Reconstruction And Rescue Of HCoV-OC43Infectious Clones

Human Coronaviruses (HCoVs) are enveloped viruses that possess a positive strand RNA genome ranged from27～31Kb, which represents the largest known genome among all RNA viruses. The enormous length of the coronavirus genome and the instability of plasmids carrying coronavirus replicase sequences have hampered the construction of a full-length cDNA clone until recently. The full-length cDNA infectious clone based on BAC system and DI-RNA was successfully obtained in2000(Almazan et.al). At present, there are three types of coronavirus rescue systems:BAC system, in vitro systematic assembly strategy and vaccinia virus vector system.HCoV-OC43belongs to the second group of coronavirus with a genome up to30.7Kb, and it has typical structure of5’ terminal cap and3’poly A. The genes are arranged in the order5’-polymerase-NS2-HE-S-NS12.9-E-M-N-3’, among which the function of NS2and NS12.9are still unknown. As HCoV-OC43and SARS-CoV belong to the same group, it is considered that HCoV-OC43could represent an excellent model for the study of SARS-CoV, without the dependence of facility of BSL-3. The first HCoV-OC43full-length infectious clone based on BAC system (pBAC-OC43FL) was constructed in2006in Canada, which provides a useful platform to study HCoV-OC43. In this study we try to insert a report gene (i.e., EGFP) into different sites or replace various ORFs based on pBAC-OC43FL, then to explore rescue of the recombinant OC43viruses. The principal results are as follows:Firstly, we have successfully rescued the HCoV-OC43virus based on the plasmid pBAC-OC43FL according to the reported protocols and the virus has been characterized by immunofluorescence assay(IFA) and western blotting(WB) after supernatant infection or passages.Secondly, the cDNA clone pBAC-OC43FL was engineered to replace different ORFs with GFP reporter gene or fused to the C terminal of N gene. Utilization of the overlapping-PCR and in vitro ligation, we modified the pBAC-OC43fL and obtained three recombinant plasmids carrying the reporter gene(GFP) designated pBAC-OC43-GFP Δ NS2, pBAC-OC43-GFPΔNS12.9and pBAC-OC43-N-GFP, which represent the replacement of NS2or of NS12.9with GFP, and the insertion of GFP next to N gene, respectively.Thirdly, two recombinant OC43virus were rescued upon transfection with pBAC-OC43-GFP Δ NS2and pBAC-OC43-GFP Δ NS12.9into BHK-21cells, and the expressions of GFP were observed via IFA and WB. But no positive result was observed from rescue with pBAC-OC43-N-GFP. Furthermore, an efficient and stable expression of GFP from recombinant OC43-GFP Δ NS2virus was observed, while low level and unstable expression of GFP for recombinant OC43-GFP Δ NS12.9virus. We did not detect the expression of GFP and N protein when the supernatant of rescued OC43-GFP Δ NS2virus was used to infect new cells. In contract, the supernatant of OC43-FL and OC43-GFP Δ NS12.9can infect new cells. However, when we lyzed the cells by freezing and thawing after transfected with pBAC-OC43-GFP Δ NS2, then infect new cells with the supernatant collected, we can detect the expression of GFP and N protein. This indicated that the function of NS2gene may be associated with the release of the OC43virus, but further study needed.In summary, we have reconstructed three HCoV-OC43infectious clones via insertion of foreign GFP gene into HCoV-OC43genome at three different positions based on a full-length cDNA infectious clone(pBAC-OC43FL), and two recombinant OC43viruses expressing GFP were rescued successfully after transfection BHK cell with pBAC-OC43-GFP Δ NS2and pBAC-OC43-GFP Δ NS12.9. Our data also suggested that NS2may contribute to the release of the OC43virus from cell. Our study might provide a platform for further study the structure and function of HCoV-OC43ORFs, also pave a way for development of coronavirus-based vectors.