Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-24Bitstream added on 2014-06-13T19:40:41Z : No. of bitstreams: 1 goncalves_ac_dr_araiq.pdf: 1427909 bytes, checksum: 13ce547164df900d11546eb7b87b39b8 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Devido a grande importância econômica da citricultura no Brasil e mundo e aos problemas sanitários enfrentados sendo alguns limitantes para o cultivo, como é o caso das doenças causadas por vírus como: a tristeza cítrica, causada pelo vírus da tristeza dos citros (CTV), pertencente ao gênero Closterovirus, família Closteroviridae, uma das maiores ameaças da citricultura mundial, mesmo com a pré imunização através de estirpes fracas do vírus e substituição de porta enxertos, estirpes fortes de CTV ainda causam prejuízos consideráveis. E com o aparecimento da doença morte súbita dos citros (MSC) de etiologia não determinada. Pelo fato de não haver ainda métodos eficientes de separação de ambos os vírus presentes em uma única amostra, levantando se as hipóteses que a causa da MSC esteja relacionada a uma estirpe do vírus CTV, a um vírus do gênero Marafivirus denominado Citrus Sudden Death-associated Virus (CSDaV), pertencente ao gênero Marafivirus, família Tymoviridae, ou a uma associação entre eles. Este trabalho vem propor um método eficaz de separação por citometria de fluxo (FC) de CTV e CSDaV em amostras semi purificadas, diluídas em tampão TE, pH7,5, utilizando marcação de ácidos nucléicos com Iodeto de Propídeo (PI) e conjugação de anticorpos policlonais anti CTV com Isotiocianato de Fluoresceína (FITC), cuja eficácia do método foi comprovada pela reação da polimerase em cadeia (PCR) / Because of high economic importance of citrus in Brazil and the world and health problems being faced some limiting factors for growing as is the case of diseases caused by viruses such as sadness citrus caused by citrus tristeza virus (CTV) belonging to Closterovirus gender, family Closteroviridae, one of the biggest threats to the citrus industry worldwide, even with the pre immunization using mild virus strains and replacement of the rootstocks, strong strains of CTV still cause considerable damage. And with the onset of the disease citrus sudden death (MSC) of undetermined etiology. Because there is not yet efficient methods of separation of the two viruses present in a single sample, raising the hypotheses that the cause of SCD is related to a strain of CTV, a virus Marafivirus group called Citrus Sudden Death-associated Virus (CSDaV) belonging to the genus Marafivirus, Tymoviridae family, or an association between them, this paper proposes an effective method of separation by flow cytometry (FC) and CTV in samples CSDaV semi purified, diluted in TE buffer, pH7, 5, using marking of nucleic acids with propidium iodide (PI) and a combination of polyclonal anti CTV with Fluorescein isothiocyanate (FITC), the effectiveness of the method was confirmed by polymerase reaction chain reaction (PCR)

Les archées sont principalement connues pour leur capacité à croître et survivre dans des conditions extrêmes de température, pression, pH, etc. qui sont hostiles à l’homme. Néanmoins, il est désormais clair que les archées sont aussi présentes de manière ubiquitaire dans divers environnements. L’étude détaillée des différents aspects de la biologie de ces microorganismes a amené à des découvertes pour le moins inattendues comme celle de la virosphère associée aux archées qui est unique. En effet, plusieurs virus infectant les archées ont été isolés et présentent une incroyable diversité tant au niveau morphologique que génomique et ne ressemblent aucunement aux virus connus de bactéries ou d’eucaryotes. Récemment, l’analyse en détails du cycle viral a mis à jour de nouveaux mécanismes d’interactions avec la cellule hôte. Au cours de mes travaux de thèse, nous nous sommes intéressés aux systèmes virus-hôtes présents dans les milieux hyperthermiques et acidophiles en sélectionnant les virus fusiforme et filamenteux SSV1 et SIRV2 en tant que modèles d’étude. Tout d’abord, nous avons défini une nouvelle classification des virus fusiformes en basée sur l’analyse comparative des protéines structurales et des génomes viraux. L’ensemble des virus considérés forme un réseau global malgré le fait qu’ils ont été isolés dans des environnements distincts ; qu’ils infectent des hôtes qui sont distant phylogénétiquement parlant et que certains de leurs virions présentent une certaine pléomorphicité. Ensuite, la caractérisation en détails de l’architecture des virions fusiformes de SSV1 a révélé qu’ils étaient enveloppés, composés de protéines de capside glycosylées et contenaient le complexe nucléoprotéique. Finalement, nous nous sommes concentrés sur la manière dont les virus d’archées interagissent avec la cellule hôte. Alors que les virions de SIRV2 semblent utiliser une stratégie pour l’entrée qui est similaire aux bactériophages dits flagellotrophiques ; on observe que les virions de SSV1 emploient un mécanisme de sortie qui rappelle le bourgeonnement des virus eucaryotes enveloppés. L’ensemble de ces recherches participent à une meilleure compréhension de la biologie des archées ainsi que de leurs virus et permettent de définir des cibles intéressantes pour de futures études. / Although, archaea were initially regarded as exotic