Dissertations / Theses on the topic 'Herpes virus 8'

Kaposi�s sarcoma (KS) is a peculiar vascular neoplasm that occurs mainly in elderly Mediterranean men and patients with acquired immunodeficiency syndrome (AIDS). The current literature indicates that KS is initiated by the human herpes virus 8 (HHV8) as a reactive polyclonal process but with deregulation of oncogene and tumour suppressor genes, it can progress to a true malignancy with monoclonality. Clinically, classical KS often presents as an indolent disease affecting mainly the lower extremities whereas AIDS-related KS has no site predilection and can progress rapidly with systemic involvement. Histologically, KS can be classified into patch, plaque and nodular stages. Interestingly, classical and AIDS-related KS are indistinguishable histologically and this suggests that AIDS-related KS and classical KS might be initiated by a common aetiology but given their different clinical courses, they may progress through different mechanisms. In view of the importance of the cell cycle proteins in the development and progression of many human malignancies, this thesis aims to examine the role of these proteins in the progression of the two main clinical subtypes of KS. The cell cycle protein expressions in a cohort of 47 patients with KS with welldocumented clinical and histological features were studied. Using a monclonal antibody against the latent nuclear antigen-1 molecule of HHV8, HHV8 was detected in 78% of the cases. The more advanced nodular lesions were found to have a higher level of proliferative activity as measured by the proliferation x marker, Ki-67. This suggests it is valid to use the histological specimens as a tumour progression model of KS. The role of the Rb/cyclin D1/p16 pathway was examined. The more advanced nodular stage KS lesions were more likely to be positive for cyclin D1, suggesting that cyclin D1 is important in the progression from patch stage to nodular stage. p16 acts as a tumour suppressor and it has an inhibitory effect on cyclin D1. The p16 expression rate was low in early stage KS but high in the more advanced lesions. It seems that reduced p16 expression occurs early in KS and may be important in its development. The rate of Rb expression, on the other hand, did not differ significantly among the histological subtypes. The results revealed the significant role of the Rb/cyclin D1/p16 pathway in the progression of KS. Of the mitotic cyclins examined, cyclin A expression was correlated with the advanced tumor stage. The rate of p34cdc2 expression was high in the lesions and there was no correlation with histological stage. This suggests that p34cdc2 is important in the early development of the tumour but not necessarily in its progression. Along the p53-apoptotic pathway, mutant p53 expression was significantly more common in the nodular stage. The cyclin G1 (a protooncogene, one of the target genes of p53) expression also paralleled that of mutant p53 with the majority of the KS lesions showing cyclin G1 expression and significant xi correlation between advanced histological stage and increasing rate of cyclin G1 expression. These findings suggest that progression along the p53 pathway may be important in the advanced stage development of KS. On the other hand, expression of the CDK inhibitor, p27, a protein that normally negatively regulates cyclin G1, was reduced in nodular KS. These findings suggest that some KS lesions may progress through a deregulated or abnormal p53 pathway. There were correlations between cyclin D1, cyclin A, cyclin G1, mutant p53 and negative HIV status. The findings suggest that components of both the Rb/cyclin D1/p16 and p53-apoptotic pathways are important in the progression of classical KS. Rb protein was the only cell cycle protein whose rate of expression correlated significantly with HHV8 status in KS. The majority of HHV8 positive lesions were also positive for Rb protein, unlike HHV8 negative lesions. This suggests that some of the HHV8 negative lesions can progress through a defective Rb pathway whereas the role of Rb in the progression may not be as important in the HHV8 positive lesions. This was an unexpected finding given that one of the postulated mechanisms of tumour initiation by the HHV8 virus is via the viral cyclin it produces. The viral cyclin produced by HHV8 acts through the Rb pathway much the same as cyclin D1 and one would have expected that HHV8 positive cases are less likely to be positive for the Rb protein. In summary, the majority of the KS lesions examined in this thesis show HHV8 infection. The Rb/cyclin D1/p16 pathway appears to be important in the progression of the different stages of KS and expression of the proteins involved in the p53 pathway were found to be important in the advanced stages of the development of KS. There were differential expressions of cell cycle proteins between AIDS-related and classical KS, and between HHV8 positive and HHV8 negative lesions. The findings also provided some clues to the possible mechanisms of development in KS lesions that were not initiated by HHV8.

