In Vivo Tracking Deciphers the Traversal of Ebola Virus across the Blood–Testis Barrier

Ebola virus (EBOV) can persist in semen to mediate sexual transmission and drive epidemic resurgence. However, the mechanism by which EBOV breaches the blood-testis barrier (BTB) to invade the male reproductive system remains poorly understood. Here, we developed a quantum dot (QD)-based virus-barrier visualization system (QD-VBVS) integrating QD-encapsulated EBOV-like particles (EBO-VLP-QDs) with two-photon microscopy to enable real-time, high-resolution visualization of viral-BTB interactions in live mice. Single-particle tracking uncovered a four-stage BTB traversal mode, with ∼2.48% of EBO-VLPs entering seminiferous tubules from the interstitium. Mechanistically, EBO-VLPs disrupt BTB integrity through the selective downregulation of junctional protein connexin 43 and vimentin, while EBOV glycoprotein (GP) is crucial for BTB disruption and viral traversal. Our study unveils key insights into the dynamic mechanism of EBOV traversal across the BTB and establishes a versatile in vivo visualization platform for virus-barrier interactions, advancing our understanding of EBOV pathogenesis in the male reproductive system.