RESUMO
The hepatitis B virus (HBV) ribonuclease H (RNase H) is an attractive but unexploited drug target. Here, we addressed three limitations to the current state of RNase H inhibitor development: (a) Efficacy has been assessed only in transfected cell lines. (b) Cytotoxicity data are from transformed cell lines rather than primary cells. (c) It is unknown how the compounds work against nucleos(t)ide analog resistant HBV strains. Three RNase H inhibitors from different chemotypes, 110 (α-hydroxytropolone), 1133 (N-hydroxypyridinedione), and 1073 (N-hydroxynapthyridinone), were tested in HBV-infected HepG2-NTCP cells for inhibition of cccDNA accumulation and HBV product formation. 50% effective concentrations (EC50s) were 0.049-0.078 µM in the infection studies compared to 0.29-1.6 µM in transfected cells. All compounds suppressed cccDNA formation by >98% at 5 µM when added shortly after infection. HBV RNA, intracellular and extracellular DNA, and HBsAg secretion were all robustly suppressed. The greater efficacy of the inhibitors when added shortly after infection is presumably due to blocking amplification of the HBV cccDNA, which suppresses events downstream of cccDNA formation. The compounds had 50% cytotoxic concentrations (CC50s) of 16-100 µM in HepG2-derived cell lines but were nontoxic in primary human hepatocytes, possibly due to the quiescent state of the hepatocytes. The compounds had similar EC50s against replication of wild-type, lamivudine-resistant, and adefovir/lamivudine-resistant HBV, as expected because the RNase H inhibitors do not target the viral reverse transcriptase active site. These studies expand confidence in inhibiting the HBV RNase H as a drug strategy and support inclusion of RNase H inhibitors in novel curative drug combinations for HBV.
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Antivirais/farmacologia , Vírus da Hepatite B , Hepatite B , Ribonuclease H/antagonistas & inibidores , DNA Circular/genética , DNA Viral/genética , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Humanos , Replicação ViralRESUMO
BACKGROUND: Out of several phases of HBV infection, the least understood phase is occult hepatitis B virus infection. The paucity of data due to non-availability of biological tissues and the prerequisite of ultra-sensitive assays for the detection of occult hepatitis B virus infection prompted us to utilize mathematical modeling in determining mechanisms that lead to occult hepatitis B virus infection and characteristics of HBV infection during occult hepatitis B virus infection. METHODS: We proposed two mathematical models (M1 and M2), considering two different phenomenon for episomal maintenance and accumulation of covalently closed circular DNA (cccDNA) in infected hepatocytes: (i) M1 - recirculation of the relaxed circular DNA/double-stranded linear DNA from cytoplasm to the nucleus, and (ii) M2 - reinfection of infected hepatocytes with virions. We further incorporated the dynamics of integrated Hepatitis B virus DNA (iHBV) to investigate its role in the development of occult hepatitis B virus infection. RESULTS: The analysis showed that the main mechanism for the spread of infection during occult hepatitis B virus infection is cell-to-cell transmission and not cell-free virus transmission. A significant viral suppression (of at least 99% from its peak production values) was essential but not sufficient in the development of occult hepatitis B virus infection under M1; however under M2, the viral suppression was neither sufficient nor essential as the inhibition of the production of HBsAg without viral suppression can also explain the development of occult hepatitis B virus infection. Our analysis also revealed that occult hepatitis B virus infection seropositive cases are more likely to progress into liver cirrhosis compared to occult hepatitis B virus infection seronegative cases. The iHBV was found to be mostly silent (by either being absent or non-productive for HBsAg) during occult hepatitis B virus infection. CONCLUSION: The viral suppression is neither essential nor sufficient to explain the development of occult hepatitis B virus infection on its own. Not only the viral suppression but the inhibition -of the production and the export of HBsAg from cccDNA and iHBV also plays an important role in the development of occult hepatitis B virus infection. This is the first study, which incorporates the dynamics of iHBV and shows that HBV primarily spreads via cell-cell transmission during occult hepatitis B virus infection.
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Vírus da Hepatite B/fisiologia , Hepatite B , Hepatócitos , Modelos Biológicos , Integração Viral , Replicação Viral , DNA Circular/metabolismo , DNA Viral/metabolismo , Hepatite B/metabolismo , Hepatite B/patologia , Hepatite B/transmissão , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , HumanosRESUMO
Hepadnavirus at very low doses establishes in woodchucks asymptomatic, serologically undetectable but molecularly evident persistent infection. This primary occult infection (POI) preferentially engages the immune system and initiates virus-specific T cell response in the absence of antiviral antibody induction. The current study aimed to determine whether POI with time may culminate in serologically identifiable infection and hepatitis, and what are, if any, its pathological consequences. Juvenile woodchucks were intravenously injected with inocula containing 10 or 100 virions of woodchuck hepatitis virus (WHV) to induce POI and followed for life or up to 5.5 years thereafter. All 10 animals established molecularly detectable infection with virus DNA in serum (<100-200 copies/mL) and in circulating lymphoid cells, but serum WHV surface antigen and antibodies to WHV core antigen remained undetectable for life. By approximately 2.5-3.5 years post-infection, circulating virus transiently increased to 103 copies/mL and virus replication became detectable in the livers, but serological markers of infection and biochemical or histological evidence of hepatitis remained undetectable. Nonetheless, typical hepatocellular carcinoma (HCC) developed in 2/10 animals. WHV DNA integration into hepatic and lymphatic system genomes was identified in 9/10 animals. Virus recovered from the liver virus-negative or virus-positive phases of POI displayed the wild-type sequence and transmitted infection to healthy woodchucks causing hepatitis and HCC. In summary, for the first time, our data demonstrate that an asymptomatic hepadnaviral persistence initiated by very small amounts of otherwise pathogenic virus, advancing in the absence of traditional serological markers of infection and hepatitis, coincides with virus DNA integration into the host's hepatic and immune system genomes, retains liver pro-oncogenic potency and is capable of transmitting liver pathogenic infection. This emphasizes the role for primary occult hepatitis B virus infection in the development of seemingly cyptogenic HCC in seronegative but virus DNA reactive patients.
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Carcinoma Hepatocelular/virologia , Hepatite B/complicações , Neoplasias Hepáticas/virologia , Animais , DNA Viral/sangue , Modelos Animais de Doenças , Hepatite B/virologia , Vírus da Hepatite B da Marmota , Marmota , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga ViralRESUMO
Antiviral therapy for chronic hepatitis B (CHB) is often required for prolonged periods. We investigated the instance of one HBV genotype switching to another during tenofovir therapy. Of the 67 patients, genotype A was present in 6 (8.9%), D in 43 (65.6%), C in 1 (1.5%), and mixed in 17 (23.8%) patients. Genotype changes were detected in 51 (76.1%) patients on therapy during a follow-up of 192 (range 52-312) weeks. Inter-genotype changes were seen in 17 (33.3%) and intra-genotype in 28 (55%) and both inter- and intra-genotype in 6 of 51 (11.7%) patients. The distribution of genotypes in patients achieving complete virological response was genotype D, 32/43 (74.4%); genotype A, 6/6 (100%); and mixed genotypes, 13/17 (76.47%). The cumulative time of genotype switch among genotype A was 12 months (range 6-18), in genotype D, 12 months (range 6-48), and mixed genotype, 18 months (range 6-24). The type of inter-genotype switch most frequently detected among genotype A1 was from A1 to D1 5/6 (83.3%), followed by mixed to genotype D3 7/13 (54%) and among intra-genotype changes, from D1 to D3 in 14/20 (70%). Pretreatment HBV genotype was the only factor predicting inter-genotype switches with genotype A or mixed genotypes more likely to undergo inter-genotype switches as compared to genotype D patients (OR 66.6 [13.6-327.0, P < 0.001]). Compared to genotype D, genotype A, and mixed genotypes are more inclined to switch while on tenofovir therapy. Genotypes tend to switch and select to a particular type possibly due to constant antiviral drug pressure. J. Med. Virol. 88:1364-1375, 2016. © 2016 Wiley Periodicals, Inc.
