Novel therapies and potential therapeutic targets in the management of chronic hepatitis B.

Chronic hepatitis B is a persistent and progressive inflammatory liver disease caused by infection with the hepatitis B virus (HBV). More than 240 million individuals are infected with HBV worldwide and hepatitis B accounts for an estimated 650 000 deaths annually. Approximately up to 30% of chronically infected patients will develop complications of HBV infection including, but not limited to, liver cirrhosis, end-stage liver disease, and hepatocellular carcinoma. Currently approved therapies have improved clinical outcomes, but have a considerable side-effect profile, elevated cost, and a finite course of treatment. This has led to a growing interest in research for new therapies. As the mechanisms for HBV replication are becoming better understood, new potential targets have been discovered, leading to the development of new therapies. In this article, we describe the promising therapies that are under evaluation, showing their mechanisms of action, effects, and stage of development.

Sofosbuvir and ABT-450: terminator of hepatitis C virus?

Combination therapy with peginterferon (pegIFN)-α and ribavirin (RBV) has been the standard of care (SOC) for chronic hepatitis C. Unfortunately, not all patients can achieve a sustained virologic response (SVR) with this regimen. SVR rates are approximately 80% in patients with hepatitis C virus (HCV) genotype 2, 3, 5 and 6 and 40%-50% in patients with genotype 1 and 4. Therefore, strategies to improve SVR rates have been an important issue for clinical physicians. Several direct acting antiviral agents (DAAs) have significantly higher SVR rates when combined with pegIFN-α and RBV than pegIFN-α and RBV alone. Treatments containing DAAs have several advantages over the previous SOC, including higher specificity and efficacy, shorter treatment durations, fewer side effects, and oral administration. Based on these advantages, treatment with pegIFN-α and RBV plus telaprevir or boceprevir has become the current SOC for patients with genotype 1 HCV infection. However, many patients are either not eligible for therapy or decline treatment due to coexisting relative or absolute contraindications as well as an inability to tolerate the hematological side effects and adverse events caused by the new SOC. These factors have contributed to the advent of pegIFN-α-free regimens. The newest therapeutic regimens containing sofosbuvir and ABT-450 have shown promising results. In this review, we summarize the development of anti-HCV agents and the clinical efficacy of sofosbuvir and ABT-450-based therapies as well as the potential for future HCV studies.

Effects of antiviral therapy on hepatitis B virus reactivation and liver function after resection or chemoembolization for hepatocellular carcinoma.

BACKGROUND: How hepatitis B virus (HBV) infection react to hepatocellular carcinoma (HCC) treatment remains unclear, and the roles of anti-HBV therapy were seldom reported in HCC. AIMS: To evaluate changes of HBV replication and liver function after hepatectomy or transarterial chemoembolization (TACE) for HCC, also the short-term effects of anti-viral therapy were analyzed. METHODS: Totally, 590 HBsAg (+) HCC patients were recruited into two groups: only surgical resection, and only TACE, and subgrouped according to anti-HBV therapy or none. Clinical data were analyzed for statistical significance and risk factors for adverse events. RESULTS: In the no antiviral therapy groups, rates of HBV reactivation were 15.7% and 17.5% in patients who underwent hepatectomy and TACE, respectively, while the rates of deterioration of liver function were 4.1% and 8.1%, respectively. In contrast, in the antivirus group, the rates of reactivation were 0% and 1.5% after hepatectomy and TACE respectively, while the liver function deterioration rates were 2.4% and 1.5%, respectively. For patients who underwent hepatectomy, no antiviral therapy, and long hepatic inflow occlusion times increased the risk of HBV reactivation. For TACE, no antivirus and HBeAg negativity were the risk factors for reactivation. HBV reactivation was significantly correlated to liver function exacerbation after hepatectomy, while HBV reactivation, baseline ALT (alanine aminotransferase), and α-fetoprotein levels were significantly correlated to liver function exacerbation after TACE. CONCLUSIONS: HBV reactivation can occur after hepatectomy or TACE. Anti-HBV therapy can reduce the risk of reactivation, thus reducing the risk of liver failure especially in patients undergoing TACE.

Hepatites virais: abordagem clínica com ênfase nos vírus A e E / Viral hepatitis: clinical approach with emphasis on A and E viruses

JUSTIFICATIVA E OBJETIVOS: As hepatites virais são condições mórbidas extremamente comuns na prática clínica. Diferentes agentes etiológicos estão implicados no desenvolvimento da doença, especialmente os vírus A, B, C, D e E. O objetivo do presente artigo foi apresentar os aspectos gerais das hepatites desencadeadas por agentes virais, enfatizando-se as hepatites causadas pelos vírus A e E. CONTEÚDO: Foram utilizadas as palavras-chaves hepatites virais, hepatite A e hepatite E como descritores na pesquisa de dados nas bases Scielo (Scientific Eletronic Library Online) e Pubmed (U. S. National Library of Medicine), assim como livros texto,consensos e diretrizes relacionados ao tema. CONCLUSÃO: A ocorrência das hepatites causadas pelos vírus A e E está intimamente relacionada ao nível socioeconômico e às condições sanitárias da população, haja vista a transmissão ocorrer predominantemente por via fecal-oral. Embora ambas as entidades nosológicas apresentem evolução benigna na maioria dos casos, a educação sanitária e a promoção da saúde representam estratégias promissoras para a melhoria das condições de vida das populações suscetíveis.

