A leptospirose bovina é uma das principais doenças reprodutivas que interfere diretamente nos índices de produção e produtividade da pecuária brasileira e mundial e por isso necessita de um aprimoramento do diagnóstico laboratorial. O objetivo do presente trabalho foi investigar a conveniência do emprego de estirpes de leptospiras autóctones isoladas no Brasil, na coleção de antígenos da microtécnica de soroaglutinação microscópica (SAM) aplicada a leptospirose. A coleção de antígenos de referência foi constituída pelos sorovares: Australis, Bratislava, Autumnalis, Butembo, Castellonis, Bataviae, Canicola, Whitcombi, Cynopteri, Grippotyphosa, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Javanica, Panama, Pomona, Pyrogenes, Hardjo, Wolffi, Shermani, Tarassovi, Patoc e Sentot. As dez estirpes isoladas no Brasil incluíram os sorovares: Bananal (duas estirpes), Brasiliensis, Canicola (três estirpes), Copenhageni, Guaricura e Pomona (duas estirpes). Foram amostradas por conveniência, 109 propriedades e 9820 bovinos, fêmeas em idade de procriar, distribuídos em 84 municípios, dos Estados de: Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Rio Grande do Sul, Santa Catarina e São Paulo. Dos 9820 animais examinados, 5806 (59,12%) foram reagentes na SAM para qualquer sorovar com a coleção de 23 sorovares de referência. Com a coleção de antígenos de referência e dez estirpes autóctones houve 6400 (65,17%) reagentes, a diferença observada foi significante (p= 0,001). O único Estado em que não houve diferença significante no número de animais reatores para qualquer sorovar foi o de Santa Catarina (p=0,522). Das 109 propriedades trabalhadas, 106 foram consideradas positivas com pelo menos um animal reagente na SAM. Não houve diferença no número de propriedades positivas segundo a coleção de antígenos empregada. Os sorovares mais prováveis identificados com a coleção de antígenos de referência foram Hardjo (43,03 %), Shermani (20 %), Wolffi (9,96%), Grippothyphosa (5,42%) e Pomona (4,28%). Com a coleção ampliada por dez estirpes isoladas no Brasil, os sorovares mais prováveis foram Hardjo (31,00%), Guaricura - M4/84 (22,50%), Shermani (15,43%), Wolffi (4,76%), Grippothyphosa (3,71%) e Autumnalis (3,24%). O sorovar Guaricura, estirpe M4/84, isolada de bovinos e búfalos no Estado de São Paulo, foi o primeiro colocado como sorovar mais provável no Estado de Mato Grosso, diferença significante do valor obtido para o sorovar Hardjo (p= 0,0001). No Mato Grosso do Sul a despeito do valor absoluto ter sido superior para o sorovar Guaricura, a diferença observada com o sorovar Hardjo foi destituída de significado estatístico (p=.0,753). Em São Paulo o Guraricura foi o segundo colocado e em Minas Gerais Goiás ocupou a terceira posição. Esta mesma estirpe foi a mais provável em 27 propriedades das 109 trabalhadas (24,77%). A introdução de estirpes autóctones na coleção de antígenos da SAM propiciou a confirmação do diagnóstico de leptospirose em 594 animais (6,00%) classificados como não reagentes pela coleção de referência (p=0,001) / Bovine leptospirosis is one of the main reproductive diseases which interferes economically on the livestock and thus it is needed the amelioration of the laboratory diagnostic procedures. The aim of this work is to investigate the adequacy to use autochthonous strains of leptospires isolated in Brazil, added into the antigen collection applied for the microscopic agglutination test (MAT). The reference antigen collection was constituted by the following serovars: Australis, Bratislava, Autumnalis, Butembo, Castellonis, Bataviae, Canicola, Whitcombi, Cynopteri, Grippothyphosa, Hebdomadis, Copenhageni, Icterohaemorrhagiae, Javanica, Panama, Pomona, Pyrogenes, Hardjo, Wolffi, Shermani, Tarassovi, Patoc and Sentot. The ten strains isolated in Brazil included the serovars: Bananal (two strains), Brasiliensis, Canicola (three strains), Copenhageni, Guaricura and Pomona (two strains). By means of non-probability sampling, 109 farms and 9,820 bovines, females at reproductive age were chosen from the 84 municipalities of the states of Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Rio Grande do Sul, Santa Catarina e São Paulo . Among the 9,820 examined animals, 5,806 (59.12%) were reactants to the MAT for any serovar using the 23 reference serovars. Using the collection of reference serovars and the ten autochthonous strains there were 6,400 (65.24%) reactants, and the difference found was significant (p=0.001). The only state with non-significant results in the number of reactants for any serovar was the State of Santa Catarina (p=0.522). Of the 109 properties analyzed, 106 were considered positive at least with one reactant animal by MAT. According to the antigens used, there was no significant difference among the properties. The most probable serovars identified by the collection of reference antigens were Hardjo (43.03%), Shermani (20.00%), Wolfi (9.96%), Grippothyphosa (5.42%) and Pomona (4.28%). With the collection amplified with the ten strains isolated in Brazil, the most probable serovars were Hardjo (31.00%), Guaricura M4/84 (22.50%), Shermani (15.43%), Wolffi (4.76%), Grippothyphosa (3.71%) and Autumnalis (3.24%). The serovar Guaricura, strain M4/84, isolated from bovines and buffaloes in the State of São Paulo, was ranked at the first place as the most probable serovar in the State of Mato Grosso and Mato Grosso do Sul, but the differences observed with the serovar Hardjo were significant only in Mato Grosso (p=0,0001). In São Paulo State, the Guaricura serovar was the second most probable and in the states of Minas Gerais and Goiás the third one. This same strain was the most probable in 27 properties among the 109 examined (24.77%). The addition of autochthonous strains into the MAT antigen collection provided the confirmation of the diagnosis of leptospirosis in 594 animals (6.00%) which have been classified as non-reactants by the reference collection

