Thesis--Universidad de Sevilla. / "Apéndice demografico": p. 134-180. Includes bibliographical references (p. 132-134).

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Jun 1.

Thesis (M.S.) University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 21, 2007) Vita. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-10Bitstream added on 2014-06-13T19:22:41Z : No. of bitstreams: 1 morais_ats_dr_sjrp.pdf: 1276143 bytes, checksum: 62a89d8b555ac92b5faf4baa19e4db2f (MD5) / O vírus da Febre Amarela (YFV) pertence ao gênero Flavivirus e causa uma importante doença. Nos últimos anos, uma alarmante ressurgência da circulação viral e expansão do vírus em áreas endêmicas têm sido detectadas na África e América do Sul. NS5 é uma proteína viral não estrutural com duas atividades essenciais para a replicação viral, uma de metiltransferase e outra de RNA Polimerase dependente de RNA (RdRp). Para o melhor entendimento dos mecanismos de replicação viral, interações entre NS5 e proteínas celulares têm sido amplamente estudadas. Assim, os objetivos desse estudo foram caracterizar a interação da proteína NS5 e eIF3L, avaliar a função de eIF3L na replicação do vírus da febre amarela, e caracterizar estruturalmente a proteína eIF3L. Métodos. Para identificar a interação de NS5 YFV com eIF3L, foi realizado ensaios em sistema duplo-híbrido usando RdRp NS5 YFV contra eIF3L. Para o mapeamento da interação, foram construídos mutantes deletantes de RNApol e analisados em sistema duplo-híbrido. A região de interação de RNApol foi segmentada em três fragmentos e analisada na presença de eIF3L. Para mapear os resíduos de NS5 críticos para a interação, foi realizada mutagênese sítio-dirigida no segmento 3 de ID. A interação foi analisada em ensaios in vitro e em cultura de células de mamíferos. A significância de eIF3L para a replicação do YFV foi investigada usando superexpressão de eIF3L em células BHK21-RepYF17D LucNeoIres. A proteína eIF3L foi purificada usando uma combinação de cromatografia de afinidade e de exclusão molecular para subsequente caracterização estrutural. Resultados. Nesse estudo, foi caracterizada a interação de NS5 com o fator eucariótico de início de tradução... / Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and expansion of the YFV endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains the methyltransferase and RNA-dependent RNA polymerase domains, which are essential during viral replication. Interactions among NS5 and cellular proteins have been studied for the understanding of viral replication. The aim of this study was to characterize the interaction of NS5 protein with EIF3L and evaluate the role of EIF3L in yellow fever replication. Methods. To identify the interaction of YFV NS5 with cellular proteins, we performed a two-hybrid screen using YFV NS5 RdRp domain as bait and a human cDNA library. For mapping the interaction, RNApol deletions mutants were performed and analyses in two-hybrid system. The RNApol region of interaction was segmented in three fragments and analyses into yeast containing eIF3L. To map residues of NS5 that are critical for its interaction, we performed a site-direct mutagenesis in segment 3 of ID. The interaction was confirmed in vitro assays and by in vivo coimmunoprecipitations. The significance of eIF3L for replication of YFV was investigated using overexpression of eIF3L in BHK21-RepYF17D LucNeoIres cells. eIF3L was purified using a combination of affinity and subsequent size exclusion chromatography for subsequent structural characterization. Results. In this work we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed by in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs in a region (Interaction Domain of RNApol domain) that is conserved in several... (Complete abstract click electronic access below)

Orientador: Carlos Augusto Araújo Valadão / Banca: Antonio Carlos Alessi / Banca: Irene Rosemir Pelá / Resumo: A endotoxemia é uma afecção importante nas diversas espécies animais decorrente da ação de lipopolissacarídeos liberados na circulação durante o choque séptico, os quais ativam a cascata inflamatória induzindo a síntese e liberação de citocinas (TNF, IL-1, IL-6), capazes de produzir febre. É bastante comum, em eqüinos, quadros de cólica gastrintestinal devido à intoxicação por amitraz (AMZ), um acaricida formamidínico. A compactação de cólon é uma das conseqüências dessa intoxicação principalmente, pela ação desta substância sobre receptores adrenérgicos a-2. Sabendo-se que