Orientadores: Clarice Weis Arns, Fernando Rosado Spilki / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T19:38:08Z (GMT). No. of bitstreams: 1 Silva_LucianaHelenaAntoniassida_D.pdf: 3352165 bytes, checksum: 1c8836441214fc41a7890899268f1163 (MD5) Previous issue date: 2013 / Resumo: Os membros da família Paramyxoviridae são vírus que causam infecções em humanos e animais de importância econômica global. Entre os membros desta família incluem patógenos de importância mundial para os humanos, como o vírus respiratório sincicial humano (hRSV), o metapneumovírus humano (hMPV) e vírus de importância em Medicina Veterinária, como o vírus respiratório sincicial bovino (bRSV) e o metapnemovírus aviário (aMPV). Os membros da família Paramyxoviridae, subfamília Pneumovirinae são vírus envelopados, não-segmentados dotados de genoma de RNA de fita simples com sentido negativo. Na primeira parte do estudo, desenvolvemos um adenovírus recombinante expressando a proteína F do aMPV. A expressão da proteína F foi determinada por Western Blot. Os níveis de transcrição do gene F foram avaliados por RT-PCR em tempo real, em células HEK-293 e células HEP-2. Foi realizada a imunização experimental de Ad-aMPV-F e foi analisada a indução de resposta de anticorpos em camundongos BALB/c. Os títulos de anticorpos neutralizantes foram detectados após a imunização com Ad-aMPV-F. Na segunda parte do trabalho o objetivo foi à construção de adenovírus recombinantes expressando a proteína F do bRSV. A proteína F parece ser um antígeno ideal para fins de diagnóstico. Utilizando anticorpo anti-V-5, uma banda de ~90 kDa foi detectada no sobrenadante de cultura de células HEK-293 infectadas com Ad-bRSV-F. Na terceira parte do estudo, o objetivo foi à construção de dois vetores adenovirais expressando as proteínas G do aMPV e bRSV, a expressão destas proteínas em células HEK-293 infectadas foi analisada pela expressão do gene repórter, da proteína verde fluorescente (GFP) / Abstract: The members of the family Paramyxoviridae are viruses that cause infectious in human and animals of importance to global economics. Among the member of this family include pathogens of importance global for humans such as human respiratory syncytial virus (hRSV), the human and metapneumovirus (hMPV) and of viruses importance in veterinary medicine, such as bovine respiratory syncytial virus (bRSV) and avian metapnemovírus (aMPV). The members of the Paramyxoviridae are enveloped, non-segmented viruses, with negative-sense single stranded genomes. In the first part of the study, we developed a recombinant adenovirus expressing the F protein of AMPV. The expression of F gene was determined by Western Blot. The levels of transcription were evaluated by RT-PCR in real time in HEK-293 cells and HEP-2 cells. Immunization experiment was carried out Ad-AMPV-F was analyzed and the induction of antibody response in BALB/c mice. The neutralizing antibody titers detected after immunization with Ad-AMPV-F. In the second part, the objective was to construct recombinant adenoviruses expressing the F protein of bRSV. Protein F appears to be an ideal antigen for diagnostic purposes. Using the anti-antibody AdV-5, a single band of ~ 90 kDa was detected in the culture supernatant in 293 cells infected with Ad-bRSV-F. In the third part of the study, the objective was to build two adenoviral vectors expressing the G protein of aMPV and bRSV and the expression of these proteins in infected HEK-293 cells were analyzed for expression of the reporter gene, green fluorescent protein (GFP) / Doutorado / Microbiologia / Doutora em Genética e Biologia Molecular

Abstract Pulmonary surfactant is a lipid-protein mixture that lines the inner surface of the lung. The main function of surfactant is to reduce surface tension at the air-liquid interface, thus preventing alveolar collapse at the end of expiration. Lack of surfactant is the main cause of respiratory distress syndrome (RDS) in preterm infants. Very preterm babies are at risk of developing a lung disease called bronchopulmonary dysplasia (BPD). The surfactant proteins SP-A, -B, -C and -D have important functions in surfactant structure, homeostasis and innate immunity of the lung. The genes of these proteins have been studied as candidates for several multifactorial lung diseases both in adults and in children. The aim of the present study was to examine the genetic variation in SP genes and to evaluate the role of SP gene polymorphism in the etiology of severe pulmonary infantile diseases, including RDS, BPD and severe respiratory syncytial virus (RSV) infection among the Finnish population. Conventional allelic association methods in combination with multiparameter analysis and family-based transmission disequilibrium test (TDT) were used. The SP-D Met11 allele was associated with a risk for severe RSV bronchiolitis in a matched case-control setting of 84 infants with severe RSV infection and 93 control infants. The variants of the SP-C gene had no detectable association with BPD. However, a modest association of SP-C Asn138 and Asn186 alleles with RDS was found. A length variation in the SP-B gene was associated with BPD among very preterm infants born before 32 weeks of gestation. The SP-B intron 4 deletion variant allele increased the risk for BPD especially in very low birth weight infants. The association was confounded by birth order, being evident only among presenting infants, who are more prone to ascending infections during a preterm birth process. The present study provides new evidence about the significance of SP gene polymorphisms in the etiology of complex infantile pulmonary diseases, including RDS, BPD and severe RSV bronchiolitis. The results help us to understand the molecular mechanisms underlying these diseases and may, in the long run, enable better treatment of these life-threatening diseases. / Tiivistelmä Keuhkosurfaktantti on keuhkon sisäpintaa peittävä kalvomainen rasva-proteiinikompleksi, jonka tärkein ominaisuus on pintajännityksen vähentäminen keuhkorakkuloissa. Surfaktantin puutos ennenaikaisesti syntyneillä lapsilla aiheuttaa hengitysvaikeusoireyhtymän, RDS-taudin (respiratory distress syndrome). Alle 30 raskausviikon iässä syntyneistä, useimmiten RDS-taudin saaneista keskosista n. 30 % sairastuu vakavaan krooniseen keuhkotautiin, BPD-tautiin (bronchopulmonary dysplasia). Surfaktanttiproteiineilla SP-A, -B, -C ja -D on osoitettu olevan tärkeä tehtävä surfaktantin toiminnassa ja keuhkon synnynnäisessä immuniteetissa. Tämän tutkimuksen tavoitteena oli selvittää surfaktanttiproteiineissa esiintyvän geneettisen muuntelun määrää ja merkitystä keskosten RDS- ja BPD-taudeissa sekä pienten lasten vakavassa respiratory syncytial -viruksen (RSV) aiheuttamassa keuhkotulehduksessa. Tutkimuksen laajin osa keskittyi tutkimaan keskosten BPD-tautia ja surfaktanttiproteiinien geenien osuutta siinä. Geneettisen muuntelun merkitystä tarkasteltiin populaatiogeneettisin keinoin tapaus-verrokkiasetelmissa ja perheaineistojen avulla. Yhteensä analysoitiin noin tuhannen lapsen ja yli kahdensadan vanhemman DNA-näytteet. Tutkimuksessa havaittiin SP-D-geenissä olevan metioniini11-geenimuodon liittyvän pienten lasten vakavaan RSV-infektioon. Lisäksi saatiin uutta tietoa SP-C-geenin populaatiotason yleisestä muuntelusta ja todettiin SP-C:n asparagiini138 ja asparagiini186 -geenimuotojen yhteys keskosten RDS-taudin esiintymiseen. Merkittävin löydös oli SP-B-geenissä olevan deleetiovariantin kytkeytyminen alle 32-viikkoisina syntyneiden keskosten BPD-tautiin. Geneettisen altistuksen lisäksi BPD-tautiin sairastumiseen vaikuttivat lukuisat keskosuudelle ominaiset seikat, kuten alhainen syntymäpaino, RDS-tauti ja syntymähetkellä todettu hapenpuute. Geneettisen tekijän vaikutus oli voimakkain erittäin pienipainoisilla keskosilla. Tutkimuksen tulokset ovat tuoneet arvokasta lisätietoa surfaktanttiproteiinien geenien osuudesta keskosten RDS- ja BPD-taudeissa sekä pienten lasten vakavassa RSV-infektiossa. Ne auttavat ymmärtämään näiden molekyylibiologisia syntymekanismeja ja voivat ajan mittaan olla edistämässä uusien hoitomuotojen kehittämistä.

