Fig. 4.

The E protein is essential for PRRSV replication. (A) Immunofluorescence of N protein in P129-ΔE inoculated cells. The ‘passage-1’ virus was prepared as culture supernatant harvested from BHK-21 cells transfected with P129-WT or P129-ΔE clone. Marc-145 cells were inoculated with ‘passage-1’ and fixed at the indicated times post-inoculation, followed by staining with N-specific MAb. (B) Detection of viral RNA in culture supernatants and in Marc-145 cells inoculated with ‘passage-1’ or ‘passage-2’ virus. Total RNA was extracted and treated with DNase I followed by RT-PCR for E gene. (C) Strand-specific detection of viral RNA in cells by RT-PCR. Marc-145 cells were inoculated with ‘passage-1’ P129-WT or ‘passage-1’ P129-ΔE, and total cellular RNA was extracted at 2 days post-inoculation. The RNA was treated by DNase I and RT-PCR was conducted to amplify the region as illustrated in the figure. Numbers in parenthesis indicate the 5′ most nucleotide position in each primer with respect to the PRRSV genome. Expected sizes of amplified products are indicated on the right of the gel.