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1.
Viruses ; 11(11)2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671829

RESUMO

The hepatitis delta virus (HDV) is a globally distributed agent, and its genetic variability allows for it to be organized into eight genotypes with different geographic distributions. In South America, genotype 3 (HDV-3) is frequently isolated and responsible for the most severe form of infection. The objective of this study was to evaluate the evolutionary and epidemiological dynamics of HDV-3 over the years and to describe its distribution throughout this continent in an evolutionary perspective. While using Bayesian analysis, with strains being deposited in the Nucleotide database, the most recent common ancestor was dated back to 1964 and phylogenetic analysis indicated that the dispersion may have started in Brazil, spreading to Venezuela and then to Colombia, respectively. Exponential growth in the effective number of infections was observed between the 1950s and 1970s, years after the first report of the presence of HDV on the continent, during the Labrea Black Fever outbreak, which showed that the virus continued to spread, increasing the number of cases decades after the first reports. Subsequently, the analysis showed a decrease in the epidemiological levels of HDV, which was probably due to the implantation of the vaccine against its helper virus, hepatitis B virus, and serological screening methods implemented in the blood banks.


Assuntos
Hepatite D/virologia , Vírus Delta da Hepatite/classificação , Vírus Delta da Hepatite/genética , Teorema de Bayes , Evolução Molecular , Variação Genética , Genótipo , Hepatite D/epidemiologia , Hepatite D/transmissão , Vírus Delta da Hepatite/isolamento & purificação , Antígenos da Hepatite delta/genética , Humanos , Filogenia , Filogeografia , RNA Viral/genética , América do Sul/epidemiologia
3.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728256

RESUMO

Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate; and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, or whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated using in vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them.IMPORTANCE Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus, and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication, and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen.


Assuntos
Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Transcrição Genética/fisiologia , Replicação Viral/fisiologia , Linhagem Celular , Antígenos da Hepatite delta/genética , Humanos , RNA Mensageiro/genética , RNA Viral/genética
4.
Biochem Cell Biol ; 97(2): 130-139, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30153423

RESUMO

Liver coinfection by hepatitis B virus (HBV) and hepatitis D virus (HDV) can result in a severe form of hepatocellular carcinoma with poor prognosis. Coinfection with HDV and HBV causes more deleterious effects than infection with HBV alone. Clinical research has shown that glutathione S-transferase P1 (GSTP1), a tumor suppressor gene, is typically downregulated in liver samples from hepatitis-infected patients. In the present study, our data indicated that small HDV antigen (s-HDAg) could specifically bind to GSTP1 mRNA and significantly downregulate GSTP1 protein expression. For the human fetal hepatocyte cell line L-02, cells transfected with s-HDAg, along with decreased GSTP1 expression, there was a significant accumulation of reactive oxygen species (ROS) and increased apoptotic ratios. Restoring GSTP1 expression through silencing s-HDAg via RNAi or overexpressing exogenous GSTP1 could largely recover the abnormal cell status. Our results revealed a novel potential mechanism of HDV-induced liver injury and hepatocarcinogenesis: s-HDAg can inhibit GSTP1 expression by directly binding to GSTP1 mRNA, which leads to accumulation of cellular ROS, resulting in high cellular apoptotic ratios and increased selective pressure for malignant transformation. To our knowledge, this is the first study to examine s-HDAg-specific pathogenic mechanisms through potential protein-RNA interactions.


Assuntos
Transformação Celular Viral , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Glutationa S-Transferase pi/biossíntese , Vírus Delta da Hepatite/metabolismo , Antígenos da Hepatite delta/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , RNA Mensageiro/metabolismo , Linhagem Celular , Glutationa S-Transferase pi/genética , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/genética , Humanos , Fígado/lesões , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , RNA Mensageiro/genética
5.
J Biol Chem ; 291(50): 26226-26238, 2016 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-27807029