Lab-on-a-chip systems for point-of-care testing demonstrate a promising development towards more accurate diagnostic tests that are of extreme importance for the future global health. This work presents an agglutination assay performed in micrometer sized well using Janus PS/Ag/AgCl micromotors to enhance the interactions between goat anti-human IgM functionalized particles and Human IgM. The fabricated microwell chips are a suitable platform to analyze the interaction between different particles and to perform the agglutination assays. The interaction between active Janus particles and passive and functionalized particles is studied, as well as the influence of ions on the motion of the Janus particles. Agglutination assays are performed with and without the presence of Janus particles, and in different PBS concentrations. Once illuminated with blue light, passive SiO2 particles were effectively excluded from Janus particles, while SiO2 NH2 particles revealed attraction. In contrast, functionalized SiO2 NH2 Ab particles suspended in PBS did not show any interaction. It was found that the optimal working conditions for antibodies and Janus particles differed and, as a result, the Janus particles did not reveal a desirable interaction between the functionalized particles and IgM. Further experiments should be performed to find the proper conditions in which the antibodies and the Janus particles maintain their activities. It is believed that an effective interaction between the functionalized and Janus particles could be achieved by modifying the parameters that affect their interaction such as the zeta potential and the medium in which the assay is being performed. This preliminary work provides the first steps towards the development of a fully integrated lab on a chip system for point of care testing.:Abstract ........................................................................................................................ iii Acknowledgments.......................................................................................................... v Table of Contents .......................................................................................................... vi List of Tables ............................................................................................................. viii List of Figures ............................................................................................................... ix Abbreviations ................................................................................................................. x 1. Introduction ............................................................................................................ 1 1.1 In vitro diagnostic tests ........................................................................................ 1 1.1.1 Point-of-care tests ......................................................................................... 2 1.2 Agglutination assay .............................................................................................. 2 1.3 Lab-on-a-chip ....................................................................................................... 5 1.4 Self-propelled particles ........................................................................................ 6 1.4.1 Light-driven Ag/AgCl micromotors ............................................................. 6 1.5 Aim ...................................................................................................................... 9 2. Materials and Methods ......................................................................................... 11 2.1 Microwell fabrication .................................................................................... 11 2.2 Microswimmers fabrication .......................................................................... 12 2.3 Functionalization of particles ........................................................................ 12 2.4.1 Scanning electron microscope ............................................................... 14 2.4.2 UV-vis spectroscopy .............................................................................. 14 2.4.3 Zeta potential ......................................................................................... 14 2.4.4 Optical microscopy ................................................................................ 15 2.5 Motion Experiments ...................................................................................... 