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The nucleotide sequencing of the protein G C-terminal region of 37 samples taken from nasopharyngeal washings of under one-year old children from some Cuban provinces was made for 5 epidemic periods (1995-2000) to find out the circulation patterns of strains of human respiratory syncytial virus that is classified in two antigenic subgroups known as A and B; each of them contains multiple variants. Subgroup A has circulated during all these years but subgroup B was detected only in the year 2000. The presence of strains with two different sizes of protein G (297 aa and 298 aa) was observed whereas subgroup B showed only one size (295 aa). Phylogenetic analysis allowed identifying 5 and 2 genotypes within subgroups A and B respectively. Viruses present in Cuba were phylogenetically related to the strains of other parts of the world. Subgroup A comprised two strains very similar to Long prototype strain. Almost all the strains of both subgroups in the year 2000 phylogenetically related with strains that circulated in South Africa during the same period

Se realizó la secuenciación nucleotídica de la región del tercio C-terminal de la proteína G de 37 muestras de exudados nasofaríngeos, de niños menores de 1 año provenientes de algunas provincias de Cuba durante 5 períodos epidémicos (1995-2000), para conocer los patrones de circulación de cepas del virus sincitial respiratorio humano; el cual se clasifica en 2 subgrupos antigénicos A y B, y cada uno contiene múltiples variantes. El subgrupo A circuló durante todos los años, el subgrupo B se detectó solamente durante el año 2000. Dentro del subgrupo A se observó la presencia de cepas con 2 tamaños diferentes de la proteína G (297 aa y 298 aa), mientras que para el subgrupo B fue observado un único tamaño (295 aa). El análisis filogenético permitió identificar 5 y 2 genotipos dentro de los subgrupos A y B, respectivamente. Los virus de Cuba se relacionaron filogenéticamente con cepas de otras partes del mundo. Dentro del subgrupo A se encontraron 2 cepas, las cuales fueron muy similares a la cepa prototipo Long. Casi todas las cepas del año 2000 de ambos subgrupos, se agruparon filogenéticamente con cepas que circularon en Sudáfrica durante ese mismo período.

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Isolation and identification of Adenovirus in hospitalized children, under five years, with acute respiratory disease, in Havana, Cuba
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Pumariega, Tania

; Savón, Clara

; Muné, Mayra

; Cancio, Reynel

; González, Grehete

; Valdivia, Angel

; González, Zoila

; Goyenechea, Angel

Memórias do Instituto Oswaldo Cruz

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dec 2000, Volume 95 N. 6 Pages 859 - 861

Abstract: En | Text: En | PDF: En

Nine Adenovirus (Ad) strains isolated in Cuba, from 128 nasopharingeal swab specimens of children below five years old, with acute respiratory diseases, during 1996 and 1997, were studied by restriction enzyme analysis of genomic DNA with two endonucleases BamH I and Sma I. All different fragment patterns were compared with the respective prototypes. The identified adenoviruses were Ad 1 (n=4), Ad 2 (n=1) and Ad 6 (n=4). Males were more frequently infected than females. The analysis of the occurrence of these Adenovirus strains of subgenus C revealed that Ad 1 and Ad 6 were the predominant serotypes in 1996 and in 1997, respectively.

1933 downloads Cited 2 times in SciELO

Antigenic and Genetic Characterization of Twenty-six Strains of Human Respiratory Syncytial Virus (Subgroup A) Isolated During Three Consecutive Outbreaks in Havana City, Cuba
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Valdivia, Angel

; González, Grehete

; Chacón, Danay

; Savón, Clara

; Otero, Anselmo

; Váldes, Odalys

; Cancio, Reynel

; Oropesa, Suset

; Melero, José A

; García-Barreno, Blanca

; Goyenechea, Angel

Memórias do Instituto Oswaldo Cruz

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jul 1999, Volume 94 N. 4 Pages 469 - 475

Abstract: En | Text: En | PDF: En

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.

