The nuclear envelope is a highly organised double membrane system that separates the activities of the nuclear and cytoplasmic compartments in eukaryotic systems. The wide range of functions recently associated with the NE and the identification of hundreds of proteins associated with this cellular structure indicates that it is a major signalling node for the cell. Recent work indicates NE functions in signalling innate immune responses to herpesviruses. The viruses, on the other hand, often target or usurp NE functions in different ways. The NE is also a physical barrier that must be overcome for viruses like the herpesviridae that assemble capsids in the nucleus. This thesis addresses two important questions: 1) How do herpesviruses cross the NE after new viral particles are produced in the nucleus? and 2) What is the nuclear envelope role of NET23/STING in the activation of immune factors upon herpesvirus infection? To address the first question, I followed two different approaches. The first used the isolation of microsomes from HSV-1 infected cells to identify possible host factors involved during herpesvirus exit through the NE on the prediction that such proteins would disperse into the ER during infection. I identified a group of vesicle fusion proteins that play a role in this herpesvirus exit through the NE. Depletion of three identified vesicle fusion proteins decreased the growth of HSV-1 in host cells, yielding accumulation of viral particles in the nucleus. The second approach was to follow the fate of nuclear envelope transmembrane proteins (NETs) during HSV-1 infection. To address the question of how NET23/STING is involved in innate immunity I tested the hypothesis that this NET acts as a transport receptor to carry signals through the peripheral channels of the NPC when central channel transport is blocked by pathogens. FRAP was used to quantify the mobility of NET23/STING upon the induction of the innate immune response, finding an increase of the mobility for this protein in the NE. To further elucidate its role within the NE I tested whether some NE-NET23/STING binding partners were being redistributed between the nucleus and cytoplasm during innate immune responses. This revealed two of these binding partners normally redistribute upon innate immune response activation and this is blocked in cells knocked down for NET23/STING. Finally, I confirmed that NET23/STING contributes to chromatin remodelling during infection involving an increase in the H3K9Me3 epigenetic mark. Collectively, these data argue the identification of novel host proteins involved in herpesvirus nuclear egress and the finding of a new role for NET23/STING within the NE.

Feline herpesviruses are endemic in free-ranging lions in South Africa. Serological surveillance among free-living felids revealed high levels of exposure to the virus. However, clinical disease in wild felids following FHV-1 infection has been only described in captive populations and reported to be similar to that in the domestic cat. To expand the epidemiological understanding of feline herpesviruses in felids and for disease control, three strains of FHV-1 isolated from a domestic cat (Felis catus) a cheetah (Acinonyx jubatus) and an African wild cat (Felis silvestris) have been compared to determine their relatedness. A region of the herpesvirus DNA polymerase gene was amplified in a nested PCR with consensus degenerate primers to confirm the identity of the isolates. The genetic relatedness were investigated by comparing patterns of genomic DNA cleaved with restriction enzymes SalI and KpnI and the DNA fingerprints generated by different RAPD primers. For antigenic relationships, a panel of nine monoclonal antibodies prepared against a vaccine strain used against domestic cats were tested in a microneutralization assay. In addition, the phenotypic characteristics of the isolates were also compared by their ability to produce plaques in CrFK monolayer cell cultures. With restriction enzyme analysis, it was not possible to make a comparison due to lack of digestion of the genomic DNA of the domestic cat isolate. However, the RAPD-PCR revealed that isolates were closely related but distinct from each other. Only two monoclonal antibodies reacted with the wild isolates; an effect similar to a toxic effect on cell was observed with the domestic isolate. No significant differences of plaque production were observed among the trains. This study provides evidence of a closer evolutionary relationship between the three isolates. The results of the relationships based on the genetic and phenotypic characterization agreed well and both indicated that the viruses from the domestic and wild felids are different but have a high degree of similarity. Copyright / Dissertation (MSc (Veterinary Tropical Diseases))--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

