This study designed and computational docked a group of ligands intended to find potent inhibitors for Neuraminidase 4 which would have strong interactions with 8 conserved amino acids in the active site. Several trials of ligands were designed based on derivatives of neuraminic acid and evaluated as inhibitors of influenza neuraminidase. Optimized geometries of those ligands were determined using HF/B3LYP/6-311++G** techniques. Binding energies of the ligands bound to the N4 subtype of the neuraminidase protein were determined using AutoDock 4.0. Currently used inhibitors for influenza viruses will also be analyzed in the exactly same way. Comparing the binding information of those candidates and current ligands can provide a useful data about the potential of these species as antiviral drugs. / Department of Chemistry

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

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 78-89) Also available in print.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Molecular Virology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 78-89) Also available online.

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

The theme of my thesis is the issue of the new strain of influenza A (H1N1 Pandemic 2009) and the associated awareness of vaccination amongst the elderly. The work is divided into two parts, a theoretical and practical part. In the theoretical part, I tried to comprehensively process the available knowledge on the origins, epidemiology and prevention of influenza with particular emphasis on vaccination, oriented towards the elderly. The practical part is focused on mapping the awareness of the elderly of the issue of vaccination against influenza and analysis of results from a research exploratory investigation. The data acquisition method I used was quantitatively oriented research using anonymus questionnaires. Altogether, I distribued 350 questionnaires; the final number for data processing was 191 questionnaires. In connection with the work I set four hypotheses: 1) More than a third of respondents were vaccinated against the new strain of influenza A (H1N1 Pandemic 2009), 2) The most common reason for not being vaccinated was a lack of information. 3) The size of the place of residence significantly contributes statistically to a sense of awareness of respondents on this issue, 4) More than half of respondents would like to obtain more information on the issue of the new strain of influenza type A (Pandemic H1N1 2009). I confirmed or refuted the formulated the hypotheses based on survey evaluation. The results of the survey showed that most respondents do not feel that they are sufficiently informed about the issue of the new strain of influenza, and therefore discard the use of vaccinations. With this work I would like to stress the importance of information, which can help people consider the risks of influenza and motivate them to be vaccinated. Vaccination is an important means of protection against influenza viruses particulary for high-risk groups including the elderly. For this reason I consider the dissemination of information as very important and I hope that my thesis also serves this purpose.

