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Human rhinoviruses (HRV) are extremely common human respiratory pathogens, most commonly associated with mild upper respiratory tract infections. The three known species of HRV (HRV-A, -B and –C) are members of the family Picornaviridae and genus Enterovirus. In contrast to the enterovirus (EV-A-D) species that commonly infect the gut, HRV are generally thought to be acid labile with replication restricted to the respiratory tract. Investigations of the clinical correlations of HRV infections detected on diagnostic screening of respiratory specimens demonstrated no specific association between HRV variant and clinical presentation. For example, similar species distributions were observed in patients admitted to the ITU and those discharged with minor illness. Unexpectedly, screening of stool specimens for HRV showed a prevalence of 10% with viral loads similar to EV infections. These findings suggested that a reappraisal of HRV tropism and disease associations may be warranted. HRV-A and -B isolates were originally classified into 100 serotypes by serological neutralisation properties. As HRV-C is difficult to isolate, no attempt had been previously made to classify the wealth of available HRV-C sequences. To facilitate definition of novel HRV types and classification of HRV-C, a system was devised to divide HRV sequences into genotypically defined types. Pairwise VP1 nucleotide p-distance analysis revealed distinct thresholds between inter- and intra- type divergence and available sequences were classified into 77 HRV-A, 29 -B and 51 -C types. This provides a standardised basis for type definition and identification, allowing consistency in studies of genetic diversity, epidemiology and evolution. It has been adopted by the ICTV Picornavirus Study Group for classification of HRV. Although the occurrence of recombination has been documented within the coding region of EV, analysis of dated HRV sequences revealed an overall lack of intra-species recombination between three coding regions of HRV-B and -C. In contrast, full HRV-A type groups appeared to have been subject to a large number of recombination events, suggesting extensive recombination during the period of its diversification into types. Putative recombination breakpoints localised to the non-structural region. Within HRV-A and HRV-B, recombination within the 5ˈUTR was infrequent. However, over 60% of analysed HRV-C strains grouped within the HRV-A clade and two recombination hotspots were identified. An additional interspecies recombination event was detected between HRV-A/C in the 2A coding region, with putative breakpoints mapping to the boundaries of the C-terminal domain of the proteinase. The studies within this thesis provide evidence for a broadened understanding of the clinical significance of HRV. In addition, the assignment of HRV sequences into genotypically defined types allowed description of the observed genetic diversity and completion of analysis which reaffirmed the sporadic nature of recombination within the coding region of HRV.

Proefschrift--Leiden, 2003. / Bibliogr. à la fin des chapitres.

Made available in DSpace on 2015-07-22T20:49:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-03-30. Added 1 bitstream(s) on 2015-08-11T03:26:02Z : No. of bitstreams: 1 Publico-12520a.pdf: 1207919 bytes, checksum: 1cbb5eb39525a466eede84c0b592aee7 (MD5). Added 1 bitstream(s) on 2015-08-11T03:26:02Z : No. of bitstreams: 2 Publico-12520a.pdf: 1207919 bytes, checksum: 1cbb5eb39525a466eede84c0b592aee7 (MD5) Publico-12520b.pdf: 1232945 bytes, checksum: dccb3be28a625e6fd226753518045ad3 (MD5) / Infecções causadas pelos rinovírus humanos (HRV) são responsáveis por 25-50% das doenças respiratórias entre indivíduos que apresentam doença semelhante à gripe (Influenza-Like Illness - ILI). Os HRVs podem ser classificados em pelo menos três espécies: HRV A, HRV B e HRV C. O HRV C tem sido frequentemente descrito entre crianças aparentemente levando a doenças mais graves e hospitalizações, no entanto a ocorrência dessa espécie entre adultos não é bem conhecida. O objetivo deste estudo foi avaliar a apresentação clínica e a distribuição das espécies de HRV que causam infecções em diferentes populações durante os anos de 2001 a 2005. Um total de 682 amostras foi coletado. As populações estudadas eram compostos por: 132 adultos da comunidade atendidos em pronto socorro e 198 adultos profissionais de saúde (2001-2003); 242 pacientes transplantados renais (2002-2004); 61 crianças portadoras de cardiopatia congênita (2005) e 49 idosos da cidade de Botucatu, São Paulo, Brasil (2003-2004). A amplificação dos genes do HRV foi realizada através da Reação em Cadeia da Polimerase precedida de Transcrição Reversa (RT-PCR), seguida de sequenciamenteo genético e análise filogenética. O HRV foi detectado em 24.05% das amostras (164/682), 15.2% (20/132) em adultos da comunidade, 29.8% (59/198) em profissionais da área da saúde, 23.6% (57/242) em transplantados renais, 22.9% (14/61) em crianças portadoras de cardiopatia congênita e 28.6% (14/49) em idosos. Um total de 85.5% (137/164) das amostras positivas foi seqüenciado, e 79.9% (131/164) foram analisadas através de filogenia. Foram identificadas 80 (61%) das amostras pertencentes à espécie A, 22 (16.8%) a espécie B e 29 (22.2%) pertencentes à espécie C. Foi encontrada uma alta taxa de