Respiratory virus infections, such as those caused by influenza, parainfluenza and respiratory syncytial virus (hRSV), continue to be a major cause of morbidity and mortality in both the developed and developing world. Currently, the main means of control of influenza virus infection is vaccination, which requires advanced knowledge of the strain that will be prevalent each year. Alternative strategies involve the use of anti-viral drugs, which function primarily as a prophylactic. Currently, there are five main drugs available against influenza, the adamantanes (amantadine and rimantidine) and the neuraminidase inhibitors (oseltamivir, zanamivir and peramivir). However, major problems exist with antivirals, notably the development of drug resistance. This means that new drugs are urgently required that also satisfy the need to intervene at specific phases of the infection. This thesis describes the development of a peptide with anti-influenza virus activity (Flupep), from which a library of closely related peptides were synthesised, with the aim of optimising antiviral efficacy. Peptides were tested in vitro using a plaque reduction assay on cultured cell lines, Vero and MDCK for parainfluenza and influenza respectively. Two strains of influenza and two of parainfluenza were used, covering the main subtypes that infect humans: Influenza A, Influenza B, PIV2 and PIV3. The plaque assay involved mixing a fixed dose of virus with dilutions of peptide and infecting the cultured cells, followed by incubation for between 3 and 14 days. The cells were then fixed, stained and plaques counted as a measure of viral infectivity. Previous work had shown that Flupep both interacts with haemagglutinin and is an antagonist of inflammatory cytokines. As a possible explanation for antiviral activity, binding affinity of the peptide to haemagglutinin was measured utilising enzyme linked immunosorbent assays. However, significant binding was not detected, suggesting non-specific binding and anti-inflammatory potential are more important routes for antiviral activity. Peptides which demonstrated greater than 90% plaque knockdown in vitro were evaluated in vivo. Anaesthetised mice were infected with influenza A and administered with the peptide concurrently. Following infection, body weights were measured daily and clinical signs, such as shortness of breath, quality of coat and posture, were monitored as indicators of overall health. Most mice were culled on the seventh day post-infection and lung viral titres were determined using a plaque assay. Two peptides were identified with high efficacy against influenza. These peptides, when used in vivo, improved clinical signs of and dramatically reduced levels of infectious virus in the lungs by 7 days post infection. The peptide with highest efficacy was PEGylated and subsequently shown to possess therapeutic potential. Intranasal administration of the PEG-peptide to anaesthetised mice, on the two days subsequent to infection with influenza A, revealed a 17-fold fall in lung viral titres by the fourth day post-infection. Overall, Flupep demonstrates great potential as a future therapeutic agent for treatment of Influenza and potentially Parainfluenza.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Mast cells are immune cells important in innate immunity. Besides their role in asthma and allergies, mast cells are critical effector cells against various pathogens. Mast cells are established to be protective against bacterial infections, but little is known about their functions in viral infections. Mast cells are abundant in the lungs where influenza A virus (FluA) enter the host. We measured mRNA transcription, protein translation, and synthesis of new viral particles in FluA-treated mast cells. While expression of FluA mRNA and proteins followed similar time courses in both mast cells and epithelial cells, mast cells released a near absence of FluA. We also studied mast cell cytokine release in response to FluA and other viral-associated stimuli such as TLR ligands and type I interferons. Mast cells released the cytokines IL-6 and TGF-, and chemokines CXCL8 and CCL5 in response to various viral stimuli. However, different stimuli were capable of inducing release of different mediator subsets, demonstrating the specificity of mast cell responses. Since FluA infection of mast cells produce little new FluA virions, we investigated whether FluA induces expression of antiviral proteins in mast cells. FluA treatment resulted in mast cell expression of antiviral proteins, namely myxovirus resistance protein A, protein kinase R, interferon stimulated gene 15, viral stress inducible protein 56, and endothelial nitric oxide synthase. Next, we performed co-culture experiments using FluA-infected epithelial cells with or without the addition of mast cells. Our results showed that mast cells in co-culture inhibited the expression of the viral hemagglutinin protein in FluA-infected epithelial cells. Also, preliminary results showed that addition of mast cells protected epithelial cells from FluA infection by limiting the release of FluA particles and reducing epithelial cell death. Our discovery that mast cells produce little virus and express antiviral proteins suggest that mast cells have antiviral mechanisms to restrict FluA infection. This concept was further supported by evidence that mast cells are protective against FluA infection in epithelial cells. This research provides a better understanding of mast cells in innate immunity and may reveal unique antiviral mechanisms valuable in the development of antiviral therapeutics.

