RESUMO
In association with hepatitis B virus (HBV), hepatitis delta virus (HDV) is a subviral agent that may promote severe acute and chronic forms of liver disease. Based on the percentage of nucleotide identity of the genome, HDV was initially classified into three genotypes. However, since 2006, the original classification has been further expanded into eight clades/genotypes. The intergenotype divergence may be as high as 35%-40% over the entire RNA genome, whereas sequence heterogeneity among the isolates of a given genotype is <20%; furthermore, HDV recombinants have been clearly demonstrated. The genetic diversity of HDV is related to the geographic origin of the isolates. This study shows the first comprehensive bioinformatic analysis of the complete available set of HDV sequences, using both nucleotide and protein phylogenies (based on an evolutionary model selection, gamma distribution estimation, tree inference and phylogenetic distance estimation), protein composition analysis and comparison (based on the presence of invariant residues, molecular signatures, amino acid frequencies and mono- and di-amino acid compositional distances), as well as amino acid changes in sequence evolution. Taking into account the congruent and consistent results of both nucleotide and amino acid analyses of GenBank available sequences (recorded as of January, 2017), we propose that the eight hepatitis D virus genotypes may be grouped into three large genogroups fully supported by their shared characteristics.
Assuntos
Biologia Computacional , Genoma Viral , Vírus Delta da Hepatite/genética , Análise de Sequência de DNA , Variação Genética , Genótipo , Vírus Delta da Hepatite/classificação , Filogenia , Recombinação Genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Hepatitis B virus (HBV) is a worldwide public health concern. The circulation of strains carrying mutations in the viral proteins implies both clinical and therapeutics challenges. HBV complete genomes (HBV-CGs) were reported from injecting drug users and HBV chronically infected patients from Argentina-including Amerindians-although no studies were conducted in blood donors. Here, we described HBV-CG sequences from the latter population. Some of the HBV sequences classified as B2 and C2 subgenotypes clustering together with Asian isolates, while others, such as D3, F1b, and F4, were homologous to European and Latin America sequences. New substitutions for all analyzed open reading frames and changes in the HBsAg hydrophobicity profiles were detected. Several HBV-CG subgenotypes are described for the first time in this population. Mutations observed in X, PreS, and P proteins have been associated with advanced liver disease, hepatocellular carcinoma, and/or natural resistance to nucleos(t)ide antiviral treatment. It deserves to be highlighted that these substitutions were detected in a population without epidemiological risk factors for viral infection, and most importantly, without any previous antiviral treatment (natural resistance). Regarding the remaining mutations, further research is warranted in order to determine their clinical and therapeutics relevance.
Assuntos
Genoma Viral , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Mutação , Proteínas Virais/genética , Argentina , Sequência de Bases , Doadores de Sangue , Genômica , Genótipo , Hepatite B/sangue , Vírus da Hepatite B/classificação , Humanos , Dados de Sequência Molecular , FilogeniaRESUMO
Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Expressão Gênica , Hepacivirus/fisiologia , Proteínas Proto-Oncogênicas c-bcr/biossíntese , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Subfamília B de Transportador de Cassetes de Ligação de ATP , Carbocianinas/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Hepatócitos/virologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Rodamina 123/metabolismoRESUMO
PCR detection of viral genomes has provided new insights into viral diagnosis. Nowadays, it is the most frequently used nucleic acid testing (qualitative and quantitative) technique. The aim of this study was to analyse the major circulating hepatitis B virus (HBV) variants PCR-amplified by three sets of primers in a patient infected with genotype E. The HBV S/Pol overlapping genomic region was amplified from the serum of an infected child using three primer sets previously described. Sequence analysis corresponding to the HBV S/Pol region revealed the presence of different viral populations depending on the set of primers used. D144A S-escape mutant was detected with two of the primer sets, while the rtL217R mutant within the Pol - conferring resistance to Adefovir - could be picked up with a different pair of primer sets. This study undoubtedly implies that the description of viral polymorphisms should be stated together with the sequence of the primers used for PCR amplification when studies of escape and/or antiviral-resistant HBV mutants are carried out.
Assuntos
Primers do DNA/genética , DNA Viral/genética , Vírus da Hepatite B/classificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Reação em Cadeia da Polimerase/métodos , DNA Viral/química , DNA Polimerase Dirigida por DNA/genética , Genótipo , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Humanos , Polimorfismo Genético , Análise de Sequência de DNARESUMO
Previous studies have revealed that hepatitis B virus (HBV)/D and HBV/F predominate among blood donors from Buenos Aires, Argentina. In the present study, blood samples from two high-risk groups were analysed: 160 corresponding to street- and hospital-recruited injecting drug users [81.2% showing the 'anti-hepatitis B core antigen (anti-HBc) only' serological pattern] and 20 to hepatitis B surface antigen (HBsAg)(+)/anti-HBc(+) men who have sex with men. HBV genotypes were assigned by polymerase chain reaction amplification followed by restriction fragment length polymorphism and confirmed by nucleotide sequencing of two different coding regions. HBV DNA was detected in 27 injecting drug users (16.9%, occult infection prevalence: 7.7%), and 14 men who have sex with men (70%). HBV/A prevailed among injecting drug users (81.8%) while HBV/F was predominant among men who have sex with men (57.1%). The high predominance of HBV/A among injecting drug users is in sharp contrast to its low prevalence among blood donors (P = 0.0006) and men who have sex with men (P = 0.0137). Interestingly, all HBV/A S gene sequences obtained from street-recruited injecting drug users encoded the rare serotype ayw1 and failed to cluster within any of the known A subgenotypes. Moreover, one of the HBV strains from a hospital-recruited injecting drug user was fully sequenced and found to be the first completely characterized D/A recombinant genome from the American continent. Data suggest that two simultaneous and independent HBV epidemics took place in Buenos Aires: one spreading among injecting drug users and another one sexually transmitted among the homosexual and heterosexual population.
Assuntos
Usuários de Drogas , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B/epidemiologia , Homossexualidade Masculina , Abuso de Substâncias por Via Intravenosa/complicações , Adulto , Argentina/epidemiologia , Análise por Conglomerados , DNA Viral/genética , Feminino , Genótipo , Vírus da Hepatite B/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Prevalência , Recombinação Genética , Análise de Sequência de DNARESUMO
The in vivo evolution of genotype F HBV variants was recorded in a chronically infected patient throughout a 3-year observation period. Fluctuating levels of HBs Ag and anti-HBs antibodies were recorded, both of them cocirculating in peripheral blood samples at given times. Fifty S gene derived clones were sequenced and phylogenetically analyzed. As expected, some amino acid replacements within the S ORF were also observed within the P ORF while others were silent for the former. Such change was statistically significant for both S and P overlapping genes, which clearly indicates the appearance of a positive selection pressure. Supporting this notion, amino acid replacements were documented at both B and T cell epitopes in samples from 1997 and 1998. Several mutations were documented within and outside the "a" determinant in the major hydrophilic region. Such substitutions might have resulted from the attempt of HBV to evade both humoral and/or cellular immune response. To the best of our knowledge this unusual profile of HBV variants in presence of usually "neutralizing" anti-HBs antibodies was examined in vivo for the first time.
Assuntos
Evolução Molecular , Genes Virais/genética , Anticorpos Anti-Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Hepatite B/genética , Sequência de Aminoácidos , Produtos do Gene pol/genética , Humanos , Dados de Sequência Molecular , Proteínas do Envelope Viral/genéticaRESUMO
An enzyme-linked immunosorbent assay (ELISA) has been developed to measure anti-tetanus toxoid antibody levels in immunized guinea-pig sera as a useful alternative to the currently used toxin neutralization test (TNT) in determining the activity of the tetanus toxoid in vaccines. The ELISA was found to measure antibody levels as low as 5.8 x 10(-5) IU/ml. Furthermore, a comparison of the results from ELISA and TNT involving 132 different commercial vaccines showed a very good correlation (r = 0.94, p < 0.001) between antibody levels measured by both methods. The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in mice for assessing the activity of tetanus toxoids in vaccines.