microorganisms capable of growing in conditions which are hostile to humans, it became clear that they are ubiquitous and abundant in various environments. Detailed studies focusing on different aspects of archaeal biology have led to many unexpected discoveries, including the unique virosphere associated with archaea. Indeed, highly diverse viruses characterized by uncommon virion shapes and mysterious genomic contents have been isolated that typically do not resemble viruses of either bacteria or eukaryotes. Recent analysis of the sequential events of the viral cycle resulted in major breakthroughs in the field. In the framework of my PhD studies, I have focused on two model hyperthermo-acidophilic virus-host systems, the spindle-shaped SSV1 and rod-shaped SIRV2, both infecting organisms of the genus Sulfolobus. Initially, we defined structure-based lineages for all known spindle-shaped viruses isolated from highly divergent hosts and residing in very different environments. Then, we provided insights into the architecture of spindle-shaped viruses by showing that SSV1 virions are composed of glycosylated structural proteins and contain a lipid envelope. Finally, we focused on virus-host interplay. Whereas SIRV2 virions appear to use a similar entry strategy as flagellotrophic bacteriophages, SSV1 virions employ an exit mechanism reminiscent of the budding of eukaryotic enveloped viruses. Collectively, these studies shed light on the biology of archaeal viruses and help to define interesting targets that should be the focus of intensive research in the next future.

Hepatitis B virus (HBV) is hyperendemic to southern Africa, the Asia-Pacific region, and the Amazon Basin. HBV causes both acute, and chronic infection of the liver that result in a wide spectrum of liver diseases ranging from acute mild subclinical infection, and an asymptomatic carrier state (ASC) to severe clinical manifestations, including, severe acute and, chronic hepatitis, which can progress to cirrhosis, and the development of hepatocellular carcinoma (HCC). Several viral factors have been implicated in the activation, and inhibition of apoptosis. The development, and progression of this wide spectrum of liver diseases are associated with an unregulated increase or decrease in hepatocyte apoptosis as well as a loss of balance between cell proliferation, and apoptosis. In southern Africa, genotype A of HBV is the predominant genotype, with subgenotype A1 prevailing. Individuals infected with subgenotype A1 have several unique characteristics, including relatively lower levels of HBV DNA, and early seroconversion of hepatitis B e antigen (HBeAg) to antibodies against HBeAg during the course of HBV infection. Infection with this subgenotype is associated with rapid disease progression, and high frequency of HCC development. Moreover, patients infected with subgenotype A1 had increased levels of apoptosis when compared to the other subgenotypes. The G1862T mutation occurs most frequently in subgenotype A1. In the clinical setting, South African HCC patients infected with G1862T subgenotype A1 strains had higher levels of apoptosis while ASCs patients infected with G1862T subgenotype A1 strains had lower levels of apoptosis, when compared to those infected with wild-type. To date, G1862T has been functionally characterized in subgenotype A2, genotype B and in a genotype D HBV genome containing a subgenotype A1 precore (PreC) region. The objectives of our study were to construct 1.28 mer replication competent clones containing an endogenous HBV promoter for wild-type subgenotypes A1, A2, and D3 as well as mutant G1862T subgenotype A1, and to functionally characterize these strains in tissue culture. Transfection of Huh7 cells was used to follow the viral replication, expression of HBeAg, activation of the unfolded protein response (UPR), and subsequent apoptosis. The strategy used for the generation of these replication competent clones had several advantages. Very few PCR errors were introduced, and carry-over of enzymes, nonspecific products, and reaction reagents downstream was prevented. The clones contained endogenous HBV promoters, and enhancers, and were generated from a single complete genome of HBV. These replication competent clones may be used in future studies for the establishment of stable cell lines that constitutively express HBV proteins without the need for further manipulation. This strategy can be used for the generation of replication competent clones belonging to genotypes A to D, and G, and with a few minor modifications, for genotypes E, F, and H. Using the newly generated clones, their replication competence was demonstrated using transfection of Huh7 cells. Even in the absence of an exogenous promoter, these clones were able to support the expression of intracellular, and extracellular HBV DNA at levels of 108 to 109 genome copies/ml. HBcAg, HBeAg, and HBsAg were expressed for a period of five days, and the order of expression was similar to that seen during acute HBV infection. Comparison of transfection with a replication competent clone containing an exogenous HBV promoter demonstrated higher expression of HBV DNA, and proteins, as well as an earlier expression, and accumulation of HBeAg in the endoplasmic reticulum (ER) relative to the clone containing an endogenous