Au cours des deux dernières décennies, il a été démontré que les maladies émergentes et réémergentes sont liées à la santé humaine, animale et environnementale. Les infections zoonotiques sont reconnues comme étant responsables d'au moins 75 % des éclosions d'agents pathogènes dans le monde. Certains virus peuvent muter et infecter un large éventail d'hôtes, qui se propagent parmi les humains et entraînent des épidémies ou des pandémies. Les chauves-souris sont connues pour être les mammifères les plus diversifiés géographiquement et le plus répandu sur Terre et peuvent être trouvées à l'intérieur structures abandonnées, ainsi que de grottes. Au Canada, il existe dix-huit espèces de chauves-souris insectivores et huit d'entre elles se perchent au Québec. Elles peuvent loger beaucoup de pathogènes pendant l'hibernation en raison de leur métabolisme distinct. Elles peuvent aussi excréter beaucoup de particules virales par différentes voies telles que la salive, les excréments et l'écholocation. Le but de ce projet de maîtrise est de caractériser les virus que les chauves-souris pourraient potentiellement transporter au Canada, en mettant l'accent sur la détection des Coronavirus. Le premier objectif est de traiter 250 échantillons (matières fécales et organes) prélevés sur des chauves-souris sauvages, puis d'utiliser d'autres techniques biomoléculaires comme le NGS pour détecter un large spectre de virus chez ces chauves-souris. Les échantillons ont été dépistés pour les Coronavirus et les Rhabdovirus à l'aide de la PCR conventionnelle. La prévalence des Coronavirus chez les chauves-souris semble actuellement être relativement faible au Canada et de nombreux facteurs, y compris le petit nombre d'échantillons prélevés jusqu'à présent, pourraient y avoir joué un rôle. Il serait important de continuer à examiner les échantillons de chauves-souris à plus grande échelle afin de caractériser pleinement les viromes que ces animaux hébergent, pour fournir un avertissement des menaces d'épidémie ou de pandémie à la première occasion. / Over the past two decades, emerging and re-emerging diseases have been shown to be interlinked between human, animal, and environmental health. Zoonotic infections are recognized to be responsible for at least 75% of pathogen outbreaks in the world. Certain viruses can mutate and infect a wide range of hosts, which spread amongst humans and lead to epidemics/pandemics. Bats are known to be the most geographically diverse and widespread mammal on Earth and can be found inside of buildings and houses or abandoned structures, as well as caves. In Canada, there are eighteen species of insectivorous bats and eight of them roost in Québec. They can also harbor plenty of pathogens during hibernation due to their distinct metabolism. They can shed plenty of pathogens through different pathways such as saliva, excreta, and echolocation. The overall goal of this master's project is to characterize the pathogens that bats could be potentially carrying in Canada, with a focus on Coronavirus detection. The first objectiveis to process 250 samples (feces and organs) collected from wild bats in the field in Canada and then using other biomolecular techniques such as NGS to detect for the presence of other bat pathogens. The samples were screened for Coronaviruses and Rhabdoviruses using conventional PCR with no positive results. The prevalence of Coronaviruses in bats currently appear to be relatively low in Canada and, many factors, including the moderately low numbers of samples collected in Canada so far, may have played a role. It will be important to continue collecting and screening bat samples on a larger scale to fully characterize the viromes that these animals harbour, to provide warning of epidemic/pandemic threats at the earliest opportunity

