Pre-existing heterosubtypic immunity provides a barrier to airborne transmission of influenza viruses.

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne infection is still unknown. Using the ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We demonstrate that a 48 hour exposure is sufficient for efficient transmission of H1N1 and H3N2 viruses. To test pre-existing immunity, a gap of 8-12 weeks between primary and secondary infections was imposed to reduce innate responses and ensure robust infection of donor animals with heterosubtypic viruses. We found that pre-existing H3N2 immunity did not significantly block transmission of the 2009 H1N1pandemic (H1N1pdm09) virus to immune animals. Surprisingly, airborne transmission of seasonal H3N2 influenza strains was abrogated in recipient animals with H1N1pdm09 pre-existing immunity. This protection from natural infection with H3N2 virus was independent of neutralizing antibodies. Pre-existing immunity with influenza B virus did not block H3N2 virus transmission, indicating that the protection was likely driven by the adaptive immune response. We demonstrate that pre-existing immunity can impact susceptibility to heterologous influenza virus strains, and implicate a novel correlate of protection that can limit the spread of respiratory pathogens through the air.

Secondary infection with Streptococcus pneumoniae decreases influenza virus replication and is linked to severe disease.

Secondary bacterial infection is a common complication in severe influenza virus infections. During the H1N1 pandemic of 2009, increased mortality was observed among healthy young adults due to secondary bacterial pneumonia, one of the most frequent bacterial species being Streptococcus pneumoniae (Spn). Previous studies in mice and ferrets have suggested a synergistic relationship between Spn and influenza viruses. In this study, the ferret model was used to examine whether secondary Spn infection (strains BHN97 and D39) influence replication and airborne transmission of the 2009 pandemic H1N1 virus (H1N1pdm09). Secondary infection with Spn after H1N1pdm09 infection consistently resulted in a significant decrease in viral titers in the ferret nasal washes. While secondary Spn infection appeared to negatively impact influenza virus replication, animals precolonized with Spn were equally susceptible to H1N1pdm09 airborne transmission. In line with previous work, ferrets with preceding H1N1pdm09 and secondary Spn infection had increased bacterial loads and more severe clinical symptoms as compared to animals infected with H1N1pdm09 or Spn alone. Interestingly, the donor animals that displayed the most severe clinical symptoms had reduced airborne transmission of H1N1pdm09. Based on these data, we propose an asymmetrical relationship between these two pathogens, rather than a synergistic one, since secondary bacterial infection enhances Spn colonization and pathogenesis but decreases viral titers.

Parallel evolution between genomic segments of seasonal human influenza viruses reveals RNA-RNA relationships.

The influenza A virus (IAV) genome consists of eight negative-sense viral RNA (vRNA) segments that are selectively assembled into progeny virus particles through RNA-RNA interactions. To explore putative intersegmental RNA-RNA relationships, we quantified similarity between phylogenetic trees comprising each vRNA segment from seasonal human IAV. Intersegmental tree similarity differed between subtype and lineage. While intersegmental relationships were largely conserved over time in H3N2 viruses, they diverged in H1N1 strains isolated before and after the 2009 pandemic. Surprisingly, intersegmental relationships were not driven solely by protein sequence, suggesting that IAV evolution could also be driven by RNA-RNA interactions. Finally, we used confocal microscopy to determine that colocalization of highly coevolved vRNA segments is enriched over other assembly intermediates at the nuclear periphery during productive viral infection. This study illustrates how putative RNA interactions underlying selective assembly of IAV can be interrogated with phylogenetics.

The viruses responsible for influenza evolve rapidly during infection. Changes typically emerge in two key ways: through random mutations in the genetic sequence of the virus, or by reassortment. Reassortment can occur when two or more strains infect the same cell. Once in a cell, viral particles 'open up' to release their genetic material so it can make copies of itself using the cell's machinery. The new copies of the genetic material of the virus are used to make new viral particles, which then envelop the genetic material and are released from the cell to infect other cells. If several strains of a virus infect the same cell, a new viral particle may pick up genetic segments from each of the infecting strains, creating a new strain via reassortment. Several factors are known to affect the success of the reassortment process. For example, if the new strain acquires a genetic defect that hinders its replication cycle, it is likely to die out quickly. Other times, this trading of genetic information can create a strain that is more resistant to the human immune system, allowing it to sweep across the globe and cause a deadly pandemic. However, a key part of the reassortment process that still remains unclear is how genome segments from two different influenza strains recognize each other before merging together to create hybrid daughter viruses. To explore this further, Jones et al. used a technique called fluorescence microscopy. They found that genome segments that evolved along similar paths were more likely to cluster in the same area inside infected cells, and therefore, more likely to be reassorted together into a new strain during assembly of daughter viruses. This suggests that assembly may guide the evolutionary path taken by individual genomic segments. Jones et al. also looked at the evolution of different genome segments collected from patients suffering from seasonal influenza, and found that these segments had a distinct evolutionary path to those in pandemic-causing strains. This research provides new insights into the role of reassortment in the evolution of influenza viruses during infection. In particular, it suggests that how the genome segments interact with one another may have a previously unknown and important role in guiding this evolution. These insights could be used to predict future reassortment events based on evolutionary relationships between influenza virus genomic segments, and may in the future be used as part of risk assessment tools to predict the emergence of new pandemic strains.