<b>Introduction:</b> Respiratory Syncytial Virus is the major viral cause of lower respiratory tract disease in young children worldwide, with the greatest burden of disease in infants aged 1-3 months. Consequently, vaccine development has centered on a vaccine to directly protect the infants in this age group. The fundamental problem is that these young infants are poor responders to candidate RSV vaccines. This thesis focuses on the use of mathematical models to explore the merits of vaccination. <b>Methods:</b> Following development and analysis of a simple non-age-structured ODE model, we elaborate this to a Realistic Age Structured model (RAS) capturing the key epidemiological characteristics of RSV and incorporating age-specific vaccination options. The compartmental ODE model was calibrated using agespecific and time series hospitalization data from a rural coastal Kenyan population. The determination of Who Acquires Infection From Whom (WAIFW) matrix was done using social contact data from 1) a synthetic mixing matrix generated from primarily household occupancy data and 2) a diary study that we conducted in the Kilifi Health and Demographic Surveillance System (KHDSS). The vaccine was assumed to elicit partial immunity equivalent to wild type infection and its impact was measured by the ratio of hospitalized RSV cases after to before introduction. of vaccination. Uncertainty and sensitivity analysis were undertaken using Latin Hypercube Sampling (LHS) and partial rank correlation respectively. Given the importance of households in the transmission of respiratory infections, an exploratory household model was developed to capture the transmission dynamics of RSV A and B in a population of households. <b>Results:</b> From the analytical work of the simple ODE model, we have demonstrated that the model has the potential to exhibit a backward bifurcation curve within realistic parameter ranges. Both the diary and the synthetic mixing matrices had similar characteristics i.e. strong assortative mixing in individuals less than 30 years old and strong mixing between children less than 5 years and adults between 20 and 50 years old. When the two matrices were jointly linearly regressed, their elements were well correlated with an R2 ~ 0.6. The RAS model was capable of capturing the age-specific disease and the temporal epidemic nature of RSV in the specified location. Introduction of routine universal vaccination at ages varying from the first month of life to the 10th year of life resulted in optimal long-term benefit at 7 months (for the diary contact model) and 5 months (for the synthetic contact model). The greatest benefit arose under the assumption of age-related mixing with the contact diary data with no great deal of effectiveness lost when the vaccine is delayed between 5 and 12 months of age from birth. Vaccination was also shown to change the temporal dynamics of RSV hospitalizations and also to increase the average age at primary infection. From the sensitivity analysis, we identified the duration of RSV specific maternal antibodies, duration of primary and tertiary infections as the most important parameters in explaining the imprecision observed in predicting both the age specific hospitalizations and the optimal month at vaccination. Results from the household model have demonstrated that the household epidemic profile may be different from the general population with strong interaction of the viruses in the household that do not necessarily reflect at the population level. <b>Conclusion:</b> The synthetic matrix method would be a preferable alternative route in estimating mixing patterns in populations with the required socio-demographic data. Retrospectively, the synthetic mixing data can be used to reconstruct contact patterns in the past and therefore beneficial in assessing the effect of demographic transition in disease transmission. Universal infant vaccination has the potential to significantly reduce the burden of RSV associated disease, even with delayed vaccination between 5 and 12 months. This age class represents the group that is being targeted by vaccines that are currently under development. More accurate data measuring the duration of RSV specific maternal antibodies and the duration of infections are required to reduce the uncertainty in the model predictions.