15 2.6 Agglutination assay ....................................................................................... 16 2.7 Effect of PBS ................................................................................................. 16 2.7.1 Janus particles ........................................................................................ 16 2.7.2 Agglutination assay ................................................................................ 17 2.7.3 Exclusion of functionalized particles ..................................................... 17 3. Results and Discussion ........................................................................................ 18 3.1 Microwell chip with integrated Janus particles ................................................. 18 3.2 Characterization of particles .............................................................................. 19 3.2.1 UV-vis spectroscopy ................................................................................... 19 3.2.2 Zeta potential .............................................................................................. 21 3.2.3 Agglutination assay in PEG-covered glass slides ....................................... 22 3.3 Motion experiments ........................................................................................... 23 3.3.1 Exclusion time ............................................................................................ 23 3.3.2 On/off light cycles....................................................................................... 26 3.4 Agglutination assay ............................................................................................ 28 3.4.1 Assay performed in wells............................................................................ 28 3.4.2 Assay performed in wells with Janus particles ........................................... 29 3.5 Effect of PBS concentration............................................................................... 30 3.5.1 Janus particles ............................................................................................. 30 3.5.2 Agglutination assay ..................................................................................... 32 3.5.3 Exclusion of functionalized particles .......................................................... 33 4. Conclusions .......................................................................................................... 35 References .................................................................................................................... 37 Declaration of Research Integrity and Good Scientific Practice ................................. 42

Orientador: Iveraldo dos Santos Dutra / Banca: Samir Issa Samara / Banca: Vera Cláudia Lorenzetti Magalhães Curci / Resumo: O carbúnculo sintomático é um problema sanitário mundial, responsável por elevados coeficientes de mortalidade em bovinos e ovinos. A imunização dos animais jovens, seguida de reforço anual até 2,5 anos de idade, é a principal medida profilática. Foram realizados três experimentos distintos com intuito de avaliar as respostas sorológicas de bovinos vacinados contra o carbúnculo sintomático, pelos testes de aglutinação em placa e Elisa, empregando-se como antígenos a cepa de referência (MT) e uma cepa de campo (SP). No primeiro experimento, os bezerros foram organizados em três grupos (G1, G2 e G3) e submetidos a três protocolos distintos de vacinação empregando-se uma vacina comercial polivalente contra clostridioses. O G1 foi primovacinado aos 4 meses de idade e recebeu o reforço na desmama (8 meses). O G2 recebeu a primeira dose na desmama e reforço 30 dias após. O G3 foi vacinado somente na desmama. As coletas de soro foram realizas aos 4, 8, 9 e 10 meses de idade dos bezerros. O G1 apresentou a melhor resposta sorológica em comparação aos outros dois protocolos. Quando a avaliação dos grupos foi realizada aos 10 meses de idade, independente do protocolo empregado, a resposta sorológica foi similar. No segundo experimento, foi avaliada a imunidade natural passiva de bezerros, filhos de vacas vacinadas até 30 dias antes do parto (2ª dose), empregando-se duas vacinas comercias polivalente contra clostridioses. As coletas de soro foram realizadas aos (±)7, 45 e 90 dias de idade dos bezerros. Independente das vacinas empregadas na imunização ativa das mães, a resposta sorológica passiva dos bezerros avaliados foi similar até os 3 meses de idade. Houve uma correlação linear da resposta sorológica passiva dos bezerros com a data de vacinação das mães e o dia do parto quando empregado o teste de Elisa. No terceiro experimento, as 30 vacas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Black leg disease is one of the most important sanitary problem, responsible for high levels of mortality observed in bovines and ovines herds. The vaccination of young animals, followed by annual booter until 2,5 years-old, is the major preventive measure against outbreaks. Three distinct experiments were conducted to measure the vaccinal response from bovines. The vaccinal strains used were the reference MT and field Clostridium chauvoei