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Antivirais/uso terapêutico , Variação Genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/virologia , Tenofovir/uso terapêutico , Adolescente , Adulto , Idoso , DNA Viral/sangue , Feminino , Variação Genética/efeitos dos fármacos , Genótipo , Hepatite B/virologia , Vírus da Hepatite B/classificação , Hepatite B Crônica/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND & AIMS: No information is available about the frequency or factors that predict spontaneous increases in alanine aminotransferase (ALT) levels in asymptomatic Indian patients with chronic hepatitis B virus (HBV) infection who are HB e antigen (HBeAg) negative and have normal ALT levels. METHODS: We followed 217 asymptomatic patients with chronic HBV who were HBeAg negative, anti-HBe antigen (anti-HBe) positive, and had normal ALT levels. Spontaneous increases in ALT levels (ALT flares) were considered to be >2-fold the upper limit of normal (ULN) and were accompanied by HBV DNA levels>or=10(5) copies/mL or a 100-fold increase from the previously measured level. RESULTS: During a median follow-up period of 69.0 months, spontaneous ALT flares occurred in 43 patients (an annual rate of 4.3%), with cumulative probabilities of 10.8% and 47.3% after 5 and 10 years, respectively. Based on multinomial logistic regression, the probability of an ALT flare correlated with age>or=30 years at presentation (odds ratio [OR], 5.31; 95% confidence interval [CI]: 1.53-18.39; P=.008), male sex (OR, 4.54; 95% CI: 1.01-20.76; P=.05), and presence of a precore mutation (OR, 10.99; 95% CI: 3.67-32.92; P<.001). The median time to spontaneous ALT flare after enrollment was 25 months (range, 1-128 months; 10th percentile=3.4 months). CONCLUSIONS: In asymptomatic patients with chronic HBV infection who have normal ALT levels and are HBeAg negative, the annual rate of ALT flares was 4.3%. Precore mutants, male sex, and age>or=30 years at presentation are independent predictors for an ALT flare. A follow-up every 3 months can capture up to 90% of flares and would help identify patients who require antiviral therapy.
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Alanina Transaminase/sangue , Vírus da Hepatite B , Hepatite B Crônica/sangue , Adulto , Biópsia , DNA Viral/genética , Feminino , Seguimentos , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/etnologia , Hepatite B Crônica/genética , Humanos , Índia , Fígado/enzimologia , Fígado/patologia , Modelos Logísticos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mutação/genética , Valor Preditivo dos Testes , Regiões Promotoras Genéticas/genética , Estudos ProspectivosRESUMO
Mechanism of initial hepatitis B virus (HBV) integrations and kinetics of DNA repair immediately after infection remain essentially unknown impairing understanding of hepadnaviral oncogenesis. WCM260 hepatocytes susceptible to HBV-compatible woodchuck hepatitis virus (WHV) were examined from 15 min to 72 h post-infection (p.i.). WHV strongly induced reactive oxygen species (ROS), transiently inducible nitric oxide (iNOS) and DNA damage from 15 min p.i. All initial WHV-host fusions had the head-to-tail format indicating their formation by non-homologous end joining (NHEJ). Transcription of poly(ADP-ribose) polymerase 1 (PARP1) and X-ray repair cross-complementing protein 1 (XRCC1), the PARP1 binding partner, were induced in 30 min p.i. and that of 8-oxyguanine DNA glycosylse (OGG1) responding to oxidative DNA damage at 12 h p.i. Nicotinamide adenine dinucleotide (NAD+), a marker of PARP1 activation, and heme oxygenase-1 (HO1), an indicator of pro-oxidative stress, were significantly augmented from 15-30 min p.i. Additionally, PARP1 cleavage activity was evident from 30 min p.i. confirming that PARP1-mediated DNA repair became operational almost instatly after hepatocyte contact with virus. By applying complementary approaches, the study showed that initial WHV integration was due to virus-induced oxidative DNA damage and implied that the NHEJ PARP1-dependent repair pathway determined format of the first virus-host DNA junctions.
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Dano ao DNA , Reparo do DNA , Genoma , Vírus da Hepatite B da Marmota/fisiologia , Hepatite B/virologia , Hepatócitos/virologia , Integração Viral , Animais , Células Cultivadas , Hepatite B/genética , Cinética , Marmota , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Replicação ViralRESUMO
BACKGROUND & AIMS: There is a paucity of data on hepatitis B virus (HBV) DNA levels and histologic lesions in patients with chronic HBV (CHBV) infection and persistently normal alanine aminotransferase (ALT) levels (PNALT). We studied the ALT, HBV DNA levels, and spectrum of histologic lesions in such patients. METHODS: One thousand three hundred eighty-seven incidentally detected asymptomatic hepatitis B surface antigen (HBsAg)-positive patients with >/=1-year follow-up and either PNALT (n = 189; hepatitis B e antigen [HBeAg(+)], 73; HBeAg(-), 116) or persistently or intermittently elevated ALT (PIEALT; n = 1198; HBeAg(+), 530; HBeAg(-), 668) were included. RESULTS: In the PIEALT and PNALT patients, baseline DNA >/=5-log copies/mL was seen in 73.8% and 60.3% in HBeAg(+) (P = .018) and 76% and 35.3% in HBeAg(-) (P < .001) patients and histologic fibrosis stage >/=2 in 65.5% and 40.2% in HBeAg(+) (P < .001) and 63.9% and 13.8% in HBeAg(-) (P < .001) patients, respectively. Approximately 21% of HBeAg(-) patients with PNALT and HBV DNA <5-log copies/mL had histologically active liver disease (histologic activity index >/=3 and/or fibrosis stage >/=2). CONCLUSIONS: A fair proportion of patients with CHBV infection with PNALT have HBV DNA >/=5-log copies/mL and significant histologic fibrosis. Use of ALT and HBV DNA levels without resorting to liver biopsy to define "inactive carrier state" in HBeAg(-) PNALT patients may miss histologically significant disease in a proportion of patients.
