The objective of this research is to identify and to investigate molecular determinants of influenza A virus host range. The hemagglutinins (HAs) on these viruses are heterogenous in biological activity and host cells, by exerting different selective pressures, promote the growth of virus populations with different biological properties. Identification of the factors involved in this selection holds promise for understanding and controlling the transfer of these viruses from animal reservoirs to the human population and for improving laboratory isolation procedures so the virus strains which will produce more effective vaccines can be obtained. This proposal describes an experimental plan which uses two variants of a well characterized H1N1 strain to identify and investigate genetically-determined differences in the HA which potentiate host-dependent selection. These substrains, a parent and a mutant virus which differ in the number of complex oligosaccharides on the HA, show host-dependent differences in cell binding properties. The location of the extra oligosaccharide on the parent HA will be determined from sequence data. Revertants of the mutant virus will be isolated and characterized in order to confirm the relationship between the molecular and biological properties of the HAs. The structure of the oligosaccharide at the attachment site found only on the parent HA will be determined in an effort to explain the effect of the host cells on the biological properties of the parent HA. Monoclonal antibodies will be used to compare the antigenic properties of the parent and mutant viruses obtained from two hosts. Virus:cell and virus:antibody equilibrium binding constants will be determined at different temperatures in an effort to detect differences in the conformational stability of the two HAs. Host cell binding constants will also be used to determine whether binding per se can explain the host-determined selection or whether host-determined differences in endocytotic processes are also involved. Lastly, influenza A (H1N1) virus strains, newly isolated from clinical samples and grown directly in a variety of laboratory hosts, will be characterized by the procedures listed above so that the role of these properties in the natural selection of virus strains can be evaluated.
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