Potato yellow dwarf virus (PYDV) is the type member of the genus Nucleorhabdovirus. The virus replicates in the nuclei of infected cells and mature virions accumulate in the perinuclear space after viral cores bud through the inner nuclear membrane. The virus was first described as an extremely destructive pathogen of potato (Solanum tuberosum) and other members of family Solanaceae. There are two different strains of PYDV based on their insect-vector specificity, namely SYDV (sanguinolenta strain) and CYDV (constricta strain). PYDV is considered a model system to study virus-vector relationship, particularly for agriculturally harmful rhabdoviruses. However, very little is known about the molecular aspects and cell biology of PYDV. Preliminary studies showed that infection of transgenic Nicotiana benthamiana plants that constitutively express GFP targeted to endomembranes with SYDV and SYNV (Sonchus yellow net virus, another member of genus Nucleorhabdovirus) results in increased accumulation of GFP and membrane within the infected nuclei, though the pattern of GFP accumulation is completely different for the two viruses. GFP accumulation was found mainly in the external and internal loci of the nucleus in SYDV-infected cells, where as, in the case of SYNV infection, the GFP accumulation was scattered throughout the nucleus of the infected cell. Molecular characterization of SYDV was undertaken to better understand the cellular difference between these two members of Nucleorhabdoviruses. This dissertation describes the determination of the complete nucleotide and ORF (open reading frame) sequences of N (nucleocapsid) and P (Phosphoprotein) gene of SYDV from cDNA clones of both viral genomic and messenger RNAs. Analyses of sequence showed that SYDV-N mRNA contains an 11 nucleotide (nt) untranslated region followed by a 1416 nt ORF encoding a 472 amino acid (aa) protein and P-mRNA contains an 18 nt 5 untranslated region followed by 840 nt ORF encoding a 280 aa protein. Characterization of SYDV-N and P protein using bioinformatic algorithms predict basic hydrophilic and coiled coil regions that may posses the putative nuclear localization signal and protein-protein interaction domain, respectively. Comparison of the SYDV-N ORF with orthologous regions from other plant and animal rhabdoviruses showed statistically significant identity. Phylogenetic analysis based on consensus N-ORFs placed SYDV into the same group with other Nucleorhabdoviruses. Localization studies of SYDV-N and P protein as autofluorescent protein fusions revealed that both proteins are exclusively nuclear localized. Taken together, this dissertation reports a detailed analysis of the biology of SYDV-N and P protein at the molecular and cellular level for the first time towards the long term goal to characterize the entire SYDV genome and to better understand SYDV-host interaction

No final da década de noventa, a mancha anular do cafeeiro voltou a manifestar seus sintomas, após muito tempo desde a sua constatação. Este fato pode estar relacionado com as condições climáticas do período, manejo nutricional e fitossanitário adotado pelos produtores. O objetivo deste trabalho foi investigar a relação entre determinados nutrientes fornecidos ao cafeeiro e a ocorrência do ácaro B. phoenicis, transmissor do vírus da mancha anular do cafeeiro, (MAC) e os sintomas dessa virose. Para tanto, foram conduzidos ensaios nas regiões de Espírito Santo do Pinhal - SP e Araguarí- MG, onde foram testados os nutrientes B, Zn e S via solo, separadamente e associados, relacionando-os à população do ácaro B. phoenicis e aos sintomas da virose, quantificados através de três avaliações mensais. Pôde-se verificar que plantas bem nutridas em enxofre e zinco com doses baixas de B podem atuar como agente controlador do ácaro avaliado. A utilização de acaricidas, interferindo na população deste vetor, também, foi observada comparando-o aos diferentes nutrientes testados. Utilizando-se plantas na ausência desses micronutrientes, com a aplicação via foliar de Carbax (dicofol 160g/l + tetradifon 60g/l) houve redução da população de ácaros, porém aumentou o número de ovos encontrados. O efeito do acaricida foi superior ao do nutriente sobre o controle do ácaro plano. Coletando-se várias amostras de solo em áreas com plantas sobre forte manifestação dos sintomas da virose (reboleiras) e adjacentes sob um grau de infestação menor e após analisá-las, pode-se observar uma menor ocorrência destes sintomas ou de seu vetor, onde era maior o nível de enxofre. A desfolha, em plantas marcadas com e sem aplicação de enxofre, foi quantificada após a eliminação das folhas caídas e com a contagem após 5 dias de novas folhas como, também, os frutos caídos. As plantas tratadas com enxofre apresentaram índice de desfolha menor. As folhas caídas estavam mais atacadas pela virose do que pelo bicho mineiro que também provoca queda de folhas e é uma praga chave do cafeeiro. A queda de frutos, diferente do ocorrido com a desfolha foi ligeiramente maior nas parcelas que receberam a aplicação de enxofre. Utilizando-se band-aids, com diferentes doses de enxofre, aplicados em plantas de café, observou-se que este elemento exerce um efeito sobre a presença do ácaro reduzindo assim a ocorrência da MAC, e nas maiores doses aplicadas foi encontrado o menor número de ácaros. Tais estudos evidenciaram que a presença de enxofre no solo, e sua aplicação em várias doses e de diferentes formas influenciaram o seu teor no tecido vegetal e consequentemente a presença do ácaro vetor e os sintomas causados, às plantas devido a ocorrência do vírus. Os resultados obtidos neste trabalho indicam que culturas bem nutridas e principalmente rica em enxofre, podem apresentar tolerância ao ataque do vetor da MAC, e suas manifestações / not available

No final da década de noventa, a mancha anular do cafeeiro voltou a manifestar seus sintomas, após muito tempo desde a sua constatação. Este fato pode estar relacionado com as condições climáticas do período, manejo nutricional e fitossanitário adotado pelos produtores. O objetivo deste trabalho foi investigar a relação entre determinados nutrientes fornecidos ao cafeeiro e a ocorrência do ácaro B. phoenicis, transmissor do vírus da mancha anular do cafeeiro, (MAC) e os sintomas dessa virose. Para tanto, foram conduzidos ensaios nas regiões de Espírito Santo do Pinhal - SP e Araguarí- MG, onde foram testados os nutrientes B, Zn e S via solo, separadamente e associados, relacionando-os à população do ácaro B. phoenicis e aos sintomas da virose, quantificados através de três avaliações mensais. Pôde-se verificar que plantas bem nutridas em enxofre e zinco com doses baixas de B podem atuar como agente controlador do ácaro avaliado. A utilização de acaricidas, interferindo na população deste vetor, também, foi observada comparando-o aos diferentes nutrientes testados. Utilizando-se plantas na ausência desses micronutrientes, com a aplicação via foliar de Carbax (dicofol 160g/l + tetradifon 60g/l) houve redução da população de ácaros, porém aumentou o número de ovos encontrados. O efeito do acaricida foi superior ao do nutriente sobre o controle do ácaro plano. Coletando-se várias amostras de solo em áreas com plantas sobre forte manifestação dos sintomas da virose (reboleiras) e adjacentes sob um grau de infestação menor e após analisá-las, pode-se observar uma menor ocorrência destes sintomas ou de seu vetor, onde era maior o nível de enxofre. A desfolha, em plantas marcadas com e sem aplicação de enxofre, foi quantificada após a eliminação das folhas caídas e com a contagem após 5 dias de novas folhas como, também, os frutos caídos. As plantas tratadas com enxofre apresentaram índice de desfolha menor. As folhas caídas estavam mais atacadas pela virose do que pelo bicho mineiro que também provoca queda de folhas e é uma praga chave do cafeeiro. A queda de frutos, diferente do ocorrido com a desfolha foi ligeiramente maior nas parcelas que receberam a aplicação de enxofre. Utilizando-se band-aids, com diferentes doses de enxofre, aplicados em plantas de café, observou-se que este elemento exerce um efeito sobre a presença do ácaro reduzindo assim a ocorrência da MAC, e nas maiores doses aplicadas foi encontrado o menor número de ácaros. Tais estudos evidenciaram que a presença de enxofre no solo, e sua aplicação em várias doses e de diferentes formas influenciaram o seu teor no tecido vegetal e consequentemente a presença do ácaro vetor e os sintomas causados, às plantas devido a ocorrência do vírus. Os resultados obtidos neste trabalho indicam que culturas bem nutridas e principalmente rica em enxofre, podem apresentar tolerância ao ataque do vetor da MAC, e suas manifestações / not available

