Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100%) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus.

One hundred and fourty eight samples from patients with a symptomatology compatible with the influenza virus were studied aimed at identifying in a fast way these viruses. A rapid MDCK-L cell culture was developed on 96 well plates, where nasopharingeal exudates or gargarisms were innoculated and incubated all night long at 37 ºC. The medium was removed and cells were washed with PBS and fixed with methanol. Viral antigens were detected through the immunoperoxidase staining by using two monoclonal antibody pools for the identification of influenza A and influenza B viruses. The HA1-71 monoclonal antibody, specific for influenza A (H3N2) and the HA2-76, which react with both A (H3N2) and A (H1N1) were used for subtyping. Of all the positive samples (136), 72 % corresponded to type A while 34.6 % and 37.5 % corresponded to subtypes H1 and H3, respectively. Influenza B was detected in 27.9 % of the 148 samples studied. Only 12 were negative (8.1 %). The use of this technique is recommended as a rapid, convenient and sensitive method that is easy to carry put and to interpretate for the detection and characterization in type and subtype of the influenza viruses starting from the nasopharyngeal exudates or gargarisms.

Con el objetivo de identificar en forma rápida los virus de influenza, se estudiaron 148 muestras de pacientes con sintomatología compatible con esta entidad. Para ello fue desarrollado un cultivo rápido de células MDCK-1, en placas de 96 pozuelos, donde fueron inoculados los exudados nasofaríngeos o gargarismos, e incubados durante una noche a 37ºC, el medio fue eliminado y las células fueron lavadas con buffer fosfato salina; posteriormente fueron fijadas con metanol y los antígenos virales detectados por la técnica de inmunoperoxidasa mediante un pool de anticuerpos monoclonales contra la influenza A y otro contra la influenza B. Para el subtipaje en A (H3N2)se utilizó el anticuerpo monoclonal HA1-71, mientras que el anticuerpo monoclonal HA2-76 reacciona con ambos subtipos A (H3N2) y A (H1N1). Del total de muestras positivas (136) correspondió 72,1 % para el tipo A y a los subtipos H1 y H3, 34,6 y 37,5 %, respectivamente. La influenza B se detectó en 27,9 % de las 148 muestras investigadas, sólo 12 resultaron negativas (8,1 %). Se recomienda la utilización de esta técnica como un método de diagnóstico rápido, sensible, conveniente y fácil de realizar e interpretar para la detección y caracterización en tipo y subtipo de los virus de influenza a partir de exudados nasofaríngeos o gargarismos.

An immunoglobulin G of mouse was purified from sera by affinity chromatography in protein A. The rabbits whose sera were able to recognize the antigen injected by double immunodiffusion were immunized with this preparation. The antibodies were precipatated from the rabbit's serum and purified by ion exchange chromatography. This preparation was conjugated to fluorescin isothiocyanate according to the conventional technique. The conjugated obtained was evaluated with the reference strains of Parainfluenza virus 1, 2, 3; Adenovirus; respiratory syncytial virus; and influenza virus A and B, by an indirect immunofluorescence technique and HIV positive samples by flow citometry. Specific monoclonal antibodies were used in both cases. Clinical specimens of patients with acute respiratory infection were evaluated.

Se purificó una inmunoglobulina G de ratón a partir de suero por cromatografía de afinidad en proteína A. Con esta preparación se inmunizaron los conejos cuyos sueros fueron capaces de reconocer al antígeno inyectado mediante inmunodifusión doble. Los anticuerpos fueron precipitados del suero de conejo y purificados mediante cromatografía de intercambio iónico. Esta preparación fue conjugada a isotiocianato de fluoresceína según la tecnología convencional. El conjugado obtenido fue evaluado con las cepas de referencia de virus Parainfluenza 1, 2, 3; Adenovirus; virus sincitial respiratorio y virus influenza A y B, por una técnica de inmunofluorescencia indirecta y muestras positivas de VIH mediante citometría de flujo. En ambos casos se utilizaron anticuerpos monoclonales específicos. Se evaluaron muestras clínicas de pacientes con infección respiratoria aguda.

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed

The polymerase chain reaction (PCR) was developed in order to identify the respiratory syncytial virus by using the reference strain. The high sensitivity and specificity obtained show the PCR utility for detecting the RSV genoma and its application on the diagnosis.

Se desarrolló la reacción en cadena de la polimerasa (RCP) para la identificación del virus sincitial respiratorio (VSR) con el uso de la cepa de referencia. La alta sensibilidad y la especificidad obtenidas en nuestro trabajo demuestran la utilidad de la RCP para la detección del genoma del VSR y su aplicación en el diagnóstico.