as substâncias agonistas de receptores a-2 produzem um aumento do tônus vagal e que a estimulação das fibras vagais, aferentes e eferentes reduz a liberação de TNF-a, o objetivo deste estudo foi avaliar os efeitos do AMZ sobre a febre, quando administrado antes da indução experimental de endotoxemia com LPS ou da administração de IL-1ß. Foram utilizados, em duas fases experimentais, 40 coelhos machos, raça Branco da Nova Zelândia, com peso entre 2,5 e 3,5 kg. Na primeira fase, para determinação da dose hipnótica 50% do AMZ (DHA50%), os animais foram separados em 4 grupos iguais de cinco animais (G1, G2, G3, G4). Na segunda fase experimental os animais foram divididos em 8 grupos iguais (GI, GII, GIII, GIV, GV, GVI, GVII, GVIII), compostos de cinco animais cada, os quais foram pré-tratados com salina (NaCl 0,9%), AMZ (0,3 mg.kg-1) ou DMSO e desafiados com salina, LPS(1,5 æg.kg-1) ou IL-1ß (100 ng.kg-1). A DHA50% e DSHA, para coelhos, foi 0,55 mg.kg-1 e 0,3 mg.kg-1, respectivamente. A febre observada no grupo AMZ-LPS foi inferior àquela demonstrada pelo grupo sal-LPS e superior àquela anotada no grupo DMSO-LPS. Concluiu-se que o AMZ, na dose de 0,3 mg.kg-1, diminui a intensidade da resposta febril ao LPS, mas o DMSO demonstra maior efetividade sobre a febre induzida por LPS. O AMZ não bloqueia a febre causada pela IL-1ß. / Abstract: Endotoxemia is an important condition in several species being induced by lipopolysaccharides. Released into the bloodstream during the septic shock, they activate the inflammatory cascade leading to syntheses and release of cytokines (TNF, IL-1, IL-6) which are able to produce fever. Colic in horses is frequently observed after treatment with amitraz, a formamidine pesticide. Large colon impactation is one of the consequences of intoxication, mainly by acting on a-2-adrenergic receptors. As a-2 agonists has been proven to produce an increase of the vagus tone, ant that the stimulation of afferent and efferent vagus has been shown to reduced TNF release, the main goal of this study was to evaluate the effects of amitraz over fever when administered before induction of experimental endotoxemia with LPS or IL-1ß administration. Forty male rabbits of the New Zealand white breed weighing 2.5-3.5 kg were used. The present study consisted of two experimental phases. In the first phase, to determine the 50% hypnotic dose of amitraz (DHA50%), the rabbits were divided into four groups (n=5). In the second phase, the rabbits were divided into eight groups (n=5). They were submited to treatment with saline (NaCl 0,9%), AMZ (0.3 mg.kg-1) or DMSO 10 minutes before intravenous injection of saline, LPS (1.5 mg.kg-1) or IL-1ß (100 ng.mg.kg-1). The DHA50% and DSHA for rabbits was 0.55 mg.kg-1 and 0.3 mg.kg-1, respectively. The results demonstrated that the fever observed in AMZ-LPS group was comparatively lower than the fever induced in the group sal-LPS and higher than the fever induced in the group DMSO-LPS. Therefore, It was concluded that amitraz, at the dose of 0,3 mg/kg, decreases the intensity of febrile response to LPS, despite the fact that DMSO is more effective over fever induced by LPS. On the other hand, amitraz does not interfere with the febrile response induced by intravenous administration of IL-1ß. / Mestre

Introdução: especula-se que os instrumentos e produtos químicos em cuja composição esteja o mercúrio sejam banidos para o uso comercial ou industrial devido à toxicidade deste metal ao meio ambiente e aos seres vivos. Como no nosso meio usa-se predominantemente o termômetro axilar de mercúrio para aferir a temperatura corporal, se de fato este termômetro deixar de ser usado, precisamos saber qual(is) termômetro(s) poderá(ão) substitui-lo; recentemente foram postos no mercado os termômetros eletrônicos instantâneos, como o auricular e o cutâneo frontal e já existia há mais tempo o axilar eletrônico; estes termômetros são confiáveis para substituírem os de mercúrio? Objetivo: avaliar a concordância entre os termômetros eletrônicos axilar e instantâneos (auricular e cutâneo frontal) com os termômetros eletrônico retal e axilar de mercúrio, este com três e cinco minutos de permanência, na aferição da temperatura corporal de crianças. Métodos: foi realizado um estudo transversal com amostra de conveniência de crianças internadas ou em consulta nas áreas pediátricas do Hospital de Clínicas de Porto Alegre. Foi aferida a temperatura corporal dos pacientes participantes do estudo com a utilização quase simultânea de cinco tipos de termômetros: axilar de mercúrio, axilar eletrônico, cutâneo frontal eletrônico, auricular eletrônico e termômetro retal eletrônico. O termômetro axilar de mercúrio foi avaliado com tempo de permanência de três e de cinco minutos. Os dados foram sumarizados em média e desvio padrão. Foi empregado o teste de Bland e Altman para avaliar a concordância entre os termômetros. Resultados: foram avaliadas 20 crianças febris e 43 eutérmicas, com idades entre um mês e dois anos incompletos, totalizando 633 aferições da temperatura corporal. A idade média dos participantes foi 7,5 meses (DP 5,7 meses), 55% deles eram do sexo masculino. Nenhuma criança foi rejeitada e também não houve recusa por parte dos responsáveis em autorizar a participação no estudo. O estudo mostrou que houve boa concordância entre os termômetros axilar de mercúrio aos três minutos e axilar eletrônico, com média das diferenças das temperaturas (MDT) de -0,027°C e intervalo de confiança de 95% (IC95%) de -0,062°C a 0,116°C; entre os termômetros eletrônico retal e axilar de mercúrio aos três minutos, com MDT de 0,096°C e IC95% de -0,175°C a 0,211ºC; e entre os termômetros eletrônico retal e axilar de mercúrio aos cinco minutos, com MDT de 0,038°C e IC95% de -0,149°C a 0,073°C. Houve apenas concordância parcial entre os termômetros eletrônicos retal e axilar, com MDT de -0,123°C e IC95% de 0,030°C a -0,245°C. Conclusões: os resultados indicaram o termômetro axilar eletrônico como o de melhor concordância nas aferições da temperatura corporal de crianças com hipertermia ou normotermia quando comparado com os termômetros axilar de mercúrio e eletrônico retal. Os termômetros eletrônicos instantâneos (auricular e cutâneo frontal) não mostraram boa concordância com termômetro eletrônico retal nem com o termômetro axilar de mercúrio. / Introduction: It is said that chemical products and instruments whose composition have mercury will be banned for commercial or industrial use due to the toxicity of this metal for the environment and beings. Since in our field the axillary mercury thermometer is mainly used for reading the body temperature, and if this thermometer is really banned, we need to know what thermometer(s) will replace it; recently, we can find instant electronic thermometers such as the ear and frontal ones in the market, and for longer, there have been the axillary electronic thermometer; are these thermometers reliable to replace the mercury one? Objective: to evaluate the agreement of the axillary electronic and instant (ear and frontal) thermometers with the rectal electronic and axillary mercury thermometers, the axillary mercury one staying three to five minutes, when reading the body temperature of children. Method: A transversal study was carried out with convenience sampling of hospitalized children or children with medical appointments in the pediatric areas of Hospital de Clínicas de Porto Alegre. The body temperature of the subject patients was taken with the use, almost simultaneously, of five types of thermometers: axillary mercury, axillary electronic, frontal electronic, ear electronic and rectal electronic. The axillary mercury thermometer was evaluated by staying three to five minutes. The data was stated by average and standard deviation. The Bland-Altman test was applied to evaluate the agreement between the thermometers. Result: 20 feverish children and 43 euthermic children, between the ages of one month to two incomplete years, were evaluated, totaling 633 body temperatures taken. The average age of the subjects was 7.5 months (5.7 standard deviation), 55% were male. No child was rejected and there was also no refusal by the responsible for the children to authorize their participation in the study. The study has shown that there is a good agreement between the axillary mercury thermometer at three minutes and the axillary electronic one, with an average of difference in temperature of -0.027°C and confidence interval of 95% (95%CI) of -0.062°C to 0.116°C; between the rectal electronic thermometer and the axillary mercury one at three minutes, with an average of difference in temperature of 0.096°C and 95%CI of -0.175°C to 0.211°C; and between the rectal electronic thermometer and the axillary mercury one at five minutes, with an average of difference in temperature of 0.038°C and 95%CI of 0.149°C to 0.073°. There was only partial agreement between the rectal and axillary electronic thermometers, with an average of difference in temperature of -0.123°C and 95%CI of 0.030°C and -0.245°C. Conclusion: the results have shown that the electronic axillary thermometer has the best agreement in reading the body temperature of children with hyperthermia or normothermia when compared with the axillary mercury and electronic rectal thermometers. The instant electronic thermometers (ear and frontal) have not shown good agreement with either the rectal electronic thermometer or the axillary mercury thermometer.

Livestock animals are an ancient, vital renewable natural resource. Many livestock species have the ability to convert inedible crops and waste food into food fit for human consumption, in the form of meat, eggs and dairy products. As the global demand for high value animal protein is ever increasing, the livestock market continues to play a major role in worldwide economics. Animal disease has the potential to be a huge burden on the livestock industry, impacting both welfare and production. Major outbreaks of transboundary diseases, such as foot and mouth disease, rinderpest and classical swine disease, have resulted in devastating global economic losses. As a result, scientific research is engaged in lowering this impact by generating effective preventative measures and treatments. One way to reduce livestock disease is to select animals that are genetically resistant, traditionally carried out through selective animal breeding programs; however, this is a time-consuming process and requires that appropriate genetic variation exists within the population. Advances in genome engineering technologies offer us an alternative approach, with the capability to make genetic improvements in livestock within a single generation. It is hypothesised that resilience to a disease, known as African swine fever (ASF), could be genetically engineered into the domestic pig. ASF is a highly contagious disease of domestic pigs and is a re-emerging global threat to the swine industry. It is a lethal haemorrhagic disease caused by a virus, known as the African swine fever virus (ASFV). At present, there is no vaccine or treatment for ASF, and disease control relies on rapid diagnosis, quarantine and the mass slaughter of animals. Unlike the domestic pig, swine indigenous to Sub-Saharan Africa, such as the warthog, show no clinical signs of disease following infection with ASFV. A comparative study was carried out to identify host genetic variation that could underlie the difference in response to ASFV, with candidate genes selected based on their potential involvement with the viral protein A238L, involved in immune evasion. Functional polymorphisms where identified in the porcine RELA gene, encoding RelA, a subunit of the NF-κB transcription factor family. This evolutionary conserved protein family plays a vital role in mediating inflammatory and immune responses. The specific RELA polymorphisms identified alter potential phosphorylation sites within the C-terminal transactivation domain of RelA which have been found to modulate NF-κB transcriptional activity in vitro. We set out to investigate whether genome editing tools could be employed to engineer the RELA sequence of domestic pigs. Initial attempts targeted the final exon of RELA, producing animals with a truncated RelA protein; modified animals lack the final 60 amino acids of the C-terminal transactivation domain. The aim of this thesis was to genotype and characterise the effects of this RELA modification at a molecular, cellular, morphological and whole organism level. The ultimate goal of this project was to investigate whether this RELA modification altered the domestic pig’s response to ASFV in vitro and in vivo. Unlike rela-/- mice which have an embryonic lethal phenotype, these RELA-edited pigs were born healthy and were fully viable when housed in a typical farm environment. Phenotypic analysis of lymphoid tissues from the RELA-edited pigs demonstrated no significant anatomical or histological changes compared to unmodified counterparts. Pigs homozygous for the RELA mutation had a significantly lower body weight compared to wild-type pigs. Molecular studies of samples from these pigs have shown that the modified RelA has an altered activity; however, the RELA modified pigs do develop the characteristic disease phenotype when challenged with ASFV. Finally, genome editors have been developed to introduce a specific warthog allele into the domestic pig RELA locus, these editors are currently being taken forward to produce a novel pig line.

