# The use of sialic acids as attachment factors is a common feature of Enterovirus-D species

## TL;DR

This study shows that Enterovirus-D species, including EV-D111, use sialic acids to attach to cells, a trait that may be unique to this group and could help explain their unusual behavior and potential zoonotic origins.

## Contribution

The study demonstrates that EV-D111, like other EV-D species, uses sialic acids for optimal replication, suggesting this is a common feature of the species.

## Key findings

- EV-D111 relies on sialic acids for optimal replication in cultured cells.
- The absence of sialic acids slows EV-D111 infection kinetics but does not prevent it.
- EV-D species may have ancestral traits involving sialic acid usage, distinguishing them from other enteroviruses.

## Abstract

Among the hundreds of enteroviruses (EVs) infecting humans, the members of the species EV-D (Enterovirus deconjuncti) display original traits. First, only five serotypes are known within this species, while other EV species have tens of serotypes each. Second, EV-Ds display a wide variety of tropisms: EV-D68s are respiratory viruses, EV-D70s have an ocular tropism, while EV-D94s, EV-D111s, and EV-D120s seem to be enteric viruses. Besides, while EV-D68s, EV-D70s, and EV-D94s have been detected in humans, EV-D120s were found exclusively in non-human primates, and the last virus type, EV-D111, was found in both. This and other observations have led to the hypothesis that EV-Ds could have a zoonotic origin. Previous studies have shown that EV-D68, EV-D70, and EV-D94 use sialic acids (Sias) as cellular attachment factors. We investigated the role of Sias in EV-D111 infection using sialidase treatments and loss-of-function experiments in human and simian cells. Assessing viral RNA yield by RT-qPCR analyses and infectious viral particle production by titration assays showed that the absence of Sias at the cell surface significantly slowed down EV-D111 infection kinetics without abolishing it. This suggests that Sia acts as an attachment factor. While EVs generally do not use Sias, EV-Ds seem to rely on them for optimal replication in cultured cells. Sia usage may therefore be an ancestral trait of this species. We also studied EV-B114, a simian enterovirus, and found that it does not use Sias. Our work provides new insight regarding an enterovirus that circulates in humans and exhibits unusual ecological traits.

Except for a few epidemics in the 1970s and 1980s, the impact of EV-Ds on human health remained modest until the 2010s. In 2014, EV-D68 was occasionally responsible for severe respiratory distress and fatal cases of muscular paralysis. EV-Ds have thus the ability to become pathogenic in humans, hence the importance of studying them. The recently discovered EV-D111, of which only a few isolates are available, has been detected in both human and simian samples, suggesting a potential zoonotic origin. We characterized the early steps of EV-D111 replication, with a focus on its ability to use Sias as attachment factors. We found that EV-D111, like other members of the EV-D species, but unlike most EVs, relies on Sia for optimal replication. Our work provides a better understanding of EV-D111 biology, which is essential to determine its tropism and its potential to emerge in humans.