RESUMEN
The monitoring of molecular systems usually requires sophisticated technologies to interpret nanoscale events into electronic-decipherable signals. We demonstrate a new method for obtaining read-outs of molecular states that uses graphics processing units made from molecular circuits. Because they are made from molecules, the units are able to directly interact with molecular systems. We developed deoxyribozyme-based graphics processing units able to monitor nucleic acids and output alphanumerical read-outs via a fluorescent display. Using this design we created a molecular 7-segment display, a molecular calculator able to add and multiply small numbers, and a molecular automaton able to diagnose Ebola and Marburg virus sequences. These molecular graphics processing units provide insight for the construction of autonomous biosensing devices, and are essential components for the development of molecular computing platforms devoid of electronics.
Asunto(s)
Técnicas Biosensibles , Gráficos por Computador , ADN Catalítico/química , Ácidos Nucleicos/análisis , ADN Catalítico/metabolismo , ElectrónicaRESUMEN
BACKGROUND: The analysis of oligonucleotide microarray data in pathogen surveillance and discovery is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied. RESULTS: Positive predictive value and false positive rates were examined to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney U, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, the chi-square proved most useful. CONCLUSIONS: The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Virus/genética , Animales , Perfilación de la Expresión Génica , Humanos , Modelos Estadísticos , Hibridación de Ácido Nucleico/métodos , Estándares de ReferenciaRESUMEN
Oligonucleotide microarrays have been applied to microbial surveillance and discovery where highly multiplexed assays are required to address a wide range of genetic targets. Although printing density continues to increase, the design of comprehensive microbial probe sets remains a daunting challenge, particularly in virology where rapid sequence evolution and database expansion confound static solutions. Here, we present a strategy for probe design based on protein sequences that is responsive to the unique problems posed in virus detection and discovery. The method uses the Protein Families database (Pfam) and motif finding algorithms to identify oligonucleotide probes in conserved amino acid regions and untranslated sequences. In silico testing using an experimentally derived thermodynamic model indicated near complete coverage of the viral sequence database.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos , Sondas de Oligonucleótidos/química , Análisis de Secuencia de Proteína/métodos , Proteínas Virales/química , Virus/aislamiento & purificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia Conservada , Bases de Datos de Ácidos Nucleicos , Bases de Datos de Proteínas , Genes Virales , Genoma Viral , VIH-1/genética , Humanos , Homología de Secuencia de Aminoácido , Proteínas Virales/genética , Virus/genéticaRESUMEN
While worldwide pandemic influenza A(H1N1) pdm case fatality rate (CFR) was 0.4%, Argentina's was 4.5%. A total of 34 strains from mild and severe cases were analyzed. A full genome sequencing was carried out on 26 of these, and a partial sequencing on the remaining eight. We observed no evidence that the high CFR can be attributed to direct virus changes. No evidence of re-assortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence was observed. Although the mutation D225G associated with severity in the latest reports from the Ukraine and Norway is not observed among the Argentine strains, an amino acid change in the area (S206T) surrounding the HA receptor binding domain was observed, the same previously established worldwide.
Asunto(s)
ADN Viral/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/virología , Mutación/genética , Adolescente , Adulto , Argentina/epidemiología , Niño , Preescolar , Análisis por Conglomerados , Femenino , Humanos , Lactante , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/mortalidad , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Viral/genética , Receptores Virales/genética , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Polymerase chain reaction (PCR) is widely applied in clinical and environmental microbiology. Primer design is key to the development of successful assays and is often performed manually by using multiple nucleic acid alignments. Few public software tools exist that allow comprehensive design of degenerate primers for large groups of related targets based on complex multiple sequence alignments. Here we present a method for designing such primers based on tree building followed by application of a set covering algorithm, and demonstrate its utility in compiling Multiplex PCR primer panels for detection and differentiation of viral pathogens.
