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Item Open Access Further screening of Venda medicinal plants for activity against HIV type 1 reverse transcriptase and integrase(2006-03-15) Bessong, Pascal O.; Rojas, Luis B; Obi, Larry C.; Tshisikhawe, Peter M.; Igunbor, Eunice O.The use of medicinal plants for AIDS-related conditions is common in South Africa. In order to establish an antiviral rationale for the use of these plants we screened fractions of the methanol extracts of medicinal plants for activity against HIV-1 reverse transcriptase (RT) and integrase (IN). The n-butanol fraction obtained from the crude methanol extracts of the roots of Bridelia micrantha (Hochst) Baill. (Euphorbiaceae) was observed to be as the most active inhibiting the RNA-dependent-DNA polymerization (RDDP) activity of HIV-1 RT with an IC50 of 7.3 g/ml. However, it had no activity on the 3’-end processing activity of HIV integrase. Bioassay-guided fractionation of the n-butanol fraction yielded friedelin and -sistosterol, which did not inhibit the RDDP of RT or 3’-end processing functions of IN even at a concentration of 500 M. An uncharacterized fraction obtained in the bioassay-guided fractionating process inhibited the RDDP with an IC50 of 9.6 g/ml, but had no inhibition on IN. Phytochemical screening indicated the presence of flavonoids and tannins in the uncharacterized fraction.Item Metadata only In-vitro bioactivity of fractions from a local medicinal plant on HIV-1 replication, and selected fungal and bacterial pathogens(2009-03) Mutshembele, Awelani Mirinda; Bessong, Pascal O.; Eloff, Jacobus N.; Obi, LarryItem Open Access Drug Resistance Mutations in Naive HIV-1 South African Patients, and Construction of Molecular Clones to Phenotype Putative Resistance Mutations(2009-03) Mavhandu, Lufuno Grace; Bessong, P. O.; Rekosh, David; Hammarskjold, Marie-LouiseIn countries such as South Africa where access to therapy is progressing data is required on patterns of resistance and evolution of resistance. Thirty protease (PR) and 31 reverse transcriptase (RT) amino acid sequences of HIV primary isolates from drug naNe patients from rural settings in South Africa were examined for resistance mutations. Samples were collected between May and August 2007. Phylogenetic analysis showed that all the sequences were HIV-1 subtype C in both the protease and reverse transcriptase genes. The mean genetic distances among the sequences were 0.0170-0.0786 for the protease, and 0.0045-0.0890 for the reverse transcriptase. However, it was noted that 3 pairs of samples 07VGNF5ZA and 07VGNF6ZA, 07VGNF7ZA and 07VGNF8ZA, 07VGNF10ZA and 07VGNF13ZA did not show any genetic variability among their protease sequences. No major resistance mutation was observed among the protease sequences. However, the following minor resistance mutations were noted: L101N (3/30), A71T (1/30), and T74S (2/30). Examination of the reverse transcriptase gene for resistance mutations reveal the presence of V118I (1/30), V179D (1/30), K103N (2/30). Most of the RT sequences were wild-type, although V118I (3.3%) and k103N (6.7%) associated with resistance to lamivudine and nevirapine, respectively, were observed. In summary, this study has shown that most of the viruses in Limpopo Province, representing the northeastern part of South Africa are HIV-1 subtype C, and that the prevalence of resistant mutations among the drug na"fve patients is still low. Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. Systems to be used in the testing of phenotypic drug resistance and susceptibility are being developed. These may intimately be used in guiding therapy to improve long term suppression of HIV replication. Two proviral chimeric clones making use of pMJ4 and pNL4-3, and two vector plasmids which deletions of sequences encoding HIV-1 protease or reverse transcriptase were constructed for cloning of HIV-1 PCR products. Growth of constructs was monitored by p24 antigen production. Susceptibility to protease and reverse transcriptase inhibitors was measured by using resistance test vectors that contain a Luciferase indicator gene. Cells were co-transfected with packaging plasmids, pluc, and pEnv, resulting in the production of virus particles that were used to infect target cells. Luciferase activity was measured following a single round of replication. The chimeric constructs MJ4 carrying the NL4-3 Apal-Hpal cassette (MJ4/NL4-3) and NL4-3 carrying the MJ4 Apal-Hpal cassette (NL4-3/MJ4) were successfully developed as shown by restriction digestion analysis. Considering growth of the constructed chimeras NL4-3/MJ4 was better than MJ4/NL4-3 although not robustly. Good p24 production was obtained from all four gap-pol plasmids. MJ4/NL4-3 worked better in delivering luciferase to the target cells while NL4-3/ML4 appeared totally devoid of any infectivity. The vectors pCMVGagPol(MJ4)-RREr and pCMVGagPol(NL4.3)-RREr were created and both expressed the viral gag-pol protein. Viral inhibition test showed that the vectors can be inhibited by NRTI, NNRTI and Pl. Inhibition was seen in all drugs in different concentrations indicating that the system works. The results showed that vector systems constructed can be used to evaluate putative drug resistant mutations, coding for resistance to protease and reverse transcriptase inhibitors, detecte� in patient viruses. In addition, the system can also be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant HIV-1 virus.Item Open Access Detection of Cryptosporidium species in stools of HIV/AIDS patients in Bela-Bela, South Africa(2010-06) Makuwa, Stenly Modupi; Bessong, P. O.; Samie, A.; Potgieter, N.Item Open Access Genetic characterization of human immunodeficiency virus from Northern South Africa(2012-12-19) Iweriebor, Benson Chuks; Bessong, Pascal Obong; Mphahlele, Jeffrey; Moyo, Sylvester RodgersGlobally, human immunodeficiency virus type 1 (HIV-1) is extraordinarily variable, and this diversity poses a major obstacle to AIDS vaccine development, diagnosis and therapy. Since HIV-1 M group began its expansion in humans roughly 70 years ago it has diversified rapidly now comprising a number of different subtypes and circulating recombinant forms (CRFs). Currently, strains belonging to the same subtype can differ by up to 20% in their envelope gene, and between subtype distances can soar to 35%. Moreover this diversity is continually growing. Although the scale of the HIV-1 pandemic makes action imperative, there is still much to learn about the extent and immunological implications of HIV-1 sequence diversity. As with other infectious agents, effective public health surveillance is essential to track the epidemic, guide research, and direct prevention activities. Significant challenges were highlighted by recent finding that some of the more divergent HIV-1 strains were not reliably detected by all antibody screening tests in current use. HIV-1 subtype C is responsible for the vast majority of the estimated 5 million infected South Africans. Thus, while subtype C viruses dominate the epidemic, the exceptional high prevalence rates in South Africa could provide significant opportunities for the spread of new genetic subtypes and/or evolution of current subtypes. Furthermore, analysis should therefore focus on areas where little information is known, such as the Limpopo Province, and where the opportunity for the introduction of new variants exists, and where prevalence rates are relatively high. The strategic location of the Province makes it imperative for regular diversity studies to be carried out. The implications of HIV-1 diversity in diagnostics and vaccine development; and the formulation of treatment regimens necessitated this study. Thus, this study therefore identified and characterized circulating HIV-1 genetic variants in sere-positive, drug na"fve populations from Limpopo Province where there is an estimated prevalence rate of about 19%. The molecular epidemiology of HIV-1 in two highly endemic areas in Limpopo Province of South Africa; mutations associated with drug resistance to protease inhibitors (Pis), nucleotide reverse transcriptase inhibitors (NRTls) and non nucleoside reverse transcriptase inhibitor (NNRTs), co-receptor usage and epitope mapping of HIV-1 isolates from drug naive individuals were investigated. Subtyping was done by phylogenetic analysis making use of ClustalX2 software while drug resistance mutation analysis, co-receptor usage prediction epitope mapping and substitution of the functional motifs were all determined by interpretation algorithms. Phylogenetic analysis of the test isolates revealed that subtype C is predominantly driving the epidemic in northern South Africa (Limpopo Province). Co-receptor usage prediction showed that majority of the isolates were R5 viruses as all had their tetrapeptide GPGQ motif characteristic of subtype C utilizing CCR5 viruses conserved. Genetic drug resistance mutation analysis of the 35 PR gene sequences did not reveal any major mutation associated with resistance but a high degree of minor mutations and polymorphisms were observed. Examination of the 44 RT genes showed a K103N substitution in two isolates. K103N change causes high level resistance to nevirapine. Epitope mapping of the gag p17 and p24 consensus sequences of the test isolates did not reveal any difference between them and the subtype C consensus sequences. They all had the same dissociation constant for the epitopes recognized by the HLA they were mapped against. Also, all the functional motifs in the PR and RT genes were conserved in majority of the test isolates. Molecular characterization of the test isolates has helped to update the baseline data on the circulating strains of HIV -1 in northern South Africa. Since all the isolates are subtype C as in other regions of South Africa and result of epitope mapping compares very well with those of subtype C consensus sequences, vaccine based on subtype C viruses could be designed and evaluated in the Province. Also, it has shown that entry inhibitor- the new class of antiretroviral drug could be of significance should the current NRTls and NNRTls begins to fail as majority of the isolates had their GPGQ motifs conserved. Since no important resistance mutation to the Pis and RTls was found among the test isolates, usage of these classes of drugs will continue to have positive impact in reducing morbidity and mortality due to AIDS in the studied area. The second element of this study focused on the amplification and analysis of a unique recombinant form composed of subtypes A1 and C subgenomic regions. Recombination plays a key role in HIV-1 genetic diversity which on the long run has a grave implication on diagnosis, therapy and vaccine development. The URF recombinant that was analysed was isolated from a female patient residing at Bela Bela which has a high HIV-1 prevalence. The amplified near-full length genome was sequenced by the 454 Genome Sequencer FLX system. The sequence generated was delineated into respective gene by the sequence locator- a web based online tool. Analysis of the recombinant virus was carried out in order to determine the subtypes that constituted the mosaic genome making use of jpHMM and REGA subtyping analytical tools. The results obtained revealed that the mosaic genome is composed of A1/C with seven breakpoints of recombination partitioning the genome into eight segments alternating between sub-subtypes A1 and C viruses. Analysis of the near-full length genome by subtyping tools showed discordant assignment of some gene regions by the different tools. Further analysis of the accessory genes did not reveal any major changes like premature termination or loss of functional motifs but deletions and insertions were observed in the tat, rev and net genes respectively. The isolation of a recombinant virus in a region where subtype C is the dominant variant shows the dynamic nature of this virus and calls for regular monitoring of the HIV-1 genetic landscape of the region. The third component of this study also involved the analysis of a recombinant virus isolated from a female patient at Mankweng Hospital near Polokwane the provincial capital of Limpopo Province. Partial fragment of 5665 nucleotides was generated, sequenced and analysed by various subtype analytical tools. Presence of drug resistance mutations in the PR, RT and IN genes was determined as well as prediction of co-receptor usage. Results revealed a mosaic recombination between subtype C and CRF11_cpx only at the RT gene of the isolate. All other gene regions analysed phylogenetically belong to pure subtype C virus. This is a novel strain as there is no known variant that has this genomic recombination in the HIV database. The epidemiological implication of this strain in Limpopo Province is not known. The frequency and pattern of polymorphisms among HIV-1 subtypes associated with resistance or resulting to a faster emergence of drug resistance once under drug pressure has been evaluated extensively on subtype B and little information exist about other subtypes. Subtype C variants are responsible for more than 50% of the global epidemic, and it is the subtype that is driving the epidemic in Southern Africa, the region with the highest HIV prevalence in the world. Antiretroviral scale up in the Limpopo region is high and therefore, it is necessary to determine whether genetic subtype differences will influence therapy outcome. The investigative theme of chapter five of this thesis is on the implications of nucleotide polymorphisms on the genetic barrier to evolution of drug resistance mutations in subtype C viruses when compared to the global subtype B consensus sequence. The protease and reverse transcriptase nucleotide sequences generated in this study were compared with the global subtype B consensus sequence at codons known to code for drug resistance mutations according to the Stanford drug resistance algorithm. The results revealed a reduced genetic barrier in subtype C viruses at codon V106M (GTA to GTG) and an increased barrier at codon L21OW (TTA/CTG/CTA to TGG) when compared to subtype B global consensus sequence. From this analysis, these are the only codons where significant differences exist between the subtypes. Apparently, there are, no major genetic barriers existing between subtypes B and C at known positions that code for drug resistance mutations. In conclusion, HIV-1 subtype C viruses is the predominant circulating variant in Limpopo Province as phylogenetic analyses of the partial gag, pol, and env C2- C3 gene fragments from HIV chronically infected patients showed that majority of the viruses are HIV-1 subtype C. The circulating HIV-1 viruses will be susceptible to the currently available protease inhibitors and reverse transcriptase inhibitors as drug resistance mutations in the naive population are very low (4.7%). Two unique recombinant forms HIV-1 A1/C and HIV-1 C/CRF11_cpx were each detected from two different individuals in the Waterberg and Capricorn districts respectively. There is no immunological difference between the HIV-1 subtype C viruses from Limpopo Province and the global consensus of subtype C viruses as epitope mapping using the consensus generated from test isolates had the same dissociation constant as the subtype C consensus sequence. Also, there is apparently no significant difference on the impact of nucleotide polymorphisms on the genetic barrier to antiretroviral drug resistance between subtype C viruses and subtype B viruses.Item Open Access Genetic analysis of HIV-1 integrase sequences from treatment naive individuals in Northeastern South Africa(Molecular Diversity Preservation International (MDPI), 2013-03-01) Bessong, Pascal Obong; Nwobegahay, JuliusRaltegravir, an integrase inhibitor, is not a component of the current South African antiretroviral treatment guidelines, but it could be introduced in the near future as cases of virological failures from current treatment regimens begin to occur. The aim of this study was to analyze the complete HIV integrase gene obtained from individuals at two treatment sites in northeastern South Africa for the presence of Raltegravir associated drug resistant mutations and viral subtypes based on the integrase gene. Examination for mutations against other integrase inhibitors, such as Elvitegravir and Dolutegravir, was also done. Viruses from 127 treatment naive individuals were analyzed. Genetic drug resistance mutations were determined using the Stanford HIV Drug Resistance Interpretation program and the International AIDS society-USA guidelines. Viral subtyping was done by phylogenetic analysis, and recombinants were determined using the REGA, jpHMM and RIP tools. No major resistance mutations were detected. However, 7% of the sequences had minor mutations and polymorphisms. The majority (99%) of the viruses were HIV-1 C. Recombination analysis showed that the polymerase gene of one virus was likely composed of HIV-1 subtype A1 and C sequences. The present study indicates that Raltegravir, Elvitegravir and Dolutegravir resistant mutations may be absent in the study communities and further indicates the presence of recombinant viruses in northeastern South Africa.Item Open Access Immunodulation of inflammation in a murine pnemococcal sepsis model(2013-10-01) Musie, Mbulaheni Edgar; Bessong, P. O.; Scheld, M. W.Mortality from pneumococcal infections remains high, despite the development of potent antibiotics. Antibiotic resistance among pathogens including S. pneumoniae calls for new therapeutics. lmmunomodulation represents a novel approach to antimicrobial therapy that depends on bolstering host immunity, rather than direct antimicrobial activity. The use of innate immune stimulation to improve survival has previously been described for Gram negative pathogens. The effect of TLR4 stimulation on survival in mice during lethal S. pneumoniae, serotype 2 infections was examined. C57BL/6, BALB/c, CBA/CaHN-Btkxid'/J, A/J, Rag-1 KO, IL-10 KO , C3H/HeN and C3H/HeJ mice were inoculated intravenously with a lethal dose of 106 cfu of S. pneumoniae serotype 2 48 hours after treatment with five doses of 10ug highly purified LPS or vehicle (PBS) at 12 hours interval. Another group of LPS or PBS treated C57bl/6 received 25 mg/kg ceftriaxone at 6 hours post infection. Survival was monitored for 5 days. Blood samples were collected at different time points (6h, 12h and 24h) after bacterial challenge for bacteriological examinations, serum cytokine measurements, and biochemical assays for liver function. Spleens were harvested for flow cytometric analysis of splenic lymphocytes or NK activation. Innate stimulation with LPS reduced systemic bacteremia by at least four logs in LPS- pretreated C57BL/6 (104 v.s 108 cfu/ml) mice compared with controls during the recorded course of infection. Death in experimental controls occurred within 48 hours. Reduced bacteremia corresponded with improved survival in all 3 strains. Survival for LPS-treated C57bl/6, Balb/c and C3H/HeN was 90% (N=29; p=0.001), 50% (N=14; p = 0.017) and 60% (N=8; p=0.009), respectively, and mortality for controls was 100% for all the strains. Mortality for ceftriaxone-treated C57bl/6 was 75% (N=12, p=0.0018). Survival for LPS-treated and non-treated mice was 80% (n=10) and O % (n= 6) in CBA/CaHN-Btkx,dJ/J mice and 85% (n=14) and O % (n=9) in A/J mice which suggests that deficiency in complement and antibodies did not ablate survival benefits. Lack of B and T cells ablated survival benefits of LPS pretreatment in RAG-1 KO mice even when mice received a high dose of LPS. These results suggest that T cells are responsible for protection and B cells are partially involved in tolerance. The level of TNF-a, IFN-g, IL-12 (p40/70), IL-6, IL-1a/b, IL-10, Eotaxin, MCP-1, and MlP-1a/b were attenuated in C57BL/6 LPS-treated mice 12 hours after infection compared to untreated group. Pretreatment with a low dose of LPS also prevented hypoglycemia (glucose level was 149 vs. 45) and liver failure (reduced the AST [139 vs. 445, p<0.025] and ALT [27 vs. 129, p<0.03] to near baseline) induced by S. pneumoniae infection. LPS pretreatment restored the splenic NK population and decrease their activation demonstrated by lower mean percentage of CD69 expression of 35.3 vs 97.3 (p<0.05) than of control infected mice. This study demonstrates a survival benefit from TLR4 stimulation prior to infection with S. pneumoniae. It provides evidence that induction of profound LPS tolerance, despite reducing cytokine production, improves host defense against infection with virulent strain of S. pneumoniae. TLR4 agonist activity can be potentially exploited to provide short-term resistance to infectious challenge such as might occur in the setting of exposure to bio-threat agents or epidemic infections. These observations have implications for prophylactic treatment after an index case is identified or as adjunctive therapies with antibiotics. It is plausible that compounds capable of stimulating early host defense and microbial clearance, but not the latter phases of inflammatory tissue injury associated with sepsis, may be advantageous. The effect of LPS postinfection, as adjunctive therapy, in a lethal murine model of systemic Streptococcus pneumoniae was investigated. Mice were inoculated intravenously with a lethal dose of 107 cfu of S. pneumoniae serotype 2; and treated with a combination of 25 mg/kg Ceftriaxone (i.p) and 10ug of LPS (i.v) at 6 hours post infection or ceftriaxone alone. The survival of mice treated with the combination of ceftrioxone and LPS was 80% in C57BL/c (n=20), compared to 40% of mice receiving ceftrioxone only (n=20) and 0% of vehicle controls (n=10) (p=0.0001). Treatment with a non-lethal dose of LPS beginning at 3 hours after infection reduced mortality by 50% in C57BL/6 mice (n=7) (p=0.03) and no survival benefits (100 % mortality) were observed when LPS alone was administered 6h after bacterial challenge (n=5) (p=0.07). Administration of LPS in combination with ceftriaxone resulted in a significant reduction of bacteremia at 7h (2.5 ± 1.6 vs 6.6 ± 1.6 log10 CFU/ml, p=0.002 and 12h (0.5 ± 1.0 vs 4.9 ±1.6 log10 CFU/ml, p=0001) after infection, compared with animals treated with ceftriaxone alone and also decreased significantly the level of TNF-alpha, IFN-gamma, IL-12p70, MIP-1 alpha, IL-1 beta, and RANTES 12h after bacterial challenge and elevated levels of IL-2, IL-3, IL4, and KC were elevated 12h after bacterial challenge compared to those treated with ceftriaxone alone (p<0.05). In this mouse model, stimulation of TLR4 by highly purified LPS as an additional therapy to the antibiotic Ceftrioxone achieved a survival benefit in the reduction of mortality in severe S. pneumoniae infections. These results show the potential for innate immune stimulation as adjunctive therapy in the treatment of pneumococcal infections and these results warrant further studies. The treatment of pneumococcal infections- such as meningitis, pneumonia, and bacteremia- with 13-lactam antibiotics can result in the paradoxical enhancement of inflammation as a result of the release of proinflammatory bacterial cell components, such as lipoteichoic acid, peptidoglycan, and bacterial toxin pneumolysin. Adenosine has strong immunosuppressive and anti-inflammatory effects mediated by A2A receptor (A2AR) expressed on immune cells. We investigated the effect of A2AR agonist, ATL313, as adjunctive therapy in a lethal C57bl/6 mouse model of systemic Streptococcus pneumoniae. Female C57bl/6 mice were inoculated intravenously with 107 colony forming units (CFU) of S. pneumoniae and treated with the A2A AR agonist ATL313 (5ug/kg or 25ug/kg), or PBS, at t=1, 6hrs and then every 24 hours after bacterial challenging spanning 48 hours. To test whether the effect of ATL313 was through functional A2A adenosine receptors and if hematopoietic cells are important in the protective effect of A2AAR, C57BL/6 A2A-receptor-deficient mice, and chimera were used and groups of C57BL/6 mice were co-injected intraperitoneally with ATL313 and ZM243185 (antagonist ligand selective for the adenosine A2Areceptor). Survival was monitored for 7 days. Blood samples were collected at 7h and 12h after bacterial challenge for bacterial counts and cytokine measurements. ATL 313 (2.5- 25ug/kg), had no survival benefits (100% mortality) when administered without antibiotic ceftriaxone. But when administered in combination with antibiotic ceftriaxone at 6h after infection, 25ug/kg ATL313 increased survival (89%, n=26) of mice compared to ceftriaxone alone treated mice (23%, n=31). Survival benefit was reversed after treatment with ZM241385 (25%; n=8/group) and after ATL313 treatment in A2A AR KO and chimeric mice (17%; N=6/group) compared to ATL 313 treated C57BL/6 mice (89%; p<0.001). Treatment with ATL313 plus ceftriaxone reduced bacteremia by 2.1 log10 and 3.3 log10 fewer bacteria at t=12h and 24h, respectively, than those of mice treated with ceftriaxone alone (p<0.05); and also decreased level of TNF-alpha (p=0.010), IL-6 (p=0.027) , IFN-gamma (p=0.001), IL- 12p70 (p=0.031), MIP-1 alpha/ beta (p=0.005), IL-10 (p=0.022), IL-1 beta (p=0.0071), IL-5 (p=0.014), and Eotaxin (p=0.023) at 12h after bacterial challenge compared to those treated with ceftriaxone alone (p<0.05). In this study, ATL313 in association with ceftriaxone reduced both the magnitude and the duration of inflammation and increased survival in a lethal murine model of systemic Streptococcus pneumoniae. The anti -inflammatory effects of A2A AR agonist ATL313 may be particularly useful for the treatment in bacterial diseases when antibiotics have been administered. Further investigations are warranted to assess the therapeutic benefit of A2A receptor agonists as an adjunctive agent to antibiotics in the treatment of pneumococcal infection and also to further understand the mechanism of protection involved in the ATL 313 protection. The mechanism of protection was investigated by characterizing the effects of A2A R activation on the NK cell activity in pneumococcal sepsis model. Natural killer (NK) cells are potent mediators of the innate immune response and their effect is attributed to both direct cytotoxiciy and indirect stimulation of macrophages by cytokine signaling. A2A adenosine receptor signaling has been implicated in adenosine-mediated inhibition of cytokine production and cytotoxic activity by activated natural killer (NK) cells, although the process of NK cell granule exocytosis is apparently suppressed via a distinct and as yet uncharacterized adenosine receptor. There is evidence that NK cells can be activated during septic events and may contribute to the pathogenesis of this condition via the secretion of IFN-y, which acts primarily to augment macrophage function. Whether modulation of NK activity during pneumococcal sepsis could improve pneumococcal sepsis outcomes and the . ability of an A2A AR agonist to modulate the NK response to improve sepsis has not been tested or available data is scanty and controversial. In this study, mice were inoculated intravenously (i.v) with a lethal dose (107 cfu) of S. pneumoniae serotype 2 and treated with a combination of ceftriaxone and 25ug/kg ATL313 or ceftriaxone alone. To assess the importance of NK cells in pneumococcal sepsis, C57BU6 and Ja281 mice were treated with a single i.p. injection of 200µg anti-NK1.1 (PK136) antibodies 2 days before bacterial challenge. Survival was monitored for seven days. Spleens were harvested and processed to assess NK cell activation (expression of markers CD69 and NK1.1, perforin and intracellular interferon gamma) using flow cytometry. Treatment with the combination of ATL and ceftriaxone (n=4) showed a high level of NK cell populations (2.063 ± 0.277) compared with ceftriaxone alone treated group (1.648 ± 0.566) (p=0.535); down regulated CD69 expression, mean percentage 32.78 ± 6.327 vs 70.03 ± 8.163) (p=0.011); reduced the release of perforin (3,4% vs 0.21% )(p<0.05); and also reduced the level of intracellular and plasma IFN-y (0.55% compared to 12.1%) (p=0.02). Blockade of NK cell activation with the treatment with PK136 and antibiotics resulted in 80% survival compared with 40% of mice that received ceftriaxone alone (p=0.01) in pneumococcal animal sepsis model. The results of the study implicate NK cells as one of the mediator of inflammation sensitive to regulation by A2AR activation. Furthermore, profound protection is imparted when this early event in the inflammatory cascade is inhibited by A2AR activation. Therapeutic agents that inhibit the activity of NK and NKT cells may therefore hold promise in the treatment of pneumococcal infection i.e. it is possible to reduce cytokine levels more substantially by targeting an upstream event in the cascade (NK cell activation). A further study to improve understanding on the negative impact of ATL313 on NK cell function during sepsis is needed. These results suggest that analysis of NK cell activation during the septic process may yield insights into the interactions that occur between NK cells and the monocytes or macrophages compartment during the course of severe bacterial infections. However, the precise molecular and functional mechanism by which the negative impact of NK cells occurs is not known and an improved understanding of NK cell function during sepsis is needed. To investigate the role of decreased NK cell activation by A2AAR treatment in the improved outcomes from experimental sepsis. We need to study the impact of A2AAR agonist on NK cell trafficking during sepsis i.e. study how NK cells impact the pathophysiology of sepsis and determine where NK cells traffic during experimental sepsis and where and to what extent they proliferate. This will permit clarity on whether organ damage corresponds to areas where NK cells traffic. Furthermore, the understanding gained in this process will potentially provide tools with which we may in turn control the immune system when it runs awry in sepsis. Growing