A Versatile Chandipura Pseudovirus System for Investigating Entry Mechanisms

Chandipura virus (CHPV) is a re-emerging neurotropic pathogen for which safe and scalableexperimental systems are urgently needed. Here, we described the development of apseudovirus-based platform using a replication-deficient vesicular stomatitis virus backbone(VSV-ΔG-TFP) encoding teal fluorescent protein (TFP) as a reporter. The system wassuccessfully pseudotyped with the CHPV envelope glycoprotein (CHPV-G) to generate (VSV-ΔG-TFP-CHPV-G particles. High-titer production was optimized in HEK293T cells, yieldingapproximately 10 8 fluorescent focus units per millilitre. Pseudovirus specificity wasdemonstrated by selective neutralization with anti-CHPV serum, with no cross-neutralisation ofVSV-G pseudotypes, while incorporation of CHPV-G was confirmed by mass spectrometry. TheCHPV pseudovirus exhibited robust thermostability and closely recapitulated wild-type CHPVentry characteristics, including a comparable neutralization profile and dependence on the hostentry factor LRP1. Importantly, this platform enabled the establishment of a high-throughputmicroneutralization assay and elicited a potent neutralizing antibody response in mice.Collectively, these findings establish the CHPV pseudovirus system as a reliable BSL-2compatible tool for studying viral entry and antibody-mediated neutralization, while alsohighlighting its potential as a subunit vaccine candidate.