Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 163-232).

The human gammaherpesviruses EBV and KSHV realize their oncogenic potential during latent infection. The species specificity of the human gammaherpesviruses has hindered the study of latency in animal models. Murine gammaherpesvirus MHV-68 (MHV-68) may be used as a representative gammaherpesvirus for the study of latency. The goal was to establish an in vitro model of MHV-68 latency using replication defective MHV-68. ORF50 has been identified as the major viral trans-activator essential for entry into the lytic replication cycle and necessary and sufficient for reactivation of MHV-68 virus from latency. ORF50 null mutants (A50) can theoretically be used to infect cells in vitro to facilitate an analysis of virus gene expression and episome maintenance during latency. In this project A50 mutants containing the luciferase or green fluorescence protein (GFP) under OW50 promoter control were used to infect a variety of cell types. 3T3 fibroblasts are a permissive cell line and were used for an initial characterization of the ability of A50 MHV-68 to establish latency. B lymphocytes and macrophages are the major reservoirs of persistence in vivo thus the ability of A50 mutants to establish latency in NSO B and RAW macrophage cell lines was explored. Latency was readily established and maintained in 3T3 and RAW cells. The low infectability of NSO B- cells restricted the utility of this cell line in studies of latency. Examination of patterns of lytic and latent transcription in 3T3 and RAW cells coordinately infected with A50 MHV-68 revealed reactivation efficiencies of 40-60%. Following long-term passage A50 exhibited stable transcription of two latency related genes M2 and ORF73, with episomal maintenance of the viral genome, in the absence of contaminating lytic infection. The results demonstrate the utility of A50 mutants for studies of gammaherpesvirus latency in vivo.

