Generation of a replication-defective rift valley fever virus and development of a single-virus quantum dot tracking platform

A replication- defective strain of Rift Valley fever virus was developed, facilitating safe investigation under BSL-2 laboratory conditions. Establishment of a single-virus tracking system for Rift Valley fever virus. Live imaging revealed that Rift Valley fever virus undergoes intracellular transport via microtubules and endocytic vesicles. Rift Valley fever virus (RVFV) is a highly virulent zoonotic pathogen posing substantial risks to global public health and livestock industries. Classified by the WHO as a priority pathogen with high pandemic potential, RVFV underscores the critical need for fundamental research to accelerate the development of vaccines and antiviral agents. In this study, we engineered a replication-defective RVFV system that preserves the capacity for host cell infection and a single round of genomic replication. Targeted deletion of the envelope glycoprotein genes Gn and Gc, together with the non-structural protein NSm resulted in a replication-incompetent virus capable of producing infectious particles only in trans-complementing cell lines engineered to express the missing components. This system enables safe experimentation under BSL-2 containment. By incorporating a biotin acceptor peptide (AP) tag into the viral L protein and leveraging biotin-streptavidin bridging for quantum dot conjugation, we developed a highly specific, protein-level labeling platform for single-virus tracking of RVFV. This advanced methodology permits real-time visualization of the viral life cycle from the point of cellular entry. Using this system, we have obtained the first live-cell imaging evidence that RVFV undergoes microtubule-dependent transport via endocytic vesicles during infection. Our findings provide unprecedented insight into the dynamic post-entry trafficking of RVFV and establish a versatile and safe strategy applicable to the study of other high-containment pathogens.