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Alanina Transaminase/sangue , DNA Viral/análise , Antígenos E da Hepatite B/análise , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/patologia , Adulto , Biópsia , DNA Viral/genética , Progressão da Doença , Feminino , Seguimentos , Genótipo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B Crônica/enzimologia , Hepatite B Crônica/virologia , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
BACKGROUND: The association and profile of surface gene mutations with viral genotypes have been studied in patients with chronic hepatitis B virus (HBV) but not in subjects with occult HBV infection. AIM: This study aimed to investigate the association of surface gene mutations with viral genotypes in occult HBV infection. MATERIALS & METHODS: Of 293 family contacts of 90 chronic HBV index patients, 110 consented for the study. Of 110 subjects, 97 were hepatitis B surface antigen (HBsAg) negative. HBV genotyping was done using direct DNA sequencing. The S-gene was also sequenced in 13 chronic hepatitis B patients to serve as controls. RESULTS: Twenty-eight (28.8%) of the 97 subjects had occult HBV infection. Bidirectional sequencing of partial S-gene was successful in 13 of them. Seven (53.8%) of the viral sequences are genotype A1, two (15.3%) each having genotypes D5&D2 and one each (7.6%) having D1&G genotypes. Seven (53.8%) of the 13 HBsAg positive patients, had genotype D&6 (46.1%) genotype A. A128V & T143M mutations were observed in 5 of 13 (38.4%) subjects and A128V & P127S in 2 of 13 (15.3%) patients (P = 0.385). A128V mutation was seen in two (15.3%) subjects with D2 genotype, while T143M mutation was seen in three (23.07%) subjects with A1genotype. At aa125, three (23.07%) subjects with D5 genotype had methionine instead of threonine. There were wild type sequences in five (38.4%) subjects, one each of D1, G genotypes (20%) and four A1 (80%) genotypes. None of the subjects had G145R mutation. CONCLUSIONS: Occult HBV infection may be common in household contacts of chronic HBV infected patients. Equal prevalence of A&D sub-genotypes was present in occult HBV subjects and in chronic HBV patients. Mutations of the S-gene are genotype specific in both occult as well as chronic HBV infection.
Assuntos
Portador Sadio , Família , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Hepatite B/virologia , Mutação , Adolescente , Adulto , Sequência de Bases , Biópsia , Estudos de Casos e Controles , Análise Mutacional de DNA , DNA Viral/sangue , Feminino , Genótipo , Hepatite B/patologia , Hepatite B/transmissão , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/imunologia , Hepatite B Crônica/patologia , Hepatite B Crônica/transmissão , Humanos , Índia , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Carga Viral , Adulto JovemRESUMO
Integration of hepatitis B virus (HBV) DNA into host's genome is evident in all stages and models of HBV infection. Investigations of the initial virus-host junctions have been just recently initiated since their nature may promote liver oncogenesis immediately following infection. We examined the time-frame and host sites at which HBV integrates in HepG2 cells overexpressing sodium taurocholate co-transporting polypeptide (NTCP) receptor mediating HBV entry. HepG2-NTCP cells were analyzed from 15â¯min to 13 days post-infection (p.i.). The results showed that except for 15â¯min p.i., HBV-host integrations were detected at all time points thereafter. At 30â¯min p.i., virus junctions with retrotransposon SINE and with neuroblastoma breakpoint family member 1 gene were detected. At one-hour p.i., HBV integration with retrotransposon THE-1B-LTR was identified, while virus insertions into proline-rich protein and protein kinase cGMP-dependent type 1 encoding genes were found at 3 h p.i. Fusion with runt-related transcription factor 1 was detected at 24 h p.i. and merges with 9 different genes at 13 day p.i. The data showed that retrotransposon elements are frequent among first-hit sites of HBV insertion. This may suggest a mechanism by which HBV DNA may spread across host's genome from earliest stages of infection.
Assuntos
Vírus da Hepatite B/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Integração Viral/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/genética , DNA Viral/genética , Genoma Humano/genética , Células Hep G2 , Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/biossíntese , Retroelementos/genética , Simportadores/biossínteseRESUMO
AIM: To confirm the presence of recombination, full-length hepatitis B virus (HBV) from chronic patients was sequenced and analyzed. METHODS: Full-length HBV genomes from 12 patients were amplified and sequenced in an automated sequencer. Phylogenetic analysis was carried out on full-length, Core and preS2/Surface regions using MEGA software. SimPlot Boot Scanning and amino acid sequence analysis were performed for confirmation of recombination. RESULTS: Eight patients were infected with genotype D strain; one patient with genotype A and three patients had genotype A and D recombination; two of them had cirrhosis and one had hepatocellular carcinoma. Phylogenetic analysis of core and preS2/surface regions separately showed evidence of genotype A and D recombination. The breakpoints of recombination were found to be at the start of preS2 and at the end of surface coding regions. CONCLUSION: We identified and characterized recombinant A and D genotype HBV in hepatitis B surface antigen (HBsAg)-positive patients.
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DNA Viral , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Mutação , Recombinação Genética , Adulto , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Carcinoma Hepatocelular/virologia , Criança , Análise Mutacional de DNA , Feminino , Genótipo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/classificação , Hepatite B Crônica/complicações , Humanos , Índia , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Precursores de Proteínas/genética , Adulto JovemRESUMO
Protein myristoylation is a key protein modification carried out by N-Myristoyltransferase (NMT) after Methionine aminopeptidase 2 (MetAP2) removes methionine from the amino-terminus of the target protein. Protein myristoylation by NMT augments several signaling pathways involved in a myriad of cellular processes, including developmental pathways and pathways that when dysregulated lead to cancer or immune dysfunction. The emerging evidence pointing to NMT-mediated myristoylation as a major cellular regulator underscores the importance of understanding the framework of this type of signaling event. Various studies have investigated the role that myristoylation plays in signaling dysfunction by examining differential gene or protein expression between normal and diseased states, such as cancers or following HIV-1 infection, however no study exists that addresses the role of microRNAs (miRNAs) in the regulation of myristoylation. By performing a large scale bioinformatics and functional analysis of the miRNAs that target key genes involved in myristoylation (NMT1, NMT2, MetAP2), we have narrowed down a list of promising candidates for further analysis. Our condensed panel of miRNAs identifies 35 miRNAs linked to cancer, 21 miRNAs linked to developmental and immune signaling pathways, and 14 miRNAs linked to infectious disease (primarily HIV). The miRNAs panel that was analyzed revealed several NMT-targeting mRNAs (messenger RNA) that are implicated in diseases associated with NMT signaling alteration, providing a link between the realms of miRNA and myristoylation signaling. These findings verify miRNA as an additional facet of myristoylation signaling that must be considered to gain a full perspective. This study provides the groundwork for future studies concerning NMT-transcript-binding miRNAs, and will potentially lead to the development of new diagnostic/prognostic biomarkers and therapeutic targets for several important diseases.
Assuntos
Aciltransferases/metabolismo , Aminopeptidases/metabolismo , Doenças Transmissíveis/diagnóstico , Metaloendopeptidases/metabolismo , MicroRNAs/metabolismo , Neoplasias/diagnóstico , Aciltransferases/genética , Aminopeptidases/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Análise por Conglomerados , Doenças Transmissíveis/enzimologia , Doenças Transmissíveis/genética , Receptores ErbB/metabolismo , Humanos , Metaloendopeptidases/genética , MicroRNAs/genética , Neoplasias/enzimologia , Neoplasias/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Chronic hepatitis B (CHB) is a major global health problem affecting an estimated 350 million people with more than 786000 individuals dying annually due to complications, such as cirrhosis, liver failure and hepatocellular carcinoma (HCC). Liver transplantation (LT) is considered gold standard for treatment of hepatitis B virus (HBV)-related liver failure and HCC. However, post-transplant viral reactivation can be detrimental to allograft function, leading to poor survival. Prophylaxis with high-dose hepatitis B immunoglobulin (HBIG) and anti-viral drugs have achieved remarkable progress in LT by suppressing viral replication and improving long-term survival. The combination of lamivudine (LAM) plus HBIG has been for many years the most widely used. However, life-long HBIG use is both cumbersome and costly, whereas long-term use of LAM results in resistant virus. Recently, in an effort to develop HBIG-free protocols, high potency nucleos(t)ide analogues, such as Entecavir or Tenofovir, have been tried either as monotherapy or in combination with low-dose HBIG with excellent results. Current focus is on novel antiviral targets, especially for covalently closed circular DNA (cccDNA), in an effort to eradicate HBV infection instead of viral suppression. However, there are several other molecular mechanisms through which HBV may reactivate and need equal attention. The purpose of this review is to address post-LT HBV reactivation, its risk factors, underlying molecular mechanisms, and recent advancements and future of anti-viral therapy.
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Hepatocellular carcinoma (HCC) is a major malignancy in the liver and has emerged as one of the main cancers in the world with a high mortality rate. However, the molecular mechanisms of HCC are still poorly understood. Long noncoding RNAs (lncRNAs) have recently come to the forefront as functional non-protein-coding RNAs that are involved in a variety of cellular processes ranging from maintaining the structural integrity of chromosomes to gene expression regulation in a spatiotemporal manner. Many recent studies have reported the involvement of lncRNAs in HCC which has led to a better understanding of the underlying molecular mechanisms operating in HCC. Long noncoding RNAs have been shown to regulate development and progression of HCC, and thus, lncRNAs have both diagnostic and therapeutic potentials. In this review, we present an overview of the lncRNAs involved in different stages of HCC and their potential in clinical applications which have been studied so far.
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Hepatocellular carcinoma (HCC), one of the leading causes of cancer deaths in the world, is offering a challenge to human beings, with the current modes of treatment being a palliative approach. Lack of proper curative or preventive treatment methods encouraged extensive research around the world with an aim to detect a vaccine or therapeutic target biomolecule that could lead to development of a drug or vaccine against HCC. Biomarkers or biological disease markers have emerged as a potential tool as drug/vaccine targets, as they can accurately diagnose, predict, and even prevent the diseases. Biomarker expression in tissue, serum, plasma, or urine can detect tumor in very early stages of its development and monitor the cancer progression and also the effect of therapeutic interventions. Biomarker discoveries are driven by advanced techniques, such as proteomics, transcriptomics, whole genome sequencing, micro- and micro-RNA arrays, and translational clinics. In this review, an overview of the potential of tissue- and serum-associated HCC biomarkers as diagnostic, prognostic, and therapeutic targets for drug development is presented. In addition, we highlight recently developed micro-RNA, long noncoding RNA biomarkers, and single-nucleotide changes, which may be used independently or as complementary biomarkers. These active investigations going on around the world aimed at conquering HCC might show a bright light in the near future.
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Resolution of chronic hepatitis C is considered when serum HCV RNA becomes repeatedly undetectable and liver enzymes normalize. However, long-term persistence of HCV following therapy with pegylated interferon-α/ribavirin (PegIFN/R) was reported when more sensitive assays and testing of serial plasma, lymphoid cells (PBMC) and/or liver biopsies was applied. Our aim was to reassess plasma and PBMCs collected during and after standard PegIFN/R therapy from individuals who became HCV RNA nonreactive by clinical testing. Of particular interest was to determine if HCV genome and its replication remain detectable during ongoing treatment with PegIFN/R when evaluated by more sensitive detection approaches. Plasma acquired before (nâ=â11), during (nâ=â25) and up to 12-88 weeks post-treatment (nâ=â20) from 9 patients and PBMC (nâ=â23) from 3 of them were reanalyzed for HCV RNA with sensitivity <2 IU/mL. Clone sequencing of the HCV 5'-untranslated region from plasma and PBMCs was done in 2 patients. HCV RNA was detected in 17/25 (68%) plasma and 8/10 (80%) PBMC samples collected from 8 of 9 patients during therapy, although only 5.4% plasma samples were positive by clinical assays. Among post-treatment HCV RNA-negative plasma samples, 9 of 20 (45.3%) were HCV reactive for up to 59 weeks post-treatment. Molecularly evident replication was found in 6/12 (50%) among PBMC reactive for virus RNA positive strand collected during or after treatment. Pre-treatment point mutations persisted in plasma and/or PBMC throughout therapy and follow-up. Therefore, HCV is not completely cleared during ongoing administration of PegIFN/R otherwise capable of ceasing progression of CHC and virus commonly persists at levels not detectable by the current clinical testing. The findings suggest the need for continued evaluation even after patients achieve undetectable HCV RNA post-treatment.
