Purified Astragalus Polysaccharide Combined with Inactivated Vaccine Markedly Prevent Infectious Haematopoietic Necrosis Virus Infection in Rainbow Trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) is experiencing a catastrophic pandemic. In recent years, infectious hematopoietic necrosis virus (IHNV) has spread nationwide, resulting in significant mortality. Currently, there are no available treatments or vaccines for IHNV in China. Here, Astragalus extract was purified and characterized. Then, we developed an inactivated IHNV vaccine with purified Astragalus polysaccharide (P-APS) as an adjuvant. Safety assays showed that IHNV was successfully inactivated. After a serious IHNV challenge, the cumulative mortality rates were 76.0%, 38.0% and 22.08% in control, vaccine and P-APS+vaccine group, respectively. P-APS+vaccine was effective in reducing head kidney damage and apoptosis after IHNV challenge by histopathological and TUNEL analyses. P-APS+vaccine group showed better results in enhancing specific antibodies (IgM) and immune enzyme activities (C3, LZM, GOT and GPT). RNA-seq revealed that many immune-related pathways were significantly enriched. TLR2, TLR7, C3, IFN-γ, IgM, MHC1, MHC2, MX1, and VIG1 identified as core genes based on RNA-seq and PPI networks. Mechanistic investigations showed that the P-APS+vaccine activates the immune pathway by upregulating the expression of these genes. P-ASP+vaccine induced effective innate and adaptive immune responses that were stronger than single vaccines after vaccination and IHNV challenged. Our findings will provide a promising vaccine candidate against IHNV. Fish were divided into three experimental groups (P-APS+vaccine, vaccine and control) to test the immunoprotective effect of the vaccine and P-APS+vaccine. Each experimental group had 150 fish, 100 of which were used for mortality measurements and the remaining 50 for sampling. The injection volume was 150 μL (intraperitoneal) in both vaccine and P-APS+vaccine groups, and 150 μL of PBS was injected under the same conditions in the control group. At 14 days (D 14) after vaccination, 150 fish per group were injected intraperitoneally with 50 µl IHNV (107 TCID50 ml-1) and monitored for mortality for two weeks. During vaccination and sampling, fish were anesthetized with 50 mg/L MS-222 (Sigma, St. Louis, MO, USA). Four fish from each treatment group were sacrificed for head kidney and blood collection after immunization at D1, D3, D7, D14, D15, D21 and D30 for assaying immune-related gene expression and non-specific enzyme activity. Notably, a total of seven fish head kidneys were collected from each group at D7 and D21, four of which were used for the gene expression assays described above, and the other three were used for RNA-seq. In addition, the head kidneys of three fish for each group were collected at D1, D3, D7, D14, D15, D21, D30 and D60 for histopathological examination. Research and Demonstration of Integrated Prevention and Control Technologies for Rainbow Trout with IHN and IPN (Grant No. GSLK-2022-11), and Discipline Team Project of Gansu Agricultural University (NO: GAU-XKTD-2022-23)