RESUMO
Southwest France is a highly endemic region for hepatitis E virus (HEV). This study examined the circulation of HEV strains between 2003 and 2014 in the Midi-Pyrénées, and compared these data with those from the rest of France. The polyproline region (PPR) of the ORF1 region of the HEV genome was also analyzed. HEV genotype was determined by sequencing a 348-nt fragment within the ORF2 gene for 333 strains in the Midi-Pyrénées and for 571 strains from the rest of France. PPR region was characterized for 56 strains. The frequency of subgenotype 3f decreased over time, whereas subgenotype 3c increased in the Midi-Pyrénées. Repartition of strains did not differ in the Midi-Pyrénées compared to the rest of France. HEV3i and HEV4 have been recently detected throughout France. PPR lengths showed that two major groups of HEV3f exist. Our study shows that HEV3 distribution in the Midi-Pyrénées was similar to the whole of France. Local dietary habits could explain the higher seroprevalence in the Midi-Pyrénées rather the circulation of a particular variant in this region.
Assuntos
Vírus da Hepatite E/classificação , Vírus da Hepatite E/isolamento & purificação , Hepatite E/virologia , Evolução Molecular , França , Genoma Viral , Genótipo , Vírus da Hepatite E/genética , Humanos , Dados de Sequência Molecular , Peptídeos/genética , Filogenia , Filogeografia , Análise de Sequência de RNA/métodosRESUMO
Little is known about virus adaptation in immunocompromised patients with chronic genotype 3 hepatitis E virus (HEV3) infections. Virus-host recombinant strains have been isolated recently from chronically infected patients. The nature and incidence of such recombinant events occurring during infections of solid-organ transplant (SOT) recipients are essentially unknown. The polyproline region (PPR) of strains isolated from SOT patients was sequenced during the acute-infection phase (n = 59) and during follow-up of patients whose infections became chronic (n = 27). These 27 HEV strains included 3 (11%) that showed recombinant events 12, 34, 48, or 88 months after infection. In one strain, parts of the PPR and the RNA-dependent RNA polymerase were concomitantly inserted. In the second, a fragment of a human tyrosine aminotransferase (TAT) gene was inserted first, followed by a fragment of PPR. A fragment of the human inter-α-trypsin inhibitor (ITI) gene was inserted in the third. All the inserted sequences were rich in aliphatic and basic amino acids. In vitro growth experiments suggest that the ITI insertion promoted more vigorous virus growth. In silico studies showed that the inserted sequences could provide potential acetylation, ubiquitination, and phosphorylation sites. We found that recombinant events had occurred in the HEV PPR in approximately 11% of the strains isolated from chronically infected transplant patients followed up in Toulouse University Hospital. These inserted fragments came from the HEV genome or a human gene and could enhance virus replication. Importance: Hepatitis E virus (HEV) can cause chronic infections in immunocompromised patients, including solid-organ transplant (SOT) recipients. Two strains that had undergone recombination with human ribosomal genes were described recently. The strains with inserted sequences replicated better in vitro. Little is known about the frequency of such recombinant events or how such an insertion enhances replication. We therefore investigated 59 SOT patients infected with HEV and found 3 strains with 4 recombinant events in 27 of these patients whose infection became chronic. The 4 inserted sequences were of different origins (human gene or HEV genome), but all were enriched in aliphatic and basic amino acids and provided potential regulation sites. Our data indicate that recombinant events occur in approximately 11% of strains isolated from chronically infected patients. The structures of the inserted sequences provide new clues as to how the inserted sequences could foster virus replication.
