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

No description available.

Vesicular stomatitis virus (VSV) is considered a prototype for studying non-segmented negative-stranded ribonucleic acid (RNA) viruses. Livestock are naturally infected by VSV, causing severe economic impact due to lack of any effective treatment. RNA interference (RNAi)-based therapeutics are promising alternatives to control viral infections. Lentiviral vector systems deliver artificial short hairpin RNA (shRNA) into the genome of cells to activate the RNAi pathway. In this study, an RNAi-based approach to generate cell lines with reduced susceptibility to VSV (Indiana) infection was tested. First, eight shRNAs targeting either the nucleocapsid (N), phosphoprotein (P), or the polymerase (L) viral genes were designed and introduced into cell systems. To test the potency of the shRNAs for silencing the target viral transcripts, semi-quantitative polymerase chain reaction (PCR) analysis of viral N, P, and L transcripts was performed. Then, supernatants from infected groups were evaluated by microtitration and immunoblot. Finally, the effect of VSV genomic variability in the target region of shRNAs was predicted by partial sequencing field and laboratory-adapted strains. Viral transcripts were significantly reduced in cells stably expressing shRNAs targeting the N viral gene (nucleotides 67-97 or 1312-1332; p<0.05) or P gene (nucleotides 1772-1792; p&lt;0.05). Reduction in viral transcripts was not observed by other VSV-shRNAs tested. Reduction of viral transcripts by the N-shRNA (sh-1312) was accompanied by a decrease in viral protein. Also, a reduction in the viral particles shed from cells expressing N-shRNAs (nucleotides 67-97, p&lt;0.05) was noted. The results also showed complementarity of target gene sequences for shRNAs in the sequence from the laboratory-adapted strain and single base substitutions in the corresponding regions from VSV field isolates. However, these mismatches did not occur within the seed region of the shRNAs. In conclusion, partial silencing of viral transcripts by a single shRNA does not block VSIV replication; however, partial impairment of VSIV replication was observed in N-shRNAs expressing cells. During infection, the naturally high level of N gene transcription may have modulated the sh-RNA effect. The combination of the most potent shRNAs identified here into a multiple shRNA vector may result in further reduction of viral replication. These data contribute to ongoing development of effective RNAi-based technologies to combat viral diseases.

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

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 118-125). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR19667

Diss. (sammanfattning) Göteborg : Univ., 2007. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 110-125).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1959. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 57-62).

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.