915 research outputs found
Fibronectin: role in viral cell association, fusion and entry of influenza A virus
No full textThe influenza A viral hemagglutinin (HA) protein binds to sialic acid (SA) groups of cellular surface glycoproteins to achieve viral attachment and entry. The SA binding specificity of HA is one of the major determinants for controlling viral tropism and host specificity.
Fibronectin (FN) is a ubiquitinious glycoprotein secreted on cell surface, either circulating in plasma, or as one of the best characterized components of the extra cellular matrix. With its binding properties towards different types of molecules and pathogens, it has been utilized by different bacterial and viral pathogens for binding, entry, propagation and pathogenesis. The binding affinity and region of plasma FN to influenza A viral glycoprotein was identified in early 1980s. Evidence also suggests the binding is SA associated. FN associates with different viral pathogens. However, evidence of FN direct involvement in influenza A pathogenesis remains unknown.
The objective of this thesis is to test the involvement of cellular FN in influenza A viral infection. To perform the study, FN siRNA and anti-FN antibody were applied. This study demonstrated possible involvement of FN in the replication of human H1N1 and highly pathogenic avian H5N1 viruses. It also discovered that FN is very important for the replication of H1N1 virus, but not H5N1 virus. Interestingly, the result suggested that FN does not affect the initial virus-host binding, but it has an effect on post-attachment events.
Key amino acid positions controlling the SA binding specificity of seasonal human or avian influenza A viruses have been identified in the HA. In this thesis, reverse genetics and mutagenic work identified that viruses with a α2,3-linked SA (SA α2,3) binding preference were not inhibited by anti-FN antibody, while viruses with a α2,6-linked SA (SA α2,6) specificity were severely inhibited. This surprising finding of SA binding preference related FN involvement in post-attachment event led to the further investigation on the structural involvement of FN and viral entry pathway analysis.
The 9th and 10th of type III repeating units of FN form the cell-binding domain of the protein for cell attachment. From site specific antibody inhibitory studies, the cell binding region of FN near the synergy adhesion site(SAS) and Arg-Gly-Asp-Ser(RGDS) cell adhesion signal was identified to be important for the replication of viruses that have a α2,6 SA binding preference, but it was also found to be independent of α5β1 integrin receptor.
After attaching to a host cell, the virus was internalized in an endosome via clathrin- or caveolin- mediated endocytosis. By application of pathway inhibitors, the FN association with viral entry pathway was evaluated. Though this study failed to identify a single specific FN mediated viral entry pathway, this pathway study indicated the possibility of FN various involvement in influenza viral entry. The study indeed indicated that viruses have difference SA binding preferences are different in their choices in viral entry pathways.
This thesis did not only introduce cellular FN as a novel host factor, but also identified possible target and brought new light in the control of influenza A viral infection.published_or_final_versionPublic HealthDoctoralDoctor of Philosoph
The analysis of 5' and 3' untranslated regions (UTRS) of influenza A virus
No full textpublished_or_final_versionabstractMicrobiologyMasterMaster of Philosoph
Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting.
No full textIntroductionWe aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR), high-resolution melting (HRM) assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.FindingsA novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m)) of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T)) for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T) was 32.98 would have an H275Y assay C(T)>30. Analysis of the TaqMan C(T) values for 609 consecutive clinical samples predicted that 207 (34%) of the samples would result in an HRM assay C(T)>30 and therefore not be amenable to the HRM assay.ConclusionsThe use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m) against C(T). Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations
Codon usage biases of influenza A viruses
No full textpublished_or_final_versionMicrobiologyMasterMaster of Philosoph
Popular participation for disempowerment? Democratic constitution making in the context of African liberal democracy
No full textThesis (LLM (Human Rights and Democratisation in Africa)) -- University of Pretoria, 2008.A Dissertation submitted to the Faculty of Law University of Pretoria, in partial fulfilment of the requirements for the degree Masters of Law (LLM in Human Rights and Democratisation in Africa). Prepared under the supervision of Orquidea Palmira Orquidea, Faculty of Law, Universidade Eduardo Mondlane, MozambiqueThe author discusses the nature and reality of the marginalisation and disempowerment of ordinary citizens. He also highlights how democratic constitution making in Africa has so far left marginalisation and disempowerment unchallengedhttp://www.chr.up.ac.za/Centre for Human RightsLL
Non-Invasive Prenatal Monitoring