Asunto(s)
Cartilla de ADN/química , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Programas Informáticos , Algoritmos , Biología Computacional , ADN Viral/análisisRESUMEN
Eukaryotes contain three essential Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin, and Smc5/6. Cohesin forms a ring-shaped structure that embraces sister chromatids to promote their cohesion. The cohesiveness of cohesin is promoted by acetylation of N-terminal lysines of the Smc3 subunit by the acetyltransferases Eco1 in Saccharomyces cerevisiae and the homologue, Eso1, in Schizosaccharomyces pombe. In both yeasts, these acetyltransferases are essential for cell viability. However, whereas nonacetylatable Smc3 mutants are lethal in S. cerevisiae, they are not in S. pombe We show that the lethality of a temperature-sensitive allele of eso1 (eso1-H17) is due to activation of the spindle assembly checkpoint (SAC) and is associated with premature centromere separation. The lack of cohesion at the centromeres does not correlate with Psm3 acetylation or cohesin levels at the centromeres, but is associated ith significantly reduced recruitment of the cohesin regulator Pds5. The SAC activation in this context is dependent on Smc5/6 function, which is required to remove cohesin from chromosome arms but not centromeres. The mitotic defects caused by Smc5/6 and Eso1 dysfunction are cosuppressed in double mutants. This identifies a novel function (or functions) for Eso1 and Smc5/6 at centromeres and extends the functional relationships between these SMC complexes.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/enzimología , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Acetiltransferasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/fisiología , Proteínas de Ciclo Celular/genética , Cromátides/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cromosomas Fúngicos/enzimología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Mitosis/fisiología , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Proteínas Nucleares/genética , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Proteínas de Schizosaccharomyces pombe/genética , CohesinasRESUMEN
Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics. IMPORTANCE : VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Microbiota , Virología/métodos , Virosis/diagnóstico , Virus/clasificación , Virus/aislamiento & purificación , Animales , Humanos , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Vertebrados , Virosis/veterinaria , Virus/genéticaRESUMEN
Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/metabolismo , Histonas/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Acetilación , Proteínas de Ciclo Celular/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Daño del ADN/genética , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Mitosis/genética , Mutación , Interferencia de ARN , ARN Interferente Pequeño , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , CohesinasRESUMEN
UNLABELLED: Deep sequencing of RNAs produced by Zaire ebolavirus (EBOV) or the Angola strain of Marburgvirus (MARV-Ang) identified novel viral and cellular mechanisms that diversify the coding and noncoding sequences of viral mRNAs and genomic RNAs. We identified previously undescribed sites within the EBOV and MARV-Ang mRNAs where apparent cotranscriptional editing has resulted in the addition of non-template-encoded residues within the EBOV glycoprotein (GP) mRNA, the MARV-Ang nucleoprotein (NP) mRNA, and the MARV-Ang polymerase (L) mRNA, such that novel viral translation products could be produced. Further, we found that the well-characterized EBOV GP mRNA editing site is modified at a high frequency during viral genome RNA replication. Additionally, editing hot spots representing sites of apparent adenosine deaminase activity were found in the MARV-Ang NP 3'-untranslated region. These studies identify novel filovirus-host interactions and reveal production of a greater diversity of filoviral gene products than was previously appreciated. IMPORTANCE: This study identifies novel mechanisms that alter the protein coding capacities of Ebola and Marburg virus mRNAs. Therefore, filovirus gene expression is more complex and diverse than previously recognized. These observations suggest new directions in understanding the regulation of filovirus gene expression.
