New technique reveals keys to success for flu virus
For the first time, scientists have been able to watch the flu virus live as it infects human airway cells. They developed a new technique which makes the viral genetic material light up under the microscope. By tracking the virus throughout the steps of its life cycle, the researchers found out that only a minority of flu infections is successful. This is mostly due to errors in reading out the viral genetic material. The new technology can be used to address unanswered questions about the flu virus life cycle, which could help in the development of better antiviral drugs and preparation for future epidemics. The study from the Tanenbaum group was published in Cell Systems.
Last week, the Dutch National Institute for Public Health and the Environment (RIVM) announced the start of a flu epidemic. This is not unusual: in the northern hemisphere, including the Netherlands, the number of flu cases peaks annually between December and April. The majority of such seasonal flu cases are caused by the influenza A virus (IAV). The virus infects airway cells, causing symptoms such as fever, nasal cold, headache, muscle pain, sore throat, coughing and fatigue. Although flu infections are usually mild or even asymptomatic, they can be more severe or even deadly for people in risk groups, including older people, young children and people with pre-existing conditions. In order to better understand and tackle severe flu infections, scientists need to understand exactly what the virus does to our cells.
Not all infections are the same
Decades of research have led to a classic model of the IAV life cycle that is quite well understood. “A virus cannot reproduce on its own, because it is not much more than a package of genetic material and a few proteins,” Huib Rabouw, one of the lead researchers of the study, explains. “It therefore needs the cells of its host, in this case human airway cells, to create new copies of itself. The IAV does so by entering a host cell and releasing its genetic material, which is called RNA. The viral RNA is then read out to make new viral components. Once this is done, the components gather near the edge of the host cell to form new viral particles, which exit the cell in great numbers to infect more host cells.”
More recent work shows, however, that infections can vary greatly from cell to cell. “One viral particle may be very successful in infecting host cells, while another is not,” says Janin Schokolowski, another lead researcher. “How the host cell responds to the infection can also vary. This variation is very relevant, because it ultimately influences the severity of the infection.” Studying this heterogeneity used to be very difficult, because it was impossible to visualize an influenza infection on a single-cell level as it occurs. The researchers from the Tanenbaum group have now developed VISUN, a technique that can do just that.
Seeing influenza at work
“VISUN works by attaching a fluorescent ‘flag’ to each individual package of viral genetic material,” says Micha Müller, the third lead researcher of the study. “This allows us to see them under the microscope as bright fluorescent spots and track their movement through the host cell, from the moment they enter the cell, through all the different steps of the influenza infection.” The researchers collaborated with the Clevers group to study influenza infection in human airway organoids, miniature airways in a lab dish. “This allowed us to study IAV in its natural host cells,” Müller explains.
Only some succeed
Using VISUN, the scientists were able to observe the complete life cycle of the IAV for the first time. They noticed a large variation between infections. “Some infections progressed slowly, others fast,” says Rabouw. “Also, most infections stalled at some point. Only a small minority successfully completed all infections steps.” By carefully analyzing the fluorescent spots over time, the researchers were able to pinpoint exactly where the failed infections went wrong. “The largest bottleneck was viral transcription. This is when the viral genetic material is read out in the host cell. We found that if this goes wrong in the early stages of the infection, the chance of a successful infection decreases greatly,” Schokolowski explains.
A single influenza virus entering a host cell, visualized with VISUN. The bright spots are individual packages of viral genetic material, which are fluorescently labeled and spreading through the host cell. Credit: Janin Schokolowski. Copyright: Hubrecht Institute.
Uncovering the weak spots in the viral life cycle will aid the development of new antiviral drugs and the optimization of existing drugs. Such drugs are sometimes administered in very severe flu cases, for example in patients with a weak immune system.
Predicting future threats
The team collaborated with the group of Ron Fouchier at the Erasmus MC, which has access to a wide variety of circulating influenza variants, and confirmed that VISUN can be used to analyze any influenza variant. “We even studied a virus isolated directly from a patient,” says Müller. “This means we are able to study not only laboratory-adapted influenza viruses, but also clinically important ones. If we can compare different variants, we can potentially find out why some variants are more severe, whereas others are mild. This could help predict the dangers of future influenza variants and prepare for epidemics,” Müller concludes.
Publication
Live-cell single-vRNP imaging identifies viral gene expression signatures that shape influenza infection heterogeneity. Huib H. Rabouw*, Janin Schokolowski*, Micha Müller*, Matthijs J.D. Baars, Antonella F.M. Dost, Theo M. Bestebroer, Jakob Püschel, Hans Clevers, Ron A.M. Fouchier and Marvin E. Tanenbaum. Cell Systems, 2026.
* Authors contributed equally