No description available.

O objetivo geral desta pesquisa foi avaliar a transferência de imunidade passiva e a sua influência na resposta vacinal para as viroses envolvidas na Doença Respiratória Bovina (DRB). Os dados obtidos nesta pesquisa estão apresentados em dois capítulos. Capítulo 1 - Objetivou-se avaliar a dinâmica de anticorpos (Acs) específicos para as viroses respiratórias e subpopulações de linfócitos em bezerros do nascimento aos 240 dias (d) de vida. Para tanto, acompanhou-se a transferência de imunidade passiva de Acs específicos para as viroses respiratórias em 19 bezerros, destes cinco foram selecionados para acompanhamento da dinâmica de Acs neutralizantes e subpopulações de linfócitos dos 14 aos 240d. O colostro fornecido era proveniente de vacas doadoras vacinadas. A análise da qualidade individual do colostro revelou índice Brix ≥21%, observando-se forte correlação desses valores com proteína total (r= 0,942 e P= 0,001). Após 48 horas da ingestão do colostro, pôde-se observar soroconversão dos 19 animais (100%) para os agentes virais envolvidos na DBR. As medianas (Log2) encontradas foram de 12,3, 9,0, 5,0 e 8,5 para BVDV, BoHV-1, BRSV e BPIV-3. Dos 14 aos 240d os 5 bezerros avaliados, demonstraram declínio gradual dos títulos de Acs (Log2) para BVDV (12,8-3,3), BoHV-1 (10,0-3,3) e BPIV-3 (10,0-2,0), embora o BVDV não tenha apresentado soronegatividade até os 240d. Manifestações clínicas de broncopneumonia foram observadas em 4/5 (80%) bezerros dos 80 aos 135 d. BRSV (11,3-2,0) apresentou perfil diferenciado na dinâmica de anticorpos em relação às demais viroses. Não foram detectadas diferenças estatísticas entre os momentos para as viroses apesar das variações detectadas (P>0,05). A meia-vida e tempo para soronegatividade foram de 36,2±6,1 e 367,01±68,7d para BVDV, 50,7±18,0 e 239,67±66,88d para BoHV-1 e, 46,8±21,1 e 303,36±60,15d para BPIV- 3. BRSV não respeitou modelo de regressão para cálculos de meia-vida e soronegatividade. Valores absolutos e relativos das populações de linfócitos não revelaram diferenças estatísticas entre os momentos (P>0,05). Contudo, a dinâmica das subpopulações de linfócitos revelou aumento de células B CD21+ até 150d; aumento nos valores relativos das subpopulações CD4+, CD8+ e CD3+CD4-CD8- principalmente aos 74-90d. Assim, pôde-se determinar uma janela de susceptibilidade a partir dos 74d especialmente ao BRSV e BoHV-1, momento que precede o aumento dos títulos de Acs após exposição natural. Capítulo 2 Objetivou-se avaliar a influência dos Acs maternos na resposta imune para DRB induzida pela vacinação. Foram selecionados 23 bezerros recém-nascidos, distribuídos aleatoriamente entre 4 grupos experimentais: G1 vacinado aos 14d e booster aos 44d; G2 aos 90d e aos 120d; G3 aos 180d e aos 210d. Além disso manteve-se um grupo controle não vacinado CG1, CG2 e CG3. Os bezerros foram vacinados com a mesma vacina comercial empregada para vacinação das doadoras de colostro. Observou-se: (1) a vacina com BVDV inativado não promoveu aumento dos títulos de Acs para nenhum dos grupos avaliados; (2) G1 não demonstrou soroconversão para nenhuma das viroses, enquanto controle CG1 exibiu decréscimo dos títulos para BVDV, BoHV-1 e BPIV-3; (3) o BRSV apresentou baixa soroconversão no G2, enquanto o controle demonstrou altos títulos dos 44-120d; (4) não foi possível distinguir entre quais tempos ocorreram diferenças entre títulos (P>0,0167); (5) linfócitos B (CD21+) aumentaram do T0-T2 para G1, diminuíram para G2, e aumentaram no G3; (6) linfócitos T CD3+ diminuíram ao longo do tempo para todos os grupos, exceto CG3; (7) apesar de oscilações, linfócitos T CD4+, CD8+ e WC1+ se mantiveram praticamente constantes até 240d, exibindo maiores proporções nos grupos vacinados; (8) a expressão do marcador CD25+foi mantida pelo grupo G1 até o T2, mas apresentou aumento no G2 e G3; (9) manifestações de broncopneumonias foram identificadas nos bezerros do grupo controle (4/5 - 80%) e podem ter exercido influência nas diferenças encontradas para as células entre os grupos. Em geral, a vacinação dos bezerros aos 90 (G2) e 180d (G3) manteve ou estimulou a produção de Acs para o BoHV-1, BRSV e BPIV-3, e a ativação das células T expressas pelo marcador CD25+ pode ter sido responsável pela proteção dos bezerros frente à DRB. Assim, com base nos resultados, concluiu-se que a intensidade da imunidade dos bezerros induzida pela vacinação aumentou de acordo com o desenvolvimento etário e diminuição dos títulos de Acs maternos. A conclusão geral desta dissertação aponta para a necessidade precoce de imunização dos bezerros, especialmente pela susceptibilidade observada para BRSV e BPIV-3 aos 74-90d de vida. Entretanto, esta pesquisa não encontrou resposta humoral induzida pela vacinação no grupo de bezerros vacinados aos 14 e 44 dias, apesar dos indícios de resposta imune celular. Assim, estudos futuros devem ser elaborados considerando estratégias para amplificar a resposta imune precoce dos bezerros para os agentes virais envolvidos na DRB / The main purpose of this research was to evaluate the transfer of passive immunity and its influence on vaccine response to the viruses involved in bovine respiratory disease (BRD). The data obtained in this study are presented in two chapters. Chapter 1 - The objective was to evaluate the dynamics of specific antibodies to the respiratory viruses and lymphocyte subpopulations in the calves from birth to 240 days (d) of life. Thus, the transfer of passive immunity of specific antibodies to the respiratory viruses were assessed in 19 calves; from these, five were selected for monitoring the dynamic of neutralizing Abs and lymphocytes subpopulations from 14 to 240d. The colostrum was provided from donor vaccinated cows. The analysis of individual quality of the colostrum revealed Brix ≥21%, observing strong correlation of these values with total protein (r = 0.942 and P = 0.001). After 48 hours of colostrum intake, was observed seroconversion of the 19 animals (100%) for the viral agents involved in the DBR. The median (Log2) ratio found was 12.3, 9.0, 5.0 and 8.5 for BVDV, BoHV-1, BRSV and BPIV-3. The 5 calves followed from 14 to 240d showed gradual decline in antibody titers (Log2) to BVDV (12.8 to 3.3), BoHV-1 (10.0 to 3.3) and BPIV-3 (10, 0-2.0), although could not be detected seronegative calves for BVDV up to eight months of age. Clinical manifestations of bronchopneumonia were observed in 4/5 (80%) calves from 80 to 135 days of life. BRSV (11.3 to 2.0) showed a distinct profile in the dynamics of antibodies compared to other viruses. There were no statistical differences between times for viruses despite variations detected (P> 0.05). The half-life and time to become seronegative were 36.2±6.1 and 367.01±68.7d for BVDV, 50.7±18.0 and 239.67± 66.88d for BoHV-1 and, 46.8±21.1 and 303.36±60.15d for BPIV-3. BRSV did not respect regression model to perform half-life and seronegative calculations. Absolute and relative values of lymphocyte populations revealed no statistical differences between times (P> 0.05). However, the dynamics of lymphocyte subpopulations showed increase in B cells CD21+ up to 150d; increase in the relative values of CD4+, CD8+ and CD3+CD4-CD8- subpopulations, mainly to 74-90d. Thus, it was possible to determine a window of susceptibility since 74d especially for BRSV and BoHV-1, moment that precede the increase in antibody titers after natural exposure. Chapter 2 - The objective was to evaluate the influence of maternal antibodies in the immune response to respiratory viruses induced by vaccination. Was selected 23 newborn calves that were randomly distributed in four groups: G1 - vaccinated at 14d and booster at 44d; G2 - vaccinated at 90d and booster at 120d; G3 - vaccinated at 180d and booster at 210d. Furthermore were kept a non-vaccinated control group - CG1, CG2 and CG3. Calves were vaccinated with the same commercial vaccine in the colostrum donors. From these, could be observed: (1) the vaccine with inactivated BVDV did not promote increase of antibodies titers in any of the assessed groups; (2) G1 did not demonstrated seroconversion for any of the viruses while CG1 control exhibited decrease in the titers for BVDV, BoHV-1 and BPIV-3; (3) BRSV had low seroconversion in G2, while the control showed high titers from 44 to 120d; (4) where the differences between times occurred could not be distinguished (P <0.0167); (5) B cells (CD21+) increased from T0 to T2 for G1, decreased for G2, and increased for G3; (6) T lymphocytes CD3+ decreased over time for all groups except for CG3; (7) despite variations, T lymphocytes CD4+, CD8+ and WC1+ remained almost constant until 240d, displaying greater proportions in the vaccinated groups; (8) the expression of the CD25+ marker was maintained in the vaccinated group G1 up to T2, whereas vaccination promoted an increase of this expression in G2 and G3; (9) clinical manifestations of bronchopneumonia were identified in the control group (4/5 calves - 80%) and may have influenceon the differences found for the cells between the groups. In general, vaccination of calves at 90 (G2) and 180d (G3) maintained or stimulated the production of Abs to BoHV-1, BRSV and BPIV-3, and the activation of T cells expressed by the CD25+ marker may have been responsible for the protection of calves from BRD. Thus, based on the results, it was concluded that the intensity of the immunity induced by vaccination of calves increased according to the age of development and decay of maternal antibody titers. The general conclusion of this research, points to the need for early immunization of calves, especially by the susceptibility observed for BRSV and BPIV-3 from 74-90d of life. However, this research didnot found humoral response induced by vaccination in the group of calves vaccinated at 14 and 44 days, despite the evidence of cellular immune response. Thus, future studies should be designed considering strategies to amplify the early immune response of calves to the viral agents involved in the BRD

Le Virus Herpès Simplex de type 1 (HSV-1) est un agent infectieux qui cause l’herpès chez une grande proportion de la population mondiale. L’herpès est généralement considéré comme une maladie bénigne dont la forme la plus commune est l'herpès labial (communément appelé « bouton de fièvre »), mais elle peut se révéler très sérieuse et causer la cécité et l’encéphalite, voir létale dans certain cas. Le virus persiste toute la vie dans le corps de son hôte. Jusqu'à présent, aucun traitement ne peut éliminer le virus et aucun vaccin n’a été prouvé efficace pour contrôler l’infection herpétique. HSV-1 est un virus avec un génome d’ADN bicaténaire contenu dans une capside icosaèdrale entourée d’une enveloppe lipidique. Treize glycoprotéines virales se trouvent dans cette enveloppe et sont connues ou supposées jouer des rôles distincts dans différentes étapes du cycle de réplication viral, incluant l'attachement, l'entrée, l’assemblage, et la propagation des virus. La glycoprotéine M (gM) qui figure parmi ces glycoprotéines d’enveloppe, est la seule glycoprotéine non essentielle mais est conservée dans toute la famille herpesviridae. Récemment, l’homologue de gM dans le Pseudorabies virus (PRV), un autre herpesvirus, a été impliqué dans la phase finale de l’assemblage (i.e. l’enveloppement cytoplasmique) au niveau du réseau trans-Golgi (TGN) en reconnaissant spécifiquement des protéines tégumentaires et d’autres glycoprotéines d’enveloppe ([1]). Toutefois, il a été proposé que cette hypothèse ne s’applique pas pour le HSV-1 ([2]). De plus, contrairement à la localisation au TGN dans les cellules transfectées, HSV-1 gM se localise dans la membrane nucléaire et sur les virions périnucléaires durant une infection. L’objectif du projet présenté ici était d’éclaircir la relation de la localisation et la fonction de HSV-1 gM dans le contexte d’une infection. Dans les résultats rapportés ici, nous décrivons tout abord un mécanisme spécifique de ciblage nucléaire de HSV-1 gM. En phase précoce d’une infection, gM est ciblée à la membrane nucléaire d'une manière virus ii dépendante. Cela se produit avant la réorganisation du TGN normalement induite par l’infection et avant que gM n’entre dans la voie de sécrétion. Ce ciblage nucléaire actif et spécifique de gM ne semble pas dépendre des plusieurs des partenaires d’interaction proposés dans la littérature. Ces données suggèrent que la forme nucléaire de gM pourrait avoir un nouveau rôle indépendant de l’enveloppement final dans le cytoplasme. Dans la deuxième partie du travail présenté ici, nous avons concentré nos efforts sur le rôle de gM dans l’assemblage du virus en phase tardive de l’infection et en identifiant un domaine critique de gM. Nos résultats mettent en valeur l’importance du domaine carboxyl-terminal cytoplasmique de gM dans le transport de gM du réticulum endoplasmique (RE) à l’appareil de Golgi, dans l’enveloppement cytoplasmique et la propagation intercellulaire du virus. Ainsi, l’export du RE de gM a été complètement compromis dans les cellules transfectées exprimant un mutant de gM dépourvu de sa région C-terminale. La délétion la queue cytoplasmique de gM cause une réduction légère du titre viral et de la taille des plaques. L'analyse de ces mutants par microscopie électronique a démontré une accumulation des nucléocapsides sans enveloppe dans le cytoplasme par rapport aux virus de type sauvage. Étrangement, ce phénotype était apparent dans les cellules BHK mais absent dans les cellules 143B, suggérant que la fonction de gM dépende du type cellulaire. Finalement, le criblage de partenaires d’interaction du domaine C-terminal de gM identifiés par le système de double-hybride nous a permis de proposer plusieurs candidats susceptibles de réguler la fonction de gM dans la morphogénèse et la propagation de virus. / Herpes Simplex Virus type 1 (HSV-1) is an infectious agent causing herpes, which affects a large population worldwide. Herpes is generally considered a benign disease whose most common form is oral herpes (commonly called "cold sores"), but it can be very serious and cause herpetic blindness and encephalitis, and even be lethal in some cases. The virus can persist throughout life in the body of its host. So far, no treatment can eliminate the virus and no vaccine has proven effective in controlling herpes infections. HSV-1 has a double-stranded DNA genome embedded in an icosahedral capsid surrounded by a lipid envelope. Thirteen viral glycoproteins are located in the envelope and are known or believed to play different roles in different stages of the viral replication cycle, including attachment, entry, assembly, and viral propagation. Among these envelope glycoproteins, glycoprotein M (gM) is the only nonessential glycoprotein but is conserved in all the herpesviridae family. Recently, the homologue of gM in Pseudorabies virus (PRV), another herpesvirus, has been implicated in the final phase of assembly (e.g. the cytoplasmic envelopment) at the trans-Golgi network (TGN) ([1]). However, it was suggested that this does not apply to HSV-1 ([2]). Moreover, unlike its TGN localization in transfected cells, HSV-1 gM localizes to the nuclear membrane and on the perinuclear virions during infection. The objective of the project presented here was to clarify the relationship of the location and function of HSV-1 gM in the context of an infection. In the results reported here, we first describe a specific and active mechanism of nuclear targeting of HSV-1 gM. In early phase of infection, gM is targeted to the nuclear membrane in a virus dependent manner. This occurs before the known reorganization of the TGN induced by the virus and before gM enters the secretory pathway. This active and specific nuclear targeting of gM seemingly does not depend on the functional interaction partners proposed in the literature. These data suggest that nuclear gM could have a new role independent of that in the final envelopment in the cytoplasm. In the second part of the work presented here, we focused iv our efforts on the role of gM in virus assembly in the late phase of infection and define an important functional domain within gM. Our results highlight the importance of the carboxyl-terminal domain of gM in the intracellular transport of gM from endoplasmic reticulum (ER) to Golgi apparatus, in the cytoplasmic envelopment of the capsids and the intercellular spread of the virus. Hence, gM ER export was completely compromised in transfected cells after deletion of its C-terminal tail. Deletion of the gM cytoplasmic tail in mutant viruses resulted in a slight reduction in viral titer and plaque size. The analysis of these mutants by electron microscopy showed an accumulation of nucleocapsids without envelope in the cytoplasm compared to wild-type virus. Interestingly, this phenotype is apparent in BHK cells but not in 143B cells, hinting that the importance of gM may be cell type specific. Finally, screening of interaction partners of C-terminal domain of gM identified by the two-hybrid system allowed us to propose several interesting candidates that may regulate the function of gM in the virus morphogenesis and propagation.

O objetivo geral desta pesquisa foi avaliar a transferência de imunidade passiva e a sua influência na resposta vacinal para as viroses envolvidas na Doença Respiratória Bovina (DRB). Os dados obtidos nesta pesquisa estão apresentados em dois capítulos. Capítulo 1 - Objetivou-se avaliar a dinâmica de anticorpos (Acs) específicos para as viroses respiratórias e subpopulações de linfócitos em bezerros do nascimento aos 240 dias (d) de vida. Para tanto, acompanhou-se a transferência de imunidade passiva de Acs específicos para as viroses respiratórias em 19 bezerros, destes cinco foram selecionados para acompanhamento da dinâmica de Acs neutralizantes e subpopulações de linfócitos dos 14 aos 240d. O colostro fornecido era proveniente de vacas doadoras vacinadas. A análise da qualidade individual do colostro revelou índice Brix ≥21%, observando-se forte correlação desses valores com proteína total (r= 0,942 e P= 0,001). Após 48 horas da ingestão do colostro, pôde-se observar soroconversão dos 19 animais (100%) para os agentes virais envolvidos na DBR. As medianas (Log2) encontradas foram de 12,3, 9,0, 5,0 e 8,5 para BVDV, BoHV-1, BRSV e BPIV-3. Dos 14 aos 240d os 5 bezerros avaliados, demonstraram declínio gradual dos títulos de Acs (Log2) para BVDV (12,8-3,3), BoHV-1 (10,0-3,3) e BPIV-3 (10,0-2,0), embora o BVDV não tenha apresentado soronegatividade até os 240d. Manifestações clínicas de broncopneumonia foram observadas em 4/5 (80%) bezerros dos 80 aos 135 d. BRSV (11,3-2,0) apresentou perfil diferenciado na dinâmica de anticorpos em relação às demais viroses. Não foram detectadas diferenças estatísticas entre os momentos para as viroses apesar das variações detectadas (P>0,05). A meia-vida e tempo para soronegatividade foram de 36,2±6,1 e 367,01±68,7d para BVDV, 50,7±18,0 e 239,67±66,88d para BoHV-1 e, 46,8±21,1 e 303,36±60,15d para BPIV- 3. BRSV não respeitou modelo de regressão para cálculos de meia-vida e soronegatividade. Valores absolutos e relativos das populações de linfócitos não revelaram diferenças estatísticas entre os momentos (P>0,05). Contudo, a dinâmica das subpopulações de linfócitos revelou aumento de células B CD21+ até 150d; aumento nos valores relativos das subpopulações CD4+, CD8+ e CD3+CD4-CD8- principalmente aos 74-90d. Assim, pôde-se determinar uma janela de susceptibilidade a partir dos 74d especialmente ao BRSV e BoHV-1, momento que precede o aumento dos títulos de Acs após exposição natural. Capítulo 2 Objetivou-se avaliar a influência dos Acs maternos na resposta imune para DRB induzida pela vacinação. Foram selecionados 23 bezerros recém-nascidos, distribuídos aleatoriamente entre 4 grupos experimentais: G1 vacinado aos 14d e booster aos 44d; G2 aos 90d e aos 120d; G3 aos 180d e aos 210d. Além disso manteve-se um grupo controle não vacinado CG1, CG2 e CG3. Os bezerros foram vacinados com a mesma vacina comercial empregada para vacinação das doadoras de colostro. Observou-se: (1) a vacina com BVDV inativado não promoveu aumento dos títulos de Acs para nenhum dos grupos avaliados; (2) G1 não demonstrou soroconversão para nenhuma das viroses, enquanto controle CG1 exibiu decréscimo dos títulos para BVDV, BoHV-1 e BPIV-3; (3) o BRSV apresentou baixa soroconversão no G2, enquanto o controle demonstrou altos títulos dos 44-120d; (4) não foi possível distinguir entre quais tempos ocorreram diferenças entre títulos (P>0,0167); (5) linfócitos B (CD21+) aumentaram do T0-T2 para G1, diminuíram para G2, e aumentaram no G3; (6) linfócitos T CD3+ diminuíram ao longo do tempo para todos os grupos, exceto CG3; (7) apesar de oscilações, linfócitos T CD4+, CD8+ e WC1+ se mantiveram praticamente constantes até 240d, exibindo maiores proporções nos grupos vacinados; (8) a expressão do marcador CD25+foi mantida pelo grupo G1 até o T2, mas apresentou aumento no G2 e G3; (9) manifestações de broncopneumonias foram identificadas nos bezerros do grupo controle (4/5 - 80%) e podem ter exercido influência nas diferenças encontradas para as células entre os grupos. Em geral, a vacinação dos bezerros aos 90 (G2) e 180d (G3) manteve ou estimulou a produção de Acs para o BoHV-1, BRSV e BPIV-3, e a ativação das células T expressas pelo marcador CD25+ pode ter sido responsável pela proteção dos bezerros frente à DRB. Assim, com base nos resultados, concluiu-se que a intensidade da imunidade dos bezerros induzida pela vacinação aumentou de acordo com o desenvolvimento etário e diminuição dos títulos de Acs maternos. A conclusão geral desta dissertação aponta para a necessidade precoce de imunização dos bezerros, especialmente pela susceptibilidade observada para BRSV e BPIV-3 aos 74-90d de vida. Entretanto, esta pesquisa não encontrou resposta humoral induzida pela vacinação no grupo de bezerros vacinados aos 14 e 44 dias, apesar dos indícios de resposta imune celular. Assim, estudos futuros devem ser elaborados considerando estratégias para amplificar a resposta imune precoce dos bezerros para os agentes virais envolvidos na DRB / The main purpose of this research was to evaluate the transfer of passive immunity and its influence on vaccine response to the viruses involved in bovine respiratory disease (BRD). The data obtained in this study are presented in two chapters. Chapter 1 - The objective was to evaluate the dynamics of specific antibodies to the respiratory viruses and lymphocyte subpopulations in the calves from birth to 240 days (d) of life. Thus, the transfer of passive immunity of specific antibodies to the respiratory viruses were assessed in 19 calves; from these, five were selected for monitoring the dynamic of neutralizing Abs and lymphocytes subpopulations from 14 to 240d. The colostrum was provided from donor vaccinated cows. The analysis of individual quality of the colostrum revealed Brix ≥21%, observing strong correlation of these values with total protein (r = 0.942 and P = 0.001). After 48 hours of colostrum intake, was observed seroconversion of the 19 animals (100%) for the viral agents involved in the DBR. The median (Log2) ratio found was 12.3, 9.0, 5.0 and 8.5 for BVDV, BoHV-1, BRSV and BPIV-3. The 5 calves followed from 14 to 240d showed gradual decline in antibody titers (Log2) to BVDV (12.8 to 3.3), BoHV-1 (10.0 to 3.3) and BPIV-3 (10, 0-2.0), although could not be detected seronegative calves for BVDV up to eight months of age. Clinical manifestations of bronchopneumonia were observed in 4/5 (80%) calves from 80 to 135 days of life. BRSV (11.3 to 2.0) showed a distinct profile in the dynamics of antibodies compared to other viruses. There were no statistical differences between times for viruses despite variations detected (P> 0.05). The half-life and time to become seronegative were 36.2±6.1 and 367.01±68.7d for BVDV, 50.7±18.0 and 239.67± 66.88d for BoHV-1 and, 46.8±21.1 and 303.36±60.15d for BPIV-3. BRSV did not respect regression model to perform half-life and seronegative calculations. Absolute and relative values of lymphocyte populations revealed no statistical differences between times (P> 0.05). However, the dynamics of lymphocyte subpopulations showed increase in B cells CD21+ up to 150d; increase in the relative values of CD4+, CD8+ and CD3+CD4-CD8- subpopulations, mainly to 74-90d. Thus, it was possible to determine a window of susceptibility since 74d especially for BRSV and BoHV-1, moment that precede the increase in antibody titers after natural exposure. Chapter 2 - The objective was to evaluate the influence of maternal antibodies in the immune response to respiratory viruses induced by vaccination. Was selected 23 newborn calves that were randomly distributed in four groups: G1 - vaccinated at 14d and booster at 44d; G2 - vaccinated at 90d and booster at 120d; G3 - vaccinated at 180d and booster at 210d. Furthermore were kept a non-vaccinated control group - CG1, CG2 and CG3. Calves were vaccinated with the same commercial vaccine in the colostrum donors. From