Introdução: especula-se que os instrumentos e produtos químicos em cuja composição esteja o mercúrio sejam banidos para o uso comercial ou industrial devido à toxicidade deste metal ao meio ambiente e aos seres vivos. Como no nosso meio usa-se predominantemente o termômetro axilar de mercúrio para aferir a temperatura corporal, se de fato este termômetro deixar de ser usado, precisamos saber qual(is) termômetro(s) poderá(ão) substitui-lo; recentemente foram postos no mercado os termômetros eletrônicos instantâneos, como o auricular e o cutâneo frontal e já existia há mais tempo o axilar eletrônico; estes termômetros são confiáveis para substituírem os de mercúrio? Objetivo: avaliar a concordância entre os termômetros eletrônicos axilar e instantâneos (auricular e cutâneo frontal) com os termômetros eletrônico retal e axilar de mercúrio, este com três e cinco minutos de permanência, na aferição da temperatura corporal de crianças. Métodos: foi realizado um estudo transversal com amostra de conveniência de crianças internadas ou em consulta nas áreas pediátricas do Hospital de Clínicas de Porto Alegre. Foi aferida a temperatura corporal dos pacientes participantes do estudo com a utilização quase simultânea de cinco tipos de termômetros: axilar de mercúrio, axilar eletrônico, cutâneo frontal eletrônico, auricular eletrônico e termômetro retal eletrônico. O termômetro axilar de mercúrio foi avaliado com tempo de permanência de três e de cinco minutos. Os dados foram sumarizados em média e desvio padrão. Foi empregado o teste de Bland e Altman para avaliar a concordância entre os termômetros. Resultados: foram avaliadas 20 crianças febris e 43 eutérmicas, com idades entre um mês e dois anos incompletos, totalizando 633 aferições da temperatura corporal. A idade média dos participantes foi 7,5 meses (DP 5,7 meses), 55% deles eram do sexo masculino. Nenhuma criança foi rejeitada e também não houve recusa por parte dos responsáveis em autorizar a participação no estudo. O estudo mostrou que houve boa concordância entre os termômetros axilar de mercúrio aos três minutos e axilar eletrônico, com média das diferenças das temperaturas (MDT) de -0,027°C e intervalo de confiança de 95% (IC95%) de -0,062°C a 0,116°C; entre os termômetros eletrônico retal e axilar de mercúrio aos três minutos, com MDT de 0,096°C e IC95% de -0,175°C a 0,211ºC; e entre os termômetros eletrônico retal e axilar de mercúrio aos cinco minutos, com MDT de 0,038°C e IC95% de -0,149°C a 0,073°C. Houve apenas concordância parcial entre os termômetros eletrônicos retal e axilar, com MDT de -0,123°C e IC95% de 0,030°C a -0,245°C. Conclusões: os resultados indicaram o termômetro axilar eletrônico como o de melhor concordância nas aferições da temperatura corporal de crianças com hipertermia ou normotermia quando comparado com os termômetros axilar de mercúrio e eletrônico retal. Os termômetros eletrônicos instantâneos (auricular e cutâneo frontal) não mostraram boa concordância com termômetro eletrônico retal nem com o termômetro axilar de mercúrio. / Introduction: It is said that chemical products and instruments whose composition have mercury will be banned for commercial or industrial use due to the toxicity of this metal for the environment and beings. Since in our field the axillary mercury thermometer is mainly used for reading the body temperature, and if this thermometer is really banned, we need to know what thermometer(s) will replace it; recently, we can find instant electronic thermometers such as the ear and frontal ones in the market, and for longer, there have been the axillary electronic thermometer; are these thermometers reliable to replace the mercury one? Objective: to evaluate the agreement of the axillary electronic and instant (ear and frontal) thermometers with the rectal electronic and axillary mercury thermometers, the axillary mercury one staying three to five minutes, when reading the body temperature of children. Method: A transversal study was carried out with convenience sampling of hospitalized children or children with medical appointments in the pediatric areas of Hospital de Clínicas de Porto Alegre. The body temperature of the subject patients was taken with the use, almost simultaneously, of five types of thermometers: axillary mercury, axillary electronic, frontal electronic, ear electronic and rectal electronic. The axillary mercury thermometer was evaluated by staying three to five minutes. The data was stated by average and standard deviation. The Bland-Altman test was applied to evaluate the agreement between the thermometers. Result: 20 feverish children and 43 euthermic children, between the ages of one month to two incomplete years, were evaluated, totaling 633 body temperatures taken. The average age of the subjects was 7.5 months (5.7 standard deviation), 55% were male. No child was rejected and there was also no refusal by the responsible for the children to authorize their participation in the study. The study has shown that there is a good agreement between the axillary mercury thermometer at three minutes and the axillary electronic one, with an average of difference in temperature of -0.027°C and confidence interval of 95% (95%CI) of -0.062°C to 0.116°C; between the rectal electronic thermometer and the axillary mercury one at three minutes, with an average of difference in temperature of 0.096°C and 95%CI of -0.175°C to 0.211°C; and between the rectal electronic thermometer and the axillary mercury one at five minutes, with an average of difference in temperature of 0.038°C and 95%CI of 0.149°C to 0.073°. There was only partial agreement between the rectal and axillary electronic thermometers, with an average of difference in temperature of -0.123°C and 95%CI of 0.030°C and -0.245°C. Conclusion: the results have shown that the electronic axillary thermometer has the best agreement in reading the body temperature of children with hyperthermia or normothermia when compared with the axillary mercury and electronic rectal thermometers. The instant electronic thermometers (ear and frontal) have not shown good agreement with either the rectal electronic thermometer or the axillary mercury thermometer.

