Amino acid variability in ORF 7b and RT-PCR analysis of ORF 7b in clinical samples versus cell culture isolate. (A) ORF 7b amino acid alignment of all SARS- and SARS-like CoV available in GenBank (sequences yielding identical alignments in the region of interest were deleted). The transmembrane domain  is shaded in black/grey. The left column shows GenBank accession numbers of representative genomes for each unique amino acid sequence, along with the starting nucleotide positions of ORF 7b in each GenBank entry. The right hand column shows strain designations and their sources (human, civet, bat). Only one sequence derived from the Frankfurt-1 strain (AB257344) shows a 45 nucleotide in-frame deletion in the predicted transmembrane domain (TMD). The drawing below the alignment panel represents the ORF 7b in recombinant virus r7bΔTMD. (B) Amplification of a 403 bp fragment of ORF 7b by RT-PCR in clinical samples taken after the initial isolation of strain Frankfurt-1 from the Frankfurt index patient (bronchoalveolar lavage sample (BAL) [lane 2], sputum sample [lane 3] stool sample [lane 4]), as well as a sputum sample from the wife of the index patient (wife, lane 5) . Lane 7 shows the corresponding amplification product in the original sputum sample that yielded the Frankfurt-1 isolate. Lane 8 depicts the PCR product of the virus isolate derived from this sample.