The Epstein-Barr virus (EBV) protein LMP1 serves as a paradigm that engages complicated ubiquitination-mediated mechanisms to activate multiple transcription factors. p62 is a ubiquitin sensor and a signal-transducing adaptor that has multiple functions in diverse contexts. However, the interaction between p62 and oncogenic viruses is poorly understood. We recently reported a crucial role for p62 in oncovirus-mediated oxidative stress by acting as a selective autophagy receptor. In this following pursuit, we further discovered that p62 is upregulated in EBV type 3 compared to type 1 latency, with a significant contribution from NF-kB and AP1 activities downstream of LMP1 signaling. In turn, p62 participates in LMP1 signal transduction through its interaction with TRAF6, promoting TRAF6 ubiquitination and activation. As expected, short hairpin RNA (shRNA)-mediated knockdown (KD) of p62 transcripts reduces LMP1-TRAF6 interaction and TRAF6 ubiquitination, as well as p65 nuclear translocation, which was assessed by Amnis imaging flow cytometry. Strikingly, LMP1-stimulated NF-kB, AP1, and Akt activities are all markedly reduced in p622/2 mouse embryo fibroblasts (MEFs) and in EBV-negative Burkitt’s lymphoma (BL) cell lines with CRISPR-mediated knockout (KO) of the p62-encoding gene. However, EBV-positive BL cell lines (type 3 latency) with CRISPR-mediated KO of the p62-encoding gene failed to survive. In consequence, shRNA-mediated p62 KD impairs the ability of LMP1 to regulate its target gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of lymphoblastic cell lines (LCLs). These important findings have revealed a previously unrecognized novel role for p62 in EBV latency and oncogenesis, which advances our understanding of the mechanism underlying virus-mediated oncogenesis. IMPORTANCE As a ubiquitin sensor and a signal-transducing adaptor, p62 is crucial for NF-kB activation, which involves the ubiquitin machinery, in diverse contexts. However, whether p62 is required for EBV LMP1 activation of NF-kB is an open question. In this study, we provide evidence that p62 is upregulated in EBV type 3 latency and, in turn, p62 mediates LMP1 signal transduction to NF-kB, AP1, and Akt by promoting TRAF6 ubiquitination and activation. In consequence, p62 deficiency negatively regulates LMP1-mediated gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of LCLs. These important findings identified p62 as a novel signaling component of the key viral oncogenic signaling pathway.

No description available.

1. Study on improving the diagnostic accuracy of treatment-naive nasopharyngeal carcinoma. / 2. Study on diagnostic accuracy of EBV-DNA on recurrent nasopharyngeal carcinoma. / 3. Studies on EBV-DNA as a screening tool for nasopharyngeal carcinoma. Part 1. To define the detection rate of NPC and the false-positive rate of IgA-VCA in an IgA-VCA-based screening problem, and to define the specificity of IgA-EA in IgA-VCA-positive screenees. Part 2. To define the specificity of EBV-DNA in IgA-VCA-positive screenees. Part 3. To define the sensitivity of IgA-EA, and EBV-DNA in IgA-positive NPC patients. / 4. Studies on pre-therapy prognostication of nasopharyngeal carcinoma Study Part 1. Objective. To assess the role of EBV-DNA in pre-therapy prognostication of early-stage NPC. / Background. The specific association between nasopharyngeal carcinoma (NPC) and the Epstein-Barr virus (EBV) had been exploited to develop a spectrum of EBV-antibodies-based blood markers. Among these markers, the Immunoglobulin A antibody against the viral capsid antigen (IgA-VCA) of the EBV has been the most popularly employed marker to assist diagnosis of NPC. There is however a relative paucity of data on the application of blood markers for screening, for detection of relapse, and for prognostification of patient cohorts managed in present-day therapy oncology protocols. Peripheral blood EBV-DNA, measured by quantitative polymerase chain reaction assay, is a newly-developed marker, and represents a prototype model of a nuclei acid-based, as opposed to antibody-based, EBV tumor marker for NPC. The present thesis describes the translation of this basic scientific advance into clinical applications, through several prospective and retrospective studies that address the diagnosis of treatment-naive NPC, the detection of recurrent NPC, the screening of individuals at risk of NPC, the pre-therapy prognostication for NPC to guide for choice of therapy. The role of integration of conventional markers and EBV-DNA in clinical applications was also examined. / Study Part 2. Objectives. To assess whether incorporation of EBV-DNA data to TNM staging improves prognostic discrimination of patients subsets within individual cancer stage, to assess if EBV-DNA is an independent prognostic factor for survival after ontological therapy. (Abstract shortened by UMI.) / Leung Sing-fai. / "February 2005." / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3695. / Thesis (M.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / School code: 1307.

