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. 2023 Sep 1;38(5):813–826. doi: 10.1016/j.virs.2023.08.010

Fig. 3.

Fig. 3

Vaccination and challenge strategies. A At 0 dpv, 4-week-old healthy piglets were inoculated with the commercial MLV-PRRSV vaccine at the indicated dose according to the manufacturer's instructions or with DMEM as a mock-vaccination control. Then, a HP-PRRSV SD-YL1712 or NADC30-like PRRSV SX-YL1806 challenge was performed in the piglets immunized with MLV vaccine or DMEM at 42 dpv. Sera was collected at the indicated time points. All piglets were euthanized at 63 dpv. B Rectal temperature of piglets was recorded daily. C Average daily gain rate of piglets was measured weekly. D PRRSV-N protein special antibodies were determined weekly by a commercial Porcine Reproductive and Respiratory Syndrome Virus AB Elisa kit (JNT, JN60415, China) according to the manufacturer's instructions, and the threshold for seroconversion was set at a S/P (sample-to-positive) ratio of 0.4. The virus load in serums (E) and lung tissue (F) were detected by real-time qPCR. A recombinant plasmid containing the ORF7 gene of PRRSV was applied to construct a standard curve. The RNA copies of PRRSV were calculated according to the standard curve. G Virus neutralization properties of immune serums from 42 dpv. Sera dilutions collected at 42 dpv were incubated with 200 TCID50 of viruses incorporating HP-PRRSV (SD-YL1712), NADC30-like PRRSV (SX-YL1806), Classical PRRSV (CH1a, VR2332), and PRRSV-1 (GZ11-G1) in DMEM medium with 3% FBS for 1 ​h. Then, the virus–antibody mixture was transferred to a 96-well plate of confluent MARC-145 ​cells or CRL2843-CD163 ​cells and incubated until the CPE was noticed. Then the immunofluorescence assay was carried out to identify the CPE induced by PRRSV. The absence of CPE at 1:8 dilution was considered positive for the presence of PRRSV neutralization. Data were shown as mean ​± ​SD ∗P ​< ​0.05; ∗∗P ​< ​0.01; ∗∗∗P < 0.001; ns, not significant.

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