Secondary infections of the dengue virus in Buenos Aires metropolitan area

El alto porcentaje de infecciones secundarias detectado en un área no endémica podría estar indicando la posible circulación críptica del virus, siendo ésta causante de infecciones inaparentes. Considerando que en el área metropolitana de Buenos Aires (AMBA) se detectó transmisión local de dengue por primera vez durante el brote de 2009, es destacable el hecho de que durante dicho año más del 20% de las muestras analizadas resultaron ser infecciones secundarias.Fil: Tittarelli, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Barrero, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Mistchenko, Alicia Susana. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Valinotto, Laura Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Wild-type Measles Virus in Brain Tissue of Children with Subacute Sclerosing Panencephalitis, Argentina

We studied eight children who had measles at 6 to 10 months of age during the 1998 Argentine measles outbreak and in whom subacute sclerosing panencephalitis developed 4 years later. We report the genetic characterization of brain tissue–associated measles virus samples from three patients. Phylogenetic relationships clustered these viruses with the wild-type D6 genotype isolated during the 1998 outbreak. The children received measles vaccine; however, vaccinal strains were not found

Secuencia nucleotídica completa del Adenovirus 7h: análisis de los polimorfismos del gen del hexón de los Adenovirus circulantes en Buenos Aires entre los años 1990-2002

Los Adenovirus circularon en el período 1999-2002 durante todo el año, registrándose un mayor número de casos en el período invernal de baja temperatura y primaveral con cambios de temperatura frecuentes. El diagnóstico virológico se realizó tempranamente luego de la internación siendo la bronquiolitis el diagnóstico clínico al ingreso más frecuente. Se registró un mayor número de casos en menores de 12 meses y la evolución en general fue favorable registrándose 9 casos fatales en un total de 362 muestras analizadas. Se encontró una correlación negativa de grado medio con respecto a la temperatura, indicando un aumento en el número de casos cuando el registro térmico desciende. En cuanto a la humedad relativa y el índice UVB, la relación no es tan clara aunque es siempre de grado menor. Este parece ser un parámetro no necesariamente asociado a la aparición de casos de Adenovirus. El análisis filogenético del hexón completo permitió agrupar las muestras fatales y las separó de las graves y leves en el periodo 1990-1999. El análisis de las regiones hipervariables en el período 1990-2002 posicionó a las muestras analizadas en el cluster GTC2 junto al AV7h. La secuencia completa del AV7h se llevó a cabo por una estrategia de ensamblado de 8 contigs que permitieron elaborar una secuencia consenso de 35259 pb. con un porcentaje G+C del 51%. Se conservaron intactos los genes para las regiones tempranas, intermedias y tardías. El análisis filogenético ubicó al genoma AV7h junto con el AVB con el que tiene una homología del 84%. Se hallaron ORFs relacionados con proteínas pulmonares que podrían inducir autoanticuerpos o daño pulmonar crónico postviral.In Argentina, Adenovirus 7h (AV7h) has been frequently associated with fatal cases and chronic lung disease. In to order to understand the characteristics of AV7h we accomplished the entire reference sequencing and analysed sample polymorphism in the hexon gene from a twelve-year period (1990-2002). Nasopharyngeal aspirates from children with acute lower respiratory infections were characterized by multiplex-PCR. Primers were designed and PCRs were performed to obtain the full genome for the reference 7h strain 87-922 and the entire hexon gene or hipervariable regions for samples. Amplicons were sequenced, contigs were aligned, a consensus full-length sequence was obtained for the reference strain 87-922 and phylogenetic relationships for hexon gene were inferred by maximum-likelihood criteria. Overall differences between AV7h hexon gene and prototype for serotype 7 (Gomen) sequence were <4% at nucleotide level and <3.5% at amino acid level. Phylogenetic analysis clustered together fatal cases apart from severe and mild cases. The entire genome was completed in 8 contigs with 44, 16, 7, 15, 14, 26, 18 and 15 primer reactions respectively. AV7h was 35263 bp in length and had a GC content of 51% and genes from the early, intermediate, and late regions were present in the expected human adenovirus locations. The genome differs from prototype strain for AVB (subspecie B2) in 16%, and 4% from vaccinal strain AV7(subspecie B1). This work constitutes the first attempt to elucidate intragenotypic variations within AV7h and to reveal particular features of this strain.Fil:Barrero, Paola Roxana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Dengue Virus 1 in Buenos Aires from 1999 to 2010: Towards Local Spread

