Dans cette étude, la bile d’un porc canadien naturellement infecté par une souche du virus de l’hépatite E (VHE) a été utilisée afin d’inoculer deux groupes de porcelets. Dans l’étude précoce (E), 4 porcelets âgés de 4 semaines et exempts de pathogènes spécifiques (SPF), ont été suivis jusqu’à 14 jours post-inoculation (pi). Dans l’étude tardive (L), 9 porcelets ont été suivis à chaque semaine jusqu’à l’abattage, soit 120 jours pi. À la nécropsie, la présence du VHE a été évaluée dans différents organes à 7, 14 et 120 jours pi. Des porcelets témoins (E=2 et L=3) ont été inoculés par de la bile exempte de VHE. Le virus a persisté chez certains animaux jusqu’à 84 à 105 jours pi dans le sérum malgré la présence d’anticorps IgG anti-VHE dans le sang, suggérant une virémie prolongée. L’excrétion virale dans les fèces s’est étalée également sur une période de 105 jours pi chez certains animaux. De plus, la détection de l’ARN viral dans les organes évalués s’est révélée presque nulle à l’âge d’abattage à l’exception de quelques vésicules biliaires, alors qu’on retrouvait l’ARN viral dans plusieurs organes à 7 et 14 jours pi. Pour évaluer la distribution du VHE chez les porcs commerciaux du Québec, un échantillonnage de porcs de trois abattoirs a été réalisé. Environ 100 échantillons de sang, fèces, foies et bile provenant des mêmes animaux en processus d’abattage ont été prélevés dans chacun des abattoirs, sur des porcs destinés à la consommation humaine. La détection de l’ARN viral et des anticorps du VHE a été réalisée à l’aide d’une RT-PCR nichée et d’un test ELISA adapté pour déceler les anticorps porcins anti-VHE. Chez les porcs d’abattoir, 12,9 % des échantillons de bile contenaient de l’ARN viral du VHE, alors que la détection virale était moindre dans les autres organes. Une séroprévalence en IgG de 26,0 % a été obtenue pour les sérums porcins analysés. Une analyse phylogénétique des différentes souches isolées pendant l’étude a démontré qu’elles sont du génotype 3. Ces données indiquent une exposition potentielle des travailleurs de l’industrie porcine au VHE porcin, notamment par les fèces, le sang et les organes et également pour les consommateurs par le biais des foies. / In this study, a strain of porcine hepatitis E virus (HEV) isolated from the bile of a naturally-infected Canadian pig was used to inoculate two groups of piglets. In the early-phase experiment (E), 4 one month-old piglets, specific pathogen free (SPF), were monitored for 14 days. In the late-phase experiment (L) 9 piglets were monitored up to slaughter (120 days post-inoculation (pi)). Controls piglets (E=2 and L=3) were inoculated with free HEV bile. The presence of HEV was monitored routinely in their blood and feces. At necropsy, viral occurence was evaluated in organs at 7, 14 and 120 days pi. Interestingly, HEV was found to persist in the serum of some animals up to 84-105 days pi, despite the presence of IgG HEV antibodies in their blood. Fecal shedding was detected until 105 days pi for a portion of pigs. In organs, HEV RNA was detected in low amount of gallbladders at killing time, while it was detected in a large number of organs at 7 and 14 days pi. To assess the distribution of HEV in commercial finishing pig in Quebec, a sampling was realised in pigs from three slaughterhouses from Quebec. Approximately a hundred samples of feces, blood, bile and liver were collected in each slaughterhouse, on pigs intended for human consumption. A sample of each type was collected on each of the chosen pigs. Detection of HEV RNA was carried out using a nested RT-PCR on each sample and a human ELISA test was adapted for the detection of swine antibodies against HEV in swine serum samples. For pigs at slaughter, 12,9 % of the bile samples were positive to HEV RNA and a seroprevalence of IgG of 26,0 % was detected in swine. A phylogenetic analysis demonstrated that all strains of the study were in the genotype 3. All results demonstrate that porcine industry workers are potentially exposed to swine HEV by feces, blood and organs.

