RESUMEN

Antiviral drug resistance hepatitis B virus (HBV) variants (HBV-DR) occur spontaneously in chronic hepatitis B (CHB) patients and after exposure to nucleos(t)ide analogues (NUCs). We determined the prevalence of HBV-DR variants among participants of the Hepatitis B Research Network (HBRN) Cohort Study conducted at 21 sites in the United States (US) and Canada. Samples obtained from 1342 CHB participants aged ≥18 years, and who were currently not receiving NUCs, were tested for HBV-DR variants by Sanger sequencing. In addition, next generation sequencing (NGS) was used to characterize HBV-DR variants from 66 participants with and 66 participants with no prior NUC exposure matched for HBV genotype and HBV DNA level. Half the participants were men, 75% Asian, 26% HBeAg positive. Primary HBV-DR variants were detected by Sanger sequencing in 16 (1.2%) participants: 2/142 (1.4%) with and 14/1200 (1.2%) without prior NUC exposure; only 1 of these 16 had a secondary variant. In total, 23 (1.7%) participants had secondary variants, including 1 with prior NUC experience. In the subset of 132 participants, NGS detected HBV-DR variants in a higher proportion of participants: primary variants in 18 (13.6%) (8 [12.1%] with, and 10 [15.2%] without prior NUC therapy) and secondary variants in 10 (7.6%) participants. Based on Sanger sequencing, prevalence of primary HBV-DR variants is low (1.2%) among adults with CHB in US/Canada. The similar low prevalence of HBV-DR variants in participants with and without NUC treatment suggests transmission of these variants is uncommon.

Asunto(s)

Antivirales/farmacología , Farmacorresistencia Viral , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/epidemiología , Adolescente , Adulto , Anciano , Antivirales/uso terapéutico , ADN Viral , Femenino , Variación Genética , Genotipo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mutación , América del Norte/epidemiología , Vigilancia de la Población , Prevalencia , Análisis de Secuencia de ADN , Adulto Joven

RESUMEN

The reason(s) that hepatitis A virus (HAV) infection may progress infrequently to acute liver failure are poorly understood. We examined host and viral factors in 29 consecutive adult patients with HAV-associated acute liver failure enrolled at 10 sites participating in the US ALF Study Group. Eighteen of twenty-four acute liver failure sera were PCR positive while six had no detectable virus. HAV genotype was determined using phylogenetic analysis and the full-length genome sequences of the HAV from a cute liver failure sera were compared to those from self-limited acute HAV cases selected from the CDC database. We found that rates of nucleotide substitution did not vary significantly between the liver failure and non-liver failure cases and there was no significant variation in amino acid sequences between the two groups. Four of 18 HAV isolates were sub-genotype IB, acquired from the same study site over a 3.5-year period. Sub-genotype IB was found more frequently among acute liver failure cases compared to the non-liver failure cases (chi-square test, P < 0.01). At another centre, a mother and her son presented with HAV and liver failure within 1 month of each other. Predictors of spontaneous survival included detectable serum HAV RNA, while age, gender, HAV genotype and nucleotide substitutions were not associated with outcome. The more frequent appearance of rapid viral clearance and its association with poor outcomes in acute liver failure as well as the finding of familial cases imply a possible host genetic predisposition that contributes to a fulminant course. Recurrent cases of the rare sub-genotype IB over several years at a single centre imply a community reservoir of infection and possible increased pathogenicity of certain infrequent viral genotypes.

Asunto(s)

Virus de la Hepatitis A/genética , Hepatitis A/mortalidad , Fallo Hepático Agudo/mortalidad , Acetaminofén/uso terapéutico , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Biomarcadores , Mapeo Cromosómico , Femenino , Genotipo , Hepatitis A/complicaciones , Hepatitis A/tratamiento farmacológico , Hepatitis A/cirugía , Virus de la Hepatitis A/aislamiento & purificación , Humanos , Fallo Hepático Agudo/virología , Trasplante de Hígado , Masculino , Persona de Mediana Edad , Mutación , Reacción en Cadena de la Polimerasa , ARN Viral/sangre , ARN Viral/genética , Factores de Riesgo , Análisis de Secuencia de ARN

RESUMEN

The detection of compensatory mutations that abrogate negative fitness effects of drug-resistance and vaccine-escape mutations indicates the important role of epistatic connectivity in evolution of viruses, especially under the strong selection pressures. Mapping of epistatic connectivity in the form of coordinated substitutions should help to characterize molecular mechanisms shaping viral evolution and provides a tool for the development of novel anti-viral drugs and vaccines. We analyzed coordinated variation among amino acid sites in 370 the hepatitis B virus (HBV) polymerase sequences using Bayesian networks. Among the HBV polymerase domains the spacer domain separating terminal protein from the reverse-transcriptase domain, showed the highest network centrality. Coordinated substitutions preserve the hydrophobicity and charge of Spacer. Maximum likelihood estimates of codon selection showed that Spacer contains the highest number of positively selected sites. Identification of 67% of the domain lacking an ordered structure suggests that Spacer belongs to the class of intrinsically disordered domains and proteins whose crucial functional role in the regulation of transcription, translation and cellular signal transduction has only recently been recognized. Spacer plays a central role in the epistatic network associating substitutions across the HBV genome, including those conferring viral virulence, drug resistance and vaccine escape. The data suggest that Spacer is extensively involved in coordination of HBV evolution.