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Antivirais/uso terapêutico , Hepacivirus/isolamento & purificação , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Antivirais/administração & dosagem , Feminino , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C Crônica/virologia , Humanos , Interferon-alfa/administração & dosagem , Limite de Detecção , Masculino , Polietilenoglicóis/administração & dosagem , RNA Viral/sangue , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Ribavirina/administração & dosagem , Replicação ViralRESUMO
Hepatitis B virus (HBV) genotypes are important in both the clinical manifestation of disease and treatment response. Although Kenya belongs to the African Region (AFR-E) characterized by high mortality and hyperendemicity of HBV, there is a paucity of HBV genotyping data. The aim of this study was to molecularly characterize the basic core promoter/precore (BCP/PC) and complete surface (S) regions of HBV isolated from 61 HBsAg-positive liver disease patients attending Kenyatta National Hospital in Nairobi. HBsAg, HBeAg and viral loads were determined. HBV DNA was amplified and sequenced from 58/61 patients. In addition to the complete genome of two isolates, the BCP/PC and the complete S regions of 43 and 38 isolates, respectively were sequenced. Following phylogenetic analysis of the S region, 38 isolates clustered with subgenotype A1, whereas two isolates clustered with genotype D, one with subgenotype D1 and another as an outlier of the clade containing subgenotype D6 and the D/E recombinant. When the complete genome of the latter isolate was sequenced it clustered with D6. The majority of isolates belonged to serological subtype adw2 and only four to ayw2. Three distinct groups of subgenotype A1, distinguished by different amino acid motifs, circulate in Kenya: two in the African cluster and a monophyletic clade in the "Asian" cluster. HBeAg-negativity was a result of G1896A in genotype D isolates, whereas in subgenotype A1, the HBeAg-negativity was a result of mutations in the Kozak region (1809-1812) or precore start codon (1814-1816). Mutations at positions 1762 and 1764 occurred more frequently in HCC patients (p<0.05). In conclusion, subgenotypes A1, D1 and D6 circulate in liver disease patients in Kenya, with A1 predominating.
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Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Cirrose Hepática/virologia , Neoplasias Hepáticas/virologia , Adulto , Sequência de Bases , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Estudos Transversais , DNA Viral/sangue , DNA Viral/genética , Feminino , Genótipo , Técnicas de Genotipagem , Antígenos de Superfície da Hepatite B/genética , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/genética , Humanos , Quênia , Cirrose Hepática/diagnóstico , Cirrose Hepática/genética , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Masculino , Filogenia , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Carga ViralRESUMO
INTRODUCTION: Hepatocellular injury is often progressive in patients with hepatitis B e antigen negative chronic hepatitis B (HBeAg -ve CHB). There is scant data on association of core mutations occurring in patients with HBeAg -ve CHB with severity of liver disease. MATERIALS AND METHODS: Hundred and eighteen patients with chronic infection who were HBeAg negative, anti-HBe, and HBV DNA positive were enrolled. Precore and core regions were amplified, sequenced, and analyzed for precore, T helper, cytotoxic T lymphocytes (CTLs), B-cell epitope, and core carboxy-terminal region mutations. RESULTS: Majority of patients were infected with HBV genotype D: 96 (81%) [D1: 16, D2: 55 and D5: 25] followed by genotype A1: 15 (13%) and genotype C: 7 (6%) [C1: 5 and unidentified subgenotype C: 2]. Classical (A1896) as well as nonclassical precore region mutations were detected in 30 (25%) and in 9 (7.6%) patients, respectively. Core immune escape, core carboxy-terminal mutations and truncations were detected in 61 (52%), 11 (9.3%), and 14 (12%) patients, respectively. Three core immune escape mutations were significantly higher in patients with coexisting precore stop codon compared with patients without precore stop codon mutation, cT12S (43 vs. 8%, p < 0.001), cS21T (16 vs. 3.4%, p < 0.026), and cE77D (30 vs. 4.5%, p < 0.002). When frequency of core immune escape mutations was compared among CHB and decompensated patients, and cT12S: (27 vs. 10%, p < 0.05), cS21T (16 vs. 1.35%, p < 0.01), cT67P/N: (20 vs. 4%, p < 0.001), cE113D (11.37 vs. 1.35%, p < 0.05), and cP130T/Q (7 vs. 0%, p < 0.001) mutations were found to be significantly higher in decompensated patients. CONCLUSION: Core immune-escape mutations cT12S, cS21T, cT67P, cE113D, and cP130T/Q are significantly higher in decompensated liver disease patients and could influence the severity of liver disease in HBeAg -ve CHB patients.
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BACKGROUND AND AIM: Virologic breakthrough (VBTH) during long-term lamivudine therapy is believed to be due to the emergence of rtYM204I/VDD mutants. We observed VBTH in 17 of 67 patients receiving long-term lamivudine therapy. The YMDD mutant at the onset of VBTH and/or subsequently was seen in eight (47%) of 17 such patients. We investigated other potential loci in the viral genome contributing to VBTH. METHODS: Chronic hepatitis B (CHB) patients (n=17) on long-term (>or=12 months) lamivudine therapy who had at least one episode of VBTH were selected (group I). Age, sex, serology and baseline viral load matched patients on long-term lamivudine without VBTH (n=12) served as controls (group II). Hepatitis B virus (HBV) DNA sequences were analyzed for pre-S, surface, polymerase, core promoter, precore and core regions and were compared with sequences of respective genotypes. RESULTS: Group I patients with VBTH (n=17) either had rtYM204I/VDD mutations (n=8, group Ia YMDD) or no rtYM204I/VDD mutations (n=9, group Ib non-YMDD). Group Ia patients had median baseline HBV DNA of 2794 pg/mL (range 3-9166 pg/mL) and a mean alanine aminotransferase (ALT) level of 86+/-33 IU/L. In group Ib, the median baseline viral DNA was 916 pg/mL (range 8.3-5787 pg/mL) and the mean ALT was 61+/-38 IU/L. The first VBTH in group Ia and Ib patients was noted at 21+/-9 months and 15.2+/-5.9 months, respectively, with a rise in HBV DNA levels from undetectable limits to 952 pg/mL (range 4.3-4875 pg/mL) and 571 pg/mL (range 1.2-1970 pg/mL), respectively. Core promoter mutations were seen in five of eight (62.5%) and in six of nine (66.6%); classic double mutations (A1762T/G1764A) of core promoter region were detected in two and three patients and novel double mutations of core promoter (G1764T/C1766G) in one patient each of group Ia and Ib patients, respectively. Compared to 11 (68%) of 17 group I patients, only three (25%) of 12 patients in group II had core promoter mutations (P<0.05). No patient in group II had double mutations of the core promoter region. No significant difference in viral mutations was seen in any other region of the viral genome between group I and group II patients. In group I, none of the 15 patients (two died of hepatocellular carcinoma), but five (42%) group II patients achieved hepatitis B e antigen (HBeAg) seroconversion and sustained response by month 24 (P<0.05). CONCLUSION: Both core promoter and YMDD motif mutation(s) are associated with VBTH in patients on long-term lamivudine therapy. Whether or not these promoter mutations in the absence of YMDD mutations confer drug resistance needs to be studied in an in vitro cell culture system, as they could create novel and stronger binding sites for hepatocyte nuclear factors.
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DNA Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Lamivudina/uso terapêutico , Mutação , Regiões Promotoras Genéticas/genética , Inibidores da Transcriptase Reversa/uso terapêutico , Adulto , DNA Viral/efeitos dos fármacos , Feminino , Seguimentos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Humanos , Masculino , Técnicas de Amplificação de Ácido Nucleico , Fatores de Tempo , Carga ViralRESUMO
Spontaneous mutations of hepatitis B virus (HBV) could influence the severity of liver disease. Since the basal core promoter (BCP) and the precore (Pc) regions are important for viral replication, these regions were examined for naturally occurring mutations and were correlated with the genotype, e antigen status, and severity of liver disease. In 82 patients with histologically confirmed chronic hepatitis B, the BCP and Pc regions were sequenced and aligned with known wild-type sequences. Sequence based HBV genotyping was done and HBV DNA was quantified. Thirty-three (40%) patients had decompensated chronic liver disease and the remaining patients had chronic hepatitis B. Forty-six (56%) patients were HBeAg positive. HBV genotype A was found in 28%, D in 65%, and B/C in 7.3%. The Pc G1896A mutation was more common in HBeAg-negative (33% vs. 2%, P < 0.01) patients and was genotype D specific. The Pc G1862T mutation was detected more often in HBeAg-positive than HBeAg-negative (37% vs. 11%, P < 0.01) patients and was genotype A specific (P < 0.01). BCP mutations at the 1,762/64 nucleotide positions were common in HBeAg negative than positive (36% vs. 13%, P < 0.05) and were equally common in different genotypes. TA 1-3 region mutations of the BCP were significantly higher in HBeAg-negative as compared to HBeAg-positive patients (78% vs. 26%, P < 0.01). BCP mutations had significantly higher HBV DNA levels. It is concluded that Pc G1862T mutant is Genotype A-specific but is not always associated with e antigen. The TA 1-3 rich mutations of BCP region are also associated with the absence of e antigen in Indian patients.