Inflammatory bowel disease (IBD), comprises Crohn’s disease (CD) and ulcerative colitis (UC), and is a chronic relapsing inflammation of gastrointestinal tract without any known cause or cure. Currently, it is accepted that IBD is a result of a dysfunctional immune response to commensal bacteria in a genetically susceptible host, and that environmental factors can trigger the onset or reactivation of the disease. This thesis considers the possibility of a specific pathogenic agent as well as an imbalance in the composition of the normal microflora in the pathogenesis of IBD. Gut biopsy tissues were taken from a population-based case-control tissue bank held at the University of Manitoba. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphisms (T-RFLP) were employed to assess the diversity of gut microbiota. The phylogenetic, virulence and biochemical characteristics of Escherichia coli isolated from IBD biopsies were examined using multi-locus sequence typing (MLST), DNA microarray technology and API 20E system. Utilizing ARISA and T-RFLP, a remarkable increase in the order of unclassified Clostridia was detected in inflamed tissues, particularly in CD patients (P < 0.05). Moreover, species richness and diversity were the highest in non-inflamed IBD biopsies. Culture-based quantification detected a significantly higher number of E. coli in IBD tissues (P < 0.05). Phylogenetic analysis revealed the tendency of E. coli isolated from IBD patients to be grouped into separate clonal clusters based on their allelic profiles (P = 0.02). A link was detected between uropathogenic E. coli (UPEC) CFT073 and strains isolated from IBD, with regards to gene distribution and virulence, using microarray technology. Amino acid substitutions N91S and S99N in FimH, the adhesive subunit of E. coli type I fimbria, were significantly associated to IBD (P < 0.05). This study demonstrated an increase in the microbial diversity of non-inflamed IBD tissues and suggested a recruitment phase of bacterial adherence and colonization, before the inflammation sets in. Furthermore, E. coli isolated from IBD tissues were distinct from commensal strains in both clonal and virulence characteristics and shared remarkable traits with extraintestinal pathogenic E. coli. Features involved in bacterial adhesion to epithelial cells may hold the key to E. coli pathogenesis in IBD.

Inflammatory bowel disease (IBD), comprises Crohn’s disease (CD) and ulcerative colitis (UC), and is a chronic relapsing inflammation of gastrointestinal tract without any known cause or cure. Currently, it is accepted that IBD is a result of a dysfunctional immune response to commensal bacteria in a genetically susceptible host, and that environmental factors can trigger the onset or reactivation of the disease. This thesis considers the possibility of a specific pathogenic agent as well as an imbalance in the composition of the normal microflora in the pathogenesis of IBD. Gut biopsy tissues were taken from a population-based case-control tissue bank held at the University of Manitoba. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphisms (T-RFLP) were employed to assess the diversity of gut microbiota. The phylogenetic, virulence and biochemical characteristics of Escherichia coli isolated from IBD biopsies were examined using multi-locus sequence typing (MLST), DNA microarray technology and API 20E system. Utilizing ARISA and T-RFLP, a remarkable increase in the order of unclassified Clostridia was detected in inflamed tissues, particularly in CD patients (P < 0.05). Moreover, species richness and diversity were the highest in non-inflamed IBD biopsies. Culture-based quantification detected a significantly higher number of E. coli in IBD tissues (P < 0.05). Phylogenetic analysis revealed the tendency of E. coli isolated from IBD patients to be grouped into separate clonal clusters based on their allelic profiles (P = 0.02). A link was detected between uropathogenic E. coli (UPEC) CFT073 and strains isolated from IBD, with regards to gene distribution and virulence, using microarray technology. Amino acid substitutions N91S and S99N in FimH, the adhesive subunit of E. coli type I fimbria, were significantly associated to IBD (P < 0.05). This study demonstrated an increase in the microbial diversity of non-inflamed IBD tissues and suggested a recruitment phase of bacterial adherence and colonization, before the inflammation sets in. Furthermore, E. coli isolated from IBD tissues were distinct from commensal strains in both clonal and virulence characteristics and shared remarkable traits with extraintestinal pathogenic E. coli. Features involved in bacterial adhesion to epithelial cells may hold the key to E. coli pathogenesis in IBD.

Abstract Feline immunodeficiency virus (FIV) is an important infectious agent of domestic cats worldwide. It has been classified into the Lentivirus genus of the Retroviridae family, together with human immunodeficiency virus (HIV). Five FIV subtypes (A, B, C, D and E) have been described based on sequence variation of the V3-V5 region of the envelope (env) gene. There is considerable sequence diversity within and between subtypes, which has been a major obstacle in the development of a successful vaccine. However, an FIV vaccine that incorporates inactivated whole viruses from subtypes A and D is now commercially available. Although the vaccine has been shown to be efficacious in protecting against challenge with homologous and a heterologous (subtype B) subtypes, its effectiveness against other viral variants is unknown. Therefore, identifying the type and diversity of FIV strains in different regions is important to establish the potential efficacy of the vaccine in areas where vaccination is to be implemented. The proviral DNA sequence of the V3-V5 region of the env gene was determined for 102 FIV-infected cats from locations in Australia, New Zealand and South Africa. Subtype A was the predominant subtype in Australia and South Africa, although subtype B and C were also identified in each of these countries, respectively. Both subtypes A and C were also present in New Zealand. Of interest, there were some samples in New Zealand and South Africa that demonstrated subtype assignment discrepancies when different regions of the genome were analysed, suggesting co-infection and/or recombination. Cats infected with FIV exhibit varying degrees of immunological impairment. Currently, prognosis for an FIV-infected cat is based on clinical signs alone, which is a relatively subjective measure. In HIV-infected patients it is recognised that viral RNA load correlates with disease stage and prognosis. This PhD research tested whether viral RNA load may be a useful prognostic marker in FIV infection. A real-time PCR assay was developed to quantify plasma viral RNA load in 42 FIV-infected cats at three different clinical stages (1:healthy, 2:unwell without signs of immunodeficiency, 3:unwell with signs of immunodeficiency). In cats older than 5 years of age, log-transformed viral RNA loads were significantly higher in cats in category 3 compared to cats in category 1. There were no significant differences in the viral RNA load of older cats in category 2 compared to category 1. There were no cats younger than 5 years of age in category 3 and there was no significant difference in viral RNA load between young cats in categories 1 and 2. Of the 15 cats for which follow-up data was available, eight showed no change in clinical signs, and seven showed a worsening of clinical signs with six of these showing a progression of clinical category including death. One of the cats in category 2 that progressed clinically had one of the highest viral RNA loads of cats in that category. Three of four cats from category 3 that were followed had either died or been euthanised. Two of these cats had among the highest viral RNA loads in the whole study, while the remaining cat (for which the definitive cause of death was not confirmed) had a relatively low viral RNA load. In summary, measurement of viral RNA load was found to be a potentially useful clinical and prognostic marker but further work is required to better assess its usefulness to veterinarians. Serum acute phase proteins were investigated as possible candidate markers of FIV disease with the aim of developing a more simplified assay that could be used as a prognostic marker for FIV infection. Blood samples from 43 FIV-infected and 25 FIV-negative cats were assayed for the concentration of four acute phase proteins. Both healthy and sick cats were included in the study. Compared to healthy cats, sick cats had significantly higher concentrations of serum amyloid A (P<0.05). Alpha 1-acid glycoprotein and haptoglobin were also found to be in higher concentrations in sick cats (P<0.1). Other variables such as age and gender were also associated with acute phase protein concentrations. With respect to FIV infection, it was found that in sick cats, serum amyloid A, in combination with the age of the cat, was the best predictor of FIV viral RNA load. Alpha 1-acid glycoprotein and haptoglobin were not significantly associated with FIV viral RNA load. Although health status did not influence albumin levels, they were found to be significantly lower in FIV-positive cats in comparison to FIV-negative cats (P<0.05). The frequent monitoring of viral RNA loads and CD4+ lymphocyte counts that is performed on HIV-infected patients is cost prohibitive in veterinary patients. This study showed that there is potential for the use of acute phase protein concentrations (in particular serum amyloid A) as alternative prognostic tools in FIV-infected cats. Further work, particularly longitudinal studies, is required to more definitively define changes in viral RNA load and acute phase protein concentrations throughout the course of FIV infection.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.