Assuntos
Vírus da Hepatite E/química , Hospedeiro Imunocomprometido , Transplante de Órgãos , Peptídeos/química , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Genoma Viral , Vírus da Hepatite E/genética , Vírus da Hepatite E/patogenicidade , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
HIV-1 subtype CRF01-AE predominates in south Asia and has spread throughout the world. The virus tropism must be determined before using CCR5 antagonists. Genotypic methods could be used, but the prediction algorithms may be inaccurate for non-B subtypes like CRF01-AE and the correlation with the phenotypic approach has not been assessed. We analyzed 61 CRF01-AE V3 clonal sequences of known phenotype from the GenBank database. The sensitivity of the Geno2pheno10 genotypic algorithm was 91%, but its specificity was poor (54%). In contrast, the combined 11/25 and net charge rule was highly specific (98%) but rather insensitive (64%). We thus identified subtype CRF01-AE determinants in the V3 region that are associated with CXCR4 use and developed a new simple rule for optimizing the genotypic prediction of CRF01-AE tropism. The concordance between the predicted CRF01-AE genotype and the phenotype was 95% for the clonal data set. We then validated this algorithm by analyzing the data from 44 patients infected with subtype CRF01-AE, whose tropism was determined using a recombinant phenotypic entry assay and V3-loop bulk sequencing. The CRF01-AE genotypic tool was 70% sensitive and 96% specific for predicting CXCR4 use, and the concordance between genotype and phenotype was 84%, approaching the concordance obtained for predicting the tropism of HIV-1 subtype B. Genotypic predictions that use a subtype CRF01-AE-specific algorithm appear to be preferable for characterizing coreceptor usage both in pathophysiological studies and for ensuring the appropriate use of CCR5 antagonists.
Assuntos
Genótipo , HIV-1/fisiologia , Tropismo Viral/genética , Adulto , Algoritmos , Sequência de Aminoácidos , Feminino , Estudos de Associação Genética , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , HIV-1/classificação , Humanos , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fenótipo , Curva ROC , Receptores CCR5/química , Receptores CCR5/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Alinhamento de SequênciaRESUMO
Hepatitis E virus (HEV) infections are responsible for chronic hepatitis in immunocompromised patients, and this can evolve to cirrhosis. Like all RNA viruses, HEV exists as a mixture of heterogeneous viruses defining quasispecies. The relationship between the genetic heterogeneity described as a quasispecies, cytokine secretion, and the outcome of acute hepatitis in immunocompromised patients remains to be elucidated. We cloned and sequenced the region encoding the M and P capsid domains of HEV from eight solid-organ transplant (SOT) patients with acute HEV infection who subsequently cleared the virus and from eight SOT patients whose infection became chronic. We analyzed the cytokines and chemokines in the sera of these SOT patients by multianalyte profiling. The nucleotide sequence entropy and genetic distances were greater in patients whose infections became chronic. A lower K(a)/K(s) ratio was associated with the persistence of HEV. The patients who developed chronic infection had lower serum concentrations of interleukin-1 (IL-1) receptor antagonist and soluble IL-2 receptor. Increased concentrations of the chemokines implicated in leukocyte recruitment to the liver were associated with persistent infection. Those patients with chronic HEV infection and progressing liver fibrosis had less quasispecies diversification during the first year than patients without liver fibrosis progression. Great quasispecies heterogeneity, a weak inflammatory response, and high serum concentrations of the chemokines involved in leukocyte recruitment to the liver in the acute phase were associated with persistent HEV infection. Slow quasispecies diversification during the first year was associated with rapidly developing liver fibrosis.
