Human intestinal enteroids platform to assess the infectivity of gastroenteritis viruses in wastewater

Fecal-orally transmitted gastroenteritis viruses, particularly human noroviruses (HuNoVs), are a public health concern. Viral transmission risk through contaminated water results underexplored as they have remained largely unculturable until recently and the robust measuring of gastroenteritis viruses infectivity in a single cell line is challenging. This study primarily aimed to test the feasibility of the human intestinal enteroids (HIE) model to demonstrate the infectivity of multiple gastroenteritis viruses in wastewater. Initially, key factors affecting viral replication in HIE model were assessed, and results demonstrated that the reagent-assisted disruption of 3D HIE represents an efficient alternative to syringe pass-through, and the filtering of HuNoV stool suspensions could be avoided. Moreover, comparable replication yields of clinical strains of HuNoV genogroup I (GI), HuNoV GII, rotavirus (RV), astrovirus (HAstV), and adenoviruses (HAdV) were obtained in single and multiple co-infections. Then, the optimized HIE model was used to demonstrate the infectivity of multiple naturally occurring gastroenteritis viruses from wastewater. Thus, a total of 28 wastewater samples were subjected to (RT)-qPCR for each virus, with subsequent testing on HIE. Among these, 16 samples (57 %) showed replication of HuNoVs (n = 3), RV (n = 5), HAstV (n = 8), and/or HAdV (n = 5). Three samples showed HuNoV replication, and sequences assigned to HuNoV GI.3[P13] and HuNoV GII.4[P16] genotypes. Concurrent replication of multiple gastroenteritis viruses occurred in 4 wastewater samples. By comparing wastewater concentrate and HIE supernatant sequences, diverse HAstV and HAdV genotypes were identified in 4 samples. In summary, we successfully employed HIE to demonstrate the presence of multiple infectious human gastroenteritis viruses, including HuNoV, in naturally contaminated wastewater samples.

The study was supported by PREVISION project (reference no. PID2019-105509RJ-I00) funded by the Spanish Ministry of Science, Innovation and Universities and AEI/FEDER, UE to WR and MCEC WATER (PID2020-116789RBC42 AEI/FEDER, UE) to GS. AM is supported by a predoctoral research grant funded by University of Bari “A. Moro” (Italy). The contract of AP-C is part of the grant IJC2020-045382-I, financed by MCIN/AEI/10.13039/501100011033, and by the European Union "NextGenerationEU/PRTR". C.S-B. is recipient of PRE2018-083315 FPI predoctoral grant (MICIN/AEI/10.13039/501100011033 and “ESF Investing in your future”). Wastewater samples were collected as part of the Spanish COVID-19 wastewater monitoring programme (VATar COVID-19) funded by the Spanish Ministry for the Ecological Transition and the Demographic Challenge and the Spanish Ministry of Health. IATA-CSIC is a Centre of Excellence Severo Ochoa (CEX2021-001189-S MCIU/AEI/10.13039/501100011033).

With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S)

Peer reviewed