<i>In vitro</i> and <i>in vivo</i> characterization of a novel HSV-1 H129 neurotracer recombinant reveals dose-dependent replication and spread dynamics

Understanding the organization and connectivity of brain circuits has been a major focus of neuroscience for decades, and viral vectors have been commonly utilized to map neural connections in animal models. The alpha-herpesvirus herpes simplex virus 1 (HSV-1) is one of the most studied and genetically modified viruses due to its bidirectional spread capabilities in the nervous system and replication dependency on the viral thymidine kinase (TK) gene in the terminally differentiated neurons. H129, a brain isolate of HSV-1, is the most promising anterograde neurotracer that has been extensively used to map neuronal projections. Replication-deficient monosynaptic H129 vectors (H129-dTK) have been previously developed. In this study, we report the development and characterization of a new H129-dTK recombinant that expresses mNeonGreen along with FlpO recombinase. We investigated the replication and spread properties of this recombinant utilizing compartmentalized primary neuron culture and FlpO/Cre reporter mouse lines. Our findings reveal that the replication, labeling, and spread phenotypes of H129-dTK recombinants strongly depend on the infecting dose of virus. At higher viral doses, H129-dTK exhibits limited replication and anterograde transsynaptic spread without requiring exogenous TK complementation, which can be visualized using Cre/FlpO reporter mice. This controlled replication reduces tissue damage at the injection site while allowing precise labeling of neuronal output projections. Together, these results provide valuable insights into the replication and spread characteristics of replication-deficient H129 recombinants in neurons <i>in vitro</i> and <i>in vivo</i>, supporting further development of H129-based neurotracers.IMPORTANCEDissecting brain circuit connectivity and organization is crucial for advancing our understanding of neural function and behavior. The herpes simplex virus 1 strain H129 is an effective neurotracer due to its predominant anterograde spread, though its replication often leads to tissue damage. To address this limitation, H129 recombinants lacking the thymidine kinase (TK) gene were developed to reduce replication in the nervous system. However, the absence of TK also limits reporter gene expression and neuron labeling. In this study, we engineered three novel H129-dTK recombinants that conditionally or constitutively express mNeonGreen along with FlpO/Cre recombinases. These recombinants showed dose-dependent replication and spread capacities, enabling efficient transsynaptic labeling without TK complementation. Additionally, recombinase-mediated labeling enables circuit tracing even under attenuated replication, providing versatility in circuit mapping. Our findings represent a significant advancement in neural circuit tracing, offering high specificity, reduced tissue damage, and precise, reliable labeling of neuronal pathways.