BACKGROUND AND OBJECTIVES: Viral hepatitis is a morbid condition extremely common in practical clinic. Different etiological agents are implicated in the development of illness, especially viruses A, B, C, D and E. The objective of this study is to present general aspects of hepatitis triggered by viral agents,with emphasis on hepatitis caused by viruses A and E. CONTENTS: We used the keywords viral hepatitis, hepatitis A and hepatitis E as descriptors in the data search Scielo (Scientific Electronic Library Online) and Pubmed (U. S. National Library of Medicine), as well as textbooks, consensus and guidelines related to the topic. CONCLUSION: The occurrence of hepatitis caused by viruses A and E is closely related to socioeconomic status and sanitary conditions of the population, since the transmission occurs predominantly via the faecal-oral route. Although both nosological entities have a benign course in most cases, health education and health promotion represent promising strategies for improving the living conditions of susceptible populations.

Hepatitis C virus genotype 1a growth and induction of autophagy.

We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes.

Quasispecies and its impact on viral hepatitis.

Quasispecies dynamics mediates adaptability of RNA viruses through a number of mechanisms reviewed in the present article, with emphasis on the medical implications for the hepatitis viruses. We discuss replicative and non-replicative molecular mechanisms of genome variation, modulating effects of mutant spectra, and several modes of viral evolution that can affect viral pathogenesis. Relevant evolutionary events include the generation of minority virus variants with altered functional properties, and alterations of mutant spectrum complexity that can affect disease progression or response to treatment. The widespread occurrence of resistance to antiviral drugs encourages new strategies to control hepatic viral disease such as combination therapies and lethal mutagenesis. In particular, ribavirin may be exerting in some cases its antiviral activity with participation of its mutagenic action. Despite many unanswered questions, here we document that quasispecies dynamics has provided an interpretation of the adaptability of the hepatitis viruses, with features conceptually similar to those observed with other RNA viruses, a reflection of the common underlying Darwinian principles.

Arginine metabolism during macrophage autocrine activation and infectionwith mouse hepatitis virus 3

Arginine metabolism during macrophage autocrine activation and infection with mouse hepatitis virus 3In contrast to BALB/c mouse macrophages (MÖ), MÖ from the A/J mouse strain, upon activation by exogenous interferon gamma (IFNã), develop an anti-mouse hepatitis virus 3 (MHV3) state which correlates with resistance to virus infection. To investigate the autocrine activation of BALB/c and A/J MÖ, we activated them with interleukin-12 (IL-12) and/or IL-18, and quantified IFNã production, the anti-MHV3 state and arginine metabolism.

Investigation of SEN virus infection in patients with cryptogenic acute liver failure, hepatitis-associated aplastic anemia, or acute and chronic non-A-E hepatitis.

SEN virus (SENV) has been tentatively linked to transfusion-associated non-A-E hepatitis. We investigated SENV's role in unexplained hepatitis in other settings. Polymerase chain reaction amplification was used to detect 2 SENV variants (SENV-D and SENV-H) in 1706 patients and control subjects. SENV was detected in 54 (22%) of 248 patients with acute or chronic non-A-E hepatitis, 9 (35%) of 26 patients with hepatitis-associated aplastic anemia, and 0 of 17 patients with cryptogenic acute liver failure, compared with 150 (24%) of 621 control subjects with liver disease and 76 (10%) of 794 healthy control subjects. When controlling for geographic region, the prevalence of SENV among case and control subjects was not significantly different. The severity of acute or chronic hepatitis A, B, or C was not influenced by coexisting SENV infection. No etiological role for SENV in the cause of cryptogenic hepatitis could be demonstrated.

Animal model, virology and gene cloning of hepatitis E.

We have developed animal models of viral hepatitis E using cynomolgus and rhesus monkeys. They developed acute biochemical and histological hepatitis after the inoculation of virus particles with identical kinetics and magnitude for the sixth subpassage. Virus particles multiplied in hepatocytes and were excreted into feces via bile. Additionally, a transient viremia was recognized. Molecular cloning of virus gene cDNA was successfully accomplished from two separate libraries (HT3 and NE). These clones were expressed into polypeptides having immunological epitopes, which were used for antibody assay of sera of monkeys and patients with positive results.

In vitro infection of woodchuck hepatocytes with woodchuck hepatitis virus and ground squirrel hepatitis virus.

Primary cultures of woodchuck hepatocytes were demonstrated to be susceptible to in vitro infection by both woodchuck hepatitis virus and ground squirrel hepatitis virus, as evidenced by the appearance of DNA species characteristic of hepadnavirus replication. Initiation of infection by woodchuck hepatitis virus was blocked by the presence of suramin, polybrene, or dideoxycytidine. Viral CCC DNA, the putative template for viral RNA transcription, was detected at 2 days postinfection. Accumulation of intracellular intermediates in virion DNA synthesis was negligible until 7-10 days postinfection, but these DNA intermediates then increased dramatically in amount over the next few weeks. Results were obtained which suggested that the prolonged accumulation of intermediates in virion DNA synthesis was an intrinsic property of the infection of individual cells, and not the result of a slow spread of virus through the cultures.

Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.

Maintenance of woodchuck hepatitis virus activity in woodchuck hepatocyte primary culture.

Primary cultures of non-proliferating hepatocytes isolated by the two-step collagenase perfusion method from woodchuck naturally infected with hepatitis virus (WHV) were used to study WHV propagation in vitro. Hepatocytes carrying WHV DNA exhibited a very high level of survival and retained their morphological characteristics for 2 to 3 months. Over this time, they were found to produce virus-specific proteins and release viral particles with DNA polymerase activity into the medium. Using Southern blot analysis and a recombinant hepatitis B virus DNA plasmid probe, intracellular and extracellular viral DNA was consistently detected. Only extrachromosomal forms of WHV DNA were observed and no integration could be demonstrated in the DNA of the cells. The WHV DNA patterns were repeatedly identical with a characteristic smear starting from 3.3 kb associated with other smaller DNA fragments which presumably represented intermediate replicative forms of viral DNA. Furthermore, dot blot hybridization of the total RNA revealed the presence of WHV-specific transcripts in cells after 3 weeks of culture. All these results are compatible with the maintenance of active WHV replication in vitro although it was somewhat reduced after the first day of culture. This provides a mammalian model for hepadnavirus replication studies in stable primary hepatocyte cultures.

Inactivation of HTLV-III/LAV, hepatitis B and non-A/non-B viruses by pasteurization in human plasma protein preparations.

Pasteurization (heat treatment at +60 degrees C for 10 hours in solution) during the production of human plasma protein preparations has proved useful 1. to inactivate a broad spectrum of viruses and 2. in combination with stabilizers to leave the nativity of the products unaffected. Their efficacy has been experimentally tested for HTLV-III/LAV, Hepatitis B and non-A/non-B viruses. The following preparations were tested: with HTLV-III/LAV: Factor VIII, Factor IX, AT III, AHC and PPSB; with Hepatitis B virus: Factor VIII, Factor XIII, AT III, PPSB and Fibrinogen; with Hepatitis non-A/non-B virus: Factor VIII and AT III.

Procedures for the inactivation of viruses in clotting factor concentrates.

A considerable body of data has accumulated on the viral inactivated clotting factor concentrates. This information strongly indicates considerable safety and expected efficacy of these products. Probably most procedures are capable of inactivating large amounts of the LAV/HTLV-III virus, but there is considerable variability between products in the ability to inactivate the hepatitis viruses and perhaps also the LAV/HTLV-III. This variability is probably related to the mechanism of viral inactivation, the time of exposure to heat or chemical additives, and also the presence and type of stabilizer. So-called "second-generation" products will probably show improved elimination of more resistant viruses including the non-A, non-B forms.

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.

Inactivation of the Hutchinson strain of non-A, non-B hepatitis virus by combined use of beta-propiolactone and ultraviolet irradiation.

A beta-propiolactone/ultraviolet irradiation procedure (beta PL/UV) has been evaluated for its ability to inactivate 30,000 chimpanzee infectious doses of the Hutchinson strain of non-A, non-B (NANB) virus. The chimpanzees were inoculated with plasma to which this dose of the titrated virus had been added prior to application of the beta PL/UV process in accordance with a procedure used for licensed blood derivatives in Germany. Neither animal developed hepatitis. When subsequently challenged with the same contaminated plasma, which had not been sterilized, both animals promptly developed typical NANB hepatitis. This study extends the high (approximately 10(7)-fold) process efficiency of the beta PL/UV procedure previously reported for hepatitis B virus to a blood-borne NANB virus.

[Spontaneous and experimental infection of alpine marmots (Marmota marmota) by the North American woodchuck hepatitis virus (Marmota monax). Initial results]. / Infection spontanée et expérimentale de la marmotte alpine (Marmota marmota) par le virus de l'hépatite de la marmotte nord-américaine (Marmota monax). Premiers résultats.

Summer's discovery in 1978 of a DNA virus, very close to human Hepatitis B virus in a woodchuck population in the U.S.A. (Pennsylvania) was a confirmation of the first description made by Snyder at Penrose Research Laboratory (Philadelphia). It was the first animal model of human B hepatitis infection. The comparative study of morphological, ecological and ethological characteristics of the marmot (Marmota marmota) and the woodchuck (Marmota monax) enables an easy distinction between these two species. The natural infection of M. monax by the WHV shows that the woodchuck is a good model for human B hepatitis and should be extended to M. marmota. A sample of 24 marmots caught in the Alpes of Haute-Provence has not revealed any spontaneous infection in these animals by the woodchuck virus. The failure of experimental inoculation of the marmot (24 animals) with the WHV confirms the refractory status of this species (no viremia and very low and short serological response with or without an immunosuppressive treatment). These preliminary results require a confirmation in other animals of different age and geographical region and also by using more specific tests such as molecular hybridization, research on DNA polymerase and direct transfection trials.