Orientador: Fátima Pereira de Souza / Banca: Karina Alves de Toledo / Banca: José Roberto Ruggiero / Resumo: O Vírus Sincicial Respiratório Humano (hRSV) foi identificado em 1957 e mesmo após vários anos de investigação, nenhuma vacina foi desenvolvida. Acredita-se que a chave de inibição da ação viral são suas glicoproteínas de membrana, em especial a proteína de fusão (F), que com auxílio da proteína de ligação (G), é responsável pela instalação do hRSV na célula hospedeira. Há evidências experimentais de que compostos como flavonóides e glicosaminoglicanos podem diminuir a infecção viral, sendo então a proteína F um bom alvo para a ação destes compostos. O presente estudo utilizou de ferramentas de bioinformática para verificar as possíveis regiões de interação da proteína F com a Heparina Sulfatada e Flavonóides. Os programas de bioinformática foram utilizados para: modelagem dos compostos, caracterização e previsão da estrutura secundária da proteína, modelagem da estrutura terciária e docking molecular entre o modelo da proteína F e as estruturas tridimensionais dos Flavonóides e da Heparina Sulfatada. Modelos válidos foram obtidos para as estruturas tridimensionais dos flavonóides e para o modelo completo da proteína F. As características da proteína incluem um alto nível de conservação na seqüência de aminoácidos e, especialmente, em seus sítios de ligação. O docking da proteína com a Heparina, e o virtual screening da biblioteca de Flavonóides e a estrutura da proteína, resultaram em sítios de interação com grande potencial de inibição, uma vez que concordam com evidências experimentais descritos na literatura. A Heparina liga-se ao sítio de clivagem II, importante região para obtenção da atividade de fusão da proteína. Os Flavonóides podem se ligar a região hidrofóbica que desestabiliza... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Human Respiratory Syncytial Virus (hRSV) was identified in 1957 and even after several years of research, no vaccine has been developed yet. It is believed that the key to the inhibition of viral action is its membrane glycoproteins, including the Fusion Protein (F), responsible for the installation of the hRSV in the host cell. There are evidences that compounds such as flavonoids and glycosaminoglycans can decrease the viral infection, and F protein can be a good target for the action of these compounds. The present study checked the possible sites of interaction between F protein and heparin and flavonoids, using computational tools. Bioinformatics programs were used for: modeling compounds, characterization and prediction of protein secondary structure, tertiary structure modeling and the docking between the protein model and the structures of flavonoids and sulfated heparin. Valid models were obtained for flavonoids structures and the complete model of F protein. The characteristics of the protein include a high level of conservation in amino acid sequence and especially in its binding sites. The heparin docking and virtual screening of flavonoids resulted in interaction sites with great potential for inhibition, since they agree with other studies and experimental evidence of F protein inhibition. This study shows that compounds such as sulfated heparin and flavonoids interact in important sites of F protein. Heparin binds to the cleavage site II and flavonoids can bind to the hydrophobic site that destabilizes the formation of the six-helix-bundle region. Both regions are important for conformational changes that F protein undergoes to get its fusion activity. Docking showed that molecular interactions are likely to occur and selected the best candidates for a possible inhibitor. These evidences... (Complete abstract click electronic access below) / Mestre

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Thesis (D.P.H.)--University of Michigan.

Dissertation (D.P.H.)--University of Michigan.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

O vírus respiratório sincicial humano (HRSV) é principal agente causador de infecções do trato respiratório inferior em crianças e lactentes. Um diagnóstico rápido e preciso evitaria o uso desnecessário de antibióticos, nos casos em que a infecção é viral. A reação em cadeia da polimerase após transcrição reversa (RT-PCR) e o ensaio de imunofluorescência indireta (IFI) são considerados ferramentas importantes na detecção do HRSV, pela alta sensibilidade e especificidade. Visando simplificar e minimizar os riscos de contaminação freqüentes, em duas etapas, foi padronizada uma reação em tubo único para detecção do HRSV em amostras clínicas. Aspirados de nasofaringe de 226 crianças de 0-5 anos de idade, com doença respiratória, atendidas no Hospital Universitário da Universidade de São Paulo (HU-USP), foram testados por imunofluorescência indireta, RT semi Nested PCR e RT-PCR em tubo único. Cento e duas amostras (45,1%) foram positivas em pelo menos uma das técnicas e 75 (33,2%) em todas. Três (1,3%) amostras foram positivas por IFI e RT semi Nested PCR, 1 (0,4%) foi positiva por IFI e RT-PCR em tubo único, 5 (2,2%) amostras foram positivas somente por IFI, 2 (0,9%) somente por RT semi Nested PCR e 16 (7,1%) amostras foram positivas pela RT semi Nested PCR e RT-PCR em tubo único. A RT-PCR em tubo único mostrou ser uma técnica rápida, sensível e específica, e o uso combinado de dois métodos aumenta a detecção do HRSV. / Respiratory Syncytial Virus is the main cause of acute lower respiratory tract infection (ALTRs) in infants, elderly and immunodepressed patients. Rapid diagnosis of Respiratory Syncytial Virus (RSV) infection is necessary to efficient treatment, avoiding the unnecessary use of antibiotics and determining patient isolation requirements. The reverse trancriptase polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay (IFA) methods have been referred as important tools for virus detection considering the high sensitivity and specificity, respectively of such methods. In order to maximize the simplicity and minimize the risk of sample cross-contamination by two steps RT-PCR, we developed a RT-PCR using a single-tube to detect HRSV in clinical samples. Nasopharyngeal aspirates (Nas) of 226 patients with acute respiratory illness, ranging 0-5 years old, were collected at the University of São Paulo Hospital (HU-USP) in São Paulo city. Samples were tested by indirect immunofluorescence assay, RT semi Nested PCR and single-tube RT-PCR. One hundred two (45,1%) of the 226 samples were positive at least by one of the three methods tested and 75 (33,2%) were positive by all methods. Three (1,3%) samples were positive only by IFI and RT semi Nested PCR, 1 (0,4%) sample were positive only by IFI and RT-PCR single-tube, 5 (2,2%) were positive only by IFI, 2 (0,9%) were positive only by RT semi Nested PCR and 16 (7,1) were positive only RT semi Nested PCR and RT-PCR single-tube. RT-PCR single-tube, showed to be fast, sensitive and specific for diagnosis of RSV and the combined use of both methods enhanced HRSV detection.