Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 42-47).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Zoology / Master / Master of Philosophy

Background:  The PDZ domain is a peptide sequence of 80-90 amino acids and can be found in e.g. bacteria, animals and plants. These domains are commonly part of the cytoplasmic and membrane adapter proteins and its function are important in protein-protein interactions. The NS1 proteins of influenza A viruses play an important role in inhibiting the IFN-β production in many ways. In the C-terminus of the NS1 protein, a peptide sequence of four amino acids had been demonstrated to bind to the PDZ domain termed as PDZ binding motif (PBM). Objective:  The aim of this study is to determine whether the PBM sequence of the NS1 protein of influenza A virus plays a key roll in the expression of interferon-β. Methods:  The open reading frame of the NS1 protein was amplified and cloned into expressing vector and transfected into A549 cells along with a reporter plasmid containing ISRE promoter, driving expression of firefly luciferase. Dual luciferase reporter assay was performed to measure luciferase activity which represented expression of IFN-β. The assay was performed only once and unfortunately the result can not be trusted since the negative control showed positive value. Therefore, to understand the interaction between the PBM sequence of NS1 proteins and the production of IFN-β, further experiments are needed.

Vaccines directed against the influenza virus become ineffective due to continuous mutation. An alternative approach might be to control replication at the genomic level by enzymatic methylation of the polymerase genes. Hence in this study, a method to locate and successfully isolate the H1N1 influenza A polymerase genes was investigated. The virus was cultured in chick embryos via the allantoic route using aseptic techniques. Following incubation, the allantoic fluid was isolated and washed to remove any contaminating blood cells. The allantoic fluid was checked for fungal and bacterial contamination using the blood agar test and the presence of the virus was established by the haemagglutination titration test. Viral particles were pelleted by ultracentrifugation. Electron microscopy verified the morphology and size of these viruses while immunofluorescence studies, using a monoclonal antibody, confirmed the influenza A strain. The ribose test verified the presence of RNA in the samples. Purified viral pellets were pooled and homogenised in buffer containing guanidine thiocyanate, mercaptoethanol and sarkosyl. The samples were incubated on ice before mechanical disruption of the virus. Viral RNA was isolated from the upper aqueous layer after a standard phenol/chloroform extraction procedure. RNA was quantified spectrophotometrically and purity assessed initially by the absorbance ratio readings at 260/280 nm. Electrophoresis of the RNA samples was performed together with RNA molecular weight markers on a 1.5% formamide agarose gel. Five bands were identified and the band containing the polymerase genes was size selected, located and excised. Purification of the polymerase genes from the agarose was achieved by using the BIO 101 RNAid kit. The three isolated polymerase RNAs were reverse transcribed using the Boehringer Mannheim cDNA synthesis kit. The results indicate that the H1N1 influenza virus was successfully grown and isolated from chick embryos. Absence of contamination and verification of viral presence at different stages of the study were indications that asepsis was successfully achieved. The RNA obtained was sufficient and suitable for cDNA synthesis. This cDNA may now be used for further molecular analysis and subsequent DNA methylation studies. Further, transfection studies may then be performed to determine, if any, the the expression of methylated and unmethylated cDNA. / Thesis (M.Med.)-University of Natal, 1992.

Thesis (D.P.H.)--University of Michigan.

DISSERTATION (PH.D.)--THE UNIVERSITY OF MICHIGAN

Thesis (M.Med.Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 42-47). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University OF MICHIGAN.