Background Rabies virus (RABV) is a neurotropic zoonotic pathogen responsible for an estimated 60,000 human deaths annually. Despite widespread vaccination programs, rabies remains a substantial public health burden, particularly in regions with limited access to post-exposure prophylaxis (PEP). Standard serological assays for evaluating rabies vaccine immunogenicity, including the Rapid Fluorescent Focus Inhibition Test (RFFIT) and the Fluorescent Antibody Virus Neutralization (FAVN) test, require live virus handling under biosafety level 3 (BSL-3) conditions, limiting their scalability and broader implementation. Methods We developed and characterized a pseudotype-based microneutralization assay using a lentiviral HIV-1 backbone to generate pseudotyped viral particles expressing glycoproteins from three antigenically distinct rabies virus strains: CTN-1 V5, CVS-11, and Pasteur. Neutralization assays were performed under BSL-2 conditions, and assay performance was evaluated in terms of sensitivity, specificity, and suitability for high-throughput applications. Results The pseudotype-based assay enabled sensitive and reproducible detection of rabies virus–neutralizing antibodies across the three glycoprotein variants. The platform demonstrated robust performance under BSL-2 containment, supporting efficient and scalable testing. Inclusion of antigenically distinct strains broadened detection capacity and improved the assessment of strain-specific neutralizing responses. Conclusions This pseudotype-based microneutralization assay represents a safe, flexible, and scalable alternative to conventional live-virus neutralization tests. By enabling high-throughput rabies serology under BSL-2 conditions, this platform supports vaccine evaluation and seroepidemiological surveillance, particularly in settings where BSL-3 infrastructure is limited.
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