Influenza virus noninfectious biologically active particle subpopulations: Detection, quantification, genetic complexity, function and their novel use as an in vitro screen for self-adjuvating live-attenuated influenza vaccines

This work investigates the functional heterogeneity of influenza virus quasispecies through quantitative analysis of cellular responses to the entry of noninfectious biologically active particles, the effect of reassortment of gene segments on the generation and function of these particle subpopulations, and the potential of these subpopulations as in vitro correlates of in vivo effectiveness of live-attenuated influenza vaccines (LAIVs).;For the first time, the clonogenic assay was used to show that populations of most influenza A viruses contained cell-killing particles in excess of infectious particles when tested in the same host cell. Thus, a new class of influenza virus particles was revealed -- noninfectious cell-killing particles which required the synthesis of a specific viral polymerase subunit to kill cells and the expression of NS1 protein to temporally delay apoptosis/cell-killing.;The noninfectious cell-killing particles were clearly distinguished from the well known defective-interfering particles by differences in their numbers in standard influenza virus populations, their temporal appearance and quantity during serial high multiplicity propagation in mammalian and chicken cells, an inability of defective-interfering particles to kill cells or interfere with the cell-killing capacity of noninfectious cell-killing particles, genetic requirements (a small DI RNA ∼350 nt and a large RNA ∼2,300 nt for defective-interfering and noninfectious cell-killing particle activities, respectively), and the extracellular T1/2 at 40.5 °C (∼40h and ∼85h for noninfectious cell-killing particles and defective-interfering particles, respectively).;Specific exchange of the NS gene segment from lethal A/HK/156/97 (H5N1) (NS1: E92, or E92D) virus for the cognate NS gene segment of A/PR/834 (H1N1) (NS1: D92) virus caused de novo generation of large defective-interfering particle subpopulations and >10-fold enhancement of interferon-inducing particle efficiency. These changes were attributed to dysfunction of the H5N1 virus NS1 gene.;Populations of two effective LAIVs (Vac+) in chickens were characterized by high defective-interfering to interferon-inducing particle ratios and induction of large amounts of interferon in chicken cells. Interferon is an antiviral cytokine that acts as a potent natural adjuvant of adaptive immune responses in chickens. Populations of two ineffective LAIVs (Vac -) in chickens had lower defective-interfering to interferon-inducing particle ratios and induced less interferon. Unexpectedly, these phenotypes were reversed in mammalian cells. Populations of Vac- (in chickens) LAIV candidates were excellent interferon inducers with high defective-interfering to interferon-inducing particle ratios in mammalian cells. In contrast, populations of Vac+ (in chickens) LAIV candidates were poor interferon inducers with low defective-interfering to interferon-inducing particle ratios in mammalian cells. As predicted by the in vitro screen, the Vac phenotypes were reversed in vivo (in mice) relative to chickens.;Overall, this study shows that the majority of noninfectious particles of influenza virus are biologically active, reassortment can change the subpopulation make of influenza virus, and a high defective-interfering to interferon-inducing particle ratio is a strong in vitro correlate of the effectiveness of self-adjuvanting LAIVs. Taken together, these attributes of an influenza virus population represent a novel ensemble of in vitro parameters that may be used to distinguish between Vac+ and Vac - LAIV candidates.