HBV promoter. This initial increased accumulation of HBeAg in the ER did not affect the level of activation of the UPR, but led to an increased level of total cell death as a consequence of necrosis. When comparing the different subgenotypes following transfection into Huh7 cells, subgenotype D3 replicated at a lower level, as measured by HBsAg, and HBV DNA levels, with HBeAg passing through the secretory pathway earlier, when compared to cells transfected with genotype A. There was no difference in the intracellular, and extracellular HBsAg between cells transfected with either subgenotype A1 or A2. However, cells transfected with subgenotype A1 had higher levels of intracellular replicative intermediates, HBeAg, and HBcAg, and lower extracellular expression of HBeAg from days 1 to 3, when compared to cells transfected with subgenotype A2. The intracellular retention of the PreC/ core (C) precursor protein in cells transfected with subgenotype A1 was clearly demonstrated by its lower expression in the secretory pathway, and its higher co-localization in the nucleus, using indirect immunofluorescence. This intracellular retention led to greater ER stress, and an earlier, and prolonged activation of the UPR. This correlated well with the higher PERK, ATF6, and IRE1/XBP1 activity seen on days 3 than on day 5. These findings suggest that the prolonged activation of the UPR in cells transfected with subgenotype A1 led to increased apoptosis, and subsequent induction of liver damage, and may therefore, be a contributing factor to the higher hepatocarcinogenic potential of subgenotype A1. Our study demonstrated that G1862T reduced replication, and led to the initial temporal retardation of intracellular core-particle-associated HBV DNA. Although, G1862T did not affect HBsAg expression, it led to a decreased expression of HBcAg, and HBeAg. The decreased expression of extracellular HBeAg was probably as a result of decreased cleavage efficiency by the signal peptide, which consequently led to the retardation of the PreC/C precursor protein in the ER, and ER-Golgi intermediate compartment (ERGIC), and its decreased expression in the nucleus. This retardation, and accumulation led to the earlier activation of all three UPR pathways, but not to increased apoptosis. Therefore, it is evident that G1862T does not completely abolish HBeAg expression, but affects the rate of HBeAg maturation, and its expression through the secretory pathway. These findings suggest that in response to the accumulation of HBeAg in the ER, the UPR was activated resulting in the alteration of the capacity to overcome this stress, consequently leading to a new homeostasis of the ER being reached. The capacity of the ER is increased, with no further activation of the UPR and apoptosis, which facilitates maturation of HBeAg. In conclusion, our study for the first time demonstrated that there are a number of factors that influence the expression of proteins in HBV transfection studies including the type of transcriptional promoter, the different genotypes/subgenotypes of HBV, the use of protein expressing as opposed to replication competent clones, and the presence, and absence of mutations, such as the G1862T. Therefore, when comparing the outcomes of various experiments these factors should be taken into consideration, and the results interpreted with caution, because experiments may not be strictly speaking comparable. Importantly, replication competent clones were generated from strains circulating in southern Africa. The generation of these clones is an important step in further functional characterization of African strains of HBV, and their comparison to strains circulating other geographical regions of the world. These strains, in particular, subgenotype A1 can develop unique mutations, such as the G1862T, which we demonstrated can influence the expression of HBeAg, in a way that it can possibly account for the higher hepatocarcinogenic potential of subgenotype A1.

Thesis (MSc Virology)--University of Limpopo, 2010. / OBJECTIVES: To date there is no effective and safe vaccine to stop the spread of human immunodeficiency virus (HIV) and provide cross protection among different subtypes. HIV accessory genes were overlooked for many years and recently they are becoming candidates for development of new anti-HIV drugs and vaccines. This is supported by their ability to elicit cytotoxic T lymphocyte response. To date, there are limited studies on accessory genes (nef, vif, vpr and vpu) on South African HIV strains. This study sought to amplify and analyse the sequences of HIV-1 subtype C accessory genes (vif, vpr and vpu) to assess the genetic diversity as well as the motifs and residues associated with key biological functions of these genes. This study further sought to compare the degree of genetic diversity between the accessory and structural genes. METHODS: The study was an exploratory study using stored (-70ºC) HIV positive plasma samples. The study population comprised of 25 HIV positive plasma samples which were already sequenced in the gag and env genes in another study. The samples were drawn from the neighbouring townships of Pretoria: Ga-Rankuwa, Soshanguve, Mamelodi, Laudium, Kalafong, Jubilee and Mabopane. For the purpose of this study, the same samples were amplified, sequenced