Assuntos
Vírus da Hepatite E/genética , Hepatite E/etiologia , Hepatite E/virologia , Transplante de Órgãos/efeitos adversos , Adulto , Sequência de Bases , Proteínas do Capsídeo/genética , Quimiocinas/sangue , Citocinas/sangue , Progressão da Doença , Feminino , Hepatite E/imunologia , Vírus da Hepatite E/patogenicidade , Humanos , Hospedeiro Imunocomprometido , Cirrose Hepática/etiologia , Cirrose Hepática/imunologia , Cirrose Hepática/virologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Viral/genética , Linfócitos T/imunologia , Proteínas Virais/genéticaRESUMO
Genotype 3 hepatitis E viruses (HEVs) are distributed across the world and are now considered to be an emerging public health concern in industrialized countries. At least 10 genotype 3 subtypes have been identified in humans and animals worldwide. It was recently reported that the sensitivities of HEV RNA assays differ greatly. We have assessed the influence of genotype 3 diversity on the performances of two HEV RNA assays: one targeting the ORF3 gene and the other targeting the ORF2 gene. We tested a panel of 5 HEV-positive reference samples of genotypes 3a, 3b, 3c, 3e, and 3f at 10-fold serial dilutions. The HEV RNA concentrations obtained with both reverse transcription (RT)-PCRs were correlated, but the RT-PCR based on ORF2 underestimated the HEV RNA concentrations. The mean [ORF3 - ORF2] difference was 1.41 log copies/ml. We also tested 34 clinical specimens of genotypes 3c (n = 15), 3e (n = 4), and 3f (n = 15), representing the most prevalent subtypes in Europe. The mean [ORF3 - ORF2] differences were 1.41 log copies/ml for genotype 3c, 0.96 log copies/ml for genotype 3e, and 0.70 log copies/ml for genotype 3f. The bias between the 2 RT-PCR assays was significantly greater for genotype 3c than for genotype 3f (P = 0.007). We therefore recommend the use of an RT-PCR protocol based on ORF3 to quantify HEV RNA of genotype 3 strains.
Assuntos
Variação Genética , Vírus da Hepatite E/genética , Vírus da Hepatite E/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Carga Viral/métodos , Animais , Europa (Continente) , Genótipo , Hepatite E/virologia , Humanos , Sensibilidade e Especificidade , Proteínas Virais/genéticaRESUMO
BACKGROUND: HIV-1 subtype D infections have been associated with rapid disease progression and phenotypic assays have shown that CXCR4-using viruses are very prevalent. Recent studies indicate that the genotypic algorithms used routinely to assess HIV-1 tropism may lack accuracy for non-B subtypes. Little is known about the genotypic determinants of HIV-1 subtype D tropism. RESULTS: We determined the HIV-1 coreceptor usage for 32 patients infected with subtype D by both a recombinant virus phenotypic entry assay and V3-loop sequencing to determine the correlation between them. The sensitivity of the Geno2pheno10 genotypic algorithm was 75% and that of the combined 11/25 and net charge rule was 100% for predicting subtype D CXCR4 usage, but their specificities were poor (54% and 68%). We have identified subtype D determinants in the V3 region associated with CXCR4 use and built a new simple genotypic rule for optimizing the genotypic prediction of HIV-1 subtype D tropism. We validated this algorithm using an independent GenBank data set of 67 subtype D V3 sequences of viruses of known phenotype. The subtype D genotypic algorithm was 68% sensitive and 95% specific for predicting X4 viruses in this data set, approaching the performance of genotypic prediction of HIV-1 subtype B tropism. CONCLUSION: The genotypic determinants of HIV-1 subtype D coreceptor usage are slightly different from those for subtype B viruses. Genotypic predictions based on a subtype D-specific algorithm appear to be preferable for characterizing coreceptor usage in epidemiological and pathophysiological studies.
