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Computer-aided drug design for influenza A virusSun, Jian, 孙健 January 2009 (has links)
published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy
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Generation of soluble, catalytically active covalent HIV-1 subtype C integrase-DNA complexes to identify novel strand transfer inhibitorsBeyleveld, Grant James January 2012 (has links)
Dissertation submitted to the Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of
Master of Science in Medicine.
Johannesburg, 2011 / The HIV-1 integrase (IN) enzyme is an integral part of the viral replication cycle
and has no known human homologues, making it an ideal target for antiretroviral
therapy. To date, only one inhibitor of IN strand transfer activity (Raltegravir,
IsentressTM) is available for human use. However, the inevitable emergence of
antiretroviral drug resistance requires ongoing research into new/novel therapies.
There are currently no assays to screen for IN inhibitors against HIV-1 subtype C
in South Africa (and worldwide), therefore, the overall objective of this study was to
generate and characterize locally relevant, soluble, functional recombinant HIV-1
subtype C IN proteins for use in strand transfer assays. Recombinant integrase
genes, including a soluble HIV-1 subtype C mutant (05ZAFV6 with C56S, C65S,
W131D, F185D and C280S) and HIV-1 subtype C Y143C mutant (05ZAFV6
soluble with Y143C) were designed, generated and cloned in frame into pET15b.
Optimal bacterial expression conditions for the expression of these constructs as
well as an HIV-1 subtype C wild type (05ZAFV6), subtype B wild type (NL4-3), and
subtype B soluble (NL4-3 with F185K and C280S; as controls) IN, in E.coli BL21
cells were determined. All five recombinant IN were successfully purified using
nickel affinity chromatography, and subsequently used to establish a strand
transfer assay to assess their activity and their response to two well-known
integrase inhibitors, L-Chicoric acid and Raltegravir. All five recombinant IN
proteins were found to be biologically active, with INY143C (116.67%) showing
equivalent activity to INBwt (117.37%), while INCsol (52.96%) was the lowest. The
IC50 values of L-Chicoric acid were higher than the expected values for all five
recombinant IN, with the subtype B and C IN solubility mutations contributing to an
increased resistance to inhibition by L-Chicoric acid.
The dose responses to Raltegravir for INCwt and INBsol were as expected, with
IC50’s in line with published data, and the INY143C mutant (known mutation
conferring resistance to Raltegravir) was resistant to inhibition of strand transfer
activity at all Raltegravir concentrations tested except the highest (50 μM).
Finally, methods to complex the INY143C mutant to thiolated-DNA were evaluated,
however definitive data could not be obtained. Future work should focus on
optimization of the purification and characterization of the IN-DNA complexes.
Overall, this study has led to the establishment of functional strand transfer assays
based on HIV-1 subtype C recombinant IN proteins, and established a framework
for screening of novel HIV-1 subtype C IN inhibitors.
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Internalization of trichosanthin initiating cellular signal transduction involved in its cytotoxicity and antiviral mechanism. / CUHK electronic theses & dissertations collectionJanuary 2003 (has links)
Huang Hai. / "May 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. (158-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.
