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A polyclonal antiserum with high titer, great specificity and sensitivity was obtained against the hemagglutinin of Influenza A/Johannesburg/33/94, belonging to subtype A (H3N2). The obtaining process of the antigen or immunogen, the immunization schedule and its administration to Wistar rats as animal model are described. All of these were validated by the hemagglutinin inhibition technique. The antiserum obtained was treated with receptor-destroying enzymes of national production (EDR/IPK) in order to eliminate the unspecific inhibitors of the hemagglutinin inhibition. Validation was carried out in front of a panel including 9 influenza reference strains belonging to subtypes A(H3N2), A(H1N1), and to type B. No unspecific reactions against strains A(H1N1) and of type B were detected. Five autochthonous isolates were satisfactorily typed and subtyped.

Se obtuvo un antisuero policlonal con alto título, alta especificidad y sensibilidad, contra la hemaglutinina de la cepa de influenza A/Johannesburg/33/94 que pertenece al subtipo A (H3N2). Se describe el proceso de obtención del antígeno o inmunógeno, el esquema de inmunización y su aplicación a las ratas Wistar como modelo animal; y se validó mediante la técnica de inhibición de la hemaglutinación (IH). El antisuero obtenido fue tratado con enzima destructora de receptores de producción nacional (EDR/IPK) para la eliminación de inhibidores inespecíficos de la IH. La validación se realizó frente a un panel que incluyó 9 cepas de referencia de influenza de los subtipos A(H3N2), A(H1N1) y el tipo B. No se detectaron reacciones inespecíficas frente a cepas A(H1N1) y del tipo B. Se determinó el tipo y subtipo de aislamientos autóctonos con resultados satisfactorios.

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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

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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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

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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Clasificación en subgrupos de cepas del virus sincitial respiratorio aisladas de un brote en Ciudad de La Habana
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CHACON, DANAY

; VALDIVIA, ANGEL

; GOYENECHEA, ANGEL

; OROPESA, ISABEL

; SAVON, CLARA

Revista Cubana de Medicina Tropical

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aug 1996, Volume 48 N. 2 Pages 136 - 137

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

A high number of acute respiratory diseases was detected among children under one year admitted in a hospital of Havana City. 25 respiratory syncytial virus strains were obtained from 93 patients studied. Viral isolations were multiplied in HEP-2 cells and after observing a cytopathic effect of 80 %, they were classified into subgroups by the indirect immunofluorescence technique, using anti-protein G antibodies from the respiratory syncytial virus. All the samples studied were classified within subgroup A. It is the first time a study like this is conducted in our country, which allowed us to deepen into the viral cause of these diseases and to know that the subgroup A of the respiratory syncytial virus circulated during the outbreak.

Se detectó un alto número de casos de enfermedades respiratorias agudas en niños menores de 1 año, ingresados en un hospital de Ciudad de La Habana. De 93 pacientes estudiados se obtuvieron 25 cepas del virus sincitial respiratorio. Los aislamientos virales fueron multiplicados en células HEP-2, y después de observarse un efecto citopático del 80 % se clasificaron en subgrupos por la técnica de inmunofluorescencia indirecta, utilizando anticuerpos anti-proteína G del virus sincitial respiratorio. Todas las muestras estudiadas se clasificaron dentro del subgrupo A. Este tipo de estudio es el primero realizado en nuestro país, lo cual nos permitió profundizar en la causa viral de estas enfermedades y conocer que durante el brote circuló el subgrupo A del virus sincitial respiratorio.