ILI (38.9%) em pacientes infectados pelo HRV. A regressão logística mostrou um aumento de ILI de três vezes quando os indivíduos estavam infectados com HRV A em comparação ao HRV C. A infecção pelo HRV causa ILI em paciente adultos não hospitalizados. O HRV A foi associado à infecção mais intensa do que o resfriado comum. A dinâmica da infecção entre as diferentes espécies de HRV merece análise no futuro. / Infections caused by Human Rhinoviruses (HRVs) account for 25%-50% of respiratory illnesses among individuals presenting influenza-like illness (ILI). HRVs could be classified in at least three species: HRV-A, HRV-B, and HRV-C. The HRV-C species has frequently been described among children and apparently has led to severe illness resulting in hospitalization; however, the occurrence among adults is unknown. The aim of this study was to assess the clinical presentation and species distribution of HRV infections in different populations during 2001-2005. A total of 682 samples were collected. Subjects consisted of 132 adults from the general community and 198 health-care workers (2001-2003), 242 renaltransplanted outpatients (2002-2004), 61 children with congenital heart disease (2005) and 49 elderly persons from Botucatu city, Sao Paulo, Brazil (2003-2004). Amplification of HRV genes was performed by Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR) and followed by sequencing and phylogenetic analysis. HRV was detected in 24.05% of samples (164/682), 15.2% (20/132) among adults from general community, 29.8% (59/198) among health-care workers, 23.6% (57/242) among renal-transplanted outpatients, 22.9% (14/61) among children with congenital heart disease and 28.6% (14/49) among elderlies. A total of 85.5% (137/164) previously positive HRV samples were sequenced and 79.9% (131/164) were analyzed. We identified 80 isolates (61.0%) of the HRV A species, 22 (16.8%) of the HRV B species and 29 isolates (22.2%) of the HRV C species. High ILI rate (38.9%) was found among HRV infected patients. Logistic regression showed a three-fold increase in prevalence of ILI in individuals with HRV A infection compared with HRV C infected patients. HRV infections caused ILI among non-hospitalized patients. HRV specie A was associated with a disease more intense than a common cold. The dynamics of infection among different species deserve further analysis. / TEDE / BV UNIFESP: Teses e dissertações

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Rationale: Chronic obstructive pulmonary disease (COPD) is one of the leading causes of deaths worldwide. Patients with COPD have episodes of aggravated symptoms called exacerbations caused by pathogens or pollution. Respiratory viruses are associated with a significant number of COPD exacerbations with the most common virus being the rhinovirus (RV). The mechanisms by which RVs trigger COPD exacerbations are still unclear. Using human whole lung tissue explants (WTE), a novel model of RV-induced COPD exacerbations is proposed. Methods: WTE from COPD patients and smokers were initially stimulated with TLR ligands that are known to activate the same receptors as RV: poly(I:C) for TLR3 and R848 for TLR7/8 activation. Pro-inflammatory cytokines and type I and III IFN gene expression was measured by ELISA and qRT-PCR, respectively. A neutralising antibody against TNFα, a corticosteroid, and a panel of inhibitors targeting TLR pathway (p38 MAPK, IKK-2 and IRAK1/4) was applied to the tissue from COPD patients to establish which signalling pathways are responsible for the inflammatory response and IFN release. Explants from COPD patients and smokers was also exposed to two RV serotypes, RV-16 and RV-1B, in order to compare findings with a clinically relevant stimulant. Results: Poly(I:C) and R848 caused a significant increase of protein and gene expression of pro-inflammatory cytokines (TNFα, CCL5 and IL-6). Type I and III IFN gene expression was also significantly increased. Using the two ligands together caused a synergistic release of TNFα and CCL5. Tissue from COPD patients released more pro-inflammatory cytokines and expressed less IFNβ when compared to smokers. TNFα neutralisation inhibited subsequent release of CCL5 and IL-6. Dexamethasone and p38 MAPK inhibitor decreased TLR3- and TLR7/8-induced pro-inflammatory response whereas IKK-2 and IRAK1/4 inhibition had little effect on cytokine release. Dexamethasone and IKK-2 showed limited effect on IFN gene expression whereas p38 MAPK inhibitor significantly decreased and IRAK1/4 inhibition enhanced IFN expression. RV-16 induced modest but significant pro-inflammatory response in lung tissue, whereas RV-1B did not induce cytokine release. Both serotypes induced type I and III IFN gene expression. Tissue from COPD patients showed a lower expression of IFNβ and IFNλ when compared to smokers. Conclusion: This tissue explant was responsive to both synthetic TLR ligands and RV. The release of pro-inflammatory cytokines in response to TLR stimulation was partially inhibited by steroid. p38 MAPK is involved in TLR-induced inflammation but it also further decreases the already impaired IFN gene expression in COPD tissue. The role of IKK-2 and IRAK1/4 in TLR-induced innate immune response remains unclear. This model is a valuable system to study the mechanisms underlying RV-induced COPD exacerbations and also to test new inhibitors in the whole tissue environment.

