Vírus da raiva provenientes de 23 morcegos de espécies hematófagas, frugívoras e insetívoras foram caracterizados geneticamente pelo seqüenciamento completo da região que codifica a nucleoproteína N. A análise filogenética das seqüências, incluindo lyssavirus e isolados de morcegos do Chile e Estados Unidos, mostrou que os diferentes isolados do vírus da raiva foram de modo geral segregados em quatro grupos genéticos distintas: morcegos hematófagos, morcegos insetívoros 1, 2 e 3. Os morcegos insetívoros 1 constituiram-se por isolados de Eptesicus furinalis: BR-EF1, BR-EF2, BREF3, BR-EF-4, BR-EA1 e BR-NL2; os morcegos insetívoros 2 consistiram de isolados de Molosssus spp: BR-MM1, BR-MM2 e BR- MA1 e os morcegos insetívoros 3 isolados de Nictinomops laticaudatus: BR-NL1 e BR-NL3. A homologia de nucleotídeos entre cada grupo de morcegos insetívoros 1, 2 e 3 foi maior que 99%, 97% e 99%, respectivamente. O grupo de morcegos hematófagos foi representado pelos isolados de: 3 morcegos hematófagos Desmodus rotundus (BR-DR1, BR-DR2 e BR-DR3); 5 morcegos frugívoros Artibeus lituratus BR-AL1, BR-AL2, BR-AL3, BR-AL4 e Artibeus planirostris BRAP1; 2 morcegos insetívoros (BR-MR1 e BR-EA2) e 2 de espécies não identificadas (BR-BAT1 e BR-BAT2). Entre as amostras seqüenciadas foram selecionadas cinco (BR-EF1, BR-NL1, BR-AL3, BR-MM1, BR-DR1) e um isolado de cão (BR-C) para os estudos de patogenicidade em camundongos albinos suíços inoculados pela vias intracerebral (IC) e intramuscular (IM). Todas as amostras quando inoculadas em camundongos pela via IC apresentaram-se patogênicas, provocando a morte dos mesmos num período de 4 a 14 dias pós-inoculação. No entanto, 500DLIC50 das mesmas amostras inoculadas pela via IM levaram a uma mortalidade de camundongos de: 60% (BR-DR1); 50% (BR-C, BR-NL); 40% (BR- AL3); 9,5% (BR-MM1); 5,2% (BR-EF10). As mesmas amostras foram utilizadas para a verificação de proteção cruzada, conferida por vacina comercial de uso animal, de camundongos que receberam uma ou duas doses de vacina pela via subcutânea (SC) e desafiados pelas vias IC e IM. Camundongos inoculados com duas doses de vacina foram protegidos quando desafiados pela via IC, com todas as amostras testadas. Quando os camundongos receberam uma dose da mesma vacina houve proteção parcial daqueles desafiados com as amostras de vírus PV e BR-C. Houve proteção de 100% dos camundongos desafiados pela via IM, com exceção daqueles vacinados com uma dose de vacina e desafiados com a amostra PV que apresentaram um índice de 66% de sobreviventes. Os resultados indicam a possibilidade de existir variantes do vírus da raiva espécies específicas circulando em morcegos. Sugerem ainda, que espécies de morcegos hematófagos, frugívoros e insetívoros compartilham o mesmo polimorfismo de vírus. A vacina comercial contra a raiva contendo vírus inativado e de uso veterinário protegeu os camundongos contra o desafio com as diferentes amostras testadas, sugerindo que as vacinas usualmente utilizadas são efetivas no tratamento profilático da raiva transmitida por morcegos, apesar da marcada diferença de neurovirulência dos diferentes isolados quando inoculados em camundongos pela via IM. / Twenty-three rabies viruses isolated from hematophagous, frugivorous and insectivorous bats were characterized genetically by complete sequencing of the region coding the nucleoprotein N. The phylogenetic analysis of the sequences, including the lyssavirus and the bat isolates from Chile and USA revealed that the isolates were segregated into four distinct genetic lineages: those related to the vampire bats and to the insectivorous bats 1, 2 and 3. The isolates related to the insectivorous bats 1 were from the Eptesicus furinalis: BR-EF1, BR- EF2, BREF3, BR-EF-4, BR-EA1 e BR-NL2; those of the insectivorous bats2 included the isolates from Molosssus spp: BR-MM1, BR-MM2 and BR-MA1 and the group 3, by the isolates from the Nictinomops laticaudatus: BR-NL1 and BR-NL3. The homology among each group of the insectivorous bats 1, 2 and 3 were greater than 99%, 97% and 99%, respectively. The lineage related to vampire bats was represented by three isolates from the D. rotundus (BR-DR1, BR-DR2 e BR-DR3); five from the fruit bats Artibeus lituratus (BR-AL1, BR-AL2, BR-AL3, BR-AL4) and Artibeus planirostris (BRAP1); two from insectivorous bats (BR-MR1 and BR-EA2) and two from unidentified species (BR-BAT1 and BR-BAT2). Among the sequenced amples, five bat isolates (BR-EF1, BR-NL1, BR-AL3, BR-MM1, BR- DR1) and one dog isolate (BR-C) were selected for the study of their pathogenicity in Swiss mice, inoculating through intracerebral (IC) and intramuscular (IM) routes. All the isolates, when inoculated via IC, were pathogenic, provoking death in 4 - 14 post inoculation days. However, mice inoculated with 500ICLD50 of the same isolates through IM route were found with different death rates: 60.0% (BR-DR1); 50.0% (BR-C, BR-NL); 40.0% (BR-AL3); 9.5% (BR-MM1) and 5.2% (BR-EF10). The same isolates were used for the assessment of cross protection conferred by a commercial vaccine of veterinary use. The mice were vaccinated subcutaneously, receiving either one or two shots of vaccine, and challenged through IC and IM routes. Mice receiving two shots were protected against all the isolates, when challenged intracerebrally. Mice receiving one shot were found only partially protected against the challenge with the fixed PV strain and BR-C isolate. Mice challenged intramuscularly showed 100.0% of protection, with the exception of those vaccinated with one dose and challenged with PV strain, which were found with 66.0% of survivors. These results indicate the possibility of the existence of rabies virus variants circulating in different species of bat population. The data also suggest that the vampires, frugivorous and insectivorous bats share the same lineage of rabies viruses. The commercial vaccine has protected the mice against the challenge with different rabies virus isolates, suggesting that the vaccines usually employed in the field are effective, although some marked difference in neurovirulence by IM inoculation was found among the isolates tested.

