Interleukin 6 (il 6)

First, hippocampus tissues were homogenized in 0.9% saline, then centrifuged at 12,000 rpm and 4 °C for 15 min. Blood was centrifuged at 3000 g and 4 °C for 5 min to isolate serum and stored at − 80 °C. The concentrations of TNF-α (FineTest, China) and IL-6 (FineTest, China) were measured at 560 nm using microplate reader (ELX450 Bio-Tek), following the manufacturer’s protocol.

The protein levels of CX3CL1 (Cloud-Clone Corp., Katy, TX, USA); CX3CR1, CD200, CD200R, IL-1β, IL-4, IL-10, IBA1 (all from Cusabio, Houston, TX, USA); IL-6, TGF-β (Fine Test, Wuhan, Hubei, China); and TNF-α (Thermo Fisher Scientific, Waltham, MA, USA) in the hippocampi and frontal cortices (n = 6–8 in each group) of male rats at PND7 were measured using commercially available ELISA kits. The procedures were performed in accordance with the manufacturer’s instructions, and the minimum detectable doses were CX3CL1 0.055 ng/ml, CX3CR1 7.8 pg/ml, CD200 11.75 pg/ml, CD200R 4.67 pg/ml, IL-1β 15.6 pg/ml, TNF-α 16 pg/ml, IL-6 37.5 pg/ml, TGF-β 18.75 pg/ml, IL-4 3.9 pg/ml, IL-10 0.78 pg/ml and IBA1 6.25 pg/ml. Intra- and inter-assay precision values were CX3CL1 < 10%, <12%, and CX3CR1, CD200, CD200R, IL-1β, TNF-α, IL-6, TGF-β, IL-4, IL-10 and IBA1 < 8%, < 10%, respectively. The protein levels of CX3CL1, CX3CR1, CD200, CD200R and IBA1 were also assessed in the hippocampi and frontal cortices (n = 6–9 in each group) of adult male offspring (PND93).

To evaluate the resistance to the African swine fever virus, we measured the production of antibodies against p54 and CD2v. PRV-Fa, PRV-ΔTK/ΔgE, and PRV-∆TK-(CD2v)-∆gE-(p54) were administered via intramuscular injections into the leg (100 μL, 5 × 10⁵ TCID₅₀) of the C57BL/6 mice. Serum samples were collected on days 7 and 14 post-immunization, and p54- and CD2v-specific antibodies were measured using an enzyme-linked immunosorbent assay (ELISA) (Southern Biotech, Birmingham, AL, USA, 5300-01 and 5300-05).
The safety of the recombinant virus was also evaluated by measuring IL-6 in serum after viral infection. The C57BL/6 mice were infected with PBS, PRV-Fa, PRV-ΔTK/ΔgE, or PRV-∆TK-(CD2v)-∆gE-(p54) via intramuscular injection into the leg (100 μL, 5 × 10⁵ TCID₅₀). Serum was collected, and the mice were subsequently sacrificed for experiments on day 3 post-immunization. Serum cytokine IL-6 (FineTest, Boulder, CO, USA, EM0121) levels were measured using an ELISA kit, following the standard protocol.