Asunto(s)
Ebolavirus/genética , Marburgvirus/genética , Edición de ARN , ARN Mensajero/metabolismo , Replicación Viral , Animales , Línea Celular , Chlorocebus aethiops , Ebolavirus/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Humanos , Marburgvirus/fisiología , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , Análisis de Secuencia de ADNRESUMEN
ABSTRACT Complete Middle East respiratory syndrome coronavirus (MERS-CoV) genome sequences were obtained from nasal swabs of dromedary camels sampled in the Kingdom of Saudi Arabia through direct analysis of nucleic acid extracts or following virus isolation in cell culture. Consensus dromedary MERS-CoV genome sequences were the same with either template source and identical to published human MERS-CoV sequences. However, in contrast to individual human cases, where only clonal genomic sequences are reported, detailed population analyses revealed the presence of more than one genomic variant in individual dromedaries. If humans are truly infected only with clonal virus populations, we must entertain a model for interspecies transmission of MERS-CoV wherein only specific genotypes are capable of passing bottleneck selection. IMPORTANCE In most cases of Middle East respiratory syndrome (MERS), the route for human infection with the causative agent, MERS coronavirus (MERS-CoV), is unknown. Antibodies to and viral nucleic acids of MERS-CoV have been found in dromedaries, suggesting the possibility that they may serve as a reservoir or vector for human infection. However, neither whole viral genomic sequence nor infectious virus has been isolated from dromedaries or other animals in Saudi Arabia. Here, we report recovery of MERS-CoV from nasal swabs of dromedaries, demonstrate that MERS-CoV whole-genome consensus sequences from dromedaries and humans are indistinguishable, and show that dromedaries can be simultaneously infected with more than one MERS-CoV. Together with data indicating widespread dromedary infection in the Kingdom of Saudi Arabia, these findings support the plausibility of a role for dromedaries in human infection.
Asunto(s)
Camelus/virología , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Genoma Viral , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/aislamiento & purificación , Animales , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/clasificación , Datos de Secuencia Molecular , Filogenia , ARN Viral , Arabia Saudita/epidemiologíaRESUMEN
Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.
Asunto(s)
Escherichia coli/genética , 5-Metilcitosina/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Biotecnología , Mapeo Cromosómico , Metilación de ADN/genética , Enzimas de Restricción-Modificación del ADN/deficiencia , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Amplificación de Genes , Eliminación de Gen , Genoma Bacteriano , Análisis de Secuencia de ADN/métodos , Compuestos de Espiro , Especificidad por SustratoRESUMEN
To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004-2005.
Asunto(s)
Enfermedades Transmisibles/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Virosis/diagnóstico , Enfermedades Transmisibles/virología , Brotes de Enfermedades , Resultado Fatal , Humanos , Malaria Falciparum/diagnóstico , Filogenia , Sensibilidad y Especificidad , Virosis/virologíaRESUMEN
Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.
Asunto(s)
Virus de la Influenza A/clasificación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Infecciones del Sistema Respiratorio/virología , Virus/aislamiento & purificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza A/genética , Neuraminidasa/genética , Rhinovirus/clasificación , Rhinovirus/genética , Proteínas Virales/genética , Virus/genéticaRESUMEN
While worldwide pandemic influenza A(H1N1) pdm case fatality rate (CFR) was 0.4%, Argentina's was 4.5%. A total of 34 strains from mild and severe cases were analyzed. A full genome sequencing was carried out on 26 of these, and a partial sequencing on the remaining eight. We observed no evidence that the high CFR can be attributed to direct virus changes. No evidence of re-assortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence was observed. Although the mutation D225G associated with severity in the latest reports from the Ukraine and Norway is not observed among the Argentine strains, an amino acid change in the area (S206T) surrounding the HA receptor binding domain was observed, the same previously established worldwide.
Mientras que la tasa de letalidad (CFR) para (H1N1)pdm en todo el mundo era del 0.4%, en la Argentina la mortalidad observada fue de 4.5%. La secuenciación del genoma completo de 26 cepas de virus argentinos de influenza A (H1N1)pdm de casos leves y graves y de 8 cepas secuenciadas parcialmente no mostró evidencia de que la elevada tasa de letalidad se pueda atribuir directamente a cambios en el virus. No se encontraron hallazgos de recombinación, de mutaciones asociadas con la resistencia a los medicamentos antivirales ni de variaciones genéticas que puedan contribuir a la virulencia observada. Si bien la mutación D225G asociada con la gravedad, comunicada en informes procedentes de Ucrania y Noruega, no se ha encontrado en las cepas argentinas estudiadas, se ha observado un cambio aminoacídico en la región (S206T) en torno al dominio del sitio de unión al receptor en la HA, el mismo hallado en cepas distribuidas alrededor del mundo.