RESUMO
The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.
Assuntos
Variação Antigênica , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B/virologia , Indígenas Sul-Americanos , Substituição de Aminoácidos , Feminino , Genótipo , Hepatite B/epidemiologia , Hepatite B/etnologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/classificação , Vírus da Hepatite B/imunologia , Humanos , Gravidez , Complicações Infecciosas na Gravidez/virologia , Análise de Sequência , Venezuela/epidemiologiaRESUMO
Recently, a new virus related to flaviviruses, the hepatitis G virus (HGV), or GBV-C virus, was discovered as a putative blood-borne human pathogen. HGV RNA (NS5 region) was amplified by reverse transcription-nested PCR in the sera of 6 of 64 (9%) hemodialysis patients; 2 of 80 (2.5%) West Yukpa Amerindians, a population with a high rate of HBV infection but negative for HCV infection; and 1 patient with an acute episode of non-A, non-B, non-C hepatitis (NABCH). The patterns of single-strand conformation polymorphism of the amplified products were unique among different specimens and similar on follow-up for hemodialysis patients. All patients tested remained HGV RNA positive 1 and 2 years later, without major sequence variation, except for the NABCH patient, for whom a double infection and an apparent clearance of the original dominant variant was observed after 2 years. The sequences of the NS5 amplified products demonstrated 85 to 90% identity with other reported HGV sequences.
Assuntos
Flaviviridae/isolamento & purificação , Hepatite Viral Humana/diagnóstico , Hepatite Viral Humana/epidemiologia , RNA Viral/sangue , RNA Viral/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Flaviviridae/genética , Hepatite C/diagnóstico , Hepatite E/diagnóstico , Hepatite Viral Humana/sangue , Humanos , Indígenas Sul-Americanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , RNA Viral/genética , Diálise Renal/efeitos adversos , Análise de Sequência de RNA , Venezuela/epidemiologiaRESUMO
Antibody reactivities to hepatitis C virus (HCV) antigens and to synthetic peptides derived from different parts of the HCV genome (core, NS4, and NS5) were evaluated in HCV-infected hemodialysis patients. In the RIBA 3 assay, NS5 was significantly less recognizable by sera of hemodialysis patients compared to other HCV-infected subjects. Among hemodialysis patients, those coinfected with hepatitis B virus (HBV) (positive for hepatitis B surface antigen [HBsAg+]) showed a reduction in reactivity to C33 and C100. Sera of only 23% of the hemodialysis patients (37 of 161) reacted with more than three of eight peptides tested, significantly fewer than the 60% (12 of 20) of the sera of other HCV-infected patients tested (P = 0.001). This immunosuppression was also manifested by a reduced frequency of recognition of additional peptides on follow-up. An even more reduced reactivity was observed among the HBV-coinfected patients (HBsAg+). The low-responder hemodialysis patients were not infected with any particular genotype of HCV, and the same HCV genotypes observed in the whole group of hemodialysis patients (1a, 1b, 2a, and 3a) were found circulating in the low-responder group. Even in this low-responder population, the good performance of two peptides (peptide 716, corresponding to a portion of the core, and peptide 59, corresponding to a portion of NS4) corroborates the immunodominance of the conserved epitopes within these peptides.