Piscicultura de água doce consiste na maior produção da aquicultura mundial e o cultivo de peixes tem crescido, devido a investimento, desenvolvimento de tecnologias e à contínua expansão de espécies cultivadas. Esse aumento gerou novas possibilidades de transmissão de vírus aquáticos, fatores limitantes à produção da aquicultura. Dentre as centenas de vírus conhecidos de peixes, um dos principais, que atualmente é de preocupação internacional, é o vírus da Viremia Primaveril da Carpa (SVCV), devido a sua importância socioeconômica considerando os países afetados e o comércio internacional de animais aquáticos e seus derivados, com base na saúde e medidas preventivas. SVC é uma doença aguda causada por um rhabdovírus do gênero Vesiculovirus, que é um vírus de ácido ribonucleico (RNA) fita simples linear de aproximadamente 11 quilobases (kb) com polaridade negativa e envelopado. Diversos ensaios diagnósticos podem ser utilizados para detectar SVCV, porém, consomem tempo e não são adaptados para diagnóstico a campo. A prevenção da disseminação do vírus é crucial; portanto, maior entendimento do vírus e de sua transmissão é necessário. No presente estudo, a padronização de uma RT-nestedPCR foi realizada, cujo limite de detecção foi de 8,62 × 10-2 cópias/reação, observado após o spiking de tecidos de peixes com diluições seriadas de um controle positivo sintético. O ensaio foi aplicado a 150 amostras teciduais provenientes de 146 peixes distintos. As amostras consistiam de fragmentos de fígado, rim e baço e foram submetidas à transcrição reversa (RT), seguida pela reação em cadeia de polimerase (PCR), em configuração nested. Duas (2) amostras foram positivas para o gene G de SVCV pela RT-nestedPCR e a identidade dos produtos obtidos foi confirmada por sequenciamento direto utilizando-se o método de Sanger. Na reconstrução filogenética, as sequências obtidas formaram clado único, separado dos demais genogrupos e mesmo de sequências derivadas de outros vesiculovírus encontrados no Brasil. Esses resultados determinam a primeira detecção e caracterização moleculares de SVCV no Brasil por RT-nestedPCR e sequenciamento nucleotídico, indicando a necessidade de adoção de medidas de biosseguridade mais restritivas na produção pesqueira nacional. / Fresh-water fish farming forms the largest portion of the world aquaculture production, and the harvest of these warm-water fish is increasing, due to investment, improvement of technologies and continued expansion of cultivated species. This increase has rendered new possibilities for the transmission of aquatic viruses, which remain limiting factors for aquaculture production. Among the hundreds of known viruses of fish, one of the main viruses that are currently of international concern is the Spring Viremia of Carp virus (SVCV) due to its socio-economic importance regarding affected countries and international trade of aquatic animals and their products, on the basis of health control and preventive measures. SVC is an acute disease, caused by a rhabdovirus, belonging to the Vesiculovirus genus, and is an enveloped virus, with a linear single-strained negative-sense ribonucleic acid (RNA) of approximately 11 kilobases (kb). Several diagnostic assays are available for the detection of SVCV, but they are time-consuming and not well adapted for field diagnosis. Prevention of spreading of the virus is crucial; therefore, more understanding of the virus and its transmission is required. In this study, standardization of a RT-nestedPCR was performed, and its detection limit was of 8.62 × 10-2 copies/reaction, observed after spiking fish tissue with serial dilutions of a synthetic positive control. The assay was applied to 150 tissue samples from 146 different fish. Samples consisted of liver, kidney and spleen fragments, and underwent reverse transcription (RT) followed by the polymerase chain reaction (PCR) of a nested configuration. Two (2) tissue samples were found positive for the G gene of SVCV by RT-nestedPCR and the identity of products was confirmed by direct sequencing using the Sanger method. By phylogenetic reconstruction, the obtained sequences formed a unique clade, separating them from the other known genogroups, and even from sequences derived from other vesiculoviruses found in Brazil. These results represent the first molecular detection and characterization of SVCV in Brazil by RT-nestedPCR and nucleotide sequencing, indicating the need to adopt more stringent biosecurity measures in national fish farming production.