Orientador: Sandra Cecilia Botelho Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T03:22:40Z (GMT). No. of bitstreams: 1 Pasquotto_Juliana_M.pdf: 2465167 bytes, checksum: dcd2cc122494bab62d8ab495927b1f0e (MD5) Previous issue date: 2008 / Resumo: O Vírus Epstein-Barr (EBV) e o Citomegalovírus (HCMV) são membros da família Herpesvírus. São encontrados em aproximadamente 90% dos indivíduos em idade adulta. A infecção ocorre, geralmente, na infância e é assintomática na maioria dos casos, persistindo de forma latente durante toda a vida do indivíduo. A transmissão destes vírus ocorre principalmente pela saliva, sangue e órgãos transplantados. O EBV está relacionado com a mononucleose infecciosa, doença linfoproliferativa (PTLD), leucemia de células pilosas em pacientes com imunodeficiência congênita ou adquirida e doença de Hodgkin. O risco de um paciente transplantado desenvolver linfoma é 28 a 50 vezes maior do que os indivíduos da população geral. Um dos fatores de risco para o desenvolvimento da PTLD são a variedade e intensidade da imunossupressão utilizada no paciente pós-transplante, idade do receptor e sorologia viral (EBV, CMV). Dependendo da idade do receptor, do tipo de transplante e dos fatores de risco, a prevalência da PTLD pode variar de 0.5% a 22%. Em transplantados pediátricos renais a prevalência chega a atingir 37%. A principal medida terapêutica para o controle da PTLD é a diminuição ou mesmo a retirada total da imunossupressão. Portanto a rejeição do enxerto se torna um problema bastante comum, que compromete a qualidade e/ou expectativa de vida dos pacientes. A introdução de testes laboratoriais rápidos e precoces permite aos clínicos detectar a replicação viral do EBV e diagnosticar, consequentemente, a infecção ativa antes do início da doença. Isso proporciona a oportunidade de iniciar o tratamento específico precocemente. Foram estudadas amostras de sangue e soro de 46 pacientes submetidos a transplantes de células hematopoéticas, acompanhados no Serviço de Transplante de Medula Óssea (STMO) do Hospital das Clínicas da UNICAMP/HEMOCENTRO. Trabalhamos no estudo para diagnosticar a infecção ativa e quantificar a carga viral do vírus Epstein-Barr (EBV) em pacientes transplantados de células hematopoéticas. Relacionar infecção ativa do vírus Epstein-Barr com o Citomegalovírus (CMV) e verificar a incidência da Doença linfoproliferativa e a Doença do enxerto contra o hospedeiro (GVHD) nos pacientes estudados. Diagnosticamos infecção ativa pelo EBV em 22 (47,8%) pacientes que apresentaram uma carga viral muito baixa (média de 29 cópias/ul). Co-infecção entre EBV e CMV ocorreu em 15/46 pacientes (32,6%). Doença por CMV ocorreu em 7/46 (15,2%) pacientes no trato gastrintestinal. Todos estes doentes apresentaram infecção ativa pelo CMV e 4/7 (57%) apresentaram infecção ativa pelo EBV. Um destes pacientes foi a óbito por doença por CMV. Verificamos que nenhum dos pacientes apresentou doença linfoproliferativa relacionada ao EBV. Os casos de co-infecção ativa EBV+CMV em relação à ocorrência de GVHD foram estatisticamente significantes (p=0.001). Concluímos que a infecção pelo CMV ainda é a maior causa de morbidade e mortalidade nos pacientes após o transplante. A qPCR é uma ferramenta útil para verificar os pacientes que reativaram pelo EBV após o transplante e pode auxiliar na prevenção da doença linfoproliferativa causada pelo EBV juntamente com a identificação dos fatores de risco associados. Verificamos a ocorrência e significância do GVHD e infecção ativa pelo CMV, mas não observamos esses mesmos resultados comparados ao EBV / Abstract: Epstein-Barr Virus (EBV) and Cytomegalovirus (CMV) are members of herpesvirus family. They are found in approximately 90% of the individuals in adult age. The infection generally occurs subclinicaly during the childhood in the major of the cases persisting in latent form during all the life of the individual. The transmission of these viruses occurs mainly for the saliva, blood and transplanted organs. EBV is related with mononucleose infectious, lynfoproliferative disease (PTLD), leukemia of hair cells in patients with congenital or acquired immunodeficiency and Hodgkin¿s disease. The risk of a transplanted patient to develop linfoma is 28 to 50 % more than other individuals of the general population. One of the risk factor for the development of the PTLD is the variety and intensity of the imunossupression used in the patient after-transplant, age of the recipient and viral serology (EBV, CMV). Depending on the age of the recipient, the type of transplant and the risk factor, the prevalence of the PTLD can vary of 0.5% 22%. In renal pediatric transplantation, the prevalence can arrives to reach 37%. The main therapeutical measure for the control of the PTLD is the reduction or total withdrawal of the imunossupression. Therefore the lost of graft is a common problem, that compromises the quality and/or life expectancy of the patients. The introduction of early and rapid laboratorial tests can permit to the physicians to detect the viral response and detect the active EBV infection before the disease. This provides the chance to initiate the specific treatment. We studied samples of blood and serum of 46 patients submitted to hematopoietic stem cell transplantation at the Bone Marrow Transplant unit of the University Hospital of the UNICAMP/HEMOCENTRO. We worked in the study to diagnosis the active infection and quantify the viral load of the Epstein-Barr virus (EBV) in transplanted patients of hematopoetic stem cells, to relate active EBV infection with active CMV infection and to verify the incidence of the lynfoproliferative disease and graft versus host disease (GVHD) in the studied patients. Active EBV infection detected by Real-Time PCR occurred in 22 patients (47,8%). The viral load found was very low (range 29 copies/ul). Co-infection between EBV and CMV occurred in 15/46 patients (32,6%). CMV disease occurred in 7/46 (15,2%) patients in the gastrointestinal tract. All these patients had active CMV infection and 4/7 patients (57%) had active EBV infection. One of these patients died by CMV disease. No patient presented lymphoproliferative disease related to the EBV. The cases of active EBV and CMV (co-infection) infection in relation to the occurrence of GVHD had been statisticaly significant (p=0.001). We conclude that the active CMV infection is already the most cause of morbidity and mortality of the patients after the transplant. The qPCR is a useful tool to verify the patients who had active EBV infection after the transplant and the identification of the risk factors associates prevention the development of the lymfoproliferative disease caused by the EBV. We verify the occurrence and significance of the GVHD and active CMV infection, but we do not observe these same results related to the EBV / Mestrado / Mestre em Farmacologia

Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2007. / Typescript. Abstract available online via ProQuest Digital Dissertations. Includes bibliographical references (leaves 207-223).

Thesis (Ph.D. in Microbiology & Immunology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 200-212).