Les rétrovirus humains tels que HTLV-I (Human T-cell Leukemia Virus) et HIV-l (Human lmmunodefiency Virus Type-l) sont associés à des pathologies mortelles et sont largement répandus à travers le monde. Actuellement, aucun vaccin ni thérapie ne sont disponibles pour prévenir ou enrayer totalement l'infection par ces virus. Le but du présent projet est de développer une nouvelle approche anti-rétrovirale se basant sur l'altération de l'ADN viral intégré dans le génome de la cellule hôte. Nous avons utilisé comme modèle le rétrovirus de la leucémie murine (MuLV) Cas-Br-E. Ce virus induit des leucémies non B et non T et engendre une maladie neurologique spongiforme chez certaines souches de souris. La cible du traitement est l'intégrase (IN), une enzyme clé du cycle réplicatif. Elle catalyse l'intégration de l'ADN du virus dans le génome de la cellule de l'hôte et est également impliquée dans la transcription inverse, le transport de l'ADN viral dans le noyau et l'assemblage des particules virales. Les traitements, des ODN mutagènes de l'lN, sont conçus de manière à introduire une mutation au début de la séquence codante de l'IN, engendrant ainsi la production de particules virales défectives et non infectieuses. Les expérimentations se sont effectuées en deux volets: avec le traitement de cellules NIH NE-8 chroniquement infectées par Cas-Br-E et, ensuite, avec celui de cellules NIH 3T3, infectées par Cas-Br-E et traitées à différentes périodes post-infection. L'impact des traitements a été déterminé par titration des virus produits en immunofluorescence, dosage de la transcriptase inverse et séquençage des produits de PCR des ADN et ARN des cellules traitées. Les résultats obtenus avec les ODN mutagènes montrent une diminution de la production de virus infectieux en fonction du nombre de traitements, de la dose utilisée et du moment où les traitements ont été effectués. Une inhibition totale de la production virale est possible avec l'utilisation d'une seule dose massive de 4,0 µM ou d'une faible dose de 1,34 µM ciblée en début d'infection, soit dans les 4 premiers jours. Des mutations du génome ont été observées au niveau de l'ADN et de l'ARN des cellules traitées. En conclusion, la diminution de la production de virus infectieux, suite aux traitements, permet d'envisager l'utilisation de cette stratégie comme nouveIle thérapie contre les infections par HlV-I et HTLV-I, d'autant plus qu'une fois la production de virus infectieux enrayée totalement, l'inhibition se maintient dans le temps.

No description available.

No description available.

No description available.

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 62-69). / viii, 69 leaves, bound ill. 29 cm