La fusion membranaire est un processus biologique courant que l'on retrouve notamment lors de l'exocytose, du trafic intracellulaire ou de l'entrée des virus dans les cellules. Dans le cas des rhabdovirus, une famille où l'on retrouve notamment le virus de la rage (RV) et le virus de la stomatite vésiculaire (VSV),cette fonction est assurée à pH légèrement acide par la glycoprotéine G. Cette protéine existe sous au moins trois conformations distinctes : à pH neutre, elle est présente sous une conformation native (N). Après une brève incubation à pH acide, elle est présente sous une conformation activée (A), sous laquelle elle est capable d'interagir avec une membrane cible par l'intermédiaire d'un peptide hydrophobe. Enfin, après une incubation prolongée à pH acide, elle apparaît sous une conformation inactivée (I) et est alors incapable d'induire la fusion membranaire. Contrairement à toutes les autres familles virales étudiées à ce jour, il existe un équilibre dépendant du pH entre ces conformations. De nombreuses données dans divers systèmes suggèrent qu'une protéine unique ne serait pas suffisante pour catalyser les processus de fusion membranaire, mais qu'au contraire une machinerie constituée d'un nombre plus ou moins important de protéines fusogènes serait nécessaire. Au cours de ce travail, nous avons étudié certaines propriétés du complexe de fusion des rhabdovirus. Nous avons ainsi montré qu'il était de grande taille et qu il était probablement plus grand que ce qui avait été proposé pour d autres familles virales. De plus, il est apparu que le complexe de fusion du virus rabique n avait pas une unique architecture possible, mais qu il existait au contraire divers types de complexe. Ensuite, l'observation par microscopie électronique de particules virales en train de fusionner avec des liposomes nous a montré que le processus de fusion induit par VSV se produisait toujours par la base et qu il s accompagnait d une redisposition complète des glycoprotéines à la surface du virus suivant un réseau hélicoïdal. Enfin, nous sommes parvenu à isoler l ectodomaine de G et à en obtenir des cristaux diffractant à 3,5 Å, ce qui permet d envisager à terme une résolution de la structure de G. L étude de l interaction entre l ectodomaine de G et des liposomes nous a permis de reproduire les réseaux hélicoïdaux observés à la surface du virus et d étudier leurs propriétés. L ensemble de ces données nous a permis de proposer un nouveau modèle pour le processus de fusion chez les rhabdovirus, mais il pourrait également être pertinent pour d autres familles virales.

Sonchus yellow net virus (SYNV), Potato yellow dwarf virus (PYDV) and Lettuce Necrotic yellows virus (LNYV) are members of the Rhabdoviridae family that infect plants. SYNV and PYDV are Nucleorhabdoviruses that replicate in the nuclei of infected cells and LNYV is a Cytorhabdovirus that replicates in the cytoplasm. LNYV and SYNV share a similar genome organization with a gene order of Nucleoprotein (N), Phosphoprotein (P), putative movement protein (Mv), Matrix protein (M), Glycoprotein (G) and Polymerase protein (L). PYDV contains an additional predicted gene between N and P, denoted as X, that has an unknown function. In order to gain insight into the associations of viral proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps using novel plant expression vectors. Sub‐cellular localization was determined by expressing the viral proteins fused to green fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta using bimolecular fluorescence complementation (BiFC). All three viruses showed Mv to be localized to the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N‐P and M‐M interactions are common to all three viruses. Associations unique to only one virus include G‐Mv for SYNV, M‐Mv, M‐G, and N‐M for PYDV and P‐M for LNYV. The cognate N‐P proteins of all three viruses exhibit changes in localization when co‐expressed. To complement the mapping data, we also mapped the functional domains in the glycoproteins of SYNV and LNYV. The truncation of the carboxy terminus has no effect on localization compared to the full‐length protein; the nuclear localization signals (NLSs) present in SYNV‐G do not interact with known importins. These data suggest that although the interactions of the three viruses differ, each protein may have similar functional domains.

As an outcome of very active arbovirus monitoring programs that began in Australia in the 1950s, some of the most diverse and unusual rhabdoviruses in the world have been isolated from this continent. These novel rhabdoviruses represent an important and valuable pool of highly diverse viruses; however, most of them have remained poorly characterised. In light of the significant disease potential of numerous rhabdoviruses, the characterisation of novel rhabdoviruses is indispensable for threat assessment to livestock, wildlife and humans and preparedness for outbreaks. The genetic characterisation of novel viruses is also an essential step for the development of molecular detection assays for improved monitoring and investigations into unidentified disease cases. In this study, the complete genomes of four novel rhabdoviruses have been sequenced and a fifth is close to completion. The substantial new data generated has significantly extended the understanding of the biology and evolution of the Rhabdoviridae. Wongabel virus (WONV), isolated from the biting midge Culicoides austropalpalis, was found to contain a unique genome structure encoding ten genes, including five novel genes (Chapter 2). Analysis by western blotting suggested that four out of the five novel genes were expressed in infected cell cultures. Ngaingan virus (NGAV), isolated from Culicoides brevitarsis, was found to have the largest genome of any rhabdovirus sequenced to date, and with thirteen genes has the largest number of genes of any (-) ssRNA virus sequenced to date (Chapter 3). Seven of the thirteen genes are novel. Similar to viruses in the genus Ephemerovirus (bovine ephemeral fever virus and Adelaide River virus), NGAV contains a second glycoprotein with an unknown function. Phylogenetic analysis places this virus alongside WONV and the north-American bird and mosquito-associated Flanders virus within the Hart Park group that remains to be classified by the ICTV. Screening of various wildlife and livestock sera collected in northern Australia indicated a strong association of NGAV with macropods. Tibrogargan virus (TIBV) and Coastal Plains virus (CPV) were isolated from cattle and Culicoides brevitarsis (TIBV). Past serological surveys reported both viruses to be highly prevalent in cattle in northern Australia and demonstrated that the two viruses share a relatively close relationship at the antigenic level. The genomic analyses revealed that these two viruses have a unique genome organization, with three additional genes (Chapter 4). These additional genes are highly diverged at the sequence level but the encoded putative proteins share a significant conservation of secondary structure elements. The sequencing of these two related viruses has provided a unique opportunity to gain insights into the characteristics and evolution of novel proteins in two different rhabdoviruses. Phylogenetic analyses showed that TIBV and CPV form an independent cluster which does not appear to belong to any of the current genera, but which is most closely related to the genus Ephemerovirus based on N protein analysis. Although neither virus has been associated with disease, a serological survey of various animal sera collected in northern Australia showed that these viruses are currently highly prevalent in sentinel cattle and buffalo. Oak Vale virus (OVRV) was isolated from mosquitoes, Culex edwardsi and Ochlerotatus vigilax, from two geographically diverse regions of Australia located approximately 3000 km apart. The genome of OVRV was found to contain only one novel gene (Chapter 5). Comparatively, the genome of this virus is much less complex than the others in this study, but this virus displays considerable divergence from all other rhabdoviruses. A high seroprevalence for this virus was found in the feral pig population in northern Australia. The data generated from this study represents a considerable increase in the quantity of genetic data available for this viral family, and has revealed the existence of a large number of previously unidentified genes, highlighting that that the potential for complexity within the prototype genomic model of a rhabdovirus is much greater than previously thought. The novel nature of the additional genes provides grounds for further research into rhabdovirus evolution. Analysis of this new data suggests that these viruses cannot be classified into existing genera under the current criteria and it is clear that the taxonomy of the Rhabdoviridae requires revision. The observation that these viruses are currently circulating in livestock and wildlife in northern Australia accentuates the need for closer monitoring of animals and the need for further study of this diverse and fascinating group of viruses.