Introdução: O surgimento de uma nova cepa do vírus Influenza A, o H1N1, determinou uma pandemia no ano de 2009, com importante repercussão global. Esse vírus infectou principalmente adultos jovens e crianças menores de dois anos com grande aumento na morbimortalidade quando comparado com as taxas anuais decorrentes do influenza. Objetivo: Avaliar as características epidemiológicas e clínicas da infecção pelo vírus influenza A (H1N1) em crianças, a fim de agregar conhecimento para melhor abordagem em futuras pandemias. Métodos: Estudo de corte transversal com revisão dos prontuários de todas as crianças, entre zero e 16 anos, hospitalizadas com quadro gripal no Hospital de Clínicas de Porto Alegre (HCPA) em 2009. A identificação do vírus H1N1 foi feita através de Reação em Cadeia da Polimerase (PCR) em laboratório de referência. Resultados: Cento e noventa e uma crianças foram internadas com suspeita de infecção pelo H1N1, destas, 83 (43%) foram submetidos à pesquisa do vírus H1N1, sendo 28 positivos (34%) e 55 negativos (66%). Os pacientes positivos para H1N1 eram mais velhos, 27 (7-108) versus sete (3-32) meses (p=0,015), todos apresentaram febre versus 70% do outro grupo (p=0,015), chegaram ao hospital com menor saturação de hemoglobina, 80% (±20%) versus 95% (±4) (p<0,001) e precisaram de maior pressão expiratória final, dez (±3) versus cinco (±1) cm H2O (p=0,001) e de maior fração inspirada de oxigênio, um (0,65-1) versus 0,4 (0,4-1) (p=0,053) quando colocados em ventilação mecânica. Não houve diferença quanto à necessidade de internação em unidade de terapia intensiva, indicação de suporte ventilatório, tempo de internação e óbito. Conclusão: As crianças acometidas pelo H1N1 apresentaram-se mais graves, embora tenham tido desfechos semelhantes às não infectadas. / Introduction: A new Influenza virus stem, H1N1, determined a pandemic in 2009 with great global repercussions. This virus infected mainly young adults and children under two years of age with marked increase in morbimortality when compared with annual rates. Objective: To analyze epidemiological and clinical characteristics of the infection by influenza A (H1N1) virus in children, in order to improve knowledge to a better approach in future pandemics. Methods: Cross section study with review of patient records for all children, between zero and 16 years, hospitalized with flu-like disease at Hospital de Clínicas de Porto Alegre (HCPA) in 2009. Identification of H1N1 virus was done through PCR technique on reference laboratory. Results: One hundred ninety-one children were hospitalized with suspected H1N1 infection. Of these, 83 (43%) patients were tested for the H1N1 virus, 28 (34%) being positive and 55 (66%) negative. H1N1 patients were older, 27 (7-108) versus seven months old (3-32) (p=0,015), all had fever versus 70% of the other group (p=0,015), they arrived at the hospital with lower oxygen hemoglobin saturation, 80% (±20%) versus 95% (±4%) (p<0,001) and when placed in mechanical ventilation they needed greater end expiratory pressures, ten (±3) to five (±1) cm H2O (p=0,001) and inspired oxygen fraction, one (0,65-1) versus 0,4 (0,4-1) (p= 0,053) . There was no difference in terms of need for hospitalization in intensive care unit, need of ventilatory support or death. Conclusion: Children infected by H1N1 were more severely ill at arrival to the hospital, although they had similar outcomes to non-infected patients.

RNA viruses have the fastest evolutionary rates amongst protein-coding organisms on the planet. Ease of sequencing, advanced techniques of analysis and global health and economic concerns have all contributed to the recognition of RNA viruses as a robust research platform. Phylogenetic methods have been at the forefront of analytical techniques used to understand the dynamics of RNA viruses - during natural circulation in populations and in individual hosts, within epidemics, across species barriers and over billions of years that viruses have been around. Most of the work presented in this thesis employs phylogenetic incongruity arising from reassortment and recombination to gain insights into the genomes and populations of RNA viruses. Chapter 2 explores the selection regimes Ebola virus has experienced following a year of circulation in humans inWest Africa, as well as its recent history. Chapter 3 investigates the extent of recombination in MERS-CoV, a novel human pathogen with an obscure epidemiology, which is suggestive of frequent co-infection of some hosts. Chapter 4, on the other hand, documents a pattern of non-intuitive linkage between some segments of the human-endemic influenza B virus genome and explores its potential to speciate. Chapter 5 builds upon chapter 4 and attempts to describe small-scale reassortment between two segments of influenza B virus and the overall migration patterns of influenza B virus in Scotland. Chapter 6 exploits the independence of segments of influenza D virus, a recently described cattle pathogen, and coalescent theory to disentangle the origins of this virus. This thesis exemplifies the success of modern sequencing methods, which, together with the use of sophisticated analytical techniques, have uncovered a wealth of information hidden away in molecular sequences of RNA viruses. The work presented herein demonstrates how reticulate evolution can be exploited as a reliable, and sometimes indispensable, marker to improve inference of evolutionary forces in RNA viruses.

