DESCRIPTION (provided by applicant): With the emergence of new human viral pathogens such as the SARS virus, Nipah virus, and the avian influenza viruses, the importance of investigations on the genetic basis of viral infections becomes clear. A major goal of this lab is to study the structure and function of Sendai virus proteins as they relate to viral pathogenicity. Sendai virus is a murine parainfluenza virus similar in structure to other paramyxoviruses that continue to be a major cause of human infection in modern man. Knowledge gained from studying Sendai virus may be applied to paramyxoviruses and other similar viruses that infect man and may lead to new therapeutic interventions. Wild-type (wt) Sendai virus causes a pneumotropic infection in mice. F1-R, a pantropic variant, causes a systemic infection. We previously hypothesized that the pantropism of F1-R is due to two phenotypic differences of the virus as compared to wt virus. One difference is the enhanced proteolytic cleavability of the fusion (F) protein of F1-R which we attributed to amino acid changes in F. Another difference is the microtubule disruption and subsequent viral bipolar budding in F1-R infections which we attributed to two amino acid differences in the matrix (M) protein of the virus. To prove our hypothesis we used reverse genetics to create recombinant viruses containing various combinations of theF1-R F and M mutations. The only virus that was able to form plaques and undergo multiple rounds of replication in LLC-MK2 cells in the absence of trypsin, or cause a systemic infection in mice was RRGV0, a virus containing all F1-R M and F changes. From these studies we could conclude that changes in the F and M genes of F1-R are sufficient to allow the virus to cause a systemic infection. The first specific aim is to determine what combination of mutations in F and M are required for the virus to form plaques and/or to undergo multiple rounds of replication in LLC-MK2 cells in the absence of trypsin and to correlate these phenotypes with virulence in mice. In tissue culture infections F1-R produces fewer viruses than wt virus or RGV0. RGV0 appeared to be more pathogenic to the mice than F1-R. We hypothesize that it is due to a higher rate of virus production in the infection caused by RGV0 as compared to the F1-R infection and that this is due to changes in the P and/or L genes of F1-R. The second specific aim of this project is to create variants of Sendai virus with all F1-R F and M mutations combined with F1-R mutations in the P and/or L genes. The purpose is to identify mutations in the F1-R that contribute to the decreased virus production and decreased pathogenicity of the virus. The third specific aim of this project is to determine if the decreased production of virus in F1-R infections is due to decreased replication/transcription rates, decreases in viral assembly and budding, or decreased inhibition of the host innate responses. PUBLIC HEALTH RELEVANCE: With the emergence of new viral human pathogens from variants of viruses that infect other animals, such as the Nipah and SARS viruses and the avian influenza virus, the importance of investigations on the genetic basis of viral infections becomes clear. The studies described in detail in this proposal provide a platform to study how changes in the genes of Sendai virus, which causes a respiratory tract infection in mice, contribute to viral pathogenesis (ability to cause disease) in tissue culture cells and in mice. Studies on the molecular mechanisms of Sendai virus infections in mice are significant in that they may provide important insights on how to more effectively control similar infections in man.

Narrative With the emergence of new viral human pathogens from variants of viruses that infect other animals, such as the Nipah and SARS viruses and the avian influenza virus, the importance of investigations on the genetic basis of viral infections becomes clear. The studies described in detail in this proposal provide a platform to study how changes in the genes of Sendai virus, which causes a respiratory tract infection in mice, contribute to viral pathogenesis (ability to cause disease) in tissue culture cells and in mice. Studies on the molecular mechanisms of Sendai virus infections in mice are significant in that they may provide important insights on how to more effectively control similar infections in man.