RESUMO
BACKGROUND: In depression, non-remission, recurrence of depressive episodes after remission and conversion to bipolar disorder are crucial determinants of poor outcome. The present study aimed to determine the cumulative incidences and clinical predictors of these long-term outcomes after the first lifetime episode of depression. METHOD: A total of 301 in- or out-patients aged 18-70 years with a validated diagnosis of a single depressive episode were assessed from 2005 to 2007. At 5 years of follow-up, 262 patients were reassessed by means of the life chart method and diagnostic interviews from 2011 to 2013. Cumulative incidences and the influence of clinical variables on the rates of remission, recurrence and conversion to bipolar disorder, respectively, were estimated by survival analysis techniques. RESULTS: Within 5 years, 83.3% obtained remission, 31.5% experienced recurrence of depression and 8.6% converted to bipolar disorder (6.3% within the first 2 years). Non-remission increased with younger age, co-morbid anxiety and suicidal ideations. Recurrence increased with severity and treatment resistance of the first depression, and conversion to bipolar disorder with treatment resistance, a family history of affective disorder and co-morbid alcohol or drug abuse. CONCLUSIONS: The identified clinical characteristics of the first lifetime episode of depression should guide patients and clinicians for long-term individualized tailored treatment.
Assuntos
Transtorno Bipolar/epidemiologia , Depressão/terapia , Adolescente , Adulto , Idoso , Ansiedade/complicações , Comorbidade , Depressão/complicações , Feminino , Seguimentos , Humanos , Pacientes Internados/psicologia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Pacientes Ambulatoriais/psicologia , Modelos de Riscos Proporcionais , Estudos Prospectivos , Escalas de Graduação Psiquiátrica , Recidiva , Indução de Remissão , Índice de Gravidade de Doença , Ideação Suicida , Adulto JovemRESUMO
We evaluated the effects of genetic variation in toll-like receptors (TLR), retinoic acid-inducible gene I (RIG-I) and their signalling pathways on spontaneous hepatitis C virus (HCV) resolution. We screened 95 single-nucleotide polymorphisms (SNPs) in 22 genes. SNPs significantly associated with resolution in the discovery cohort were genotyped in a validation cohort. Multivariate logistic regression adjusted for sex, hepatitis B surface antigen, HIV infection and the interleukin-28B rs12979860 SNP was performed in the combined cohort. Haplotype reconstruction and linkage disequilibrium analysis were performed. srs2233437, rs730775 and rs28362857 in Inhibitor of NF-kB ε (IkBε) and rs352140 in TLR9 were associated with spontaneous HCV resolution (P ≤ 0.05) in the discovery cohort (n = 308). In the validation cohort (n = 216), we replicated a significant association with HCV resolution for two SNPs in the IkBε, rs2233437 and rs730775. Presence of one or two of the variant allele in rs2233437 had more than twofold higher odds of resolution in adjusted logistic regression (adjusted odds ratio (aOR), 2.6; (95% CI, 1.4, 4.8) P = 0.002). We identified polymorphisms in the IkBε gene associated with spontaneous HCV resolution in two independent cohorts.
