Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Ziel der Arbeit war die Entwicklung und Implementierung einer internen Extraktions-Kontrolle für die quantitative Echtzeit-PCR humanpathogener DNA-haltiger Viren der Routinediagnostik des Instituts für Virologie der Universität Leipzig, basierend auf einem artfremden Vollvirus. Durch Verwendung einer internen Kontrolle sollen falsch-negative PCR-Ergebnisse aufgrund unzureichender DNA-Extraktion aus humanem Probenmaterial identifiziert werden. Die interne Kontrolle soll zur Zeitersparnis als Multiplex-PCR durchgeführt werden. Verwendet wurde das Baculoviurs Autographa californica multicapsid nucleopolyhedrovirus, da keine Baculovirus-Infektionen beim Menschen vorkommen. Es wurden Primer und Sonden so entwickelt, dass sowohl das Amplifikat als auch Primer und Sonden eine hohe Übereinstimmung zu den Amplifikaten, Primern und Sonden der humanpathogenen Viren zeigen. Dadurch soll die PCR-Effizienz der internen Kontrolle möglichst ähnlich zu der PCR-Effizienz der humanpathogenen Viren sein. In Vorversuchen wurde gezeigt, dass eine Hybridisierung der Baculovirus-Primern und -Sonden an das Genom humanpathogener Viren und das humane Genom minimal war. Des Weiteren wurde nachgewiesen, dass sich die Baculovirus-DNA als interne Kontrolle vemischt mit Lysispuffer über mindestens eine Woche bei 4°C ohne signifikante Verluste lagern lässt. Im vorgestellten und validierten Protokoll wird die interne Kontrolle automatisch während der maschinellen DNA-Extraktion zu jeder Probe hinzugegeben. An bereits positiv getesteten Patientenproben konnte gezeigt werden, dass Konzentrationen von 10-20 Kopien Baculovirus pro Ansatz sicher nachweisbar sind und gleichzeitig auch geringe DNA-Mengen an humanpathogener Viren sicher nachgewiesen werden.

Banana bunchy top virus (BBTV) is a circular, single-stranded (css) DNA virus that belongs to the genus Babuvirus in the family Nanoviridae. BBTV is responsible for the most devastating virus disease of banana known as "bunchy top", for which conventional control measures are generally ineffective. Genetically engineered resistance appears to be the most promising strategy to generate BBTV-resistant bananas but the success of this strategy is largely dependent upon the molecular characterisation of the target virus and knowledge of the virus life cycle, particularly the replication strategy. This PhD study was aimed at the molecular characterisation of the intergenic regions of BBTV, in order to complement the molecular information currently available and to potentially contribute to the development of transgenic resistance strategies against BBTV in banana. Three putative iterative sequences (iterons; GGGAC) previously identified in the BBTV intergenic regions were initially characterised. In order to determine their role in the binding of the master BBTV replication initiation protein (M-Rep), the putative iterons (F1 and F2 in the virion sense, and R in the complementary sense) were independently mutated in a BBTV DNA-6 greater-than-genome-length clone (1.1 mer). The DNA-6 1.1 mers (native and mutants) and the M-Rep-encoding component (DNA-1) were co-bombarded into banana (Musa spp. cv."Lady finger") embryogenic suspension cells and transient replication was evaluated by Southern hybridisation. Analysis of the DNA-6 replicative forms showed a significant decrease of approximately 41% for the F1 iteron mutant and 61% for the R iteron mutant in comparison with native levels. However, the mutation in the F2 iteron caused the most dramatic effect, decreasing replication to levels barely detectable by Southern hybridisation. These results suggest that the three iterons all play a role in BBTV replication, most likely as recognition and binding sites for the M-Rep, but that the F2 iteron appears to be the most important in replication. Following the observation that all BBTV isolates sequenced to date have identical iteron sequences, the extent to which the M-Rep would recognise, bind and initiate replication of heterologous components from geographically diverse BBTV isolates (the South Pacific and the Asian groups) was evaluated. Cross replication assays revealed that heterologous M-Reps from Fiji, Hawaii (South Pacific group) and Vietnam (Asian group) were able to initiate replication of the coat protein-encoding component (DNA-3) from the Australian BBTV isolate (South Pacific group). However, replication of DNA-3 from the Vietnamese isolate was not initiated by