Assuntos
Hepatite B/imunologia , Anticorpos Anti-Hepatite C/biossíntese , Antígenos da Hepatite C/imunologia , Antígenos da Hepatite C/farmacologia , Hepatite C/imunologia , Diálise Renal/efeitos adversos , Proteínas não Estruturais Virais/farmacologia , Genoma Viral , Hepatite B/sangue , Hepatite B/complicações , Hepatite C/sangue , Hepatite C/complicações , Anticorpos Anti-Hepatite C/sangue , Antígenos da Hepatite C/genética , Humanos , Epitopos Imunodominantes/imunologia , Fatores de Tempo , Proteínas não Estruturais Virais/imunologiaRESUMO
Infection by hepatitis C virus (HCV)*, the aetiologic agent responsible for the majority of non-A-non-B posttransfusion hepatitis, is detected by assaying for antibodies against structural and nonstructural recombinant proteins or synthetic peptides. The aim of this study was to characterize the antibody reactivity of selected sera against antigenic peptides spanning immunodominant regions of the core, NS4 and NS5 HCV proteins. Reactivity to synthetic peptides was determined by enzyme immunoassay (EIA) for 11 selected sera from blood donors (good responders), for 27 selected sera from hemodialysis patients (poor responders), all positive for HCV antibodies (tested by different second and third-generation assays), and for 7 negative sera. Some peptides from the core and the NS4 region were widely recognized by the tested sera. Sera not reactive with core, NS4, or NS5 region by some immunoblot assays exhibited reactivity against peptides from these proteins. Autoimmune reactivity associated with HCV infection was evaluated by using a synthetic peptide derived from the GOR peptide; 8/11 HCV-positive sera were found reactive against this peptide. No correlation was found between reactivity to any of the peptides tested and the presence of HCV RNA in the serum or with HCV genotype. The EIA reactivity of peptides from the core region suggested a multideterminant antigenic structure, where reactivity of each epitope may be differentially affected by neighboring amino acids depending on individual sera. This situation was particularly evidenced in selected sera from poor responder specimens where a more restricted antibody response to core peptides was observed. Reactivity of sera from HCV-infected patients with synthetic peptides from the core, NS4, and NS5 regions indicated the presence of multiple linear epitopes (particularly in the core region) that may be used in a mixture for immunodiagnosis; however, the length and exact position of the synthetic peptides must be chosen carefully.
Assuntos
Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Antígenos da Hepatite C/imunologia , Hepatite C/imunologia , Epitopos Imunodominantes/imunologia , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Genoma Viral , Hepacivirus/genética , Hepatite C/sangue , Anticorpos Anti-Hepatite C/sangue , Humanos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologiaRESUMO
Monoclonal antibodies (mAbs) were raised against hepatitis B virus core produced by a recombinant clone of Escherichia coli (rHBc). The three mAbs recognized rHBc by Western blot, suggesting that they reacted with non-conformational epitopes. Competition experiments between mAbs and human anti-HBc sera confirmed the existence of an immunodominant HBc epitope within the viral antigen. A monoclonal competition enzyme immunoassay using an IgM mAb conjugated to biotin and streptavidin-peroxidase as the detection system yielded 99% sensitivity and 100% specificity, when compared to other commercial assays.
Assuntos
Anticorpos Anti-Hepatite B/análise , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Epitopos Imunodominantes/imunologia , Técnicas Imunoenzimáticas , Animais , Anticorpos Monoclonais/imunologia , Ligação Competitiva/imunologia , Western Blotting , Humanos , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
To analyse the effect of strain-specific sequence variation on the antigenic properties of the protein encoded by the open reading frame 3 (ORF 3) of hepatitis E virus (HEV), two sets of short overlapping peptides spanning amino acids 91 to 123 of this protein from Burmese and Mexican strains were synthesized and tested with sera obtained from outbreaks of enterically transmitted non-A, non-B hepatitis in three different regions of the world (Mexico, Turkmenistan and Kenya). The data suggest strain-specific variation in the antigenic reactivity of the ORF 3 protein. The C-terminal region of this protein contains several antigenic epitopes located in the most variable positions. Individual sera were found to interact with different groups of epitopes from each set of peptides. The antigenic epitopes of the Mexican strain appear to be less conformation-dependent than those of the Burmese strain. The most immunoreactive epitope of the ORF 3 protein from the Mexican strain was localized at amino acid positions 95 to 101. The ORF 3 protein of the Burmese strain contains an immunodominant epitope at amino acid positions 112 to 117. Some of these short peptides may be useful for the development of a diagnostic assay to discriminate between the Burmese and Mexican strains.