Assuntos
RNA Helicases DEAD-box/genética , Resistência à Doença , Hepatite C/genética , Hepatite C/imunologia , Quinase I-kappa B/genética , Transdução de Sinais/genética , Receptores Toll-Like/genética , Adulto , Estudos de Coortes , Proteína DEAD-box 58 , Feminino , Estudos de Associação Genética , Variação Genética , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Receptores ImunológicosRESUMO
Twenty-five per cent of individuals infected with hepatitis C virus (HCV) are able to clear HCV spontaneously. Differences in host genetics are believed to affect the outcome of HCV infection. We analysed an exonic, a promoter and an intronic single nucleotide polymorphism (SNP) of the interferon-λ3 coding interleukin (IL)-28B gene to study the relationship between IL28B SNPs and outcome of HCV infection. Among 206 HIV-1-infected Europeans with evidence of HCV infection, 47 (23%) individuals had cleared HCV and 159 (77%) had developed chronic infection. The exonic rs8103142 CT, the promoter rs12979860 CT and the intronic rs11881222 AG genotypes were associated with a decreased HCV clearance rate with adjusted odds ratios (aOR) of 0.3 (95% CI, 0.1-0.7), 0.4 (95% CI, 0.2-0.8) and 0.4 (95% CI, 0.2-0.8), respectively. The haplotype block TCG CTA was associated with a decreased HCV clearance rate (aOR 0.4, 95% CI, 0.2-0.8). Further, we found significant differences in HCV RNA levels among individuals chronically infected with HCV genotype 1 for rs8103142 and rs12979860 (P ≤ 0.05). Chronically infected individuals with HCV genotype 3 and with the favourable haplotype block CTA CTA had higher median HCV RNA levels than individuals with unfavourable haplotype blocks (P ≤ 0.05). Our findings suggest that IL28B may account for some differences in HCV outcome but that other factors including the viral genotype, host genetics and the host-virus interaction are likely to influence the outcome of HCV infection.
Assuntos
Hepacivirus/imunologia , Hepacivirus/isolamento & purificação , Hepatite C/imunologia , Hepatite C/virologia , Interleucinas/genética , Polimorfismo de Nucleotídeo Único , Carga Viral , Adulto , Feminino , Frequência do Gene , Infecções por HIV/complicações , Haplótipos , Humanos , Interferons , Masculino , Resultado do TratamentoRESUMO
Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid substitutions appeared to disrupt necessary interactions between E1 and E2. For some mutants, reductions in HCVpp-associated E1 were associated with the incorporation of a high molecular weight, hyperglycosylated E2 that displayed decreased CD81-binding. Other entry-deficient mutants displayed normal E1E2 incorporation into pseudoparticles and normal CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production. However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry.
Assuntos
Vetores Genéticos , Hepacivirus/fisiologia , Retroviridae/genética , Proteínas do Envelope Viral/genética , Vírion/genética , Internalização do Vírus , Substituição de Aminoácidos , Antígenos CD/metabolismo , Linhagem Celular , Hepacivirus/genética , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Ligação Proteica , Recombinação Genética , Tetraspanina 28 , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/fisiologiaRESUMO
Predictive factors for initiation of antiviral therapy in chronically infected hepatitis C virus (HCV) patients are not fully elucidated. The aim of this study was to determine predictive factors for initiation of treatment with standard or pegylated interferon either alone or combined with ribavirin. A Danish cohort of individuals chronically infected with HCV was used and observation time was calculated from the date of inclusion in the cohort to date of death, last clinical observation, 1 January 2007, or start of HCV antiviral treatment in treatment-naïve patients. Kaplan-Meier survival analysis was used to construct time to event curves. Cox regression was used to determine the incidence rate ratios as estimates of relative risk (RR) and 95% confidence intervals (CI). A total of 1780 patients were enrolled in the study. The cumulative chance of treatment initiation over 5 years was 33.0%. We found several strong predictors of treatment initiation: elevated alanine aminotransferase [>2 times upper limit (RR = 2.17, 95% CI 1.64-2.87), >3 times upper limit (RR = 3.64, 95% CI 2.75-4.81)], genotype 2 or 3 (RR = 1.86, 95% CI 1.49-2.31) and HIV co-infection (RR = 0.28, 95% CI 0.15-0.53). To our knowledge, this study is the first to estimate factors predicting initiation of antiviral treatment in patients with chronic HCV infection on a nationwide scale. We found that several of the factors predicting initiation of antiviral treatment correlate with factors known to predict a better response to treatment and factors known to increase the progression of liver disease.
Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/tratamento farmacológico , Adulto , Biomarcadores , Estudos de Coortes , Dinamarca , Feminino , Humanos , Interferons/uso terapêutico , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Prognóstico , Ribavirina/uso terapêutico , Adulto JovemRESUMO
BACKGROUND: The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5â¯B proteins. OBJECTIVE: To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5â¯B RASs without prior knowledge of sample genotype. STUDY DESIGN: In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (â¼â¯9â¯kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. RESULTS: The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. CONCLUSIONS: The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.
Assuntos
Farmacorresistência Viral/genética , Técnicas de Genotipagem , Hepacivirus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fases de Leitura Aberta , Proteínas não Estruturais Virais/genética , Variação Genética , Genótipo , Hepatite C/sangue , Humanos , Filogenia , Análise de Sequência de DNARESUMO
Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. This RNA virus circulates as a quasispecies and its genetic heterogeneity has been implicated in the lack of protective immunity against HCV and in its persistence following infection. HCV might escape from immune surveillance by developing mutations in proteins that are subject to immune pressure.
Assuntos
Variação Genética , Hepacivirus , Hepatite C/virologia , Animais , Variação Antigênica , Hepacivirus/genética , Hepacivirus/imunologia , Hepacivirus/patogenicidade , Hepatite C/imunologia , Humanos , Carga ViralRESUMO
The serotonin-transporter-linked polymorphic region (5-HTTLPR) is one of the most extensively investigated candidates to be involved in gene-environment interaction associated with depression. Nevertheless, the interaction remains controversial. In an original study, we tested the hypothesis that risk for use of antidepressants following a diagnosis of colorectal cancer is associated with bi- and triallelic genotypes of 5-HTTLPR. In addition, in an inclusive meta-analysis, we tested the hypothesis that depression following a diagnosis of cancer is associated with biallelic 5-HTTLPR genotype. We created an exposed-only cohort of 849 colorectal cancer patients from the Danish Diet, Cancer and Health cohort study. The hypothesized association was investigated with Cox regression models and competing risk analyses. Five studies comprising a total of 1484 cancer patients were included in the meta-analysis. Nationwide registries provided information on dates of diagnosis of colorectal cancer and use of antidepressants. Unadjusted odds ratios of depression according to the biallelic 5-HTTLPR genotype were included in the meta-analysis. 5-HTTLPR genotypes were not associated with use of antidepressants after colorectal cancer. Estimated hazard ratios ranged 0.92-1.08, and we observed no statistically significant associations across biallelic and triallelic genotypes in crude as well as adjusted models. The meta-analysis showed no statistically significant associations of 5-HTTLPR biallelic genotype with depression after cancer. Our findings in an original study and a meta-analysis do not support the hypothesis of an association between the 5-HTTLPR genotype and depression after cancer.
Assuntos
Antidepressivos/uso terapêutico , Neoplasias Colorretais/complicações , Neoplasias Colorretais/psicologia , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Transtorno Depressivo/complicações , HumanosRESUMO
Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. HCV is a positive-strand genotype RNA virus with extensive genetic heterogeneity; HCV isolates define 6 major genotypes, and HCV circulates within an infected individual as a number of closely related but distinct species, termed a quasispecies. This article reviews characteristic aspects of HCV molecular biology and their implications for treatment and vaccine development.
Assuntos
Hepacivirus/genética , Regiões 3' não Traduzidas/fisiologia , Variação Genética , Genoma Viral , Genótipo , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Proteínas não Estruturais Virais/fisiologia , Proteínas Estruturais Virais/fisiologia , Vacinas Virais/imunologiaRESUMO
On the basis of research in Guinea-Bissau, this paper re-analyses a severe measles epidemic which occurred in 1885 in Sunderland (England). In both England and Guinea-Bissau, acute measles mortality was higher in households with multiple cases than in families with only a single case of measles. Secondary cases (infected in the house) had higher mortality and higher frequency of severe complications than index and single cases. In Sunderland, severe complications were associated with a history of previous respiratory infection and with greater number of siblings. Since cases with severe complications had significantly prolonged prodromal symptoms and shorter periods of incubation, it is suggested that high dose of infection may be an essential mechanism in the pathogenesis of severe disease. Overcrowding may be a major determinant of severe measles because it increases the risks of intensive exposure, intercurrent infections, and previous respiratory infection.
Assuntos
Sarampo/complicações , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Aglomeração , Inglaterra , Guiné-Bissau , Humanos , Imunidade Materno-Adquirida , Lactente , Sarampo/epidemiologia , Sarampo/mortalidade , Distúrbios Nutricionais/complicações , Infecções Respiratórias/complicações , Fatores de TempoRESUMO
A chimeric cDNA genome was constructed in which the core, E1 and E2 genes of hepatitis C virus (HCV) replaced the core, E(rns), E1 and E2 genes of bovine viral diarrhea virus (BVDV). High levels of HCV structural proteins were expressed in a small number of human or bovine cells following transfection with chimeric RNA. However, in one cell line, bovine embryonic trachea cells [EBTr(A)], the number of cells expressing HCV proteins increased to greater than 70% following serial passage of culture medium. These cells were persistently infected with a non-cytopathogenic BVDV helper virus. In these cells, the chimeric genome was packaged into infectious particles that accumulated in the culture medium at a titer as high as 10(7)-10(9) genome equivalents per ml. The virus particles were pseudotypes, because they were neutralized by anti-BVDV but not by anti-HCV.
Assuntos
Vírus da Diarreia Viral Bovina/genética , Regulação Viral da Expressão Gênica , Engenharia Genética , Hepacivirus/genética , Proteínas Estruturais Virais/biossíntese , Animais , Anticorpos Antivirais/imunologia , Western Blotting , Bovinos , DNA Recombinante/genética , Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/fisiologia , Genoma Viral , Vírus Auxiliares/genética , Vírus Auxiliares/fisiologia , Hepacivirus/fisiologia , Humanos , Soros Imunes/imunologia , Microscopia de Fluorescência , Ensaio de Radioimunoprecipitação , Transfecção , Células Tumorais Cultivadas , Proteínas Estruturais Virais/genética , Replicação ViralRESUMO
In an urban area of Guinea-Bissau, where more than 80% of the children have been vaccinated, measles continues to be a major cause of child mortality. Compared with the period before the introduction of vaccination, more cases occur outside the community, while more cases within the district are now guests and newcomers. Half of the new introductions of measles into the community and 30% of the measles deaths can be traced back to the paediatric ward. Contact with health care institutions plays an important role in the transmission of measles, particularly among the youngest children. This consequence of health care may be avoidable, however, since several studies suggest that sick children can be vaccinated safely and effectively.
Assuntos
Infecção Hospitalar/epidemiologia , Sarampo/transmissão , Adolescente , Pré-Escolar , Feminino , Guiné-Bissau , Hospitais , Humanos , Lactente , Masculino , Sarampo/epidemiologia , Sarampo/prevenção & controle , VacinaçãoRESUMO
Hepatitis C virus (HCV) has a significant, albeit varied, presence around the world. This virus is primarily transmitted parenterally, although sexual and perinatal transmission does appear to occur. However, no risk factor for transmission could be identified in a significant proportion of infected individuals. It was found that individuals became viremic early after the primary HCV infection, whereas seroconversion and hepatitis occurred several weeks later. It was demonstrated that less than 20% of patients cleared their viremia, with the majority of patients becoming chronically infected. A significant proportion of chronically infected individuals developed chronic hepatitis and liver cirrhosis, and a strong association has been found with the development of hepatocellular carcinoma. Finally, HCV seems to be associated with autoimmune diseases and type II cryoglobulinemia. Thus, significant morbidity and mortality is caused by HCV infection worldwide. In a single infected individual the genome population of HCV has been found to comprise a quasispecies that consists of a number of identical sequences (i.e., the master sequence) and other closely related sequences. The master sequence of this quasispecies population changes during infection. In particular, it has been found that the sequence of the hypervariable region I changes rapidly in infected individuals. It is possible that the quasispecies nature of HCV constitutes a mechanism by which HCV escapes immune surveillance and establishes a persistent infection in the host. It is now well established that HCV exists as distinct genotypes among different HCV isolates. According to the currently used classification these can be divided into a number of major genetic groups (or types) and subgroups (or subtypes). The extensive genetic heterogeneity of HCV has important implications for diagnosis, pathogenesis, treatment and vaccine development.
Assuntos
Hepacivirus/fisiologia , Heterogeneidade Genética , Genótipo , Hepacivirus/genética , Hepatite C/epidemiologia , Hepatite C/transmissão , HumanosRESUMO
Earlier studies have suggested that general measles vaccination programmes should not be made a priority in developing countries because the presumably malnourished children saved from measles are likely to die from something else. Recent community studies indicate, however, that malnutrition is not the cause of high measles mortality. In an urban community in Guinea-Bissau, child mortality has been registered for a period of 3 years; 1 year before and 2 years after the introduction of a general measles vaccination program. In the years following the introduction of measles vaccination, mortality for children aged 6 to 35 months has significantly diminished. Though this is not a controlled study of vaccinated and unvaccinated children, much of the reduced mortality can apparently be attributed to the protective effect of measles vaccination. Children with a history of earlier measles infection had a significantly higher mortality rate than children vaccinated against measles. Rather than being a mechanism of natural selection taking the weakest children, measles apparently aggravates the condition of many children, leading to delayed excess mortality. In areas where the case fatality rate is high, vaccination against measles should be made an indispensable part of primary health care.
Assuntos
Vacina contra Sarampo , Sarampo/mortalidade , Mortalidade , Vacinação , Criança , Pré-Escolar , Guiné-Bissau , Humanos , LactenteRESUMO
Cell-mediated immunity is generally regarded as the essential factor in recovery from measles infection. In other viral infections humoral immunity has been considered a critical factor when antibody titres were correlated with outcome or when serum therapy proved protective. A review of available studies of severe-to-fatal cases of measles infection having non-neurological symptoms indicate that the antibody response is depressed in virtually all cases. The current view of immune globulin being an ineffective therapeutic agent is based on treatment of measles encephalitis; in fact, the least effect should be expected among encephalitis cases since some already have antibodies from the onset of symptoms. Larger examinations of measles with other than neurological symptoms suggest that immune globulin has a beneficial impact on the clinical course of infection. There are indications that hyperimmune globulin increases the efficacy of this form of treatment. Since measles is still a major cause of hospitalization and mortality, further studies of the therapeutic effect of specific immune globulin are warranted. From our current knowledge, both the humoral and cell-mediated immunity seem to be critical factors in recovery from measles infection.