Piscicultura de água doce consiste na maior produção da aquicultura mundial e o cultivo de peixes tem crescido, devido a investimento, desenvolvimento de tecnologias e à contínua expansão de espécies cultivadas. Esse aumento gerou novas possibilidades de transmissão de vírus aquáticos, fatores limitantes à produção da aquicultura. Dentre as centenas de vírus conhecidos de peixes, um dos principais, que atualmente é de preocupação internacional, é o vírus da Viremia Primaveril da Carpa (SVCV), devido a sua importância socioeconômica considerando os países afetados e o comércio internacional de animais aquáticos e seus derivados, com base na saúde e medidas preventivas. SVC é uma doença aguda causada por um rhabdovírus do gênero Vesiculovirus, que é um vírus de ácido ribonucleico (RNA) fita simples linear de aproximadamente 11 quilobases (kb) com polaridade negativa e envelopado. Diversos ensaios diagnósticos podem ser utilizados para detectar SVCV, porém, consomem tempo e não são adaptados para diagnóstico a campo. A prevenção da disseminação do vírus é crucial; portanto, maior entendimento do vírus e de sua transmissão é necessário. No presente estudo, a padronização de uma RT-nestedPCR foi realizada, cujo limite de detecção foi de 8,62 × 10-2 cópias/reação, observado após o spiking de tecidos de peixes com diluições seriadas de um controle positivo sintético. O ensaio foi aplicado a 150 amostras teciduais provenientes de 146 peixes distintos. As amostras consistiam de fragmentos de fígado, rim e baço e foram submetidas à transcrição reversa (RT), seguida pela reação em cadeia de polimerase (PCR), em configuração nested. Duas (2) amostras foram positivas para o gene G de SVCV pela RT-nestedPCR e a identidade dos produtos obtidos foi confirmada por sequenciamento direto utilizando-se o método de Sanger. Na reconstrução filogenética, as sequências obtidas formaram clado único, separado dos demais genogrupos e mesmo de sequências derivadas de outros vesiculovírus encontrados no Brasil. Esses resultados determinam a primeira detecção e caracterização moleculares de SVCV no Brasil por RT-nestedPCR e sequenciamento nucleotídico, indicando a necessidade de adoção de medidas de biosseguridade mais restritivas na produção pesqueira nacional. / Fresh-water fish farming forms the largest portion of the world aquaculture production, and the harvest of these warm-water fish is increasing, due to investment, improvement of technologies and continued expansion of cultivated species. This increase has rendered new possibilities for the transmission of aquatic viruses, which remain limiting factors for aquaculture production. Among the hundreds of known viruses of fish, one of the main viruses that are currently of international concern is the Spring Viremia of Carp virus (SVCV) due to its socio-economic importance regarding affected countries and international trade of aquatic animals and their products, on the basis of health control and preventive measures. SVC is an acute disease, caused by a rhabdovirus, belonging to the Vesiculovirus genus, and is an enveloped virus, with a linear single-strained negative-sense ribonucleic acid (RNA) of approximately 11 kilobases (kb). Several diagnostic assays are available for the detection of SVCV, but they are time-consuming and not well adapted for field diagnosis. Prevention of spreading of the virus is crucial; therefore, more understanding of the virus and its transmission is required. In this study, standardization of a RT-nestedPCR was performed, and its detection limit was of 8.62 × 10-2 copies/reaction, observed after spiking fish tissue with serial dilutions of a synthetic positive control. The assay was applied to 150 tissue samples from 146 different fish. Samples consisted of liver, kidney and spleen fragments, and underwent reverse transcription (RT) followed by the polymerase chain reaction (PCR) of a nested configuration. Two (2) tissue samples were found positive for the G gene of SVCV by RT-nestedPCR and the identity of products was confirmed by direct sequencing using the Sanger method. By phylogenetic reconstruction, the obtained sequences formed a unique clade, separating them from the other known genogroups, and even from sequences derived from other vesiculoviruses found in Brazil. These results represent the first molecular detection and characterization of SVCV in Brazil by RT-nestedPCR and nucleotide sequencing, indicating the need to adopt more stringent biosecurity measures in national fish farming production.