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Evaluation of methods of susceptibility testing for laribacter hongkongensisPoon, Wing-shan, Rosana., 潘穎珊. January 2005 (has links)
published_or_final_version / Medical Sciences / Master / Master of Medical Sciences
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Development of a high-throughput screening platform to identify small molecule inhibitors targeting influenza A virusTsui, Heung-wing, Wayne, 徐向榮 January 2006 (has links)
published_or_final_version / Medical Sciences / Master / Master of Medical Sciences
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The role of antivirals and vaccines in the control of influenza epidemics and pandemicsNg, Sophia., 吳鈺陪. January 2012 (has links)
Influenza vaccination is the best preventive measure against influenza virus infection, and antivirals including oseltamivir are effective treatments. From a public health point of view, it is important to evaluate whether vaccination and antiviral treatment reduces transmission of the virus. I analyzed data from a community-based study of influenza virus transmission in households, and identified effectiveness of antiviral treatment in reducing duration of illness and some evidence that treatment reduced transmission to household contacts. I also analyzed data from a community-based placebo-controlled trial of influenza vaccination and confirmed efficacy of vaccination against seasonal influenza but differential efficacy against pandemic influenza possibly because of timing and mediation of seasonal influenza epidemics. In further analyses I found that antibody titers of 1:40 correlated with 50% protection against infection, and repeated vaccination with the same strains tended to be associated with reduced responses to those strains although there was no evidence of reduced efficacy. In the study, one child in each household was randomly allocated to receive vaccine or placebo and I did not identify any evidence of indirect benefits to the household members of vaccinated children. I reviewed vaccine target groups in different countries, and noted that some countries now include school-age children in their target groups based mainly on the principle of herd immunity. My findings did not support the inclusion of school-age children as a target group for vaccination in Hong Kong. Further studies should examine the indirect as well as direct benefits of vaccination in different settings in order to guide optimal influenza vaccination policies. / published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy
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Mechanism study of novel CCR5 antagonists and their potential as anti-HIV-1 microbicidesKang, Yuanxi., 康元曦. January 2012 (has links)
R5-tropic HIV-1 is predominantly transmitted during unprotected sexual contacts, rendering CCR5 antagonist as an attractive agent not only for antiretroviral therapy but also for prevention. Here, we report two 1,3,3,4-tetrasubstituted pyrrolidine embodied compounds, TD-0232 and TD-0680, as novel small molecule CCR5 antagonists and investigate their specificities, potencies and underlying mechanisms. We found that both TD-0232 and TD-0680 inhibited a diverse group of R5-tropic HIV-1 and SIV strains in both single-cycle infectivity assays and live viral PBMC assays. When compared to other CCR5 antagonists, such as TAK-779 and the only FDA-approved Maraviroc, TD-0680 displayed the highest potency with EC50 values at the subnanomolar levels (range 0.09nM-2.29nM). TD-0232 and TD-0680, but not Tenofovir, a nucleoside reverse transcriptase inhibitor, completely blocked envelope-mediated cell-cell fusion and subsequent viral transmission. Critically, TD-0680 was potent at inhibiting the replication of a TAK-779/Maraviroc-resistant HIV-1 variant in PBMCs at a subnanomolar concentration.
Interestingly, despite binding to a similar transmembrane pocket of CCR5, TD-0232 and TD-0680 functioned differently as revealed by site-directed mutagenesis and drug combination assays. Based on the sequence homology, we constructed a CCR5 molecule model using the crystallized CXCR4 as a template. By docking of CCR5 antagonists with CCR5, we identified a unique binding mode of TD-0680, which has not been described previously. TD-0680, with an exo-configuration, extended its interaction with the ECL-2 region of CCR5 in a protruding manner, thereby interrupting the interaction between the virus and its co-receptor more effectively. In an antibody recognition assay, we confirmed that TD-0680 had an enhanced inhibitory activity against the anti-ECL2 monoclonal antibodies binding.
Furthermore, we investigated the antiviral activities of TD-0232 and TD-0680 that were formulated into a thermo-reversible acidic microbicide gel. Both drugs were stable in the acidic gels and could be released rapidly for long lasting and potent antiviral activities. Although human semen could enhance the infection of HIV-1, it did not seem to affect the potencies of the TD-0232 and TD-0680 gels.
In summary, our findings suggest that TD-0232 and TD-0680 can be further developed not only as anti-HIV-1 agents for therapeutic purposes but also as potent microbicides for the prevention of sexual transmission of R5-tropic HIV-1. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy
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Structural characterization of H1N1 nucleoprotein-nucleozin binding sitesPang, Bo, 龐博 January 2013 (has links)
Although influenza is usually acute self-limiting respiratory infection, influenza viruses are among the most common pathogens that threaten the health of humans and animals worldwide. Various anti-viral therapeutic agents are currently used for treatment and prophylaxis of influenza virus, but the problem is that the targets of these drugs are easily mutated and result in resistance. Therefore, medications that have broad spectrum coverage are urgently needed to combat with the disease. Since nucleoprotein (NP), which is encoded by influenza virus genome, is regarded as a druggable target due to its conserved sequence and important functions during influenza virus life cycle, numerous studies are focused on this protein in attempts to develop broad-spectrum anti-influenza therapeutics. Recently, Kao et al. found that the addition of a novel small molecule nucleozin could lead to large aggregates of NP, which in turn caused cessation of virus replication. Give that the interaction between NP and nucleozin is still not unveiled, it is crucial to identify the binding sites using X-ray crystallography.
