Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon.

The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1-72aa) is sufficient for viral transcript elongation and second exon (73-101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-kappaB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells.

Oligonucleotide array for simultaneous detection of respiratory viruses using a reverse-line blot hybridization assay.

The interest in developing new diagnostic methods based on arrays of multiple probes to detect and type simultaneously a wide range of different infectious agents is increasing. This becomes a necessity in the case of infectious agents such as respiratory viruses that cause diseases with very similar signs and symptoms. Such tools will permit rapid and accurate diagnosis of different agents causing respiratory infection leading to the most adequate prevention and/or treatment measures. In this article a reverse-line blot hybridization (RLB) assay for the detection of a wide range of respiratory viruses is presented and evaluated for its usefulness in routine diagnosis. This assay employs an array of 18 oligonucleotide probes immobilized on a nylon membrane. Biotin-labeled PCR products obtained with two multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assays described previously, which allow for the detection of fourteen different groups of respiratory viruses, were hybridized to the oligonucleotide array. Detection was performed using a chemiluminescent method. The standardization of the method showed that the RLB assay could be an alternative to the nested PCR assay for enhancing the sensitivity in the detection of the amplified products, avoiding the problem of cross-over contamination, increasing the specificity, and therefore simplifying the method. This is of main interest in laboratories with few facilities. The feasibility and accuracy of the RT-PCR-RLB assay for detecting respiratory viruses proves that such approach could be a first stage to develop a microarray assay for routine diagnosis of infectious diseases.

[Respiratory infections due to metapneumovirus in hospitalized infants]. / Infecciones respiratorias por metapneumovirus en lactantes hospitalizados.

BACKGROUND: Human metapneumovirus (hPMV) is a recently identified virus that is recognized as a cause of respiratory tract illness in the pediatric population. OBJECTIVES: To determine the incidence of respiratory tract infections caused by hPMV in hospitalized infants and to describe the clinical characteristics and possible presence of coinfection with other viral agents. PATIENTS AND METHODS: We performed a prospective study from September to June 2003 in all children aged less than 24 months who were admitted to the Severo Ochoa Hospital (Leganés, Madrid) with a respiratory tract infection. Virological diagnosis was made with a direct immunofluorescent assay and/or reverse transcriptase-polymerase chain reaction on specimens obtained from nasopharyngeal washing. Demographic and clinical data from patients with an hPMV respiratory tract infection were analyzed. RESULTS: During the study period, 200 infants were admitted with a respiratory tract infection, of which 18 (9 %) had an hPMV infection. HPMV was the viral agent isolated in 13.8 % of positive nasopharyngeal washings. All patients were admitted between March and April. The mean age was 6.7 +/- 6.1 months. The most common diagnoses were recurrent wheezing (55.5 %) and bronchiolitis (38.8 %). Oxygen therapy was required by 55.5 % of infants during hospitalization. Coinfection with other respiratory viruses was confirmed in 33.3 % of the patients. CONCLUSIONS: Human metapneumovirus is a major cause of respiratory tract illness in hospitalized infants. This virus causes mainly bronchiolitis and recurrent wheezing and is more frequent in spring. Coinfection with other respiratory viruses is frequent.

Infecciones respiratorias por metapneumovirus en lactantes hospitalizados / Respiratory infections dur to metapneumovirus in hospitalized infants

Antecedentes El metapneumovirus humano es un virus de reciente descripción al que se atribuyen infecciones respiratorias que afectan fundamentalmente a la población infantil. Objetivos Conocer la incidencia de infecciones por metapneumovirus en lactantes hospitalizados, así como sus características clínicas y la posible presencia de coinfecciones con otros agentes virales. Pacientes y métodos Estudio prospectivo realizado de septiembre a junio de 2003 en todos los niños menores de 2 años ingresados en la Unidad de Lactantes del Hospital Severo Ochoa de Leganés (Madrid), por infección respiratoria. La detección de agentes virales se realizó mediante recogida de aspirado nasofaríngeo y realización de inmunofluorescencia directa y/o reacción en cadena de la polimerasa-transcripción inversa (RT-PCR). Descripción de las características clínicas y epidemiológicas de los procesos respiratorios de los pacientes con detección positiva para metapneumovirus humano. Resultados Ingresaron 200 lactantes con patología respiratoria durante el período mencionado, de los cuales en 18 se detectó infección por metapneumovirus humano (9 por ciento de los pacientes). El metapneumovirus humano supuso un 13,8 por ciento de los aislamientos virales positivos. El 100 por ciento de estos niños ingresaron en marzo-abril. La edad media fue de 6,7+/-6,1 meses. El 38,8 por ciento desarrolló una bronquiolitis y en el 55,5 por ciento se objetivó un episodio recurrente de sibilancias. El 55,5 por ciento de los niños precisó oxigenoterapia durante el ingreso. Se encontraron coinfecciones con otros agentes virales en el 33,3 por ciento de estos pacientes. Conclusiones El metapneumovirus humano es un agente viral muy frecuente en los lactantes afectados de enfermedad respiratoria, causando fundamentalmente bronquiolitis y episodios recurrentes de sibilancias. Es más frecuente en primavera y tiene una alta tendencia a la coinfección con otros virus (AU)