RESUMO

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198-210), containing the 10-amino acid TAT peptide and HDAg-L(198-210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Núcleo Celular/metabolismo , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , Sinais de Localização Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Vírion/metabolismo , Montagem de Vírus/fisiologia , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Núcleo Celular/virologia , Células Hep G2 , Antígenos da Hepatite delta/genética , Humanos , Sinais de Localização Nuclear/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Peptídeos/farmacologia , Domínios Proteicos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Vírion/genética , Montagem de Vírus/efeitos dos fármacos
6.
Virus Res ; 224: 6-11, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27515509

RESUMO

Hepatitis Delta virus (HDV) is not well known, even though HDV and Hepatitis B virus (HBV) co-infection leads to severe forms of acute and chronic liver diseases. HDV is endemic in the Western Amazon region. Recently, the HDV genotype 8 was found in chronic patients followed at the center for liver studies in the Northeast Brazil, Maranhão. Previous studies suggested that this genotype was introduced in Maranhão during the slave trade. The presence of HDV in that study, which was done outside the Amazon region, led us to investigate whether the virus is found infecting individuals in other regions of Maranhão as well. Thus, we screened ninety-two HBsAg positive individuals from five Municipalities of Maranhão for anti-HD antibody and eight were found positive (8.7%). These eight positive individuals were submitted to polymerase chain reaction (PCR) to investigate active HDV infection. Half of them were positive for a fragment sequence of the delta antigen; their sequence samples were submitted to genotype characterization by phylogenetic analysis. All sequences clustered in a unique branch of the tree separated from the other branch described in Africa. Our study confirmed the presence of HDV-8 in Maranhão. These infected individuals had no evidence of contact with African people. Furthermore, we found individuals infected with HDV-8 in two more different municipalities. More studies like ours are urgent because the co-infection HBV/HDV is more difficult to treat. Identification of the endemic regions and implementation of healthy policies for preventing this infection are urgent in this region.


Assuntos
Doenças Endêmicas , Pessoas Escravizadas , Hepatite D Crônica/epidemiologia , Hepatite D Crônica/virologia , Vírus Delta da Hepatite/classificação , Antígenos da Hepatite delta/genética , Adulto , África/epidemiologia , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Coinfecção/virologia , Pessoas Escravizadas/história , Feminino , Genótipo , Vírus da Hepatite B/genética , Hepatite B Crônica/epidemiologia , Hepatite B Crônica/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/isolamento & purificação , Antígenos da Hepatite delta/sangue , História do Século XVI , Humanos , Fígado/patologia , Fígado/virologia , Masculino , Pessoa de Meia-Idade , Filogenia , Análise de Sequência de DNA , Adulto Jovem
7.
J Virol ; 88(13): 7402-11, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24741096

RESUMO

UNLABELLED: The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2'OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg. IMPORTANCE: RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex.


Assuntos
Antígenos da Hepatite delta/química , Antígenos da Hepatite delta/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Bases , Antígenos da Hepatite delta/genética , Humanos , Microscopia de Força Atômica , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Cadeia Dupla/genética , RNA Viral/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Homologia de Sequência do Ácido Nucleico
8.
J Virol Methods ; 197: 34-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24140186

RESUMO

Hepatitis D virus (HDV) infection is often accompanied by hepatitis B virus (HBV) infection. Co-infection of HDV and HBV may lead to more severe symptoms and even death. Current methods for HDV diagnosis have high false-positive rates and show significant result discrepancies. The Abbott AxSYM AUSAB test, currently a standard test for HDV detection, is too laborious and expensive for routine application. Therefore, new sensitive and cost-efficient methods for HDV diagnosis are urgently needed. In this study, S-HDAg protein was produced in high yield in Escherichia coli. Optimal protein production was achieved by optimization of S-HDAg gene codons according to the codon preference of E. coli and using host cells with appropriate cell density. Under optimal expression conditions, the S-HDAg protein expression yield (30mg/l) was the highest among any proteins expressed in E. coli. A standard enzyme-linked immunosorbent assay (ELISA) for HDV was developed using the purified S-HDAg protein, which showed high specificity against hepatitis B, C, D and E viruses. Overall, the ELISA had superior specificity and sensitivity compared with the Abbott AxSYM AUSAB test and was also more convenient and cost-efficient.


