Isolation of largemouth bass virus (LMBV) and dominant immunogenic epitope screening of the major capsid protein of LMBV.

Increasing disease challenges represent a critical bottleneck for the sustainable development of largemouth bass farming. Vaccination can effectively protect fish from pathogens, with dominant immunogenic epitope identification being essential for vaccine development. This research documents a case of largemouth bass virus (LMBV) infection and identifies the dominant immunogenic epitope of the major capsid protein of LMBV (LMBV-MCP). Samples showing clinical signs, such as skin lesions and hepatic bleeding, were collected from naturally infected largemouth bass. Histopathological analysis showed numerous tissue pathological changes, including cell necrosis, hemorrhage and cytoplasmic vacuolization. Aeromonas hydrophila was isolated from the gills and ulcers due to a secondary infection resulting from surface injuries. All the samples were LMBV-positive, and LMBV was isolated using EPC cells. Experimental infection with LMBV resulted in >90 % mortality, recapitulating natural outbreak symptoms. Tissue tropism analysis showed that the heart, liver, and spleen were the primary target organs. Based on structural and antigenic epitope analysis, the LMBV-MCP gene was divided into four segments for prokaryotic expression, designated M1, M2, M3, and M4. Affinity detection revealed that M2 and M3 exhibit strong binding affinity with specific antibodies in positive serum. Moreover, the counts of blood cells (erythrocytes, leukocytes, lymphocytes and neutrophils), immune-related enzyme activities (AKP, SOD, ACP and T-AOC) and gene expression levels (TNF-α, IgM, MHCI-α, IL-10, IL-8 and CD-8) are significantly increased following vaccination with M2 and M3. For largemouth bass immunized with M1, M2, M3, M4 and LMBV-MCP, the relative percentage survival (RPS) values were 30.8 %, 64.9 %, 70.7 %, 29.8 % and 61.4 %, respectively. Results collectively indicate that M2 and M3 have the potential to induce strong immune reactions against LMBV, and M3 is recognized as the dominant immunogenic epitope of LMBV-MCP.