Vectorial release of novel MLB human astrovirus in polarized intestinal enteroids in association with extracellular vesicles

While human astroviruses (HAstVs) are well-recognized as a significant cause of gastroenteritis in children, the elderly, and immunocompromised individuals, they have also been linked to severe neurological complications, such as encephalitis and meningitis, particularly in immunocompromised hosts. Disease onset begins with viral replication in the intestinal epithelium, but in some cases the infection may disseminate to the central nervous system. For other enteric viruses, association with extracellular vesicles (EVs) upon cell release has been identified as a factor promoting dissemination beyond the intestine and immune evasion. In addition to classic HAstVs, two novel HAstV clades containing neurotropic strains (VA and MLB) were described, with a less clear association with gastroenteritis.

Our work aims to explore the effects of novel MLB HAstV on the intestinal epithelium using human intestinal enteroids (HIEs) and determine the process of virus egress and the phenotype of released virions. MLB2 HAstV may infect differentiated HIEs, and released virions are strongly associated with EVs, without causing cell lysis. Infection of polarized HIEs on transwells shows that virus release predominantly occurs through the apical membrane as EV-associated virions, with a density of 1.10-1.13 g/cm³. Additionally, accumulation of virions with higher densities (1.17-1.19 g/cm³) is also observed in the basolateral compartment, even without a significant disruption of barrier permeability at the used MOI.

Overall, our results indicate that MLB2 HAstVs have the ability to infect and cross the epithelial barrier without causing significant cell damage. The HIE model may help to better elucidate MLB pathogenesis and understand which factors promote extraintestinal dissemination.

Funding: Spanish Ministry of Science and Innovation (PID2021-124023OB-I00; MICIU/AEI/10.13039/501100011033 and FEDER, UE) and Generalitat Valenciana (CIAPOS/2023/356, co-funded by European Social Fund ESF+). INSA-UB is María de Maeztu Unit of Excellence (CEX2021-001234-M; MICIN/AEI/FEDER, UE).