Assuntos
Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Tropismo Viral , Sequência de Aminoácidos , Genótipo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/classificação , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Alinhamento de SequênciaRESUMO
We used ultra-deep pyrosequencing and the Toulouse Tropism Test phenotypic assay to determine the prevalence of CXCR4-using viruses in 21 patients with primary HIV-1 infections. We found X4-containing virus populations in 9% of patients by ultra-deep pyrosequencing using position-specific scoring matrices (PSSM(X4/R5)) or geno2pheno(5.75) and in 14% using the combined 11/25 and net charge rule. The phenotypic assay identified 9% of CXCR4-using viruses. This confirms that R5 viruses are predominant in primary HIV-1 infections.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Receptores CCR5/genética , Receptores CXCR4/genética , Adulto , França , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , RNA Viral , Análise de Sequência de DNA/métodos , Proteínas Virais/genéticaRESUMO
Infections with hepatitis E virus (HEV) in solid-organ transplant recipients can lead to chronic hepatitis. However, the incidence of de novo HEV infections after transplantation and risk for reactivation in patients with antibodies against HEV before transplantation are unknown. Pretransplant prevalence of these antibodies in 700 solid-organ transplant recipients at Toulouse University Hospital in France was 14.1%. We found no HEV reactivation among patients with antibodies against HEV at the first annual checkup or by measuring liver enzyme activities and HEV RNA. In contrast, we found 34 locally acquired HEV infections among patients with no antibodies against HEV, 47% of whom had a chronic infection, resulting in an incidence of 3.2/100 person-years. Independent risk factors for HEV infection were an age <52 years at transplantation and receiving a liver transplant. Effective prophylactic measures that include those for potential zoonotic infections should reduce the risk for HEV transmission in this population.
Assuntos
Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite E/imunologia , Hepatite E/epidemiologia , Transplante de Rim/efeitos adversos , Transplante de Fígado/efeitos adversos , Ativação Viral , Adolescente , Adulto , Idoso , Feminino , França/epidemiologia , Hepatite E/imunologia , Hepatite E/virologia , Vírus da Hepatite E/fisiologia , Hospitais Universitários/estatística & dados numéricos , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Adulto JovemRESUMO
OBJECTIVE: Cysteine-cysteine receptor 5 (CCR5)-using viruses classically predominate during HIV-1 primary infection but the frequency of cysteine-X-cysteine receptor 4 (CXCR4)-using viruses varies between studies and could be different between plasma and peripheral blood mononuclear cells (PBMCs). Thus, we determined HIV-1 tropism in both these compartments during primary infection and evaluated the impact of CXCR4-using viruses on disease progression. DESIGN: One hundred and thirty-three patients with primary HIV-1 infection were screened for HIV-1 coreceptor usage in plasma and PBMCs using both genotypic and phenotypic methods. The impact of CXCR4-using viruses' transmission on subsequent disease progression was assessed in a case-control study. METHODS: HIV-1 coreceptor usage was determined using a recombinant virus phenotypic entry assay and V3-based genotypic algorithms. We also monitored CD4(+) T-cell count, clinical events and therapeutic intervention. RESULTS: There was 6.4% of CXCR4-using HIV-1 in plasma during primary infection as measured by a phenotypic assay and combined criteria from the 11/25 and net charge genotypic rules. Geno2pheno10 overestimated the prevalence of CXCR4-using viruses (12%). HIV-1 tropism in plasma and PBMCs was 98% concordant. The HIV-1 RNA load and CD4(+) T-cell count during primary infection were not related to virus tropism. Primary infection with CXCR4-using viruses was associated with an accelerated rate of disease progression, estimated by a faster decline of CD4 T-cell count under 350 cells/microl and by a reduced delay in initiating a first antiretroviral treatment. CONCLUSIONS: Plasma or PBMC samples can be used for determining HIV-1 tropism during primary infection. CXCR4-using viruses are rare during primary infection but increase the risk of disease progression.