Oncolytic viruses (OVs) are a class of cancer therapy that is currently undergoing clinical trials on its way to full regulatory approval. At present, the downstream processing of OVs relies on a combination of chromatography and membrane-based processes to remove process-related (e.g. host-cell proteins and nucleic acids) and product-related impurities (e.g. aggregated virus particles). This thesis explores various methods that can potentially be used to address the challenges associated with downstream processing during the production of OVs. To this end, the Rhabdoviral vector, which is currently undergoing clinical trials (phase I/II) for use in treating advanced or metastatic solid tumors, was selected as a promising oncolytic virus. One potential improvement in the downstream process that was investigated was the use of monolithic column chromatography for Rhabdovirus purification. Two monolithic anion-exchange columns (2 and 6 µm pore size) and one hydrophobic interaction column (6 µm pore size) were used to examine how column pore size affects virus recovery and contaminant removal. This investigation ultimately inspired the development of a purification process based on monolithic hydrophobic interaction column chromatography. Furthermore, this work is also the first to investigate how additives, namely glycerol, impact the hydrophobic interaction chromatography of virus particles. The developed process could be readily implemented for the scaled-up purification of the Rhabdoviral vector. Another challenge associated with the downstream processing of OVs is membrane fouling, which is characterized by a dramatic rise in transmembrane pressure (TMP) and low virus recovery. Indeed, membrane fouling poses a significant challenge, as some recent studies have reported that it can result in viral vector titer losses of over 80%. One critical use of membranes in downstream processing is for the sterile filtration of OVs, which is a required final step that is conducted right before vialing and involves passing the virus particles through a validated sterile filter. One of the main objectives of this thesis was to develop a fundamental understanding of the sterile filtration process and to optimize it in order to achieve higher throughput and lower losses, which are both essential to the large-scale production of OVs. To this end, a dead-end sterile filtration setup was designed, and various commercially available filters were evaluated to examine how membrane morphology affects fouling and product recovery. The results of these tests showed that double-layered composite filters enabled higher virus recovery and filtration capacity compared to single-layered sterile filters. Another cause of membrane fouling is the aggregation of virus particles, which is mediated by various interactions in the solution. To study this, the above-described setup was re-designed to create an effective procedure that utilizes minimal volumes of virus solution, while also enabling the rapid assessment of microscale filtration performance and a comprehensive understanding of virus-virus and virus-membrane interactions. This setup was used to study how different additives, including various proteins (bovine serum albumin and α-lactalbumin) and polymers (polyethylene glycol and polyvinylpyrrolidone), affect the microfiltration of the Rhabdoviral vector and, consequently, the TMP profile. Furthermore, the correlation between the membrane fouling rate (via TMP profiles) and virus recovery was also investigated. This investigation revealed that proteins significantly increase virus transmission and that polymers are incapable of mimicking the effects of the proteins. To explain this phenomenon, a theory based on the biophysical structure of proteins, mainly heterogenicity in charge distribution, was proposed. Moreover, membrane surface modification tests were conducted using bovine serum albumin, with the results indicating that this approach has considerable potential for enhancing virus transmission. Due to the similarities between the test setup and actual downstream processing unit operations, the results from this part of the thesis could be easily and accurately applied to process optimization. / Thesis / Candidate in Philosophy / There is considerable interest in the development of oncolytic viruses for cancer immunotherapy. Indeed, at the time of this thesis’ writing, a Canadian team of researchers is conducting the world’s first clinical trial using a combination of two viruses to kill cancer cells and stimulate an immune response. The process of manufacturing oncolytic viruses is generally divided into two major steps: upstream processing and downstream processing. While upstream processing focuses on virus propagation, downstream processing aims at removing process-related and product-related impurities. However, research into downstream process design and optimization has largely been neglected in favour of a focus on upstream processing, aimed at increasing bioreactor yields and achieving high viral titers. Consequently, downstream processing has become the main bottleneck in virus manufacturing processes, accounting for as much as 70% production costs. This thesis aims to identify and develop a fundamental understanding of the main challenges associated with the downstream processing of oncolytic viruses and to investigate methods for addressing them. Specifically, the present work focuses on the purification and final sterile filtration steps in the manufacturing of oncolytic Rhabdoviral vectors.