Abstract Enterovirus-D68 (EV-D68) often causes mild respiratory infections, but can also cause severe respiratory infections and extra-respiratory complications, including acute flaccid myelitis (AFM). Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 viremia occurs. We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells (PBMC) inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, were productively infected. In BLCL, neuraminidase treatment to remove α2,6- and α2,3-linked sialic acids resulted in a significant decrease of EV-D68 infected cells, suggesting that sialic acids are the functional receptor on B cells. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, play an important role in the development the systemic dissemination of EV-D68 during an infection, which is an essential step towards the development of extra-respiratory complications. Author summary Enterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells and the infection commonly results in mild respiratory diseases. However, EV-D68 infection is also associated with complications outside the respiratory tract, including a polio-like paralysis. Despite the severity of these extra-respiratory complications, it is unclear how EV-D68 is able to spread outside the respiratory tract and infect other organs, like the central nervous system (CNS). To understand this, we investigated if immune cells play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, i . e . B cells and dendritic cells (DCs), and that the virus can be transferred from DCs to B cells. Our findings suggest that lymphoid tissues, which harbor many immune cells, can be a secondary replication site for EV-D68, from where virus is released in the circulation. Our data reveal the importance of immune cells in the systemic spread of EV-D68, which is essential for infection of extra-respiratory tissues. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent virus spread from the respiratory tract to other organs.

The potential role of human immune cells in the systemic dissemination of enterovirus-D68