No full textEmbodiments Of The Present Invention Are Directed To The Detection Of Fetal Or Maternal Rna In A Blood Sample From A Pregnant Subject, And May Involve Subjecting The Sample To A Test For Fetal Or Maternal Analysis Indicative Of A Fetal Or Maternal Condition Or Characteristics. For Instance, The Rna Analysis May Involve The Assessment Of The Gene Expression Pattern Of An Unborn Fetus By Analyzing A Blood Sample From The Mother. The Prenatal Monitoring Technology Allows, For The First Time, The Detection Of Genes Which Are Expressed By The Fetus, Just By Analysis Of A Sample Of Maternal Blood. In Specific Embodiments, The Prenatal Monitoring Technology Is Based On The Discovery Of Circulating Rna Of Fetal Origin In The Plasma Of Pregnant Women.; In General, The Detection Method Performed On A Maternal Serum Or Plasma Sample From A Pregnant Female Comprises Detecting The Presence Of Rna Of Fetal Or Maternal Original In The Sample.published_or_final_versio
Where are the mothers? Interrogating maternal mortality as a violation of the rights to life and health : a Nigerian and Ethiopian perspective
No full textA Dissertation submitted to the Faculty of Law University of Pretoria, in partial fulfilment of the requirements for the degree Masters of Law (LLM in Human Rights and Democratisation in Africa). Prepared under the supervision of Dr. Salah Hammad, Faculty of Law, Addis Ababa University, AddisThesis (LLM (Human Rights and Democratisation in Africa))--University of Pretoria, 2009.The author argues that maternal mortality can easily be avoided and that the right to health and life is as much a developmental issue as it is one of human rights. Focuses on the maternal mortality ratio and relevant laws protecting women’s right to life and health in Nigeria and Ethiopia.http://www.chr.up.ac.za/Centre for Human RightsLL
Synthetic RNA interference against influenza A virus
No full textpublished_or_final_versionabstractMicrobiologyMasterMaster of Philosoph
Non-Invasive Methods For Detecting Non-Host Dna In A Host Using Epigenetic Differences Between The Host And Non-Host Dna
No full textIn A First Aspect, The Present Invention Features Methods For Differentiating Dna Species Originating From Different Individuals In A Biological Sample. These Methods May Be Used To Differentiate Or Detect Fetal Dna In A Maternal Sample Or To Differentiate Dna Of An Organ Donor From Dna Of An Organ Recipient. In Preferred Embodiments, The Dna Species Are Differentiated By Observing Epigenetic Differences In The Dna Species Such As Differences In Dna Methylation. In A Second Aspect, The Present Invention Features Methods Of Detecting Genetic Abnormalities In A Fetus By Detecting Fetal Dna In A Biological Sample Obtained From A Mother. In A Third Aspect, The Present Invention Features Methods For Differentiating Dna Species Originating From An Organ Donor From Those Of An Organ Recipient.; In A Fourth Aspect, The Present Invention Features Kits For Differentiating Dna Species Originating From Different Individuals In A Biological Sample.published_or_final_versio
Identification of human annexin A6 as a novel cellular interactant of influenza A virus M2 protein and regulator of virus budding andrelease
No full textInfluenza viruses exploit sophisticated host cell machinery to replicate, causing both seasonal epidemics and unpredictable pandemics. Studying the host cellular factors interacting with conserved domains of viral proteins will help us to identify key host proteins for the virus infection. This will not only strengthen our understanding of the precise mechanisms of the virus life cycle, but also pave new avenues for anti-viral development.
The cytoplasmic tail of M2 ion channel (M2/CT) is one of these highly conserved domains. It is fully accessible to the host cell machinery after fusion of the virus envelope with the endosomal membrane and during the trafficking, assembly, and budding processes. I hypothesized that recruitment of host cellular factors by M2/CT may regulate the M2-dependent stages of the virus life cycle. Through a large scale yeast two-hybrid (Y2H) screen with the M2/CT used as bait, the human annexin A6 was identified as a novel host cell interactant and this interaction was further confirmed by both GST pull-down assay on purified proteins and co-immunoprecipitation assay on virus infected cells.
A functional characterization of this novel interaction demonstrated that depletion of annexin A6 could enhance the virus production, while its overexpression could reduce the virus propagation, which indicates that annexin A6 is a negative regulator of the virus infection. However, I found that the virus infection could not induce any changes of annexin A6 expression. Therefore, the annexin A6-mediated regulation may depend on the subcellular localization where the interaction with M2/CT occurs. To decipher which step of the virus replication is regulated, we dissected the virus life cycle and found that modulation of annexin A6 expression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected and annexin A6-overexpressing cells during a transmission electron microscopy study. To further decipher the underlying molecular mechanisms, the contribution of annexin A6-mediated plasma membrane lipid rafts reorganization through cholesterol homeostasis modulation and cortical actin cytoskeleton remodeling was also investigated.
In conclusion, here I have identified the human annexin A6 as a novel host cell interactant of M2/CT that negatively modulate the influenza virus infection by impairing the virus budding and release. This work further supports the idea that M2 is a multifunctional protein and is also consistent with the discovery by Rossman et al. that M2/CT mediates the virus budding process (Rossman et al., 2010). This study further emphasizes the importance of host cell interactants of M2/CT in this process. Regarding the biology of annexins, this study also adds a new member of this protein family in the list of regulators of influenza virus infection.published_or_final_versionPublic HealthDoctoralDoctor of Philosoph
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