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Objective@#To investigate the molecular evolution characteristics of human coronavirus (HCoV) subtypes in patients with fever and respiratory tract infection in Guangzhou from 2010 to 2012.@*Methods@#Partial fragments of NP, RdRp and S genes of HCoV-OC43, HCoV-229E and HCoV-NL63 positive samples were amplified by RT-PCR and sequencing. Bioinformatics software, including Bio-edit, Mega4.0 and Clustal1.83 were used for comparison and analysis of NP, RDRp and S gene sequences. Molecular evolutionary tree of different gene regions of HCoV-OC43, HCoV-229E and HCoV-NL63 were built.@*Results@#No remarkable variation or recombinant strain of HCoV-OC43, HCoV-229E and HCoV-NL63 was found in Guangzhou during 2010—2012. The HCoV-OC43 substrains were genetically closest to the strains found in Belgium and Hong Kong (GenBank accession number JN129834 and AY903460). HCoV-229E substrains were genetically closest to those found in Amsterdam (GenBank accession number JX503060) and HCoV-NL63 most genetically close to those in Amsterdam and Beijing (GenBank accession number JX104161 and DQ445911). The NP and RDRp genes of all subtypes were highly conserved, while S gene was more variable.@*Conclusions@#There were at least 3 substrains of HCoV-OC43, HCoV-229E and HCoV-NL63 epidemic in Guangzhou during 2010—2012, and no remarkable variation or recombinant viral strain was found. The NP and RDRp genes of all subtypes were highly conserved and can be used in virus detection, while S gene was more variable and suitable for phylogenetic and variation study.
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Objective:To study the possible immune escape mechanisms of HCoV-OC43 from human dendritic cells(DC).Methods:HCoV-OC43 was isolated from clinical specimen using BSC-1 cells and identified by Real-time PCR,and the cytopathic effect was observed by phase contrast microscope.DCs were induced in vivo using hu-GM-CSF and IL-4 cytokines,and after 7 days of differentiation,DCs were infected by HCoV-OC43.The morphology of HCoV-OC43 infected DC was observed by transmission electron microscope,and the cytokines related to DC functions were detected by Real-time PCR after infection.DC proportion and function related co-stimulatory molecules were analyzed by flow cytometry.Results:In vitro HCoV-OC43 infected human DC model was successfully built.HCoV-OC43 can infect DC and generate immune response of DC in vitro,but no virus nucleonic acid could be detected in culture supernatant.The DC expression of IFN-α,IFN-β,CCL3 and CCL5 were significant decreased when infected with HCoV-OC43,but the expression of costimulatory molecules including HLA-DR,CD1c and CD86 were not affected by HCoV-OC43 infection.Conclusion:Human DC could be infected by HCoV-OC43 and generate immune response,but could not produce progeny virus.HCoV-OC43 may escape from immune response by suppressing the expression of IFN-α and other inflammatory cytokines and chemokines in DC.
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Objective To investigate the feasibility of using recombinant infectious clones of hu-man 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 en-coding 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 res-cued virus strains were characterized by immunofluorescence assay ( IFA) and Western blot ( WB) assay af-ter 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 ob-tained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infec-ted 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-OC43 FL 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.
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The incidence and clinical features of human coronaviruses (HCoVs) among Brazilian patients with respiratory illness are not well known. We investigated the prevalence of HCoVs among Brazilian outpatients and hospitalised patients with respiratory illnesses during 2009 and 2010. To identify the HCoVs, pancoronavirus and species-specific reverse-transcriptase polymerase chain reaction assays were performed. Five of 394 samples were positive for HCoVs (1.2%): 1/182 (0.5%) outpatients and 4/212 (1.8%) hospitalised patients. The OC43 and NL63 HCoVs were identified. Two patients were admitted to the intensive care unit. Underlying chronic disease was reported in cases and one diabetic adult died. HCoVs can cause lower respiratory infections and hospitalisation. Patients with pre-existing conditions and respiratory infections should be evaluated for HCoV infections.