O vírus respiratório sincicial humano (HRSV) é o principal agente viral causador de doença respiratória em bebês e crianças em idade pré-escolar. A fim de estudar a variabilidade genética de HRSV, grupo B, com inserção de 60 nucleotídeos no gene G, selecionamos amostras de aspirado de nasofaringe de crianças menores de 5 anos de idade, com doença respiratória aguda, admitidas no hospital universitário da Universidade de São Paulo. Testamos 521 amostras, das quais 35,3% foram positivas para HRSV. A região G2 da glicoproteína G foi utilizada para genotipar essas amostras. Todas as amostras do grupo B apresentaram a inserção de 60 nucleotídeos no gene da proteína G, como descrito anteriormente em Buenos Aires, em 1999. As modificações de aminoácidos e nucleotídeos dessas amostras foram comparadas com outras amostras com inserção de 2001-2005. A seqüência de nucleotídeos duplicados foi a cópia exata dos 60 nucleotídeos precedentes em vírus mais antigos, mas as cópias do segmento duplicado acumularam substituições de nucleotídeos em vírus mais recentes. / Human respiratory syncytial virus (HRSV) is the leading viral cause of respiratory illness in infants and young children. In order to study the genetic variability of HRSV group B, with 60-nucleotide duplication in the gene G, we selected nasopharyngeal aspirates samples of children less than five years of age, with acute respiratory illness admitted in the university hospital of São Paulo (USP). We tested 521 samples and the HRSV-detection test positivity rate was 35.3%. The G2 region of glycoprotein G was used as genotyping default. All type B HRSV had a 60-nucleotide duplication in the attachment protein gene like previously described in Buenos Aires, in 1999. Changes in aminoacids and nucleotides in these samples were compaired with other samples with duplication from 2001-2005. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but copies of the duplicated segment accumulated nucleotide substituions in more recent viruses.

Le virus respiratoire syncytial (VRS) est le plus important pathogène respiratoire du jeune enfant. Dans une étude prospective menée au cours de deux saisons hivernales, parmi les enfants de 0-3 ans hospitalisés pour infections des voies respiratoires (IVR) , au moitis un virus respiratoire a été retrouvé chez 74%. Le VRS était présent chez 55,6% des enfants. Deux sousgroupes (A et B) du VRS et plusieurs génotypes ont été identifiés basé sur la séquence du gène G. Le séquençage de la glycoprotéine G et l'analyse phylogénétique ont été effectués sur les souches détectées. La comparaison des données cliniques des 106 enfants avec le sous-groupe A du VRS (96 génotypes GA2) et 94 enfants avec le sous-groupe B du VRS (62 génotypes GB3) a montré que le sous-groupe A et le génotype GAI étaient associés à une plus grande sévérité de la maladie en comparaison avec les souches du sous-groupe B. Parmi les enfants atteints de VRS qui ont reçu des antibiotiques (AB) de façon empirique à l'admission, la réception d'un résultat d'un test rapide confirmant la présence du VRS n'a pas réduit subséquemment l'utilisation des AB. La proportion des hospitalisations attribuables au VRS et à l'influenza estimée à partir des banques administratives par six méthodes statistiques connues a été comparée aux résultats de l'étude prospective chez les enfants de 6 à 23 mois. Les estimés obtenus variaient de façon considérable selon la saison et la méthode. Les méthodes statistiques usuelles ne semblent pas en mesure d'estimer correctement les hospitalisations attribuables aux virus respiratoires.

No description available.

This dissertation is related to mathematical modelling of the spread of respiratory syncytial virus (RSV) in Valencia and is still causing a large number of hospitalizations of children in this community. A mathematical model based on a system of nonlinear differential equations of first order has been built. This model considers the population divided into two age groups to pay particular attention to children under one year who are the most affected by this disease. This model has been fitted with hospitalizations data of Valencia and has been used to perform a cost analysis of a potential vaccination strategy. We also propose a complete network model to study the seasonal evolution of RSV epidemics in which seasonal parameters were fitted with the previous continuous model. Both developments are contrasted. On the complete network model we propose a strategy for vaccination of children based on the administration of three doses, and develop a cost-effectiveness study for different vaccination rates. Finally we have defined a SIRS model for RSV epidemics on a random social network of contacts among individuals. In this model has not forced the seasonality. The seasonality arises naturally for certain values of the duration of immunity of a patient recovered, the number of contacts and the likelihood of infection from a contact in a day. In this social network model only a narrow range of parameters can support RSV epidemic seasons. / Moraño Fernández, JA. (2010). Mathematical modelling of Respiratory Syncytial Virus spread in the Spanish region of Valencia. Preventive applications [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8638

La bronchiolite à virus respiratoire syncytial (VRS) est en augmentation [3,9,12,25], affectant de nombreux nourrissons et pouvant mener à l'asthme. Le taux de césarienne augmente également [106,108], entraînant des problèmes respiratoires dont les mécanismes sont mal cernés. Nous avons évalué l'association entre le mode d'accouchement et la bronchiolite à VRS sévère. Nous avons réalisé une étude cas-témoin non appariée comparant des enfants nés à terme et hospitalisés au Centre Hospitalier de l'Université Laval (CHUL) pour bronchiolite à VRS entre 2004 et 2007 à des enfants du même âge nés à terme au CHUL. Une régression logistique non conditionnelle a été appliquée en tenant compte des facteurs de risque établis dans la littérature. Les facteurs de risque de bronchiolite sévère suivants ont été retenus: la césarienne en urgence (RC 1.97 (IC: 1.16-3.36)); la présence d'un(e) frère/soeur (RC 3.27 (IC:2.26-4.73)); la naissance entre septembre et février (RC 1.83 (IC: 1.30-2.58)); le sexe masculin (RC 1.5 (IC: 1.07-2.13)).

A incidência de doença respiratória pelo vírus sincicial respiratório humano (VSRH) avaliada em uma coorte de recém-nascidos, entre dezembro/2002 a setembro/2005, foi de 9,84/1000 criança-mês.Um total de 316 amostras de lavado de nasofaringe, foram processadas por três diferentes técnicas (isolamento viral, imunofluorescência direta e PCR) para detecção de vírus respiratório sincicial humano (VSRH). Destas, 36 (11,4%) foram positivas para o VSRH. A PCR foi à técnica mais sensível, sendo positiva em 35 (11,1%) das amostras, seguido da imunofluorescência direta (25/316, 7,9%) e isolamento viral (20/315, 6,3%) (p < 0,001). Os dados do presente estudo sugerem que o conceito de isolamento viral como "padrão ouro" no diagnóstico do VSRH seja revisto. / The incidence of respiratory illnesses caused by the human respiratory syncytial virus (HRSV) in a cohort of neonates between December 2002 and September 2005 was 9.84/1000 children/month. A total of 316 samples of nasopharyngeal lavage were processed using three different techniques (viral isolation, direct immunofluorescence and PCR) to detect the human respiratory syncytial virus (HRSV). Of these, 36 (11.4%) were positive for HRSV. PCR was the most sensitive technique. It was positive in 35 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). The findings of this study suggest that the view that viral isolation is the "gold standard" for diagnosis of HRSV should be reconsidered.

