http://hdl.handle.net/10386/196 2013-05-20T12:07:15Z http://hdl.handle.net/10386/673 Title: Full length genome characterisation of Hepatitis B virus isolates at Dr George Mukhari Hospital in Pretoria, South Africa Authors: Magobo, Rindidzani Edith Abstract: Introduction: Sub-Saharan Africa is a region with hepatitis B virus (HBV) hyperendemicity with more than 8% HBsAg prevalence. An estimate of two billion people has been reported to carry HBV markers. HBV was associated with about 25% of annual deaths in Africa. HBV possesses a DNA polymerase which lacks proofreading mechanism. This results in highly variability and genetic diversity which poses a challenge for the diagnosis and therapeutic management of HBV infection. High mutation rate of HBV also has great implications on the development of drug resistant mutations. Moreover, HBV diversity represents a challenge for the sensitivity of immunological and molecular diagnostic assays. A number of studies on HBV full length genome have been conducted particularly in developed countries. Limited studies are available in Africa and South Africa. In South Africa, few studies have been done analysing the complete genome of HBV isolates from patients with asymptomatic carriers and fulminant hepatitis B (Owideru et ai, 2001 a; Owideru et ai, 2001 b; Kimbi et ai, 2004; Kramvis et ai, 2002).This study was aimed at characterising the full-length genome of HBV isolates at Dr George Mukhari Hospital, Pretoria, South Africa, with a view of developing a PCR-based technology for amplification and characterisation of HBV strains with different serological profiles. The technology, if successfully developed, will contribute in understanding the molecular mechanisms resulting in various HBV variants or isolates. Methods: The study design was exploratory. Four stored serum samples collected from HBV infected patients at Dr George Mukhari hospital, Pretoria, were used to develop the molecular technology and test the hypothesis. HBV serology was previously performed targeting 5 HBV serological markers; HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe using Elecsys version; HBV DNA quantification was done using Cobas Amplicor HBV DNA monitor assay, HBV DNA was extracted and subjected to nested PCR assay targeting HBV full length genome as two overlapping fragments: fragment A (1670 bp) and fragment B (1868 bp). The generated PCR products for both fragments were cloned into the pGEM T easy vector and 2 clones were selected from each sample. The plasm ids were purified using Invisorb@ Spin Plasmid Mini Two and the clones were recovered by PCR assay. The sample PCR products and the clone PCR products were purified and sequenced using SpectruMedix SCE2410 genetic analysis system. HBV genotyping was performed using the NCBI web-based genotyping tool. Phylogenetic analysis was done using MEGA 4 software to confirm HBV genotypes. Results: Serology results were as follows: All samples were HBsAg positive, Anti-HBs negative, anti-HBc positive and anti-HBe negative. Sample B1121 and sample 6 were HBeAg positive while samples B452 and 5 were HBeAg negative. A total of 12 PCR products were sequenced (4 study samples and 8 clones [2 clones each sample]). In total, 7 HBV full length genome sequences were deduced from this study, with 3 sequences belonging to genotype A, 2 to genotype C and 2 to genotype D. 3 HBV genotypes were detected from this study; genotype A, C and D with subgenotype A2, C1 and D1 respectively. Mutations were observed throughout the genome. In the pre-S/S open reading frame (ORF), the most significant findings were the detection of mutations within the "a" determinant site and major hydrophilic region (MHR). These mutations included Y161F,E164G observed in sample B1121 and B1121C1 belonging to subtype A1; 2 amino acid insertion at aa 161-162 in sample 5 belonging to subtype C1. Drug resistance associated mutations were identified in the polymerase gene, these included M204T and L217R which are associated with adefovir resistance, M204T also resulted in a change from tryptophan (W) to arginine (R) at aa 196 on the overlapping surface gene on sample B452 C1. Basal core promoter (BCP) and pre core/core mutations were detected in study isolates; specifically the BCP double mutation (1762/1764) was seen in 8 isolates which belonged to subtype C1 (5) and D1 (3) and the pre-core stop codon mutations (G1896A) in 4 isolates. (2 belonging to subtype C1 and the other 2 to D1). Other changes observed included a 48 nucleotides deletion in the pre-core gene, 6 nucleotides insertion in the HBx gene of all subtype D1 isolates and a 3 nucleotides deletion in subtype C1 clone. Conclusion: This study successfully optimised a PCR-based technology for the amplification and characterisation of HBV full length genome. 3 HBV genotypes were detected with subtypes A2, C1 and D1. However, the detected subtypes are rarely detected in South Africa. The detection of subtype A2 may confirm its Southern African origin. Drug resistance associated mutations were observed in this study. These included the adefovir resistance mutation which the current study confirmed it is a naturally occurring mutation as it was detected in adefovir therapy na'ive patient. The BCP and pre-core/core mutations were detected in genotype C and D isolates; however, their association with serological profile and clinical outcomes could not be deduced. Unique or novel mutations were seen in the study isolates, these included 48 nucleotides deletion in the pre core gene, 3 amino acids insertion in the RNase H and 8 amino acids deletion in the RT domain of polymerase gene. To our knowledge, these mutations have not been identified or reported in the literature. The detection of 6 nucleotide insertion in the HBx gene was reported for the first time in South African isolates. Further analysis is required to ascertain the biological significance of the unique mutations detected in this study. Description: Thesis (MSc. (Medical Virology))-- University of Limpopo, 2011. 2011-01-01T00:00:00Z http://hdl.handle.net/10386/672 Title: Burden of rotavirus disease and molecular characterization rotaviruses at Dr George Mukhari Hospital from 2003-2005 Authors: Seheri, Luyanda Mapaseka Abstract: Background: Rotavirus infection remains a significant clinical problem throughout the world, infecting almost every child younger than 5 years of age, despite socio-economic status or environmental conditions. Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants and young children. Implementation of an effective vaccine programme could reduce the incidence and severity of rotavirus disease. Decisions about new candidate rotavirus vaccines require reliable data on disease impact in both developed and developing countries. The aim of this study was to assess the burden of rotavirus associated disease at the tertiary care Dr George Mukhari Hospital, Ga-Rankuwa and the secondary care hospital Brits Hospital, Madibeng and to describe the genetic diversity of rotavirus strains circulating in Ga-Rankuwa and Brits communities over a similar time period as the testing of Rotarix@ vaccine. The broad objectives included; to perform a hospital-based burden of rotavirus disease in two different hospitals in the North West of Pretoria area, to conduct molecular characterization of rotaviruses circulating in the Pretoria region and lastly to devise an alternative molecular typing method to detect rotavirus VP6 subgroups. Materials and Method: To investigate the hospital-based burden of rotavirus disease, diarrhoeal stool samples were collected at Dr George Mukhari and Brits Hospitals from children less than 5 years of age. Group A rotavirus antigen was detected from the samples using commercially available rotavirus enzyme immunoassay IDEIA TM Rotavirus test (DAKO, Dakocytomation, Denmark). Genetic analyses of rotavirus strains were determined by polyacrylamide gel electrophoresis (PAGE) to characterize the electrophoretic patterns followed by analysis of the P and G genotypes by RT -PCR and multiplex PCR amplification of specific sequences of VP7 and VP4 genes. To devise an alternative molecular typing method to detect rotavirus VP6 subgroups, with subgroup specificities determined by both VP6 monoclonal antibodies and restriction fragment length polymorphism using restriction endonuclease Acil, Odel and Rsa I. Selected PCR amplicons (VP7 and VP6 genes) were purified, cloned and sequenced. Consensus sequences of the VP7 and VP6 genes were aligned and analysed manually with Chromaslite and BioEdit software packages. Multiple sequence alignment was implemented by Mafft software packages. The nucleotide and deduced amino acid sequences of the VP7 and VP6 genes were compared with reference strains available from GenBank. Multiple methods were used to construct phylogenetic trees and included neighbor¬ joining, maximum parsimony analysis and maximum likelihood distance. Bootstrap values were computed using 1000 replicates with Phylip and the MEGA softwares. The graphic representation of each phylogenetic tree was displayed with the Treeview program. Results: Between 2003 and 2005, a total of 2 514 diarrhoeal stool samples were collected. Of these, 527 (21%) were positive for group A rotavirus and the majority of children hospitalized were less than 2 years of age. The annual peak prevalences of group A rotavirus were 56%, 59% and 56% for 2003, 2004 and 2005, respectively and were observed during the autumn and winter months. The estimated incidence of gastroenteritis associated with rotavirus indicates that