Les phlébovirus présentent sont présents dans toutes les régions du globe. Certains phlébovirus transmis par phlébotomes provoquent une maladie fébrile et des infections du système nerveux central. Depuis, de plus en plus de données montrent que la péninsule des Balkans joue un rôle majeur dans l'émergence de maladies à transmission vectorielle. Au début de ce travail, on comptait un nombre très limité de phlébovirus identifiés et isolés dans cette région. Une étude intégrée et transdisciplinaire en vue d'un inventaire des virus circulant dans pays des Balkans. (i) Un total de 3,850 phlébotomes sont été recueillis dans sept pays des Balkans en 2014 et 2015. Ils ont été testés pour la présence d'ARN viral et inoculé sur des cellules VERO afin d'isoler le virus détecté; (ii) des études de séroprévalence utilisant des tests de neutralisation ont été effectuées sur des échantillons de bovins et de moutons pour évaluer à deux agents pathogènes humains : le virus Toscana (TOSV) et le virus Sandfly fever Sicilian virus (SFSV). Nos résultats se composent de (i) la découverte et le séquençage de 3 nouveaux phlébovirus appartenant à 2 espèces différentes, (ii) la première identification du genotype B de TOSV en Croatie, (iii) la preuve de la co-circulation de deux genotypes (B et C) de TOSV, (iv) des taux d'anticorps neutralisants qui sont beaucoup plus élevés chez les bovins et les moutons pour le SFSV que pour TOSV. En conclusion, les résultats obtenus au cours de ce travail démontrent qu’es les Balkans représentent une zone de très importante activité pour les phlebovirus et donc mérite une surveillance particulière à cause du risque d’émergence et de dissémination. / Phleboviruses have a worldwide distribution. In the areas where sand flies are present, some of the sandfly-borne phleboviruses cause febrile illness and central nervous system infections. Sandfly fever was first reported in the Balkan Peninsula at the end of the 19th century. Since there is accumulating data showing that the Balkan peninsula plays a major role in the emergence of vector-borne diseases. At the outset of this work, a very limited number of phleboviruses had been identified and isolated in this region. To fill this gap, an integrated and transdisciplinary study was designed aiming at an inventory of viruses circulating in Balkans and associated seroprevalence studies using domestic animals: (i) a total of 3,850 sandflies were collected in seven Balkan countries (Albania, Bosnia-Herzegovina, Croatia, Kosovo, Montenegro, Republic of Macedonia and Serbia) in 2014 and 2015. They were tested for the presence of viral RNA and inoculated on VERO cell for virus isolation; (ii) seroprevalence studies using neutralisation tests were performed on cattle and sheep samples to assess the level of exposure to two human pathogens, Toscana virus (TOSV) and Sandfly fever Sicilian virus (SFSV). Our results consist of (i) the discovery and sequencing of 3 novel phleboviruses belonging to 2 different species, (ii) the identification for the first time of TOSV lineage B in Croatia, (iii) evidence of co-circulation of two lineages (Lineage B and C) of TOSV, (iv) rates of neutralising antibodies that are much higher in cattle and sheep for SFSV than for TOSV. Together the findings obtained during this work demonstrate that the Balkan area is a hot spot for phleboviruses.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / African swine fever is a hemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV), a double-stranded DNA virus and the only member of the family Asfarviridae. The structure of this multilayer virion contains more than 34 proteins including the protein p72 which is the major capsid protein. A single conformational neutralizing epitope has been identified on p72, but information on the other antigenic regions (epitopes) is lacking. The objective of this study was to identify p72 epitopes using polyclonal swine sera and a panel of monoclonal antibodies with the ultimate goal being the development of a blocking ELISA assay for the detection of anti-ASFV antibodies. The segment of the p72 protein from amino acids 1 to 345 was divided into five overlapping fragments which were then commercially synthesized. These fragments were cloned into the pHUE expression vector and transformed into Escherichia coli competent cells. The recombinant proteins were expressed in vitro, purified, and used as antigens in indirect ELISAs and western blots to test monoclonal antibodies and polyclonal swine sera. The monoclonal antibodies were produced against the p72 protein based on the ASFV Georgia/07 strain. The polyclonal sera were obtained from pigs immunized with a defective alphavirus replicon particle, RP-sHA-p72, expressing a recombinant protein composed of the extracellular domain of the ASFV HA protein together with the whole p72 protein. The polyclonal sera reacted to p72 in two distinct regions: between amino acids 1 and 83 and between amino acids 250 and 280. The anti-p72 reactive monoclonal antibodies reacted with p72 in three regions: between amino acids 100 and 171, amino acids 180 and 250, and amino acids 280 and 345. Fine mapping with oligopeptides allowed for the identification of six different linear epitopes. Among the monoclonal antibodies selected for blocking assay development, two have been shown to be promising candidates for further evaluation using sera from ASFV-infected pigs.