Cryo-electron tomography reveals lineage-specific replication features of Zika virus

Zika virus (ZIKV), a member of the Flavivirus genus, can cause severe neurodevelopmental disorders when infection occurs during pregnancy. Phylogenetic analyses classify ZIKV into two major lineages: African and Asian. Despite their genetic similarity, only the Asian lineage has been linked to congenital anomalies. ZIKV possesses a single-stranded RNA genome and replicates by remodelling the host endoplasmic reticulum (ER) to form membrane-bound replication complexes, which support viral RNA synthesis, virion assembly, and immune evasion. ZIKV also induces the formation of convoluted membranes (CMs), whose function remains unclear. Most ultrastructural studies of ZIKV replication have relied on transmission electron microscopy, which, despite its value, can alter membrane architecture due to sample preparation. To overcome these limitations, we employed cryo-focused ion beam milling combined with electron tomography to visualize ZIKV replication in a near-native state. By comparing infections with African and Asian ZIKV lineages, we uncovered lineage-specific ultrastructural features, despite similar replication dynamics and growth curves. In cells infected with the Asian strain, virions at different maturation stages were often co-packaged within the same compartment. In contrast, the African lineage ZIKV showed distinct compartmentalization of virions and vesicles. Furthermore, the ER-virion spacing was uniform in African-lineage-infected cells, while morphological heterogeneity was evident in the Asian lineage, including irregular viral sacs. Vesicle diameter was also significantly larger in cells infected with the Asian lineage. Together, these findings reveal both conserved and distinct replication structures between ZIKV lineages. The Asian lineage induces more extensive membrane remodelling and CM formation, potentially contributing to its association with congenital disease.