heterologous M-Reps from the two South Pacific isolates tested (Australia and Hawaii). These results suggest that a broad-range transgenic resistance strategy based on replication using Australian BBTV intergenic regions may be successful as this region will be recognised by the M-Reps from both Asian and South Pacific BBTV isolates. However, a Rep protein-mediated resistance strategy will more likely be specific to geographical isolates and, therefore, less suitable as a broad-range control strategy. To further characterise the BBTV intergenic regions and to gain a better understanding of the BBTV transcription process, the 5' untranslated regions (UTRs) of the major open reading frames (ORFs) associated with each of the six BBTV DNA components were mapped. In all cases, the transcription start sites were located 3' of a putative TATA box and the 5' UTRs varied in length from 23 nucleotides (DNA-6) to 5 nucleotides (DNA-3). Two potential transcription start sites (nt 84 and 87) were mapped for DNA-1, but whether these represent the transcription start sites of the two genes associated with DNA-1 remains to be determined. Two start sites were also associated with DNA-2 which is thought to be monocistronic. Whether one of these start sites is an artefact or whether they are due to natural sequence variability of BBTV is unknown. These results now enable us to define the transcribed regions of each BBTV DNA component and accurately predict their promoter regions in an attempt to gain a fundamental understanding of BBTV gene expression patterns.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Mastreviruses (family Geminiviridae) are plant-infecting viruses with circular single-stranded (ss) DNA genomes (~2.7kb). The genus Mastrevirus is comprised of thirty-two species which are transmitted by leafhoppers belonging to the genus Cicadulina. Mastreviruses are widely distributed and have been found in the Middle East, Europe, Asia, Australia, Africa and surrounding islands. Only one species, dragonfly-associated mastrevirus has so far been identified in the Americas, isolated from a dragonfly in Puerto Rico. Species can be group based on the host(s) they infect, those which infect monocotyledonous (monocot) plants and those which infect dicotyledonous (dicot) plants. In recent years many new mastrevirus species have been discovered. Several of these new discoveries can largely been attributed to the development of new molecular tools. The current state of sequencing platforms has made it affordable and easier to characterise mastreviruses at a genome level thus allowing scientists to delve deeper into understanding the dynamics of mastreviruses. A few mastrevirus species have been identified as important agricultural pathogens and as a result have been the focus of much of the mastrevirus research. Maize streak virus, strain A (MSV-A) has been the most extensively studied due to the devastating impact it has on maize production in Africa. Studies have shown that MSV-A likely emerged as a pathogen of maize less than 250 years following introduction of maize in Africa by early European settlers. There is compelling evidence to suggest that MSV-A is likely the result of recombination events between wild grass adapted MSV strains. It therefore is equally important to monitor viruses infecting non-cultivated plants in order to gain a greater understanding of the epidemiological dynamics of mastreviruses, which in turn is essential for implementing disease management strategies. The objective of the research undertaken as part of this PhD thesis was to investigate global mastrevirus dynamics focusing on diversity, host and geographic ranges, mechanisms of evolution, phylogeography and possible origins of these viruses. In addition to this a viral metagenomic approach was used in order to identify novel mastreviruses or mastrevirus-like present in New Zealand. The dynamics of the monocot-infecting mastreviruses are investigated in Chapter Two and Three. The work described in these two chapters focus mainly on mastreviruses which infect non-cultivated grasses in Africa and Australia, a total of 161 full mastrevirus genomes were recovered collectively in the two studies. Chapter Two reveals a high level of mastrevirus diversity present in Australia with the discovery of four new species and several new strains of previously characterised species. An extensive sampling effort in Africa undertaken in Chapter Three reveals a broader host range and geographic distribution of the African monocot-infecting mastreviruses than previously documented. Mosaic patterns of recombination are evident among both the Australian and African monocot-infecting mastreviruses. In Chapters Four, Five and Six a comprehensive investigation was undertaken focusing on the dicot-infecting mastreviruses. The study undertaken in Chapter Four entailed the recovery of 49 full mastrevirus genomes from Australia, the Middle East, Africa, Turkey and the Indian Subcontinent to investigate the diversity of dicot-infecting mastreviruses from a global context. Analyses revealed a high degree of CpCDV strain diversity and extended the known geographic range of CpCDV. For the first time phylogeographic analysis was able to investigate the origins of the dicot-infecting mastreviruses. Results revealed the likely origin of the most recent common ancestor (MRCA) of these viruses is likely closer to Australia than anywhere else that dicot-infecting mastreviruses have been sampled and illuminated a supported series of historical movements following the emergence of the MRCA. In Chapter Five two novel mastreviruses Australian-like mastreviruses were isolated from chickpea material from Pakistan. A comprehensive analysis of CpCDV isolates in the major pulse growing regions of Sudan in Chapter Six reveals that this region harbours a high degree of strain diversity. Complex patterns of intra-species recombination indicate these strains are evidently circulating in these regions and infecting the same hosts, driving the emergence of new CpCDV strains. Collectively the results discussed in Chapters Two through Six extended the current knowledge of mastrevirus diversity. The natural host range of many mastreviruses has proven to be more extensive than previously documented, with many species having overlapping host ranges and hence these hosts could be acting as ‘mixing vessels’ enabling inter-species recombination. Patterns of recombination and selection were observed in both the monocot-infecting and the dicot-infecting mastreviruses further elucidating the mechanisms these viruses employ to evolve rapidly. Extensive sampling in a wide range of geographic regions provides insights into the true geographic range of species such as MSV and CpCDV. Given that mastreviruses have been able to move globally and Australia has been identified as a major mastrevirus diversity hotspot it is conceivable that mastreviruses are also present in New Zealand. In Chapter Seven and Eight this is explored by using a viral metagenomic approach to investigate the ssDNA viral populations associated with wild grasses and sewage material in New Zealand. Although no mastreviruses were recovered, this endeavour resulted in the discovery of more than 50 novel circular Rep-encoding ssDNA (CRESS DNA) viruses associated with non-cultivated grasses and treated sewage material, many of which are similar to mastreviruses and other geminiviruses. These discoveries expand current knowledge on the diversity of ssDNA viruses present in New Zealand and further highlight this viral metagenomic approach as an effective method for ssDNA virus discovery. Overall the results discussed in this thesis provide insights into mastrevirus diversity and dynamics as well as revealing a wealth of novel CRESS DNA viruses, some of which share similarities to geminiviruses.

No description available.

Epstein-Barr virus (EBV) BKRF4 gene product is a tegument protein encoded by a gene with no sequence homology outside of the gamma subfamily of Herpesviridae. Its positional homologs are necessary for an efficient viral lytic program, in particular viral progeny egress and primary infection. To characterize BKRF4 in this regard, EBV recombinant viruses deficient for BKRF4 were developed using site-directed mutagenesis and a bacterial artificial chromosome (BAC)-based recombineering system. Stable human embryonic kidney (HEK) 293 cell lines containing these genomes were generated and the phenotypes of these mutants were analyzed following stimulation of the viral lytic cycle. During the lytic program, BKRF4-null cell lines showed decreased protein expression of various EBV lytic genes that were analyzed using immunostaining and flow cytometry. Reduced amounts of extracellular viral progeny were observed when quantified by real-time PCR and infectivity assays as compared to wild type. These findings suggest an active role of BKRF4 in EBV infection, possibly in viral egress.