Assuntos
Infecções por HIV/imunologia , HIV-1/fisiologia , Receptores CCR5/imunologia , Receptores CXCR4/imunologia , Tropismo Viral/fisiologia , Adulto , Contagem de Linfócito CD4 , Estudos de Casos e Controles , Progressão da Doença , Feminino , Genótipo , Infecções por HIV/genética , Humanos , Masculino , Fenótipo , Receptores CCR5/genética , Receptores CXCR4/genéticaRESUMO
BACKGROUND: Hepatitis E virus (HEV) infections can lead to chronic hepatitis in immunocompromised patients. We have investigated the risk factors for HEV infection among solid-organ transplant recipients and the characteristics of these infections. METHODS: We performed serological tests, quantified the virus, and genotyped the virus in plasma samples. We performed a case-control study with HEV-infected patients and control participants matched for sex and age who were recruited from a population of solid-organ transplant recipients with no markers of HEV infection. RESULTS: We investigated 38 consecutive cases of HEV genotype 3 infection. Twenty-two (58%) of these 38 patients developed a chronic infection. The acute-phase aminotransferase levels were higher in the patients who cleared the virus than in those who developed chronic infections. The anti-HEV immunoglobulin G and immunoglobulin M profiles and HEV RNA concentration in patients who cleared the virus were similar to those in patients who developed a chronic infection. A logistic regression analysis of 37 case patients and 148 control participants indicated that the only factor independently associated with HEV infection was the consumption of game meat (68% of case patients vs 47% of control participants; odds ratio, 2.32; 95% confidence interval, 1.04-5.15). CONCLUSION: Immunocompromised patients should avoid eating insufficiently cooked game meat or pork products so as to reduce the risk of HEV infection and chronic liver disease.
Assuntos
Vírus da Hepatite E/isolamento & purificação , Hepatite E/virologia , Transplantes/efeitos adversos , Adulto , Idoso , Animais , Aves/virologia , Estudos de Casos e Controles , Doença Crônica , Comportamento Alimentar , Feminino , França/epidemiologia , Genótipo , Anticorpos Anti-Hepatite/sangue , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Carne/virologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Viral/sangue , RNA Viral/genética , Fatores de Risco , Análise de Sequência de DNA , Suínos/virologiaRESUMO
BACKGROUND: Genotypic predictions of HIV-1 tropism could simplify CCR5 antagonist usage. However, the genotypic algorithms built from subtype B viruses could be inadequate for non-B subtypes. We therefore performed paired genotypic and phenotypic determination of subtype C tropism. METHODS: We studied 52 patients recruited in Malawi and 21 patients recruited in France. We directly sequenced the V3 env region and performed a recombinant virus phenotypic entry assay in parallel. RESULTS: The Malawi patients had 29% of CXCR4-using subtype C viruses compared with only 5% in the patients from France. For detecting CXCR4-using subtype C viruses, the genotypic rule combining the amino acids at positions 11/25 and the net charge of V3 was 93.3% sensitive and 96.4% specific. The Geno2pheno tool was 86.7% sensitive and 89.1% specific. The WebPSSM tool with the SI/NSI matrix was 80% sensitive and 98.2% specific in its subtype B version and 93.3% sensitive and 81.8% specific in its subtype C version. CONCLUSIONS: The genotypic determinants of coreceptor usage for HIV-1 subtype C were mainly in V3 and were globally similar to those previously reported for subtype B viruses. The main genotypic algorithms built from subtype B viruses perform well when applied to subtype C viruses.
Assuntos
Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Tropismo Viral/genética , Algoritmos , Sequência de Aminoácidos , França/epidemiologia , Regulação Viral da Expressão Gênica , Genótipo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/epidemiologia , Humanos , Malaui/epidemiologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores CCR5 , Sensibilidade e Especificidade , Internalização do VírusRESUMO
BACKGROUND: Clinical trials of CCR5 antagonists have relied on the phenotypic determination of HIV-1 coreceptor usage. Few phenotypic assays are available, with few data on their concordance, and none has been designed to determine tropism from cell-associated HIV-1 DNA. OBJECTIVES: To assess the performance of the new Toulouse Tropism Test (TTT) phenotypic assay to characterize HIV-1 tropism using blood plasma and peripheral blood mononuclear cells (PBMC). STUDY DESIGN: 434 plasma and 168 PBMC samples were tested with the TTT assay. We determined the correlation between our assay results on plasma samples and those of the commercial Trofile assay. RESULTS: The TTT assay determined the tropism of 97% of samples after successful amplification of the env gene. It performed well on both cell samples and plasma samples with various HIV-1 loads and subtypes. It detected 0.5% of minor CXCR4-using variants in the virus population. The TTT and the Trofile assays were >90% concordant for predicting HIV-1 tropism. CONCLUSION: We have validated a new recombinant virus phenotypic assay for determining HIV-1 tropism using both plasma and cell samples from patients who are candidates for treatment with CCR5 antagonists.