Assuntos
Variação Genética/genética , Vírus da Hepatite E/química , Epitopos Imunodominantes/análise , Proteínas Virais/química , Sequência de Aminoácidos , Vírus da Hepatite E/genética , Vírus da Hepatite E/imunologia , Humanos , Epitopos Imunodominantes/genética , México , Dados de Sequência Molecular , Mianmar , Fases de Leitura Aberta/genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Antibodies against hepatitis E virus (HEV) were detected in sera by a synthetic peptide-based enzyme immunoassay (EIA) from different populations in Venezuela. Antibodies against HEV were found in 1.6% (3/184) of urban pregnant woman (Caracas), in 3.9% (8/204) of rural populations (San Camilo, Edo Apure), and in 5.4% (12/223) of rural Amerindians (Padamo, Edo Amazonas). Positivity was confirmed by a neutralization EIA based on the use of competing soluble free peptides. The prevalence of antibodies in the Amerindian group was significantly higher than in urban pregnant women. No relation was found between age and HEV prevalence in rural populations. Three of 21 positive sera were also weakly positive by Western blot for IgM antibodies. This result, together with the low optical density values observed by EIA, suggested that the presence of antibodies in these sera reflects past infections. Based on these results, Venezuela does not seem to be highly endemic for hepatitis E. This is the first report of serological evidence of infection by HEV in South America.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Hepatite E/imunologia , Hepatite E/epidemiologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Hepatite E/etnologia , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina M/sangue , Indígenas Sul-Americanos , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/microbiologia , Prevalência , População Rural , Estudos Soroepidemiológicos , População Urbana , Venezuela/epidemiologiaRESUMO
Viral hepatitis serological markers were analyzed in two groups of pregnant women residing in Caracas from: 1) a maternity unit at the moment of delivery (106 sera, low income population), and 2) a private clinic during the third trimester of pregnancy (105 sera, medium-high economic class population). A higher percent positivity was observed in the maternity unit compared to the private clinic for hepatitis A virus (HAV) as measured by anti-HAV activity (96% vs 48%; p < 0.01%), for hepatitis B virus (HBV) as measured by anti-HBc activity (13% vs 2%; p < 0.01%), but not for HBV carriage, as measured by HBsAg (3.8% vs 0%; p = 0.06 %). These differences appear to correlate with the socio-economic level. All the HBsAg positive sera were HBeAg negative and negative for the presence of DNA by PCR, confirming the low rate of perinatal transmission observed in Venezuela. Two out of 106 sera (1.9%) were positive for HCV antibodies in the maternity unit and 0/105 in the private clinic, although these differences were non significant (N.S.). Two out of 106 sera (1.9%) were positive for HEV antibodies in the maternity unit and 1/80 (1.3%) in the private clinic (N.S.). The anti-HEV seropositivity probably reflects a past infection. The importance of testing these viral markers during pregnancy is discussed.
Assuntos
Anticorpos Anti-Hepatite/sangue , Hepatite Viral Humana/imunologia , Complicações Infecciosas na Gravidez/imunologia , Feminino , Hepatite A/imunologia , Hepatite B/imunologia , Hepatite B/transmissão , Anticorpos Anti-Hepatite B/sangue , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Hepatite C/imunologia , Hepatite C/transmissão , Hepatite E/imunologia , Hepatite Viral Humana/epidemiologia , Humanos , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Fatores Socioeconômicos , Venezuela/epidemiologiaRESUMO
A series of synthetic peptides derived from proteins encoded by open reading frames 2 and 3 (ORF2 and ORF3) of the hepatitis E virus was used in an enzyme immunoassay to determine the localization of epitopes in these proteins. Five peptides spanning almost the entire ORF3 protein sequence and 12 peptides from the ORF2 protein were synthesized. Serum samples collected from outbreaks in three different regions of the world (Turkmenistan, Kenya, and Mexico) were analyzed by a peptide-based enzyme immunoassay. Primary analysis of the peptides was accomplished with the use of serum samples obtained from Middle Asia. Four of 5 peptides from the ORF3 protein and 4 of 12 peptides from the ORF2 protein specifically reacted with antibody from sera of HEV-infected patients. Peptides representing immunodominant epitopes were used for the analysis of serum samples from outbreaks in Kenya and Mexico. The data indicate that these synthetic peptides may be used to develop a diagnostic test to detect antibody to the hepatitis E virus.