Kaposi�s sarcoma (KS) is a peculiar vascular neoplasm that occurs mainly in elderly Mediterranean men and patients with acquired immunodeficiency syndrome (AIDS). The current literature indicates that KS is initiated by the human herpes virus 8 (HHV8) as a reactive polyclonal process but with deregulation of oncogene and tumour suppressor genes, it can progress to a true malignancy with monoclonality. Clinically, classical KS often presents as an indolent disease affecting mainly the lower extremities whereas AIDS-related KS has no site predilection and can progress rapidly with systemic involvement. Histologically, KS can be classified into patch, plaque and nodular stages. Interestingly, classical and AIDS-related KS are indistinguishable histologically and this suggests that AIDS-related KS and classical KS might be initiated by a common aetiology but given their different clinical courses, they may progress through different mechanisms. In view of the importance of the cell cycle proteins in the development and progression of many human malignancies, this thesis aims to examine the role of these proteins in the progression of the two main clinical subtypes of KS. The cell cycle protein expressions in a cohort of 47 patients with KS with welldocumented clinical and histological features were studied. Using a monclonal antibody against the latent nuclear antigen-1 molecule of HHV8, HHV8 was detected in 78% of the cases. The more advanced nodular lesions were found to have a higher level of proliferative activity as measured by the proliferation x marker, Ki-67. This suggests it is valid to use the histological specimens as a tumour progression model of KS. The role of the Rb/cyclin D1/p16 pathway was examined. The more advanced nodular stage KS lesions were more likely to be positive for cyclin D1, suggesting that cyclin D1 is important in the progression from patch stage to nodular stage. p16 acts as a tumour suppressor and it has an inhibitory effect on cyclin D1. The p16 expression rate was low in early stage KS but high in the more advanced lesions. It seems that reduced p16 expression occurs early in KS and may be important in its development. The rate of Rb expression, on the other hand, did not differ significantly among the histological subtypes. The results revealed the significant role of the Rb/cyclin D1/p16 pathway in the progression of KS. Of the mitotic cyclins examined, cyclin A expression was correlated with the advanced tumor stage. The rate of p34cdc2 expression was high in the lesions and there was no correlation with histological stage. This suggests that p34cdc2 is important in the early development of the tumour but not necessarily in its progression. Along the p53-apoptotic pathway, mutant p53 expression was significantly more common in the nodular stage. The cyclin G1 (a protooncogene, one of the target genes of p53) expression also paralleled that of mutant p53 with the majority of the KS lesions showing cyclin G1 expression and significant xi correlation between advanced histological stage and increasing rate of cyclin G1 expression. These findings suggest that progression along the p53 pathway may be important in the advanced stage development of KS. On the other hand, expression of the CDK inhibitor, p27, a protein that normally negatively regulates cyclin G1, was reduced in nodular KS. These findings suggest that some KS lesions may progress through a deregulated or abnormal p53 pathway. There were correlations between cyclin D1, cyclin A, cyclin G1, mutant p53 and negative HIV status. The findings suggest that components of both the Rb/cyclin D1/p16 and p53-apoptotic pathways are important in the progression of classical KS. Rb protein was the only cell cycle protein whose rate of expression correlated significantly with HHV8 status in KS. The majority of HHV8 positive lesions were also positive for Rb protein, unlike HHV8 negative lesions. This suggests that some of the HHV8 negative lesions can progress through a defective Rb pathway whereas the role of Rb in the progression may not be as important in the HHV8 positive lesions. This was an unexpected finding given that one of the postulated mechanisms of tumour initiation by the HHV8 virus is via the viral cyclin it produces. The viral cyclin produced by HHV8 acts through the Rb pathway much the same as cyclin D1 and one would have expected that HHV8 positive cases are less likely to be positive for the Rb protein. In summary, the majority of the KS lesions examined in this thesis show HHV8 infection. The Rb/cyclin D1/p16 pathway appears to be important in the progression of the different stages of KS and expression of the proteins involved in the p53 pathway were found to be important in the advanced stages of the development of KS. There were differential expressions of cell cycle proteins between AIDS-related and classical KS, and between HHV8 positive and HHV8 negative lesions. The findings also provided some clues to the possible mechanisms of development in KS lesions that were not initiated by HHV8.

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O herpesvírus associado ao sarcoma de Kaposi (KSHV) é um gammaherpesvírus associado ao sarcoma de Kaposi (SK). A proteína K1 do KSHV é conhecida por aumentar a sobrevivência e proliferação celular em células infectadas. A ORF-K1 viral apresenta elevada variabilidade, de modo a discriminar diferentes genótipos do KSHV. Até o momento não se sabe se diferentes genótipos virais apresentam características biológicas próprias. Assim, vetores recombinantes contendo a ORF-K1 de genótipos virais A e B foram gerados. A ORF-K1 foi obtida a partir do DNA de linhagens de linfoma de efusão primária (PEL) constitutivamente infectadas pelo KSHV. O amplicon da ORF-K1 e vetor comercial foram digeridos, ligados e clonados em bactéria E. coli DH5α. Clones selecionados foram então submetidos à sequenciamento automatizado de DNA. As sequências geradas dos vetores recombinantes foram alinhadas e comparadas com sequências-protótipo de ORF-K1 do KSHV. Sequência de aminoácidos dos vetores recombinantes foram geradas e analisadas quanto a região codificadora do ITAM de K1. Um clone validado de cada vetor recombinante foi transfectado estavelmente em células HEK293 e a expressão de K1 foi avaliada por Western blot (WB) O sequenciamento de DNA demonstrou que a ORF-K1 dos vetores recombinantes de genótipo A, B e C corresponde a de sequências-protótipo depositadas de linhagens de PEL. A presença de K1 no lisado de células HEK293 transfectadas foi demonstrada por WB. A análise da sequência de aminoácidos de K1 codificada nos vetores recombinantes revelou que o domínio ITAM de K1 do genótipo B apresenta aminoácidos distintos em relação ao ITAM de K1 de genótipos A e C. Portanto, vetores recombinantes de ORF-K1 foram produzidos e validados, e serão úteis para se estabelecer modelo experimental para análises das propriedades biológicas da proteína K1 do KSHV
Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus associated with the development of Kaposi's sarcoma. The K1 protein of KSHV has been shown to induce increases the survival and proliferation of infected cells. The viral ORF-K1 shows high variability, so it is possible to distinguish different KSHV genotypes (genotypes A, B, C, D). So far, it is unclear whether different viral genotypes have their own biological characteristics. To this intent, recombinant vectors were generated containing the ORFK1 genotypes A and B. ORF-K1 was obtained from the DNA of primary effusion lymphoma (PEL) cell lines. The amplicon generated and the commercial vector were digested, bonded and cloned in E.coli DH5α. Selected clones underwent automated DNA sequencing. The generated sequences were compared with prototype sequences of ORF-K1. The amino acid sequences from vectors were generated and analyzed in the K1 ITAM-coding region. Validated clones were stably transfected into HEK293 cells and K1 expression was evaluated using Western blot (WB). DNA sequencing showed that the ORF-K1 recombinant vectors corresponds to the prototype sequences deposited from PEL cell lines. WB demonstrated the presence of K1 in the lysate of HEK293 transfected cells. Analysis of the K1 amino acid sequence encoded in the vectors revealed that ITAM domain of K1 (genotype B) has distinct amino acids from the ones in the ITAM domain of K1 (genotypes A and C). Therefore ORF-K1 recombinant vectors were produced and validated, and will be useful to establish an experimental model for analysis of the biological properties of the K1 protein

Transplantation-associated Kaposi's sarcoma (KS), causatively associated with human herpes virus 8 (HHV-8), is particularly prevalent in Saudi Arabia, although the prevalence rate of HHV-8 infection in the general population there is comparable to that in other geographical regions. The serologic and genomic prevalences of HHV-8 in samples representing the general population (n=238), patients with end-stage renal disease (n=78), and renal allograft recipients (n=66) were investigated. To evaluate if oral shedding of HHV-8 might play a role in transplantation KS, the extent of HHV-8 shedding in the mouth compared to other anatomical compartments, and the presence of multiple HHV-8 infection were also studied. PCR protocols were applied to amplify 3 fragments of the viral genome (from open reading frames 26 and K1) from whole-mouth saliva, parotid saliva, buccal and palatal exfoliates, plasma, sub sets of peripheral blood cells, and KS lesional tissue, and to quantify the salivary viral load. Demographic and clinical data were analysed to identify risk factors for HHV-8 infection. A higher HHV-8 seroprevalence was observed in patients with renal disease compared to the general population, but no significant difference in HHV-8 DNA detection rates in CD45+ cells was found. The oral cavity was identified as a major site of HHV-8 shedding in renal disease patients regardless of a previous history of KS. In patients with end-stage renal disease, HHV-8 DNA was more frequently detectable in oral samples than in blood. They and renal allograft recipients showed evidence of being multiply infected by HHV-8. These findings suggest that iatrogenic, salivary HHV-8 transmission between patients with renal disease prior to transplantation accounts for the relatively high prevalence of HHV-8 infection. Implementation of measures to minimise contamination of oral fluid between renal disease patients may play a role in controlling HHV-8 transmission and reduce the incidence of transplantation-associated KS.

Le Ketosis-Prone Diabetes (KPD) est un phénotype de diabète intermédiaire entre le diabète de type 1 et le diabète de type 2, fréquemment rencontré chez le sujet d’origine noire africaine. Cette forme de diabète suscite un intérêt certain de par son évolution clinique marquée notamment par la restauration de l’insulinosécrétion initialement altérée. Sobngwi et coll. en 2008 ont établi une association entre le virus HHV8 et le KPD chez des sujets Africains vivant en France. Nulle part ailleurs l’étude n’a été reproduite. L’objectif de cette thèse était de rechercher la potentielle association entre l’infection à HHV8 et le KPD ; puis d’évaluer l’impact de l’infection à HHV8 sur le profil inflammatoire des phénotypes du diabète de type 2. L’étude s’appuie sur une population de patients Africains vivant en Afrique admis consécutivement pour une décompensation hyperglycémique (glycémie à jeun≥2,5g/l) au Centre National d’Obésité de l’Hôpital Central de Yaoundé. Plus spécifiquement, il était question de :• étudier la fréquence du diabète non auto-immun à tendance cétosique (KPD); • étudier l’association entre le virus HHV8 et le KPD;• rechercher si l’infection à HHV8 est associée à un profil inflammatoire pouvant participer aux phénotypes de diabète. Etait inclus dans l’étude tout patient diabétique âgé de plus de 18 ans présentant un diabète aigu avec syndrome cardinal et cétonurie (KPD1), ceux ayant présenté un diabète inaugural aigu avec syndrome cardinal et cétonurie et en rémission depuis plus de trois mois et sans cétonurie à l’inclusion (KPD2), et ceux présentant un diabète de type 2 connu sans cétonurie (DT2). Etait exclu de l’étude tout patient présentant des stigmates d’auto-immunité du diabète de type 1 A, un diabète « MODY », une endocrinopathie, une maladie du pancréas, ou un diabète de type auto-immun. Chez l’ensemble des participants admis, nous avons collecté les données cliniques (le poids, la taille, l’IMC, le rapport tour de taille sur tour de hanche, la pression artérielle, et le pourcentage de graisse) et des prélèvements à jeun ont été effectués (sérum et cellules mononuclées du sang périphérique) pour les analyses biologiques : la glycémie par glucose oxydase, l’HbA1c par HPLC, les paramètres du profil lipidique par des methodes enzymatiques, les concentrations d’insuline et de peptide-C par électrochimiluminescence, les anticorps anti-HHV8 par immunofluorescence, l’ADN viral HHV8 par PCR en temps réel, et les marqueurs de l’inflammation par le Luminex. Les indices HOMA-β et HOMA-IR ont été utilisés pour évaluer l’insulinosécrétion et la sensibilité à l’insuline respectivement. Les marqueurs sérologiques de l’inflammation recherchés étaient : TNF-α, MCP-1, IL-8, MIP-1β, VEGF et MIP-1α
Ketosis-Prone Diabetes (KPD) is a diabetes phenotype intermediate between type 1 and type 2 diabetes, frequently encountered in populations of African origin. This form of diabetes arouses some interest because of its clinical course marked in particular by restoring the initial impaired insulin secretion. Sobngwi et al. in 2008 established an association between HHV-8 virus and KPD in African population living in France. Nowhere else has the study been replicated. The objective of this thesis was to investigate the potential association between HHV-8 infection and KPD; then evaluate the impact of HHV-8 infection on the inflammatory profile of type 2 diabetes phenotypes. The study is based on African patients living in Africa consecutively admitted for hyperglycemic decompensation (Fasting blood glucose≥2,5g/l) at the National Obesity Centre of the Yaounde Central Hospital. More specifically, the issue was:• study the frequency of non-immune ketosis-prone diabetes (KPD);• investigate the association between HHV8 and KPD;• investigate whether HHV-8 infection is associated with an inflammatory profile that may participate in diabetes phenotypes.Was included in this study all diabetic patients old more than 18 years with acute diabetes with syndrome cardinal and ketonuria (KPD1), those who presented with an acute inaugural cardinal syndrome and diabetes ketonuria and in remission for more than three months and without ketonuria at baseline (KPD2), and those with type 2 diabetes experienced without ketonuria (T2D). Was excluded from the study all patient with stigmata of autoimmunity of type 1 A diabetes, diabetes "MODY", endocrinopathy, pancreatic disease or an autoimmune diabetes.Among all participants admitted, we collected clinical data (weight, height, BMI, waist to hip ratio, blood pressure, and the percentage of fat) and levies fasting were made (serum and peripheral blood mononuclear cells) for biological testing: glyceamia by glucose oxidase, HbA1c by HPLC, lipid profile by enzymatic methods, the insulin and C-peptide concentrations by electrochemiluminescence, anti-HHV8 antibodies by immunofluorescence, HHV8 viral DNA by real-time PCR, and markers of inflammation by Luminex. HOMA-β and HOMA-IR indices were used to assess insulinsecretion and insulinsensitivity respectively. Serological markers of inflammation investigated were : TNF-α, MCP-1, IL-8, MIP-1β, MIP-1α and VEGF

Thesis (Ph. D.)--Southern Illinois University Carbondale, 2008.
"Department of Molecular Biology, Microbiology and Biochemistry." Includes bibliographical references (p. 110-131). Also available online.

Determina la seroprevalencia del HHV-8 en donantes de sangre que acuden al Servicio de Medicina Transfusional del Centro Médico Naval “Cirujano Mayor Santiago Távara”. Se recolectan 350 muestras de sangre de los donantes admitidos, de noviembre del 2007 a marzo del 2008, siendo el 77.4% de sexo masculino y el 22.6% de sexo femenino. Éstas son evaluadas mediante la prueba de inmunofluorescencia indirecta para la detección de anticuerpos contra antígenos líticos y latentes del HHV-8. Los resultados revelan que la seroprevalencia fue de 14.0%, 13.3% en la población masculina y 16.5% en la femenina.
Tesis

La dérégulation de l’expression des microARN peut induire des cancers. De plus, ils jouent un rôle crucial dans la pathogénèse et la survie des virus. L’herpès virus humain de type 8 (HHV-8 ou KSHV) est l’agent étiologique du sarcome de Kaposi et est impliqué dans la génération de lymphomes agressifs de type B. De manière intéressante, le génome ce virus code 12 pré-miARN localisés dans la région de latence et exprimés sur un même pri-miARN. Les miARN du KSHV sont importants pour le maintien de la latence, l’inhibition de l’apoptose ou encore la régulation du cycle cellulaire de l’hôte. Nous nous intéressons à leur expression et leur régulation durant l’infection virale. Nous avons résolu la structure secondaire de l’ARN codant ces miARN afin d’identifier les critères structuraux responsables de leur accumulation différentielle. Nous avons initié une analyse cinétique de la première étape de maturation et enfin nous essayons d’identifier des co-facteurs modulant leur expression
It is now well known that modulation of microRNAs expression is linked to the development of cancers. Moreover, they play a crucial role in the pathogenesis and the survival of some viruses. Kaposi’s sarcoma associated herpes virus (KSHV) is the etiologic agent of Kaposi’s sarcoma and is involved in human aggressive B lymphomas generation. Its genome encodes 12 precursor miRNAs that are clustered in a latency region and expressed on a single long primary transcript. KSHV miRNAs are important to maintain the virus latency and to regulate or inhibit the host cell cycle or apoptosis, respectively. Therefore, understanding the regulation of KSHV miRNA accumulation is of prime importance. In this respect, we resolved the secondary structure of them iRNA cluster to identify structural criteria responsible of their differential accumulation. In addition, we started to analyse the mechanism of their maturation by kinetics studies. Finally we tried to identify some cofactors of miRNA expression

Le virus humain herpès-8 (HHV-8) est un virus oncogene associé au développement de cancers dont le sarcome de Kaposi (KS) et le lymphome primitif des séreuses (PEL). Plusieurs études mettent en évidence l'importance de différents facteurs dont les cytokines pour la survie et la prolifération des cellules tumorales infectées par HHV-8. Les médiateurs lipidiques bioactifs, provenant de l'hydrolyse des phospholipides, jouent un rôle important dans plusieurs formes de cancer en influençant le développement, la progression ainsi que le potentiel métastatique. En effet, la littérature scientifique décrit de plus en plus clairement que l'autotaxine (ATX)/acide lysophosphatidique (LPA) ainsi que la cyclooxygénase-2 (COX-2)/prostaglandine E2 (PGE2) sont intimement liés au développement du cancer. Par conséquent, l'utilisation d'inhibiteurs ciblant la synthèse ou l'action biologique de ces médiateurs lipidiques représente une avenue thérapeutique fort prometteuse. À ce jour, le rôle des médiateurs lipidiques dans la tumorigénèse associée à HHV-8 n'est pas clairement défini. Cette thèse présente donc deux projets directement reliés à la surexpression de d'ATX et de COX-2 suivant une infection par HHV-8. Nos résultats démontrent que l'ATX, une enzyme responsable de la production de LPA, est surexprimée dans les lignées cellulaires, dans l'ascite et les biopsies cutanées provenant respectivement de patients atteints du lymphome primitif des séreuses et du sarcome de Kaposi. L'augmentation de l'activité enzymatique de l'autotaxine in vitro favorise la prolifération et/ou la survie des cellules infectées. Notre étude permet aussi de confirmer l'induction de la COX-2 suivant une infection des cellules endothéliales par HHV-8. De plus, nous avons étudié les effets de la protéine Kl5 d'HHV-8 sur l'expression et la régulation du gène de la COX-2. Nos résultats ont permis de démontrer que la protéine Kl5 induit l'expression du gène de la COX-2 via les facteurs de transcription NFAT et AP-1. L'ensemble des résultats obtenus au cours de mes études de doctorat met en évidence une conséquence importante et spécifique de l'infection par HHV-8 soit l'induction des enzymes permettant la production de lipides bioactifs. Le LPA mène à une augmentation de la prolifération et/ou de la survie des cellules infectées alors que la COX-2 est essentielle à une infection efficace in vitro ainsi qu'au maintient de la latence virale.

Thesis (Ph.D. (Public Health))--University of the Witwatersrand, Faculty of Health Sciences, 2012
Factors associated with the transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV) are inconclusive. In countries where KS and KSHV are confined to men who have sex with other men (MSM), KSHV is associated with sexual risk factors. In countries where KSHV is endemic, it affects adults and children of all ages and irrespective of sexual orientation, suggesting the existence of non-sexual risk factors for KSHV infection. In this thesis, three distinct cross sectional studies aiming to define the seroprevalence of KSHV in South African populations and to identify plausible risk factors for KSHV infection were undertaken. The studies measured KSHV seropositivity in relation to sociodemographic factors and HIV status. In children, factors associated with horizontal mother to child transmission were also explored. In adults KSHV seropositivity was also measured in relation to sexually transmitted infections and/or measures of sexual behaviour. Calculated risk factors were expressed as odds ratios (95% confidence interval) for KSHV. Methods Mother to Child KSHV seroepidemiology Study: KSHV seroprevalence (reactive to either lytic K8.1 or latent Orf73) was measured in 1287 children and their 1179 biological mothers. Association between KSHV seropositivity in children was measured against KSHV seropositivity and HIV status of their mothers. KSHV seroepidemiology in women attending antenatal clinics: Antibodies to KSHV lytic K8.1 and latent Orf73 antigens were tested in 1740 pregnant women attending antenatal clinics in South Africa in 2001. Information on HIV and syphilis serology, age, education, residential area, gravidity, and parity was anonymously linked to evaluate risk factors for KSHV seropositivity. Clinics were grouped by municipal regions and their proximity to the two main river catchments defined. Carletonville Community KSHV seroepidemiology Study: Sera from 2103 South African individuals (862 miners, 95 sex workers, 731 female and 415 male township residents) were tested for antibodies to KSHV lytic K8.1 and latent Orf73, HIV gonococcus, herpes simplex virus type 2 (HSV-2), syphilis and chlamydia. Information on social, demographic and highrisk sexual behaviour was linked to laboratory data. Results Mother to Child KSHV seroepidemiology Study: KSHV seroprevalence (reactive to either lytic K8.1 or latent Orf73) was 15.9% (204 of 1287 subjects) in children and 29.7% (350 of 1179 subjects) in mothers. The risk of KSHV seropositivity was significantly higher in children of KSHV seropositive mothers compared with those of KSHV-seronegative mothers. The HIV status of mothers was marginally associated with an increased risk of KSHV seropositivity in their children (AOR = 1.6, 95% CI: 1.0 to 2.6; P = 0.07). KSHV seroprevalence was significantly higher in HIV-infected subjects (P = 0.0005), and HIV-infected subjects had significantly higher lytic and latent KSHV antibody levels than HIV-negative subjects. KSHV seroepidemiology in women attending antenatal clinics: KSHV seroprevalence was nearly twice that of HIV (44.6% vs. 23.1%). HIV and syphilis seropositivity was 12.7% and 14.9% respectively in women without KSHV, and 36.1% and 19.9% respectively in those with KSHV. Women who were KSHV seropositive were 4 times more likely to be HIV positive than those who were KSHV seronegative (AOR 4.1 95%CI: 3.4 - 5.7). Although, women with HIV infection were more likely to be syphilis seropositive (AOR 1.8 95%CI: 1.3 - 2.4), no association between KSHV and syphilis seropositivity was observed. Those with higher levels of education had lower levels of KSHV seropositivity compared to those with lower education levels. KSHV seropositivity showed a heterogeneous pattern of prevalence in some localities. Carletonville Community KSHV seroepidemiology Study: Overall KSHV and HIV prevalences were 47.5 and 40%, respectively (P<0.43). The risk of HIV infection was highest in sex workers followed by female residents and miners, compared with male residents (P<0.001). HSV-2 infection was highly prevalent (66%) and lower, but still substantial, prevalence (6–8%) was observed for other sexually transmitted infections (STI). No significant difference in KSHV infection was observed among the residential groups (P>0.05). KSHV was not associated with any of the STI or any measures of sexual behaviour. Conclusion The findings of these three studies contribute substantially to global KSHV seroepidemiology and show that in Southern African settings KSHV is associated with non-sexual mode of transmission. Firstly KSHV is common in very young children up to ten years of age and increases with age until adulthood. The high prevalence of KSHV in the South African populations remained evident in all populations. In children, the risk of acquisition of KSHV was higher among children of KSHV-seropositive mothers than if the mother was KSHV negative. The association between KSHV and HIV was also noted in the study of pregnant women attending antenatal clinics and in the mother to child study. However this association was not evident in the Carletonville population where both KSHV and HIV were highly prevalent. In both the adult studies the lack of association between KSHV and syphilis was evident. KSHV infection was also not associated with other sexually transmitted infections and measures of sexual behaviour. As expected, the pattern of HIV and STI in sex workers suggests high rates of high-risk sexual behaviour in this population; however KSHV seropositivity was the same amongst sexworkers and all the other community groups. This pattern of the lack of association with high-risk sexual behaviour, particularly in sex workers and with any markers of STI strongly suggests that the sexual mode does not play a significant role in KSHV transmission in this South African population. This may also suggest that KSHV transmission may involve geographical and cultural factors other than sexual transmission.

Factors associated with the transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV) are inconclusive. In countries where KS and KSHV are confined to men who have sex with other men (MSM), KSHV is associated with sexual risk factors. In countries where KSHV is endemic, it affects adults and children of all ages and irrespective of sexual orientation, suggesting the existence of non-sexual risk factors for KSHV infection. In this thesis, three distinct cross sectional studies aiming to define the seroprevalence of KSHV in South African populations and to identify plausible risk factors for KSHV infection were undertaken. The studies measured KSHV seropositivity in relation to sociodemographic factors and HIV status. In children, factors associated with horizontal mother to child transmission were also explored. In adults KSHV seropositivity was also measured in relation to sexually transmitted infections and/or measures of sexual behaviour. Calculated risk factors were expressed as odds ratios (95% confidence interval) for KSHV. Methods Mother to Child KSHV seroepidemiology Study: KSHV seroprevalence (reactive to either lytic K8.1 or latent Orf73) was measured in 1287 children and their 1179 biological mothers. Association between KSHV seropositivity in children was measured against KSHV seropositivity and HIV status of their mothers. KSHV seroepidemiology in women attending antenatal clinics: Antibodies to KSHV lytic K8.1 and latent Orf73 antigens were tested in 1740 pregnant women attending antenatal clinics in South Africa in 2001. Information on HIV and syphilis serology, age, education, residential area, gravidity, and parity was anonymously linked to evaluate risk factors for KSHV seropositivity. Clinics were grouped by municipal regions and their proximity to the two main river catchments defined. Carletonville Community KSHV seroepidemiology Study: Sera from 2103 South African individuals (862 miners, 95 sex workers, 731 female and 415 male township residents) were tested for antibodies to KSHV lytic K8.1 and latent Orf73, HIV gonococcus, herpes simplex virus type 2 (HSV-2), syphilis and chlamydia. Information on social, demographic and highrisk sexual behaviour was linked to laboratory data. Results Mother to Child KSHV seroepidemiology Study: KSHV seroprevalence (reactive to either lytic K8.1 or latent Orf73) was 15.9% (204 of 1287 subjects) in children and 29.7% (350 of 1179 subjects) in mothers. The risk of KSHV seropositivity was significantly higher in children of KSHV seropositive mothers compared with those of KSHV-seronegative mothers. The HIV status of mothers was marginally associated with an increased risk of KSHV seropositivity in their children (AOR = 1.6, 95% CI: 1.0 to 2.6; P = 0.07). KSHV seroprevalence was significantly higher in HIV-infected subjects (P = 0.0005), and HIV-infected subjects had significantly higher lytic and latent KSHV antibody levels than HIV-negative subjects. KSHV seroepidemiology in women attending antenatal clinics: KSHV seroprevalence was nearly twice that of HIV (44.6% vs. 23.1%). HIV and syphilis seropositivity was 12.7% and 14.9% respectively in women without KSHV, and 36.1% and 19.9% respectively in those with KSHV. Women who were KSHV seropositive were 4 times more likely to be HIV positive than those who were KSHV seronegative (AOR 4.1 95%CI: 3.4 - 5.7). Although, women with HIV infection were more likely to be syphilis seropositive (AOR 1.8 95%CI: 1.3 - 2.4), no association between KSHV and syphilis seropositivity was observed. Those with higher levels of education had lower levels of KSHV seropositivity compared to those with lower education levels. KSHV seropositivity showed a heterogeneous pattern of prevalence in some localities. Carletonville Community KSHV seroepidemiology Study: Overall KSHV and HIV prevalences were 47.5 and 40%, respectively (P<0.43). The risk of HIV infection was highest in sex workers followed by female residents and miners, compared with male residents (P<0.001). HSV-2 infection was highly prevalent (66%) and lower, but still substantial, prevalence (6–8%) was observed for other sexually transmitted infections (STI). No significant difference in KSHV infection was observed among the residential groups (P>0.05). KSHV was not associated with any of the STI or any measures of sexual behaviour. Conclusion The findings of these three studies contribute substantially to global KSHV seroepidemiology and show that in Southern African settings KSHV is associated with non-sexual mode of transmission. Firstly KSHV is common in very young children up to ten years of age and increases with age until adulthood. The high prevalence of KSHV in the South African populations remained evident in all populations. In children, the risk of acquisition of KSHV was higher among children of KSHV-seropositive mothers than if the mother was KSHV negative. The association between KSHV and HIV was also noted in the study of pregnant women attending antenatal clinics and in the mother to child study. However this association was not evident in the Carletonville population where both KSHV and HIV were highly prevalent. In both the adult studies the lack of association between KSHV and syphilis was evident. KSHV infection was also not associated with other sexually transmitted infections and measures of sexual behaviour. As expected, the pattern of HIV and STI in sex workers suggests high rates of high-risk sexual behaviour in this population; however KSHV seropositivity was the same amongst sexworkers and all the other community groups. This pattern of the lack of association with high-risk sexual behaviour, particularly in sex workers and with any markers of STI strongly suggests that the sexual mode does not play a significant role in KSHV transmission in this South African population. This may also suggest that KSHV transmission may involve geographical and cultural factors other than sexual transmission.

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.

MSc (Microbiology)
Department of Microbiology
Background: Prostate cancer (PCA) is a major health concern in males, particularly those above 40 years old. It is the most common form of cancer in males worldwide, including South Africa. In South Africa, the rate of histologically diagnosed prostate cancer is 40 per 100 000 in whites and 14 per 100 000 in blacks, and 1 in 8 men will develop PCA in their lifetime. Several reports have suggested the association of viruses in the pathogenesis of prostate cancer. Objectives: This study was aimed at identifying Hepatitis B virus (HBV), human papilloma virus (HPV) and human herpes virus type 8 (HHV-8), implicated in other forms of cancer, in a cohort of South African patients with either PCA or benign prostatic hyperplasia (BPH); and to seek possible associations thereof. Methods: The study group comprised 187 male patients recruited from Polokwane Hospital presenting with either PCA (staged by Gleason scores) or BPH. Enzyme-linked immunosorbent assay was used to detect antibodies to HHV-8 and HPV; and to detect hepatitis B surface antigen (HBsAg) in the plasma of the study subjects. Total DNA was extracted from plasma and targeted for the identification of HBV and HHV-8 DNA by nested PCR protocols. The HBV nested PCR protocol amplifies a 336bp fragment of the overlapping surface polymerase gene of HBV. The HHV-8 nested protocol amplifies a 233bp fragment of the ORF 26 gene of HHV-8. Amplified DNA products were purified, sequenced by the Sanger protocol and phylogenetically analysed for viral genotypes. The Chi-square test was used to infer statistically significant differences in the level of detection of viruses and the stage of prostate cancer development. Results: Of the 187 participants, a seroprevalence of 4.8% (9/187, HBsAg), 5.3% (10/187, HPV IgG antibody) and 27% (33/124, HHV-8 IgG antibody) were observed. HBsAg was detected more in individuals with BPH than those without and this was statistically significant at ( 2=6.0, p< 0.05). HHV-8 DNA was detected more in individuals in the 60-79 years age range and this was statistically significant at ( 2=61.1, p< 0.05). Occult HBV infection (that is the presence of HBV DNA in the absence of HBsAg) was detected in 23/178 (12.9%) of patients. Taking into account occult HBV infection, the overall prevalence of HBV was 17.7%. HBV genotype E was more prevalent (86.7%) followed by genotype A (13.3%). HHV-8 genotypes K and R were inferred. Apparently, this is the first report on the identification of HHV-8 genotypes K and R from South Africa. Conclusion: The current study has demonstrated for the first time, the presence of genotypes K and R of HHV-8 in South Africa. This study also suggests that there is a high level of occult genotype E HBV infection. Future studies will explore the virome in prostate cancer biopsies.

Department of Microbiology
PhD (Microbiology)
Human herpes virus type 8 (HHV-8), also known as Kaposi’s sarcoma associated herpes virus (KSHV), is the etiologic agent of Kaposi’s sarcoma (KS), and AIDS related Kaposi’s sarcoma (AIDS-KS). HHV-8 which is a member of the Herpesviridae family, exhibits extensive genetic diversity globally. In endemic regions, infection with HHV-8 occurs very early on in life, which is an indication of both environmental and vertical routes of transmission. The advent of HIV leads to the classification of an AIDS-KS defining condition in HIV infections. This suggests that in regions where HIV and HHV-8 are endemic, KS may become common in a mature HIV epidemic. Just like the prevalence of HIV in Northern South Africa is generally high as in most regions of the country, as the HIV epidemic matures in South Africa, it is important to understand the burden and distribution of HHV-8 infection, and the likely genotypes infecting the population. The main objective of the thesis was to establish the epidemiology and infecting genotypes of HHV-8 in Northern South Africa (Limpopo Province), where no data exists. First, a systematic review of the literature was carried out for the entire African continent to determine the seroprevalence and genotype distribution of HHV-8 in all African countries (n=53). In this review, Sudan and South Sudan were considered as one country. Articles were searched using the PRISMA guideline and exported using an article grid. More than two-thirds (64%) of the studies reported on seroprevalence, 29.3% on genotypes; and 9.5% were on both seroprevalence and genotypes. About 45% (24/53) of the African countries had data on HHV-8 seroprevalence exclusively, and more than half (53%) had data on either seroprevalence or genotypes. Almost half (47%) of the countries had no data on HHV-8 infection. There was high heterogeneity in the types of tests and interpretation algorithms used in determining HHV-8 seropositivity across the different studies. Generally, seroprevalence ranged from 2.0% in a group of young children in Eritrea to 100% in a small group of individuals with KS in the Central Africa Republic and a larger group of KS in individuals in Morocco. Approximately, 16% of all the studies reported on children. The difference in seroprevalence across the African region was not significant (95% CI, X2 =0.86; p =0.35), although specifically, a relatively significant ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|VIII level of infection was observed in HIV-infected children. About 38% of the countries had data on K1 genotypes A, A5, B, C, F and Z which occurred at frequencies of 5.3%, 26.3%, 42.1%, 18.4%, 5.3% and 2.6% respectively. Twenty-three percent of the countries had data for K15 genotypes, whereas genotypes P, M and N occurred at frequencies of 52.2%, 39.1% and 8.7% respectively. Data on HHV-8 inter-genotype recombinant is scanty. Our finding suggests that HHV-8 is endemic on the entire African continent, and in HIV endemic regions, but there is need for a harmonized testing protocol for better understanding of HHV-8 seropositivity. HHV-8 genotype A5 and B for K1 gene and genotype P and M for K15 gene are the most predominant genotypes in Africa. The review, for the first time, has provided information on HHV-8 burden on the entire African continent, and suggests that vaccine development efforts for Africa should focus on genotypes B and P. The second component of the investigation focused on the burden of HHV-8 in an HIV population in Northern South Africa (Limpopo Province). Plasma from 3501 HIV infected individuals from 5 districts in Limpopo Province were assessed for antibodies to both the lytic antigen (ORFK8.1) and the latent antigen (ORF73). The distribution of infection was analyzed based on demographic, socioeconomic, and immunological parameters. Statistical inferences for significant differences were determined by Chisquare at a confidence interval of 95%. P-values less than 0.05 were considered significant. About 19.0% of the study population was positive for antibodies to either the lytic or latent antigens or both. Prevalence of antibodies to the lytic antigen was significantly higher than prevalence of antibodies to the latent antigen (17.3% vs 4.1%; p=0.0001). Significant differences were observed for age groups, racial population groups, districts and year of sample collection (p=<0.0001, p=<0.0001, p=<0.0001 and p=0.0385) respectively. Associations were found between both antigens in comparison to the different variables such as age group, racial population groups and districts (R2 value ranging between 0.886 and 1.0). The burden of HHV-8 has now been established for the first time in Northern South Africa. The third aspect of the investigation was a meta-analysis of HHV-8 seroprevalence in Southern Africa in order to understand the impact of geographical location (urban vs rural) on infection. The analysis revealed a significant association between urban settings and HHV-8 infection (p=0.0001). ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|IX The fourth component of the thesis examined the detection of HHV-8 antigen through polymerase chain reaction (PCR) in 534 participants in HIV infected and HIV noninfected populations. A selection of mouthwash DNA samples were subjected to Next Generation Sequencing (NGS) for subsequent genotype inference. Mouth wash samples were obtained from each consenting individual before eating or smoking, and their DNA was purified. A 233bp fragment of the ORF26 gene of HHV-8 was amplified by PCR. HHV-8 was detected in 150 of the 534 participants (28.1%). A significant difference in detection was observed for gender, HIV status, district and the level of education (p=0,0003; p=0.0094; p=0.0002 and p=0.0095) respectively. Consensus sequences were derived from NGS reads for 13 samples. The genotyping results revealed that genotype Q, B, E and N are the genotypes predominant in the study population. As such no mixed infections were detected. Therefore, from the investigations foregoing have demonstrated for the first time the following: (1) HHV-8 is endemic in the entire African continent, which suggest a coendemicity in regions already endemic for HIV; (2) HHV-8 is endemic in Northern South Africa; (3) Urban settings in Southern Africa are associated with high HHV-8 infection; (4) HHV-8 genotypes Q, B, E and N may be predominant in Northern South Africa, with B and P common on the entire African continent. Hence, studies should focus on the generation of full length HHV-8 genomes of the common genotypes to support the selection of genes for vaccine design and development.
NRF

博士
國立陽明大學
公共衛生研究所
101
The aim of this study was to conduct serological and molecular epidemiology of Human herpes virus type 8( HHV-8), human parvovirus type B19( B19) and human influenza virus type B( Flu B) infections in different populations in Taiwan. HHV-8, the causal agent of Kaposi’s sarcoma, is transmitted sexually among homosexual men, but little is known of its transmission among injection drug users (IDUs). In contrast, B19, a causative agent for anemia, is most frequently detected in IDUs. A total of 744 serum samples among them 515 from IDUs and homosexual men attending a sexually transmitted disease (STD) clinic or from 229 HIV-based hospital unit were analyzed for anti-HHV-8 and anti-B19 antibodies. The presence of antibodies against HHV-8 and B19 were tested with ELISA, indirect immunofluorescence assay (IFA) and immunoblot assay (IBA). HHV-8 viral load was analyzed using a K6 gene real-time PCR assay. We used K1 gene nested-polymerase chain reaction (N-PCR) assay to amplify DNA of HHV-8 in HIV-1/AIDS patients. Furthermore, the DNA sequences were analyzed to determine inter-patient specific mutations and subtypes of HHV-8. The results showed that, among HIV-1/AIDS patients, men who have sex with men (MSM) had significantly higher HHV-8 infection rate (32.8% vs. 5.5%, P<0.0001) and significantly lower B19 infection rate (35.20% vs. 82.03%, P<0.001) than IDUs. The CD4 counts were not significantly different between HIV-1/AIDS patients with and without HHV-8. Phylogenetic analysis of 23 HHV-8 strains showed that there were 2 (8.7%)subgroup A, 17 (74%) subgroup C, 2 (8.7%) subtype E and 2 (8.7%) subgroup D. In summary, MSM had significantly higher prevalence rate of HHV-8 than IDUs in Taiwan. The independent association of HHV-8 infection with IDUs suggests that HHV-8 is transmitted through needle sharing, albeit less efficiently than B19. The results of this study are useful for designing preventive medicine and intervention strategies of those infectious diseases. Concerning Flu B study, the antigenic and genetic character of the virus was analyzed in patients registered to a sentinel surveillance site located in the northern region of Taiwan from 2006 to 2007. Both hemagglutinin (HA) test and fluorescence antibody assay were used to determine the isolation rate of Flu B in MDCK cells. Besides, antigenic analysis of HA in circulating Flu B was performed using hemagglutinin inhibition (HI) test. Finally, the phylogentic analysis and gene variation of Flu B were analyzed using one-step reverse transcription (RT)-PCR assay. The results showed that 143 of 452 clinical respiratory samples (32 %) were positive for B/Malaysia /2506/04-like strain ( It was recognized as a B reassortant lineage). Finally, we have confirmed the molecular evolution of Flu B gene and prospects for serum antibody responses after intradermal vaccination against virus. In the summary, The goal of this study is to determine the antigenic and genetic characterization of the hemagglutinins of influenza B viruses in Taiwan. Further investigation may be beneficial to improve strategies for the treatment and disease control of influenza B virus, and also helpful to the development of antiviral drug and vaccine.