Thesis (Ph.D. in Biochemistry) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 191-200). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--Duke University, 2008. / Includes bibliographical references.

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease, which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1) of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several techniques were attempted to correct this mutation, but none were successful. Even though the second mutation could not be corrected, in vitro transcriptions were performed on three clones followed by subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA- mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and provided preliminary evidence that the mutated clones were still capable of systemic infection and viral movement. These results are still inconclusive, and several post-infection studies will have to be performed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will be investigated further and post-infection analysis performed to deduce whether the viral progeny are devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable of systemic infection in N. benthamiana plants were constructed in this study and have laid the foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.

Made available in DSpace on 2014-08-27T14:36:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-08-23Bitstream added on 2014-08-27T15:57:19Z : No. of bitstreams: 1 000723028_20150823.pdf: 1028906 bytes, checksum: 197bceaec267086e9658eb5a35574fe1 (MD5) Bitstreams deleted on 2015-08-24T10:46:34Z: 000723028_20150823.pdf,. Added 1 bitstream(s) on 2015-08-24T10:47:09Z : No. of bitstreams: 1 000723028.pdf: 1485800 bytes, checksum: aaac9adf54c11e7df2321502f879e475 (MD5) / A hepatite C é consequência da infecção pelo vírus da hepatite C (HCV) e estima-se que cerca de 150 milhões de pessoas em todo o mundo estejam cronicamente infectadas. Estudos têm demonstrado interações entre proteínas virais e do hospedeiro durante o ciclo de replicação do HCV e estas interações podem ser utilizadas para o desenvolvimento de novas terapias contra a hepatite C. As proteínas de choque térmico (Hsp) são proteínas celulares que interagem com proteínas do HCV e a inibição destas proteínas poderiam reduzir a replicação viral. Neste estudo, as proteínas celulares Hsp90 e Hsp27 foram inibidas por siRNA isoladamente ou em combinação com a inibição das regiões virais 5'UTR, NS3 e NS5A. Foi utilizada uma cultura celular estável Huh7 expressando um replicon subgenomico do HCV e todas as moléculas de siRNA dirigidas ao genoma do vírus mostraram eficiência na redução da replicação viral. A melhor resposta foi obtida pelo siRNA 5'UTR, que apresentou bons resultados também nos estudos à longo prazo. A inibição da proteína celular Hsp27 aumentou os níveis de replicação do vírus, entretanto a supressão de Hsp90 mostrou redução da replicação viral e a utilização desta molécula juntamente com a molécula de siRNA dirigida para 5'UTR mostraram uma diminuição de mais de 90% da replicação viral. Entretanto a inibição prolongada de Hsp90 levou à morte celular, evidenciando o importante papel desta proteína na sobrevivência das células. Portanto o presente trabalho sugere que a terapia combinada de siRNAs pode ser uma alternativa eficiente no combate à hepatite C em pacientes com HCC uma vez que a inibição de Hsp90 atua tanto na supressão tumoral quanto na replicação do HCV / Hepatitis C is a consequence of infection by hepatitis C virus (HCV) and it is estimated that approximately 150 million people are chronically infected worldwide. Several studies have demonstrated interactions between viral and host proteins during the HCV replication cycle and these interactions might be used for development of new therapies against hepatitis C. The heat shock proteins (Hsp) are cellular proteins that interact with proteins of HCV and inhibition of these proteins could reduce viral replication. In this study, cellular proteins Hsp90 and Hsp27 were inhibited by siRNA targeting the mRNA of these proteins in combination with siRNA to viral 5'UTR region, NS3 and NS5A. We used a stable cell line expressing HCV subgenomic replicon and all siRNA molecules targeting the viral genome showed effectiveness in reducing viral replication. The best response was achieved by siRNA 5'UTR, which showed good results on long term studies. The inhibition of cellular protein Hsp27 increased virus replication, but knockdown of Hsp90 showed reduced viral replication. Use of this molecule together with the siRNA molecule directed to 5'UTR showed a decrease of more than 90% viral replication. However, the prolonged inhibition of Hsp90 led to cell death, demonstrating the important role of this protein in cell survival. Finally this work suggests that the combination therapy of siRNAs can be an effective alternative to treat hepatitis C in patients with HCC since reduction of Hsp90 expression if effective on tumor suppression and in HCV replication

RNA viruses have the fastest evolutionary rates amongst protein-coding organisms on the planet. Ease of sequencing, advanced techniques of analysis and global health and economic concerns have all contributed to the recognition of RNA viruses as a robust research platform. Phylogenetic methods have been at the forefront of analytical techniques used to understand the dynamics of RNA viruses - during natural circulation in populations and in individual hosts, within epidemics, across species barriers and over billions of years that viruses have been around. Most of the work presented in this thesis employs phylogenetic incongruity arising from reassortment and recombination to gain insights into the genomes and populations of RNA viruses. Chapter 2 explores the selection regimes Ebola virus has experienced following a year of circulation in humans inWest Africa, as well as its recent history. Chapter 3 investigates the extent of recombination in MERS-CoV, a novel human pathogen with an obscure epidemiology, which is suggestive of frequent co-infection of some hosts. Chapter 4, on the other hand, documents a pattern of non-intuitive linkage between some segments of the human-endemic influenza B virus genome and explores its potential to speciate. Chapter 5 builds upon chapter 4 and attempts to describe small-scale reassortment between two segments of influenza B virus and the overall migration patterns of influenza B virus in Scotland. Chapter 6 exploits the independence of segments of influenza D virus, a recently described cattle pathogen, and coalescent theory to disentangle the origins of this virus. This thesis exemplifies the success of modern sequencing methods, which, together with the use of sophisticated analytical techniques, have uncovered a wealth of information hidden away in molecular sequences of RNA viruses. The work presented herein demonstrates how reticulate evolution can be exploited as a reliable, and sometimes indispensable, marker to improve inference of evolutionary forces in RNA viruses.

Orientador: Paula Rahal / Coorientador: Bruno Moreira Carneiro / Banca: Maurício Lacerda Nogueira / Banca: Isabel Maria Vicente Guedes de Carvalho Mello / Resumo: A hepatite C é consequência da infecção pelo vírus da hepatite C (HCV) e estima-se que cerca de 150 milhões de pessoas em todo o mundo estejam cronicamente infectadas. Estudos têm demonstrado interações entre proteínas virais e do hospedeiro durante o ciclo de replicação do HCV e estas interações podem ser utilizadas para o desenvolvimento de novas terapias contra a hepatite C. As proteínas de choque térmico (Hsp) são proteínas celulares que interagem com proteínas do HCV e a inibição destas proteínas poderiam reduzir a replicação viral. Neste estudo, as proteínas celulares Hsp90 e Hsp27 foram inibidas por siRNA isoladamente ou em combinação com a inibição das regiões virais 5'UTR, NS3 e NS5A. Foi utilizada uma cultura celular estável Huh7 expressando um replicon subgenomico do HCV e todas as moléculas de siRNA dirigidas ao genoma do vírus mostraram eficiência na redução da replicação viral. A melhor resposta foi obtida pelo siRNA 5'UTR, que apresentou bons resultados também nos estudos à longo prazo. A inibição da proteína celular Hsp27 aumentou os níveis de replicação do vírus, entretanto a supressão de Hsp90 mostrou redução da replicação viral e a utilização desta molécula juntamente com a molécula de siRNA dirigida para 5'UTR mostraram uma diminuição de mais de 90% da replicação viral. Entretanto a inibição prolongada de Hsp90 levou à morte celular, evidenciando o importante papel desta proteína na sobrevivência das células. Portanto o presente trabalho sugere que a terapia combinada de siRNAs pode ser uma alternativa eficiente no combate à hepatite C em pacientes com HCC uma vez que a inibição de Hsp90 atua tanto na supressão tumoral quanto na replicação do HCV / Abstract: Hepatitis C is a consequence of infection by hepatitis C virus (HCV) and it is estimated that approximately 150 million people are chronically infected worldwide. Several studies have demonstrated interactions between viral and host proteins during the HCV replication cycle and these interactions might be used for development of new therapies against hepatitis C. The heat shock proteins (Hsp) are cellular proteins that interact with proteins of HCV and inhibition of these proteins could reduce viral replication. In this study, cellular proteins Hsp90 and Hsp27 were inhibited by siRNA targeting the mRNA of these proteins in combination with siRNA to viral 5'UTR region, NS3 and NS5A. We used a stable cell line expressing HCV subgenomic replicon and all siRNA molecules targeting the viral genome showed effectiveness in reducing viral replication. The best response was achieved by siRNA 5'UTR, which showed good results on long term studies. The inhibition of cellular protein Hsp27 increased virus replication, but knockdown of Hsp90 showed reduced viral replication. Use of this molecule together with the siRNA molecule directed to 5'UTR showed a decrease of more than 90% viral replication. However, the prolonged inhibition of Hsp90 led to cell death, demonstrating the important role of this protein in cell survival. Finally this work suggests that the combination therapy of siRNAs can be an effective alternative to treat hepatitis C in patients with HCC since reduction of Hsp90 expression if effective on tumor suppression and in HCV replication / Mestre

Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.

The Tetraviridae are a family of ss (+) RNA viruses that specifically infect lepidopteran insects. Their icosahedral capsids are non-enveloped and approximately 40 nm in diameter with T=4 quasi-equivalent symmetry. The omegatetraviruses, which are structurally the best characterised in the family, include Helicoverpa armigera stunt virus (HaSV) and Nudaurelia capensis omega virus (NwV). The omegatetravirus procapsid is composed of 240 identical copies of the capsid precursor proteins, which undergo autoproteolytic cleavage at its carboxyl-terminus generating the mature capsid protein (b) and γ-peptide. This process occurs in vitro following a shift from pH 7.6 to pH 6.0. The viral capsid encapsidates two ss genomic RNAs: The larger RNA1 encodes the viral replicase as well as three small ORFs while RNA2 encodes the capsid precursor protein together with an overlapping ORF designated P17. While a wealth of structural data pertaining to the assembly and maturation of omegatetraviruses is available, little is known about how this relates to their lifecycle. The principle aim of the research described in this thesis was to use an experimental system developed in the yeast, Saccharomyces cerevisiae, to investigate the assembly of HaSV and NwV virus-like particles (VLPs) in terms of maturation and encapsidation of viral RNAs, in vivo. The yeast expression system used two promoter systems for expression of capsid precursor protein: in the first, a hybrid promoter (PGADH) was used for high-level expression, while the second, PGAL1, produced substantially lower levels of the virus capsid protein precursors. An increase in the level of HaSV capsid protein precursor (p71) via the PGADH promoter resulted in a dramatic increase in VLP assembly as compared with the PGAL system. A protein equivalent to the mature capsid protein (p64) appeared at later time intervals following induction of transcription. Transmission electron microscopic studies showed that p64 correlated with the presence of mature VLPs as opposed to procapsids in cells containing p71. This confirmed that the presence of p64 denoted maturation of VLPs in vivo. Further investigation indicated that maturation correlated with cell aging and the onset of apoptosis. It was shown that induction of apoptosis resulted in VLP maturation while inhibition of apoptosis prevented maturation. These results suggested that the process of apoptosis might be the trigger for maturation of virus procapsids in their host cells. The increase in the efficiency of VLP assembly observed in the high-level expression system was proposed to be due to an increase in the cellular concentrations of viral RNA. To test this hypothesis, HaSV P71 was co-expressed with either P71 mRNA or full length RNA2. An increase in the solubility of p71 was observed in cells expressing increased levels of both RNAs, but there was no increase in the efficiency of VLP assembly. Northern analysis of encapsidated RNAs revealed that there was no selective encapsidation of either P71 mRNA or viral RNA2. This data indicated that the increase in viral RNA was not the reason for increased efficiency of VLP assembly, but most likely resulted from higher concentrations of p71 itself. It was decided to determine whether a highly efficient nodavirus replication system developed in yeast for heterologous production of proteins, could be used as a method for expressing the capsid protein precursor. The aim of using this system was to determine if VLPs assembled in a replication system specifically encapsidated viral RNA. Transcripts encoding the NwV capsid protein precursor (p70) were generated in yeast cells by replication of a hybrid RNA template by the Nodamura virus (NoV) replicase. Western analysis confirmed the presence of p70 as well as a protein of 62 kDa corresponding to the mature NwV capsid protein. Northern analysis of purified VLPs showed that NoV RNA1 and RNA3 were encapsidated, but no RNA2 was detected. Taken together, the data lead to the conclusion that specific encapsidation of tetraviral RNAs required more than close proximity of the viral RNAs and assembling virus-like particles. Encapsidation specificity in the omegatetraviruses may require additional viral proteins such as p17 during encapsidation or specific viral RNA encapsidation was replication-dependent. Replication-dependent assembly has been shown in the nodaviruses.