Assuntos
Formação de Anticorpos , Sarampo/imunologia , Humanos , Imunoterapia , Sarampo/mortalidade , Panencefalite Esclerosante Subaguda/imunologia , Panencefalite Esclerosante Subaguda/mortalidadeRESUMO
In previous studies, human hepatitis viruses have been experimentally transmitted to New World monkeys of the genus Saguinus (tamarins). Recently, two Flaviviridae-like agents (GBV-A and GBV-B) were identified in tamarins that developed hepatitis following inoculation with serum of the 11th tamarin passage of a potentially new human hepatitis agent. However, it was not shown that these viruses originated from the initial inoculum. We here report the discovery of indigenous species-specific viruses related to GBV-A in several species of New World monkeys and suggest that GBV-A virus was fortuitously acquired during passage in tamarins. Sera or plasma from 98 wild-caught New World monkeys representing 10 different species was tested by RT-PCR with conserved degenerate primers to the 5' noncoding region of the genome. Viral sequences were identified in 33 animals and sequence analysis was performed on the amplicons. In addition, the genomic region corresponding to the putative NS3 RNA helicase of GBV-A was amplified from most positive animals and sequenced. We detected GBV-A-like viruses in 13 (35%) of 37 S. mystax, 7 (78%) of 9 S. nigricollis, 3 (25%) of 12 S. labiatus, 2 (50%) of 4 S. oedipus, 2 (100%) of 2 Callithrix jacchus, and 6 (50%) of 12 Aotus trivirgatus monkeys. Each positive animal was infected with a unique strain of the GBV-A-like viruses. Analysis of the 5' NC and NS3 helicase sequences revealed that these viruses could be classified into 5 major genetic groups with genetic distances equivalent to or greater than those found among major genetic groups of hepatitis C virus. Species-specific GBV-A-like viruses were found in S. mystax, S. nigricollis, S. oedipus, C. jacchus, and A. trivirgatus species. The viruses specific for S. nigricollis were closely related to GBV-A, suggesting that GBV-A was acquired by passage through this species during the initial transmission studies. The natural history of the GBV-A-like viruses was studied in serial serum samples from 9 S. mystax and 2 A. trivirgatus monkeys. Each animal was chronically infected and the viral strain did not vary during 9-27 months of follow-up. Finally, we demonstrated that four S. mystax were positive upon arrival to the United States from the country of origin. No apparent disease was associated with chronic infection of the GBV-A-like viruses. In conclusion, many New World monkeys are persistently infected with indigenous species-specific viruses that may represent a new genus within the virus family Flaviviridae.
Assuntos
Aotus trivirgatus/virologia , Flaviviridae/enzimologia , Hepatite Viral Animal/virologia , Doenças dos Macacos/virologia , Saguinus/virologia , Proteínas não Estruturais Virais/genética , Animais , Sequência de Bases , Cebidae/virologia , DNA Viral/análise , Flaviviridae/classificação , Flaviviridae/genética , Hepatite Viral Animal/sangue , Dados de Sequência Molecular , Filogenia , RNA Helicases , Homologia de Sequência do Ácido Nucleico , Serina EndopeptidasesRESUMO
We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.
Assuntos
Hepacivirus/genética , Hepatite C/diagnóstico , Reação em Cadeia da Polimerase/métodos , RNA Viral/análise , Sequência de Bases , Genética Populacional , Anticorpos Anti-Hepatite/análise , Hepatite C/imunologia , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/químicaRESUMO
In a previous study we sequenced the 5' noncoding (NC) region of 44 isolates of hepatitis C virus (HCV) and identified heterogeneous domains that provided evidence for additional genetic groups of HCV not previously recognized. In this study we have determined the complete nucleotide sequence of the putative envelope 1 (E1) gene in 51 HCV isolates from around the world and found that they could be grouped into at least 12 distinct genotypes. The E1 gene sequence of 8 of these genotypes has not been reported previously. Although the genetic relatedness of HCV isolates determined by the previous analysis of the 5' NC region predicted the relationships observed in the E1 gene, analysis of the 5' NC sequence alone did not accurately predict all HCV genotypes. The nucleotide and amino acid sequence identities of the E1 gene among HCV isolates of the same genotype were in the range of 88.0-99.1% and 89.1-98.4%, respectively, whereas those of HCV isolates of different genotypes were in the range of 53.5-78.6% and 49.0-82.8%, respectively. The latter differences are similar to those found when comparing the envelope gene sequences of the various serotypes of the related flaviviruses as well as other RNA viruses. We found that some genotypes of HCV were widely distributed around the world, whereas others were identified only in discreet geographical regions. Four genotypes were identified exclusively in Africa and comprised the majority of HCV isolates on that continent. The E1 gene was exactly 576 nucleotides in length in all 51 HCV isolates with no in-frame stop codons. Analysis of the predicted E1 protein identified several conserved domains that may be important for maintaining its biological function: (i) eight invariant cysteine residues, (ii) three potential N-linked glycosylation sites, (iii) a domain of nine amino acids (GHRMAWDMM), and (iv) an amino acid doublet (GV) near the putative cleavage site at the C terminus of the protein. In conclusion, the discovery of at least 12 genotypes of HCV has important implications for HCV diagnosis and vaccine development.
Assuntos
Genes Virais , Hepacivirus/genética , Filogenia , Sequência de Aminoácidos , Sequência de Bases , DNA Viral/genética , DNA Viral/isolamento & purificação , Genótipo , Hepacivirus/isolamento & purificação , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Homologia de Sequência de Aminoácidos , Terminologia como AssuntoRESUMO
Recently, the principal etiological agent of parenterally transmitted non-A, non-B hepatitis was molecularly cloned from the plasma of an experimentally infected chimpanzee and has been named hepatitis C virus. Determination of the complete nucleotide sequence of the hepatitis C virus genome was a crucial step in preparing the way for future study of this medically important human pathogen. Due to the very low concentration of virus in serum, amplification of viral RNA sequences by reverse transcription and polymerase chain reaction is the only practical method currently available for demonstrating viremia in patients with hepatitis C virus infection. This review examines the pivotal role of the polymerase chain reaction in understanding the biology of hepatitis C virus.
Assuntos
Hepacivirus/isolamento & purificação , Hepatite C/microbiologia , Doença Aguda , Animais , Doença Crônica , DNA Complementar , Genoma Viral , Hepatite C/transmissão , Humanos , Reação em Cadeia da Polimerase , RNA Viral/análiseRESUMO
We previously sequenced the 5' noncoding region of 44 isolates of hepatitis C virus (HCV), as well as the envelope 1 (E1) gene of 51 HCV isolates, and provided evidence for the existence of at least 6 major genetic groups consisting of at least 12 minor genotypes of HCV (i.e., genotypes I/1a, II/1b, III/2a, IV/2b, 2c, V/3a, 4a-4d, 5a, and 6a). We now report the complete nucleotide sequence of the putative core (C) gene of 52 HCV isolates that represent all of these 12 genotypes as well as two additional genotypes provisionally designated 4e and 4f that we identified in this study. The phylogenetic analysis of the C gene sequences was in agreement with that of the E1 gene sequences. A major division in the genetic distance was observed between HCV isolates of genotype 2 and those of the other genotypes in analysis of both the E1 and C genes. The C gene sequences of 9 genotypes have not been reported previously (i.e., genotypes 2c, 4a-4f, 5a, and 6a). Our analysis indicates that the C gene-based methods currently used to determine the HCV genotype, such as PCR with genotype-specific primers, should be revised in light of these data. We found that the predicted C gene was exactly 573 nt long in all 52 HCV isolates, with an N-terminal start codon and no in-frame stop codons. The nucleotide and predicted amino acid identities of the C gene sequences were in the range of 79.4-99.0% and 85.3-100%, respectively. Furthermore, we mapped universally conserved, as well as genotype-specific, nucleotide and deduced amino acid sequences of the C gene. The predicted C proteins of the different HCV genotypes shared the following features: (i) high content of proline residues, (ii) high content of arginine and lysine residues located primarily in three domains with 10 such residues invariant at positions 39-62, (iii) a cluster of 5 conserved tryptophan residues, (iv) two nuclear localization signals and a DNA-binding motif, (v) a potential phosphorylation site with a serine-proline motif, and (vi) three conserved hydrophilic domains that have been shown by others to contain immunogenic epitopes. Thus, we have extended analysis of the predicted C protein of HCV to all of the recognized genotypes, confirmed the existence of highly conserved regions of this important structural protein, and demonstrated that the genetic relatedness of HCV isolates is equivalent when analyzing the most conserved (i.e., C) and the most variable (i.e., E1) genes of the HCV genome.