Asunto(s)

ADN Polimerasa Dirigida por ADN/química , Evolución Molecular , Virus de la Hepatitis B/enzimología , Proteínas Virales/química , ADN Polimerasa Dirigida por ADN/genética , Estructura Terciaria de Proteína , Proteínas Virales/genética

RESUMEN

Hepatitis C Virus sequence studies mainly focus on the viral amplicon containing the Hypervariable region 1 (HVR1) to obtain a sample of sequences from which several population genetics parameters can be calculated. Recent advances in sequencing methods allow for analyzing an unprecedented number of viral variants from infected patients and present a novel opportunity for understanding viral evolution, drug resistance and immune escape. In the present paper, we compared three recent technologies for amplicon analysis: (i) Next-Generation Sequencing; (ii) Clonal sequencing using End-point Limiting-dilution for isolation of individual sequence variants followed by Real-Time PCR and sequencing; and (iii) Mass spectrometry of base-specific cleavage reactions of a target sequence. These three technologies were used to assess intra-host diversity and inter-host genetic relatedness in HVR1 amplicons obtained from 38 patients (subgenotypes 1a and 1b). Assessments of intra-host diversity varied greatly between sequence-based and mass-spectrometry-based data. However, assessments of inter-host variability by all three technologies were equally accurate in identification of genetic relatedness among viral strains. These results support the application of all three technologies for molecular epidemiology and population genetics studies. Mass spectrometry is especially promising given its high throughput, low cost and comparable results with sequence-based methods.

Asunto(s)

Genoma Viral/genética , Hepacivirus/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Espectrometría de Masas/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

Genotyping of hepatitis B virus (HBV) is important for tracking HBV infections, prognosticating the development of severe liver disease, and predicting outcomes of therapy. Current genotyping methods can be laborious and costly and rely on subjective data interpretation. To identify less expensive but equally reliable alternatives, we compared "gold standard" sequencing to a novel mass spectrometry approach. Sera from individuals with acute or chronic HBV infection (n = 756), representing all genotypes, were used to PCR amplify the HBV S gene. All amplicons were subjected to base-specific cleavage and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The resulting mass peak patterns were used to identify HBV genotype by automated comparison to peak patterns simulated from reference sets of HBV sequences of known genotypes. The MALDI-TOF MS data and phylogenetic analysis of HBV sequences produced completely concordant results. Several parameters such as genetic relatedness of tested HBV variants to the reference set, chronic infections, and the quality of PCR products can lower the MS score but never affected the accuracy of the genotype call. This new streamlined MS-based method provides for rapid and accurate HBV genotyping, produces automated data reports, and is therefore suitable for routine use in diagnostic settings.

Asunto(s)

Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/genética , Hepatitis B/virología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Virología/métodos , ADN Viral/química , ADN Viral/genética , Genotipo , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Filogenia , Reacción en Cadena de la Polimerasa

RESUMEN

Numerous hepatitis A outbreaks were linked to the consumption of raw molluscan shellfish in the United States between 1960 and 1989. However, there had been no major molluscan shellfish-associated hepatitis A outbreaks reported in the United States for more than a decade (1989 to 2004). Beginning in late August 2005, at least 10 clusters of hepatitis A illnesses, totaling 39 persons, occurred in four states among restaurant patrons who ate oysters. Epidemiologic data indicated that oysters were the source of the outbreak. Traceback information showed that the implicated oysters were harvested from specific Gulf Coast areas. A voluntary recall of oysters was initiated in September. Hepatitis A virus (HAV) was detected in multiple 25-g portions in one of two recalled samples, indicating that as many as 1 of every 15 oysters from this source was contaminated. Comparing 315 nucleotides within the HAV VPl-2B region, 100% homology was found among four amplicons recovered from a total of six independent experiments of the implicated oysters, and an identical HAV sequence was detected in sera from all 28 patient serum specimens tested. Ten percent heterogeneity over 315 nucleotides (31 variants) was observed between the outbreak strain (subgenotype 1A) and an HM-175 strain (subgenotype 1B) used in the laboratory where the oysters were processed. To our knowledge, this investigation is the first in the United States to identify an HAV-identical strain in persons with hepatitis A as well as in the food that was implicated as the source of their infections.