Orientador: Paulo Cesar Giraldo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T21:42:54Z (GMT). No. of bitstreams: 1 EleuterioJunior_Jose_D.pdf: 17867889 bytes, checksum: 0d9e1c3f2399c5be86d6fbf719dc98c4 (MD5) Previous issue date: 2007 / Resumo: Objetivos: Estudar a importância de determinados marcadores de diagnóstico e prognóstico de lesões escamosas genitais, com ênfase nos estudos de p16INK4a e HPV de alto risco. Material e Métodos: Marcadores tumorais foram revisados em 21 estudos publicados entre 1994 e 2005, no sentido de identificar aqueles que teriam melhor valor diagnóstico e/ou prognóstico das lesões intra-epiteliais escamosas. Revisão mais apurada avaliou os marcadores p16INK4a e HPV de alto risco em lesões do colo uterino (36 publicações entre 1994 e 2006). Estudou-se a associação do p16INK4a e HPV de alto risco em 96 amostras de colo utenno (13 casos de lesão intra-epitelial escamosa de alto grau (HSIL), 26 casos de lesão intra-epitelial escamosa de baixo grau (LSIL) e 57 biópsias normais. O p16INK4a foi identificado por imuno-histoquímica, usando-se o p16INK4a kit (E6H4 clone, DakoCytomation, Carpinteria, CA) e o DNA-HPV foi classificado por captura híbrida (Digene®). Associações foram avaliadas pelo índice KAPPA. No artigo foram envolvidos 54 homens, parceiros sexuais assintomáticos de mulheres com lesão intra-epitelial escamosa de baixo grau associada com HPV de alto risco, com a finalidade de verificar se a presença do HPV de alto risco poderia ajudar a identificar os casos com maior risco de ter lesões intra-epiteliais penianas, devendo submeter-se à biópsia. O DNA-HPV foi testado por captura híbrida (Digene®) em raspados do pênis. Peniscopia identificou lesões suspeitas que resultaram em biópsias. Resultados: As revisões demonstraram uma clara potencialidade clínica no uso da associação do p16INK4a e do HPV de alto risco no diagnóstico das SIL do colo uterino, e um possível uso como fator prognóstico. O p16INK4a foi detectado em 92,3% das HSIL, em 15,4% das LSIL e em nenhum caso de histologia normal. Encontrou-se respectivamente sensibilidade, especificadade, valor preditivo positivo e valor preditivo negativo de 92,3%, 100%, 100% e 98,3%, de p16INK4a para HSIL e 100%, 70,42%, 43,3% e 100% do HPV de alto risco para HSIL. No segundo estudo o HPV de alto risco estava presente em 25,9% dos parceiros. A peniscopia levou a 13 biópsias (24,07%) com os seguintes diagnósticos: condiloma (2 casos), PIN I (2 casos), PIN II (1 caso) e histologia normal (8 casos). O teste de HPV de alto risco revelou 80% de sensibilidade, 100% de especificidade, 100% de valor preditivo positivo e 88,9% de valor preditivo negativo para identificação de lesões penianas, mostrando que homens com HPV de alto risco positivo têm maior chancer de ter lesões escamosas penianas em biópsias guiadas pela peniscopia que aqueles com lesões aceto-brancas com teste de HPV negativo, (p = 0.007); OR = 51 (Cl 1.7-1527.1). Conclusões: Marcadores como o HPV de alto risco têm um potencial muito grande para aumentar o poder diagnóstico das HSIL e, principalmente, supor o prognóstico da evolução destas lesões, principalmente quando associado ao p16INK4a / Abstract: Objectives: To study the importance of the diagnostic and prognostic markers of genital squamous lesions, meanly p16INK4a and high risk HPV. Material And Methods: Squamous intra-epithelial lesion tumoral markers were revised in 21 publications between 1994 and 2005 to identify those with diagnostic and prognostic value. More accurate revision assessed the markers p16INK4a and high risk HPV (36 publications between 1994 and 2006). The p16INK4a and high-risk Human papillomavirus were investigated in 96 samples of the cervix (13 cases of high grade squamous intraepithelial lesions, 26 cases of low grade intraepithelial lesions and 57 normal tissues). The p16INK4a was identified by immunohistochemistry using the p16INK4a kit (E6H4 clone, DakoCytomation, Carpinteria, CA). and Human papillomavirus DNA was classified by hybrid capture (Digene®). Associations were evaluated by the KAPPA index. In the other report fifty four asymptomatic male sexual partners of women with low grade squamous intraepithelial lesions (LSIL) associated to high risk HPV were examined, between April 2003 and June 2005, to verify if the high risk HPV could help to identify those with more risk to have a squamous penile lesion. The DNA-HPV was tested by second generation Hybrid Capture (Digene ®) in penile scraped samples. Peniscopy identified suspicious lesions leading to biopsy. Results: The revisions showed the clinical potentiality of the concomitant use of high risk HPV and p16INK4a in diagnosis of cervical SIL and a possible utility in prognosis of genital squamous intra-epithelial. In 96 cervical biopsies, p16INK4a was detected in 92.3% of the high-grade squamous intraepithelial lesions, in 15.4% of the low-grade and in none of the normal tissues. The sensitivity, specificity, positive predictive value and negative predictive value for high-grade lesion were 92.3%, 100%, 100%, and 98.3%, respectively when considering p16INK4a expression, and 100%, 70.2%, 43.3% and 100%, respectively when considering high-risk HPV. In the male partner study high risk HPV was present in 25.9% (14/54) of the cases. Peniscopy led to 13 biopsies (24.07%). Condyloma (2 cases), PIN I (2 cases), PIN II (1 case) and normal tissue (8 cases) were found. The high risk HPV test presented 80% sensitivity, 100% specificity, 100% positive predictive value and 88.9% negative predictive value for the identification of penile lesions. So, there was a greater chance in finding HPV lesions in the biopsy in the positive cases for high risk HPV with abnormal peniscopy than in the negative cases for high risk HPV with anormal peniscopy (p = 0.007); OR = 51 (CI 1.7-1527.1). Conclusions: Markers as high risk HPV have a potential to increase the diagnostic of HPV induced lesions and maybe indicate the evolution, meanly associated with p16INK4a / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

Orientador: Laura Sterian Ward / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T04:46:06Z (GMT). No. of bitstreams: 1 Garbim_JanainaLuisaLeite_D.pdf: 8361906 bytes, checksum: b8f6cf0d2690bfc52710d97eef61e003 (MD5) Previous issue date: 2007 / Resumo: Interações entre vírus, o sistema imunológico e a ação de enzimas detoxificantes têm sido associadas à etiologia de muitas doenças incluindo o câncer e várias doenças auto-imunes. Investigamos o papel dos herpes vírus tipo 6 (HHV-6) e tipo 1 (HHV-1) e das variantes do códon 72 (P72) e códon 47 do éxon 4 de TP53, responsáveis por uma diminuição da atividade antiapoptótica de TP53, na suscetibilidade ao câncer de pele e à Doença de Basedow-Graves. Utilizamos PCR para a detecção dos vírus, polimorfismos de TP53 e GSTs, com restrição enzimática para alguns dos polimorfismos. Quando estudamos 120 pacientes com lesões de pele, comparados com 41 controles mostramos que a infecção por HHV-6 aumenta o risco de um indivíduo apresentar carcinoma basocelular (OR=3,182;95%IC:1,125-8,997). O risco para indivíduos infectados por HHV-1 foi aumentado em seis vezes (OR=6,078;95%IC:1,365-27,061). Observamos que este risco tendia a ser maior entre os indivíduos imunosuprimidos. Estudamos 78 pacientes transplantados renais comparados com 151 controles. A infecção por HHV-6 foi realmente mais freqüente nos transplantados renais (35,89%) do que nos controles (11,25%) (F; p<0,0001). Indivíduos positivos para HHV-6 apareciam com maior freqüência entre os transplantados renais que possuíam variantes de P72 (60,71%) do que nos que apresentaram o genótipo selvagem Arg/Arg (22%) (F; p=0,001). Para estudarmos a relação entre o HHV-6 e as doenças auto-imunes, analisamos 127 pacientes com diagnóstico da Doença de Basedow-Graves, observando que indivíduos que estavam infectados pelo HHV-6 tinham maior risco de desenvolver esta doença (OR=2,225;95%IC=1,197-4,135). O genótipo Pro/Pro de TP53 estava presente em 11,8% dos pacientes com Doença de Basedow-Graves (p<0,001), aumentando significativamente o risco para a doença (OR=28,395; 95%IC=1,658-486,36). Assim, nossos estudos mostram que a presença do HHV-6 e HHV-1 aumenta o risco para o câncer de pele, sugerindo que esses vírus podem ter um papel na suscetibilidade a malignidades da pele; a herança germinativa de P72 aumenta o risco para a infecção de HHV-6 e há uma tendência para o aumento de risco para desenvolvimento da Doença de Basedow-Graves quando associamos a infecção por HHV-6 e a presença do alelo prolina do códon 72 de TP53 / Abstract: Interactions between viruses, the immune system and detoxifying enzymes have been associated to the etiology of many conditions including cancer and various autoimmune diseases. We investigated the role of herpes viruses type 6 (HHV-6) and 1 (HHV-1) and the codon 72 (P72) and codon 47 (S47) variants of exon 4 of TP53, responsible for a diminished antiapoptotic activity of TP53, in the susceptibility to skin cancer and to Graves-Basedow Disease. We used PCR for virus detection, TP53 polymorphisms and GSTs, with restriction fragment length polymorphism analysis of some polymorphisms. When we studied 120 patient with skin lesions, compared with 41 controls we showed that the HHV-6 infection increases the risk of a patient to present basal cell carcinoma (OR=3.182; 95%CI: 1.125-8.997). The risk for HHV-1 infected patients was increased six times (OR=6.078;95%CI:1.365-27.061). We observed that this risk tended to be higher among immunocompromized patients. Were studied 78 kidney recipients compared with 151 controls. Was observed that HHV-6 infection was more frequent among kidney recipient patients (35.89%) than among the controls (11.25%) (F;p<0.0001). We also observed that HHV-6 positive patients appeared more frequently among kidney recipients patients that presented P72 variants (60.71%) than among those presenting the wild-type genotype Arg/Arg (22%) (F; p=0.001). To study the relationship between HHV-6 and autoimmune diseases, we analyzed 127 patients with Graves-Basedow Disease, observing that HHV-6 infected patients had a higher risk to developed this disease (OR=2.225;95%CI=1.197-4.135). The Pro/Pro of TP53 genotype was present in 11.8% of Graves-Basedow Disease patients (p<0.001), increasing significantly the risk to this disorder (OR=28.395; 95%CI=1.658-486.36). Therefore, our studies indicate that the presence of HHV-6 and HHV-1 increase the risk to skin cancer, suggesting that this virus can play a role in the susceptibility to skin malignancies; the germline inheritance of P72 increases the risk to HHV-6 infection and there is a tendency to a higher risk of Graves-Basedow Disease development in patients that have both an HHV-6 infection and the proline allele of codon 72 of TP53 / Doutorado / Doutor em Farmacologia

Malignant catarrhal fever (MCF), a disease that is usually fatal in cattle, is caused by two distinct but related bovine herpesviruses which are members of the genus Macavirus. The wildebeest-associated alcelaphine herpesvirus-1 (AlHV-1) occurs mainly in East and southern Africa, whereas the sheep-associated ovine herpesvirus-1 (OvHV-2) has an almost worldwide distribution. The natural hosts or carriers of these two viruses are subclinically infected. The 130 kilobase pair (kbp) AlHV-1 double stranded DNA genome consists of 18 open reading frames (ORFs) coding for structural proteins and approximately 50 ORFs coding for non-structural proteins. The 18 structural ORFs encode for 4 capsid proteins, 5 tegument proteins, 8 glycoproteins and a minor capsid scaffold protein. ORF8 encoding for glycoprotein B, is the most conserved of the proteins amongst gammaherpesviruses, whereas the minor capsid protein encoded by ORF65, is amongst the most variable. Thus, the minor capsid protein is one of the antigens of choice for the development of an ELISA for detection of AlHV-1 reactive antibodies and glycoprotein B could be of importance in developing a cross-protective vaccine for gammaherpesviruses. The naming and annotation of most of the AlHV-1 ORFs is based on comparison with related gammaherpesviruses and bioinformatics. Most of these ORFs are putative as there is no direct experimental evidence confirming that they code for any particular protein. In order to investigate whether the ORFs code for any proteins, two ORFs were targeted for in vitro heterologous expression. AlHV-1, isolate C500, was grown in fetal bovine turbinate (BT) cell culture and viral genomic DNA extracted. ORF8, the putative glycoprotein B, was amplified with a PCR assay and inserted into a mammalian expression vector, pCI. VERO cells were transfected with the recombinant vector. Expression of ORF8 was confirmed by an indirect immunofluorescence assay (IFA) with AlHV-1 polyclonal sera and rabbit anti-bovine IgG (whole molecule) FITC conjugate. Truncated forms of ORF8 were further expressed as baculovirus recombinants using the Bac-to-Bac baculovirus expression system. Expression of the truncated ORF8 was confirmed by SDS-PAGE and Western blot. AlHV-1 ORF65, the