these, could be observed: (1) the vaccine with inactivated BVDV did not promote increase of antibodies titers in any of the assessed groups; (2) G1 did not demonstrated seroconversion for any of the viruses while CG1 control exhibited decrease in the titers for BVDV, BoHV-1 and BPIV-3; (3) BRSV had low seroconversion in G2, while the control showed high titers from 44 to 120d; (4) where the differences between times occurred could not be distinguished (P <0.0167); (5) B cells (CD21+) increased from T0 to T2 for G1, decreased for G2, and increased for G3; (6) T lymphocytes CD3+ decreased over time for all groups except for CG3; (7) despite variations, T lymphocytes CD4+, CD8+ and WC1+ remained almost constant until 240d, displaying greater proportions in the vaccinated groups; (8) the expression of the CD25+ marker was maintained in the vaccinated group G1 up to T2, whereas vaccination promoted an increase of this expression in G2 and G3; (9) clinical manifestations of bronchopneumonia were identified in the control group (4/5 calves - 80%) and may have influenceon the differences found for the cells between the groups. In general, vaccination of calves at 90 (G2) and 180d (G3) maintained or stimulated the production of Abs to BoHV-1, BRSV and BPIV-3, and the activation of T cells expressed by the CD25+ marker may have been responsible for the protection of calves from BRD. Thus, based on the results, it was concluded that the intensity of the immunity induced by vaccination of calves increased according to the age of development and decay of maternal antibody titers. The general conclusion of this research, points to the need for early immunization of calves, especially by the susceptibility observed for BRSV and BPIV-3 from 74-90d of life. However, this research didnot found humoral response induced by vaccination in the group of calves vaccinated at 14 and 44 days, despite the evidence of cellular immune response. Thus, future studies should be designed considering strategies to amplify the early immune response of calves to the viral agents involved in the BRD

A proteína quinase dependente de RNA (PKR) é uma molécula sentinela ativada em situações de estresse celular, incluindo infecções virais. A ativação de PKR por meio de sua fosforilação aciona cascatas de sinalização intracelular envolvidas em respostas inflamatórias e inibição da síntese protéica. Dados prévios do nosso laboratório sugerem que PKR está envolvida na hiperalgesia térmica de origem inflamatória. No presente estudo, foi investigado o papel da PKR na hiperalgesia térmica induzida pelo vírus da herpes simples tipo 1 (HSV1), durante as fases herpética e pós-herpética, combinando métodos comportamentais, genéticos, farmacológicos e moleculares. Camundongos C57bl/6, PKR+/+ e PKR-/- machos foram inoculados com HSV1. Os grupos controle foram inoculados com HSV1 inativo. Alodínia mecânica e hiperalgesia térmica foram monitoradas antes da inoculação do vírus e 8, 14, 21 e 28 dias após a inoculação. A curva dose e temporesposta e o teste da capsaicina foram realizados no 8º e 21º dias após a inoculação do vírus. Também nos períodos herpético e pós-herpético, foi investigado o perfil de expressão de proteínas envolvidas nas vias de sinalização de PKR (PKR, eIF2?, PACT, IKK e PP2A?), assim como o efeito da inibição de PKR pelo monitoramento da fosforilação de PKR, IKK?/?, P38, JNK, ERK1,2 e STAT3, e expressão de CaMKII? e TRPV1 nos GRD (L3-L6) ipsilateralmente à pata inoculada. Alodínia mecânica e hiperalgesia térmica ficaram evidentes até 28 dias após a inoculação. Camundongos PKR-/- desenvolveram alodínia mecânica, mas não hiperalgesia térmica, quando comparados com animais PKR+/+. A inibição sistêmica de PKR reverteu a hiperalgesia térmica de modo tempo- e dose-dependente e preveniu o comportamento nocifensivo induzido por capsaicina, enquanto PKR-/- apresentaram resposta nocifensiva praticamente ausente em ambas as fases herpética e pósherpética. Houve aumento da expressão de PP2A? e da fosforilação de PKR, IKK?/? e eIF2?, durante os períodos herpético e pós-herpético, e de PACT na fase pósherpética. A inibição de PKR promoveu o aumento da fosforilação de P38 em ambas as fases, e redução da fosforilação de PLC?1 acompanhada do retorno da fosforilação de Akt e STAT3 ao nível do grupo controle e o aumento da expressão de Ca-MKII? na fase herpética. Já na fase pós-herpética, reduziu a fosforilação de JNK e Akt e a expressão de Ca-MKII?, retornou a fosforilação de ERK1,2, PLC?1 e STAT3 ao nível do grupo controle e aumentou a expressão de TRPV1. Nossos resultados indicam que a atividade de PKR desempenha papel essencial na hiperalgesia térmica induzida por infecção pelo HSV1 / Double stranded RNA-activated protein kinase (PKR) is a sentinel molecule activated by cellular stress conditions, including viral infections. PKR activation by phosphorylation triggers cascades involved in inflammatory response and protein synthesis suppression. Our previous data suggest that PKR is involved in the inflammatory thermal hyperalgesia. Here we investigated the role played by PKR on thermal hyperalgesia induced by herpes simplex virus type-1 (HSV-1), during herpetic and post-herpetic phases, by combining behavioral, genetic, pharmacological, and molecular methods. Adult male C57bl/6, PKR+/+ and PKR-/- mice were inoculated with HSV-1. Control groups were inoculated with inactive (mock) HSV1. Mechanical allodynia and thermal hyperalgesia were monitored before virus inoculation and 8, 14, 21, and 28 days post-inoculation. The dose- and timeresponse curve and the capsaicin test were performed at 8th and 21st days post virus inoculation. Also in the herpetic and post-herpetic periods, was investigated the expression profile of proteins involved in the PKR signaling pathways (PKR, eIF2?, PACT, IKK and PP2A?), and the effect of PKR inhibition by monitoring PKR, IKK?/?, P38, JNK, ERK1,2, and STAT3 phosphorylation, and Ca-MKII? and TRPV1 expression in the dorsal root ganglia (L3-L6) ipsilaterally to the inoculated paw. Mechanical allodynia and thermal hyperalgesia became evident until 28 days postinnoculation. PKR-/- mice developed mechanical allodynia but not thermal hyperalgesia, when compared with PKR+/+ mice. Systemic PKR inhibition reversed thermal hyperalgesia in a dose and time-dependent manner, and prevented the capsaicin-induced nocifensive behavior, whereas PKR-/- showed no nocifensive behavior almost absent in both herpetic and post-herpetic phases. There was increased expression of PP2A? and the phosphorylation of PKR, IKK?/?, and eIF2?, during herpetic and post-herpetic periods, and PACT in the post-herpetic phase. PKR inhibition increased P38 phosphorylation in both phases, and reduction of PLC?1 phosphorylation together with the return of the Akt and STAT3 phosphorylation to the control group level, and enhanced Ca-MKII? expression in the herpetic phase. At the post-herpetic phase, suppressed JNK and Akt, and Ca-MKII? expression returned ERK1,2, PLC?1 and STAT3 phosphorylation to control group level and increased TRPV1 expression. The data indicate that PKR activity plays an essential role in the HSV-1 infection-induced thermal hyperalgesia

