Competition between different PB2 segment deletions within a viral population over time.
VIRAL HETEROGENEITY
Influenza virus populations are highly heterogeneous at multiple levels. In addition to physical and sequence diversity, individual influenza virus virions vary greatly in the specific viral genes they encode, with most virions lacking the full set of viral genes needed for replication. The mechanisms that give rise to the enormous amount of genomic heterogeneity within viral populations, and more importantly the consequences for viral replication, transmission, and pathogenesis, remain unknown.
COLLECTIVE INTERACTIONS
Most influenza virus virions can’t replicate on their own because they are missing functional copies of one or more essential genes. While this seems like it would be a big problem for the virus, incomplete virions can complement each other through cellular co-infection, collectively encoding a productive infection. We and others have shown that cellular co-infection is common in multiple animal models of infection. This suggests that the prevalence of cellular co-infection is an important determinant of viral replication efficiency, one that influenza viruses may have evolved to depend upon.
We want to understand how the prevalence of cellular co-infection influences viral replication and evolutionary dynamics as well as the host response to infection. We are especially focused on identifying the specific host and viral factors that influence patterns of cellular co-infection.
Illustration of how variation in viral genomic input gives rise to phenotypic heterogeneity at the single cell level.
Each dot is a cell infected with influenza virus (A/California/07/09); Red dots are cells mounting a detectable interferon response.
HETEROGENEITY IN ANTI-VIRAL RESPONSES
We and others have shown that the induction of critical innate immune factors such as interferon is surprisingly rare and highly stochastic at the single cell level during viral infection. The inherent stochasticity of cellular responsiveness to infection is largely ignored in efforts to dissect models of viral pathogenesis, which often conceptualize the host anti-viral response as a series of deterministic pathways and signaling cascades.
This represents a fundamental gap in our understanding of infection biology and raises broader questions about why so many cells fail to mount interferon responses and how hosts have evolved to rely upon such highly stochastic systems for survival and fitness. We are working to define the central role that stochastic processes play in initiating the innate immune response and exploring the potential benefits of stochastic circuitry in host defense.
INFECTION DYNAMICS ACROSS SCALES
Due to the interplay between viral heterogeneity, host cell heterogeneity, variation in multiplicity of infection, and random chance, no two infected cells behave exactly the same. We want to understand how the diverse infection outcomes that occur at the single cell level collectively give rise to infection outcomes at the organismal and host population levels. Gaining a better understanding of how infection dynamics interact across scales will both shed light on the evolutionary forces that act on viral populations as well as help identify novel host determinants of infection outcome. We are addressing these fundamental questions through a combination of single-cell and single-virion experimental approaches and multiscale mathematical modeling (through collaborators).
Longitudinal single cell RNAseq data showing how transcriptional patterns diverge over time within subsets of infected cells
Plot of SNP frequencies showing the emergence of compensatory substitutions following antigenic escape.
ANTIGENIC EVOLUTION
The persistence of influenza virus (as well as many other viruses) within the human population depends upon the ability of the virus to continually evolve to evade adaptive immune memory elicited by prior infection. Unfortunately, we still know very little about the fundamental rules that govern the antigenic evolution of influenza viruses, a limitation that has hobbled efforts to develop next-generation “universal” influenza vaccines that elicit responses that the virus cannot escape from.
We are working to define the specific constraints that govern the process of antigenic evolution. We are especially interested in understanding how interactions between the viral genome segments and constraints imposed by specific host environments influence the specific evolutionary pathways taken by viral populations.
VIRAL GENE REGULATION
Genome segmentation significantly complicates the viral infection process. The individual RNA segments of the influenza virus genome differ significantly in replication and expression kinetics and magnitude for reasons that are not well understood. We are defining how the replication and expression of individual viral gene segments are regulated both in cis and in trans and exploring how functional interactions between segments are altered when divergent viral genotypes co-infect the same cell.
SHAPE-MaP data showing predicted regions of secondary structure within the NA gene segment for two different viral mutants.