Assuntos
Anticorpos Anti-Hepatite/sangue , Hepatite D/diagnóstico , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Expressão Gênica , Antígenos da Hepatite delta/genética , Humanos , Imunoglobulina G/sangue , Proteínas Recombinantes/genética , Sensibilidade e Especificidade
9.
Antivir Ther ; 18(3 Pt B): 541-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23792471

RESUMO

Hepatitis delta is an inflammatory liver disease caused by infection with HDV. HDV is a single-stranded circular RNA pathogen with a diameter of 36 nm. HDV is classified in the genus Deltavirus and is still awaiting a final taxonomic classification up to the family level. HDV shares similarities with satellite RNA and viroids including a small circular single-stranded RNA with secondary structure that replicates through the 'double rolling circle' mechanism. The HDV RNA genome is capable of self-cleavage through a ribozyme and encodes only one structural protein, the hepatitis delta antigen (HDAg), from the antigenomic RNA. There are two forms of HDAg, a shorter (S; 22 kDa) and a longer (L; 24 kDa) form, the latter generated from an RNA editing mechanism. The S form is essential for viral genomic replication. The L form participates in the assembly and formation of HDV. For complete replication and transmission, HDV requires the hepatitis B surface antigen (HBsAg). Thus, HDV infection only occurs in HBsAg-positive individuals, either as acute coinfection in treatment-naive HBV-infected persons, or as superinfection in patients with pre-existing chronic hepatitis B (CHB). HDV is found throughout the world, but its prevalence, incidence, clinical features and epidemiological characteristics vary by geographic region. There are eight genotypes (1 to 8) distributed over different geographic areas: HDV-1 is distributed worldwide, whereas HDV-2 to 8 are seen more regionally. Levels of HDV viraemia change over the course of HDV infection, being significantly higher in patients with early chronic hepatitis than in cirrhosis. Chronic HDV infection leads to more severe liver disease than chronic HBV monoinfection with an accelerated course of fibrosis progression, an increased risk of hepatocellular carcinoma and early decompensation in the setting of established cirrhosis. Current treatments include pegylated interferon-α and liver transplantation; the latter of which can be curative. Further studies are needed to develop better treatment strategies for this challenging disease.


Assuntos
Hepatite D/terapia , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/fisiologia , Coinfecção , Genótipo , Hepatite B/complicações , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite D/diagnóstico , Vírus Delta da Hepatite/patogenicidade , Vírus Delta da Hepatite/ultraestrutura , Antígenos da Hepatite delta/química , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/metabolismo , Humanos , Transplante de Fígado , Filogenia , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Vírus Satélites/genética , Vírus Satélites/patogenicidade , Replicação Viral
10.
J Virol ; 87(15): 8665-74, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23740973

RESUMO

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.


Assuntos
Motivos de Aminoácidos , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arginina/genética , Arginina/metabolismo , Linhagem Celular , Análise Mutacional de DNA , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/genética , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas de Ligação a RNA/genética , Deleção de Sequência
11.
Expert Rev Anti Infect Ther ; 11(5): 489-98, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23627855

RESUMO

Hepatitis delta virus (HDV) is a defective RNA virus that depends on hepatitis B virus (HBV) for its lifecycle. Treatment of chronic HDV infection is difficult as it does not have an enzymatic function as a target, such as polymerases and proteases of HBV and hepatitis C virus. Recently, it has been suggested that farnesyl transferase could be an enzymatic target. Currently, interferon is the only agent against HDV infection. Virological response has risen to 20-47% with pegylated interferon. Monotherapy of nucleos(t)ide analogs are ineffective against the HDV infection, but adefovir and pegylated interferon combination therapy have had some advantages for reduction of HBV surface antigen (HBsAg) levels. Recent studies suggest that measuring HBsAg levels during treatment could be more meaningful than HDV RNA negativity to predict virological response. Prenylation inhibitors that can affect the interactions between the large HDV antigen and HBsAg in the HDV virion are expected treatments for HDV infection. More studies are needed to understand the molecular mechanisms of HDV to manage the disease.


Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Hepatite D Crônica/tratamento farmacológico , Vírus Delta da Hepatite/efeitos dos fármacos , Interferon-alfa/uso terapêutico , Organofosfonatos/uso terapêutico , Polietilenoglicóis/uso terapêutico , Adenina/uso terapêutico , Quimioterapia Combinada , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Farnesil-Difosfato Farnesiltransferase/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/virologia , Hepatite D Crônica/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/metabolismo , Antígenos da Hepatite delta/genética , Humanos , Prenilação de Proteína/efeitos dos fármacos , Proteínas Recombinantes/uso terapêutico , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos
12.
Virus Res ; 170(1-2): 75-84, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23022530

RESUMO

Hepatitis delta virus (HDV) is an RNA virus and eight clades of HDV have been identified. HDV clade 3 (HDV-3) is isolated only in the northern area of South America. The outcome of HDV-3 infection is associated with severe fulminant hepatitis. Variations in the large delta antigen (LDAg) between HDV clade 1 (HDV-1) and HDV-3 have been proposed to contribute to differences in viral secretion efficiency, but which changes might be relevant remains unclear. The control of subcellular localization of LDAg has been reported to be associated with post-translational modifications, such as phosphorylation and isoprenylation. We have observed evidence for acetylation on the LDAg of HDV-3 (LDAg-3) and LDAg of HDV-1 (LDAg-1). Green fluorescent protein-fused LDAg-3 (GFP-LD3) was used to investigate the cellular distribution and secretion of the protein. Sequence alignment of LDAg amino acids suggested that lysine-71 of LDAg-3 could be an acetylation site. Expression of a mutant form of LDAg-3 with an arginine-substitution at lysine-71 (GFP-LD3K71R) showed a distribution of the protein predominantly in the cytoplasm instead of the nucleus. Western blot analyses of secreted empty viral particles (EVPs) revealed a higher amount of secreted GFP-LD3K71R compared to GFP-LD3. Furthermore, the ectopic expression of p300, a histone acetyltransferase, led to a reduction of GFP-LD3 in EVPs. By contrast, expression of three histone deacetylases (HDAC-4, -5, and -6) facilitated the secretion of GFP-LD3. Combined, our observations support the hypothesis that the acetylation status of LDAg-3 plays a role in regulating LDAg-3's localization inside the nucleus or cytoplasm, and its secretion.


Assuntos
Genótipo , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/química , Antígenos da Hepatite delta/metabolismo , Lisina , Acetilação , Linhagem Celular , Núcleo Celular/metabolismo , Genes Reporter , Antígenos de Superfície da Hepatite B/metabolismo , Antígenos da Hepatite delta/genética , Histonas/metabolismo , Humanos , Transporte Proteico , Fatores de Tempo , Transfecção , Liberação de Vírus , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
13.
Artigo em Chinês | MEDLINE | ID: mdl-23002540

RESUMO

OBJECTIVE: To prepare HDAg with biological activities as a candidate of diagnostic reagent. METHODS: To synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA: RESULTS: Plasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies. CONCLUSION: HDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.


Assuntos
Hepatite D/diagnóstico , Antígenos da Hepatite delta/imunologia , Ensaio de Imunoadsorção Enzimática , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/isolamento & purificação , Humanos , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
14.
Virol J ; 9: 162, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22894717

RESUMO

BACKGROUND: In spite of a high occurrence of Hepatitis Delta in the province of Sindh in Pakistan, no genetic study of Hepatitis Delta virus (HDV) isolates from this region was carried out. The aim of this study is to analyze the genetic proximity within local HDV strains, and relationship with other clades of HDV, using phylogenetic analysis. RESULTS: Phylogenetic analysis of nucleotide sequences of the Hepatitis Delta Antigen (HDAg) R0 region obtained in this study, showed considerable diversity among the local strains with a potential subgroup formation within clade I. The multiple sequence alignment of predicted amino acids within clade I showed many uncommon amino acid substitutions within some conserved regions that are crucial for replication and assembly of HDV. CONCLUSIONS: The studied strains showed a range of genetic diversity within HDV clade I. There is clustering of sequences into more than one group, along with formation of potential subgroup within clade I. Clustering shows the genetic closeness of strains and indicates a common origin of spread of HDV infection. Further phylogeny-based studies may provide more information about subgroup formation within clade I and may be used as an effective tool in checking and/or preventing the spread of hepatitis D virus infection in this region.


