Parasite and host factors associated with malaria parasite liver stage latency

Plasmodium vivax (Pv) forms dormant liver stage hypnozoites that cause relapsing malaria, a major barrier to Pv eradication. However, parasite-intrinsic mechanisms and host factors governing hypnozoite persistence are mostly unknown. Using purified, cryopreserved Pv Chesson strain sporozoites and Thai field isolates, we established reproducible liver stage infections in human hepatocyte-chimeric mice and primary human hepatocytes, generating persistent and activating hypnozoites. Transcriptomic profiling and RNA-FISH identified the RNA-binding protein PvPUF1 as a conserved hypnozoite-associated marker downregulated upon activation. Liver stage expression of PvPUF1 in P. yoelii (Py) induced quasi-persistent, growth-restricted liver stages in vivo. Comparative in silico analyses identified a PUF1-associated 9-mer RNA motif across Pv and Py liver stage expressed genes, linking PUF1-associated transcripts to the CCR4-NOT deadenylation complex. In addition, spatial transcriptomics revealed that hypnozoites occupy a transcriptionally quiescent host microenvironment distinct from replicating schizonts. Together, these findings identify a molecular regulator underlying hypnozoite persistence, providing a framework for therapeutic strategies targeting hypnozoites.