antibiotic resistance and paradoxical enhancement of lethality as a result of the release of proinflammatory cell components after antibiotic treatment prompted our study of Etanercept (a tumor necrosis factor alpha neutralizing agent) as an adjunctive therapy against systemic S. pneumoniae. TNF is a major therapeutic target since it appears early and is related to disease severity. Balb/c mice were inoculated intravenously with a lethal dose (107 cfu) of S. pneumoniae serotype 2 and then treated with 25mg/kg ceftriaxone with/out 100ug of Etanercept (i.v) at 4 hours after bacterial challenge. Survival was monitored for several days. Serum samples from mice were analyzed for inflammatory cytokines with bead-based multi-analyte flow cytometry at 24 hours after challenge. Etanercept as adjunctive therapy significantly improved bacterial clearance and survival (70%, n=16) compared with 25% (n=20) survival of ceftriaxone alone treated mice (p=0.001). Inflammatory cytokines, TNF-alpha (787 v/s 5801 pg/ml), IFN- gamma (504 v/s 2642 pg/ml), interleukin-6 (IL-6) (255 v/s 2806 pg/ml), were reduced in Etanercept treated mice compared with ceftriaxone treated mice (p<0.05). In conclusion, this study indicates that early TNF-alpha is a critical component of antibacterial host defense and late neutralization of that same endogenous TNF alpha provides survival benefits when combined with ceftriaxone. Moreover, the current wave of enthusiasm regarding the treatment of patients with anti- TNF antibodies or soluble receptors must be tempered by the awareness of potential infectious complications that may occur as a result of this specific therapy. The study outlines the potential usefulness of Etanercept as an adjunctive therapy for pneumococcal infection and underlines the need for further research in the field. Therefore, more investigations are warranted, to further assess the therapeutic benefit of etanercept as an adjunctive agent to antibiotics in the treatment of pneumococcal infections in a clinical set-up.Item Open Access Prevalence and molecular characterization of enteric viruses and their association with malnutrition in children less than two years old in the Vhembe Region of Limpopo Province, South Africa(2014-01-10) Shivambu, Nurse; Samie, Amidou; Bessong, PascalItem Open Access Studies on human immunodeficiency virus genetic drug resistance and subtype distribution in Northern South Africa(2014-01-10) Nwobegahay, Julius; Bessong, Pascal O.; Selabe, GloriaItem Open Access Genetic Variants of APOC3 Promoter and HLA-B genes in an HIV Infected Cohort in Northern South Africa: A Pilot Study(Molecular Diversity Preservation International (MDPI), 2014-06-26) Masebe, Tracy; Bessong, Obong Pascal; Ndip, Roland Ndip; Meyer, DebraMetabolic disorders and hypersensitivities affect tolerability and impact adherence to highly active antiretroviral therapy (HAART). The aim of this study was to determine the prevalence of C-482T/T-455C variants in the Apolipoprotein C3 (APOC3) promoter gene and Human leukocyte antigen (HLA)-B*57:01, known to impact lipid metabolic disorders and hypersensitivity respectively; and to correlate genotypes with gender, CD4+ cell count and viral load in an HIV infected cohort in northern South Africa. Frequencies of C-482 and T-455 polymorphisms in APOC3 were determined by restriction fragment length polymorphism analysis. Allele determination for HLA-B was performed with Assign SBT software in an HLA library. Analysis of APOC3 C-482 site revealed a prevalence of 196/199 (98.5%) for CC, 1/199 (0.5%) for CT and 2/199 (1.0%) for TT genotype (p = 0.000 with 1° of freedom; χ2 = 126.551). For the T-455 site, prevalences were: 69/199 (35%) for TT and 130/199 (65%) for the CC genotype (p = 0.000 with 1° of freedom; χ2 = 199). There was no association between gender and the presence of −482 (p = 1; χ2 = 0.00001) or −455 genotypes (p = 0.1628; χ2 = 1.9842). There was no significant difference in the increase in CD4+ cell count irrespective of genotypes. Significant increases in CD4+ cell count were observed in males and females considering the −455C OPEN ACCESS Int. J. Mol. Sci. 2014, 15 11404 genotype, but not in males for the −455T genotype. Viral load decreases were significant with the −455C and −482C genotypes irrespective of gender. HLA-B*57:01 was not identified in the study cohort. The apparently high prevalence of APOC3 T-455CC genotype needs confirmation with a larger samples size and triglyceride measurements to support screening of patients to pre-empt HAART associated lipid disorders.Item Open Access Molecular diagnosis and characterization of clinical isolates of entamoeba histolytica, giadia lamblia and cyptosporidium species from the United Arab Emirates and South Africa(2014-11-03) ElBakri, Ali Mohammed Kamal; Bessong, P. O.; Potgieter, N.; AbuOdeh, R. OItem Open Access Studies on HIV-1 subtype c drug susceptibility : development of a phenotypic resistance assay and evaluation of plant-derived compounds(2014-11-03) Mavhandu, Lufuno Grace; Bessong, Pascal. Obong; Rekosh, David.; Hammarskjold, Marie-LouItem Open Access Expression of an active HIV-1 subtype C protease(2014-11-03) Tambani, Tshifhiwa; Bessong, Pascal. O.; Tebit, Denis.Item Open Access Prevalence and molecular identification of candida oral infections in HIV patients attending treatment centres, Vhembe District, Limpopo Province(2014-11-03) Mashao, Mmbangiseni Beauty; Samie, A.Item Open Access HIV co-infections with cytomegalovirus, hepatitis c virus and human papillomavirus in northern South Africa(2014-11-03) Rikhotso, Mikateko; Bessong, P. O.; Tebit, Denis.Item Open Access Molecular detection of norovirus GI ang GII genotypes in children less than two years of age and impact on child growth(2014-11-03) Moloro, Glenton Thabo; Samie, A.; Bessong, P. O.Background: Noroviruses (NoVs) are, after Rotaviruses, the second most common causative agents of acute gastroenteritis in young children. In South Africa, NoV was first reported in 1993 in gastroenteritis associated with the consumption of salad. NoV antibody prevalence was later reported in both urban and rural South African populations, and Norwalk and Hawaii strains were detected. However very few studies have been conducted to identify strains of oV in the country and their impact on child growth is not well understood. This study aims to identify common NoV genogroups and the strains that are more prevalent in diarrhoeal and non-diarrhoeal stool samples of children less than 2 years of age in rural areas in Vhembe district, South Africa, using reverse transcriptase Real Time PCR. Moreover, an inve tigation of the impact of different genotypes on child group was determined. Methodology: In the present study, 185 children were recruited of whom 88 were males and 97 females. Of all the children 141 had experienced diarrhoea at least once while 44 never had diarrhoea. Samples were treated with Sodium Chloride (NaCl), and RNA was purified from them using the QiaAmp viral RNA purification kit and stored at -20°C. Following RNA purification, samples were subjected to One step reverse transcriptase real-time PCR to detect oV genotypes. Positive samples were further run in reverse transcriptase PCR using specific primers that amplify genogroup-specific sequences of the N-terminal and shell (N/S) region of the NoV VPI gene, and the cDNA synthesized was run in a conventional PCR. uccessfully amplified conventional PCR products for NoV GI and NoV GII were sequenced and the sequences aligned and compared with the existing sequences in the GeneBank, in order to determine the genetic relationship and variability of strains of NoV in Vhembe district, South Africa. Results: In the study, 708 samples were tested, of which 256 (36.2%) were diarrhoeal samples and 453 (63.8%) were non-diarrhoeal samples. Norovirus GIi was the most common genogroup detected in the present study. Among the 256 diarrhoeal samples, 34 (24.1%) were GI, and 93 (66.0%) were GIi. Five (13.2%) of the children who were born stunted presented with NoV GI, whilst 22 (57.9%) of them presented with NoV GIi at 12 months. The number of infection increased with the children's age. About 12 (61.7%) of the children who were stunted presented with NoV GI and 29 (61.7%) of the children who were stunted presented with NoV GIi. Norovirus GIi infections showed to be the highest in June (48.1%), October (48.0%) and November (50.6%) whilst infection with Norovirus GI was the highest in October (20.0%). Analysis of sequences showed that the NoV strains differed in their sequences up to 40%. Comparison of study strains with reference has shown the difference in the sequences of the strains, indicating a high mutation rate among Norovirus strains. Discussion: The present study shows that infection with GIi is the most common variant among children and contracted easily during the winter months of the year. Reverse-transcriptase RT-PCR can be recommended as a rapid and sensitive method that can be used to detect NoV, in order to give a quick response to eliminate and prevent infection. Though sequences detected are of similar strains, their nucleotide alignment varies due to the high mutation rate of NoV. Mutations in NoV can cause a problem with vaccine development or an alteration to confer vaccination in order to prevent infection, and spread ofNoV. Conclusion: The present study shows how NoV affects the children's health and has an effect on the child's nutritional state and growth. It also shows that Norovirus strains differ from one country to another, this also include seasonal variation of Norovirus genogroups, which calls for a lot of attention on vaccine development. Norovirus has shown to be more active during winter season and has NoV GII as a predominant strain of which other studies also agree with these results.Item Open Access Characterization of heat shock protein 70-z (PfHsp70-z) from plasmodium falciparium(2015) Zininga, Tawanda; Shonhai, Addmore; Burger, A.Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually, mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae, P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is central to protein homeostasis and determines the fate of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70 genes encode for proteins that are localised in different sub-cellular compartments. Of these two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum, PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in prevention of protein aggregation and facilitates protein folding. Little is known about PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the folding of proteins possessing asparagine rich repeats. However, based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study, PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was investigated and its association with PfHsp70-1 was explored using a co-immuno precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress and the protein is heat stable based on investigations conducted using Circular Dichroism. Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1 were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro. Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum iii PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of functions of this protein, could explain this PfHsp70-z is an essential protein in malaria parasite survival. This is the first study to show that PfHsp70-z possess independent chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable molecule and that it is capable of forming high order oligomers.Item Open Access Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED)(Lancet Glob Health, 2015) Platts-Mills, James A.Background Most studies of the causes of diarrhoea in low-income and middle-income countries have looked at severe disease in people presenting for care, and there are few estimates of pathogen-specifi c diarrhoea burdens in the community. Methods We undertook a birth cohort study with not only intensive community surveillance for diarrhoea but also routine collection of non-diarrhoeal stools from eight sites in South America, Africa, and Asia. We enrolled children within 17 days of birth, and diarrhoeal episodes (defi ned as maternal report of three or more loose stools in 24 h, or one loose stool with visible blood) were identifi ed through twice-weekly home visits by fi eldworkers over a follow-up period of 24 months. Non-diarrhoeal stool specimens were also collected for surveillance for months 1–12, 15, 18, 21, and 24. Stools were analysed for a broad range of enteropathogens using culture, enzyme immunoassay, and PCR. We used the adjusted attributable fraction (AF) to estimate pathogen-specifi c burdens of diarrhoea. Findings Between November 26, 2009, and February 25, 2014, we tested 7318 diarrhoeal and 24 310 non-diarrhoeal stools collected from 2145 children aged 0–24 months. Pathogen detection was common in non-diarrhoeal stools but was higher with diarrhoea. Norovirus GII (AF 5·2%, 95% CI 3·0–7·1), rotavirus (4·8%, 4·5–5·0), Campylobacter spp (3·5%, 0·4–6·3), astrovirus (2·7%, 2·2–3·1), and Cryptosporidium spp (2·0%, 1·3–2·6) exhibited the highest attributable burdens of diarrhoea in the fi rst year of life. The major pathogens associated with diarrhoea in the second year of life were Campylobacter spp (7·9%, 3·1–12·1), norovirus GII (5·4%, 2·1–7·8), rotavirus (4·9%, 4·4–5·2), astrovirus (4·2%, 3·5–4·7), and Shigella spp (4·0%, 3·6–4·3). Rotavirus had the highest AF for sites without rotavirus vaccination and the fi fth highest AF for sites with the vaccination. There was substantial variation in pathogens according to geography, diarrhoea severity, and season. Bloody diarrhoea was primarily associated with Campylobacter spp and Shigella spp, fever and vomiting with rotavirus, and vomiting with norovirus GII. Interpretation There was substantial heterogeneity in pathogen-specifi c burdens of diarrhoea, with important determinants including age, geography, season, rotavirus vaccine usage, and symptoms. These fi ndings suggest that although single-pathogen strategies have an important role in the reduction of the burden of severe diarrhoeal disease, the eff ect of such interventions on total diarrhoeal incidence at the community level might be limited.Item Open Access Application of cloning in the detection of HIV-1 and drug resistant minority populations(2015-01-14) Hatyoka, Luiza Miyanda; Bessong, T. M.; Masebe, Tracy. M.Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). The virus is genetically highly diversified with different genetic strains infecting different populations worldwide. Highly active antiretroviral therapy (HAART) reduces the morbidity and mortality due to AIDS. Unfortunately, the efficiency of these drugs is limited by the development of drug resistance usually caused by mutations in the protease and reverse transcriptase genes which complicates patient management. Sequencing direct PCR products reveals the properties of major viral populations but has the potential to miss minority viral populations. This clearly means that resistant strains of minority populations may not be detected in direct sequencing of PCR products. Furthermore, if multiple drug resistance mutations are detected as mixtures in a given sample, it is not possible to determine whether the individual drug resistance mutations are present together on a single virus or exist in different viral sub-populations. Therefore, the general objective of this study was to design and evaluate a single genome sequencing protocol for the detection of HIV-1 drug resistance mutations in the protease and reverse transcriptase genes. In this study the HIV Pol gene ( for protease and reverse transcriptase genes respectively) was amplified from 8 samples of HIV-1 infected individuals (7 treatment experienced and 1 treatment na·ive). Subsequent cloning was done on all 8 samples using the Topo TA cloning vector, 35 and 33 clones were obtained for protease and reverse transcriptase respectively. Sequencing of purified PCR products and viral clones was done using a BigDye terminator sequencer. A comparison of sequences obtained from direct sequencing of PCR products and cloned PCR sequences was done. Phylogenetic analysis confirmed that all sequences were of HIV-1 subtype C and amino acid determination helped revealed amino acid substitutions in comparison to the HIV-1 subtype B and C global consensus. Using the Stanford drug resistance interpretation program, sequences obtained by direct sequencing of PCR products revealed mutations V11I (a minor resistant mutation for the protease region) and M184V (a NRTI resistant mutation), K101E and Y181C (NNRTI resistant mutations) from samples MARBB14 and MARBB73. Viral clones of samples MARBB14 and MARBB73 revealed the same mutations as those observed by direct sequencing of PCR products. In addition viral clones of sample MARBB73 also revealed reverse transcriptase mutations T215Y (a NRTI resistant mutation) and Y181V (a NNRTI resistant Mutation). Polymorphisms such as K20R, M361, M36V, L63P, H69K, V771 and V821 selected by protease inhibitors were also observed in the protease region from both direct sequences of PCR products and cloned PCR sequences. Using the student t-test, p values of 0.03 and 0.0002 for protease and reverse transcriptase respectively were obtained from comparisons of resistant mutations observed in sequences of PCR products and clone sequences for both protease and reverse transcriptase genes and the difference was considered significant. The cloning technique was used as an approach to detect minority variants. Important resistant mutations were detected in clone sequences and not in sequences of PCR products. This suggests that mutations of interest could be missed in the direct sequencing of PCR products which is currently the norm in many settings.Item Metadata only Screening of herbal preparation (Pheko) for anti HIV-1 replication properties(2015-01-14) Matume, Nontokozo Daphney; Bessong, P. O.; Sekhoacha, M.