and characterised in the pol and accessory (vif, vpr and vpu) genes in order to obtain near full length sequences of the HIV isolates from Pretoria region. Six samples were cloned for accessory genes. Five clones from each sample were selected. Sequence analysis was performed for all the PCR amplicons and clones. Base calling for the sequences generated was performed on Chromas Pro program. Computing of phylogenetic tree was performed with MEGA 4 program. ClustalW software was used for sequence alignment and translation of nucleotides to amino acids was performed with BioEdit. The amino acid alignments were analysed on graphic view. RESULTS: All 25 samples were successfully amplified for accessory genes (vif, vpr and vpu) and pol gene. All the 25 pol PCR amplicons were successfully sequenced, while all but one accessory PCR amplicons were successfully sequenced. A number of conserved motifs and residues were observed in all the four genes (vif, vpr, vpu and pol). Vif and vpr showed to harbour most of these conserved motifs and residues; 144-SLQYLA-149 and H71 respectively. In addition, the R77Q mutation associated with long term non-progressors was observed in the vpr gene of 15 sequences. Drug resistant mutations were evaluated in both protease and RT regions. Nine samples had one or two drug resistant mutations i.e T74S, L10I, V179D, E138A/D, Y318F,Y181C and K108N. Phylogenetic analysis confirmed the 25 HIV positive samples to be HIV-1 subtype C in both structural and accessory genes. The genetic diversity of HIV-1 subtype C was compared between accessory (vif, vpr and vpu) and structural (pol, gag and env) genes. The gag and env sequences were available from a previous project (Musyoki, 2009). The gag and vif gene sequences were highly conserved (89% to 96% and 88% to 96%, respectively), as compared to vpr gene (84% to 94%), the pol gene (79% to 95%), the env gene (83% to 93%) and finally the vpu gene (73% to 92%). CONCLUSION: This study found that amplification of clones was more sensitive as compared to direct samples and analysis of clone sequences was more clear than analysis of direct PCR products. Functional motifs and residues observed in all accessory genes were highly conserved. Vif was more conserved, followed by vpr and vpu, respectively. Genetic analysis of pol gene revealed that there were drug resistant strains in circulation. This indicates that the patients were infected with drug resistant viruses; this cannot be verified from the study population. And that most of the strains in this study had mutations associated with long term non-progressors (LTNP’s). However, it is not known whether these patients were indeed LTNP’s. Comparison of genetic diversity between structural and accessory genes demonstrated that, gag, vif and vpr were more conserved than pol, env and vpu.

Thesis (MSc. (Medical Virology))-- University of Limpopo, 2011. / Introduction: Sub-Saharan Africa is a region with hepatitis B virus (HBV) hyperendemicity with more than 8% HBsAg prevalence. An estimate of two billion people has been reported to carry HBV markers. HBV was associated with about 25% of annual deaths in Africa. HBV possesses a DNA polymerase which lacks proofreading mechanism. This results in highly variability and genetic diversity which poses a challenge for the diagnosis and therapeutic management of HBV infection. High mutation rate of HBV also has great implications on the development of drug resistant mutations. Moreover, HBV diversity represents a challenge for the sensitivity of immunological and molecular diagnostic assays. A number of studies on HBV full length genome have been conducted particularly in developed countries. Limited studies are available in Africa and South Africa. In South Africa, few studies have been done analysing the complete genome of HBV isolates from patients with asymptomatic carriers and fulminant hepatitis B (Owideru et ai, 2001 a; Owideru et ai, 2001 b; Kimbi et ai, 2004; Kramvis et ai, 2002).This study was aimed at characterising the full-length genome of HBV isolates at Dr George Mukhari Hospital, Pretoria, South Africa, with a view of developing a PCR-based technology for amplification and characterisation of HBV strains with different serological profiles. The technology, if successfully developed, will contribute in understanding the molecular mechanisms resulting in various HBV variants or isolates. Methods: The study design was exploratory. Four stored serum samples collected from HBV infected patients at Dr George Mukhari hospital, Pretoria, were used to develop the molecular technology and test the hypothesis. HBV serology was previously performed targeting 5 HBV serological markers; HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe using Elecsys version; HBV DNA quantification was done using Cobas Amplicor HBV DNA monitor assay, HBV DNA was extracted and subjected to nested PCR assay targeting HBV full length genome as two overlapping fragments: fragment A (1670 bp) and fragment B (1868 bp). The generated PCR products for both fragments were cloned into the pGEM T easy vector and 2 clones were selected from each sample. The plasm ids were purified using Invisorb@ Spin Plasmid Mini Two and the clones were recovered by PCR assay. The sample PCR products and the clone PCR products were purified