Dengue virus (DENV) is a public health problem representing the most important arthropod-borne viral disease in humans. In Argentina, Northern provinces have reported autochthonous cases since 1997, though these outbreaks have originated in bordering countries, where co-circulation of more than one serotype has been reported. In the last decade, imported dengue cases have been reported in Buenos Aires, the urban area of Argentina with the highest population density. In 2009, a dengue outbreak affected Buenos Aires and, for the first time, local transmission was detected. All cases of this outbreak were caused by DENV-1. In this report, we present the full-length sequences of 27 DENV-1 isolates, corresponding to imported cases of 1999–2000, as well as local and imported cases of the 2009 and 2010 outbreaks. We analyzed their phylogenetic and phylodynamic relationships and their global and local spread. Additionally, we characterized their genomic and phenotypic features. All cases belonged to DENV-1 genotype V. The most recent ancestor for this genotype was dated ∼1934, whereas that for the 2009 outbreak was dated ∼2007. The mean rates of nucleotide substitution were 4.98E-4 and 8.53E-4 subs./site/yr, respectively. We inferred an introduction from Paraguay in 1999–2000 and mainly from Venezuela during 2009–2010. Overall, the number of synonymous substitutions per synonymous site significantly exceeded the number of non-synonymous substitutions per site and 12 positively selected sites were detected. These analyses could contribute to a better understanding regarding spread and evolution of this pathogen in the Southern Cone of South America.Fil: Tittarelli, Estefanía. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Barrero, Paola Roxana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Secondary dengue virus infections during the 2009 outbreak in Buenos Aires

Objectives: To evaluate the occurrence of secondary dengue virus (DENV) infections during the 2009 outbreak in a non-endemic area. Viral loads were evaluated in serum from acute-phase patients, comparing primary and secondary infection. Methods: Serum samples from patients with clinical diagnosis of suspected dengue were referred to the Virology Laboratory at 'Ricardo Gutiérrez' Children's Hospital. Dengue-positive samples were classified as primary or secondary DENV infections through serological methods (anti-DENV IgM and IgG). Viral loads were measured by quantitative real-time PCR (qRT-PCR) in samples obtained in the first 5 days of infection. Statistical analyses were performed to evaluate factors that might correlate with differences in the viral load of primary or secondary infection. Results: A total of 229 DENV cases were confirmed; among them, 22.7% were secondary infections. No significant differences were found between the viral load of primary and secondary infections. Conclusion: We detected a high percentage of secondary DENV infections in a non-endemic area; this finding might correspond to socio-demographic characteristics of the group under study or indicate a previous cryptic DENV circulation causing inapparent infections.Fil: Tittarelli, Estefanía. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barrero, Paola Roxana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mistchenko, Alicia Susana. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; ArgentinaFil: Valinotto, Laura Elena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Emergence of intratreatment resistance to oseltamivir in pandemic influenza A H1N1 2009 virus

Background: Pandemic influenza A H1N1 2009 virus presents a new challenge to health authorities and communities worldwide. In Argentina, the outbreak was at its peak by the end of June 2009, during the southern winter. A systematic analysis of samples from patients with pandemic H1N1 2009 studied in our laboratory (Virology Laboratory, Hospital de Niños R Gutiérrez, Buenos Aires, Argentina) detected two patients presenting intratreatment emergence of the H275Y neuraminidase mutation, which confers resistance to oseltamivir. Methods: Complementary DNAs, including the 275 codon, were obtained by reverse transcriptase PCR using viral RNAs extracted from nasopharyngeal or tracheal aspirates. Conventional sequencing and pyrosequencing were performed on each sample. In order to measure the virus susceptibility to oseltamivir, 50% inhibitory concentration determinations were performed by chemiluminescence. Results: Sequential samples of two paediatric patients under oseltamivir treatment were analysed. Pretreatment samples were composed of 100% oseltamivir-sensitive variants. In case 1, the oseltamivir-resistant variant was found 8 days after the beginning of treatment. In case 2, the viral population became resistant on the second day of treatment, with 83% of the viral population bearing the mutation and this reached 100% on the seventh day. Conclusions: We describe the intratreatment emergence of oseltamivir resistance in two paediatric patients. Pyrosequencing allowed us to detect variant mixtures, showing the transition of the viral population from sensitive to resistant.Fil: Valinotto, Laura Elena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Diez, Roberto Alejandro. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Biosidus S. A.; ArgentinaFil: Barrero, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Farías, Julio A.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: López, Eduardo L.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentin

Molecular evidence of St. Louis encephalitis virus infection in patients in Buenos Aires, Argentina

We report two cases of St. Louis encephalitis where the virus was detected in patients? sera directly by molecular techniques allowing subsequent typing.. Phylogenetic analysis of both samples showed that NS5 sequences clustered with viruses previously classified as genotype III.Fil: Valinotto, Laura Elena. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barrero, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; ArgentinaFil: Viegas, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alvarez López, María C.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; ArgentinaFil: Mistchenko, Alicia Susana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentin

Whole genome analysis of the action of interferon-β

Objectives: To characterize the IFN1a-regulated gene expression on leukocytes of Multiple Sclerosis (MS) patients using microarrays with whole human genome representation. Methods: Genes differentially expressed by interferon- were identified by a microarray in vitro study performed in leukocytes obtained from 5MS relapsing-remitting patients. Results: Following the culture of peripheral blood mononuclear cells from MS relapsing-remitting patients for 24 hs with IFN1a, the expression of 868 genes was modified: 545 increased (including CXCL11, CCL8, INDO, IFI27, CFB, CXCL10 and IFIT1) and 323 diminished (including RBP7, SEPT5, RNF8, ADORA2B and FOS). Conclusions: Since many of them were previously recognized as involved in MS pathogenesis, the IFNb1a mechanism of action could imply a compensatory regulation of systems deregulated in MS.Fil: Kauffman, Marcelo Andres. Biosidus S. A.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Yankilevich, Patricio. Biosidus S. A.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barrero, Paola Roxana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez". Laboratorio de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ricardo Bello. Biosidus S. A.; ArgentinaFil: Marangunich, L.. Biosidus S. A.; ArgentinaFil: Vidal, A.. Biosidus S. A.; ArgentinaFil: Criscuolo, M.. Biosidus S. A.; ArgentinaFil: Diez, Roberto Alejandro. Biosidus S. A.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sterin Prync, Aída Edith. Biosidus S. A.; Argentin

Two recombinant human interferon-beta 1a pharmaceutical preparations produce a similar transcriptional response determined using whole genome microarray analysis

Objectives: Recombinant human interferon-beta (IFN-b) is a well-established treatment for multiple sclerosis (MS). The regulatory process for marketing authorization of biosimilars is currently under debate in certain countries. In the EU, EMEA has clearly defined the process including overarching and product-specific guidelines, which includes clinical testing. Biosimilarity needs to be based on comparability criteria, including at least molecular characterization, biological activity relevant for the therapeutic effect and relative bioavailability (“bioequivalence”). In the case of such complex diseases as MS, where the effect of treatment is not so directly measurable, in vitro tools can provide additional data to support comparability. Genomic microarrays assays might be useful to compare multisource biopharmaceuticals. The aim of the present study was to compare the pharmacodynamic genomic effects (in terms of transcriptional regulation) of two recombinant human IFN-β1a preparations on lymphocytes of multiple sclerosis patients using a whole genome microarray assay. Methods: We performed an ex vivo whole genome expression profiling of the effect of two preparations of IFN-β1a on non-adherent mononuclears from five relapsing-remitting MS patients analyzing microarrays (CodeLink™ Human Whole Genome). Patients blood was drawn, PBMCs isolated and cultured in three different conditions: culture medium (control), 1,000 U/ml of IFN-β1a (BLA- (STOFERON™, Bio Sidus) and 1,000 U/ml of IFN-β1a (REBIF™, Serono) RNA was purified from non-adherent cells (mostly lymphocytes), amplified and hybridized. Raw data were generated by CodeLink™ proprietary software. Data normalization, quality control and analysis of differential gene expression between treatments were done using linear model for microarray data. Functional annotation analysis of IFN-β1a MS treatment transcription was done using DAVID. Results: Out of the approximately 45,000 human sequences examined, no evidence of differential regulation was found when both treatments were compared (minimum adjusted p-value > 0.999). The IFN-β1a effect differentially regulated the expression of 868 genes. The expression of standard markers such as GTP cyclohidrolase, MxA, and OAS isoenzymes A and B changed as a consequence of the action of IFN-β1a. Conclusions: This exhaustive and highly sensitive assay did not show differences in the genomic expression profile of these two products under the assayed experimental conditions. These results suggest that this technology might be useful for the initial comparison of biosimilars, being part of a comprehensive comparability program that includes clinical testing.Fil: Sterin Prync, Aída Edith. Bio Sidus S.A.; ArgentinaFil: Yankilevich, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Bio Sidus S.A.; ArgentinaFil: Barrero, Paola Roxana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Bello, R.. Bio Sidus S.A.; ArgentinaFil: Marangunich, L.. Bio Sidus S.A.; ArgentinaFil: Vidal, A.. Bio Sidus S.A.; ArgentinaFil: Criscuolo, M.. Bio Sidus S.A.; ArgentinaFil: Benasayag, L.. Centro Neurológico Integral ; ArgentinaFil: Famulari, A. L.. Fundación Argentina contra las Enfermedades Neurológicas del Envejecimiento; ArgentinaFil: Domínguez, R. O.. Fundación Argentina contra las Enfermedades Neurológicas del Envejecimiento; ArgentinaFil: Kauffman, Marcelo Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Bio Sidus S.A.; ArgentinaFil: Diez, Roberto Alejandro. Bio Sidus S.A.; Argentin