Specific extrusion of Enterovirus-A71-infected cells from human colonoids and consequences for viral spread

Enterovirus-A71 (EV-A71) is a causative agent of Hand Foot and Mouth Disease and occasionally progresses to life-threatening encephalitis and viral meningitis; it is currently endemic in East Asia. There are no antiviral treatment options and no FDA-approved vaccine. EV-A71 is transmitted via fecal-oral routes and thus first enters the body through the GI tract. The Kirkegaard laboratory has cultured colonic epithelial organoids (colonoids) so as to present their apical surface to the medium, mimicking the lumen of the intestine, to study EV-A71. We have found, after eight hours of infection, infected cells are extruded from the epithelium via mechanosensitive pathways. Given that this both liberates virus-infected cells from the epithelium and is likely to preserve epithelial integrity, we hypothesize that the specific extrusion of infected cells benefits both the virus and the host.
To test this hypothesis, I have employed colonoids to investigate the fate of extruded cells and the virus within, as well as how extrusion affects overall viral burden. Extruded cells initially sequester virus, but significantly release virus by sixteen hours post infection. Additionally, viral Stability was equivalent in cell-associated and free virus, indicating that infected extruded cells can propagate infection. Cell-associated virus displayed decreased levels of secondary infection as compared to equivalent titers of cell free virus; clusters of secondarily-infected cells were observed in RD monolayers when intact extruded cells were used to propagate infection, indicating that extruded cells could propagate infection en bloc. When extrusion was blocked by inhibiting force-sensitive ion channels with GsMTx4, virus was still released as free-virus, which ultimately increased viral burden. Thus, blocking extrusion led to more severe infection. Overall extrusion establishes a limited and persistent infection, which benefits both the host and the virus.
Extrusion of infected cells has shown to be a host response to a growing number of pathogens, including poliovirus, measles, Salmonella enterica serovar Typhimurium, and Listeria. Understanding extrusion and its role in the EV-A71 infectious cycle can illuminate the factors that promote or prevent severe disease and elucidate targets for antiviral treatments.