Rabies is an almost always fatal disease caused by rabies virus (RABV). Once RABV enters the central nervous system, the mortality rate is almost 100%. Therefore, preventing RABV from entering the nervous system is crucial for the development of anti-RABV therapeutics. Here, we found that the L-type calcium channel Cav1.2 pore-forming subunit (Cav1.2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the internalization of RABV using an siRNA approach. RABV glycoprotein interacts with Cav1.2, a process that is required for the subsequent activation of the channel. The activated Cav1.2 then interacts with and activates KCa1.1 to regulate the endocytosis of RABV by promoting the F-actin polymerization. Importantly, diltiazem, a blocker of Cav1.2 and an FDA-approved drug, has been shown to substantially reduce mortality in mice infected with RABV. Approximately 70% of mice survived following treatment with diltiazem at 50 mg/kg, whereas the 25 mg/kg dose resulted in 26% survival. Our findings suggest that Cav1.2 is a promising target for the development of antiviral drugs against rabies and enhances our comprehension of the RABV entry mechanism.

Lyssaviruses cause rabies in mammals, a viral encephalomyelitis that is almost always fatal once clinical symptoms develop and can be transmitted to humans. Given this high fatality rate, strict biosafety measures are essential during laboratory handling of potentially infectious materials. This review critically assesses current biosafety practices in rabies diagnostic and research laboratories by examining principal risk factors, transmission pathways, and the effectiveness of existing preventive measures. We conducted a comprehensive review of the literature on biosafety risks associated with handling rabies virus and related lyssaviruses, including the use of personal protective equipment, sample handling procedures, and chemical and physical methods for virus inactivation. Particular attention was given to documented cases of laboratory-acquired rabies and their implications for laboratory safety. Although laboratory-acquired infections remain rare, their occurrence highlights important gaps in current biosafety practices. The effectiveness of disinfection and inactivation methods varies widely, especially in resource-limited settings, and key diagnostic procedures, such as the direct fluorescent antibody test, require validation under biosafety constraints. Significant knowledge gaps remain regarding infectious dose, the effectiveness of decontamination methods, and diagnostic standardisation. Strengthening evidence-based biosafety protocols and validating inactivation methods are essential to ensure safer rabies diagnostics and to support effective rabies control, particularly in low-resource settings.

Domestic cats are highly prolific, contributing to overpopulation that negatively affects animal welfare, wildlife biodiversity, and public health. Although surgical sterilization is widely used to prevent cat reproduction, it has important practical, economic, and ethical limitations. We studied the safety, immune response, and reproductive effects of a contraceptive anti-GnRH vaccine (Gonacon), administered concurrently with a rabies vaccine, in female pet cats living under normal home conditions. The combined vaccination was generally safe, and most queens maintained protective immunity against rabies. However, the contraceptive effects varied considerably among animals. Indoor queens developed stronger, longer-lasting immune responses and experienced fewer signs of heat and fewer pregnancies than outdoor queens. In contrast, many outdoor queens showed weaker and shorter-lasting immune responses. These findings suggest that the combined vaccination may provide a practical non-surgical approach to controlling reproduction in some pet cats, but that living conditions can strongly influence its effectiveness.

Mechanistic understanding of how networks of neurons carry out neural computations requires knowledge of their underlying synaptic connectivity. However, current methods for reconstructing synaptic connections are extremely laborious and typically limited to mapping connections between nearby neurons. Here we developed Barcoded Rabies In Situ Connectomics (BRISC), which uses rabies viruses carrying random molecular barcodes to map both local and long-range monosynaptic inputs of hundreds of neurons in parallel. To ensure that the majority of post-synaptic starter neurons are uniquely labeled with distinct barcode sequences, we generated libraries of rabies viruses with sufficient diversity to label >1000 neurons and controlled the probability of barcode transmission between starter neurons by limiting their density. We applied BRISC to map inputs of single neurons in the mouse primary visual cortex. We read out the expression of viral barcodes in rabies-infected neurons in situ, preserving spatial information. We then matched barcode sequences between starter and presynaptic neurons, mapping the inputs of 385 neurons and identifying 7,814 putative synaptic connections, revealing layer- and cell-type-specific local connectivity rules and topographic organization of long-range inputs. These results show that BRISC can simultaneously resolve the synaptic connectivity of hundreds of neurons, enabling reconstruction of neural circuits at an unprecedented scale.

Reliable detection of low-abundance rabies viral RNA (RABV RNA) requires sensitive readouts that tolerate interfacial and matrix-associated signal variation. We developed a DNA tetrahedron-confined G4-nanotweezer biosensor combining electrochemiluminescence (ECL) and surface-enhanced Raman scattering (SERS). A Ti3C2/CsPbBr3@PDA@AuNPs nanocomposite (PAM) provided the dual-mode transducing interface, with hemin serving as both an ECL-modulating species and a SERS reporter. CsPbBr3 served as the ECL emitter, PDA improved interfacial stability, AuNPs provided SERS hotspots and DNA anchoring sites, and Ti3C2 acted as a conductive scaffold. Without target RNA, the bridging strand (ST) promoted a closed G4/hemin configuration, producing high SERS and low ECL signals. RABV RNA-triggered binding-induced isothermal amplification generated single-stranded DNA activators for CRISPR-Cas12a. Subsequent ST cleavage prevented nanotweezer closure and reduced interfacial hemin enrichment, producing an ECL-on/SERS-off response. Control experiments linked hemin-associated ECL modulation to the interfacial configuration without establishing a unique microscopic quenching pathway. The ratiometric signal R = IECL/ISERS enabled quantification over 1 aM to 50 fM. Additional low-concentration calibration yielded an estimated detection limit of 0.0675 aM by blank-threshold inverse prediction, extrapolated below the lowest measured nonzero standard of 0.10 aM. At 10 fM, matched-channel comparisons gave ratiometric relative standard deviations of 2.49% for ten successive readings and 2.87% across six independently prepared electrodes. In tenfold-diluted human serum at 10 fM, the ratio showed an 8.20% absolute deviation from phosphate-buffered saline, indicating partial matrix-effect compensation. This shared-reporter architecture links programmable DNA switching to complementary optical readouts for sensitive ratiometric viral RNA analysis.

The retina serves as a uniquely accessible entry point to the central nervous system, enabling both cell type-specific manipulation and circuit-level interrogation of visual pathways. Viral vectors have emerged as indispensable tools in this context, facilitating precise gene delivery, functional perturbation, and connectivity mapping across retinal and brain circuits. Among retinal neurons, retinal ganglion cells (RGCs) represent a major focus due to their role as the sole output neurons conveying visual information to central targets. Accordingly, recent advances in viral engineering including capsid design, transcriptional targeting, and synthetic regulatory elements have significantly improved the efficiency and specificity of RGC-directed gene delivery, while also enabling targeting of diverse retinal cell populations. Beyond retinal targeting, viral tools have transformed the study of RGC connectivity with downstream brain nuclei. In particular, transsynaptic and projection-specific viral strategies now enable mapping of RGC inputs and outputs across visual pathways with increasing precision. Approaches using retrograde AAVs, monosynaptic rabies systems, and anterograde tracers have revealed complex, cell type-dependent wiring patterns linking the retina to thalamic, midbrain, and cortical circuits. In this review, we highlight current viral technologies for targeting RGCs and other retinal cell types and subsequently discuss viral approaches for dissecting retinal-brain connectivity. Together, these complementary strategies provide a powerful framework for linking molecular identity, circuit architecture, and visual function, thereby advancing both basic neuroscience and translational efforts in vision research.