mAb-PN9cx3 neutralizes HP-PRRSV-SD16 infectivity in primary PAMs. A PRRSV-N protein transcript levels detected by RT-qPCR. PAMs were infected with PRRSV-SD16 virus at an MOI of 0.1 or PRRSV-SD16 virus pretreated (1-h incubation time) with mAb-PN9cx3 at different concentrations or with mAb-2G8 (isotype control, IC) at a concentration of 2 μM. After inoculation with the mAb-virus mixtures, the PAMs were incubated for 1 h, and the unbound PRRSV virions were removed by washing the cells with fresh medium. The replication of PRRSV at 24 h post-infection was determined by RT-qPCR analysis of the PRRSV-N mRNA transcripts. The experiment was repeated three times. The significant differences between the mAb-PN9cx3-treated PRRSV-SD16-challenged group and the untreated PRRSV-SD16-infected group are marked by asterisks: * (p < 0.05) and ** (p < 0.01). B Virus yields in culture supernatants of PAMs infected with the PRRSV-SD16 strain after preincubation with mAb-PN9cx3 (from 0 to 2 μM). Supernatants were collected at 24 hpi and titrated in MARC-145 cells. The significant differences between the 0 μM mAb-PN9cx3-treated group and the indicated groups are marked by asterisks: * (p < 0.05) and ** (p < 0.01). C Western blotting of PAMs infected with PRRSV-SD16 virus at an MOI of 0.1 or PRRSV-SD16 virus pretreated (1-h incubation time) with mAb-PN9cx3 at the indicated concentrations or mAb-2G8 (isotype control, IC) at a concentration of 2 μM. After inoculation with the mAb-virus mixture, the PAMs were incubated for 1 h, and the unbound PRRSV virions were removed by the washing cells with fresh medium. The PAMs were then incubated for 24 h at 37 °C prior to lysis for SDS-PAGE and Western blotting to determine the PRRSV N protein levels. mAb-2G8 was included as an antibody-isotype control. D PRRSV-SD16 was used as the target virus in the assay at an MOI of 0.1 and incubated with mAb-PN9cx3 and mAb-2G8 at different doses for 1 h at 37 ℃ prior to the inoculation of MARC-145 cells. An IFA with the PRRSV-N-specific mAb-PP7EF11 was conducted 18 h after inoculation, and the florescence spots were counted and compared with those found in the no-mAb control group.