Let's dive into our new exciting findings on Enterovirus-D68! EV-D68 is pretty crafty about how it gets into cells! We wanted to understand the receptor requirements of EV-D68. Using glycan arrays, we looked at two glycan types: heparan sulfate (HS) and sialosides.

Previous work found two EV-D68 viruses using HS to enter cells. Utilising a HS array, we confirmed this and found a third! The secret? They all share a special change at position 271 in their VP1 protein - switching from a negatively/neutrally charged amino acid to a positive one

Why is it important to know the HS binding profile? HS-binding is linked to cell-culture adaptation, and we did not find this polymorphism in naturally circulating strains. It might still matter for disease, as lab-adapted viruses differ phenotypically compared to other viruses.

Now for the most exciting part - sialic acids! We knew EV-D68 uses these, but we wanted to understand the sialoside receptor specificity. We used a complex glycan array containing α2,3-linked, α2,6-linked N-linked glycans and ganglioside structures.

As expected, we found that EV-D68 binds to α2,6-linked SIA, but some strains were fond of specific disialylated gangliosides GD3, GD1c, GT1a, and GQ1b. We confirmed a functional relevance for viral entry using enzymatic inhibition and by using glycolipid mimetics as inhibitors.

What are the consequences of glycolipid binding? The exact role of gangliosides in viral entry we do not know, but we speculate that they serve as attachment and/or uncoating receptors. Likely similar to porcine sapelovirus and hepatitis A virus which also use glycolipids.

What does this mean for disease? While glycolipids might help the virus infect nerve cells, previous work shows neurotropism doesn't depend on sialic acids. The exact role of glycolipids here remains a mystery!

Lastly, we investigated the relationship between receptor binding and viral stability. We show that receptor preference does not influence capsid stability. We show that the requirement for acidic pH during entry varies and is also independent of their receptor preference.

Key Takeaways: 1. Stronger evidence for HS binding as cell culture adaptation 2. EV-D68 is promiscuous towards various glycans 3. Glycolipids serve as a new receptor group 4. Different strains show remarkable variety in receptor use 5. Big question: How does this affect disease?

My special thanks go to Ashley and Anouk, as they worked incredibly hard on their Master's and Bachelor's thesis bringing this project together. Our former PhD student @Syriam0803 who started this project with me and was involved in generating the viruses for the glycan arrays

And to @RobertPdeVries1 and his PhD student Jaap for exploring with us the glycan specificities of enteroviruses and helping us with the glycan parts, and lastly big thanks to @DebbyvanRiel for giving me the opportunity to lead this fun project!