Persistent Japanese encephalitis virus infection in fetuses of an amplifying pig host affects virus genetic heterogeneity and drives transcriptional footprints in offspring

Japanese encephalitis virus (JEV) poses a global threat to public health. This study demonstrates that during fetal infection in natural amplifying hosts, pigs, the in utero environment is conducive to the emergence of new intra-host JEV variants. Transplacental and fetal infection in pigs with JEV resulted in the emergence of virus variants that at least partially resembled variants reported in field samples from JEV endemic regions. The JEV genetic heterogeneity acquired during fetal infection persisted in expelled fetal membranes and offspring. Like in fetuses, JEV mutations identified in afterbirth fetal membranes and offspring were previously reported in the field. Several selected fetal- and offspring-specific mutations that emerged during persistent in utero infection were stable during passage in cell culture. This comparative analysis with field-reported mutations, together with the stability of emerged mutations during serial passages, may suggest the potential for onward transmission of in utero-emerged JEV variants from infected fetal membranes or offspring. Exposure to JEV during fetal life also induced transcriptional footprints in blood cells and tonsils of live offspring, including alterations in pathways associated with epigenetic regulation and memory. Altogether, we generated new knowledge on JEV biology and pathogenesis in its natural amplifying hosts, pigs, during pregnancy. Our findings provide directions for future studies investigating pathways linking fetal infection to offspring and subsequent environmental dissemination, fetal infection as an additional source of JEV genetic diversification, and the mechanisms underlying molecular footprints and their long-term sequelae in JEV-affected offspring, including increased offspring susceptibility to zoonotic pathogens.