Le virus de l'herpès simplex de type 1 (VHS-1) est l'un des agents pathogènes humains les plus anciens et les plus efficaces. On estime que 3.7 milliards de personnes dans le monde vivent avec le VHS-1. Le virus persiste à l'état latent dans les neurones sensoriels, réapparaissant occasionnellement sous la forme d'une infection lytique qui endommage l'épithélium. Même si le VHS-1 provoque une maladie bénigne connue sous le nom de feu sauvage dans la majorité des cas, l'infection peut entraîner des conséquences catastrophiques telles que l'encéphalite et la kératite chez les personnes immunodéprimées les nouveau-nés. Compte tenu de la présence généralisée des infections à VHS-1, le virus représente une menace potentielle pour le système de santé. Le génome à ADN du VHS-1 est protégé par une cage protéique appelée capside. Bien que l'assemblage de la capside du VHS-1 et l'encapsidation du génome aient lieu à l'intérieur du noyau de l'hôte, les étapes finales de la maturation doivent être achevées dans le cytoplasme. Ainsi, pour la sortie du noyau, le virus a développé un mécanisme connu sous le nom d’enveloppement-déenveloppement-réenveloppement. La première étape de ce processus est principalement régulée par le complexe de sortie nucléaire (pUL31 et pUL34) et entraîne le bourgeonnement de la capside alors enveloppée dans l'espace périnucléaire. Par la suite, le déenveloppement de ces capsides périnucléaires et leur libération dans le cytoplasme seraient largement modulés par la kinase virale pUs3. Ce processus est sélectif, car les capsides remplies d'ADN (capsides C) sortent préférentiellement du noyau au détriment des intermédiaires viraux sans génome (capsides A et B). Cependant, nous ne savons pas pourquoi les capsides C sont favorisées lors de ce processus. En aval, le virus mûrit, recrute de nombreuses protéines puis acquiert une enveloppe à partir d'un compartiment cytoplasmique. Il sort ensuite de la cellule sous forme de virions enveloppés matures. Outre les facteurs viraux mentionnés et quelques protéines hôtes, l'implication de nombreuses autres protéines virales et cellulaires dans cette voie n'a pas été entièrement caractérisée. Pour élucider davantage ce processus de sélection de la capside C, nous avons profité de l'analyse MS/MS des capsides nucléaires du VHS-1 pour définir les facteurs hôtes et viraux spécifiques à chaque intermédiaire de capside nucléaire (Chapitre 2; Article 1). Nous avons trouvé deux protéines virales (pUL42 et pUL46) et sept facteurs de l'hôte (glycogène synthase, quatre protéines différentes liées à la kératine, fibronectine 1 et PCBP1) qui étaient spécifiques des capsides C matures. Fait intéressant, toutes ces protéines semblent posséder des fonctions qui ont le potentiel de médier la sortie nucléaire préférentielle des capsides C. Par conséquent, l'analyse fonctionnelle future de ces protéines pourrait nous fournir des informations inestimables sur la sortie nucléaire actuellement énigmatique des capsides du VHS-1. Les travaux en cours d'un collègue de laboratoire avec lequel je collabore impliquent PCBP1 en tant que modulateur de la sortie nucléaire (mémoire de Mackenzie Thornbury). Nous nous sommes ensuite concentrés sur un ensemble de données protéomiques déjà existantes des virions extracellulaires matures, qui a identifié jusqu'à 49 protéines hôtes incorporées dans le virus, y compris une hélicase à ARN humaine appelée DDX3X qui s'est avérée être un modulateur actif de la propagation virale (Chapitre 2; Article 2). Nous avons remarqué que cette protéine se déplace vers le noyau tard lors de l'infection, coïncidant avec la majeure partie de la sortie nucléaire virale. Par conséquent, nous avons émis l'hypothèse que DDX3X serait impliqué dans la sortie nucléaire virale. Nous avons découvert que, tardivement au cours de l'infection, pUL31 interagit avec DDX3X au niveau du noyau. Nous avons également constaté que DDX3X stimule de grandes agrégations de capsides virales matures dans la périphérie nucléaire. Fait intéressant, la redirection de DDX3X vers le bord nucléaire dépend de la présence de la machinerie de sortie nucléaire virale (pUL31, pUL34 et pUs3) et de capsides matures. Enfin, nos données ont montré qu'en l'absence de DDX3X, les capsides C s'accumulent entre les deux membranes nucléaires, probablement à la suite d'une incorporation inefficace de pUs3 au site de sortie. Ces résultats ont élucidé une nouvelle fonction de DDX3X et pourraient ouvrir de nouvelles voies passionnantes de recherche pour développement d’antiviraux en ciblant cette hélicase à ARN cellulaire. / Herpes simplex virus type 1 (HSV-1) is one of the oldest and most successful human pathogens. It is estimated that 3.7 billion people worldwide are living with HSV-1. The virus latently persists in sensory neurons, occasionally recurring as a lytic infection which damages the connected epithelium. Even though HSV-1 causes a mild disease known as the cold sore in majority of cases, the infection can have catastrophic consequences such as encephalitis and keratitis in immunocompromised individuals, newborns and, more rarely, in immune competent adults. Considering the widespread presence of HSV-1 infections, the virus poses a potential threat to the healthcare system. The DNA genome of HSV-1 is protected by a protein cage called a capsid. Although HSV-1 capsid assembly and genome packaging take place inside the host nucleus, the final steps of maturation must be completed inside the cytoplasm. Since the large diameter of these viral capsids (~125 nm) far exceeds the 30 nm cut-off of the nuclear pore complex, the virus has evolved a mechanism known as envelopment-deenvelopmentreenvelopment. The first step of this complex process is mainly regulated by the components of the nuclear egress complex (pUL31 and pUL34) and results in the budding of enveloped capsid into the perinuclear space. Subsequently, deenvelopment of these perinuclear capsids and their release into the cytoplasm is thought to be largely modulated by the viral kinase pUs3. This process is selective as DNA-filled capsids (C-capsids) preferentially exit the nucleus compared to genome-free viral intermediates (A- and Bcapsids). However, it is unclear how C-capsids are preferentially selected for the nuclear egress. Further downstream, the virus matures and recruit numerous proteins onto the viral capsids and acquire an envelope from a cytoplasmic compartment. It then exits the cell as mature enveloped virions. Apart from the mentioned viral factors and a handful of host proteins, implication of many other viral and cellular proteins in this pathway have not been fully characterized. To further resolve this process of C-capsid selection, we took advantage of MS/MS analysis of HSV-1 nuclear capsids to define host and viral factors specific to each nuclear capsid intermediate (Chapter 2; Article 1). We found two viral proteins (pUL42 and pUL46) and seven host factors (glycogen synthase, four different keratin-related proteins, fibronectin 1, and PCBP1) that were specific to mature C-capsids. Interestingly, all these proteins seem to possess functions that have the potential to mediate the preferential nuclear exit of C-capsids. Therefore, future functional analysis of these proteins might provide us with invaluable insights into the currently enigmatic nuclear egress of HSV-1 capsids. Ongoing work by a lab colleague with which I collaborate implicates PCBP1 as a modulator of nuclear egress (memoir of Mackenzie Thornbury). We then focused on an existing proteomics data set of mature extracellular virions, which revealed 49 virus-incorporated host proteins, including a human RNA helicase called DDX3X that we found to be an active modulator of viral propagation (Chapter 2; Article 2). We observed that DDX3X relocates to the nuclear rim late during infection, coinciding with the bulk of viral nuclear egress, and leading us to hypothesize that DDX3X is involved in the process. We discovered that, late during the infection, pUL31 interacts with DDX3X at the nuclear rim. We also found that DDX3X stimulates large aggregations of mature viral capsids in the nuclear periphery. Unexpectedly, redirection of DDX3X to the nuclear rim was dependent on the presence of the viral nuclear egress machinery (pUL31, pUL34 and pUs3) and mature capsids. Lastly, our data showed that in the absence of DDX3X, C-capsids accumulate in the perinuclear space, likely as the result of inefficient incorporation of pUs3 to the site of egress. These results have elucidated a novel function for DDX3X and may open new and exciting paths to produce antivirals by targeting this cellular RNA helicase.