Made available in DSpace on 2016-01-26T12:51:26Z (GMT). No. of bitstreams: 1 joaobatistasalomaojunior_tese.pdf: 726701 bytes, checksum: be09a3fcaa3d491c8ac2601eb72d7961 (MD5) Previous issue date: 2008-02-08 / Acute lower respiratory tract disease (ALRTD) accounts for high infantile mortality and morbidity rate worldwidely. Respiratory syncytial virus (RSV) is frequently found among pathogens. Objectives: The objectives were: 1) to evaluate the RSV frequency in children from 0 to 6 years hospitalized due to acute lower respiratory disease in São José do Rio Preto, SP; 2) to characterize the virus seasoning in this city and 3) to verify possible association among epidemiologic, clinical and diagnostic data with this viral agent. Casuistic and Method: From May 2004 to September 2005, 278 children aged from 0 to 6 years with ALRTD were studied. They have contracted the disease in the community, hospitalized in the children s ward, emergency room and Pediatric Intensive Care Unit of Hospital de Base, São José do Rio Preto. They were asymptomatic in a 7-day period before the beginning of the disease. Questionnaires were used for the children's characterization and their clinical presentation. Samples of nasopharyngeal secretion were collected to identify RSV, using reverse transcription polymerase chain reaction (RT-PCR). Results: The results showed that in the 290 hospitalizations of ALRTD, RSV was positive in 29.3%. ALRTD was more frequent in infants (average = 13.5 months) and male (57.6%). RSV was more frequent in bronchiolitis cases (64%). RSV+ infections were more frequent in the first year of life (35%). In RSV + infections, pneumonia frequency varied from 19.5 to 26.2% in the studied age groups; acute wheezing was observed in 31.8% of children aged over 2 years; bronchiolitis was registered in 62.5% of the children younger than 1 year; pneumonia with pleural effusion was noticed in 18.7% of the children aged over 2 years. Conclusions: The frequency of RSV in children from 0 to 6 years hospitalized due to ALRTD was 29.3% in São José do Rio Preto, SP. The ALRTD were more frequent between June and November 2004. In 2005, the hospitalizations occurred mainly starting from March decreasing in September. There was RSV prevalence in children younger than 2 years, male and with bronchiolitis. The RSV frequency in the hospitalizations was higher in 2004 than in 2005. In the RSV+ infections, the cases of pneumonia had similar occurrence in the studied age groups. There was reduction of the RSV frequency as age increases in the cases of pneumonia with pleural effusion and increase in the cases of acute wheezing; in bronchiolitis most of the RSV+ cases occurred in children younger than1 year. The clinical and radiological data obtained did not allow the proper identification of the infection by RSV. Laboratory examination by means of RT-PCR was necessary to identify it. / As doenças agudas do aparelho respiratório inferior (DARI) são responsáveis por altos índices de mortalidade e morbidade infantil em todo mundo. Dentre os patógenos predominantes encontra-se o vírus sincicial respiratório (VSR). Objetivos: Os objetivos foram: 1) avaliar a freqüência do VSR em crianças de 0 a 6 anos hospitalizadas por DARI em São José do Rio Preto, SP; 2) caracterizar a sazonalidade do vírus nessa cidade e 3) evidenciar possível associação de dados epidemiológicos, clínicos e diagnósticos e este agente viral. Casuística e Método: No período de maio de 2004 a setembro de 2005 foram estudadas 278 crianças de 0 a 6 anos com DARI adquirida na comunidade, internadas na enfermaria, emergência e Unidade de Terapia Intensiva Pediátrica do Hospital de Base de São José do Rio Preto, que estavam assintomáticas por um período de 7 dias antes do início da doença. Foram utilizados questionários para caracterização das crianças e do quadro clínico. Para identificação do VSR foram coletadas amostras de secreção de nasofaringe, utilizando-se a técnica de Reverse Transcription Polymerase Chain Reaction (RT-PCR). Resultados: Os resultados mostraram que nas 290 internações por DARI, o VSR foi positivo em 29,3%. DARI foi mais freqüente em lactentes (mediana = 13,5 meses) e do gênero masculino (57,6%). O VSR foi mais freqüente nos casos de bronquiolite (64%). As infecções VSR+ foram mais freqüentes no primeiro ano de vida (35%). Nas infecções VSR+, a freqüência de pneumonia variou de 19,5 a 26,2% nas faixas etárias estudadas; em 31,8% das crianças maiores de 2 anos observou-se sibilância aguda; bronquiolite foi registrada em 62,5% das crianças menores de 1 ano; pneumonia com derrame pleural foi notada em 18,7% das crianças maiores de 2 anos. Conclusões: A freqüência do VSR em crianças de 0 a 6 anos hospitalizadas por DARI em São José do Rio Preto, SP, foi 29,3%. As DARI foram mais freqüentes entre junho e novembro de 2004. Em 2005, as internações ocorreram principalmente a partir de março, com queda em setembro. Houve predomínio do VSR em crianças de 0 a 2 anos, do gênero masculino e com bronquiolite. A freqüência do VSR nas internações foi maior em 2004 que em 2005. Nas infecções VSR+ os casos de pneumonia tiveram ocorrência semelhante nas faixas etárias estudadas. Houve redução da freqüência do VSR com aumento da idade nos casos de pneumonia com derrame pleural e aumento nos casos de sibilância aguda; na bronquiolite a maioria dos casos VSR+ ocorreu em crianças menores de 1 ano. Os dados clínicos e radiológicos encontrados não permitiram a identificação correta da infecção pelo VSR, havendo necessidade do exame laboratorial pela técnica RT-PCR para sua identificação.

A incidência de doença respiratória pelo vírus sincicial respiratório humano (VSRH) avaliada em uma coorte de recém-nascidos, entre dezembro/2002 a setembro/2005, foi de 9,84/1000 criança-mês.Um total de 316 amostras de lavado de nasofaringe, foram processadas por três diferentes técnicas (isolamento viral, imunofluorescência direta e PCR) para detecção de vírus respiratório sincicial humano (VSRH). Destas, 36 (11,4%) foram positivas para o VSRH. A PCR foi à técnica mais sensível, sendo positiva em 35 (11,1%) das amostras, seguido da imunofluorescência direta (25/316, 7,9%) e isolamento viral (20/315, 6,3%) (p < 0,001). Os dados do presente estudo sugerem que o conceito de isolamento viral como "padrão ouro" no diagnóstico do VSRH seja revisto. / The incidence of respiratory illnesses caused by the human respiratory syncytial virus (HRSV) in a cohort of neonates between December 2002 and September 2005 was 9.84/1000 children/month. A total of 316 samples of nasopharyngeal lavage were processed using three different techniques (viral isolation, direct immunofluorescence and PCR) to detect the human respiratory syncytial virus (HRSV). Of these, 36 (11.4%) were positive for HRSV. PCR was the most sensitive technique. It was positive in 35 (11.1%) of the samples, followed by direct immunofluorescence (25/316, 7.9%) and viral isolation (20/315, 6.3%) (p < 0.001). The findings of this study suggest that the view that viral isolation is the "gold standard" for diagnosis of HRSV should be reconsidered.