Fig. 3.

Identification of the leader-body junctions for all SeACoV subgenomic mRNAs. (A) Alignment of the leader sequences between SeACoV (upper line) and PEDV (lower line; US-MN strain, GenBank accession no. KF4687752). The sequence of the leader primer LF is underlined. Each of the leader transcription regulatory sequence (TRS) is marked in red. (B) Genomic and subgenomic organizations of SeACoV. The 5’-leader region is represented by a solid box, and the 3’-poly (A) tail is depicted by A(n). Positions of forward (LF) and reverse primers (S1-R, sgORF3-R, sgE-R, sgM-R, sgN-R and NS7a-R/NS7-R) used for PCR amplification of distinct subgenomic mRNAs (sgRNAs) are indicated by arrows under the genome. The seven small black boxes at the 5’ ends of the genomic RNA (gRNA) and sgRNAs depict the common leader sequence. Genomic and subgenomic RNA numbers (1 for gRNA and 2 to 7 for sgRNAs) are also indicated. (C) Detection of SeACoV sgRNAs by RT-PCR. Different combinations of the forward primer (LF) and one of the six reverse primers (indicated below each lane) were used. The bands representing RT-PCR products of specific SeACoV sgRNAs are marked with white arrowheads. The numbers above each lane represent specific sgRNAs amplified with the corresponding reverse primers. Lane M: DNA markers. (D) Leader-body fusion sites of sgRNAs and their corresponding intergenic sequences. The TRS is indicated in red. The junction sequences in sgRNAs (left panel) and the body sequences fused with the 5’ leader (right panel) are underlined. (E) Detection of the unique SeACoV NS7 sgRNA (arrowhead) by RT-PCR (left panel) and further determination of the sequence containing the leader-body junction site (right panel). The leader sequence is italicized, with the forward primer LF underlined. The body TRS is shown in red. The putative start and stop codons of NS7a and NS7b are boxed by solid and dashed lines, respectively. The binding site for the reverse primer NS7-R used in RT-PCR is also underlined.