Construction of recombinant infectious clones of HCoV-OC43 expressing green fluorescent protein and viral rescue

Objective To investigate the feasibility of using recombinant infectious clones of human coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids (pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12.9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12.9 genes with the reporter gene encoding the green fluorescent protein (GFP) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the rescued virus strains were characterized by immunofluorescence assay (IFA) and Western blot (WB) assay after transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12.9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12.9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12.9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was obtained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infected with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors. Key words: Human coronavirus OC43; Infectious clone; Viral rescue; Green fluorescent protein; Viral vector