FIG. 1.

Stability of rTGEV-Δ3 and rTGEV-Δ3-TRS3a-GFP recombinant viruses. Northern blot analysis of intracellular RNAs extracted at passages 1 (p1), 10 (p10), and 20 (p20) from cells infected with the wild-type (wt), rTGEV-Δ3, or rTGEV-Δ3-TRS3a-GFP virus. Hybridization was performed with a probe complementary to the 3′ untranslated region of the genome. Numbers and letters on the left indicate the position of TGEV mRNAs. S, spike; 3a, gene 3a; E, envelope; M, membrane; N, nucleoprotein; 7, gene 7. The positions of the new sgmRNAs generated from recombinant viruses are indicated by arrows on the right side of the figure. GFP, sgmRNA encoding GFP. Δ3, sgmRNA synthesized from the TRS of ORF 3a in the deletion mutant rTGEV-Δ3. The mobility shift noted in mRNA 7 of wild-type virus is a product of running the gel and does not represent size differences in the sgmRNA. The transfer efficiency of mRNAs during the Northern blotting is highly dependent on mRNA size, and some variability may be observed, especially in the transfer efficiency of RNAs of the larger (gRNA) or smaller (mRNA 7) size. Genomic RNA is not shown because in this experiment, the transfer conditions of RNAs during Northern blotting were set up to optimize the transfer of non-genome-size mRNAs.