Caracterización molecular de variantes del virus Junín con diferentes grados de virulencia

El objetivo principal de este trabajo, consistió en el estudio de la información contenida en el genoma del virus Junín, en relación al fenómeno de atenuación de la virulencia. - Como objetivo secundario, se estudió la filogenia en la familia de los arenavirus y se analizaron las relaciones evolutivas entre los mismos. - Además, se diseñó un método para la detección de nuevos arenavirus a nivel molecular, en base al análisis de secuencias mencionado arriba. - Finalmente, se estudió el papel de la proteína de la nucleocápside en la regulación de los procesos de transcripción y replicación en el virus Junín. Con el fin de organizar el contenido y favorecer la presentación de este estudio resultó conveniente la división en capítulos, a saber: introducción, materiales y métodos, análisis y discusión de los resultados, conclusiones generales y referencias bibliográficas. A continuación, se resume brevemente el contenido de cada capítulo. 1. En el primer capítulo, se realiza una descripción introductoria sobre la estructura del virus Junín y su relación con la fiebre hemorrágica. En particular, se analiza la información disponible y los antecedentes bibliográficos que forman el contexto para el desarrollo de este estudio. 2. En el segundo capítulo se describen los materiales y los métodos utilizados en el transcurso de este estudio. 3. En el tercer capítulo se aborda específicamente, el problema de la atenuación de la virulencia en las cepas relacionadas genealógicamente con el virus vacunal, Junín Candid #1. 4. En el cuarto capítulo, se analiza la filogenia en la familia de los arenavirus y las posibles relaciones evolutivas entre los mismos. 5. En el quinto capítulo, se describe el diseño de un procedimiento experimental que permitió caracterizar un nuevo arenavirus. Este trabajo se realizó en colaboración con el Dr. M.E. Lozano y el Lic. D.M. Posik. 6. En el sexto capítulo, se analiza el control de la transcripción en el virus Junín. Este trabajo se realizó en colaboración con el Dr. R.V. Rivera Pomar y el Dr. M.E. Lozano. 7. En el séptimo capítulo, se establecen las conclusiones generales del presente estudio. 8. En el último capítulo, se listan las referencias bibliográficas relacionadas con los capítulos anteriores.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

The cis-acting replication signal at the 3′ end of Flock House virus RNA2 is RNA3-dependent

AbstractThe nodavirus Flock House virus has a bipartite positive-sense RNA genome consisting of RNAs 1 and 2, which encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp catalyzes replication of both genome segments and produces from RNA1 a subgenomic RNA (RNA3) that transactivates RNA2 replication. Here, we replaced internal sequences of RNAs 1 and 2 with a common heterologous core and were thereby able to test the RNA termini for compatibility in supporting the replication of chimeric RNAs. The results showed that the 3′ 50 nt of RNA2 contained an RNA3-dependent cis-acting replication signal. Since covalent RNA dimers can direct the synthesis of monomeric replication products, the RdRp can evidently respond to cis-acting replication signals located internally. Accordingly, RNA templates containing the 3′ termini of both RNAs 1 and 2 in tandem generated different replication products depending on the presence or absence of RNA3

Rift Valley fever virus lacking NSm proteins retains high virulence in vivo and may provide a model of human delayed onset neurologic disease

AbstractRift Valley fever virus is a significant human and veterinary pathogen responsible for explosive outbreaks throughout Africa and the Arabian Peninsula. Severe acute disease in humans includes rapid onset hepatic disease and hemorrhagic fever or delayed onset encephalitis. A highly efficient reverse genetics system was developed which allowed generation of recombinant RVF viruses to assess the role of NSm protein in virulence in a rat model in which wild-type RVF virus strain ZH501 (wt-ZH501) results in 100% lethal hepatic disease 2–3 days post infection. While extensive genomic analysis indicates conservation of the NSm coding capability of diverse RVF viruses, and viruses deficient in NSs proteins are completely attenuated in vivo, comparison of wt-ZH501, a reverse genetics generated wt-ZH501 virus (R-ZH501), and R-ZH501 virus lacking the NSm proteins (R-ΔNSm-ZH501) demonstrated that the NSm proteins were nonessential for in vivo virulence and lethality. Surprisingly, while 44% of R-ΔNSm-ZH501 infected animals quickly developed lethal hepatic disease similar to wt- and R-ZH501, 17% developed delayed onset neurologic disease (lethargy, head tremors, and ataxia) at 13 days post infection. Such infections may provide the basis for study of both RVF acute hepatic disease and delayed onset encephalitic disease in humans

Development of a RVFV ELISA that can distinguish infected from vaccinated animals

<p>Abstract</p> <p>Background</p> <p>Rift Valley Fever Virus is a pathogen of humans and livestock that causes significant morbidity and mortality throughout Africa and the Middle East. A vaccine that would protect animals from disease would be very beneficial to the human population because prevention of the amplification cycle in livestock would greatly reduce the risk of human infection by preventing livestock epizootics. A mutant virus, constructed through the use of reverse genetics, is protective in laboratory animal models and thus shows promise as a potential vaccine. However, the ability to distinguish infected from vaccinated animals is important for vaccine acceptance by national and international authorities, given regulations restricting movement and export of infected animals.</p> <p>Results</p> <p>In this study, we describe the development of a simple assay that can be used to distinguish naturally infected animals from ones that have been vaccinated with a mutant virus. We describe the cloning, expression and purification of two viral proteins, and the development of side by side ELISAs using the two viral proteins.</p> <p>Conclusion</p> <p>A side by side ELISA can be used to differentiate infected from vaccinated animals. This assay can be done without the use of biocontainment facilities and has potential for use in both human and animal populations.</p

Building a Sentiment Corpus of Tweets in Brazilian Portuguese

The large amount of data available in social media, forums and websites motivates researches in several areas of Natural Language Processing, such as sentiment analysis. The popularity of the area due to its subjective and semantic characteristics motivates research on novel methods and approaches for classification. Hence, there is a high demand for datasets on different domains and different languages. This paper introduces TweetSentBR, a sentiment corpora for Brazilian Portuguese manually annotated with 15.000 sentences on TV show domain. The sentences were labeled in three classes (positive, neutral and negative) by seven annotators, following literature guidelines for ensuring reliability on the annotation. We also ran baseline experiments on polarity classification using three machine learning methods, reaching 80.99% on F-Measure and 82.06% on accuracy in binary classification, and 59.85% F-Measure and 64.62% on accuracy on three point classification.Comment: Accepted for publication in 11th International Conference on Language Resources and Evaluation (LREC 2018

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

Zinc-binding properties of Junín virus nucleocapsid protein

The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX2HX23CX4C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.Instituto de Biotecnologia y Biologia Molecula

On the Bank Stocks Return and Volatility: Tale of a South Asian Economy

This paper examines Pakistani Banks stock return and volatility relationship with market, interest rate and foreign exchange rate. The study extensively applies different statistical approaches to model return and volatility relation. First, Ordinary Least Square (OLS) multiple regression model is applied for estimation of return relation. Further, Generalize Method of Movement (GMM) is applied to cater the endogeniety issue. Secondly, Due to presence of Conditional Heteroskedasticity, Weighted Least Square (WLS) and Generalize Auto Regression Conditional Heteroskedasticity - GARCH (1,1) estimation model is applied to estimate conditional return and volatility. Interest rate and foreign exchange rate have significant impact on unconditional and conditional bank stock returns under different model specifications. Market return is a determining factor in bank stock pricing. The results infer that bank volatility is significantly related with interest rate and foreign exchange rate risk. The volatility of bank stock returns is persistent with slower decay over time

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula