Objective To sequence and analyze the complete genome of two human coronavirus NL63 (HCoV-NL63) strains collected from Beijing Children Hospital .Methods Eighteen pairs of primers were designed according to the gene sequences of HCoV-NL63 reference strain ( HCoV-NL63_Amsterdam 1) and used to amplify the target fragments covering the complete genome of HCoV-NL63 strains.Rapid ampli-fication of cDNA ends ( RACE) and RT-PCR assays were used to amplify the full length genome of HCoV-NL63 strains.Phylogenetic analysis was conducted by using Mega 5.0 software.Results The complete ge-nome sequences of the two HCoV-NL63 strains were 27 538 bp in length, showing a homology of 99.1%in nucleotide sequences .There were 15 consecutive bases deleted from 1a region.The systematic phylogenetic analysis demonstrated that four genotypes of NL 63 virus including A , B, C and D have been identified , and two domestic strains were belonged to the new genotype D .Conclusion The complete genome sequences of two domestic HCoV-NL63 isolates were identified for the first time .This study provided evidence for further investigation on molecular epidemiology of HCoV-NL63 in China .

A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63.
Colorimetry ; methods ; Coronavirus Infections ; diagnosis ; virology ; Coronavirus NL63, Human ; genetics ; isolation & purification ; DNA Primers ; genetics ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Reverse Transcription ; Sensitivity and Specificity

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.
Cell Line ; Coronavirus ; genetics ; pathogenicity ; Genes, Viral ; Humans ; Interferons ; antagonists & inhibitors ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Regulatory and Accessory Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism

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.