O Passion flower little leaf mosaic virus (PLLMV) foi encontrado causando danos severos em plantios de maracujazeiro (Passilora edulis f. flavicarpa) em dois municípios do Estado da Bahia no ano de 2001. Nesses locais foi constatada que a incidência deste begomovirus estava relacionada à colonização das plantas por Bemisia tabaci, cujo biótipo não foi identificado. Até o momento este vírus não parece constituir grave ameaça a cultura do maracujazeiro, o que aparentemente esta relacionado ao fato de P. edulis f. flavicarpa não ser preferida para a alimentação desse aleyrodídeo. Assim, os objetivos deste trabalho foram: a) selecionar espécies silvestres de Passiflora resistentes a este begomovirus que possam ser úteis em futuro programa de melhoramento genético; b) identificar possíveis hospedeiros alternativos do patógeno entre algumas espécies da vegetação espontânea e cultivadas e c) avaliar se adultos de B. tabaci biótipo B presentes no Estado de São Paulo são capazes de transmitir esse vírus. A reação de espécies silvestres de Passiflora foi avaliada por enxertia em maracujazeiro amarelo infectado que serviu como de fonte de inóculo. As avaliações foram feitas por meio da expressão de sintomas, análise de PCR e teste de recuperação do vírus para maracujazeiro amarelo. As espécies P. alata, P. quadrangularis, P. morifolia, P. serrato-digitata, P. suberosa e P. foetida foram suscetíveis ao PLLMV, enquanto P. caerulea, P. cincinnata, P nitida, P. mucronata e P. giberti se mostraram resistentes a este vírus. No estudo de hospedeiros alternativos, primeiramente o PLLMV foi inoculado mecanicamente nas seguintes espécies vegetais: Capsicum annuum, Chenopodium quinoa, Solanum lycopersicon, S. tuberosum, P. edulis f. flavicarpa, Phaseolus vulgaris, Nicotiana benthamiana e Sida sp.. Somente N. benthamiana foi infectada sistemicamente. Posteriormente foram feitas tentativas de transmissão desse begomovirus por meio de enxertia, usando-se como fonte de inóculo (porta-enxerto), plantas infectadas de N. benthamiana. Foram avaliadas as seguintes espécies vegetais: S. pimpinellifolium, S. lycopersicon, S. tuberosum, N. benthamiana, D. stramonium, C. annuum, N. glutinosa e Sida rhombifolia. O vírus foi transmitido somente para plantas de S. pimpinellifolium. As sucessivas transmissões mecânicas do PLLMV em N. benthamiana permitiram a seleção de uma estirpe fraca e protetora deste begomovirus. B. tabaci biótipo B não foi capaz de transmitir o PLLMV. / The Passion flower little leaf mosaic virus (PLLMV) was found causing severe damage in passion flower (Passilora edulis f. flavicarpa ) orchards in two counties of Bahia state, in 2001. The high incidence of this begomovirus was related to the colonization of plants by Bemisia tabaci, whose biotype was not indentified. To date this virus doesnt seem to be a serious threat to the passion flower cultivation, which apparently is related to the fact that P. edulis f. flavicarpa is not a preferred specie for whitefly feeding. The aim of this work were: a) to select wild species of Passifloraceae resistant to PLLMV, that may be useful in future breeding program; b) to indentify some possible alternative hosts of the pathogen among some weed and cultivaded species and c) to evaluat whether adults of B. tabaci biotype B are capable of transmitting this virus. The reaction of wild species of Passifloracea was evaluated by grafting on infected yellow passion fruit that served as a source of inoculum. The evaluation of these plants were done by means of symptoms, PCR test and biological recovery of the virus to yellow passion fruit. The sepecies P. alata, P. quadrangularis, P. morifolia, P. serrato-digitata, P. suberosa and P. foetida were susceptible to PLLMV, while P. caerulea, P. cincinnata, P nitida, P. mucronata and P. giberti were resistant to this virus. In the study of alternative hosts, at first, PLLMV was mechanically inoculated in the following species: Capsicum annuum, Chenopodium quinoa, Solanum lycopersicon, S. tuberosum, Passiflora edulis f. flavicarpa, Phaseolus vulgaris, Nicotiana benthamiana and Sida sp. Only N. benthamiana was systematically infected. Subsequently, graft transmission of PLLMV was carried out using infected N. benthamiana as source of inoculum (root stock). The following species were evaluated: Solanum pimpinellifolium, S. lycopersicon, S. tuberosum, N. benthamiana, D. stramonium, C. annuum, N. glutinosa and Sida rhombifolia.The virus was transmited only to S. pimpinellifolium. Successive mechanical transmissions of PLLMV in N. benthamiana led to the selection of a mild and protective strain of this begomovirus. B. tabaci biotype B was not able to transmit the PLLMV.