1933 downloads Cited 1 time in SciELO

Incidencia de los Adenovirus en las conjuntivitis virales
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Torres Rojas, Gisset

; Goyenechea, Ángel

; Savón, Clara

; Valdés, Odalys

; Oropesa, Isabel

Revista Cubana de Medicina Tropical

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dec 1998, Volume 50 N. 3 Pages 182 - 185

Abstract: En Es | Text: En Es | PDF: En | PDF: Es

A study about the incidence of Adenovirus on viral conjunctivitis was conducted. A sample design was made and samples of conjunctival exudate were taken from 150 patients with diagnosis of apparently viral conjunctivitis at the "Ramón Pando Ferrer" Ophthalmological Hospital from July to December, 1994. Samples were innoculated in cell culture and the indirect immunofluorescence technique was applied to those with a cytopathogenic effect that suggested infection due to Adenovirus. Monoclonal antibodies were used against Adenovirus allowing to identify them as part of the Adenoviridae family. The hemoagglutination technique was used with erythrocytes of monkeys and rats as an indicator system in order to group the isolates previously identified by the indirect immunofluorescence technique. Later on, it was made an analysis by restriction enzymes of the viral genome to enable typing. The results of this study showed an incidence of Adenovirus on viral conjunctivitis of 20 %, with a confidence interval between 14 and 26 % and a reliability index of 95 %. It was proved that serotype 37 caused conjunctitivis more frequently.

Se llevó a cabo un estudio de incidencia de los Adenovirus en las conjuntivitis virales, para lo cual se realizó un diseño muestral y se tomaron muestras de exudado conjuntival a 150 pacientes con diagnóstico de conjuntivitis de presuntiva causa viral, en el Hospital Oftalmológico "Ramón Pando Ferrer" en el período comprendido entre julio y diciembre de 1994. Las muestras fueron inoculadas en cultivo celular y a las que presentaron un efecto citopatogénico sugestivo de infección por Adenovirus, se les realizó la técnica de inmunofluorescencia indirecta (IFI). Se usaron anticuerpos monoclonales contra Adenovirus, lo que permitió identificarlas como pertencientes a la familia Adenoviridae. Se utilizó la técnica de hemaglutinación, con hematíes de monos y ratas como sistema indicador, para agrupar los aislamientos previamente identificados mediante la técnica de IFI, después se les realizó el análisis por enzimas de restricción del genoma viral, lo cual posibilitó la clasificación en tipos. Los resultados de este estudio mostraron una incidencia de los Adenovirus en las conjuntivitis virales de 20 %, con un intervalo de confianza del 14-26 %, y un índice de confiabilidad de 95 %. Se demostró que el serotipo 37 fue el que produjo conjuntivitis con mayor frecuencia.

Evaluación de un conjugado anti-IgG de ratón-fluoresceína mediante técnicas de inmunofluorescencia indirecta y citometría de flujo
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VILASECA, JUAN CARLOS

; PÉREZ, LILIANA

; SAVÓN, CLARA

; CHACÓN, DANAY

; OROPESA FERNÁNDEZ, SUSET

; RODRÍGUEZ, HERMIS

; GOYENECHEA, ÁNGEL

; OTERO, ANSELMO

Revista Cubana de Medicina Tropical

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aug 1997, Volume 49 N. 2 Pages 120 - 124

Abstract: En Es | Text: En Es | PDF: En | PDF: Es

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.

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Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping
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Valdivia, Angel

; Savón, Clara

; Chacón, Danay

; Sarmiento, Luis

; Morier, Luis

; Otero, Anselmo

; Soto, Yudira

; Oropesa, Suset

; Goyenechea, Angel

Memórias do Instituto Oswaldo Cruz

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may 1997, Volume 92 N. 3 Pages 389 - 393

Abstract: En | Text: En | PDF: En

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

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Aplicación de la reacción en cadena de la polimerasa para la identificación del virus sincitial respiratorio
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SARMIENTO, LUIS

; CHACÓN, DANAY

; VALDIVIA, ÁNGEL

; SAVÓN, CLARA

; GOYENECHEA, ÁNGEL

Revista Cubana de Medicina Tropical

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apr 1997, Volume 49 N. 1 Pages 21 - 23

Abstract: En Es | Text: En Es | PDF: En | PDF: Es

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.