Assuntos
Anticorpos Antibacterianos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Testes de Neutralização/métodos , Toxoide Tetânico/análise , Animais , Feminino , Cobaias , Masculino , Camundongos , Toxoide Tetânico/imunologiaRESUMO
GBV-C/HGV RNA was investigated in serum samples from 70 HIV(+) intravenous drug users (IVDU), as well as from 200 blood donors from Buenos Aires, Argentina. Viral RNA was demonstrated in 21 IVDU by reverse transcription-nested PCR of the 5' UTR. c-DNA amplified products were analyzed and their sequences compared with those downloaded from GenBank. A phylogenetic tree based on 171 sequences demonstrated the presence of three major genogroups, including two subgroups, within local samples, i.e. group 1 (n=1), 2a (n=11), 2b (n=4) and 3 (n=5). These results agreed entirely with those obtained by a novel RFLP (J. Clin. Microbiol. 37, 1340-1347, 1999) of the same 5' UTR amplicons. As expected, GBV-C/HGV RNA prevalence was significantly higher among IVDU than among blood donors (P<0.0001), although within the latter group an unexpectedly high rate was also detected, since 11 of 200 sera (5.5%) proved positive. These viral isolates were ascribed either to subgroup 2a (n=5), subgroup 2b (n=5) or genogroup 3 (n=1). Briefly, this partial view of GBV-C/HGV molecular epidemiology in Argentina shows: (i) different rates of GBV-C/HGV infection within both IVDU and blood donors; (ii) a high prevalence of viral RNA among blood donors; and (iii) a predominant circulation of genogroup 2, with minor contribution of groups 3 and 1.
Assuntos
Doadores de Sangue , Flaviviridae/genética , Infecções por HIV/complicações , Abuso de Substâncias por Via Intravenosa/complicações , Adulto , Antígenos Virais/genética , Argentina , Feminino , Flaviviridae/isolamento & purificação , Testes Genéticos , Variação Genética , Infecções por HIV/virologia , Humanos , Masculino , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genéticaRESUMO
HCV genomic characterization was performed by nucleotide sequence analysis (n=50) combined with restriction fragment length polymorphism (RFLP) of the 5' UTR region in 82 isolates corresponding to different Argentine groups. Genotype 1 was detected in 70.7% of the samples (58 out of 82), genotype 2 in 21.9% (18 of 82) and genotype 3 in the remaining 6 sera (7.3%). HCV 1b subtype contributed with 35.3% to the whole population studied (29 to 82) and was detected in 6 out of 21 sporadic cases. Besides their epidemiological significance, these results should be taken into account when future vaccines are considered on the basis of geographical HCV genotypic prevalence.
Assuntos
Hepacivirus/genética , Hepatite C Crônica/sangue , Filogenia , Polimorfismo de Fragmento de Restrição , Adolescente , Adulto , Idoso , Argentina , Sequência de Bases , Criança , Pré-Escolar , Feminino , Genótipo , Hepatite C Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Fatores de Risco , Análise de Sequência de RNARESUMO
With the aim of analyzing the properties of the waves that are scattered by nanoslits on metallic surfaces, we provide a direct observation of the near-field in a slit-doublet experiment at optical wavelengths. We show that two distinct waves are involved: a surface plasmon polariton and another wave with a free-space character. From the recorded data, we have extracted the amplitudes and phases of these waves, their damping characteristic lengths and their relative weights as a function of the separation distance from the slit. The analysis is fully supported by a quantitative agreement with vector-theory computational results.
RESUMO
Typing of hepatitis C virus (HCV) isolates from Argentine patients was performed by using different methodologies in a population of 243 patients. HCV subtype was assigned based upon restriction fragment length polymorphism (RFLP). HCV RNA genomes obtained from serum samples were classified as belonging to clade 1 (53.5%), 2 (23. 0%), or 3 (8.6%); 14.8% of samples showed HCV mixed infections, more frequently implying different subtypes within the same clade. In addition to RFLP typing, phylogenetic relatedness among sequences from both 5' untranslated region (n = 50) and nonstructural 5B coding region (n = 15) was established.
Assuntos
Hepacivirus/genética , Regiões 5' não Traduzidas/química , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Genótipo , Hepacivirus/classificação , Humanos , Fígado/fisiopatologia , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Estudos Retrospectivos , Abuso de Substâncias por Via Intravenosa/complicaçõesRESUMO
The immunogenicity of the diphtheria component of 73 commercial vaccines from five different manufacturers was tested by the toxin neutralization test (TNT) and the enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. A comparison of the antibody levels measured by both assays showed a very good correlation (r = 0.95, p < 0.001). The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in guinea pigs. Also, the ELISA was found to measure IgG antibody levels as low as 5.5 x 10(-5) IU ml-1. To evaluate the possibility of accelerating the active immunization during the activity test of vaccines, an alternative schedule using one single human dose was assayed. A very good correlation was observed between the IgG antibody response obtained with this schedule and with the traditional programme. Therefore, the cost and the time required to perform the activity test may be considerably reduced when both the rapid immunization schedule and the ELISA are used.
