No description available.

Subtype C accounts for the majority of HIV infections and in South Africa, is the dominant subtype. The Gag cleavage sites of subtype C viruses show a high degree of natural variation compared to subtype B and group M sequences, with the p2/NC site having the highest degree of variation among all cleavage sites and between all subtypes. This study therefore aimed to determine the functional effect of this variation on viral fitness. A library of drug naïve subtype C sequences were screened using computational analysis to predict binding affinity between HIV protease and the Gag substrate at the p2/NC site. Ligands with high predicted affinity had hydrophobic cleavage sites with substantial diversity at positions P5-P3. Lower ranking ligands were mostly similar to the consensus subtype C. Three ligands were selected for fitness assays from each the high ranking and low ranking groups. Chimeric viruses expressing selected cleavage sites were generated by site directed mutagenesis. Replication capacity assays of these viruses showed moderate differences in fitness but failed to demonstrate a correlation with computational estimates of binding affinity. Enzymes assays were performed to further investigate substrate preferences and the binding mechanism of protease. To this end, recombinantly expressed HIV-1 protease was tested against a range of substrates the matching the p2/NC cleavage sites used in the replication capacity assay. Results of the enzyme assay did not correlate with either the computation studies or the replication capacity assay results, suggesting a sequence independent binding and recognition mechanism of HIV-1 protease. Taken together the results suggest that processing of Gag is determined by tertiary folding of the polyprotein and not amino acid sequence at the cleavage site. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2012.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 212-238). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 212-238). Also available online.

No description available.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Introdução: As arboviroses causam graves problemas de saúde pública no Brasil e em muitos dos países da América Latina. A epidemiologia molecular é um instrumento valioso na compreensão da dispersão, persistência e diversidade desses patógenos virais. Objetivos: Neste projeto, buscamos investigar a dinâmica epidemiológica molecular dos arbovíroses (com especial enfoque aos vírus da dengue-DENV, chikungunya - CHIKV e zika -ZIKV) que circularam no estado do Amapá entre os anos de 2013 e 2016. Métodos: 824 amostras de plasma humano foram coletadas pelos laboratórios de Saúde Publica (LACEN) no estado do Amapá entre os anos de 2013 e 2016; essas amostras foram obtidas de pacientes que apresentavam sintomas consistentes com uma das arboviroses. O material genético viral presente nestas amostras foi extraído e os ensaios de qPCR foram realizados. Todas as amostras foram submetidas inicialmente a um ensaio triplex (ZIKV/DENV/CHIKV), as amostras negativas foram posteriormente submetidas a um ensaio de pan-flavivírus. As amostras positivas para um dos ensaios foram submetidas a NGS (sequenciamento de nova geração). Resultados: Das 824 amostras testadas, 36 foram positivas para DENV, ZIKV ou CHIKV; desses 36 positivos, 24 foram para DENV, 11 para CHIKV e 1 para ZIKV. Foram obtidos 27 genomas completos: 16 de DENV (15 DENV1, genótipo V e 1 DENV2, genótipo III) e 11 de CHIKV (genótipo asiático / caribenho). Das 788 amostras testadas com o ensaio de pan-flavivírus, 22 amostras foram positivas; porem apenas uma amostra produziu genoma completo pela técnica de NGS. Este genoma foi relacionado com um flavivírus com semelhante em 76,81% com o vírus Long Pine Key - LPKV, que anteriormente só tinha sido descrito em mosquitos. Árvores de Maximum likelihood e Maximum clade credibility foram construídas utilizando os genomas do DENV1 obtidos neste estudo. Essas árvores exibiam duas linhagens distintas de DENV1, genótipo V presentes na América Latina. Uma destas linhagens tem um padrão de circulação que inclui países do Caribe, América Central e América do Sul (incluindo Brasil); a outra linhagem distinta circula dentro das fronteiras do Brasil. As árvores também indicam que o DENV1 presente no estado do Amapá é da linhagem que tem o padrão de circulação que inclui o Caribe e as Américas Central e do Sul e que essa linhagem surgiu no Amapá entre 2005 e 2010. Conclusão: Este estudo fornece dados importantes sobre as arboviroses no Amapá e os dados genômicos mais recentes disponíveis para a região, bem como o contexto brasileiro e latino-americano para esses dados. Dados dessa natureza são inestimáveis nos esforços das autoridades de saúde pública para a prevenção e controle de epidemias por estes agentes. / Introduction: Arboviral febrile illnesses plague the nation of Brazil and many of its surrounding Latin America countries. Molecular epidemiology is a growing and increasingly invaluable tool in the field of public health for understanding the dispersal, persistence, and diversity of these impactful viral pathogens. Objectives: In this project, the identities and molecular epidemiological dynamics of arboviruses circulating in the Brazilian state of Amapá between the years 2013 and 2016, with special focus on DENV, CHIKV and ZIKV, were investigated and given Brazilian and Latin American geographical and temporal context via molecular epidemiological analyses. Methods: 824 human blood plasma samples were collected from LACEN laboratories in the state of Amapá between the years 2013 and 2016; these samples originated from patients showing symptoms consistent with any of the common arboviral febrile illnesses. The viral genetic material present in these samples was extracted and qPCR diagnostics assays were performed; all samples first underwent a triplex assay (ZIKV/DENV/CHIKV - ZDC), then the samples yielding negative results for the triplex assay underwent a pan-flavivirus assay. The samples yielding positive results for either assay were submitted for NGS and all whole viral genomes subsequently obtained underwent phylogenetic molecular epidemiological analyses. Results: Of the 824 samples tested, 36 tested positive for the ZDC assay; of those positives, 24 tested positive for DENV, 11 for CHIKV, and 1 for ZIKV. 27 full genomes were obtained from these ZDC positives: 16 of DENV (15 DENV1, genotype V and 1 DENV2, genotype III) and 11 of CHIKV (Asian and Caribbean genotype). Of the 788 samples tested with the pan-flavivirus assay, 22 samples yielded positive results, from only one of which a genome was obtainable. This genome was found to be closely related to a flavivirus previously only found in mosquitoes (76.8% identity with Long Pine Key Virus - LPKV). Maximum likelihood and maximum clade credibility trees were constructed using the DENV1 genomes obtained from this study. These trees displayed two distinct lineages of DENV1, genotype V present in Latin America, one of which has a circulation pattern spanning widely across the Caribbean and Central and South America (including Brazil), while the other circulates within Brazilian borders. The trees also indicate that the DENV1 present in the state of Amapá is of the lineage having the wider circulation pattern and that this lineage emerged in Amapá between 2005 and 2010. Conclusion: This study provides important data concerning the range of the arboviral landscape in Amapá and the most recent genomic data available for the region as well as Brazilian and Latin American context to that data. Data of this nature are invaluable in the efforts of public health officials for the prevention and control of epidemics of these impactful arboviral pathogens.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references. Also available in print.