Human Rhinoviruses (HRVs) utilize Internal Ribosome Entry Sites (IRES) to drive viral protein synthesis. IRESs are specialized RNA elements present within the 5’ UTR of mRNAs that recruit ribosomes independently of the 5’ m7G cap structure. hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), a multifunctional RNA binding protein, is required for the IRES-dependent translation of many specific RNAs within the cell cytoplasm. The phosphorylation of hnRNP A1 is required for its cytoplasmic accumulation. I have identified and validated the role of HK2 in hnRNP A1 cellular localization by immunofluorescence microscopy, by analysis of HRV infection and by siRNA-based screening. These studies show that decreased HK2 protein levels lead to decreased cytoplasmic accumulation of hnRNPA1 during osmotic shock and HRV infection, to a decrease in HRV-infected cells and to decreased caspase activation in osmotically stressed and HRV-infected cells. Thus, HK2 may regulate hnRNP A1 cytoplasmic localization following HRV infection.

Cytotoxiques en lysant différents types de cellules et régulent la réponse immunitaire. Leur rôle dans l’asthme et ses exacerbations reste encore à identifier même si des modifications phénotypiques ont été observées chez des patients asthmatiques et qu’il a été récemment montré dans un modèle murin qu’elles n’intervenaient pas dans le développement de l’asthme allergique. L’objectif de la thèse était de mieux comprendre la place des cellules NK dans la pathologie asthmatique en se focalisant sur deux aspects : l’exacerbation viro-induite et l’inhibition par des composants microbiens.L’hypothèse pour la 1ère partie de la thèse était que les cellules NK de patients asthmatiques pouvaient présenter une dysfonction dans leur réponse à des agents microbiens qui pourrait favoriser l’exacerbation de la réaction asthmatique. Pour cela, nous avons analysé l’activation, la cytotoxicité et la production de cytokines de cellules NK provenant de patients asthmatiques sévères stimulées avec des molécules mimant des microorganismes ou un rhinovirus vivant (HRV), en comparaison avec des donneurs sains. Nous avons montré que les cellules NK de patients sévères étaient moins cytotoxiques que les cellules NK de donneurs sains en réponse à la stimulation avec un agoniste de Toll-Like Receptor 3 ou du TLR7/8 et avec HRV. En outre, lorsqu’elles sont stimulées avec de l’IL-12 et de l’IL-15, des cytokines produites pendant l’infection virale, les cellules NK de patients asthmatiques sévères expriment moins d’IFN-γ que les cellules NK de donneurs sains. Nos résultats suggèrent que l’activation des cellules NK de patients asthmatiques pourrait être insuffisante pendant les infections respiratoires et pourraient participer à l’aggravation de l’asthme.L’hypothèse pour la 2ème partie de la thèse était que les cellules NK pourraient participer à l’inhibition de la réaction asthmatique allergique dans un modèle murin. Dans des souris C57BL/6 sensibilisées à l’ovalbumine, l’instillation de FSL1, un agoniste de TLR2/6 inhibe la réaction asthmatique allergique. Cette inhibition étant associée à des modifications de la population des cellules NK, nous avons analysé leur rôle grâce à des souris déficientes en cellules NK. En l’absence de cellules NK, les souris développent un asthme allergique, et l’inhibition par FSL1 est maintenue. Par conséquent, les cellules NK ne jouent pas de rôle dans le développement de l’asthme allergique expérimental, ni dans son inhibition induite par un agent microbien. Cependant, elles pourraient être modifiées par l’environnement allergique, et avoir ainsi un rôle dans les exacerbation viro-induites. Cette question cruciale rejoint le travail réalisé dans la première partie de la thèse.En conclusion, nos résultats suggèrent que les fonctions des cellules NK seraient modifiées dans la pathologie asthmatique, qu’elle soit allergique ou non. Notre hypothèse est que le défaut d’activation des cellules NK participerait aux exacerbations viro-induites de l’asthme. Les perspectives de ces travaux sont de poursuivre la caractérisation des cellules NK chez les patients asthmatiques sévères et d’évaluer le rôle des cellules NK dans un modèle murin d’exacerbation de la réaction asthmatique.L’hypothèse pour la première partie de la thèse était que les cellules NK de patients asthmatiques pouvaient présenter une dysfonction dans leur réponse à des agents microbiens qui pourrait favoriser l’exacerbation de la réaction asthmatique. Pour cela, nous avons analysé l’activation, la cytotoxicité et la production de cytokines de cellules NK provenant de patients asthmatiques sévères stimulées avec des molécules mimant des microorganismes ou un rhinovirus vivant (HRV), en comparaison avec des donneurs sains. Nous avons montré que les cellules NK de patients sévères étaient moins cytotoxiques que les cellules NK de donneurs sains en réponse à la stimulation avec un agoniste de Toll-Like Receptor 3 ou du TLR7/8 et avec HRV [...] / Asthma is a chronic inflammatory disease of the airways affecting 334 million of people worldwide. Among asthma patients, 5% suffers from severe asthma. Severe asthma represents a major unmet need because, despite heavy treatments, patients still suffer from uncontrolled asthma symptoms, frequent exacerbations and a dramatic decrease in their respiratory capacity. The role of microorganisms in asthma is complex. On one hand, a group of epidemiologic and experimental studies have shown that chronic exposure with bacteria or microbial compounds, particularly during early childhood, would provide protection against allergic asthma. On the other hand, respiratory viruses are responsible for 80% of exacerbations and are associated with an increasing risk of developing asthma in children, whether allergic or not. Natural Killer cells (NK) are lymphocytes involved in innate antiviral immunity. They have cytotoxic functions by lysing different types of cells but also regulatory functions by producing cytokines and activating other immune cells. Their role in asthma and its exacerbations has yet to be identified, although phenotypic changes have been observed in asthmatic patients and it has recently been shown in a mouse model that they are not involved in the development of allergic asthma. The objective of the thesis was to better understand the role of NK cells in asthmatic pathology by focusing on two aspects : virus-induced exacerbation and inhibition by microbial compounds.The hypothesis for the first part was that NK cells from asthma patients may present a dysfunction in their response to microbial agents that could promote the exacerbation of the asthmatic reaction. To do this, we analysed NK cell activation, cytotoxicity and production of cytokines from severe asthmatic patients stimulated with molecules mimicking microbes or a human rhinovirus (HRV), compared to healthy donors. We showed that NK cells from severe asthma patients were less cytotoxic than NK cells from healthy donors in response to stimulation with a Toll-like Receptor 3 or TLR7/8 agonist and HRV. Moreover, when stimulated with IL-12 and IL-15, cytokines produced during viral infections, NK cells from severe asthma patient express less IFN-γ than NK cells from healthy donors. Our results suggest that the activation of NK cells in asthma patients may be insufficient during respiratory infections and may contribute to the worsening of asthma.The hypothesis for the second part was that NK cells may participate to the inhibition of a mouse model of allergic asthma. In C57BL/6 mice sensitized with ovalbumin, instillation of FSL1, agonist of TLR2/6, inhibits the features of experimental asthma. Since this inhibition is associated with changes in the population of NK cells, we analysed their role using mice deficient in NK cells. In the absence of NK cells, mice develop allergic asthma, and inhibition by FSL1 is maintained. Therefore, NK cells do not play a role in the development of experimental allergic asthma or in its inhibition induced by a microbial agent. However, they may be modified by the allergic environment, and thus have a role in viro-induced exacerbations. This crucial question is in line with the work done in the first part of the thesis.In conclusion, our results suggest that the functions of NK cells may be modified in asthmatic pathology, whether allergic or not. Our hypothesis is that the defect in NK cell activation may participate to virus-induced asthma exacerbations. Perspectives of this work are to further characterize NK cells in severe asthma patients and to evaluate the role of NK cells in a mouse model of asthma exacerbation.