Assuntos
Anticorpos Antibacterianos/sangue , Toxoide Diftérico/imunologia , Animais , Toxoide Diftérico/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Cobaias , Esquemas de Imunização , Imunoglobulina G/sangue , Masculino , Testes de Neutralização , Reprodutibilidade dos TestesRESUMO
A phylogenetic tree based on 150 5' untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that HinfI and either AatII or AciI could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out.
Assuntos
Regiões 5' não Traduzidas/química , Flaviviridae/classificação , Polimorfismo de Fragmento de Restrição , Sequência de Bases , DNA Complementar/química , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We imaged magnetic domains in Pt/Co/Pt multilayers using an apertureless scanning near-field optical microscope operating in reflection mode. As the magneto-optical effects are weak for this kind of structure, a polarization modulation technique with a photoelastic modulator was used to reveal the contrast between magnetic domains. In the case of a Pt/Co/Pt trilayer structure, a strong improvement in lateral resolution is observed compared with far-field magneto-optical images and good sensitivity is achieved. In the case of a Pt/[Co/Pt]Pt multilayer structure, stripe domains of 200 nm width could be resolved, in good agreement with images obtained by magnetic force microscopy on the same structure.
RESUMO
By ion irradiation through a lithographically made resist mask, the magnetic properties of cobalt-platinum simple sandwiches and multilayers were patterned without affecting their roughness and optical properties. This was demonstrated on arrays of 1-micrometer lines by near- and far-field magnetooptical microscopy. The coercive force and magnetic anisotropy of the irradiated regions can be accurately controlled by the irradiation fluence. If combined with high-resolution lithography, this technique holds promise for ultrahigh-density magnetic recording applications.
RESUMO
To investigate hepatitis C virus (HCV) and GBV-C/hepatitis G virus (HGV) genotype prevalence among HCV-infected porphyria cutanea tarda (PCT) patients, 19 HCV-infected patients with associated PCT were studied. A control group of 53 age-matched HCV-infected patients without associated PCT was selected. Eighteen of the 19 serologically positive HCV-PCT patients showed HCV RNA in serum. Genotype 1b was the most prevalent among both HCV-PCT patients (72.2%; 13/18) and age-matched HCV controls (50.9%; 27/53). Such different genotypic prevalence failed to reach statistical significance (chi(2) with Yates' correction, p = 0.19). The single HCV-PCT patient without detectable HCV RNA was also infected with genogroup 3 GBV-C/HGV. This GBV-C/HGV RNA prevalence (5.3%) among HCV-PCT patients is not statistically different from that observed among Argentine blood donors (5.5%; 11/200). To our knowledge, these results show for the first time the molecular epidemiology of both HCV and GBV-C/HGV associated to PCT in America.
Assuntos
Flaviviridae/genética , Hepacivirus/genética , Hepatite C/complicações , Hepatite Viral Humana/complicações , Porfiria Cutânea Tardia/complicações , Adulto , Idoso , Argentina/epidemiologia , Feminino , Genótipo , Hepatite C/epidemiologia , Hepatite Viral Humana/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , RNA Viral/genéticaRESUMO
An adult male farmer with chronic active hepatitis and cirrhosis despite previous circulating anti-HBs antibodies was studied. No markers of other hepatotropic viral infection were observed. HBV DNA was detected in serum by PCR and was characterized further by restriction fragment length polymorphism (RFLP) and sequencing of cloned PCR products derived from the S gene. The HBV DNA was ascribed to genotype F, and single-strand conformational polymorphism (SSCP) demonstrated the co-circulation of multiple quasispecies. Some of the variants exhibited changes located within the neutralizing "a" determinant, located between amino acids 124-147 of the S protein. Within this region, two clones showed either C124R or C124Y mutations. Other mutations were Q129R, C138R, C139R, and S140T (one clone each). Outside the "a" determinant several substitutions were documented. The high degree of the quasispecies variability was probably linked to the severity of the infection. Most members of the patient's family were infected with HBV, all with genotype F.