and sequenced using SpectruMedix SCE2410 genetic analysis system. HBV genotyping was performed using the NCBI web-based genotyping tool. Phylogenetic analysis was done using MEGA 4 software to confirm HBV genotypes. Results: Serology results were as follows: All samples were HBsAg positive, Anti-HBs negative, anti-HBc positive and anti-HBe negative. Sample B1121 and sample 6 were HBeAg positive while samples B452 and 5 were HBeAg negative. A total of 12 PCR products were sequenced (4 study samples and 8 clones [2 clones each sample]). In total, 7 HBV full length genome sequences were deduced from this study, with 3 sequences belonging to genotype A, 2 to genotype C and 2 to genotype D. 3 HBV genotypes were detected from this study; genotype A, C and D with subgenotype A2, C1 and D1 respectively. Mutations were observed throughout the genome. In the pre-S/S open reading frame (ORF), the most significant findings were the detection of mutations within the "a" determinant site and major hydrophilic region (MHR). These mutations included Y161F,E164G observed in sample B1121 and B1121C1 belonging to subtype A1; 2 amino acid insertion at aa 161-162 in sample 5 belonging to subtype C1. Drug resistance associated mutations were identified in the polymerase gene, these included M204T and L217R which are associated with adefovir resistance, M204T also resulted in a change from tryptophan (W) to arginine (R) at aa 196 on the overlapping surface gene on sample B452 C1. Basal core promoter (BCP) and pre core/core mutations were detected in study isolates; specifically the BCP double mutation (1762/1764) was seen in 8 isolates which belonged to subtype C1 (5) and D1 (3) and the pre-core stop codon mutations (G1896A) in 4 isolates. (2 belonging to subtype C1 and the other 2 to D1). Other changes observed included a 48 nucleotides deletion in the pre-core gene, 6 nucleotides insertion in the HBx gene of all subtype D1 isolates and a 3 nucleotides deletion in subtype C1 clone. Conclusion: This study successfully optimised a PCR-based technology for the amplification and characterisation of HBV full length genome. 3 HBV genotypes were detected with subtypes A2, C1 and D1. However, the detected subtypes are rarely detected in South Africa. The detection of subtype A2 may confirm its Southern African origin. Drug resistance associated mutations were observed in this study. These included the adefovir resistance mutation which the current study confirmed it is a naturally occurring mutation as it was detected in adefovir therapy na'ive patient. The BCP and pre-core/core mutations were detected in genotype C and D isolates; however, their association with serological profile and clinical outcomes could not be deduced. Unique or novel mutations were seen in the study isolates, these included 48 nucleotides deletion in the pre core gene, 3 amino acids insertion in the RNase H and 8 amino acids deletion in the RT domain of polymerase gene. To our knowledge, these mutations have not been identified or reported in the literature. The detection of 6 nucleotide insertion in the HBx gene was reported for the first time in South African isolates. Further analysis is required to ascertain the biological significance of the unique mutations detected in this study.

Thesis (M Cur)-- University of Limpopo, 2012. / ABSTRACT Background and problem statement The increase in AIDS related deaths of parents leave many children orphaned and some of these children live with HIV. These children are cared for by primary caregivers who are mostly elderly women. The primary caregivers experiences challenges when caring for the children living with HIV. These challenges may be physical, spiritual, psychological and social. The researcher conducted a study in order to explore the experiences of primary caregivers caring for children living with HIV. Aim and objectives The aim of the study was to promote the mental health of primary caregivers who provide care for children living with HIV attending the Wellness Clinic at the Jubilee Hospital in Hammanskraal. The objectives of this study were to: i) describe the biographical data of primary caregivers who provide care for children living with HIV; ii) explore and describe the experiences of primary caregivers who provide care for children living - with HIV; and iii) make recommendations which are based on the findings of this study in order to assist the nursing personnel at the Wellness Clinic in the promotion of the mental health of primary caregivers based on the findings of this study. Research Design and method A qualitative, exploratory, descriptive and contextual design was utilised to enable the primary caregivers to share with the researchers their experiences of caring for children living with HIV. The setting was the Wellness Clinic at the Jubilee hospital, Hammanskraal. Ethical principles were adhered to in order to protect the rights of the primary caregivers. Throughout the process, the methods to ensyre trustworthiness of the study were foll9wed. A purposive sample of eight primary caregivers was chosen for the unstructured interviews. Data were analysed by the researcher and an independent coder using the Tescn method. Research Findings Consensus was reached after consultation with an independent coder,- about the following categories