Molecular characterization of rabbit hemorrhagic disease virus type 2 and long-lasting protection conferred by a VLP-based vaccine

Rabbit hemorrhagic disease virus (RHDV) type 2 (GI.2) is an emerging pathogen genetically distinct from classic RHDV (GI.1). It can infect young, wild, and even vaccinated rabbits, and may also contract RHD. This study investigated the prevalence of RHD in China. Furthermore, a virus-like particle (VLP) vaccine was developed based on the prevalent strains, and the vaccine’s protective efficacy against different strains as well as the duration of antibody persistence were evaluated. From 2020 to 2024, 205 clinical samples were collected from 16 cities in China. Among them, 86 samples tested positive for RHDV-2, while no classical RHDV-1 was detected. Furthermore, the VP60 gene of RHDV-2 was sequenced from 18 positive samples. These sequences showed greater than 95% nucleotide identity with the reference strain of RHDV-2 (GenBank accession number: MT383749). However, recombination analysis indicated a potential risk of recombination between RHDV-2 and RHDV-1. A recombinant baculovirus expressing the VP60 gene from the RHDV2 YC05 strain was constructed, and molecular characterization confirmed that the VP60 capsid protein was efficiently expressed in baculovirus-infected Sf9 cells. Hemagglutination activity reached a titer of 1:214 at 4 days post-infection, and the expressed protein self-assembled into virus-like particles (VLPs) in the infected Sf9 cells. Immunization with VLPs containing RHDV2-VP60 at an HA titer of 1:28 induced specific antibodies against RHDV2-VP60 in rabbits, with hemagglutination inhibition titers ensured at 1:25 or higher. All rabbits immunized with the VLP vaccine survived challenge with different clinical RHDV2 strains and remained fully protected for at least 180 days. Our results demonstrated that RHDV2 has become widely prevalent and is now the dominant pathogen in China. Recombinant RHDV-2 VP60 protein expressed in the baculovirus-insect system demonstrates robust immunogenicity and confers long-term protection, making them a promising vaccine candidate for preventing RHDV2 infection in rabbits.