Influenza is an infectious disease of birds and mammals (including humans) caused by influenza viruses. In humans the disease has two outcomes; seasonal influenza epidemics (3-5 million severe cases and 250,000-500,000 deaths worldwide each year) and human influenza pandemics (even greater public health and economic loss). Molecular characterization of pandemic viruses of 20th (1918 H1N1, 1957 H2N2, and 1968 H2N3) and 21st centuries (2009 H1N1pdm) have revealed that these pandemic viruses evolved from avian influenza viruses. Among avian influenza viruses, highly pathogenic H5N1 and H7 viruses pose the greatest pandemic potential. The risk posed by these viruses is encountered by pandemic preparedness strategies; constant surveillance of virus evolution, risk assessment and preparation of pre-pandemic vaccine seeds. I identified two major needs in the pandemic preparedness; the first was the evolution of novel PB1-F2 truncations in H5N1 viruses and the second was the poor immunogenicity of H7 viruses and vaccines. To fulfill the needs, I worked on two research projects which are compiled as two chapters of this dissertation. Firstly, we studied evolution and prevalence of PB1-F2 truncations in mammalian versus avian influenza viruses along with their risk assessment. We for the first time found independent occurrence and evolution of PB1-F2 truncations in multiple clades of H5N1 viruses. In our risk assessment, we found a virus expressing C-terminal (N-terminal truncated) PB1-F2 (A/chicken/Vietnam/NCVD-296/2009; VN/296) 1000 fold more virulent than another very similar virus (A/chicken/Vietnam/NCVD-281/2009; VN/281) expressing full length PB1-F2. In vitro studies with cloned PB1-F2 expressing proteins of 0, 24 and 90 amino acids did not show any significant difference on cell apoptosis and IFNâ expression. However protein expression from full length and deleted (ATG-ACG) mutants were obvious in transfected A549 cells with fractional mitochondrial localization. We propose the model that C-terminal fragment of PB1-F2 as in virus VN/296 is responsible for the higher virulence and the phenotype is countered by N-terminal fragment as present in VN/281.Secondly, we evaluated and compared the immunogenicity of purified recombinant hemagglutinin (HA) proteins of two H7 and two seasonal influenza viruses. Our studies showed that H7 HAs are weaker immunogens than seasonal influenza virus HAs. This suggests the use of higher doses of HA in H7 vaccines or possible use adjuvants. To summarize, the PB1-F2 risk assessment showed that virus expressing C-terminal fragment is 1000 times more virulent than another very similar virus having full length PB1-F2. These findings highly recommend including PB1-F2 truncations in pre-pandemic virus surveillance and risk assessment. The H7 HA immunogenicity study concludes that the poor immunogenicity of H7 viruses and vaccines is due to intrinsic properties of their HA protein. Further recommendations include using either higher doses of HA in H7 prepandemic vaccines or adjuvants to achieve accepted level of protection. These recommendations will strengthen the current pandemic preparedness studies.
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