Browsing by Author "Abrahams, Melissa-Rose Hilda"

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    An investigation into the specific function of the vaccinia virus :13.8 kDa protein encoded by the N1L gene
    (2005) Abrahams, Melissa-Rose Hilda; Kotwal, Girish J
    Vaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies.
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    Open Access
    An investigation into the specific function of the vaccinia virus 13.8 kDa protein encoded by the N1
    (2005) Abrahams, Melissa-Rose Hilda; Kotwal, Girish J
    Vaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies.
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    The transmitted HIV-1 subtype C: characterization of the transmitted/founder full-length virus genome and the influence of early immune selective pressure on virus replication
    (2014) Abrahams, Melissa-Rose Hilda; Williamson, Carolyn
    The identification of targets of early immune responses associated with control of HIV-1 infection will inform immunogen design for vaccine interventions. The early evolution of transmitted/founder subtype C virus sequences was investigated to determine the location and frequency of immune selection, and the impact of early immune escape mutations on viral replicative capacity. Single-genome amplified env sequences from 26 acutely-infected women were evaluated for conformance to a model of random evolution to elucidate multiplicity of infection. Near fulllength genome sequences from the first six months of infection were generated for five women and sites evolving under immune selection were mapped. CD8+ cytotoxic Tlymphocyte escape mutations in HLA-B-restricted epitopes were introduced into infectious molecular clones of cognate transmitted/founder viruses by site-directed mutagenesis and their impact on viral replicative fitness was evaluated using parallel replication assays. In 77% of women (n=20) a single transmitted/founder variant established infection and two to five variants in the remaining 23% (n=6). Near full-length genome sequencing in five women confirmed single variant/low-diversity transmission and identified fifty-five genome regions evolving under immune selection, 40% of which was attributed to CD8+ cytotoxic Tlymphocyte pressure, 35% to antibody-mediated pressure, 16% to reversion and 9% could not be classified. The rate of sequence diversification and number of sites evolving under immune selection was highest in nef. The majority of evolving CD8+ cytotoxic T-lymphocyte epitopes (82%) contained shuffling/toggling mutations. A novel B*15:10-associated mutation, A164T, combined with a V85A Pol mutation reduced viral replication capacity in one individual. In a second individual, the attenuating HLA-B*58:01-associated mutation, T242N, enhanced viral replication capacity due to pre-existing compensatory polymorphisms in the transmitted/founder virus. A third individual, who had extremely rapid disease progression, was infected with the virus with the highest replication capacity. This thesis describes the complex nature of early immune selection and escape in transmitted/founder viruses. Although attenuating escape mutations were identified in viruses from two individuals, this was not associated with clinical benefit. The extensive variability of epitopes evolving under early selection may implicate many early immune targets as poor candidates for vaccine immunogens; however some early targets may be useful if clinical benefit is conferred through attenuating escape mutations.