Cyanobacterial secondary metabolites, often identified as toxins such as microcystin, have also demonstrated biological functions including inhibition of bacterial and viral growth. In this study, 10 cyanobacterial strains were isolated from field sites around Adelaide and laboratory cultures and assessed for bioactivity against bacterial, viral and fungal pathogens. A comprehensive literature search identified a number of screening assays employed by research groups to identify cyanobacterial strains with biological activity. Within the review, methods to optimise extraction of the compounds were also noted. Combinations of extraction methods, solvents and assay procedures were investigated to optimise the success of this phase of the study. Bioactivity was confirmed by development of agar disc diffusion and microtitre plate assays to analyse cyanobacterial biomass extracts. Result of the assays indicated a methanolic extract of one species, Microcystis flos-aquae (Wittr Elenkin), inhibited growth of bacterial cells and viral infectivity and was selected for further analysis. The bioactive compound was isolated by HPLC and mass spectrometric analysis. Separation of the bioactive extract into component peaks indicated only one that was likely to represent the metabolite of interest, at a retention time of approximately 18 min. A second profile was constructed of a methanolic extract of the same species in a later growth stage that did not inhibit growth of either the bacterial or viral test organisms. Comparison of the profiles exposed the absence of the peak at 18 min retention time in the second profile. Accumulation of the fraction was conducted using a semi-preparative HPLC column for analysis by mass spectrometry. A sample of the isolated peptide was submitted to Proteomics International, a subsidiary of Murdoch University, WA, for identification and structural characterisation. Proteomics International analysed the data by electronspray ionisation time of flight mass spectrometry (LC/MS/TOF) followed by LC. De novo sequence analysis of the data was carried out using Analyst QS software; however, PI was unable to provide a readily interpretable, continuous amino acid sequence, despite their admission that some gaps in the fragmentation ladder corresponded to known amino acids. Interpretation of the data generated by Proteomics International by a research chemist within the University of Adelaide proposed the following amino acid sequence and subsequent structure for the compound: [Figures omitted] Proposed (a) amino acid sequence and (b) structure for the bioactive compound isolated from non-toxic M. flos-aquae. Comparison of the proposed sequence with those contained in peptide databases was unable to classify the compound (B Neilan, personal communication, April 2008), suggesting the bioactive metabolite is perhaps previously undetected and therefore may be considered a novel compound, or has undergone a modification and is thus a variant of a known compound. Taxonomic classification of the strain used during this study was completed by PCR amplification of 16S ribosomal RNA, using primers from alternative cyanobacterial sources. The sequence was classified in the following taxonomic hierarchy (with 100% assignment detail, for a confidence threshold of 95%): Domain: Bacteria Phylum Cyanobacteria Class Cyanobacteria Family Family 1.1 Genus Microcystis This classification confirms that the species investigated during this research is of the genus Microcystis. Synthesis of cyanobacterial metabolites is generally accepted to be a result of nonribosomal synthetic pathways. The presence of non-ribosomal peptide synthetase and polyketide synthetase genes in Microcystis flos-aquae was confirmed by PCR amplification using degenerate primers from other cyanobacterial sources. Analysis of sequence data identified the presence of an NRPS gene demonstrating significant similarity (98%) to the NRPS cyanopeptolin gene of Microcystis sp. However, the PKS (polyketide) gene identified verified only a 63% similarity to a known sequence, that of the PKS (mcyG) gene of M. aeruginosa PCC 7806 (Koch). Results of the molecular investigation imply this compound may belong within the cyanopeptolin family. Researchers have speculated that the majority of cyanobacteria possess genes for production of toxins, though in many instances the gene cluster may be incomplete or one or more genes may be absent or mutated. The presence of microcystin genes was confirmed by PCR amplification using primers from previously characterised cyanobacterial genes. Analysis of the sequence data identified the presence of several mcy genes generally found in toxic strains of cyanobacteria noted for synthesis of the toxin microcystin. The DNA sequences show significant similarity to the mcyA, mcyC, mcyD and mcyE genes described for Microcystis sp. and Microcystis aeruginosa PCC 7806. However, analysis of the sequence data for the mcyB gene revealed that this gene was not present. Further PCR amplification of the region between mcyA and mcyC using the reverse complements of the original primers indicated that a sequence was present that may have been a truncated variant of mcyB or another gene entirely. Time constraints prohibited submission of this region for sequence analysis. The primary objective of this research project was to screen a field strain of cyanobacteria for synthesis of biologically active secondary metabolites, and to isolate those compounds using a combination of analytical chemistry and molecular biotechnology. This study forms part of a collaborative project between the University of Adelaide, South Australian Research and Development Institute (Aquatic Sciences) and the Environmental Biotechnology Cooperative Research Centre, entitled “P6: Commercial scale integrated biosystems for organic waste and wastewater treatment for the livestock and food processing industries”. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1352988 / Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering, 2009

Thesis (M. Med. Sc.)--University of Hong Kong, 2005. / Also available in print.