one in every 50 to 70 children in the area is likely to be hospitalized with rotavirus diarrhoea between birth and 2 years of age. During the three-year study period, ten, six and seven different RNA electrophoretic patterns were identified in 2003, 2004 and 2005, respectively. The VP6 genes of the representative strains (G1, G2, G3, G9, G8 and G12) were ana lysed with restriction endonuclease Acil, Ode! and Rsa!. The restriction endonucleases produced 11 unique restriction profiles (A-K). The VP6 RFLP results correlated well with strains displaying long RNA electropherotypes and VP6 subgroup 1/ specificity and also with strains displaying short RNA electropherotypes and exhibiting VP6 subgroup I specificity as determined with VP6 monoclonal antibodies. The genotypic distribution varied remarkably and major rotavirus strains detected in circulation during the study period included G2P[4] in 2003, G1 P[8] in 2004 and G3P[8]/ G3P[6] in 2005. Rotavirus strains carrying G8P[8] specificities and unusual G 12P[6] strains were also detected at low frequency The consensus VP7 nucleotide sequences, exhibited the greatest homology and identity (97-99%), when compared against corresponding international reference strains. The nucleotide sequence datasets were closely related to strains from South Africa, Vietnam, Bangladesh, East India, Republic of Congo, China, Russia, Thailand and Japan. The phylogenetic tree revealed the South African strains (G1-G3, G8-G9 and G12) clustered with international strains whereas the G1 strains clustered into two different lineages. Phylogenetic analysis of the VP6 gene revealed four lineages with international reference strains. The VP6 gene showed 97-99% identity at the deduced amino acids level with strains from Taiwan, Bangladesh, the United States and Brazil. Conclusion: This is the first study to estimate the disease burden associated with rotavirus diarrhoea in South Africa. The overall results confirm that rotavirus is the most common cause of severe diarrhoea. The epidemiology of rotavirus diarrhoea in South Africa correlates well with what has been reported in other countries. The proportion of hospitalization of rotavirus infection in children less than 5 years was estimated to an annual prevalence of 22.8% (95%CI 21.2%, 24.5%) at Dr George Mukhari Hospital, while at Brits Hospital was estimated at 18.2% (95%CI 14.9%, 22.1 %). Rotavirus genotypes circulating at Dr George Mukhari Hospital showed a high degree of diversity and the emergence of uncommon rotavirus strains such as G12. The emergence of novel rotaviruses in the region needs to be taken into account where vaccine efficacy is concerned. It is, thus, important to continue with such studies to monitor the rotavirus strains associated with severe gastroenteritis in a hospital setting before and after the introduction of a rotavirus vaccine. Results also indicated that RFLP analysis of VP6 amplicons might be a simple and reliable, alternative to MAb subgrouping. The sequence analysis of the partial length VP6 gene confirmed the location and the recognition sites of the restriction enzymes The RFLP analysis proved to have more potential to accurately detect different rotavirus subgroups. Description: Thesis (PhD (Medical Virology))-- University of Limpopo, 2010. 2013-01-01T00:00:00Z http://hdl.handle.net/10386/670 Title: Intraventricular haemorrhage in premature babies at Dr George Mukhari Hospital, Pretoria Authors: Lentsoane, Tiisotso Lenake Abstract: Background: Intraventricular hemorrhage (IVH) is a known complication occurring in the first week of life in premature neonates. The exact time of its occurrence and the ideal time to perform diagnostic imaging investigation remain controversial. Objectives: 1. To determine the incidence of intraventicular hemorrhage in premature babies at Dr George Mukhari Hospital, Pretoria. 2. To determine the timing at which bleeding occurs. 3. To determine if the rate of diagnosing intraventicular hemorrhage improves when performing ultrasound via the posterior fontanelle. 4. To determine the risk factors for intraventricular haemorrhage Materials and methods: The study included 60 premature babies of gestational age of less than 32 weeks that were admitted to our neonatal Intensive Care Unit over a two months period and screened for IVH. They were grouped into three categories according to their weight at birth, and according to their gestational age. All babies had a cranial ultrasound on day 1, 3 and 7. Results: We found that the overall incidence of IVH among premature babies was 28%. Although it did not reach statistical significance, the incidence was found to be inversely related to the birth weight and gestational age. The majority of the bleeds occurred within the first day of life and were mostly grade I and II according to Papile’s classification. The use of inotropes was found to be significantly associated with development of IVH. We also found that scanning through the posterior fontanelle did not significantly increase the rate of diagnosis for IVH. Description: Thesis MSc(Med)(Virology)-- University of Limpopo, 2011. 2011-01-01T00:00:00Z http://hdl.handle.net/10386/417 Title: Molecular characterization of the hepatitis B virus X gene Authors: Malinga, Lesibana Anthony Abstract: Introduction: Hepatitis B virus (HBV) is a serious problem worldwide causing various liver diseases such as chronic hepatitis and hepatocellular carcinoma (HCC). The pathogenesis of HBV related HCC is not well established. Hepatitis B X protein (HBx) plays an important role in the pathogenesis of HCC. HBx coded by HBV X gene enhances several cellular pathways in hepatocytes which may lead to HCC. The genetic variability of other HBV genomic regions plays a significant role in diagnosis, vaccine development and drug resistance. However, the genetic variability of HBV X gene is not well understood. In addition the dual basal core promoter mutations found within the X gene have been implicated in the inhibition of hepatitis B e antigen (HBeAg) expression. Studies focusing on HBV X gene are scarce in South Africa. Consequently HBV X gene variability may reveal interesting mutations and substitutions that are important in chronic liver diseases or HCC. This study aimed at characterising HBV X gene at a molecular level isolated from patients with different serological profiles. Methods: This was an exploratory study which used 20 stored sera (-70°C) collected from adult patients at Dr George Mukhari hospital, Pretoria. The samples were already tested for HBsAg, anti-HBs, anti-HBc and HBeAg serological markers (Elecsys, Roche Diagnostics, Penzburg, Germany). HBV DNA extraction was performed from serum using High Pure Viral Nucleic Acid Assay (Roche Diagnostics, Penzburg, Germany). Nested PCR assay was used for the amplification of 465 nucleotide HBV X gene. Sequencing of PCR positive samples was done using spectruMedix SCE2410 genetic analysis system. Six samples selected, were cloned into the pGEM®-T Easy vector system (Promega, Madison, USA). Three clones of each sample were selected and their plasmids purified using Pure Yield™ Plasmid Miniprep System (Promega, Madison, USA). The plasmid DNA was recovered using optimised nested PCR assay and sequenced. A total of 38 sequences were generated from the study and compared with reference strains retrieved from GenBank. Phylogenetic analysis based on HBV X gene sequences was done using MEGA 4 software to determine different genotype clusters. vi Results: HBV X gene was successfully detected and amplified in 20 study samples. The sequenced HBV X gene products revealed mutations and insertions. Particularly a six nucleotide insertion, GCATGG between nucleotides 1611 and 1618 which was detected in five samples. In addition, the six cloned samples confirmed the six nucleotide insertion and other mutations associated with inhibition of hepatitis B e antigen (HBeAg) detected in the study. The substitutions within HBx were detected in the N (1-50 amino acids) and C (51-154) terminals by comparing our sequences with archival sequences from GenBank. Important substitutions found within the N and C terminals were S31A, P38S, A42P, F73L, H94Y, P101S, K118T, D119N, I127T/N, K130M and V131I. These substitutions are associated with various biological functions and pathogenesis. Other substitutions with unknown functions detected in the study include A2G, A3G, A4G, C6W, P42S and V116L. Further mutations of T1753M, A1762T and G1764A associated with inhibition of HBeAg expression were detected in most samples and only one sample had C1766T mutation. Phylogenetic analysis resulted in A, C and D HBV genotypes. Five samples and 11 clones clustered with genotype D, two samples and four clones clustered with genotype C and finally 13 samples and 3 clones clustered with genotype A. Conclusion: HBV X gene was successfully characterised using various molecular methods. HBx substitutions detected are involved in various pathogenic effects and may present a risk of HCC for patients infected with HBV. Genotype D samples displayed most mutations/substitutions and this can be regarded as an important genotype with high risk of HCC. The detection of a six nucleotide insertion (GCATGG) in 5 samples may emerge as a new variant of genotype D. Furthermore triple mutations of T1753M/A1762T/G1764A within basal core promoter region were detected mostly in HBeAg negative samples. However further analysis of HBV X gene variability is needed. Description: Thesis ( M Med (Virological Pathology))--University of Limpopo, 2010. 2010-01-01T00:00:00Z