Ziel der Arbeit war die Entwicklung und Implementierung einer internen Extraktions-Kontrolle für die quantitative Echtzeit-PCR humanpathogener DNA-haltiger Viren der Routinediagnostik des Instituts für Virologie der Universität Leipzig, basierend auf einem artfremden Vollvirus. Durch Verwendung einer internen Kontrolle sollen falsch-negative PCR-Ergebnisse aufgrund unzureichender DNA-Extraktion aus humanem Probenmaterial identifiziert werden. Die interne Kontrolle soll zur Zeitersparnis als Multiplex-PCR durchgeführt werden. Verwendet wurde das Baculoviurs Autographa californica multicapsid nucleopolyhedrovirus, da keine Baculovirus-Infektionen beim Menschen vorkommen. Es wurden Primer und Sonden so entwickelt, dass sowohl das Amplifikat als auch Primer und Sonden eine hohe Übereinstimmung zu den Amplifikaten, Primern und Sonden der humanpathogenen Viren zeigen. Dadurch soll die PCR-Effizienz der internen Kontrolle möglichst ähnlich zu der PCR-Effizienz der humanpathogenen Viren sein. In Vorversuchen wurde gezeigt, dass eine Hybridisierung der Baculovirus-Primern und -Sonden an das Genom humanpathogener Viren und das humane Genom minimal war. Des Weiteren wurde nachgewiesen, dass sich die Baculovirus-DNA als interne Kontrolle vemischt mit Lysispuffer über mindestens eine Woche bei 4°C ohne signifikante Verluste lagern lässt. Im vorgestellten und validierten Protokoll wird die interne Kontrolle automatisch während der maschinellen DNA-Extraktion zu jeder Probe hinzugegeben. An bereits positiv getesteten Patientenproben konnte gezeigt werden, dass Konzentrationen von 10-20 Kopien Baculovirus pro Ansatz sicher nachweisbar sind und gleichzeitig auch geringe DNA-Mengen an humanpathogener Viren sicher nachgewiesen werden.

Les virus géants sont un groupe de virus ADN double brin caractérisés par une taille géante du virion et du génome, et un répertoire de gènes qui comprend environ 450 à 2500 gènes prédits. Une proportion importante de ces gènes (jusqu'à 93%) sont des 'ORFans', ou codent pour des protéines de fonction inconnue. Acanthamoeba polyphaga mimivirus est le premier virus géant découvert, il y a une décennie, par co-culture sur Acanthamoeba spp. Il est le membre prototype de la famille Mimiviridae. Le génome de Mimivirus code pour environ 1000 protéines, parmi lesquelles ~50% n'ont pas d'homologue connu dans les banques de séquences publiques. La capside de Mimivirus a un diamètre d'environ 500 nm et est couverte par une couche dense de fibres, à l'exception de l'un de ses sommets. Ces fibres sont d'environ 130 nm de longueur et se composent d'une tige souple et d'une tête de forme globulaire.Dans ce travail de thèse, nous avons cherché à étudier les gènes impliqués dans la formation des fibres de Mimivirus. Dans ce but, nous avons notamment exprimé des gènes candidats dans E. coli, et nous avons mis au point une stratégie qui a utilisé l'interférence ARN afin d'étudier la fonction et la structure des protéines de Mimivirus. Nous avons annoté quatre protéines associées aux fibres. La stratégie utilisant les petits ARN interférant appliquée ici est originale et a été utilisée pour la première fois pour les virus géants qui infectent les amibes. Elle pourrait permettre de décrypter la fonction des gènes des mimivirus et d'annoter potentiellement des centaines de protéines présentes dans les bases de données publiques, et de différencier l'ADN poubelle des gènes réellement utilisés. / Giant viruses are a group of double stranded DNA viruses that are characterized by a giant virion and genome size, and gene repertoires encompassing approximately 450 to 2500 predicted genes. A substantial proportion of these genes (up to 93%) consists in ORFans, or encodes proteins with unknown functions. Acanthamoeba polyphaga mimivirus is the first giant virus that was discovered, a decade ago, after co-culturing on Acanthamoeba spp. It is the prototype member of the family Mimiviridae. Mimivirus encodes about 1000 proteins, among which ~50% have no known homolog in public sequence databases. The Mimivirus capsid is about 500 nm in diameter and is covered by a dense layer of fibers, except at one of its vertices. These fibers are about 130 nm in length and consist of a soft shaft and a globular shaped head.In this thesis work, we aimed to study the genes involved in the formation of the Mimivirus fibers. For this purpose, we have expressed candidate genes in E. coli, and implemented a strategy that used RNA interference to study the function and structure of Mimivirus proteins. We then succeeded in annotating four proteins as fiber associated proteins. The short interfering RNA strategy that we applied here is original and has been used for the first time in giant viruses that infect amoeba. It could allow deciphering the function of the mimivirus gene repertoires and help annotating hundreds of proteins without known function found in public databases and differentiate between junk DNA and truly used genes.

No description available.