The Japanese encephalitis serogroup of the neurogenic Flavivirus has a specific feature that expresses a non-structural protein NS1\'produced through a programmed-1 ribosomal frameshifting (-1 PRF).Herein,C19orf66,a novel member of interferon-stimulated gene (ISG) products,exhibited significant activity of antagonizing Japanese encephalitis virus (JEV)infection.Overexpression of C19orf66 in 293T cells significantly inhibited JEV replication,while knock-down of endogenous C19orf66 in HeLa cells and A549 cells significantly increased virus replication.Notably,C19orf66 had an inhibitory effect on frameshift production of JEV NS1\'.The inhibition was more significant when C19orf66 and JEV NS1-NS2A were co-expressed in the 293T cells.Both C19orf66-209 and C19orf66-Zincmut did not significantly change the NS1\'to NS1 ratio and had weaker antiviral effects than C19orf66.Similarly,C19orf66-209 and C19orf66-Zincmut had no significant effect on the expression of the JEV NS3 protein,whose expression was down-regulated by C19orf66 via the lysosome-dependent pathway.These findings suggest that C19orf66 may possess at least two different mechanisms of antagonizing JEV infection.This study identified C 19orf66 as a novel interferon-stimulated gene product that can inhibit JEV replication by targeting-1 PRF and the NS3 protein.The study provides baseline information for the future devel-opment of broad-spectrum antiviral agents against JEV.