Identification and epitope mapping of swine acute diarrhea syndrome coronavirus accessory protein NS7a via monoclonal antibodies

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric coronavirus that causes vomiting, severe diarrhea, dehydration and death in suckling piglets. NS7a is putative accessory protein that is predicted to be encoded by SADS-CoV, but still to be confirmed experimentally. In the present study, recombinant NS7a protein was expressed in a prokaryotic expression system and used as an antigen to prepare monoclonal antibodies (mAbs) specific to NS7a protein. We obtained two anti-NS7a mAbs, termed AH5 and EH3, that were shown by western blotting to react with the natural NS7a protein in Vero E6 cells infected with SADS-CoV. Using the produced mAbs, we observed by confocal microscopy that NS7a protein was expressed in the cytoplasm. Further studies revealed that the motif (31)VNTWQEFA(38) was the minimal unit of the linear B-cell epitope recognized by mAb AH5, and the motif (82)FDLFERF(88) was the minimal unit of the linear B-cell epitope recognized by mAb EH3. Alignment of amino acids showed that these two epitopes were highly conserved among different SADS-CoV strains and SADS-related coronaviruses from bats, but with one substitution in these two motifs in bat coronavirus HKU2. In summary, we generated and characterized two mAbs against SADS-CoV NS7a protein, and demonstrated NS7a expression in SADS-CoV-infected cells for the first time.