57 downloads Cited 1 time in SciELO

Comparación de la línea NCI-H292 con otras líneas continuas para la multiplicación de virus respiratorios
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MORIER, LUIS

; PÉREZ, LISSETTE

; CANCIO, REYNEL

; SAVÓN, CLARA

; GONZÁLEZ, ZOILA

; GOYENECHEA, ANGEL

Revista Cubana de Medicina Tropical

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dec 1996, Volume 48 N. 3 Pages 171 - 173

Abstract: En Es | Text: En Es | PDF: En | PDF: Es

The NCI-H292 continual line of mucoepidermoid cells of the human lungs has been reported to be useful for the propagation of many viruses, mainly Adenovirus and Paraxymovirus. It is stated the possible substitution of primary cultures of monkey kidney for NCI-H292 in order to isolate such agents. In the present paper it is evaluated the utility of this line for multiplying the respiratory syncytial viruses Adenovirus 3 and 7, and the parainfluenza viruses 1, 2, and 3, in comparison with the continual cellular lines traditionally used for the propagation of these viruses, whose strains were inoculated this time in the Vero, HEp-2, and HeLa lines, according to their know sensitivities as well as in NCI-H292 simultaneously. The viral multiplication was detected by the appareance of the cytopathic effect or by hemaadsorption. As a result, it was demostrated the multiplication capacity of the NCI-H292 line for Adenoviruses 3 and 7 and parainfluenza 3, being more useful for their multiplication than the tradicionally used lines.

La línea continua NCI-H292 de células mucoepidermoides de pulmón humano ha sido reportada ser de utilidad para la propagación de muchos virus, principalmente Adenovirus y Paramyxovirus. Se plantea la posible sustitución de cultivos primarios de riñón de mono por NCI-H292 para el aislamiento de dichos agentes. En el presente trabajo se evalúa la utilidad de esta nueva línea para la multiplicación de los virus sincitial respiratorio, Adenovirus 3 y 7 y los virus parainfluenza 1, 2 y 3 en comparación con las líneas celulares continuas utilizadas tradicionalmente para la propagación de éstos; para lo cual se inocularon cepas de los virus en cuestión en las líneas Vero, HEp-2 y HeLa, según sus sensibilidades conocidas, y en NCI-H292 paralelamente. La multiplicación viral se detectó por aparición de efecto citopático o por hemadsorción. Como resultado se corroboró la capacidad de multiplicación de la línea NCI-H292 para los Adenovirus 3 y 7 y el parainfluenza 3, siendo más útil para la multiplicación de éstos que las líneas tradicionalmente usadas.

90 downloads Cited 1 time in SciELO

10.

Normalización de un ensayo de ultramicroELISA para la detección de anticuerpos IgG al virus sincitial respiratorio
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SAVÓN, CLARA

; LAFERTÉ, JOSÉ

; GOYENECHEA, ANGEL

; VALDIVIA, ANGEL

; MORIER, LUIS

; TEJEIRO, YAISEL

Revista Cubana de Medicina Tropical

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dec 1996, Volume 48 N. 3 Pages 161 - 162

Abstract: En Es | Text: En Es | PDF: En | PDF: Es

An ultramicroELISA assay of double antibody for the detection of IgG antibodies to the respiratory syncytial virus (RSV) wasstandardized. It was used a RVS antiprotein F monoclonal antibody produced by the Genetic Engineering and Biotechnology Center (GEBC) in Havana. The use of this antibody allowed to include crude antigenic preparations instead of purified fractions, which caused a significant reduction of the reactivity obtained with the antigen control. The assay conditions were determined by crossed titration. It was obtained a sensitivity of 97.2 %, a coincidence of 91 %, and a specificity of 83.3 % of the UMELISA as regards the complement fixation. The results may be qualitatively expressed or by antibody titres using only one serum dillution (1:40) and a pattern curve.

Se normalizó un ensayo de ultramicroELISA de doble anticuerpo para la detección de anticuerpos IgG al virus sincitial respiratorio (VSR), para ello se dispuso de un anticuerpo monoclonal antiproteína F del VSR, producido por el Centro de Ingeniería Genética y Biotecnología de La Habana (CIGB). La utilización de este anticuerpo posibilitó la inclusión de preparaciones antigénicas crudas en lugar de fracciones purificadas, lo que disminuye notablemente la reactividad obtenida con el control de antígeno. Las condiciones del ensayo fueron determinadas mediante titulación cruzada y se obtuvo una sensibilidad de 97,2 %, un 91 % de coincidencia y una especificidad 83,3 % del UMELISA con respecto a la fijación del complemento. Los resultados pueden ser expresados cualitativamente o en títulos de anticuerpos empleando una sola dilución de suero (1:40) y una curva patrón.

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