Entre as cucurbitáceas cultivadas no Brasil, a abobrinha de moita (Cucurbita pepo L.) apresenta grande importância econômica, notadamente no Estado de São Paulo, a maior área de plantio. No Brasil, já foram encontrados nove vírus capazes de infectar esta espécie. Os mais freqüentes e responsáveis por prejuízos significativos à produção da abobrinha de moita são os do mosaico do mamoeiro – estirpe melancia (Papaya ringspot virus - type W – PRSV-W) e do mosaico amarelo da abobrinha (Zucchini yellow mosaic virus – ZYMV), devido principalmente à sua alta sensibilidade. A premunização das plantas com estirpes fracas vem sendo investigada há mais de 10 anos no Brasil e surge como uma alternativa interessante para controle destas viroses. A premunização de abobrinha de moita só foi estudada até o momento em plantas da variedade Caserta. O presente trabalho teve o objetivo de dar continuidade a estes estudos avaliando o efeito de estirpes premunizantes PRSV-W-1 e ZYMV-M na produção quantitativa e qualitativa de quatro variedades comerciais de C. pepo: Samira, Novita Plus, AF-2847 e Yasmin. Os resultados obtidos em experimentos conduzidos em estufa plástica mostraram que as plantas destas variedades infectadas com a estirpe fraca ZYMV-M, não exibiram sintomas foliares acentuados e não apresentaram alterações na produção quantitativa e qualitativa de frutos, quando comparada com a produção das plantas sadias. No entanto, as plantas dessas mesmas variedades infectadas com a estirpe PRSV-W-1, só ou em mistura com a estirpe ZYMV-M, exibiram sintomas acentuados de mosaico foliar e alteração significativa na qualidade dos frutos, caracterizada pelo escurecimento da casca. Não houve alteração significativa na quantidade de frutos produzidos por essas plantas e no peso médio dos frutos. Quando plantas dessas variedades foram infectadas somente com a estirpe fraca PRSV-W-1 e conduzidas paralelamente em condições de campo e estufa plástica, constatou-se intensificação dos sintomas nos frutos e nas folhas das plantas infectadas conduzidas principalmente em estufa, porém a produção quantitativa mais uma vez não foi alterada. A variedade Samira mostrou-se a mais sensível ao PRSV-W-1 em ambas as condições. Os resultados sugerem que fatores ambientais, além da interação da estirpe fraca com a variedade de abobrinha, parecem interferir na expressão de sintomas nos frutos das plantas infectadas com a estirpe PRSV-W-1. Também foi objeto de estudo desse trabalho a identificação da melhor hospedeira, entre algumas cucurbitáceas, para a multiplicação e manutenção das duas estirpes fracas, e a avaliação da eficiência dos afídeos Myzus nicotianae e M. persicae na transmissão da estirpe ZYMV-M. Os resultados mostraram que a estirpe ZYMV-M parece atingir as maiores concentrações em C. pepo cv. Caserta e em C. melo cv. Casca-de-Carvalho. Para a estirpe PRSV-W- 1, as melhores hospedeiras foram C. pepo cv. Caserta, seguida de C. lanatus cv. Crimson Sweet e C. sativus cv. Safira. M. nicotianae e M. persicae transmitiram a estirpe fraca ZYMV-M para 11,7% e 0% das plantas de abobrinha de moita cv. Caserta, respectivamente. Enquanto a estirpe severa foi transmitida para 47,0% e 80% das plantas teste, respectivamente. / Zucchini squash (Cucurbita pepo L.) is widely cultivated in Brazil, especially in the State of São Paulo, which is the major producer. Nine viruses capable to infect this species have already been described in Brazil. The most frequent and responsible for significant yield losses are the potyviruses Papaya ringspot virus - type W (PRSV-W) and Zucchini yellow mosaic virus (ZYMV), for which zucchini squash is highly susceptible and intolerant. Preimmunization with mild strains of both viruses have been investigated in Brazil for the last 10 years and appears as and interesting option for the control of these viruses. As preimmunization have been evaluated only for zucchini squash cv. Caserta, the purpose of the present work was to investigate the effect of the mild strains PRSV-W-1 and ZYMV-M on the quantitative and qualitative yield of four other commercial zucchini squash varieties: Samira, Novita Plus, AF-2847 e Yasmin. The results of an experiment carried out under plastic-house showed that mild strain ZYMV-M induced mild symptoms on the leaves of the plants of all varieties, but did not affect the yield of marketable fruits, as compared to those from the respective healthy controls. On the other hand, plants from all varieties exhibited accentuated mosaic and leaf malformation when infected with mild strain PRSV-W-1, alone or in mixture with ZYMV-M. The amount of fruits harvested (number and average weight) from these plants was similar to that from the respective healthy controls. However, the quality of the fruits was severely affected, since the mild strain PRSV-W-1 induced alteration on the texture and color of the skin. When plants infected only with mild strain PRSV-W-1 were grown simultaneously under field and plastic-house conditions, it was