Development of in vitro and in vivo neutralization assays for New World alphaviruses based on the VSV pseudovirus system

The New World alphaviruses, including Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus (WEEV), and Venezuelan Equine Encephalitis Virus (VEEV), are known to cause neurological diseases that pose a significant threat to public health concerns and bioterrorism preparedness challenges due to their potential for aerosol transmission. Currently, no FDA-approved vaccines or antiviral drugs are available for humans, although ongoing studies are exploring potential solutions. Most vaccine evaluation methods rely on live virus models, which require handling in biosafety level 3 (BSL-3) facilities. In this study, we constructed pseudoviruses for NW alphaviruses using the vesicular stomatitis virus (VSV) system by expressing the glycoproteins E3-E2-E1 on the surface of the VSV vector. In vitro cell infection experiments revealed that the pseudovirus titres of EEEV and VEEV were comparatively higher. Bioluminescence imaging in a mouse model was used to assess infection in vivo. When injected into the brain, this was the main site of infection for NW alphavirus-based pseudoviruses. When administered via the tail vein, the pseudovirus primarily infected the spleen, while intraperitoneally injected pseudoviruses mainly infected the intestines and thymus. Furthermore, we systematically evaluated the correlation between neutralizing antibody titres induced by DNA immunization and the protection against homologous virus strains. This study establishes a safe, convenient, and efficient system for evaluating the protective effects of vaccines against NW alphaviruses, which can be operated in a BSL-2 facility.