RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is caused by a respiratory virus with a wide range of manifestations, varying from asymptomatic to fatal cases, with a generally short outcome. However, some individuals present long-term viral shedding. We monitored 38 individuals who were mildly affected by the SARS-CoV-2 infection. Out of the total studied population, three (7.9%) showed atypical events regarding the duration of positivity for viral RNA detection. In one of these atypical cases, a previously HIV-positive male patient presented a SARS-CoV-2 RNA shedding and subgenomic RNA (sgRNA) detected from the upper respiratory tract, respectively, for 232 and 224 days after the onset of the symptoms. The SARS-CoV-2 B.1.1.28 lineage, one of the most prevalent in Brazil in 2020, was identified in this patient in three serial samples. Interestingly, the genomic analyses performed throughout the infectious process showed an increase in the genetic diversity of the B.1.1.28 lineage within the host itself, with viral clearance occurring naturally, without any intervention measures to control the infection. Contrasting widely spread current knowledge, our results indicate that potentially infectious SARS-CoV-2 virus might be shed by much longer periods by some infected patients. This data call attention to better adapted non-pharmacological measures and clinical discharge of patients aiming at preventing the spread of SARS-CoV-2 to the population.
RESUMO
BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Genoma Viral , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral , Análise de Sequência de RNA/métodos , Montagem de Vírus , Alphavirus/genética , República Centro-Africana , Biologia Computacional , Mapeamento de Sequências Contíguas , Mengovirus/genética , Valores de Referência , Reprodutibilidade dos Testes , SoftwareRESUMO
BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Viral , Genoma Viral , Análise de Sequência de RNA/métodos , Montagem de Vírus , Técnicas de Amplificação de Ácido Nucleico/métodos , Valores de Referência , Software , República Centro-Africana , Reprodutibilidade dos Testes , Alphavirus/genética , Mengovirus/genética , Biologia Computacional , Mapeamento de Sequências ContíguasRESUMO
La hepatitis C aguda es una enfermedad generalmente subclínica, de ahí que no se incluya en el diagnóstico diferencial de los pacientes con un cuadro agudo. Además diagnosticarla presenta dificultades ya que los anticuerpos contra el virus tardan en aparecer, pudiendo ser negativos cuando el paciente manifiesta los síntomas; en este punto la enfermedad podría diagnosticarse con el RNA viral, pero éste no es fácil que sea solicitado inicialmente. Se presenta un paciente que ingresó por una hepatitis aguda en el que se descartaron causas virales como hepatitis A-B, Ebstein Barr, Citomegalovirus (CMV) hepatitis autoinmune, hepatotoxicidad y enfermedad hipoxicoisquémica, que explicaran la sintomatología y los hallazgos bioquímicos del paciente, en quien se demostró seroconversión contra el virus de la hepatitis C asociado a una carga viral elevada. Todo lo anterior es consistente con un diagnóstico de hepatitis C aguda. Se describe el manejo del paciente y las características de la enfermedad.
Acute hepatitis C is usually a sub-clinical disease, thus it is not included in the differential diagnosis of patients with acute disease. Making the diagnosis is also difficult because the virus antibodies appear at later stages and many even be negative even if the patient has symptoms; at this point the diagnosis of the disease could be made with the viral RNA, but it is not easy to ask for it initially. A patient is admitted because of acute hepatitis where viral causes such as hepatitis A-B, Epstein Barr, Cytomegalovirus (CMV), auto-immune hepatitis, hepatoxitiy and hypoxic-isquemic disease, that would explain the symptoms and bio-chemical findings were discarded. The patients seroconversion against Hepatitis C virus associated to a high viral load was demonstrated. All this is consistent with an acute Hepatitis C diagnosis. Patients management and disease characteristics are described. (Acta Med Colomb 2008; 33: 28-32).
RESUMO
Se introdujo la técnica de la reacción en cadena de la polimerasa para la caracterización intratípica de Poliovirus. Se usaron cebadores que sólo promueven la ampliación de las cepas vacunales de Sabin, comprobada por corrida electroforética de los productos de ADN amplificados (Sabin 1-97 pb, Sabin 2-71 pb, Sabin 3-44 pb) y cuya especificidad se verificó satisfactoriamente. Se estudiaron por esta técnica 23 cepas cubanas de Poliovirus aisladas e identificadas en el Laboratorio de Enterovirus del Instituto de Medicina Tropical "Pedro Kourí" de 1993 a 1994, y todas resultaron ser del tipo vacunal. Se observó cómo el Poliovirus vacunal de Sabin puede ser causa de meningoencefalitis viral como complicación neurológica más leve. Este estudio aportó una evidencia más a favor de la no circulación del Poliovirus salvaje en Cuba.
The polimerase chain reaction techniques was introduced for the intratypic characterization of Poliovirus. Primers were used only to promote the amplification of the Sabin vaccine strains proved by electrophoretic run of the amplified DNA products (Sabin 1 - 97 pb, Sabin 2 - 71 pb, Sabin 3 - 44 pb) and whose specificity was satisfactorily verified. 23 Cuban poliovirus strains isolated and identified at the Laboratory of Enterovirus of the "Pedro Kourí" Tropical Medicine Institute from 1993 to 1994 were studied by this technique. All of them were of the vaccine type. It was observed how the Sabin vaccine poliovirus may be the cause of viral meningoencephalitis as a milder neurological complication. Tghis study provided one more evidence about the non circulation of the wild poliovirus in Cuba.
Assuntos
Humanos , Poliovirus/classificação , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Poliovirus/genética , Poliovirus/isolamento & purificação , Reação em Cadeia da Polimerase/estatística & dados numéricos , RNA Viral/genéticaRESUMO
No período de 1984 a 1986, 285 amostras de fezes de crianças com sintomatologia diarreica foram submetidas às provas diagnósticas de ensaio imunoenzimático, eletroforese em gel de poliacrilamida e microscopia eletrônica. Destas amostras, 15,4% foram positivas para rotavírus e 3,2% para adenovírus. Das 44 (15,4%) amostras positivas para rotavírus pelo método imunoenzimático, 37 apresentaram perfil eletroforético do RNA característico dos rotavírus. Destas últimas, 27 foram analisadas segundo o esquema de Lourenço et alii, 1981, tendo sido verificada grande heterogeneidade de perfis e predominância dos rota vírus do subgrupo 1 foi detectada (AU).