Assuntos
HIV-1/fisiologia , Receptores de HIV/análise , Virologia/métodos , Internalização do Vírus , Células Cultivadas , Humanos , Leucócitos Mononucleares/virologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
We assessed the performance of genotypic algorithms for predicting the tropism of human immunodeficiency virus type 1 coreceptor usage in 52 patients infected with the CRF02-AG subtype. The combined criteria of the 11/25 and net charge rules accurately detected CXCR4-using CRF02-AG viruses, whereas the Geno2pheno tool lacked sensitivity and the position-specific scoring matrix (PSSM) tool WebPSSM lacked specificity.
Assuntos
Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Receptores Virais , Ligação Viral , Algoritmos , Genótipo , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
OBJECTIVE: Replacing phenotypic assays with simple genotypic predictions of HIV-1 coreceptor usage would make the clinical use of CCR5 antagonists easier. DESIGN: Paired genotypic and phenotypic determination of HIV-1 coreceptor usage was performed to assess several genotypic approaches for detecting CXCR4-using and CCR5-using viruses in a clinical setting. METHODS: HIV-1 coreceptor usage was prospectively assessed using plasma samples from 103 patients who were candidates for treatment with a CCR5 antagonist. Direct sequencing of the V3 region and a sensitive recombinant virus phenotypic entry assay were performed in parallel for each patient from the same bulk env PCR product. RESULTS: The 103 patients had a median CD4+ T lymphocyte count of 268 x 10(6)cells/l and nadirs of 98 x 10(6)cells/l. Paired genotypic and phenotypic data were obtained for 98 of the 103 patients. For detecting CXCR4-using viruses, the genotypic rule based on amino-acid residues at positions 11/25 and the overall net charge of V3 was 77% sensitive and 96% specific. The Geno2pheno bioinformatic tool was 88% sensitive and 87% specific. The WebPSSM tool prediction with the SI/NSI matrix was 77% sensitive and 94% specific. The global concordance between genotypic and phenotypic data was 91% with the rule combining the amino-acid residues at positions 11/25 and V3 net charge. CONCLUSION: Genotypic predictions performed well in paired genotypic and phenotypic assessment of HIV-1 coreceptor usage. Multicenter studies analyzing the correlations between the genotypic determination of HIV-1 tropism and clinical response to CCR5 antagonists are needed to validate this approach in clinical practice.
Assuntos
Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , HIV-1/metabolismo , Fragmentos de Peptídeos/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Adulto , Sequência de Bases , Biologia Computacional , Primers do DNA/genética , Feminino , Engenharia Genética , Vetores Genéticos/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , Estudos Prospectivos , Receptores CCR5/genética , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de ProteínaRESUMO
Hepatitis C virus infection is a significant problem in hemodialysis units. HCV is very variable genetically with six genotypes. Clinical and epidemiological investigation of a new infection requires the determination of both the genotype and the strain of the HCV involved. A prospective, epidemiologic study of 395 dialysis patients in Tunisia was conducted from November 2001 to November 2003 to identify the source of nosocomial transmission using phylogenetic analysis of NS5b and E2 sequences. Hepatitis C infection was diagnosed by screening for anti-HCV antibodies and HCV RNA in sera using third generation ELISA and a qualitative RT-PCR assay. HCV strains were genotyped by sequencing the NS5b region. The genetic relatedness of the HCV strains was studied by sequencing the NS5b and the HVR-1 regions of the HCV genome. Two de novo cases of HCV infection were detected during the follow-up. One of them has been described previously. The case described in this study occurred in a center in which 12 patients were already infected with HCV strains belonging to genotypes 1b (n = 8) and 1a (n = 4). Phylogenetic analysis of the NS5b region from the HCV strains circulating in this center disclosed four clusters, confirmed by analysis of the HVR-1 region, providing strong evidence for nosocomial infection. Epidemiological data showed that these patients were dialyzed during the same shift and in the same area. Phylogenetic analysis of NS5b sequences is useful for determining the HCV genotype and providing evidence of nosocomial transmission.