A influenza suína (IS) é uma doença altamente contagiosa, de curso rápido e pronta recuperação, causada pelo vírus Influenza tipo A (VIS). Os principais sinais clínicos são tosse, febre, anorexia e baixo desenvolvimento. A doença está presente em outros países e, geralmente, está associada com outros agentes infecciosos. Porém, no Brasil, a sua primeira descrição ocorreu em 2011 e foi associada ao vírus H1N1 pandêmico (pH1N1). O principal objetivo deste estudo foi caracterizar as alterações histológicas mais importantes em casos de doença respiratória suína sugestiva de IS e estudar a associação dessas alterações com os resultados de imuno-histoquímica (IHQ) anti-vírus da influenza A (VIA), anti-circovírus suíno tipo 2 (PVC2) e anti-vírus da síndrome reprodutiva e respiratória suína (PRRSV). Para tanto, foram estudadas 60 amostras de pulmões suínos selecionadas dos materiais do arquivo do Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (SPV-UFRGS), de casos de doença respiratória remetidos no período de 2009 a 2010 e que apresentavam alterações histopatológicas compatíveis com pneumonia viral causada pelo VIS. Trinta e uma amostras (52%) foram provenientes do estado do Rio Grande do Sul, 14 (23%) do Paraná, 11 (18%) de Santa Catarina e quatro (7%) do Mato Grosso do Sul. A IHQ para IA confirmou a presença do agente viral em 45% das amostras analisadas. Os achados histológicos mais significativos associados à IHQ positiva para IA foram bronquiolite necrótica, atelectasia, broncopneumonia purulenta e hiperemia. Por outro lado, as alterações histológicas dos pulmões estudados mais significativamente associadas à IHQ negativa para IA foram hiperplasia dos pneumócitos tipo II, estruturas alveolares e bronquiolares similares a pólipos, hiperplasia de tecido linfoide associado a brônquios (BALT) e pleurite, que são alterações associadas a processos crônicos. Somente dois casos apresentaram marcação positiva na IHQ para PCV2 e nenhum pulmão foi positivo para PRRSV. Esses resultados sugerem que as lesões histológicas encontradas no presente estudo foram compatíveis com as causadas pelo VIS. Os casos negativos de IHQ para IA (55%) podem ser explicados pela baixa frequência do antígeno viral nos tecidos estudados. Como o curso da doença é muito rápido, o teste de IHQ é mais indicado para diagnóstico no início da infecção. Este estudo evidenciou novas alterações em amostras de pulmões de suínos com problemas respiratórios enviadas para o SPV UFRGS a partir de 2009, com ênfase para bronquiolite necrótica, e reforça a importância do estudo histopatológico dos casos de campo para auxiliar na monitoria da sanidade dos rebanhos. / Swine influenza is caused by swine influenza type A virus (SIV). It is a highly contagious disease with a rapid course and recovery. The main clinical signs are cough, fever, anorexia and poor performance. Usually, it is associated with other infectious agents in many countries; however, it has not been described yet in Brazil. The first report of pandemic H1N1 influenza A virus in Brazilian swine herd occurred in 2011. The main aim of this study was to characterize histological features in association with immunohistochemical (IHC) results for influenza A (IA), porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) from lung samples from 60 pigs with lesions suggestive of viral pneumonia and collected during the period 2009-2010 and diagnosed at the Setor de Patologia Veterinária of Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil. All the pigs in this study had clinical respiratory disease. Sample distribution was 31 (52%) from Rio Grande do Sul, 14 (23%) Paraná, 11 (18%) from Santa Catarina State and four (7%) from Mato Grosso do Sul State. Positive anti-IA IHC was observed in 45% of the cases and was associated with necrotizing bronchiolitis, atelectasia, purulent bronchopneumonia and hyperemia. Moreover, type II pneumocyte hyperplasia, alveolar and bronchiole polyp-like structures, BALT (bronchus-associated lymphoid tissue) hyperplasia and pleuritis were the significant features of negative samples by anti-IA IHC, which were associated with chronic lesions. Only two cases were positive to PCV2 and none to PRRSV, supports the hypothesis that SIV was the viral agent infecting swine’s lungs. Negative IHC to IA (55%) cases could be explained due to the absence of viral antigens associated with the rapid progress of SI; hence, IHC should be requested in the beginning of the infection. This work has shown how important a careful histological evaluation should be done in order to give the diagnosis. Since 2009, a new histological feature of swine pneumonia from animals with respiratory clinical sign has been observed at samples submitted to SPV-UFRGS. In addition, these results described here proved the importance of histological evaluation in swine herd health management.