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable du syndrome de l’immunodéficience acquise (SIDA). Il faut identifier de nouvelles cibles pour le développement d’agents anti-VIH-1, car ce virus développe une résistance aux agents présentement utilisés. Notre but est d’approfondir la caractérisation de l’étape du changement de cadre de lecture ribosomique en -1 (déphasage -1) nécessaire à la production du précurseur des enzymes du VIH-1. Ce déphasage est programmé et effectué par une minorité de ribosomes lorsqu’ils traduisent la séquence dite glissante à un endroit spécifique de l’ARN messager (ARNm) pleine-longueur du VIH-1. L’efficacité de déphasage est contrôlée par le signal stimulateur de déphasage (SSF), une tige-boucle irrégulière située en aval de la séquence glissante. La structure du SSF est déroulée lors du passage d’un ribosome, mais elle peut se reformer ensuite. Nous avons montré que des variations de l’initiation de la traduction affectent l’efficacité de déphasage. Nous avons utilisé, dans des cellules Jurkat-T et HEK 293T, un rapporteur bicistronique où les gènes codant pour les luciférases de la Renilla (Rluc) et de la luciole (Fluc) sont séparés par la région de déphasage du VIH-1. La Rluc est produite par tous les ribosomes traduisant l’ARNm rapporteur alors que la Fluc est produite uniquement par les ribosomes effectuant un déphasage. L’initiation de ce rapporteur est coiffe-dépendante, comme pour la majorité des ARNm cellulaires. Nous avons examiné l’effet de trois inhibiteurs de l’initiation et montré que leur présence augmente l’efficacité de déphasage. Nous avons ensuite étudié l’effet de la tige-boucle TAR, qui est présente à l’extrémité 5’ de tous les ARNm du VIH-1. TAR empêche la liaison de la petite sous-unité du ribosome (40S) à l’ARNm et module aussi l’activité de la protéine kinase dépendante de l’ARN double-brin (PKR). L’activation de PKR inhibe l’initiation en phosphorylant le facteur d’initiation eucaryote 2 (eIF2) alors que l’inhibition de PKR a l’effet inverse. Nous avons étudié l’effet de TAR sur la traduction et le déphasage via son effet sur PKR en utilisant TAR en trans ou en cis, mais à une certaine distance de l’extrémité 5’ afin d’éviter l’interférence avec la liaison de la 40S. Nous avons observé qu’une faible concentration de TAR, qui active PKR, augmente l’efficacité de déphasage alors qu’une concentration élevée de TAR, qui inhibe PKR, diminue cette efficacité. Nous avons proposé un modèle où des variations de l’initiation affectent l’efficacité de déphasage en modifiant la distance entre les ribosomes parcourant l’ARNm et, donc, la probabilité qu’ils rencontrent un SSF structuré. Par la suite, nous avons déterminé l’effet de la région 5’ non traduite (UTR) de l’ARNm pleine-longueur du VIH-1 sur l’efficacité de déphasage. Cette 5’UTR contient plusieurs régions structurées, dont TAR à l’extrémité 5’, qui peut interférer avec l’initiation. Cet ARNm a une coiffe permettant une initiation coiffe-dépendante ainsi qu’un site d’entrée interne des ribosomes (IRES), permettant une initiation IRES-dépendante. Nous avons introduit cette 5’UTR, complète ou en partie, comme 5’UTR de notre ARNm rapporteur bicistronique. Nos résultats démontrent que cette 5’UTR complète inhibe l’initiation coiffe dépendante et augmente l’efficacité de déphasage et que ces effets sont dus à la présence de TAR suivie de la tige-boucle Poly(A). Nous avons aussi construit un rapporteur tricistronique où les ribosomes exprimant les luciférases utilisent obligatoirement l’IRES. Nous avons observé que cette initiation par l’IRES est faible et que l’efficacité de déphasage correspondante est également faible. Nous avons formulé une hypothèse pour expliquer cette situation. Nous avons également observé que lorsque les deux modes d’initiation sont disponibles, l’initiation coiffe dépendante est prédominante. Finalement, nous avons étudié l’effet de la protéine virale Tat sur l’initiation de la traduction et sur l’efficacité de déphasage. Nous avons montré qu’elle augmente l’initiation de la traduction et que son effet est plus prononcé lorsque TAR est située à l’extrémité 5’ des ARNm. Nous proposons un modèle expliquant les effets de Tat sur l’initiation de la traduction par l’inhibition de PKR ainsi que par des changements de l’expression de protéines cellulaires déroulant TAR. Ces résultats permettent de mieux comprendre les mécanismes régissant le déphasage du VIH-1, ce qui est essentiel pour le développement d’agents anti-déphasage. / The human immunodeficiency virus type 1 (HIV-1) is responsible for the acquired immune deficiency syndrome (AIDS). HIV-1 develops a resistance towards the inhibitors used to treat infected patients. It is thus important to identify new targets for the development of novel antiretroviral agents. The aim of our work was to better characterize the programmed -1 ribosomal frameshift which generates the precursor of HIV-1 enzymes. The frameshift occurs at a specific sequence of HIV-1 full-length messenger RNA (mRNA), the slippery sequence, and is performed by a minority of the ribosomes translating this mRNA. The frameshift efficiency is controlled by the frameshift stimulatory signal (FSS), an irregular stem-loop located downstream of the slippery sequence. FSS structure is unfolded by every ribosome translating this region and can refold afterwards. We showed that HIV-1 frameshift efficiency is affected by changes in the rate of translation initiation. We transfected Jurkat-T and HEK 293T cells with a bicistronic reporter that contains the frameshift region of HIV-1 between the Renilla luciferase (Rluc) and the firefly luciferase (Fluc) genes. Rluc is produced by all ribosomes translating this reporter whereas only ribosomes that make a –1 frameshift produce Fluc. The translation of the reporter is initiated via a cap-dependant mode, like the majority of cellular mRNAs. We first determined the effect of three inhibitors of translation initiation. We showed that their presence increases the frameshift efficiency. We next determined the impact of the TAR stem loop, which is located at the 5’end of every HIV-1 mRNA. TAR is known to impair the binding of the small subunit of the ribosome (40S) to the mRNA. TAR also modulates the activity of the double-stranded RNA-dependent protein kinase (PKR). When PKR is activated, it phosphorylates the eukaryotic initiation factor 2 (eIF2), inhibiting translation initiation. The inhibition of PKR has the opposite effect. We studied the effect of TAR on PKR by positioning TAR at a distance of the 5’ end where it cannot interfere with the binding of the 40S. Our results showed that a small amount of TAR, which activates PKR, increases the frameshift efficiency whereas a large amount of TAR, which inhibits PKR, decreases it. A model is presented where the variations of translation initiation modulate HIV-1 frameshift efficiency by altering the distance between the elongating ribosomes. This influences the probability that these ribosomes encounter or not a folded FSS. We next observed the effect of the 5’ untranslated region (UTR) of HIV-1 full length mRNA on its frameshift efficiency. This 5’UTR contains several structured parts, including TAR at the 5’end, which can inhibit translation initiation. This mRNA has a cap and an internal ribosome entry site (IRES) and could then use a cap dependent and an IRES-dependent mode of translation initiation. We replaced the 5’UTR of our bicistronic reporter mRNA by the complete 5’UTR of HIV-1 full-length mRNA or a part of it. Our results showed that the presence of the complete 5’UTR inhibits cap-dependent initiation of translation and increases the frameshift efficiency. Those effects are mostly due to the presence of TAR followed by a Poly(A) stem-loop. We also constructed a tricistronic reporter where the ribosomes translating the luciferases have to use an IRES-dependent initiation mode. The rate of this initiation was low and the frameshift efficiency obtained was also low. We proposed a hypothesis accounting for this situation. We also observed that when both initiation modes are available, the cap-dependent mode seems to be highly favored. Finally, we studied the impact of the Tat viral protein on translation initiation and frameshift efficiency. We showed that the presence of Tat increases translation initiation and decreases the frameshift efficiency. Those effects are more important when TAR is present at the 5’end of mRNA. We propose a model explaining the effects of Tat on translation initiation by the inhibition of PKR and by changes in the expression of cellular proteins that are able to unfold TAR. Our results allow us to better understand the mechanisms controlling HIV-1 frameshift, which will help in the development of drugs targeting the HIV-1 frameshift.

Le virus humain T lymphotrope de type 1 (HTLV-1) est l’agent étiologique de la leucémie/lymphome T de l’adulte (ATLL) qui se développe après plusieurs décennies et pour laquelle il n’existe à ce jour pas de traitement efficace. Parmi les protéines virales de HTLV-1, Tax et HBZ jouent un rôle déterminant dans le développement de l’ATLL. Si Tax participe au processus leucémogène dès les étapes précoces, HBZ jouerait plutôt un rôle dans le maintien du phénotype tumoral dans les étapes tardives. Dans ce contexte, là nous nous sommes intéressés à la régulation de l’expression des protéines anti-apoptotiques Bfl-1 et Bcl-xL, par les protéines virales Tax et HBZ. Nous avons montré que Tax induit l’expression des protéines anti-apoptotiques Bfl-1 et Bcl-xL de la famille Bcl-2 via la voie NF-κB, alors que HBZ n’a aucun effet sur leur expression. De plus, Tax coopère avec les facteurs de transcription c-Jun et JunD de la voie AP-1 pour augmenter l’expression de ces gènes anti-apoptotiques. En revanche, HBZ module uniquement la trans-activation de bfl-1 induite par Tax. L’ensemble de nos résultats indique donc que Tax joue un rôle prépondérant dans l’activation de l’expression de Bfl-1 et de Bcl-xL et suggère que Bfl-1 et Bcl-xL sont exprimées au cours des étapes précoces et tardives du développement de l’ATLL. Par une stratégie d’ARN interférence, nous avons ensuite montré que Bfl-1 et/ou Bcl-xL sont impliquées dans la survie de lignées cellulaires T infectées par HTLV-1, suggérant que Bfl-1 et Bcl-xL représentent des cibles thérapeutiques potentielles pour traiter l’ATLL. Actuellement, il existe des petites molécules ciblant les membres anti-apoptotiques de la famille Bcl-2, mais aucune ne cible spécifiquement Bfl-1. En collaboration avec la société IMAXIO, nous avons identifié par deux cribles à haut débit 83 molécules capables d’inhiber l’activité anti-apoptotique de Bfl-1. L’une de ces molécules induit spécifiquement la mort de lignées cellulaires T infectées par HTLV-1 pour lesquelles Bfl-1 représente un gène de survie. Ainsi, ce travail doit permettre à terme de développer de futurs médicaments dirigés contre Bfl-1 et de proposer une nouvelle stratégie thérapeutique ciblée contre l’ATLL / Human T lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL) that develops after several decades and for which there is no effective treatment. Among the viral proteins of HTLV-1, Tax and HBZ play a major role in the development of ATLL. If Tax participates in the initiation of leukemogenesis from the early stages, HBZ rather plays a role in maintaining the tumor phenotype in the late stages. The aims of our study were to better understand the regulation of Bfl-1 and Bcl-xL anti-apoptotic protein expression by Tax and HBZ viral proteins, as well as their role in the survival of HTLV-1-infected T-cells to propose new therapeutic strategies. We showed that Tax induces Bfl-1 and Bcl-xL expression via the NF-κB pathway, whereas HBZ has no effect on their expression. Tax also cooperates with c-Jun and JunD transcription factors of AP-1 family to increase the expression of these anti-apoptotic genes. By contrast, HBZ modulates the Tax-induced bfl-1 trans-activation. Altogether, our data indicate that Tax plays a key role in activating Bfl-1 and Bcl-xL expression and suggests that Bfl-1 and Bcl-xL are potentially expressed during the early and the late stages of ATLL development. Using short hairpin RNA strategy, we then showed that Bfl-1 and/or Bcl-xL are involved in HTLV-1-infected T-cell line survival, indicating that Bfl-1 and Bcl-xL represent potential therapeutic targets in the case of ATLL. One approach currently being developed in anti-cancer drug discovery is to search for small inhibitory compounds targeting anti-apoptotic proteins of the Bcl-2 family. But so far, no drug specifically targeting Bfl-1 is available. In collaboration with the IMAXIO Company, we have identified 83 molecules able to inhibit Bfl-1 anti-apoptotic activity using two high-throughput screening. One of these molecules specifically induced the death of HTLV-1-infected T-cell for which Bfl-1 represents a survival gene. This work provides new insight for long-term development of future drugs directed against Bfl-1 and should allow us to propose new therapeutic strategy for ATLL treatment

Herpes simplex virus Typ 1 (HSV-1) ist ein humanpathogenes Virus der Familie Herpesviridae. Für eine erfolgreiche Virusreplikation besitzt HSV-1 mehrere Gene, die in den meisten infizierten Zelltypen Apoptose verhindern. Im Gegensatz dazu führt die HSV-1 Infektion eines zentralen Zelltyps des Immunsystems, den unreifen dendritischen Zellen (iDCs), zu Apoptose. Dies könnte ein Aspekt der HSV-1 Immunevasion sein. Bisher waren die Ursachen der Apoptose von HSV-1 infizierten iDCs unzureichend aufgeklärt. Es wurde jedoch gezeigt, dass das antiapoptotische zelluläre Protein c-FLIP in HSV-1 infizierten iDCs reduziert ist. In dieser Arbeit wurde die c-FLIP Menge in iDCs erstmalig mit Hilfe von RNA Interferenz erfolgreich reduziert. Dies bestätigte die Bedeutung von c-FLIP für die Lebensfähigkeit von iDCs. Folglich könnte auch die Reduktion der c-FLIP Menge nach HSV-1 Infektion iDCs für Apoptose empfindlich machen. Die HSV-1 induzierte c-FLIP Reduktion erfolgte in späten Stadien der Infektion, abhängig von der ordnungsgemäßen Expression viraler „early“ und „leaky late“ Gene. Sie fand nicht auf RNA Ebene statt und war unabhängig vom Proteasom und der Bindung an den „death inducing signaling complex“. Stattdessen wurde c-FLIP wahrscheinlich von einer viralen oder zellulären Protease abgebaut. In dieser Arbeit wurde erstmals gezeigt, dass zusätzlich zu Veränderungen im zellulären Apoptosesignalnetzwerk der Mangel an einem antiapoptotischen viralen Faktor zur Apoptose von HSV-1 infizierten iDCs beiträgt. Eine Microarray Analyse der HSV-1 Genexpression ergab, dass HSV-1 Latenz-assoziierte Transkripte (LATs) in apoptotischen iDCs signifikant geringer exprimiert waren als in nicht-apoptotischen epithelialen Zellen. LATs besitzen in Neuronen und epithelialen Zellen eine antiapoptotische Aktivität. Diese könnte den Mangel an c-FLIP kompensieren. Übereinstimmend mit dieser