The full length influenza A/WSN/33 (H1N1) NP gene was cloned into pET28 vector, with His-tag in its C-terminus and overexpressed in E.coli strain Rosetta 2. Cell culture was purified by HisTrap HP and Superdex-200 16/60 gel filtration columns. Crystals were grown using the vapour diffusion method and the NP-nucleozin complex was prepared by soaking native crystal in solution containing 0.25 mM nucleozin for 2h. Crystals of the complex can diffract to 3.0 Å at the Shanghai Synchrotron Radiation Facility. The structure of NP was determined by molecular replacement and it belongs to space group C121 with two NP trimers per asymmetric unit. After further refinement, two nucleozin molecules were found in each asymmetric unit, and each of them could bind with two NP molecules at the same time. The ligand binding pockets were formed by the combination of Y289/N309 pocket from one NP molecule, and R382 pocket from another NP molecule. Therefore, the function of nucleozin is to bridge two NP molecules and lead to NP aggregation, which are in agreement with functional studies on nucleozin. Furthermore, computational models of the NP-nucleozin binding are provided to reveal the mechanism of nucleozin induced aggregation. In addition, recent work on interaction between NP and another novel molecule named compound A has also been briefly described and compared with NP-nucleozin complex at the end of this thesis. Collectively, this study presents a new paradigm for better understanding of how NP and nucleozin interact with each other and hence result in NP aggregates, which is envisaged to accelerate the development of anti-influenza therapeutic agents. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy
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An investigation of the antiviral effect of several Arizona plant extracts on influenza virus A in miceFried, Mary Lakritz, 1925- January 1954 (has links)
No description available.
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Characterization of interferon and retroposon-like repetitive elements in salmonid fishTengelsen, Leslie A. 11 August 1992 (has links)
Hatchery-reared salmonid fish routinely encounter stress due to
handling, barging, tagging, and overcrowding. It has been demonstrated that
there exists a direct correlation between stress and transient immune
suppression which can last for many days in fish. Epizootic viral infections
routinely appear in hatcheries and can have a devastating effect on the fish
population. The major viral pathogens in salmon and trout are the fish
rhabdovirus, infectious hematopoietic necrosis virus (IHNV), and the fish
birnavirus, infectious pancreatic necrosis virus (IPNV). Vaccines for these
viral pathogens are under investigation; however, the fish immune system
becomes virtually nonresponsive during episodes of immune suppression. It
was necessary to develop a nonantibody mediated, nonimmune method for
preventing viral infections.
An interferon-like substance has been described for fish which
possesses antiviral activity against both IHNV and IPNV. Since interferon
administered to cattle has been very effective against vesicular stomatitis
virus, a cattle rhabdovirus, an examination of interferon-like activity in fish was
initiated. We report here the establishment of in vitro interferon assays. In
addition, the salmonid genome contains a multigene family of p-interferon-like
genes, much like those in the bovine, equine and porcine genome. The
rainbow trout interferon-like genes were found to be inducible in a manner
which parallels those seen with bovine and human interferons.
In addition to the multigene interferon-like family, it was found that
rainbow trout also contain a retroposon multigene family. Retroposons are
repetitive elements which appear to have arisen by a reverse transcription
event. Two Ll like repetitive elements have been cloned, one of which
contains a Drosophila retroposon polymerase sequences never before
described for salmonid fish. A number of retroviruses have been described in
fish including the walleye dermal sarcoma virus and the Atlantic salmon
swimbladder sarcoma virus. Interferon shows prophylactic promise both in
vivo and in vitro, against the human retrovirus, HIV. Therefore, research into
fish interferon may be even more important if it demonstrates not only anti-
IHNV and anti-IPNV, but also anti-fish retrovirus properties. / Graduation date: 1993
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