Browsing by Author "Marie, Veronna."

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The HIV-1 gag and protease: exploring the coevolving nature and structural implications of complex drug resistance mutational patterns in subtype C.
(2019) Marie, Veronna.; Gordon, Michelle Lucille.
Due to the high prevalence of HIV-1 subtype C infection coupled with increasing antiretroviral (ARV) drug treatment failure, the elucidation of complex resistance mutational patterns arsing through protein coevolution is required. Despite the inclusion of LPV and DRV in second- and third-line, many patients still fail treatment. In this study, protease (PR) inhibitor resistance mutations were identified by comparing treatment versus naïve sequences datasets in Gag and PR. Thereafter, to investigate Gag-PR coevolution and pathways to LPV resistance, phylogenetic analyses and Bayesian networks were constructed. Following this, structural analyses combining homology modelling, molecular docking and molecular dynamic simulations were carried out on specific patterns of protease resistance mutations (PRMs). To complement these analyses, the structural impact of a mutated Gag cleavage site on PR resistance dynamics was also evaluated. Accordingly, this study identified 12 major PRMs and several resistance combinations. Of these, the M46I+I54V+V82A pattern frequently occurred. The second most frequently recurring pattern included L76V as a fourth mutation to the above triplet. Coevolution analyses revealed correlations between positions 10, 46, 54 and 82 in PR. Of these, minor PRM L10F occurred in 6.4% of the dataset and was involved in pathways to LPV resistance. Additionally, Gag cleavage site (CS) mutation A431V was also correlated with L10F and the major PRMs. Distinct changes in PR’s active site, flap and elbow regions due to the PRMs (L10F, M46I, I54V, L76V, V82A) were found to alter LPV and DRV drug binding. When the PRMs were combined with the mutant Gag CS binding was greatly exacerbated. While the A431V Gag CS mutation coordinated several amino acid residues in PR, the L76V mutation was found to have a significant role in substrate recognition rather than directly inhibiting the drugs. These data show that the co-selection of mutations in Gag-PR greatly contributes to resistance outcomes and that our understanding on drug resistance is largely lacking, particularly where structure is concerned.
Investigating the presence of microbial pathogens in the Umhlangane River, Durban, South Africa.
(2015) Marie, Veronna.; Lin, Johnson.
The use of rivers for recreational and domestic practices makes it imperative to scrutinize the water quality circulating within the surrounding communities. The presence of potential pathogens in the Umhlangane River was monitored at five points (Phoenix industrial: P1; upstream KwaMashu wastewater/residential: P2; natural wetlands: P3; Riverhorse Valley industrial/business estate: P4; and Springfield industrial: P5) on a monthly basis from October 2013 to September 2014. Commonly measured physico-chemical parameters were determined according to standard protocols. Bacterial indicators were enumerated using the membrane filtration technique. A tangential flow filtration process was set up to remove the bacteria and to concentrate the virus populations from 25 ℓ of river water samples. Somatic and F⁺RNA coliphages were enumerated using plaque assays. The virus-like particles (VLPs) were estimated using epifluorescence microscopy and viral morphology was observed using transmission electron microscopy (TEM). The potential infectious nature of the concentrated viruses was assessed using cytopathic effect (CPE) of tissue culture. The specific detection of some virus populations was determined by a two round nested-PCR reaction using virus-specific primer sets and confirmed by sequencing. Chemical and biological oxygen demand (COD; BOD) fluctuated at all sampling points and months with BOD ranging from 0.48 mg/ℓ (Riverhorse Valley; April 2014) to 12.4 mg/ℓ (Phoenix industrial; June 2014), respectively. The highest COD content was recorded at the Phoenix industrial site in May with 269 mg/ℓ. The total dissolved solid (TDS) content and electrical conductivity (E.C.) fluctuated throughout all sampling months and points with all measurements exceeding the Department of Water Affairs recommended limits of 0 – 100 mg/ℓ and 0 – 15 mS/m, respectively. High counts of E. coli (EC), total and faecal coliform (TC; FC) and Shigella (SHIG) were recorded at the industrial sites in Phoenix and Springfiled and upstream of the KwaMashu WWTP in Phoenix while the total heterotrophic bacteria (THB) depicted the highest and lowest counts at the Phoenix industrial natural wetland sites ranging from 14.9 x 10⁶ cfu/100mℓ to 1.3 x 10⁶ cfu/100mℓ, respectively. Somatic and F⁺RNA coliphages produced its highest counts at the industrial site in Phoenix ranging from 765 pfu/mℓ and 585 pfu/mℓ in January 2014, respectively. Direct VLP counts were substantially lower (105 vlp/mℓ; April 2014) than the plaques produced by the somatic and F⁺RNA coliphages. Morphological changes of HEK293, Vero and Hep-G2 cell lines were indicative of a positive CPE for viral concentrates. Apart from visualization of bacteriophages belonging to the Siphoviridae, Myoviridae and Podoviridae families, presumptive Picornaviridae, Adenoviridae, Herpesviridae, Coronaviridae, Reoviridae, Polyomaviridae and Orthomyxoviridae VLPs were revealed based on size and comparisons to electron micrographs of known viruses. Adenovirus, polyomavirus, and hepatitis A and C virus-specific nested primers revealed the detection of these waterborne pathogens in the Umhlangane River. Moreover, sequence data confirmed the presence of these virus populations by comparisons made in GenBank. An increase in the amount of chemical pollutants entering the water would allow for the high COD, BOD and changing E.C. and TDS levels. Elevated THB populations at all sampling points and months indicate poor water quality. High EC, TC, FC and SHIG are indicative of possible faecal pollution, which could be attributed to faecal contamination entering the catchment. The presence of these indicators as well as the somatic and F⁺RNA coliphages could be due to anthropogenic activities, changing climatic conditions and the excreta of infected and non-infected individuals entering the river. Viruses or phages in the river water samples are morphologically diverse. Phage diversity further indicates diversity in their bacterial counterparts. The presence of various VLPs revealed by TEM together with substantial CPE on human tissue cell lines and the confirmation of adenoviruses, polyomaviruses as well as hepatitis A and C viruses by molecular detection and sequencing data raise the health concerns of the river system. The present study highlights the importance of routine environmental surveillance of human enteric viruses for a better understanding of the actual burden of these viral infections on those who might be using the water directly without treatment.