minor capsid protein gene, was amplified with a PCR assay from the viral genomic DNA and cloned in frame with a histidine tag in a bacterial expression vector, pCOLD I. Expression of the minor capsid protein was confirmed by SDS-PAGE and Western blot with the histidine tag monoclonal as well as AlHV-1 polyclonal sera. Orf65 was expressed in large quantities and column purified using the histidine tag. Orf65 was also expressed as a baculovirus recombinant using the Bac-to-Bac baculovirus expression system. Expression of the protein was confirmed by SDS-PAGE and Western blot with the histidine tag and AlHV-1 polyclonal sera. ORF65 expression in the baculovirus Bac-to-Bac expression system was up-scaled and the expressed protein column purified. Antibodies raised in chicken against the purified antigen were used successfully in an indirect immunoassay to detect AlHV-1 infected cells. An indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against AlHV-1 was developed. It is based on the use of the AlHV-1 minor capsid protein as the capture antigen for antibodies. The primary antibodies are detected by the addition of enzymelabelled (horseradish peroxidase) protein G which detects bovid, ovid and wildebeest antibodies. Addition of a substrate of the enzyme, in this case, 3,3’,5,5’- tetramethylbenzidine (TMB), results in a colour reaction which is measured using spectrophotometric procedures. At a selected cut-off point of 18, the ELISA test has a sensitivity of 100% and a specificity of 100% and has been shown to detect AlHV-1 antibodies in cattle and wildebeest. The ELISA showed no cross-reactivity with sera raised in cattle against related viruses such as ovine herpesvirus 2, bovine herpesvirus 1, 2 and 4. The two expressed proteins used in this study were found to be amongst the antigens expressed in cattle suffering from malignant catarrhal fever. The experimental AlHV-1 indirect ELISA needs further validation and this research may be extended to determine the performance of these antigens as candidate subunit vaccines. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

Orientador: Clarice Weis Arns / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T02:57:43Z (GMT). No. of bitstreams: 1 Barros_AlyneVieira_M.pdf: 608733 bytes, checksum: 93e4d814c0f99c65741a343e76d35fc4 (MD5) Previous issue date: 2011 / Resumo: Estudos de plantas medicinais com conhecimento tradicional têm sido uma fonte potencial de substâncias com atividades farmacológicas e biológicas significantes. Guettarda angelica Mart. ex Müll. Arg. (Rubiaceae) é uma planta medicinal no qual suas raízes são popularmente utilizadas para diversos fins terapêuticos, incluindo veterinário. Estudos antimicrobianos com raízes desta planta também relataram uma atividade in vitro contra bactérias. Como as infecções virais ainda continuam sendo um sério problema mundial, a etnofarmacologia fornece uma abordagem alternativa para descoberta de novos agentes antivirais. O objetivo do presente trabalho foi o estudo antiviral de extratos da casca das raízes, folhas e sementes de G. angelica frente aos herpesvírus bovino (BoHV-1), suíno (SuHV-1) e equino (EHV-1), reovírus (ARV) e metapneumovírus aviário (aMPV). A atividade antiviral foi testada in vitro em células Vero e MDBK utilizando os ensaios de redução do título viral e o ensaio quantitativo colorimétrico através do MTT. Inicialmente, a concentração máxima não-citotóxica (MNCC) dos extratos foi determinada nas células através da observação de suas alterações morfológicas. Estudos realizados através da redução dos títulos virais mostrou que apenas o extrato aquoso de sementes (AEs) apresentou uma atividade antiviral contra o BoHV-1, SuHV-1, EHV-1 e ARV. Assim, esse extrato foi posteriormente avaliado pelo método MTT para determinação do CC50 (concentração citotóxica a 50%), IC50 (concentração inibitória a 50%) e o SI (índice de seletividade). Os valores de CC50 do extrato AEs foram 400,60 e 920,50 para células Vero e MDBK, respectivamente. E os valores de IC50 e SI foram 22, 79 e 40,39 para BoHV-1; 91,30 e 10,08 para SuHV-1; 19,95 e 20,08 para EHV-1; e 23,59 e 17,00 para ARV. Esses resultados indicam que a semente de G. angelica contém compostos com atividade antiviral promissora e baixa toxicidade / Abstract: Study of medicinal plants with traditional knowledge has been a potential source of substances with significant pharmacological and biological activities. Guettarda angelica M. (Rubiaceae) is a medicinal plant where its roots are popularly used for various therapeutic purposes including veterinary. In vitro antimicrobial studies also related antibacterial activity of these roots against bacteria. Like viral infections still remain a serious worldwide problem, the ethnopharmacology provides an alternative approach for discovery of new antiviral agents. The aim of the present work was the antiviral study of extracts from roots bark, leaves and seeds of G. angelica against bovine (BoHV-1), swine (SuHV-1) and equine (EHV-1) herpesviruses, avian reovirus (ARV) and metapneumovirus (aMPV) The antiviral activity was tested in vitro on Vero and MDBK cells using the viral titer assay and the quantitative colorimetric assay through MTT. Initially, the maximum non-citotoxic concentration (MNCC) of extracts was determined in cells by observation of their morphological alterations. Studies through the reduction of viral titers showed that only the aqueous extract from seeds (AEs) presented an antiviral activity against BoHV-1, SuHV-1, EHV-1 and ARV. Then, this extract was further evaluated by MTT method to determine the CC50 (50% cytotoxic concentration), IC50 (50% inhibitory concentration) and SI (selectivity index). The values of CC50 of AEs extract were 400.60 and 920.60 to Vero and MDBK cells, respectively. And the values of IC50 and SI were 22.79 and 40.39 for BoHV-1; 91.30 e 10.08 for SuHV-1; 19.95 and 20.08 for EHV-1; 23.59 and 17.00 for ARV. These results indicate that the seeds from G. angelica contain composts with promising antiviral activity and low toxicity / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

MSc (Microbiology) / Department of Microbiology / Background: Human Herpes Virus-8 (HHV-8) and Human Immunodeficiency Virus (HIV) are endemic in sub-Saharan Africa. However, the prevalence of HHV-8 in HIV-infected individuals in sub-Saharan Africa has not been fully described and characterized. Objectives: The objective of this study was to determine the seroprevalence and genetic subtypes of HHV-8 in an HIV-infected population in Cameroon. Methodology: KSHV/HHV-8 Enzyme-linked Immunosorbent Assay (ELISA) kit (Advanced Biotechnologies Inc., USA) was used to detect IgG antibodies in the plasma of 406 HIV-infected outpatients of the Mutengene Baptist Health Centre, Cameroon. To detect the viral presence, a 233 bp fragment of the ORF 26 gene of HHV-8 was targeted by polymerase chain reaction (PCR) in total DNA purified from patients’ whole blood. A 453 bp of the K1 gene was amplified by nested PCR, sequenced and phylogenetically analysed to infer subtypes. The online tool, Synonymous Non-synonymous Analysis Program (SNAP), was used to determine the rate of synonymous and nonsynonymous mutations in the K1 gene. The genetic variability among the derived K1 nucleotide sequences was determined by mean genetic distance analysis. Results: Of the 406 participants, an HHV-8 seroprevalence of 79.1% was obtained. There was a statistically significant association of seroprevalence with age (p= 0.00), CD4+ cell count (p= 0.02), marital status (p= 0.02) and ownership of a transistor radio set (p= 0.00). Seventy samples (23.3%) were successfully amplified for ORF 26 gene confirming the presence of replicating virus. K1 sequences were obtained for 14 of the 20 (70%) K1 amplified DNAs. The mean genetic diversity of K1 sequences ranges from 0.0%-22.3%. Phylogenetic analysis revealed two infecting viral subtypes in the study cohort: subtype A5 (57.1%), and subtype B (35.7%). Greater positive selection and genetic diversity were observed in A5 subtype compared to B subtype of K1. Interestingly, one sample (BM 547) clustered with an unclassifiable sequence from South Africa. Conclusions and recommendation: This study revealed the endemicity of HHV-8 infection in the studied population, with subtypes A5 and B as the most important epidemiological genetic variants. In addition, targeting the ORF 26 region by PCR could be an approach to detect replicating virus in individuals. Further studies should investigate the association between HHV-8 infection and KS development in the study area which is endemic for HIV. This study contributes data to the HIV/HHV-8 co-infection landscape in the study area and in Africa at large.