This was a big team effort by the bsky-less @Huib, @janinschoko.bsky.social, @baarsmatthijs.bsky.social and Jakob in @marvintanenbaum.bsky.social lab. Also, a great collaboration with @RonFouchier and @antonelladost.bsky.social & @hansclevers.bsky.social on the patient samples and airway organoids!

We envision that the tools described in this manuscript open up new avenues to study IAV biology in unprecedented detail. Conceptually, with virus-specific modifications our imaging systems are even broadly applicable to many different (-)RNA viruses.

(3) Finally, we found that even when vRNPs are present, they often lack transcriptional activity. As a result, most infected cells only transcribe very few vRNPs. We conclude that viral transcription itself is a highly limiting factor in determining the successful outcome of IAV infections.

(2) Mitosis causes vRNPs to be distributed over two separate sister cells. Therefore, these sister-cells often end up having incomplete sets of genome segments. We termed this “viral aneuploidy”, akin to chromosome segregation errors occurring during host cell division.

(1) As previously reported, due to the segmented nature of the IAV genome, virions can lack one or multiple genome segments. We confirmed this both by using live-cell imaging of incoming vRNPs and by performing smFISH on viral particles.

Finally, we wondered what the underlying reason for the observed defects in viral gene expression could be and found three mechanisms:

We analyzed naturally occurring single-gene KOs (cells expressing all but one viral gene) - which are otherwise hard to generate because most viral genes are essential - to study viral protein function and gained insights into which proteins are important for viral replication and nuclear export.

We wondered why so many infections fail to progress through all life-cycle stages. We combined our live-cell imaging technologies with multiplexed smFISH and found that many viruses fail to transcribe one or multiple genes.

Using single-cell traces of many hundreds of cells, we constructed a kinetic map of IAV infections, revealing large heterogeneity in the timing and success rates of the individual steps in the viral life cycle. Infections are very unsuccessful, with only ~4% of them resulting in progeny production.

To capture the late-stage event of new virions budding off, we developed a second, orthogonal technique that visualizes the build-up of HA-protein on the cell surface of infected cells and even labels budding virions.

Further we can follow IAV over time and observed vRNPs replicating and later getting exported from the nucleus to allow assembly of new virions.

Using this technology we show for the first time the moment that virions fuse with the endosomal membrane and release vRNPs step-by-step into the host cell

The NP nanobody recognizes a broad range of IAV strains, including swine, avian, and human strains, and even viruses directly isolated from patient samples! Since there is no need to genetically modify the viruses, it is very easy to do experiments with new strains and isolates.

Influenza viruses are segmented, negative-sense RNA viruses that, like other (-)RNA viruses, have their genomes encapsidated by nucleoproteins (NP). We exploited this characteristic to visualize single, unmodified IAV genomes by expressing a fluorescently labelled nanobody that binds to NP.

Link to preprint:
www.biorxiv.org/content/10.1...

Congrats! 🥳