i) primary caregivers' experiences in caring for a child living with HIV related to the self of the caregiver; ii) primary caregivers' experiences related to the decision to disclose the child's HIV status to various role-players were influenced by stigmatisation and discrimination related to HIV and AIDS; iii) primary caregivers' challenges when caring for a child living with HIV; and iv) the mobilisation of resources by primary caregivers to assist them in caring for a child living with HIV. Findings were contexualised by implementing a literature control and recommendations were made to promote their mental health. Conclusions Primary caregivers who cared for children living with HIV in this study were mostly elderly women who were related to the children. They took over the care of the children living with HIV after the children's parents had died. Although they were faced with many challenges, their concern for the children's wellbeing made them to give the children loving care. They found strength and support from prayer, faith and hope in God. The primary caregivers also appreciated the support they received from the health care workers at the Well"ness Clinic. Key words: caring, mental health, HIV, children, primary caregiver

Lassa virus (LASV) is a member of the Old World Complex of arenaviruses infecting an estimated 300,000-500,000 people each year primarily in the endemic region of West Africa. Lassa fever (LF), a neglected tropical disease, puts a heavy burden on the communities in which it is endemic. Reportedly, 15%-20% or up to 50% during epidemics succumb to LASV infection. Without U.S. Food and Drug Administration approved diagnostics, therapeutics, or vaccines, knowledge of this disease must advance in order to create affordable and reliable care. Unlike other viral infections, LF does not stimulate a robust humoral immune response. Many in vitro studies have demonstrated the ability of LASV nucleoprotein (NP) and Z protein to interrupt innate immune activation of important cytokines and costimulatory molecules, which can impair the adaptive immune response. To this end, we investigated the antibody and cytokine responses in LF confirmed patients. We then investigated the role NP plays in subverting the innate immune response, suggesting a possible mechanism for humoral immune impairment. Our findings demonstrate a prolonged IgM response and a dysregulated cytokine response in vivo. Additionally, we show LASV NP is capable of modulating the immune system by masking pathogen pattern signals, namely dsRNA, that initiate immune cascades via the Toll Like Receptor 3 pathway. / 1 / Jessica Nicole Hartnett

Ein vielversprechender neuer Ansatz zur Behandlung von Krebs beim Menschen ist die Verwendung von onkolytischen Viren, die einen Tumor-spezifischen Tropismus aufweisen. Einer der Top-Kandidaten in diesem Bereich ist das onkolytische Vaccinia Virus (VACV), das bereits vielversprechende Ergebnisse in Tierversuchen und in klinischen Studien gezeigt hat. Aber die von den in vivo in tierischen Modellen erhaltenen Resultate könnten ungenaue Informationen wegen der anatomischen und physiologischen Unterschiede zwischen den Spezies liefern. Andererseits sind Studien in Menschen aufgrund ethischer Erwägungen und potenzieller Toxizität nur limitiert möglich. Die zahlreichen Einschränkungen und Risiken, die mit den Humanstudien verbunden sind, könnten mit der Verwendung eines humanisierten Mausmodells vermieden werden. Die LIVP-1.1.1, GLV-2b372, GLV-1h68, GLV-1h375, GLV-1h376 and GLV-1h377 VACV Stämmen wurden von der Genelux Corporation zur Verfügung gestellt. GLV-2b372 wurde durch Einfügen der TurboFP635 Expressionskassette in den J2R Genlocus des parentalen LIVP-1.1.1-Stammes konstruiert. GLV-1h375, -1h376 and -1h377 kodiert das Gen für den menschlichen CTLA4-blockierenden Einzelketten-Antikörper (CTLA4 scAb). Befunde aus Replikations- and Zytotoxizitätsstudien zeigten, dass alle sechs Viren Tumorzellen infizieren, sich in ihnen replizieren und sie in Zellkultur schließlich ebenso dosis- und zeitabhängig effizient abtöten konnten. CTLA4 scAb und β-Glucuronidase (GusA) Expression sowie Virus Titer in GLV-1h376-infizierten A549-Zellen wurde anhand von ELISA-, β-Glucuronidase- and Standard Plaque-Assays bestimmt. Hierbei zeigte sich eine ausgezeichnete Korrelation mit Korrelationskoeffizienten R2>0.9806. Der durch das GLV-1h376 kodierte CTLA4 scAb wurde erfolgreich aus Überständen von infizierten CV-1-Zellen gereinigt. CTLA4 scAb hat eine hohe in-vitro-Affinität zu seinem menschlichen CTLA4-Zielmolekül sowie abwesende Kreuzreaktivität gegenüber murine CTLA4 gezeigt. CTLA4 scAb Funktionalität wurde in Jurkat-Zellen bestätigt. LIVP-1.1.1, GLV-2b372, GLV-1h68 und GLV-1h376 wurden auch in nicht-tumorösen und/oder tumortragenden humanisierten Mäusen getestet. Zunächst wurde gezeigt, dass die Injektion von menschlichen CD34+ Stammzellen in die Leber von vorkonditionierten neugeborenen NSG Mäusen zu einer erfolgreichen systemische Rekonstitution mit menschlichen Immunzellen geführt hat. CD19+-B-Zellen, CD4+- und CD8+-CD3+-T-Zellen, NKp46+CD56- und NKp46+CD56+-NK-Zellen sowie CD33+-myeloischen Zellen wurden detektiert. Die Mehrheit der nachgewisenen humanen hämatopoetischen Zellen im Mäuseblut in den ersten Wochen nach der Humanisierung waren CD19+-B-Zellen, und