Objective:To establish and optimize the in vitro enrichment method for human coronavirus NL63 reference strain (HCoV-NL63-NC_005831).Methods:HCoV-NL63 was cultured in rhesus monkey kidney cell line (LLC-MK2). Then at 135 000× g, the virus culture supernatant was centrifuged for 4 h, 6 h, 8 h, 10 h and 12 h to enrich the virus, and compared with the enrichment method of commercial PEG Precipitation Kit. Real time reverse transcription polymerase chain reaction (qRT-PCR), TCID 50 assay and plaque assay were used to quantitatively detect the viral nucleic acid and viral biological activity of the original virus solution and the enriched virus solution respectively. The virus morphology before and after enrichment was observed after negative staining under a transmission electron microscope. Results:After 100∶1 volume enrichment by ultracentrifugation and PEG precipitation, both of the concentration of virus nucleic acid and live virus were higher than that of the original virus solution ( P < 0.001), and before and after the enrichment of the two method, the normal negative staining morphology of virus particles can be observed under the electron microscope. After 4 h, 6 h, 8 h, 10 h and 12 h ultracentrifugation, the concentration of live virus was (7.35±1.62) times, (13.98±1.71) times, (36.36±10.41) times, (48.16±9.38) times, (48.16±9.38) times and (54.26±7.02) times that of the original virus solution, respectively; the concentration of live virus after PEG precipitation was (3.39±0.16) times that of the original virus solution, and the nucleic acid concentration and live virus concentration of concentrated virus solution obtained by ultracentrifugation were significantly higher than those obtained by PEG precipitation ( P < 0.05). After 8 h, 10 h or 12 h ultracentrifugation, the virus concentration was significantly higher than that of 4 h or 6 h ( P < 0.05). However, there was no significant difference in virus content between 8 h, 10 h and 12 h ( P >0.05). Conclusions:Compared with commercial PEG precipitation method, ultracentrifugation method can enrich HCoV-NL63 virus more effectively; when the relative centrifugal force is 135 000× g, the better enrichment effect in vitro can be obtained by selecting the super separation time of 8 h.