Estimación del título de anticuerpos IgG a Adenovirus utilizando una curva patrón y un ensayo de ultramicroELISA indirecto
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LAFERTE, JOSE

; SAVON, CLARA

; GOYENECHEA, ANGEL

; VAZQUEZ, VALEXIS

; OTERO, ANSELMO

; TEJERO, YAISEL

; HERNANDEZ, BARBARA

Revista Cubana de Medicina Tropical

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aug 1996, Volume 48 N. 2 Pages 102 - 108

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

It was possible to standardize a procedure which combined an indirect microELISA assay with a standard curve and that allowed to estimate the titre of IgG antibodies to Adenovirus in samples of human serum, using only one dilution of these. Based on the end-point titre previously determined for a panel of 117 serum samples, we selected 90 of these samples (r² = 0,98) to build 4 standard curves that related the natural logarithm of the fluorescence responses to the natural logarithm of the end-point titre for a wide range of serum dilutions (1:40=1:320). It was selected the curve corresponding to serum dilution 1:40 (r² = 0,81), which made possible an optium utilization of those accessories designed to handle the volumes in the ultramicro range and, therefore, the automation of the whole procedure. The results obtained as regards the complement fixation test (100 % of sensitivity and 97,3 % of specificity) support the use of this method in our laboratory as a complementary tool to carry out seroepidemilogical studies on a large scale and with diagnostic ends.

Se logró normalizar un procedimiento que combinaba un ensayo de ultramicroELISA indirecto con una curva patrón y que permitió estimar el título de anticuerpos IgG a Adenovirus en muestras de suero humano utilizando una sola dilución de éstos. Basándose en el título a punto final previamente determinado para un panel de 117 muestras de suero, se seleccionaron 90 de éstas (r² = 0,98) para construir 4 curvas patrones que relacionaron el logaritmo natural de las respuestas de fluorescencia y el logaritmo natural del título a punto final para un rango amplio de diluciones del suero (1:40 hastra 1:320). Se seleccionó la curva correspondiente a la dilución 1:40 del suero (r² = 0,81), la cual posibilitó una óptima utilización de los accesorios diseñados para la manipulación de los volúmenes en el rango ultramicro y, por tanto, la automatización de todo el procedimiento. Los resultados obtenidos con respecto a la técnica de fijación del complemento (100 % de sensibilidad y 97,3 % de especificidad) respaldan la utiización de este método como una herramienta complementaria en la ejecución de estudios seroepidemiológicos a gran escala y con fines diagnósticos.

Cited 1 time in SciELO

10.

Rapid Detection of respiratory syncytial virus by indirect immunofluorescence in infants hospitalized with acute respiratory disease
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Goyenechea, Angel

; Razon, Rafael

; Savon, Clara

; Valdivia, Angel

; Chacon, Danay

; Sarmiento, Luis

Memórias do Instituto Oswaldo Cruz

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aug 1996, Volume 91 N. 4 Pages 489 - 490

Abstract: En | Text: En | PDF: En

1676 downloads Cited 1 time in SciELO

11.

Neuropatía epidémica cubana.: Parte IV. Relación antigénica de los aislamientos virales con estructuras del sistema nervioso central. ¿Posible mecanismo etiopatogénico?
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MAS LAGO, PEDRO

; GUZMAN, MARIA G

; CAPO, VIRGINIA

; ALVAREZ, MAYLING

; RESIK, SONIA

; GOYENECHEA, ANGEL

; KOURI, GUSTAVO

Revista Cubana de Medicina Tropical

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jun 1995, Volume 47 N. 1 Pages 26 - 31

Abstract: Es | Text: Es | PDF: Es

Se presentan resultados que permiten plantear la existencia de relaciones antigénicas entre los virus aislados del líquido cefalorraquídeo de pacientes con neuropatía epidémica y estructuras del sistema nervioso central humano. Estas evidencias se han obtenido por 2 vías distintas e independientes: 1) por método de doble difusión en agarosa, inmunoblot e inmunohistoquímicos, comprobándose que los anticuerpos inducidos por los virus aislados reaccionan con antígenos del sistema nervioso central y periférico, 2) el suero obtenido por inmunización de un conejo con extracto de cerebro humano neutraliza los mismos virus que los neutralizados por los sueros hiperinmunes obtenidos con los aislamientos. Se discute la posible participación de los virus como mediadores de un proceso autoinmune en la etiopatogenia de la enfermedad.

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