Assuntos
Infecção Hospitalar/transmissão , Unidades Hospitalares de Hemodiálise/normas , Hepacivirus/genética , Hepatite C/transmissão , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Hepatite C/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Tunísia/epidemiologiaRESUMO
A 9.2-kb sequence from a hepatitis C virus (HCV) strain found in southwest France was compared to sequences from reference strains in HCV sequence databases. We found a recombinant virus with genotype 2 at the 5' end and genotype 5 at the 3' end. The crossover point was located between genes NS2 and NS3. Recombination between HCV genotypes must now be considered in studies on HCV epidemiology and evolution and in predictions of the virus response to antiviral therapy. Knowing the location of the recombination point may also be useful for constructing infectious chimeric viruses.
Assuntos
Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/virologia , Recombinação Genética , Sequência de Bases , França , Genoma Viral , Hepacivirus/isolamento & purificação , Humanos , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Homologia de SequênciaRESUMO
Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver disease worldwide. The genetic heterogeneity of HCV and its spread among infected patients can be examined accurately by nucleotide sequencing. The diversity of HCV genotype 2 strains (HCV-2) was studied in a large cohort of patients in the Midi Pyrénées area of southern France. Phylogenetic analysis was performed on 344 NS5B sequences from patients infected with HCV-2. These included 145 strains whose E2 region was also analyzed, and epidemiological data were collected for the corresponding patients. HCV-2 accounts for 11.3% of HCV infections in this area. Phylogenetic analysis of NS5B sequences revealed eight subtypes, while that of the E2 region provided congruent results for 100% of strains. The most frequent subtypes were 2i (24.7%), 2k (22.4%) 2c (17.4%), and 2a (10.8%). The mean age of HCV-2-infected patients was 55.5 years. Epidemiological data showed that blood transfusion is the major route of infection, but it was not associated with any particular subtype. By contrast, intravenous drug users were infected predominantly with genotype 2a. HCV-2a-infected patients were younger than patients infected with other subtypes (48 vs. 56.5 years, P < 0.01). This study shows substantial genetic diversity of HCV-2 subtypes in the south of France and the spread of 2a strains via intravenous drug users.
Assuntos
Variação Genética , Genoma Viral , Hepacivirus/genética , Hepatite C/epidemiologia , Estudos de Coortes , Feminino , França/epidemiologia , Genótipo , Hepacivirus/classificação , Hepacivirus/isolamento & purificação , Humanos , Masculino , Filogenia , Proteínas não Estruturais Virais/genéticaRESUMO
The reasons for poor CD4+ T-cell recovery in some human immunodeficiency virus (HIV)-infected subjects despite effective highly active antiretroviral therapy (HAART) remain unclear. We recently reported that CXCR4-using (X4) HIV-1 could be gradually selected in cellular reservoirs during sustained HAART. Because of the differential expression of HIV-1 coreceptors CCR5 and CXCR4 on distinct T-cell subsets, the residual replication of R5 and X4 viruses could have different impacts on T-cell homeostasis during immune reconstitution on HAART. We examined this hypothesis and the mechanisms of CD4+ T-cell restoration by comparing the virological and immunological features of 15 poor and 15 good immunological responders to HAART. We found a high frequency of X4 viruses in the poor immunological responders. But the levels of intrathymic proliferation of the two groups were similar regardless of whether they were infected by R5 or X4 virus. The frequency of recent thymic emigrants in the poor immunological responders was also similar to that found in the good immunological responders, despite their reduced numbers of naïve CD4+ T cells. Our data, rather, suggest that the naïve T-cell compartment is drained by a high rate of mature naïve cell loss in the periphery due to bystander apoptosis or activation-induced differentiation. X4 viruses could play a role in the depletion of naïve T cells in poor immunological responders to HAART by triggering persistent T-cell activation and bystander apoptosis via gp120-CXCR4 interactions.