Assuntos
Variação Genética , Antígenos de Superfície da Hepatite B/sangue , Hepatite B/complicações , Hepatite D/virologia , Vírus Delta da Hepatite/classificação , Vírus Delta da Hepatite/genética , Filogenia , Adulto , Análise por Conglomerados , Feminino , Vírus Delta da Hepatite/isolamento & purificação , Antígenos da Hepatite delta/genética , Humanos , Masculino , Dados de Sequência Molecular , Paquistão , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
15.
J Clin Virol ; 54(3): 223-8, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22608280

RESUMO

BACKGROUND: Guidelines suggest that all HBsAg-positive patients should be tested for anti-HDV IgG antibodies and to confirm active hepatitis D virus (HDV) infection by detection of HDV RNA by reverse transcriptase (RT) polymerase chain reaction (PCR). OBJECTIVES: The aim of this study was to determine the serological prevalence and molecular features of HDV within an Amerindian community from Argentina exhibiting positivity for HBsAg and/or anti-HBc total Ig. STUDY DESIGN: Forty-six plasma samples were tested for the detection of total anti-HDV antibodies by ELISA. Concomitantly, a partial RNA region coding for the delta antigen (HDAg) was amplified by RT-nested PCR (RT-nPCR). In silica translation of DNA sequences into the amino acid (aa) sequence of HDAg-S (aa110-195) and HDAg-L (aa110-214) was performed. RESULTS: Out of 46 HDV non-reactive samples by ELISA, 3 were HDV RNA positive by RT-nPCR. These samples were anti-HBc-only positive, 2 of them identified as cases of occult hepatitis B infection (OBI). The 3 cases were HBeAg-negative and showed normal ALT/AST levels. All sequences were ascribed to HDV genotype 1, but exhibited nucleotide differences in HDAg-L coding region, among which, mutations at codons 197 and 201 - reportedly known to promote in vitro an unsuitable interaction with HBsAg - were observed. CONCLUSIONS: These results provide evidence of covert HDV infection even among OBI, highlighting the need to reevaluate the currently applied guidelines for HDV diagnostic algorithms, as well as to explore if the observed mutations promote any effect on HDV pathogenesis.


Assuntos
Hepatite B/complicações , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/sangue , Antígenos da Hepatite delta/genética , Adolescente , Adulto , Idoso , Argentina , Doenças Assintomáticas , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Anticorpos Anti-Hepatite/sangue , Humanos , Imunoglobulina G/sangue , Índios Sul-Americanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Adulto Jovem
16.
J Med Virol ; 84(3): 445-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22246830

RESUMO

Co-infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) has been shown to be associated with a more severe form of acute and chronic hepatitis. Cloning and expression of recombinant HDV antigen (rHDAg) in Escherichiacoli are described. Using purified rHDAg, a cost-effective indirect anti-HDV enzyme-linked immunosorbent assay (ELISA) kit was developed. Direct comparison of 15 known HDV-positive sera and 15 HDV-negative sera showed concordance agreement between the new assay kit and the Abbott Murex Anti-Delta (total) kit. In addition, 1,486 hepatitis B surface antigen (HBsAg) positive blood samples collected from various areas of China were tested using this indirect anti-HDV ELISA. It was found that 1.2% (95% CI: 0.7-1.9%) of the samples were anti-HDAg positive. It is suggested that the prevalence of HDV and HBV co-infection in China is relatively low.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Anti-Hepatite/imunologia , Hepatite B/complicações , Hepatite D/epidemiologia , Vírus Delta da Hepatite/imunologia , China/epidemiologia , Coinfecção , Hepatite B/virologia , Vírus da Hepatite B/imunologia , Hepatite D/complicações , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/imunologia , Antígenos da Hepatite delta/isolamento & purificação , Humanos , Prevalência
17.
Artigo em Chinês | MEDLINE | ID: mdl-23547452

RESUMO

OBJECTIVE: To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. METHODS: Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. RESULTS: SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. CONCLUSION: Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.


