The use of human intestinal enteroid cell cultures for detection of multiple gastroenteric viruses

Human norovirus (HuNoV) and human astrovirus (HAstV) are viral enteric pathogens and known causative agents of acute gastroenteritis. Identifying the presence of these viruses in environmental samples such as irrigation water, or foods exposed to virus contaminated water (e.g., shellfish, agricultural crops), remains an important goal in the field of food safety. Determining if a virus species present in a sample is infectious is complicated by the recalcitrance of many enteric virus species to grow in culture, and/or the lack of a common cell culture system(s). Human intestinal enteroids (HIEs) can support the replication of HuNoV and HAstV, and thus hold promise as a platform for demonstrating the replication of multiple enteric virus species within a single sample. The objective of this study was to determine if HIEs can support co-replication of two genetically distinct human enteric viruses, HAstV3 and HuNoV GII.4[P16]. In single virus infections, HuNoV GII.4[P16] RNA levels were highest at 48-72 hpi (6.3-9.1 x 10⁶ genome copy equivalents [gce]/well) and HAstV3 RNA levels were highest at 24 hpi (3.4 ×10⁸ gce/well). HAstV3-infected cells stained positive for viral capsid protein at 24 hpi and induced the synthesis of RNA and protein expression of interferon (IFN)-beta, -lambda 1 and 2/3, peaking at 24 hpi and 48 hpi respectively. HuNoV GII.4[P16] replication was negatively impacted by HAstV3 co-infection, but HAstV3 was unaffected by HuNoV. A reduction in HuNoV GII.4[P16] RNA during co-infections was observed at 72 hpi, with partial restoration achieved using neutralizing anti-IFN-antibodies. Human intestinal enteroids can support the co-infection and replication of HuNoVGII.4[P16] and HAstV3, even at more than 100-fold excess in one virus over the other, and compounds (e.g., anti-IFN antibodies) that interfere with HIE antiviral mechanism(s) can aid in maximizing HuNoV replication during co-infection.