Fig 8.

Evidence of SARS-CoV replication in PESU-B5L cells. PESU-B5L cells were infected with the mouse-adapted strain of SARS-CoV, MA15, and with chimeric SARS-CoV viruses bearing the spike genes of GD03 and HC/SZ/61/03 at an MOI of ∼1. Total RNA was harvested from the cells at 48 and 72 h postinfection and subjected to RT-PCR using primers designed to detect leader-containing subgenomic transcripts indicative of replication. (A) Subgenomic (sg) transcripts for X1, envelope (E), membrane (M), and open reading frame 6 [ORF6 (X3)] were detected at both time points for all three strains. Lane 1, MA15 at 48 h; lane 2, MA15 at 72 h; lane 3, Invitrogen 1-kb Plus ladder; lane 4, SARS+GD03 at 48 h; lane 5, SARS+GD03 at 72 h; lane 6, mock-infected cells; lane 7, SARS+HC/SZ/61/03 at 48 h; and lane 8, SARS+HC/SZ/61/03 at 72 h. (B) The sg M bands for all three strains were excised, and the DNA was purified and sequenced to verify that the SARS-CoV leader sequence and the 5′ end of the M gene were present. Shown here is sg M from SARS-CoV strain GD03.