isolated. Sera from vaccinated animals were tested by agglutination and Enzyme Linked Immunosorbent Assay (Elisa), both standardized for the present study. First experiment, calves were divided into three groups (G1, G2 and G3); and submitted to three vaccination schedule with a polyvalent vaccine. The G1 received first vaccine at 4 months of age and a subsequent booster after calving (8 month-old). The G2 received first vaccine dose after calving and booster at 30 days after. The G3 received only one vaccine dose at 8 months. The sera were colleted at 4, 8, 9 and 10 months for all groups studied. The G1 group showed the best serological response at 10 months of age in comparison to G2 e G3 and control. Moreover, at 10 months of age all groups presented similar levels of serological response. The second experiment, the natural immunity of calves, separated from their mothers vaccinated 30 days before calving with two polyvalent vaccines. The respective serum was colleted at (±) 7, 45 and 90 days of age. All calves presented similar serological response at 3 months of age, independent of vaccinal strain used. The third experiment, 30 heifers, Nelore race, aged above 4 years-old, without vaccination against black leg, were vaccinated with two Clostridium strains. When the SP strain was used the serological response was considered good in G3 (first experiment), second and third experiment for agglutination assay. To compare both techniques, agglutination... (Complete abstract click electronic access below) / Mestre

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-03-15T12:30:54Z No. of bitstreams: 2 Tese - Juliana Bonifacio de Alcantara - 2015.pdf: 1563501 bytes, checksum: a96b02c76ef8d17bbaa4c2f2bf2838fa (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-16T11:22:38Z (GMT) No. of bitstreams: 2 Tese - Juliana Bonifacio de Alcantara - 2015.pdf: 1563501 bytes, checksum: a96b02c76ef8d17bbaa4c2f2bf2838fa (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-16T11:22:38Z (GMT). No. of bitstreams: 2 Tese - Juliana Bonifacio de Alcantara - 2015.pdf: 1563501 bytes, checksum: a96b02c76ef8d17bbaa4c2f2bf2838fa (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-08-13 / Salmonella sp. might cause food deseases in humans and it be isolated from the gastrointestinal tract of different animal species, especially birds. In this study, we aimed to point out the main characteristics of non-conventional poultry farm systems at Central and South regions of the State of Goiás and to determine the frequency of Salmonella sp. Isolation in conventional and non-conventional poultry farms, as well as the frequency of positivity for antigen-antibody reaction at non-conventional poultry farms. On paper 1, we studied 190 non-conventional systems of broiler rearing; we collected 3,040 blood and organ samples (heart, liver, crop and cecum) from 760 birds. Rapid Plate Agglutination Test was used for the detection of anti-Salmonella sp. antibodies in blood serum, and conventional bacteriology and biochemistry tests were used for bacterium culture and isolation. Data on the characteristics of the properties were obtained through medical records. The poultry rearing systems were classified as semi-intensive (49.0%) and extensive (42.6%). In these breeding systems, 48.0% were of specific breed free-range chickens and 42.0% of rustic rustic free-range chickens; 74.2% of the farms had commercial purpose. The frequency of properties with chicken seropositive to the anti-Salmonella sp. antigen was 16.3%, and 12.0% of the samples. Salmonella was detected in 4.7% of the properties and the identified serotypes were Anatum, Infantis, Mbandaka, Schwarzengrund and Panama. For Paper 2, we studied 44 flocks of chickens from nine poultry slaughterhouses, three with over 51,000 birds slaughtered/day and six with up to 50,000 birds slaughtered/day. On the slaughter line 1,232 organ samples and feathers were harvested. A total of 21 feather samples and 21 samples of each organ (spleen, crop and cecum) was collected, both of them were analyzed by conventional bacteriology and biochemistry tests. The organ samples were processed in groups of three, in a total of seven samples of each organ. Of the 44 flocks of chickens, 22 were positive for Salmonella. The feathers presented a higher positivity frequency (12.3%). The spleen presented the highest frequently of isolates (8.1%). The frequency of positive samples in both crop and cecum was 3.8%. Among 88 Salmonella isolates, the serovars Schwarzengrund (29.5%), Agona (25.2%), Mbandaka (12.6%), Anantum (8.0%) and Infantis (3.4%) were predominant. In conclusion, the non-conventional designs are characterized as semi-intensive, and use improved lineage of chickens for commercial purposes. Regardless of the levels of contamination, Salmonella sp. is present in chickens from both conventional and non-conventional production systems. The pathogen was identified in greater numbers in chicken organs at conventional farm. In non-conventional breeding samples, Salmonella sp. isolation was low, but the number of seropositive chickens was higher. The serovars identified in samples of both conventional and non-conventional systems were similar, and some of relevance to