Made available in DSpace on 2014-06-11T19:32:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-12Bitstream added on 2014-06-13T19:03:33Z : No. of bitstreams: 1 dias_fc_dr_jabo.pdf: 950154 bytes, checksum: 170fddcca2fc4c7004161e93f093a97f (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A ocorrência de anticorpos neutralizantes contra os genótipos do vírus da diarréia viral bovina (BVDV 1 e BVDV 2) foi verificada, pelo teste de virusneutralização (VN), em 260 amostras de soro sangüíneo de 26 rebanhos não vacinados contra o BVDV, provenientes dos Estados de Minas Gerais e São Paulo. Do total de amostras, 102 (39,2%) reagiram ao BVDV, das quais 81 (31,1 %) foram reagentes ao BVDV 1 e ao BVDV 2, sete (2,7%) reagiram apenas ao BVDV 1 e 14 (5,4%) reagiram apenas ao BVDV 2. Nos mesmos rebanhos, verificou-se também a presença sugestiva de animais persistentemente infectados (PI) por meio da pesquisa de anticorpos neutralizantes em cinco amostras de soro sangüíneo de bezerros sentinelas com idade entre 6 e 12 meses. A ocorrência de animais PI foi pesquisada em três dos 26 rebanhos, dos quais foram colhidas amostras pareadas de sangue de todos os animais do rebanho. Todas as amostras foram submetidas ao teste de VN contra o BVDV 1 e o BVDV 2, e naquelas não reagentes a pelo menos um dos genótipos, bem como nas amostras provenientes de bovinos com menos de seis meses de idade, foi realizada a pesquisa do vírus pela RT-PCR. Em um dos rebanhos foram detectados dois animais PI, cujas estirpes foram caracterizadas geneticamente como pertencentes ao subgenótipo eVOV 1 b. A infecção natural pelo BVDV foi monitorada nos três rebanhos selecionados para a pesquisa de animais PI, sendo que em dois deles constatou-se a eliminação do BVDV. No entanto, a infecção permaneceu no rebanho em que haviam sido detectados dois animais PI, pois foram diagnosticados posteriormente um animal transitoriamente infectado (TI) e novos animais reagentes ao vírus. Foram submetidos à análise estatística os resultados dos testes de VN contra o BVDV 1 e o BVDV 2, realizados em 1925 amostras de soro sangüíneo obtidas no período de execução... / The occurrence of neutralizing antibodies against to bovine viral diarrhoea virus genotypes (BVDV 1 and BVDV 2) has been confirmed by virusneutralization test (VN) in 260 samples of blood serum undertaken in the states of Minas Gerais and São Paulo, Brazil. Samples were retrieved from 26 cattle herds which were not BVDV vaccinated. One hundred and two samples (39.2%) were reagents to BVDV, or rather, 81 (31.1%) were reagents to BVDV 1 and BVDV 2, seven (2.7%) were reagents to BVDV 1 only and 14 (5.4%) were reagents to BVDV 2 only. In the same herds, the significant presence of persistently infected (PI) animais was also verified by a research involving neutralizing antibodies in five samples of blood serum in 6 to 12¬month-old calves. The occurrence of PI animais was researched in three out of 26 herds by paired blood samples of total animais in the herdo Ali samples were analyzed by VN test against to BVDV 1 and BVDV 2, and virus research was undertaken by RT-PCR in samples which were not reagent to at least one of the genotypes and in samples of less than 6-month-old cattle. Two PI animais, genetically characterized as belonging to BVDV 1 b subgenotype, were detected in one of the herds. BVDV natural infection was monitored in three herds selected for PI animais research. BVDV was eliminated in two herds. However, infection remained in the herd in which two PI animais were diagnosed, because a transiently infected animal (TI) and other virus-reagent animais were detected later on. Statistical analysis evaluated VN test results against BVDV 1 and BVDV 2 with 1925 samples of blood serum obtained during the research period. Although no significant ditterences were found between BVDV 1 reagent bovines and BVDV 2 reagent ones (p>0.05), a significant ditterence was detected among titles of antibodies from samples which were reagent to both genotypes (p<0.0001).

