<italic>Nkx2-3</italic> and <italic>Tbx15</italic> mediate organelle dysfunction in testicular cells during early Zika virus infection

<sec><p indent="0mm">Zika virus (ZIKV) infection is associated with various reproductive and neurological disorders; however, the precise molecular mechanisms by which it affects the male reproductive system, particularly spermatogenesis, remain poorly understood. A key challenge in this research is the heavy reliance on interferon receptor knockout (Ifnar^−/−) mouse models. Although these models are widely used due to their heightened susceptibility to ZIKV, their severe testicular pathology significantly limits the ability to examine early-stage and temporally dynamic molecular alterations. To address this, we used human <italic>STAT2</italic> knock-in (hSTAT2 KI) mice with intact immune function as a more physiologically relevant model to systematically examine early molecular and cellular responses in testicular tissue following ZIKV infection. </sec><sec> We found that during the early phase of infection, ZIKV selectively targets S100A4-positive monocytes and macrophages within the testicular interstitium, while other known susceptible cell types, such as Sertoli cells and germ cells remain uninfected at this stage. Despite this restricted viral tropism, bulk RNA sequencing revealed widespread transcriptional reprogramming in the testes, characterized by aberrant activation of pathways related to ribosome biogenesis, mitochondrial function, and cellular stress. These results indicate that even localized infection can elicit broad molecular disturbances within the testicular microenvironment. </sec><sec> To obtain higher-resolution insights into cell type-specific responses, we performed single-cell RNA sequencing (scRNA-seq) at 14 days post-infection. The results revealed widespread dysregulation of gene expression across multiple testicular cell populations, including spermatogonia, Sertoli cells, and Leydig cells, regardless of whether they were directly infected by the virus. A core set of differentially expressed genes showed consistent alterations across these cell types. Through transcriptional network analysis and <italic>in</italic> <italic>vivo</italic> validation, we identified <italic>Nkx2-3</italic> and <italic>Tbx15</italic> as candidate transcription factors that may drive these transcriptomic changes, particularly within spermatogenic cells. These regulators are likely to function downstream of immune or stress-related signals originating from infected interstitial immune cells. </sec><sec> Furthermore, CellChat analysis of intercellular communication indicated that immune cells may influence germ cell function through paracrine mechanisms, particularly via the PSAP signaling pathway. This interaction was associated with transcriptional signatures indicative of DNA damage and cellular stress in spermatogenic cells, suggesting that immune-mediated signaling rather than direct viral invasion plays a pivotal role in early-stage testicular injury. </sec><sec> Although ZIKV remains largely restricted to the interstitial compartment and does not invade the seminiferous tubules, we observed significant transcriptomic changes in both germ cells and supporting somatic cells. These findings underscore the critical role of the testicular interstitial microenvironment in mediating the wider impact of localized viral infection. </sec><sec> In summary, our study offers a comprehensive molecular and cellular characterization of the early testicular response to ZIKV infection in a physiologically relevant immunocompetent mouse model. These findings highlight the critical role of non-cell-autonomous mechanisms, particularly the interactions between immune and germ cells, in mediating ZIKV-induced testicular dysfunction and provide novel insights into potential therapeutic targets for preserving male fertility. </sec>