Assuntos
Engenharia Genética/métodos , Antígenos da Hepatite delta/genética , Proteínas Recombinantes/biossíntese , Eletroforese em Gel de Poliacrilamida , Hepatite D/diagnóstico , Antígenos da Hepatite delta/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação
18.
Arch Virol ; 156(12): 2215-20, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21984217

RESUMO

Hepatitis delta virus (HDV) is a subviral agent of hepatitis B virus (HBV), and its life cycle is dependent on HBV. It is commonly accepted that HDV has eight distinct genotypes. In this study, the complete nucleotide sequences of HDV genomes isolated from nine Turkish patients were obtained by RT-PCR using two pairs of primers that cover the entire HDV genome. PCR products were sequenced directly. The results showed that these 9 isolates were approximately 1680 base pairs in length and clustered in the genotype HDV-1 branch when phylogenetic analysis was done with the sequences together with the complete sequences of HDV genomes representing each genotype retrieved from GenBank. Analysis of a portion of the large hepatitis D antigen (L-HDAg) gene showed that sequence similarity among these Turkish isolates is between 87.4 and 97.1%, and the Turkish isolates have the most sequence similarity to HDV-1 (90.5%), while they have the least sequence similarity to HDV-3 (64.1%). Full-genome analysis indicates that the sequence similarity is between 80.7 and 95.4%, and the highest sequence similarity is 84.8% (between the Turkish isolates and HDV-1). The lowest sequence similarity is 56.4% (between the Turkish isolates and HDV-3). In conclusion, phylogenetic analysis shows that the Turkish HDV isolates belong to HDV-1.


Assuntos
Hepatite D Crônica/virologia , Vírus Delta da Hepatite/classificação , Vírus Delta da Hepatite/genética , Adulto , Sequência de Bases , Primers do DNA/genética , Genoma Viral , Vírus Delta da Hepatite/isolamento & purificação , Antígenos da Hepatite delta/genética , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Turquia
19.
Dig Liver Dis ; 43 Suppl 1: S15-8, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21195366

RESUMO

The hepatitis D virus (HDV) was discovered in Italy in the mid-1970s during a major outbreak of hepatitis D in the Mediterranean basin. The outbreak has been brought under control in Europe and throughout the industrialized world in the last twenty years; in parallel, the clinical pattern of HDV disease has consistently changed. Though the decline of hepatitis D has diminished attention to this problem and at present testing for HDV is not seldom neglected, hepatitis D is not eradicated in Europe and its circulation did not decline further in the last decade. Fresh new cases are cumulating in migrants from HDV endemic areas of the developing world. Hepatitis D remains a major health problem in many developing areas with outbreaks of the disease continuing to be reported from Asia, Africa and South Africa.


Assuntos
Hepatite B/complicações , Hepatite D/epidemiologia , Hepatite D/etiologia , Vírus Delta da Hepatite/genética , RNA Viral/genética , Animais , Antígenos de Superfície da Hepatite B/química , Hepatite D/genética , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/patogenicidade , Antígenos da Hepatite delta/química , Antígenos da Hepatite delta/genética , Humanos
20.
J Virol ; 84(2): 918-27, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19889771

RESUMO

Hepatitis delta antigen (HDAg) is a nuclear protein that is intimately involved in hepatitis delta virus (HDV) RNA replication. HDAg consists of two protein species, the small form (S-HDAg) and the large form (L-HDAg). Previous studies have shown that posttranslational modifications of S-HDAg, such as phosphorylation, acetylation, and methylation, can modulate HDV RNA replication. In this study, we show that S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhanced HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. This result supports our previous proposition that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that for genomic RNA synthesis and mRNA transcription, requiring different modified forms of S-HDAg. Sumoylation represents a new type of modification for HDAg.


Assuntos
Genoma Viral , Vírus Delta da Hepatite/metabolismo , Antígenos da Hepatite delta/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteína SUMO-1/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/genética , Humanos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Viral/genética , Proteína SUMO-1/genética , Transfecção , Técnicas do Sistema de Duplo-Híbrido
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