Hypothese induzierte eine HSV-1 LAT-Deletionsmutante mehr Apoptose in iDCs im Vergleich zum Wildtyp-Virus. / Herpes simplex virus type 1 (HSV-1) is a human pathogen which belongs to the family Herpesviridae. HSV-1 encodes several genes, which serve to efficiently prevent apoptosis in most infected cell types, thereby ensuring successful virus replication. In contrast, HSV-1 infection of one central cell type of the immune system, immature dendritic cells (iDCs), results in apoptosis. This could be one aspect of HSV-1 immunevasion. So far, the mechanisms underlying apoptosis of HSV-1 infected iDCs were poorly defined. However, it has been shown that the antiapoptotic cellular protein c-FLIP is reduced in HSV-1 infected iDCs. In this work, the amount of c-FLIP was for the first time successfully reduced in iDCs by RNA interference. This confirmed the importance of c-FLIP for viability of iDCs. Therefore, it is likely that c-FLIP reduction after HSV-1 infection also sensitizes iDCs to apoptosis. HSV-1 induced c-FLIP reduction occurred at late stages of infection and was dependent on proper expression of early and leaky late virus genes. Furthermore, it was not operative at the RNA level and was independent from the proteasome and binding to the death inducing signaling complex. Rather, c-FLIP was presumably degraded by a viral or cellular protease. In this work it was shown for the first time, that in addition to changes in the cellular apoptosis signaling network, the lack of one antiapoptotic viral factor contributes to apoptosis of HSV-1 infected iDCs. HSV-1 latency-associated transcripts (LATs) were significantly lower expressed in apoptotic iDCs compared to non-apoptotic epithelial cells, determined by microarray analysis of HSV-1 gene expression. It is known that in neurons and epithelial cells, LATs possess a potent antiapoptotic activity. This could compensate the lack of c-FLIP. Consistent with this hypothesis, a LAT deletion mutant of HSV-1 induced more apoptosis in iDCs compared to the respective wild type virus.

Nach dem Eintritt in den Wirtsorganismus und initialer Replikation infizieren Alphaherpesviren Neuronen zur weiteren Ausbreitung im Nervensystem und zur Etablierung einer Latenz. Dazu werden die Viruspartikel innerhalb der Axone retrograd von der Peripherie zum neuronalen Zellkörper transportiert. Die umgekehrte Richtung beschreibt den Weg des anterograden Transports vom Zellkörper zur Synapse für weitere Infektionen von Neuronen höherer Ordnung oder zurück zur Peripherie. Der retrograde intraaxonale Transport ist gut untersucht. Dagegen wird über den anterograden Transport kontrovers diskutiert. Zwei verschiedene Transportmodelle werden vermutet. Das „Married Model“ postuliert, dass umhüllte Virionen innerhalb von Vesikeln entlang des Axons transportiert werden. Die Freisetzung der Partikel erfolgt an der jeweiligen Synapse durch Endocytose. Das „Subassembly Model“ geht dagegen davon aus, dass einzelne Virusstrukurkomponenten (Nukleokapsid, Hülle) entlang des Axons transportiert werden. Der Zusammenbau und die Freisetzung erfolgt am Axonterminus bzw. an der Synapse (in vivo) oder am Wachstumskegel (in vitro) oder an speziellen Auftreibungen des Axons, den sogenannten Varicosities. Nach Infektion eines neuronalen Explantatsystems mit dem Pseudorabiesvirus (PrV) konnten ultrastrukturell umhüllte Virionen in Vesikeln detektiert werden und so der Nachweis der Gültigkeit des „Married Model“ als vorherrschendes Transportmodell geführt werden. Dagegen ist die Situation beim prototypischen Alphaherpesvirus, dem Herpes Simplex Virus Typ 1 (HSV-1), weiterhin ungeklärt. Aufgrund der zahlreichen unterschiedlichen Analysemethoden und -systeme war ein direkter Vergleich der beiden Viren bislang nicht möglich. Daher sollte in dieser Arbeit ein standardisiertes neuronales Kultursystem genutzt werden, um vier verschiedene HSV-1 Stämme im Vergleich zu PrV zu untersuchen. Für die Infektionen wurden sowohl Neuronen aus dem oberen Cervikalganglion als auch aus Spinalganglien genutzt. So konnte gezeigt werden, dass in Neuronen, welche mit den HSV-1 Stämmen HFEM, 17+ und SC16 infiziert waren ca. 75% als umhüllte Virionen in Vesikeln und ca. 25% als nackte Kapside vorlagen. Ingesamt war die Anzahl der Viruspartikel in HSV-1 infizierten Neuronen signifikant geringer als in PrV infizierten Kulturen. Überraschenderweise zeigten mit HSV-1 KOS infizierte Neuronen ein reverses Bild. Hier lagen nur 25% der Viruspartikel als umhüllte Virionen in Vesikeln vor, während 75% als nackte Kapside detektiert wurden. Dieser unerwartete Phänotyp sollte auf molekularbiologischer Ebene genauer untersucht werden. Dabei wurde auf die Genregion von US9 fokussiert. Das von US9 codierte Membranprotein spielt eine wichtige Rolle während des Zusammenbaus der Virionen und bei anschließenden axonalen anterograden Transportvorgängen. In dieser Arbeit konnte gezeigt werden, dass das HSV-1 KOS Genom durch verschiedene Basenaustausche an der vorhergesagten TATA-Box von US9 eine Mutation aufweist. Zusätzlich trägt das offene Leseraster durch eine weitere Mutation ein vorzeitiges Stopcodon auf und wird dadurch auf 58 Kodons reduziert, im Gegensatz zu anderen HSV-1 Stämmen, wo es 91 Kodons umfasst. Die Mutation an der TATA-Box verändert auch das ursprüngliche Stopcodon vom US8a Gen, was zur einer Verlängerung von ursprünglich 161 zu 191 Kodons führt. In Northern Blot Analysen konnte eine reduzierte Transkription von US9 in HSV-1 KOS infizierten Zellen detektiert werden. In HSV-1 KOS infizierten Zellen konnten mittels eines spezifischen Antiserums gegen US9 im Western Blot kein Genprodukt nachgewiesen werden. Auch Immunfluoreszenzanalysen zeigten, dass das abgeleitete verkürzte Protein offenbar nicht stabil exprimiert wird. Dagegen konnten Western Blot Analysen die Vergrößerung des pUS8a bestätigen. Der beobachtete auffällige intraaxonale Phänotyp könnte somit durch die Mutation des US9 Protein erklärt werden. Zusammenfassend wurde in dieser Arbeit gezeigt, dass auch bei HSV-1 vorwiegend das „Married Model“ für den anterograden intraaxonalen Transportweg bevorzugt wird und somit beide Alphaherpesviren, HSV-1 und PrV, denselben Transportweg nutzen.