No description available.

The odorous house ant, Tapinoma sessile, is a widespread, common pest in North American residential areas, yet the understanding of how viral pathogens shape its behavior, management, and interactions with other insects remains limited. Over the course of my thesis, I characterized four novel viruses in T. sessile using metatranscriptomic analysis, investigated their infectivity, and explored how colony social organization influences viral infection patterns. Inoculation experiments confirmed active infection in T. sessile colonies, suggesting trophallaxis as a route for horizontal transmission. These viruses were prevalent in urban populations with polygyne, unicolonial traits, indicating a potential association between colony social structure, colony location, and virus presence and diversity. Additionally, common honey bee viruses including Deformed Wing Virus and Kashmir Bee Virus were detected in T. sessile colonies, with higher prevalence near beehives, indicating a route for virus spillover between the species. Furthermore, the impact of viral infection on foraging behavior was assessed, revealing altered activity and diet preferences in infected colonies and highlighting the need for modified control strategies. Finally, I explored virus transmission between T. sessile and associated arthropods and found shared viruses and active replication in arthropods within ant nests, indicating a novel case of virus spillover between ants and myrmecophiles. These findings offer insights into viral pathogen interactions within ant colonies, the influence of social organization on infection dynamics, and the potential for pathogen transmission between ants and associated arthropods and offer a deeper understanding of an important native ant species that may shape future pest management strategies. / Master of Science in Life Sciences / In my thesis research I studied the odorous house ant or sugar ant, a common pest in homes and businesses in North America, to understand how viral pathogens shape its behavior, management, and interactions with other insects. I discovered four new odorous house ant viruses infecting these ants using molecular analysis techniques and found that they can pass from ant to ant when they share food. Interestingly, these viruses were more common in urban ant colonies with multiple queens, suggesting a link between the ants' social structure and the viruses they carry. Additionally, I found viruses typically seen in honey bees in these ant colonies, especially when those colonies were close to beehives, indicating that the odorous house ant can pick up viruses from honey bees. Furthermore, I studied how these viruses affect ant behavior and found that infected ant colonies change their activity and food preferences. Lastly, I investigated if these viruses are able to jump from ants to other insects and found evidence that insects living in their nests shared the same or similar virus infections. These findings provide important insights into how viruses spread among ant colonies and nearby insects, how their social structure affects transmission of viruses, and how viruses can move between different species of insects. Understanding these interactions is crucial for developing better ways to control ant pests and for gaining a deeper understanding of this important native ant species.