noticed that leaf and fruit symptoms were more intense on those maintained in the plastic-house. Once more the quantitative fruit yield was not affected by this mild strain. Zucchini squash cv. Samira was the most sensitive to PRSV-W-1 under both conditions. Together theses results suggest that environmental variables, besides the interaction between the variety and the mild strain, might influence the expression of symptoms shown by plants infected with PRSV-W-1. In addition to this, the present work also tried to identify the most appropriate host, among some cucurbit species/varieties, for multiplication of both mild strains, and the efficiency of two species of aphids (Myzus nicotianae and M. persicae) on the transmission of the mild strain ZYMV-M. The mild strain ZYMV-M attained the highest concentration in C. pepo cv. Caserta and Cucumis melo cv. Casca-de-Carvalho. The most appropriate hosts for multiplication of mild strain PRSV-W-1 were C. pepo cv. Caserta followed by Citrullus lanatus cv. Crimson Sweet and C. sativus cv. Safira. M. nicotianae and M. persicae transmitted the mild strain ZYMV-M to 11.7% and 0% of the test-plants of zucchini squash cv. Caserta. The transmission of the severe strain of ZYMV by both species of aphids occurred for 47% and 80% of the test-plants, respectively.

Alphaviruses are a group of medically relevant arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that are maintained by mosquito vectors. These zoonotic viruses are clustered into two groups: New World and Old World, depending on their geographical origin/distribution and clinical manifestations. Both of these groups cause disease symptoms of an acute febrile illness; however, each group has a distinct, hallmark disease symptom; New World alphaviruses, such as Eastern, Western, and Venezuelan equine encephalitis viruses (EEEV, WEEV, and VEEV, respectively), present with severe encephalitis while Old World alphaviruses, such as Sindbis, chikungunya, and Mayaro viruses (SINV, CHIKV, and MAYV, respectively) present with an incapacitating polyarthralgia that can persist for years following initial infection. To date, the most effective means of controlling these arboviral infections is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. Given the close phylogenetic and antigenic relationship between MAYV and CHIKV, we hypothesized that prior CHIKV immunity may affect the outcome of MAYV disease and/or limit its emergence in humans. Our work has shown that anti-CHIKV neutralizing antibodies can provide cross-protective immunity against MAYV disease. Alongside these studies, we have characterized the potency of a camelid-derived single-domain antibody (sdAb) that neutralizes a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express two anti-CHIKV sdAbs to target infection, dissemination, and transmission of MAYV and CHIKV within this deadly vector. These findings are particularly significant because they highlight the ability to co-target two emerging alphaviruses that are crippling public health and obliterating quality of life around the globe within a single defense strategy. / Doctor of Philosophy / Alphaviruses are arthropod-borne viruses (arboviruses) belonging to the Togaviridae family that infect millions of people annually via the bite of female mosquitoes. These viruses are major public health threats due to their ability to infect humans and animals and infections resulting in a range of debilitating diseases. Viruses within this genus are clustered into two groups: Old World and New World, based on geographical origin and distribution. While New World alphaviruses are known for inducing severe encephalitis (i.e., swelling in the brain), a hallmark symptom of the Old World alphaviruses is the development of incapacitating polyarthralgia (i.e., widespread joint pain) that can persist for years following initial infection. To date, the most effective means of combatting these viruses is through mosquito control programs. However, these programs have crucial limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens. Given the close genetic relationship between chikungunya virus (CHIKV) and Mayaro virus (MAYV), our research has focused on harnessing cross-reactive immunity between these emerging alphaviruses. We discovered this cross-reactivity provides protective immunity to both viruses (i.e., CHIKV and MAYV) after exposure to only one (i.e., CHIKV) of the viruses. Next, we characterized the potency of a small, single-domain antibody (sdAb) to neutralize a breadth of alphaviruses, including CHIKV and MAYV. With these data, we have designed and generated transgenic Aedes aegypti mosquitoes that express this sdAb to target both CHIKV and MAYV within this deadly mosquito vector. These findings are particularly significant because they provide the foundation for a novel approach to controlling and preventing outbreaks of these emerging alphavirus pathogens that obliterate quality of life in public health settings around the globe.

Influenza virus infects the human population worldwide and causes acute respiratory disease. Currently, the primary strategy for preventing influenza is seasonal vaccination which is capable of providing protection in most populations. However, seasonal vaccines are less efficacious to immunize the elderly and poorly induce cross-protective immunity against the reassorted pandemic virus in the recipients. Neuraminidase (NA) inhibitors have also been widely utilized to limit disease outcome. The currently used NA inhibitors, nonetheless, generate the drug-resistant progeny viruses; moreover, they are unable to directly target the host immune responses which cause immunopathology in severe cases. Therefore, new strategies that provide more effective immunogenicity, cross-protection and therapies against influenza infection must be developed. In this thesis, the adjuvanticity of CpG oligodeoxynucleotide (ODN), type I interferon (IFN) and Complete Freund’s adjuvant (CFA) when coadministered with seasonal influenza vaccines in ferrets is presented. It has been found that the adjuvanted vaccines are efficacious to induce neutralizing antibody responses. Several common and distinguished signaling pathways leading to dendritic cell (DC) maturation and B cell activation have been discovered from their adjuvanticity. Furthermore, it was determined that seasonal H1N1 prior infection more effectively induces cross-protection against the newly emerged 2009 pandemic H1N1 (H1N1pdm) virus in ferrets and mice than the seasonal vaccines. The prior infection-induced cross-reactive but non-neutralizing antibodies are capable of providing substantial protection in the H1N1pdm infected mice when CD8 T cells are absent. Lastly, function of different vaccine adjuvants for controlling H1N1pdm infection in mice has been investigated. Unlike other adjuvants, CFA is capable of protecting the mice from infection through enhancement of Treg cell suppressive molecules galectin-1 and CTLA-4 which downregulated DC costimulation and effector T cell responses. Overall, this thesis has provided novel mechanistic insights for developing protective strategies against influenza infection.

Influenza virus infects the human population worldwide and causes acute respiratory disease. Currently, the primary strategy for preventing influenza is seasonal vaccination which is capable of providing protection in most populations. However, seasonal vaccines are less efficacious to immunize the elderly and poorly induce cross-protective immunity against the reassorted pandemic virus in the recipients. Neuraminidase (NA) inhibitors have also been widely utilized to limit disease outcome. The currently used NA inhibitors, nonetheless, generate the drug-resistant progeny viruses; moreover, they are unable to directly target the host immune responses which cause immunopathology in severe cases. Therefore, new strategies that provide more effective immunogenicity, cross-protection and therapies against influenza infection must be developed. In this thesis, the adjuvanticity of CpG oligodeoxynucleotide (ODN), type I interferon (IFN) and Complete Freund’s adjuvant (CFA) when coadministered with seasonal influenza vaccines in ferrets is presented. It has been found that the adjuvanted vaccines are efficacious to induce neutralizing antibody responses. Several common and distinguished signaling pathways leading to dendritic cell (DC) maturation and B cell activation have been discovered from their adjuvanticity. Furthermore, it was determined that seasonal H1N1 prior infection more effectively induces cross-protection against the newly emerged 2009 pandemic H1N1 (H1N1pdm) virus in ferrets and mice than the seasonal vaccines. The prior infection-induced cross-reactive but non-neutralizing antibodies are capable of providing substantial protection in the H1N1pdm infected mice when CD8 T cells are absent. Lastly, function of different vaccine adjuvants for controlling H1N1pdm infection in mice has been investigated. Unlike other adjuvants, CFA is capable of protecting the mice from infection through enhancement of Treg cell suppressive molecules galectin-1 and CTLA-4 which downregulated DC costimulation and effector T cell responses. Overall, this thesis has provided novel mechanistic insights for developing protective strategies against influenza infection.

Einleitung Streptococcus (S.) suis verursacht bei Schweinen unter anderem Meningitis, Arthritis, Serositis und Endokarditis und ist eine der größten Herausforderungen für die Schweineindustrie. Von 29 beschriebenen Serotypen sind die Serotypen 2, 7 und 9 unter invasiven Isolaten weltweit besonders prävalent, vor allem in Europa. Bis heute gibt es keinen zugelassenen Impfstoff zur Prävention von S. suis-Erkrankungen in Europa, daher ist im Feld die Anwendung stallspezifischer Impfstoffe verbreitet. Diese bieten jedoch höchstens homologen Schutz und ihre Wirkung kann durch prädisponierende Faktoren wie eine Infektion mit dem porcine reproductive and respiratory syndrome Virus (PRRSV) beeinträchtigt werden. Daher hat sich die Forschung auf Antigene fokussiert, die potentiell heterologen Schutz vermitteln. Zielstellung Ziele der Studie waren die Charakterisierung vernachlässigter invasiver S. suis Pathotypen der wichtigen Serotypen 7 und 9 und die Etablierung neuer Infektionsmodelle im Schwein. Des Weiteren sollte die immunogene und protektive Wirkung des Immunoglobulin (Ig) M-degradierenden Enzyms von S. suis, IdeSsuis, im Serotyp 9 Infektionsversuch untersucht werden. Material und Methoden In dieser Arbeit wurden in vitro Versuche und experimentelle Infektionen im Schwein durchgeführt. Dazu gehörte die Geno- und Phänotypisierung von 22 S. suis Serotyp 7 Stämmen und vier Serotyp 9 Stämmen. Die Genotypisierung erfolgte mittels multiplex (MP) Polymerase-Kettenreaktion (PCR), einer PCR zur Differenzierung verschiedener Genvarianten des muramidase-release protein (MRP) sowie mittels multilocus sequence typing (MLST). Zur Phänotypisierung der S. suis Stämme wurden bactericidal assays eingesetzt, die als Bakteriämiemodell fungierten. Auf diese Weise konnten die Empfänglichkeit gegenüber S. suis Stämmen sowie deren Virulenz beurteilt werden. Durch Zugabe von rekombinantem (r) IdeSsuis wurde die Rolle adaptiven IgMs in der Begrenzung der Bakteriämie untersucht. Anhand von Western Blot Analysen erfolgte die Untersuchung der Expression und Funktionalität von IdeSsuis sowie die Expression von MRP in S. suis Serotyp 7 Stämmen. Enzyme-linked immunosorbent assays (ELISA) kamen zum Einsatz, um die Entwicklung von IgM und IgG Spiegeln in Ferkeln im zeitlichen Verlauf und die IgG Spiegel nach rIdeSsuis Immunisierung zu messen. Durch rIdeSsuis Immunisierung induzierte Antigen-spezifische T-Helferzellen (Th-Zellen) wurden mithilfe der Durchflusszytometrie untersucht. Schließlich erfolgte die Durchführung zweier S. suis Serotyp 7 Etablierungsversuche mit je 18 bzw. 5 Ferkeln sowie zwei S. suis Serotyp 9 Impf- und Infektionsversuche mit je 18 Ferkeln. Sektionsproben wurden histologisch untersucht. In einem Fall wurde eine Endokarditis mittels fluorescence in situ hybridization (FISH) charakterisiert. Ergebnisse Die meisten untersuchten Serotyp 7 Stämme gehörten dem Sequenztyp (ST) 29 an, einem emerging pathotype in Europa. Trotz der engen phylogenetischen Verwandtschaft, war mrp in den Stämmen sehr variabel. Phänotypisch bildeten alle Stämme gleichermaßen eine kleine MRP Variante, MRPs. Für vier ausgewählte Serotyp 7 Stämme wurde die Expression von IdeSsuis gezeigt, jedoch mit Unterschieden in Größe und Funktionalität. Bactericidal assays dieser vier Stämme zeigten starke Proliferation im Blut von Absatzferkeln, aber Abtöten im Blut von Läuferschweinen aus zwei Herden mit unterschiedlichem S. suis Status. Dieses Überlebensmuster unterschied sich deutlich von dem eines Serotyp 9 Stammes. Durch Zugabe von rIdeSsuis, konnte gezeigt werden, dass das Abtöten der Serotyp 7 Stämme im Blut von Läuferschweinen des infizierten Bestandes IgM-vermittelt war. Unabhängig von der Herkunft entwickelten sich die IgM Spiegel in den Ferkeln im zeitlichen Verlauf fast synchron. Schließlich konnte die Virulenz eines Serotyp 7 Stammes in einem intravenösen Infektionsversuch mit 5 Ferkeln gezeigt werden. Alle Tiere entwickelten schwere Symptome einer S. suis Erkrankung. In einem Impf- und Infektionsversuch mit einem hoch virulenten Serotyp 9 Stamm wurde die immunogene und protektive Wirkung einer rIdeSsuis Immunisierung untersucht. Neun Ferkel wurden mit rIdeSsuis oder einem Placebo prime-boost-boost immunisiert und zwei Wochen später infiziert. Neunzig Prozent der Placebotiere entwickelten schwere Symptome einer S. suis Erkrankung und starben oder mussten aus Tierschutzgründen euthanasiert werden. Alle geimpften Tiere überlebten den Versuch, fielen jedoch mit Fieber und Lahmheiten auf. Es konnte also gezeigt werden, dass eine rIdeSsuis Immunisierung vor Mortalität, nicht aber Morbidität durch den Infektionsstamm schützt. Alle immunisierten Ferkel serokonvertierten und Antigen-spezifische Th-Zellen wurden nachgewiesen. Weder die IgG Antwort noch die Th-Zell Antwort wurde jedoch durch die Infektion verstärkt. In einem weiteren Impf- und Infektionsversuch mit einem anderen Serotyp 9 Stamm fiel ein Tier am 11. Tag nach der Infektion mit Zeichen einer akuten Leptomeningitis auf, nachdem es zuvor klinisch völlig unauffällig war. In der Sektion des Tieres wurde eine Endokarditis der Mitralklappe diagnostiziert, die mit Biofilm-Bildung assoziiert war, was mithilfe von Histologie und FISH gezeigt werden konnte. Zusätzlich wurde eine fibrinopurulente Leptomeningitis diagnostiziert. Das Ferkel hatte Antikörper gegen rIdeSsuis und tötete den Infektionsstamm ex vivo im Blut ab. Schlussfolgerungen In dieser Arbeit habe ich zwei wichtige S. suis Pathotypen charakterisiert: S. suis Serotyp 7 Stämme des ST29 und einen hoch virulenten Serotyp 9 Stamm vom ST94. Die Serotyp 7 Stämme stellten einen besonderen Pathotypen dar, bei dessen Bekämpfung IgM eine wichtige Rolle spielt. Bei einer Serotyp 9 Infektion konnten trotz des Abtötens der Bakterien im Blut und opsonisierender Antikörper Biofilmbildung und eine folgende akute Leptomeningitis nicht verhindert werden. Letztlich wurde Schutz vor einem hoch virulenten Serotyp 9 Stamm durch rIdeSsuis Immunisierung und damit zum ersten Mal für ein Antigen Protektion vor den wichtigen Serotypen 2 und 9 aufgezeigt. Dies ist ein vielversprechendes Ergebnis hinsichtlich der Entwicklung eines Impfstoffes gegen S. suis. / Introduction Streptococcus (S.) suis causes meningitis, arthritis, serositis and endocarditis and is one of the biggest challenges for the swine industry. Of 29 described serotypes, the serotypes 2, 7 and 9 are highly prevalent amongst invasive S. suis isolates worldwide, especially in European countries. To date, no commercially produced vaccine is available in Europe for prevention of S. suis disease, thus the use of autogenous vaccines in the field is common. However, bacterins may at most confer homologous protection and their efficacy may be influenced by predisposing factors such as an infection with the porcine reproductive and respiratory syndrome virus (PRRSV). Research has therefore focused on subunit vaccines with the potential to elicit cross-protection against various serotypes. Aim of the study The objective of this study was to characterize neglected invasive S. suis pathotypes of the important serotypes 7 and 9 and to establish infection models in the main host of S. suis, the pig. A further aim was to investigate immunogenicities and protective efficacies of the immunoglobulin (Ig) M-degrading enzyme of S. suis, IdeSsuis, in a serotype 9 challenge experiment. Materials and methods In vitro experiments and experimental challenges in swine were conducted as part of this thesis. This included the geno- and phenotyping of 22 S. suis serotype 7 strains and four serotype 9 strains. Genotyping was conducted using a multiplex (MP) polymerase chain reaction (PCR), a PCR for differentiation of variants of the muramidase-released protein (MRP) gene and multilocus sequence typing (MLST). For phenotyping of the S. suis strains, bactericidal assays were carried out which served as a model for bacteraemia. This way, susceptibility to S. suis strains and virulence of different strains was assessed. Further, through addition of recombinant (r) IdeSsuis, the role of adaptive IgM in limiting bacteraemia was elucidated. Western blot analyses were conducted to investigate expression and functionality of IdeSsuis as well as expression of MRP in S. suis serotype 7 strains. Enzyme-linked immunosorbent assays (ELISA) were used to determine the development of IgM and IgG levels in piglets over time and to assess IgG levels following rIdeSsuis immunization. Antigen-reactive T-helper (h) cells induced by rIdeSsuis immunization were investigated using flow cytometry. Finally, two experiments to establish a serotype 7 infection model with 18 and 5 piglets each and two vaccination and challenge experiments using different S. suis serotype 9 strains (n=18 piglets/ experiment) were conducted. Samples of dissected animals were examined histologically. In one case, an endocarditis was analysed using fluorescence in situ hybridization (FISH). Results Most of the investigated serotype 7 strains belonged to sequence type (ST) 29 which was thus shown to be an emerging pathotype in Europe. Despite the close phylogenetic relation of the strains, mrp was highly variable. Phenotypically, all strains expressed a small variant of MRP, MRPs. Four selected serotype 7 strains were shown to express IdeSsuis with differences in size and functionality. Bactericidal assays of these four strains revealed high proliferation in blood of weaning piglets but killing in blood of growing piglets of two herds which differed in their S. suis infection status. This survival pattern was distinct from a serotype 9 strain. Addition of rIdeSsuis revealed that killing of the serotype 7 strains in blood of growing piglets of the infected herd was IgM-mediated. Independent of the originating herd, IgM levels of the piglets rose almost synchronous over time. Finally, the virulence of a serotype 7 strain was proven in an intravenous challenge experiment with five pigs which all developed severe clinical signs of S. suis disease. In a vaccination and challenge experiment using a highly virulent serotype 9 strain, immunogenicities and protective efficacies of rIdeSsuis immunization were investigated. Nine piglets were prime-boost-boost vaccinated with rIdeSsuis or placebo-treated and challenged two weeks later. Ninety per cent of the placebo-treated piglets developed severe clinical signs of S. suis disease and died or had to be euthanized due to animal welfare reasons. All vaccinated piglets survived the experiment, however elevated body temperatures and lameness were also noted in this group. Accordingly, rIdeSsuis vaccination protected from mortality but not morbidity caused by the challenge strain. Seroconversion of the immunized piglets and antigen-reactive Th cells were detected. Neither the IgG response nor the Th cell response was boosted through the challenge. In a further vaccination and challenge experiment with a different serotype 9 strain, one animal was clinically unobtrusive following infection and then developed an acute leptomeningitis on the 11th day post infection and had to be euthanized. Dissection of the animal revealed an endocarditis on the mitral valve which was proven to be associated with biofilm formation by histology and FISH. In addition, a fibrinosuppurative leptomeningitis was diagnosed. The piglet had specific antibodies against rIdeSsuis and mediated killing of the challenge strain in a bactericidal assay. Conclusions In this thesis, I characterized two important pathotypes: S. suis serotype 7 strains of ST29 and a highly virulent serotype 9 strain of ST94. The serotype 7 strains represent a distinct pathotype and IgM plays a significant role in their control. Following a serotype 9 infection, biofilm formation and a subsequent acute leptomeningitis could not be prevented despite blood bactericidal activity and opsonizing antibodies. Finally, protection against challenge with a highly virulent serotype 9 strain through rIdeSsuis immunization was demonstrated. Thereby, for the first time an antigen was shown to confer cross-protection against the important serotypes 2 and 9, which is highly encouraging regarding the development of a vaccine against S. suis.

Microbes typically form highly complex and diverse communities that account for a significant portion of life's genetic diversity. Analysis of living systems, e.g. bacterial or cell population, plays a significant role in detecting and identifying pathogens, testing antibiotic susceptibility, and the fundamental research of population diversity and evolution. This work focuses on the analysis of bacterial communities using droplets based millifluidics. To monitor the bacteria growth, we designed an optofluidic system, combining the encapsulation of bacteria in numerous emulsion droplets to monitor their long-term behavior and relationship in a co-culture environment using fluorescent signals. In the first part of this work, we co-encapsulated and cultured two isogenic strains of Escherichia coli (E. coli) in numerous emulsion droplets to reveal their competition and cooperation relationship. Since two strains of E. coli express blue and yellow fluorescent proteins (BFP and YFP, respectively), we quantified their growth by integrating a fluorescence detection system. We analyzed the following parameters: doubling time, population yield, final biomass ratio, correlation map of doubling time and competition coefficient to characterize and compare the bacterial growth kinetics and behavior in mono and co-cultures. In addition, the experimental observations were compared with the predictions from a single growth model. Finally, we employed the millifluidic device to verify the appearance of cross-protection between antibiotic-sensitive bacteria and antibiotic-resistant bacteria. It is one of the mechanisms by which different bacteria, sharing the same environment, protect each other to survive in the presence of antibiotics. For this purpose, the E.coli YFP strain was chosen as an antibiotic-sensitive group. Simultaneously, the E.coli BFP strain with β-lactam and its mutations were selected as resistant strains. Combining the millifluidic droplet reactor method with other detection strategies, e.g. fluorescence microscopy, fluorescence flow cytometry, and plate reader, we proved the appearance of cross-protection by detecting the filamentary cells, the fluorescence of cell-free media, viable cell rates, cell shape and size, as well as β-lactamase activity. All these results obtained by millifluidic devices proved that this strategy could be used in a high-throughput bacterial coexistence study. In addition, the research of these general fields, such as bacterial community and antibiotic impact, can help us to reveal the interaction between microbial species and determine the right dose of antibiotics to inhibit bacterial growth in a co-existent environment efficiently.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are closely related Flaviviruses that are important arthropod-borne human pathogens. Both of these viruses can cause encephalitis with significant morbidity and mortality after infection. Flaviviruses co-circulate in many areas of the world, which raises the risk for sequential infection between heterologous viruses. Sequential infection between dengue virus serotypes can lead to cross-protection, but in some cases, it leads to a severe outcome, dengue hemorrhagic fever. Previous work in hamsters and non-human primates demonstrated that prior JEV immunity protects against a lethal WNV infection. However, the ability of prior WNV immunity to protect against a lethal JEV infection has been inconclusive. WNV-immune hamsters were fully protected from JEV viremia, but in non-human primates, prior WNV-immunity only reduced disease severity, with symptoms of encephalitis still observed. These differences in cross-protection led to further investigation on the directionality as well as the underlying mechanisms for this phenomenon. Previous work in our lab found that JEV-immune C57BL/6J (B6) mice were fully protected against a lethal WNV infection, and JEV-immune CD4+ and CD8+ T cells were required for this cross-protection. In other mouse models, memory cross-reactive CD4+ and CD8+ T cell responses may induce protection or immunopathology upon secondary heterologous viral challenge. We hypothesize that JEV/WNV cross-reactive CD4+and CD8+ T cells preferentially expand upon 2o infection and contribute to cross-protection. To elucidate the potential role of T cells in sequential flavivirus infection, we identified and characterized cross-reactive CD4+ and CD8+ T cell responses between JEV and WNV. A previously reported WNV NS4b CD8+ T cell epitope and its JEV variant elicited CD8+ T cell responses in both JEV- and WNV-infected mice. Despite similarities in viral burden for pathogenic JEV and WNV viruses, CD8+ T cells from pathogenic JEV-infected mice exhibited functional and phenotypic profiles similar to those seen for the attenuated JEV strain. We believe the differences in the CD8+ T cell responses during primary JEV and WNV infection are due at least in part to the low levels of peripheral replication seen in JEV-infected mice compared to WNV-infected mice. We also found that WNV-immune B6 mice were protected against a lethal JEV infection. Cross-reactive CD8+ T cells in JEV-immune mice rapidly expanded after WNV infection. Even though WNV-immune mice had higher frequencies of memory CD8+ T cells, cross-reactive CD8+ T cells did not expand after secondary JEV infection. Neutralizing antibodies to JEV were detected in WNV-immune mice; however, cross-reactive CD8+ T cells did not expand even in the absence of these cross-reactive neutralizing antibodies. We did not detect any differences in the CD8+ T cell repertoires between JEV- and WNV-infected mice nor were WNV-immune CD8+ T cells functionally exhausted. In fact, proliferation of memory CD8+ T cells did not correlate with the ability of WNV-immune CD8+ T cells to restrict recombinant vaccinia viruses expressing the cross-reactive epitope or lyse peptide-coated targets. These data suggest that the higher frequency of memory CD8+ T cells and cross-reactive antibodies in WNV-immune mice are better able to prevent neuroinvasion following 2o JEV infection.

No description available.