RESUMO
Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function.
Assuntos
Vírus Junin/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas da Matriz Viral/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/metabolismo , Infecções por Arenaviridae/virologia , Western Blotting , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Spodoptera/genética , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Assuntos
Fracionamento Celular/métodos , Separação Celular/métodos , Fezes/parasitologia , Giardia/isolamento & purificação , Oocistos , Oocistos/isolamento & purificação , Animais , DNA de Protozoário/isolamento & purificação , Cães , Eletroforese em Gel de Ágar , Endopeptidase K/farmacologia , Giardia/citologia , Giardia/genética , Temperatura Alta , Humanos , Oocistos/química , Oocistos/efeitos dos fármacos , Pressão Osmótica , Cloreto de Sódio/farmacologia , Soluções , Estresse Mecânico , Sacarose/farmacologiaRESUMO
El objetivo de este trabajo fue optimizar y evaluar las técnicas de purificación, aislamiento y ruptura de quistes de Giardia spp a partir de heces formoladas para la obtención de ADN. La materia fecal filtrada fue sometida a 3 técnicas de purificación, utilizando soluciones de formol-éter, sacarosa y formol-éter más sacarosa. La solución de sacarosa permitió aislar los quistes con menos detritos. Los quistes purificados fueron tratados con 3 técnicas para la ruptura de los mismos: shock osmótico y calor, degradación química y shock térmico, acción enzimática y efecto mecánico. Solamente con la técnica de shock térmico, acción enzimática y efecto mecánico se observaron bandas fluorescentes en geles de agarosa. Los resultados de este trabajo permiten contar con una metodología de rutina, simple, que podría ser usada en los pasos previos a la técnica de PCR para la genotipificación de este parásito.
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
Assuntos
Animais , Cães , Humanos , Fracionamento Celular/métodos , Separação Celular/métodos , Fezes/parasitologia , Giardia/isolamento & purificação , Oocistos , Oocistos/isolamento & purificação , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Ágar , Endopeptidase K/farmacologia , Giardia/citologia , Giardia/genética , Temperatura Alta , Pressão Osmótica , Oocistos/química , Oocistos/efeitos dos fármacos , Soluções , Estresse Mecânico , Cloreto de Sódio/farmacologia , Sacarose/farmacologiaRESUMO
The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.
RESUMO
OBJECTIVE: To compare patients with Coagulase Negative Staphylococcus bacteremia (CoNS-B) and pseudobacteremia (CoNS-PB) in a pediatric hospital. METHODS: Descriptive and comparative study between children diagnosed with CoNS-B and CoNS-PB. RESULTS: A total of 159 children with CoNS positive blood cultures were evaluated. 66 children were classified as CoNS-B (41.5%) and 93 as CoNS-PE3 (58.5%). On the average CoNS was isolated on day 21 among children with bacteremia (B) and on day 2 in those with PB (p < 0.01). Excluding newborns, patients with B and PB had on average 2.6 and 1.1 positive cultures respectively. Most children with bacteremia were at the intensive care unit (67.2%), while patients with PB were mostly detected at the emergency room. Using logistic regression analysis, we found four factors independently associated with CoNS bacteremia: total parenteral nutrition (OR 5.4; 95% CI 2.2-12.9), low birth weight (OR 2.6; 95% CI 1.1-5.9), catheters placed by cut-down technique (OR 1.9; 95% CI 1.1-3.8), and inmuno-compromised patients (OR 2.7; 95% CI 1.1-6.7). Resistance to oxacilin was reported in 71.7% of the CoNS isolated. The overall mortality associated to CoNS-B was 6%. Among children with CoNS-PB, 10% received antibiotics, half of them vancomycin. CONCLUSIONS: These results show that CoNS-B occurs mainly as a nosocomial episode. CoNS-PB more likely resulted from specimen contamination at collection, being responsible for almost 60% of all positive blood cultures. The false-positive results caused unnecessary administration of antibiotics in a significant proportion of CoNS-PB events and have a potential impact upon the emergence of resistant pathogens.
Assuntos
Bacteriemia/microbiologia , Infecções Estafilocócicas/microbiologia , Adolescente , Bacteriemia/epidemiologia , Criança , Pré-Escolar , Coagulase/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Infecções Estafilocócicas/epidemiologia , Staphylococcus/enzimologiaRESUMO
RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junín virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.
Assuntos
Arenavirus/fisiologia , Células Eucarióticas/virologia , Proteínas do Nucleocapsídeo/fisiologia , Animais , Arenavirus/genética , Arenavirus/metabolismo , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular , Cricetinae , Vírus Junin/química , Vírus Junin/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Viral/análise , RNA Viral/genética , Transcrição Gênica , Ativação Transcricional , Transfecção , Replicação Viral/genéticaRESUMO
DNA fingerprints of lactic acid bacteria were generated by polymerase chain reaction using a primer based on the repetitive elements found in the genome of Streptococcus pneumoniae (BOX-PCR). The method made it possible to identify 37 isolates from raw milk. industrial starters and yogurt. Differentiation at species, subspecies and strain level was possible for Lactobacillus delbrueckii subsp. lactis, Lb. delbrueckii subsp bulgaricus and Str. thermophilus. BOX-PCR was also applied to studying the strain composition of a starter culture and the direct detection of strains in commercial fermented milk.
Assuntos
Impressões Digitais de DNA/métodos , Lactobacillus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Streptococcus/isolamento & purificação , Animais , Sequência de Bases , DNA Bacteriano , Lactobacillus/classificação , Lactobacillus/genética , Leite/microbiologia , Dados de Sequência Molecular , Fenótipo , Sequências Repetitivas de Ácido Nucleico , Streptococcus/classificação , Streptococcus/genética , Iogurte/microbiologiaRESUMO
Bread from wheat flour is one of the most widely consumed products by the Mexican population. In this study, proximate composition, and mineral content were determined, and cost per/gram of protein and energy of traditional and industrial bread were compared. Seven types of bread were analyzed: bolillo, virginia, white bread and pastries conchita, croissant, breadroll and donuts. Products were analyzed for proximate composition by official methods, and content of Na, K, Ca, Mg, Fe, y Zn by atomic absorption spectrometry. Based on 24 hour recall interviews, the consumption of each type of bread as well as the percent of protein and energy allowances were calculated. Significant differences (p < 0.05) were found with respect to nutrient content between traditional and industrial breads. White bread and pastries from industrial origin showed higher costs (p < 0.05) per gram of protein and energy than traditional products in most cases. Both industrial and traditional breads showed higher fat content than that established by Official Mexican regulations. A high content of Ca was found in bread from the industrial origin and K, Mg, Fe y Zn were higher (p < 0.05) than those from the traditional bakery.
Assuntos
Pão/análise , Ingestão de Energia , Indústria Alimentícia , Proteínas/química , México , Valor NutritivoRESUMO
Arenaviridae is a worldwide distributed family, of enveloped, single stranded, RNA viruses. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as the geographic distribution. In this study the phylogenetic relationship among the members of the Arenaviridae was examined, using the reported genomic sequences. The comparison of the aligned nucleotide sequences of the S RNA and the predicted amino acid sequences of the GPC and N proteins, together with the phylogenetic analysis, strongly suggest a possible kinship of Pichindé and Oliveros viruses, with the Old World arenavirus group. This analysis points at the evolutive relationships between the arenaviruses of the Americas and can be used to evaluate the different hypotheses about their origin.
Assuntos
Arenavirus/genética , Filogenia , Arenavirus/classificação , Sequência de Bases , Evolução Molecular , Genes Virais , Vírus Pichinde/genética , Análise de Sequência de RNA , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
The Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper we report the nucleotide sequences of S RNA of Candid #1 and its more virulent ancestors XJ#44 and XJ (prototype). Their relationship to Junin virus wild-type MC2 strain and other closely and distantly related arenaviruses was also examined. Comparisons of the nucleotide and amino acid sequences of N and GPC genes from Candid #1 and its progenitor strains revealed some changes that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype.
Assuntos
Vírus Junin/genética , Vacinas Virais/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Humanos , Dados de Sequência Molecular , Nucleocapsídeo/genética , RNA Viral , Homologia de Sequência de Aminoácidos , Vacinas Atenuadas/genética , Proteínas do Envelope Viral/genéticaRESUMO
Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.
Assuntos
Arenavirus/genética , Arenavirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Animais , Arenavirus/classificação , Sequência de Bases , Linhagem Celular , Sequência Conservada , Cricetinae , Primers do DNA , Evolução Molecular , Genoma Viral , Rim , Dados de Sequência Molecular , Filogenia , RNA Viral/química , RNA Viral/genética , Alinhamento de SequênciaRESUMO
BLAD (Bovine Leukocyte Adhesion Deficiency) and DUMPS (Deficiency of Uridine Monophosphate Synthase) are monogenic autosomal, recessive inherited diseases of Holstein cattle. Single nucleotide changes (point mutations) responsible for the genetic disorders were detected by polymerase chain reaction coupled with restriction fragment length polymorphism assays (PCR-RFLP). Using oligonucleotide primers, DNA fragments of predicted sizes were amplified, and the products' specificity was assessed by nucleotide sequencing. Mutations were detected in DNA samples from bovine blood and semen by the presence or absence of restriction sites within the PCR amplification products (Taq I, Hae III for BLAD, Ava I for DUMPS). The test included 104 bulls and 950 cows of Argentinean Holstein breed. Defective alleles frequencies were as follows: 2.88% BLAD in bulls used in artificial insemination, 1.79% in cows; 0.96% DUMPS in bulls and 0.11% in cows.
Assuntos
Doenças dos Bovinos/diagnóstico , Síndrome da Aderência Leucocítica Deficitária/veterinária , Programas de Rastreamento/veterinária , Complexos Multienzimáticos/deficiência , Orotato Fosforribosiltransferase/deficiência , Orotidina-5'-Fosfato Descarboxilase/deficiência , Reação em Cadeia da Polimerase/veterinária , Animais , Argentina/epidemiologia , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/genética , DNA/genética , Feminino , Genes Recessivos , Síndrome da Aderência Leucocítica Deficitária/diagnóstico , Síndrome da Aderência Leucocítica Deficitária/genética , Masculino , Programas de Rastreamento/métodos , Complexos Multienzimáticos/genética , Orotato Fosforribosiltransferase/genética , Orotidina-5'-Fosfato Descarboxilase/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , PrevalênciaRESUMO
Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%.
Assuntos
Febre Hemorrágica Americana/diagnóstico , Vírus Junin/genética , Reação em Cadeia da Polimerase/métodos , Virologia/métodos , Argentina , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , Erros de Diagnóstico , Método Duplo-Cego , Febre Hemorrágica Americana/virologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/estatística & dados numéricos , RNA Viral/sangue , RNA Viral/genética , DNA Polimerase Dirigida por RNA , Sensibilidade e Especificidade , Fatores de Tempo , Virologia/estatística & dados numéricosRESUMO
Argentine hemorrhagic fever (AHF) is an endemoepidemic disease with cardiovascular, renal and neurologic alterations acquired in the richest farming land in Argentina. It is caused by Junín virus, one of the few human pathogenic arenaviruses. The S RNA of Junín virus has been molecularly cloned and its nucleotide sequence determined in our laboratory. This information was used to develop a rapid nucleic acid-based diagnostic test commensurate with the low viraemia detected in AHF patients. Junín virus-specific cDNA probes labeled using various methods proved insensitive in dot-hybridizations. Therefore, a RT polymerase chain reaction (PCR) was developed using a pair of oligonucleotide primers to reverse-transcribe and amplify the viral S RNA. The amplification of the target sequences was measured by ethidium bromide staining of the DNA fragments after agarose gel electrophoresis. This type of assay allowed the specific detection of Junín virus RNA sequences present in a single infected BHK21 cell over a background of 10(4) uninfected cells. Control reactions were performed on RNA samples extracted from uninfected cells or cells infected with a high multiplicity of LCMV, another arenavirus present in the AHF endemic area. The PCR was first adapted to detect viral RNA in peripheral blood mononuclear cells, described to harbor most of the virus. A simplification of this assay allows the detection of Junín virus in RNA extracted from 100 microliters of whole blood using guanidium thiocyanate disruption and acid phenol extraction. Under the conditions described in this paper, it is possible to detect up to 0.01 pfu of Junín virus in a blood sample. An early and rapid laboratory diagnostic test for AHF is important since the only effective therapy that reduces the mortality rate from 30% to less than 1% consists of early treatment with immune plasma.
Assuntos
Arenavirus do Novo Mundo/genética , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Animais , Arenavirus do Novo Mundo/isolamento & purificação , Sequência de Bases , Linhagem Celular , DNA Viral/genética , Estudos de Avaliação como Assunto , Febre Hemorrágica Americana/diagnóstico , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , RNA Viral/sangue , Sensibilidade e EspecificidadeRESUMO
In this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5' end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3'-terminal sequences and may form a very stable panhandle structure (delta G-242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (delta G -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function.