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Bibliography: leaves 127-138. This thesis studies the minor double stranded RNAs (dsRNA) and single stranded RNAs (ssRNA) which are consistently associated with plants infected with Q strain of cucumber mosaic virus (Q-CMV). The investigations are focused on the structural elucidation of new RNAs which have been observed in single stranded and double stranded RNA profiles of Q strain of CMV.

Three closely related isolates belonging to the A��� serotype of infectious pancreatic necrosis virus (IPNV) were selected for comparison, to provide insight into the nature of variation in the virulence of IPN viruses. Brook trout fry (Salvelinus fontinalis) were experimentally infected with the three isolates by immersion. Cumulative mortalities over a 62 day period for the three isolates were 67%, 78%, and 93%. The negative control was 3%. Virus titers from whole fish homogenates sampled at peak mortality for each isolate were statistically similar, indicating that quantity of virus does not account for virulence differences. For the two least virulent isolates, the virus titer was inversely correlated with fish weight, whereas for the most virulent isolate, no correlation was observed. Amino acid sequences of the viral capsid protein VP2 were determined using the reverse transcriptase polymerase chain reaction (RT-PCR). There were two amino acid changes at residue 217 and 288 between the two least virulent isolates and the most virulent isolate. These changes might provide a specific molecular basis for the variations in virulence among isolates. The progression of IPN virus infection in the experimentally infected fry was followed using histopathology, in situ cDNA hybridization, and alkaline phosphatase immunohistochemistry (APIH). While microscopic lesions were limited almost exclusively to necrosis of the pyloric caeca and pancreas, positive reactions with in situ hybridization and APIH were observed in tissues throughout infected fish. An IPNV infection appeared to be established in the fish by two routes: by entering the skin/lateral line and diffusing through the muscle, and from the oral region into the gastrointestinal tract by ingestion. In a second experiment, within a group of experimentally infected brook trout fry, external and behavioral signs of IPN disease in moribund fish disappeared, with the fish becoming healthy in appearance. Several of these fish were sampled, along with dead, moribund, and asymptomatic fish (never showed signs of IPN disease). Very few differences were observed among the fish sampled, using histopathology and in situ hybridization. Fish that appeared to recover after displaying signs of IPN disease died within a 2 week period. / Graduation date: 1997

Thesis (Ph. D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed Jan. 26, 2010). Additional advisors: Asim K. Bej, Gail E. Christie, Peter E. Prevelige, Jr., R. Douglas Watson. Includes bibliographical references.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Influenza infections continue to be a global health concern that causing both seasonal epidemics and unpredictable pandemics. Hemagglutinin (HA) and Neuraminidase (NA) are the two major surface glycoproteins of influenza viruses, which are important for their host cell sialic acid (Sia) receptor binding and cleaving activities. Although numerous methods have been developed to study the HA and NA interactions with sialic acid, x-ray crystallography remained the only method to provide detailed information at atomic resolution. The aim of this study is to develop and evaluate a novel strategy for the investigation of influenza virus-receptor interactions, which is able to provide information about an interaction down to atomic resolution. Influenza virus-like particles (VLPs) containing HA and NA separately were developed and it was reported here for the first time that sole expression of NA in mammalian cell led to VLP formation. Characterization of these VLPs demonstrated that they are non-infectious, but morphologically and biochemically mimic the native viruses. Therefore the VLPs can be regarded as an ideal research model to study the HA-Sia interaction without the interference of NA, or vice versa. Saturation transfer difference (STD) NMR spectroscopy is a state-of-the-art technology to determine how a binding-ligand interacts with its target protein. Modification of STD-NMR methodology was performed to adapt the technique to influenza VLP system. HA-Sia interaction was investigated in great detail and group epitope mapping of the interacting ligands was performed by analyzing the STD-NMR spectra. The data obtained are in a good agreement with the well established crystallography technique, reflecting the reliability of the STD-NMR technology. Regarding the NA-Sia interaction, my data demonstrated that substrate-hydrolysis specificity of NA is dependent on the binding of NA to those ligands. In addition, using competition experiments with NA inhibitor, a secondary sialic acid binding site was detected. It is the first direct experimental evidence that confirms avian, seasonal human and human pandemic swine-origin influenza virus N1 neuraminidases exhibit a distinct secondary binding site. In conclusion, here I presented a novel interdisciplinary strategy using VLP and NMR technology to study the interaction of influenza virus with its receptor. This method is unique in its ability to provide detailed information on the HA and NA interactions with sialic acid leading to group epitope mapping of the binding ligands, which will help us not only to understand the virus tropism but also to define new therapeutic targets. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy