance of the precore/core protein being present in the nucleus is not clear, but suggests that it may be important in the replicative cycle of the virus. Finally, HBeAg produced by some of the constructs could not be detected because amino acid substitutions affected antibody-binding epitopes.