Assuntos
Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Receptores CXCR4/fisiologia , Adulto , Contagem de Linfócito CD4 , Citometria de Fluxo , Proteína gp160 do Envelope de HIV/genética , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Humanos , Ativação Linfocitária , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência de DNA , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Timo/virologia , Carga ViralRESUMO
Although polymorphisms of chemokine genes (SDF1, stromal cell-derived factor-1 and RANTES, regulated on activation, normal T cell expressed and secreted) and chemokine-receptor genes (CCR5, CCR2, CX(3)CR1) were shown to be associated with sensitivity to HIV infection and untreated HIV disease progression, their association with the response to highly active antiretroviral therapy (HAART) remains unclear. To explore the possible influence of such polymorphisms on the evolution of AIDS in treated patients, we have studied SDF1-3'A, CCR5Delta32, CCR2-64I, CX(3)CR1-249I, and CX(3)CR1-280M polymorphisms in HIV-infected patients under HAART (n = 169). We studied the evolution of plasma virus load and peripheral T lymphocyte counts in these patients up to 3 years after the initiation of HAART. We observed that some of the genetic polymorphisms studied had an impact on the evolution of these two parameters. After 1 year of HAART, patients with a virological response (undetectable plasma HIV-1 RNA) have a higher frequency of the homozygous SDF1-3'A genotype than other patients (p = 0.005). Similarly, patients with a CD4 increase of over 200/mm(3) from baseline after 1 year of HAART display higher frequencies of homozygous SDF1-3'A (p = 0.035) and homozygous CX(3)CR1-280M genotypes (p = 0.04) than other patients. Moreover, we showed that the CX(3)CR1- 280M allele was associated with higher peripheral CD4+ T cell counts not only in HIV+ patients but also in healthy controls (p = 0.003).
Assuntos
Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Quimiocinas CXC/genética , Infecções por HIV/tratamento farmacológico , Polimorfismo Genético , Receptores CCR5/genética , Receptores de Quimiocinas/genética , Adulto , Sequência de Bases , Receptor 1 de Quimiocina CX3C , Quimiocina CXCL12 , Primers do DNA , Feminino , Infecções por HIV/genética , Humanos , Masculino , Pessoa de Meia-Idade , Receptores CCR2RESUMO
Numerous studies reported that patients infected by the genotype 1 of hepatitis C virus (HCV) and/or with a high baseline viral load responded poorly to antiviral therapy. Study of viral kinetics has provided clues to the understanding of non-response to alpha-interferon (IFN-alpha)-based therapy. The objective of this study was to clarify the influence of viral factors such as the genotype and baseline viral load on HCV resistance to treatment through the study of their impact on the first phase of viral decline. HCV RNA levels were determined frequently following the administration of 3 million units of IFN-alpha in 22 chronic HCV carriers. The evolution of HCV RNA level over 24 hr was different in genotype 1-infected patients, compared to that in patients infected by other genotypes. The viral load decline at 24 hr was lower in patients with genotype 1. Patients with a high baseline viral load exhibited a viral dynamics different from patients with a lower level of viremia; the extent of the first phase was also lower in these patients. Non-responder patients had a slower viral decay on day 1 of therapy than patients who cleared the virus under treatment. In conclusion, 24 hr HCV dynamics is regulated by genotype and baseline viral load. Genotype 1 strains and those that produce high viral loads are the most resistant to the antiviral action of IFN-alpha. Resistant HCV strains could be distinguished from sensitive viruses as early as a few hours after the beginning of the treatment.