A Promising Attenuated Rhabdovirus Vaccine Candidate Conferring Dual-Route Protection Against MSRV Disease in Largemouth Bass (Micropterus salmoides).

Background/objectives: Largemouth bass rhabdovirus (Micropterus salmoides rhabdovirus, MSRV) disease causes high mortality in largemouth bass farming. Therefore, vaccine development is critical for largemouth bass prevention against MSRV.

Methods: An attenuated strain, denoted as MSRV-0509, was selected through intraperitoneal injection and immersion challenge assays, followed by plaque purification. The biological characteristics of MSRV-0509, including optimal inoculation dose, replication kinetics, thermostability, pH resistance, chloroform tolerance, and storage viability, were determined via viral titration. Spatiotemporal distribution patterns in largemouth bass post-intraperitoneal injection or immersion infection were quantified by qPCR. Immunoprotective efficacy was evaluated through intraperitoneal and immersion vaccination. Mechanistic insights were explored via relative qPCR and serum neutralization assays. Safety was assessed by single-dose overdose immunization and virulence reversion experiments.

Results: An attenuated strain MSRV-0509 was screened through a challenge assay, exhibiting complete avirulence in largemouth bass compared to the virulent strain SCRV-T6. MSRV-0509 demonstrated optimal replication at low MOI (0.0001) in CPB cells, with peak titers (10^8.3 TCID₅₀/mL) at 96 h post-infection. The virus showed susceptibility to high temperatures, lipid solvents and acidic conditions, with prolonged stable storage viability at -80 °C. Tissue distribution revealed the spleen as the primary target after intraperitoneal injection, while immersion restricted infection to gills, with rapid clearance by 3-6 dpi. Vaccination trials identified 5 × 10² TCID₅₀/fish via intraperitoneal injection and 10^6.0 TCID₅₀/mL via immersion as effective immunizing doses, providing 100% relative survival post-challenge. Immune gene expression and serum neutralization showed Th1 and Th2 activation via intraperitoneal injection (elevated IL-12, IFN-γ, IL-10, IgM), whereas only the Th1 response was activated after vaccine immersion. No abnormality and mortality were observed in single overdose vaccination and virulence reversion experiments, confirming that MSRV-0509 was safe.

Conclusions: These results proved that MSRV-0509 could be a promising vaccine candidate to protect largemouth bass from MSRV disease.