| Human coronavirus NL63(HCoV-NL63) was first identified in Netherlands in2004. Epidemiological studies have shown that this virus is globally distributed. HCoV-NL63mainly infects infants and immunocompromised adults, causing both upper and lower respiratory tract diseases. Clinical symptoms usually manifest as the common cold such as fever, cough and croup. However, it might also cause bronchiolitis, pneumonia even resulting in acute respiratory distress symptoms in some cases. HCoV-NL63is known as one of the six important human coronaviruses.Update to December of2012, there are only five complete genome sequences available in GenBank:three from the Netherlands and two from America. No information is available on the complete genome sequences and phylogenetic analysis of HCoV-NL63derived from China. In this study, our first aim is to sequence and characterize the complete genome sequence of HCoV-NL63derived from China. This may pay a way for molecular epidemiological studies of HCoV-NL63in China.On the other hand, studies have demonstrated that HCoV-NL63share the same receptor (angiotensin converting enzyme2, ACE2) with SARS-CoV for cell entry. However, the clinical manifestations of both viruses are different. We plan to construct the full-length infectious cDNA clone based on the complete genome sequence of HCoV-NL63strains in this study, which might lay a foundation for further study of virus gene functions, replication and virus-host interactions of HCoV-NL63derived from Chinese patients. Furthermore, this reverse genetics platform might be tool or model to characterize the pathogen biology of SARS-CoV infection.The main results of this study are summarized as follows:Firstly, RNAs from32HCoV-NL63positive specimens collected from Beijing Children’s hospital were extracted and quantified respectively, and two positive specimens with high copies were selected for clone and sequencing of full-length genome. Total18pairs of primers were designed according to NL63_Amsterdam reference strains, which covered the complete genome of NL63. By RT-PCR together with3’end and5’end of rapid amplification of cDNA ends (3’/5’RACE), two complete full-length genome sequences were obtained directly from clinical NL63specimens. The genomic structure and phylogenetic analysis of these domestic NL63strains was also characterized. Systematic phylogenetic analysis demonstrated that all known sequences of HCoV-NL63can be genotyping as A, B, C and D, while two domestic HCoV-NL63strains cloned in this study were genotyped as novel group, genotype D. Bootscan analysis indicated that recombination with other strains of HCoV-NL63might be occured during virus circulation and evolution of Chinese HCoV-NL63strains.Secondly, the complete full-length cDNA of NL63domestic strains was assembled by strategy of overlapping PCR and in vitro ligation. The recombined virus, which named as ic-NL63, was successfully rescued after in vitro transcription and electronic transfection of viral mRNAs based on the full-length infectious cDNA clone. The CPE of rescued virus was significantly evident at8d.p.i. Western blot and IFA assays also demostrated that N protein was expressed in infected cells. The introduced molecular marker was identified and genetic stability of rescued ic-NL63during rescued virus propogation was also confirmed.Thirdly, the open reading frame3(ORF3) of HCoV-NL63was replaced with heterologous green fluorescent protein (GFP) for ic-NL63by molecular cloning, which was named as ic-NL63-gfp. And we demonstrated that the ORF3was not essential for the replication of NL63virus in vitro. The growth kinetics of both ic-NL63and ic-NL63-gfp were similar and reached peak at titirs of105/TCID50/mL8d.p.i., which was lower and delayed compared to reference HCoV-NL63strains (NL63_Amsterdam I).In Summary, two full-length genome were cloned and sequenced directly from clinical specimens of Chinese patients. The genomic structure and phylogenetic analysis of these HCoV-NL63strains was also characterized. Systematic phylogenetic analysis demonstrated that NL63virus can be genotyping as A, B, C and D. While domestic strains were formed as a novel group as genotype D. The full-length infectious cDNA clone was successfully assembled and ic-NL63virus was rescued by in vitro transcription and electronic transfection. Furthermore, a novel reporter virus, ic-NL63-gfp in which ORF3was substituted with GFP was also successfully assembled and developed. Our data demonstrated that the ORF3was not essential for the replication of NL63virus in vitro, which might provide a novel good platform for further study of gene functions, replication and pathogenesis of HCoVs. |
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