Everybody knows the flu. It is what keeps you home for a week in winter times, in bed with the chills, a cough and a snotty nose. You got it from your dears, from your colleagues or from a stranger in the metro. Flu is caused by influenza viruses, which transmit from an infected person to a non-infected person via the air. Although influenza viruses are among the most studied respiratory viruses, there are still a lot of unknown about how they transmit. Among these unknowns is the part of the respiratory tract, and more precisely the tissue, from which influenza viruses are expelled to travel via the air and infect other people.
We started to collaborate with Dr. Anice Lowen, Emory University, who studies the reassortment of influenza viruses (exchange of genetic material between two influenza viruses) using genetically tagged viruses. These viruses can be tracked as they have different genetic “bar codes” but yet have an undistinguishable behaviour. We thought of using these tagged viruses to hit two birds with the same stone: understanding how viral tropism would restrict reassortment (resulted in a joint publication which can be found here) and understanding where in the respiratory tract influenza viruses are generated for transmission.
Ferrets are commonly used to study influenza viruses because they can be readily infected with influenza viruses and they develop a similar disease as humans. To study influenza virus transmission via the air, we use a set-up in which a ferret (the “donor”) is experimentally infected with an influenza virus. Subsequently, a non-infected ferret (the “recipient”) is placed in the opposite cage, separated from the “donor” by two steel grids. The only way the “recipient” can become infected is upon transmission of the virus via the air. In this setting, human influenza viruses (the viruses that keep you in bed in winter times) transmit from the donor to the recipient and avian influenza viruses (the “bird flu” viruses) do not, which is what is also seen in humans. In this study we used this ferret transmission set-up, but instead of infecting the “donor” with one influenza virus, we infected it with two genetically tagged influenza viruses. One was deposited in the lower respiratory tract and the other one in the upper respiratory tract. This double infection resulted in replication of the viruses in separate parts of the respiratory tract. We then tracked which virus transmitted to the “recipient”. In each transmission case, the virus that was transmitted was the one that was replicating in the upper respiratory tract.
More or less at the same time, we were working with Dr. Judith van den Brand, veterinary pathologist (at the time working in our lab who is now at Utrecht University, the Netherlands). She had done great work looking at differences in tropism, kinetics and pathogenesis of human and avian influenza viruses in ferrets (you can find her study here). In that experiment, she had already noticed that human influenza viruses infect cells in nasal epithelium faster and better than avian influenza viruses. We went back to this data and quantified the phenomena. We also made use of an avian influenza virus which was modified to transmit via the air in our experimental transmission set-up. Interestingly, this virus also infected better the nasal epithelium than the original avian influenza virus, similar to human influenza viruses. When we subsequently investigated which of the mutations in this modified avian influenza virus were responsible for the increased infection efficiency in the nose, mutations in one of the surface proteins, the hemagglutinin, were identified. These results, combined with our previous observations, enabled us to conclude that influenza viruses transmit via the air from the nasal epithelium of ferrets.
Finally, we sought to determine the relevance of these findings for humans. We used primary human nasal cells isolated from people who underwent nasal polypectomy. Through contact with the air (like the cells in our nose are), these cells differentiate to form a “mini-tissue”: a 3D epithelium with multiple layers of different cell types, similar to the ones found in our nose. There are the "hairy" cells, epithelial cells which have cilia, tiny “hairs” that help moving the mucus around (it is actually possible to see the cilia beating through the microscope). There are the “snotty” cells, the goblets cells that produce mucus (it is actually possible to harvest mucus from these cultures). And finally, there are the basal cells that are like “reserve” cells that can differentiate into epithelial cells or goblet cells upon damage to regenerate the epithelium. We infected these cells with human influenza viruses (which transmit via the air) and avian viruses (which do not transmit via the air). Similar to our results in ferrets, the human influenza viruses infected abundantly the human nasal cells and the avian influenza viruses did not. These results suggest that the nasal respiratory epithelium is also important for the generation and expulsion of influenza viruses in humans.
The results of our study bring an additional fundamental understanding of how influenza viruses transmit, which is crucial to develop intervention strategies to prevent influenza virus transmission, for instance in health care settings. Based on this work and previous work, we propose a model of how influenza viruses transmit: influenza viruses are expelled from the nasal respiratory epithelium of the donor and are deposited in the oropharyngeal cavity of the recipient. Virus replication is then initiated in the oropharyngeal cavity of the recipient after which the virus spreads to the nasal respiratory epithelium, from where it can be expelled for onwards transmission via the air. Should this model be correct, simple measures that target the URT to block transmission of influenza A viruses could be implemented in health care settings.