Overcoming host restrictions to enable continuous passaging of human noroviruses in human intestinal enteroids

Abstract Human noroviruses (HuNoVs), the leading cause of viral gastroenteritis, can now be cultivated in human intestinal enteroids (HIEs). However, indefinite passaging of HuNoVs in HIEs remained a challenge, necessitating the use of patient stool samples as viral inocula. Using RNA-seq, we identified host restriction factors that might limit viral passaging. CXCL10, CXCL11, and CCL5 were among the most upregulated chemokines, suggesting their potential as host restriction factors. TAK-779, a CXCR3/CCR5/CCR2 antagonist, enhanced GII.3 HuNoV replication and viral spread in a dose- and time-dependent manner, enabling successful passaging of GII.3 HuNoV in two different HIE lines and generation of viral stocks. TAK-779 also enhanced replication of GI.1 and GII.17 strains, but not GII.4, suggesting strain-specific host interactions or immune evasion. This breakthrough in passaging provides critical insight into HuNoV-host interactions, establishes a scalable in vitro system for virus propagation, and opens avenues for structural, biochemical and therapeutic studies.