public health. / Salmonella sp. podem causar toxinfecção alimentar no homem e ser isolada do trato gastrintestinal de diferentes espécies animais, principalmente das aves. Objetivou-se com este estudo caracterizar os sistemas alternativos de criações avícolas nas regiões Central e Sul do Estado de Goiás e determinar a frequência de isolamento de Salmonella sp. em aves de criação alternativa e industrial, assim como a frequência de positividade da reação antígeno-anticorpo em soro de aves de criação alternativa. No artigo 1 estudou-se 190 propriedades de criações alternativas de frangos e em 760 aves colheram sangue e 3.040 amostras de órgãos, sendo coração, fígado, ceco e inglúvio. Realizou-se o teste de Soroaglutinação Rápida em Placa para detecção de anticorpos anti Salmonella sp. em soro sanguíneo e para cultura e isolamento da bactéria ultilizou-se bacteriologia convencional e provas bioquímicas. Os dados sobre as características das propriedades foram obtidos através de ficha de atendimento. As criações avícolas foram classificadas em semi-intensivas 49,0% e 42,6% extensivas. Observou-se que nestas criações as aves criadas são do tipo caipira melhorado 48,0% e 42,0% do tipo caipira rústico, e 74,2% das explorações têm a finalidade de comercialização. A frequência de propriedades com aves sororreagentes ao antígeno anti Salmonella sp. foi de 16,3%, e em amostras de 12,0%. Detectou-se Salmonella em 4,7% das propriedades e os sorovares identificados foram Anatum, Infantis, Mbandaka, Schwarzengrund e Panama. Para o artigo 2 o estudo foi realizado em 44 lotes de frangos de criação industrial de nove abatedouros, três com abate dia acima de 51.000 aves e seis com abate dia de até 50.000 aves dia. Na linha de abate colheram-se 1.232 amostras de órgão e penas. Num lote foram colhidas 21 amostras de penas e 21 amostras de cada órgão, sendo baço, ceco e inglúvio, ambas analisadas por bacteriologia convencional e provas bioquímicas. As amostras de órgãos foram processadas em pool de três, totalizando sete amostras para cada órgão. Dos 44 lotes de frangos, 22 foram positivos para Salmonella. As penas apresentaram maior frequência de amostras positivas (12,3%) e o baço foi o órgão com maior frequência de isolados (8,1%). A frequência de amostras positivas tanto para inglúvio quanto para ceco foi de 3,8%. Dentre os 88 isolados de Salmonella houve predominância do sorovar Schwarzengrund (29,5%), Agona (25,2%), Mbandaka (12,6%), Anantum (8,0%) e Infantis (3,4%). Conclui-se que as criações alternativas se caracterizam como do tipo semi-intensiva, utilizam aves de linhagem caipira melhorado com finalidade comercial. Independente dos níveis de contaminação, salmonella sp. está presente em aves dos sistemas industriais e alternativos de produção. O patógeno foi identificado em maior número em órgãos de frango de criação industrial. Em aves de criações alternativas obteve-se baixa frequência de isolamento do patógeno, mas obteve-se maior número de aves sororreagente. Observa-se que os sorovares identificados tanto em amostras de frango do sistema alternativo como do sistema industrial foram semelhantes, e alguns de relevância em saúde pública.

<p>Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. </p><p>The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. </p><p>Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.</p>

Prostasomes are extracellularly occurring organelles which are secreted in human semen by the prostate gland. Prostasomes have several known biological activities, but their physiological function is still unclear. In this thesis some new aspects were studied on the biological role of the prostasomes. The motility-stimulatory effect of prostasomes on cryopreserved spermatozoa was further studied by supplementing the swim-up medium with seminal prostasomes, and with prostasomes purified from a PC-3 prostate cancer cell line (PC-3 prostasomes), on fresh spermatozoa. The recovery of motile spermatozoa after swim-up increased by 50% when the swim-up medium was supplemented with prostasomes. The PC-3 prostasomes bore a functional resemblance to seminal prostasomes as regards various expressions of sperm motility promotion. Prostasomes proved to have potent antibacterial effects. The effects were not strictly confined to Bacillus megaterium since a few other bacteria were also sensitive. The high percentage of patients with anti-prostasome antibodies showed that prostasomes could be one of the major targets for antisperm antibodies (ASA). The results demonstrate that ASA in serum of infertile men and women recognise prostasomes as antigens, and that polyclonal antibodies raised against prostasomes agglutinate human spermatozoa. This suggests that prostasomes contribute at least partly to immunological infertility. Three types of prostasomes (seminal-, native- and metastasis-derived prostasomes) demonstrated similarities regarding a high cholesterol/phospholipid ratio and some marker enzymes. The conclusion is that prostasomes have a common and exclusive prostatic origin in man and that they are internalised in storage vesicles of the secretory cells and released in toto by an ordinary exocytotic event.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.