Green algae are quite important primary producers in fresh waters. The genus Chlorella represents one of algae most frequently utilized in algal biotechnologies to produce biomass, using either autotrophic or heterotrophic cultivation systems. It is than exploited as a food supplement for humans or animals. However, particular species within the genus are morphologically indistinguishable and molecular markers should be used to characterize production strains. This work is aimed to molecularly characterize three production strains of Chlorella for patent protection purposes and to specify their phylogenetic and taxonomic position.

Habenaria é um dos maiores gêneros da família Orchidaceae, e estimativas atuais pressupoem a existência de aproximadamente 835 espécies. Habenaria seção Pentadactylae com 34 espécies é a maior entre as 14 seções do gênero existente no novo mundo e compreende um conjunto de espécies morfologicamente bastante heterogênea. A fim de investigar a monofilia da seção e sua relação com outras seções do gênero, foram executadas análise Bayesiana e de Máxima Parcimônia com o emprego de um marcador nuclear (ITS) e três marcadores plastidiais (matK, intron trnK, rps16-trnk). Os resultados demonstraram que a seção Pentadactylae é altamente polifilética. Baseado nas análises filogenéticas e reavaliação de caracteres morfológicos, a seção Pentadactylae foi recircunscrita neste trabalho e sete espécies são aceitas: H. dutraei, H. ekmaniana, H. exaltata, H. henscheniana, H. megapotamensis, H. montevidensis e H. pentadactyla, enquanto outras 32 espécies foram excluídas. Habenaria crassipes é reconhecida como um sinônimo de H. exaltata. Lectótipos são designados para H. crassipes e H. recta. Todas as espécies da seção habitam pântanos ou locais bastante úmidos; com área de distribuição passando pelo norte da Argentina, Uruguai, Paraguai, sul, sudeste e centro do Brasil. O estado do Rio Grande do Sul (sul do Brasil), possivelmente, constitui um centro de diversidade da seção onde todas as espécies podem ser encontradas. A biologia reprodutiva de duas espécies da seção Pentadactylae, H. megapotamensis e H. montevidensis; e duas espécies da seção Macroceratitae, H. johannensis e H. macronectar, foram estudas. Todas as espécies estudadas oferecem néctar como recompensa floral aos polinizadores, produzido no interior de um prolongamento do labelo denominado esporão. Habenaria montevidensis é polinizada por borboletas da família Hesperiidae, enquanto as demais espécies são polinizadas por mariposas da família Sphingidae. Todas as espécies estudadas são auto-compatíveis mas dependentes de agentes polinizadores para a produção de frutos. O sucesso reprodutivo é alto (69,48 - 93%). Na área de estudo, todas as quatro espécies estudadas são reprodutivamente isoladas devido a um conjunto de fatores tais como diferenças na morfologia floral e diferentes polinizadores. / Habenaria is one of the largest genus of Orchidaceae family and current stimates accounts to the existence of 835 species. Habenaria section Pentadactylae with 34 species is the largest among the 14 New World sections of the genus and comprises a morphologically heterogeneous group of species. To investigate the monophyly of the section and the relation with other sections of the genus, Bayesian and parsimony analyses using one nuclear marker (ITS) and three plastid markers (matK, trnK intron, rps16-trnK) were performed. The results demonstrated that sect. Pentadactylae is highly polyphyletic. Based on the phylogenetic analyses and re-evaluation of morphological characters, Habenaria sect. Pentadactylae is re-circumscribed and seven species are accepted for the section: H. dutraei, H. ekmaniana, H. exaltata, H. henscheniana, H. megapotamensis, H. montevidensis and H. pentadactyla, while other 32 species were excluded. Habenaria crassipes is included under the synonym of H. exaltata. Lectotypes are designated for H. crassipes and H. recta. All species in the section are from marshes or wet grasslands and range from Northern Argentina, Uruguai, Paraguai and south, southeast and center of Brazil. The Rio Grande do Sul state (south Brazil), possibly constitute a diversity center of the section where every species can be founded. Most are rare, known by few populations, and threatened due to loss of habitat and population decline. The reproductive biology of two species from the section Pentadactylae, H. megapotamensis and H. montevidensis; and two species from the section Macroceratitae, H. johannensis and H. macronectar, were studied. All studied species offer nectar as floral reward concealed in a labellar process termed spur. Habenaria montevidensis is pollinated by Hesperiidae butterflies, while the remaining species are pollinated by Sphingidae moths. All studied species are self-compatible, but pollinator-dependent. The reproductive success is high (69.48 - 93%). At the study site, every four studied species are reproductively isolated by a set of factors that includes differing floral morphologies and different pollinators.

Submitted by Valquíria Barbieri (kikibarbi@hotmail.com) on 2018-04-18T20:33:59Z No. of bitstreams: 1 DISS_2015_Belgath Fernandes Cardoso.pdf: 3865065 bytes, checksum: 3b7d79b4224429f58cb6ca5c3d96d7e9 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2018-04-27T17:29:40Z (GMT) No. of bitstreams: 1 DISS_2015_Belgath Fernandes Cardoso.pdf: 3865065 bytes, checksum: 3b7d79b4224429f58cb6ca5c3d96d7e9 (MD5) / Made available in DSpace on 2018-04-27T17:29:40Z (GMT). No. of bitstreams: 1 DISS_2015_Belgath Fernandes Cardoso.pdf: 3865065 bytes, checksum: 3b7d79b4224429f58cb6ca5c3d96d7e9 (MD5) Previous issue date: 2015-03-26 / CAPES / O gênero Orthobunyavirus, família Bunyaviridae, alberga arbovírus de importância médica. Estes estão envolvidos em epidemias de doença febril em áreas tropicais. No Brasil, o vírus Oropouche (OROV) é considerado o arbovírus mais frequente após o vírus da dengue (DENV). O objetivo deste estudo foi investigar a circulação de orthobunyavírus em Mato Grosso (MT). 529 amostras de soro obtidas entre outubro de 2011 e julho de 2012 de pacientes com doença febril aguda suspeita de dengue com até cinco dias do início dos sintomas em MT e 387 pools de mosquitos Cx quinquefasciatus capturados entre janeiro e abril de 2013 foram submetidos à nested RT-PCR para o segmento S de orthobunyavírus pertencentes ao sorogrupo Simbu. Amostras positivas foram testadas em pelo menos duas reações independentes e submetidas a sequenciamento nucleotídico para análise filogenética. Inoculou-se as amostras positivas em células vero. Dentre os 529 pacientes, 5 (0,94%) foram positivos para orthobunyavirus do sorogrupo Simbu por nested RT-PCR e isolamento viral. O vírus foi isolado de 3/8 pools. O segmento S do OROV foi identificado em cinco pacientes, quatro do sexo feminino, com 14-62 anos. Dois pacientes encontravam-se co-infectados com DENV-4. Estes pacientes são oriundos das cidades de Cuiabá (n=3), Várzea Grande (n=1) e Nova Mutum (n=1), todos residentes em área urbana, que apresentavam febre, cefaleia, dor retroorbital, mialgia, artralgia, prostação e náuseas. 8/387 pools de Cx. quinquefasciatus foram positivos para o segmento S do OROV por nested-RT-PCR, com taxa mínima de infecção (MIR) de 2,3 por 1000 mosquitos. As sequências obtidas do segmento S apresentaram 98% a 100% de homologia com o mesmo segmento das cepas do OROV verificadas no GenBank. A análise filogenética indica que as amostras de humanos e mosquitos pertencem ao subgenótipo Ia, similares a cepas do Pará obtidas de humanos, preguiças, Aedes (Ochlerotatus) serratus e Cx. quinquefasciatus. O genótipo I é o mais conservado e frequente no Brasil dentre os genótipos do OROV. Cx. quinquefasciatus, culicídeo de maior abundância em Cuiabá, é considerado vetor secundário do OROV em área urbana. Culicoides paraensis, principal vetor do OROV em áreas urbanas na região amazônica, não foi capturado neste estudo. Sorologia para o OROV foi identificada em residentes de cidades do Pará afetadas pela rodovia Cuiabá-Santarém e em primatas do Pantanal Sul-matogrossense, corroborando com a identificação do OROV em cidades do MT geograficamente interligadas por esta mesma rodovia. Infecções esporádicas por um orthobunyavirus do sorogrupo Simbu, possivelmente o OROV, foram identificadas em pacientes de MT, além de oito pools de Cx. quinquefasciatus em Cuiabá, indicando que este mosquito possui capacidade vetorial e pode estar envolvido no ciclo urbano de transmissão do vírus no estado. / The genus Orthobunyavirus, family Bunyaviridae, contains medically importante arboviruses. These are involved in epidemics of febrile illness in tropical areas. In Brasil, Oropouche virus (OROV) is considered the most frequent arbovirus after dengue virus (DENV). The aim of this study was to investigate the circulation of orthobunyaviruses in Mato Grosso (MT). 529 serum samples collected between October, 2011 and July, 2012 of patients with acute febrile illness suspected of dengue for up to five days of symptom onset from MT and 387 pools of Cx. quinquefasciatus mosquitoes captured between January and April, 2013, were subjected to nested RT-PCR for the segment S of orthobunyaviruses belonging to Simbu serogroup. Positive samples were tested in at least two independent reactions and subjected to nucleotide sequencing for phylogenetic analysis. Positive samples were inoculated in vero cells. Among the 529 patients, five (0.94%) were positive for serogroup Simbu by nested RT-PCR and viral isolation. The virus was isolated from 3/8 pools. The OROV segment S was identified in five patients, four females, with 14-62 years-old. Two patients were co-infected with DENV-4. These patients are from Cuiabá (n=3), Várzea Grande (n=1) and Nova Mutum (n=1), all residents in urban areas, presenting fever, headache, retroorbital pain, myalgia, arthralgia, prostration and nausea. 8/387 pools of Cx. quinquefasciatus were positive for OROV segment S by nested-RT-PCR with a minimum infection rate (MIR) of 2.3 per 1,000 mosquitoes. The segment S nucleotide sequences presented 98% to 100% of homology with the same segment of OROV strains available in GenBank. Phylogenetic analysis indicate the human and mosquito samples belong to genotype Ia, similar to strains obtained in Pará from humans, sloths, Aedes (Ochlerotatus) serratus and Cx. quinquefasciatus. The genotype I is the most conserved among OROV genotypes. Cx. quinquefasciatus, the most abundant culicidae in Cuiabá, is considered a secondary vector for OROV in urban areas. Culicoides paraensis, the main vector for OROV in urban areas in the Amazon region, was not captured in the study. Serology for OROV was identified in humans in cities of Pará affected by the Cuiabá-Santarém highway and in primates in South Pantanal, corroborating for the findings in cities of MT geographically linked by the same highway. Sporadic infections by an orthobunyavirus from Simbu serogroup, possibly OROV, were identified in patients from MT, also eight Cx. quinquefasciatus pools from Cuiabá, indicating that has vectorial capacity and may be involved in the urban cycle of virus transmission in the state.