Asunto(s)

Contaminación de Alimentos/análisis , Virus de la Hepatitis A/aislamiento & purificación , Hepatitis A/epidemiología , Ostreidae/virología , Mariscos/virología , Animales , Secuencia de Bases , Análisis por Conglomerados , Brotes de Enfermedades , Reservorios de Enfermedades , Virus de la Hepatitis A/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia , Estados Unidos/epidemiología

RESUMEN

Bacteria employ autoinduction systems to sense the onset of appropriate cell density for expression of developmental genes. In many gram-negative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families. Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium that appears to autoregulate its virulence genes, produces compounds that promote expression of several heterologous acyl-HSL-responsive reporter gene constructs. High-pressure liquid chromatography of highly concentrated ethyl acetate extracts revealed that culture supernatants of strain AW1 contained two compounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL. To investigate the role of these acyl-HSLs in R. solanacearum virulence gene expression, transposon mutants that were deficient for inducing an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were generated. Three loci involved in normal acyl-HSL production were identified, one of which was shown to contain the divergently transcribed solR and solI genes, the luxR and luxI homologs, respectively. A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardless of the locus inactivated) and a naturally acyl-HSL-defective strain of R. solanacearum to produce acyl-HSLs. Inactivation of solI abolished production of all detectable acyl-HSLs but affected neither the expression of virulence genes in culture nor the ability to wilt tomato plants. AW1 has a functional autoinduction system, because (i) expression of solI required SolR and acyl-HSL and (ii) expression of a gene linked to solR and solI, designated aidA, was acyl-HSL dependent. Because AidA has no homologs in the protein databases, its discovery provided no clues as to the role of acyl-HSLs in R. solanacearum gene regulation. However, expression of solR and solI required the global LysR-type virulence regulator PhcA, and both solR and solI exhibited a cell density-associated pattern of expression similar to other PhcA-regulated genes. The acyl-HSL-dependent autoinduction system in R. solanacearum is part of a more complex autoregulatory hierarchy, since the transcriptional activity of PhcA is itself controlled by a novel autoregulatory system that responds to 3-hydroxypalmitic acid methyl ester.

Asunto(s)

4-Butirolactona/análogos & derivados , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Bacilos y Cocos Aerobios Gramnegativos/genética , Bacilos y Cocos Aerobios Gramnegativos/metabolismo , Ácidos Palmíticos/metabolismo , Proteínas Represoras , Transactivadores , 4-Butirolactona/análisis , 4-Butirolactona/aislamiento & purificación , 4-Butirolactona/metabolismo , Agrobacterium tumefaciens/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Bacilos y Cocos Aerobios Gramnegativos/patogenicidad , Datos de Secuencia Molecular , Plásmidos , Recombinación Genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Virulencia/genética

RESUMEN

Susceptible plants infected by Pseudomonas solanacearum usually will, largely due to extracellular proteins (EXPs) and the high-molecular-mass extracellular polysaccharide (EPS I) this pathogen produces. Circumstantial evidence suggested that a 28-kDa protein, the single most abundant EXP made by P. solanacearum in culture, is associated with production of EPS I, and thus might have a role in pathogenesis. The 28-kDa EXP was purified and, based on its N-terminal amino acid sequence, an oligonucleotide mixture was made and used as a hybridization probe to clone the gene encoding it. DNA sequence analysis suggested that the coding sequence for the 28-kDa EXP is within a gene, designated tek, that encodes a 58-kDa membrane-associated precursor protein that is processed by signal peptidase II during export. Analysis of radiolabeled polypeptides expressed from tek confirmed that it encodes a 58-kDa precursor protein, which is exported out of the cells as a 55-kDa preprotein and processed extracellularly to release the very basic 28-kDa EXP from its C terminus. The position, transcriptional direction, and regulated expression of tek suggest that it is cotranscribed with xpsR, a gene essential for regulating biosynthesis of EPS I, and reinforces the association of the 28-kDa EXP with virulence. However, since P. solanacearum mutants lacking only the 28-kDa EXP produced wild-type amounts of EPS I and were fully virulent, the function of this protein remains unclear.

Asunto(s)

Proteínas Bacterianas/genética , Genes Bacterianos , Proteínas de la Membrana/genética , Pseudomonas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Polisacáridos Bacterianos/biosíntesis , Pseudomonas/patogenicidad , Virulencia/genética

RESUMEN

Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis. It is a member of the response regulator superfamily of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of sporulation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-containing surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spoIVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria.

Asunto(s)

Bacillus/genética , Bacillus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clostridium/genética , Clostridium/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Bacillus/fisiología , Secuencia de Bases , Sitios de Unión/genética , Clostridium/fisiología , Secuencia Conservada , Cartilla de ADN/genética , ADN Bacteriano/genética , Genes Bacterianos , Secuencias Hélice-Asa-Hélice , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Esporas Bacterianas/genética , Esporas Bacterianas/fisiología