nur ein kleiner Teil waren CD3+-T-Zellen. Mit der Zeit wurde eine signifikante Veränderung in CD19+/CD3+-Verhältnis beobachtet, die parallel zur Abnahme der B-Zellen und einem Anstieg der T-Zellen kam. Die Implantation von A549-Zellen unter die Haut dieser Mäuse führte zu einem progressiven Tumorwachstum. Bildgebende Verfahren zur Detektion von Virus-vermittelter TurboFP635- und GFP-Expression, Standard Plaque Assays sowie immunohistochemische Analysen bestätigten die erfolgreiche Invasion der Viren in die subkutanen Tumoren. Die humane CD45+-Zellpopulation in Tumoren wurde hauptsächlich durch NKp46+CD56bright-NK-Zellen und einen hohen Anteil von aktivierten CD4+- und zytotoxische CD8+-T-Zellen dargestellt. Es wurden jedoch keine signifikanten Unterschiede zwischen den Kontroll- und LIVP-1.1.1-infizierten Tumoren beobachtet, was darauf hindeutete, dass die Rekrutierung von NK- und aktivierten T-Zellen, mehr Tumorgewebe-spezifisch als Virus-abhängig waren. Die GLV-1h376-vermittelten CTLA4 scAb-Expression in den infizierten Tumoren war ebenfalls nicht in der Lage, die Aktivierung von Tumor-infiltrierenden T-Zellen im Vergleich zur Kontrolle und GLV-1h68-behandelten Mäusen, signifikant zu erhöhen. ELISA-, β-Glucuronidase- and Standard Plaque-Assays zeigten eine eindeutige Korrelation mit den Korrelationskoeffizienten R2>0,9454 zwischen CTLA4 scAb- und GusA-Konzentrationen und Virus Titer in Tumorproben von GLV-1h376-behandelten Mäusen. T-Zellen, die aus der Milz dieser Tumor-tragenden Mäuse isoliert wurden, waren funktionell und konnten erfolgreich mit Beads aktiviert werden. Mehr CD25+ und IFN-ɣ+ T-Zellen wurden in der GLV-1h376-Gruppe gefunden, wahrscheinlich aufgrund der CTLA4-Blockade durch die Virus-vermittelte CTLA4 scAb-Expression in den Mäusen. Außerdem wurde eine höhere Konzentration von IL-2 in dem Kulturüberstand von diesen Splenozyten im Vergleich zu Kontrollproben nachgewiesen. Im Gegensatz zu der Aktivierung mit Beads konnten T-Zellen von allen drei Maus-Gruppen nicht durch A549 Tumorzellen ex vivo aktiviert werden. Unser Mausmodell hat den besonderen Vorteil, dass sich Tumoren unter der Haut der humanisierten Mäuse entwickeln, was eine genaue Überwachung des Tumorwachstums und Auswertung der onkolytischen Virotherapie ermöglicht. / A promising new approach for the treatment of human cancer is the use of oncolytic viruses, which exhibit tumor tropism. One of the top candidates in this area is the oncolytic vaccinia virus (VACV), which has already shown promising results in animal studies and in clinical trials. However, due to discrepancies in both innate and adaptive immunity between mice and men the evaluation of the vaccinia virus’ interactions with the host immune system in mice are not fully conclusive of what is actually happening in human cancer patients after systemic administration of vaccinia virus. Also, ethical and legal concerns as well as risk of potential toxicity limit research involving human patients. Therefore, a good in vivo model for testing interactions between vaccinia virus and human immune cells, avoiding the numerous limitations and risks associated with human studies, could be a humanized mouse model. LIVP-1.1.1, GLV-2b372, GLV-1h68, GLV-1h375, GLV-1h376 and GLV-1h377 VACVs were provided by Genelux Corporation. GLV-2b372 was constructed by inserting TurboFP635 expression cassette into the J2R locus of the parental LIVP-1.1.1. GLV-1h375, -1h376 and -1h377 VACVs encode the human CTLA4-blocking single-chain antibody (CTLA4 scAb). Performed replication and cytotoxicity assays demonstrated that all six viruses were able to infect, replicate in and kill human tumor cells in virus-dose- and time-dependent fashion. CTLA4 scAb and β-glucuronidase (GusA) expression as well as viral titers in GLV-1h376-infected cells were analyzed by ELISA, β-glucuronidase assay and standard plaque assay, respectively, and compared. An excellent correlation with correlation coefficients R2>0.9806 were observed. GLV-1h376-encoded CTLA4 scAb was successfully purified from supernatants of infected CV-1 cells and demonstrated in vitro affinity to its human CTLA4 target and lack of cross-reactivity to mouse CTLA4. CTLA4 scAb functionality was confirmed in Jurkat cells. LIVP-1.1.1, GLV-2b372, GLV-1h68 and GLV-1h376 were next studied in non-tumorous and/or tumor-bearing humanized mice. It was demonstrated that injection of human CD34+ stem cells into the liver of preconditioned newborn NSG mice let to a successful systemic reconstitution with human immune cells. CD19+ B cells, CD4 and CD8 single positive CD3+ T cell, NKp46+CD56- and NKp46+CD56+ NK cells as well as CD33+ myeloid cells developed. At early time points after engraftment, majority of the human hematopoietic cells detected in the mouse blood were CD19+ B cells and only a small portion were CD3+ T cells. With time a significant change in CD19+/CD3+ ratio was reported with a decrease of B cells and an increase of T cells. Implantation of A549 cells under the skin of those humanized NSG mice resulted in a progressive tumor growth, described for the first time in this thesis. Successful colonization of subcutaneous A549 tumors with VACVs was visualized and demonstrated by detection of virus-mediated TurboFP635 and GFP expression as well as by standard plaque assay and immunohistochemistry. The human CD45+ cell population in tumors was represented mainly by NKp46+CD56bright NK cells and a large portion of activated CD4+ and cytotoxic CD8+ T cells. However, no significant differences were observed between control and LIVP-1.1.1-infected tumors, suggesting that the recruitment of NK and activated T cells were more tumor tissue specific than virus-dependent. Unfortunately, virus-mediated CTLA4 scAb expression in the GLV-1h376-infected tumors was also not able to significantly increase activation of T cells compared to control and GLV-1h68-treated mice. Importantly, ELISA, β-glucuronidase and standard plaque assays showed an excellent correlation with correlation coefficients R2>0.9454 between CTLA4 scAb, GusA concentrations and viral titers in tumor samples from those GLV-1h376 treated mice. T cells isolated from the spleens of such control or GLV-1h68- or -1h376-treated A549 tumor-bearing mice were functional and could successfully be activated with human T cells activation beads. However, although no significant difference was observed between the three mouse groups, a slightly higher percentage of the GLV-1h376-treated mice-derived T cells were expressing CD25 and producing IFN-ɣ after ex vivo activation, probably due to the CTLA4 blockade by the virus-encoded CTLA4 scAb in the GLV-1h376-treated mice. Also, slightly higher levels of IL-2 were detected in the culture supernatant of those splenocytes compared to control samples. In contrast, T cells from all three mouse groups were not able be activated by A549 tumor cells ex vivo. Our model has the specific advantage that tumors develop under the skin of the humanized mice, which allows accurate monitoring of the tumor growth and evaluation of the oncolytic virotherapy. Therefore it is important to choose the right approaches for its further improvement.

The emergence of viruses in the family Paramyxoviridae, especially those such as Hendra virus and Nipah virus (NiV) that are zoonotic, highlighted the severity of disease that could be caused by infection with viruses belonging to this family. In addition to causing disease outbreaks, several newly discovered paramyxoviruses were found to have unique genetic features, which provoked renewed interest in the study of previously unclassified or uncharacterised viruses in this family. J-virus (JPV) was isolated from wild mice, in Queensland, Australia, in 1972, and has been suggested to be a natural respiratory pathogen of mice. Beilong virus (BeiPV), another paramyxovirus, was first isolated from human mesangial cells in Beijing, China, in 2003, and was subsequently detected in rat mesangial cells. Following initial characterisation, the genomes of JPV and BeiPV were found to contain two genes, SH and TM, not common to other paramyxoviruses, as well as an extended attachment protein gene. BeiPV has the largest genome in the family Paramyxoviridae, which is, in fact, larger than that of any other virus within the order Mononegavirales. The genetic material of paramyxoviruses is not amenable to manipulation via classical genetics; a reverse genetics approach was therefore employed to study the evolution and classification of JPV and BeiPV. Minireplicon systems utilising green fluorescent protein as a reporter were established for JPV, BeiPV and NiV, and were used to better assess the taxonomic status of JPV and BeiPV, and to determine the relationship between these viruses and henipaviruses, which also have exceptionally large genomes. These studies indicate that JPV and BeiPV are closely related and should be classified in the same genus and their replication and transcription machinery is different from that of the henipaviruses. / To gain an understanding of the biology of JPV and BeiPV, viral surface proteins from JPV were expressed and evaluated. Chimeric JPV virions containing recombinant surface proteins were generated and electron microscopy was used to determine the localisation of the proteins encoded by those JPV genes which are uncommon in other paramyxoviruses. Analysis of the attachment protein gene of JPV indicated that the virus was able to assemble an exceptionally large protein (156 kDa) into the virion structure, providing evidence in support of the hypothesis that JPV and BeiPV may represent an ancient lineage of viruses within the family Paramyxoviridae. In order to determine tissue tropism of JPV during experimental infection and to aid future work with a full-length JPV infectious clone, a real-time PCR assay for JPV was developed and assessed on tissues collected from mice infected with JPV. A multiplex microsphere assay for JPV and BeiPV was developed and used to analyse the seroprevalence of these viruses in Australian and Malaysian rodents. Although there is currently no evidence for disease caused by JPV or BeiPV, this does not preclude the emergence of a zoonotic rodent paramyxovirus related to these viruses. If this were to occur, the tools for virus detection and serological monitoring are now established.

Includes corrigendum attached to back leaf. Bibliography: leaves 114-132. This thesis describes the complete nucleotide sequence and genome organisation of SCSMaV-RNA, examines the taxonomy of SCSMaV by phylogenetic analysis, and describes the development of diagnostic tests for application to field study. (abstract)