African horsesickness virus (AHSV), a member of the Orbivirus genus in the family Reoviridae, is the aetological agent of an economically important disease affecting equids. One of the outer coat proteins of the virus, VP5, has the important function of interacting with the conserved core proteins as well as adapting to facilitate changes in the major outer coat protein, VP2. VP5 plays a role in destabilizing the endosomal membrane following viral attachment to the host cell, thereby allowing access of the viral core into the host cytoplasm. Furthermore, VP5 contributes to the immune response in infected animals and has relevance to recombinant vaccine development. The main aim of this study was to compare the genetic variation between VP5 proteins of field isolates of AHSV, isolated from dead animals, to that of laboratory strains, likely to be avirulent. This comparison was done in order to determine if any significant/common amino acid changes could be linked to the virulence phenotype. The VP5 genes of six AHSV isolates of serotypes 3, 6, 8 and 9 respectively were cloned, sequenced and the amino acid sequences deduced. This study represents the first report on the sequencing analysis of the VP5 gene and protein of an AHSV serotype 8. All sequences were aligned and compared to those of published laboratory/vaccine strains, and other available sequence data. Interserotype amino acid identities of between 95% and 99% were observed between isolates of serotypes 3, 6 and 9 (apart from a laboratory strain of AHSV9), indicating significant conservation between these serotypes. The VP5 protein of AHSV8 showed less homology and differed from the other serotypes by between 5.2 and 7%. The AHSV4 protein differed significantly from the rest, showing between 15 and 19% amino acid differences. The identity between the VP5 proteins of different isolates/strains of the same serotype was very high in most instances, at approximately 97% for different AHSV3 strains and 94 % for different AHSV6 strains. The VP5 protein of a laboratory adapted strain of AHSV9, however, differed from an AHSV9 field isolate by nearly 10%. In the analyses of these amino acid differences, no common amino acid change, indicative of a virulence marker, could be identified between the assumedly virulent field isolates and their respective laboratory adapted strains. Furthermore, no distinctive differences in regions supposedly involved in membrane destabilization, or particular patterns of variation were observed. The results indicate that virulence of a particular AHSV serotype is unlikely to be influenced by a common amino acid change in VP5, and more likely to be multi-faceted. The second aim of this study was to investigate whether VP5 has a cytotoxic effect when expressed in a bacterial system, in light of similar effects reported for the analogous protein of bluetonguevirus (BTV), a related orbivirus. Computer analyses were performed and indicated that amphipatic helices found in the N-terminus of BTV VP5, proposed to be responsible for cytotoxicity, were also present in AHSV VP5. A full-length copy of the AHSV4 VP5 gene was cloned into the pET41 transfer plasmid and expression induced in bacterial cells. Optical density measurements of the bacterial cultures, indicative of cell growth, were taken at time intervals post induction. The expression of AHSV VP5 clearly had a severe negative effect on cell density (hence growth) over time, in contrast to a control where the cell density continued to increase. The results therefore clearly indicated that expression of AHSV VPS was cytotoxic to bacterial cells. Lastly, a neutralizing epitope present on VPS was expressed on the surface of a particulate display system, based on crystalline particles formed when the VP7 protein of AHSV9 is expressed in insect cells. Expression of the VP7/epitope A chimeric protein was confirmed by polyacrylamide gel electrophoresis and partially purified by sucrose gradient separation. Hexagonal crystals with a unique surface structure, but morphologically quite similar to VP7 crystals, were observed by scanning electron microscopy. The VP7 display vector could therefore tolerate insertion of the epitope without losing its ability to form crystals. The chimeric particles are now available for further evaluation as to its immunological properties. The results of this study should contribute to the development of a possible vaccine strategy against AHS in future. / Dissertation (MSc (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted