| Coronavirus belongs to GenusCoronavirusin the family Coronaviridaein the system classification system which is named by its shape like a corona. Coronavirus is one of the major pathogens causing common sneezing and cough in adults, as well as upper respiratory infection in children and acute gastroenteritis in newborns; however, acute respiratory syndrome, organ failure and even death can be seen in some severe cases of coronavirus infections. Up to now, six coronaviruses had been confirmed causing human infection by WHO. They are human coronavirus229E (hCoV-229E), human coronavirus OC43(hCoV-OC43), Severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63(hCoV-NL63), human coronavirus HKU1(hCoV-HKU1), and Middle East Respiratory Syndrome coronavirus (MERS-CoV). hCoV-229E, hCoV-OC43, hCoV-NL63, hCoV-HKU1, which are less pathogenic compared to SARS-CoV and MERS-CoV, are circulating continuously around the world, especially in winter and early spring seasons. The viruses are mainly associated with respiratory tract illness with high incidence of pneumonia and bronchiolitis. SARS-CoV is much pathogenic and contagious, mainly causing infectious atypical pneumonia of human being which is also named severe acute respiratory syndrome (SARS). In the large outbreak of SARS in2002-2003, over8000people were infected in more than30countries across five continents and more than900people died of it. Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a novel coronavirus found in September,2012, which causes disease named Middle East Respiratory Syndrome (MERS) similar to atypical pneumonia with acute renal failure. Until February7th,2014, a total182cases including79deaths has been confirmed by WHO with a mortality of43%. It is unknown whether MERS is as contagious as SARS, which means a new threat to public health. Therefore, we should keep alert to coronavirus infection and surveille the circulation. Research on biological characteristic and pathogenicity of known coronaviruses should go on. The molecular biological and serological assays for detection of four coronavirus and the unknown lineages should be developed.Coronavirus is spherical and pleomorphic with a diameter of60-220nm and envelop. The spike on the envelop surface is arranged radially, while it is like petals and arranged in a circle in human coronavirus. In inside, the virus particle is composed of RNA and nucleocapsid protein with a spiral structure. Coronavirus is a positive single strain RNA with base pair of27-S3kb, which possess the largest genomes among all known RNA. viruses. The major structural proteins of coronavirus are nucleocapsid (N), membrane (M), small envelope (E) and spike (S) proteins; there is another structural glycoprotein, haemagglutinin-esterase (HE), in β-B group coronavirus such as hCoV-OC43.SARS-CoV nucleocapsid protein is the second encoded and another abundant protein in the major structural proteins, which is encoded by the ninth ORF with the number of amino acid residues of422and relative molecular mass of approximately 48kDa. The nucleocapsid protein is also a phosphoprotein with high hydrophilia and poorer reliability without cysteine residues and disulfide linkages. SARS-CoV nucleocapsid protein can be phosphorylated as soon as it was generated in the host cytoplasm and forms the nucleocapsid with RNA to implicate in viral replication. It can also reacted with the C terminus of the membrane protein, which may be associated with viral mature and budding. The N protein only shares20-30%homology with the N proteins of other CoVs. It can induce strong humoral and cellular immune responses, making it a major antigen, an optimum diagnostic target and a potential vaccine candidate, according to the relative researches.Many SARS-associated serological diagnostic methods have been developed based on recombinant N protein since2003. We expressed SARS-CoV N protein with GST label and N proteins of hCoV-229E and hCoV-OC43with His label inE.coli expression system, developed a monoclonal antibody-based N protein antigen-capture ELISA to diagnose SARS early infection as well in our preliminary study. However, these recombinant N proteins are difference from natural N proteins in structure and biological functions, because of the inherent deficiency of E.coli expression system. The N protein from E.coli expression system may possess less antigenicity and may react with negative serum in a certain extent on account of absence of phosphorylation.In this study, we expressed the nucleocapsid protein of SARS-CoV\hCoV-229E\hCoV-OC43(SARS-NP\229E-NP\OC43-NP) in Bac-to-Bac Baculovirus system, analyzed the antigenicity of SARS-NP and develop a generic nest PCR assay for detection of human coronaviruses.1. Expression of SARS-NP\229E-NP\OC43-NP in Bac-to-Bac Baculovirus system(1) Expression of SARS-NP in Bac-to-Bac Baculovirus system and antigenic specificity analysisWe amplified SARS-NP gene with pGEX-5X-3/N plasmid as template and primers with BamH I and Sal I restriction endonucleases loci. The PCR product digested with BamHI and Sal I restriction endonucleases was cloned intovector pFastBac HTC of Bac-to-BacBaculovirus expression system.Recombinant plasmid (pFastBac HTC-SARS-NP) was transformedDH10Bac cells to obtain therecombinant Bacmid DNA. Recombinant Bacmid DNA was transferred into Sf9cells which were inducted to express the recombinant protein in High Five cells. After purified by Ni affinity chromatography, the confirmation and antigenicity of the recombinant protein SARS-NP was analyzed by antigen-capture ELISA and Western blot.The recombinant plasmid pFastBac HTC-SARS-NP was constructed correctly, and SARS-NP solution was obtained with the expression lever of0.2mg/mL, the relative molecular mass of48kDa and a part of product with the relative molecular mass of35kDa that was considered to be the degradation product. The antigen-capture ELSA and Western blot showed that the SARS-NP reacted with monoclonal antibody to SARS-NP, immune serum of SARS-NP from rabbits and serum form SARS patients, not with immune serum of229E-NP and OC43-NP from rabbits, cultural supernatant of229E-NP and OC43-NP and serum from healthy people. It meant that SARS-CoV nucleocapsid was expressed successfully in the Bac-to-BacBaculovirus Expression System, and displayed a well antigenic specificity.(2) Expression of229E-NP\OC43-NP in Bac-to-Bac Baculovirus systemWe amplified229E-NP gene and OC43-NP gene with pQE30/hCoV-229E-N\and pQE30/hCoV-OC43-N plasmid as template and primers with BamH I andNot I restriction endonucleases loci; and then did the following procedure including the construction of recombinant plasmid, recombinant Bacmid DNA and protein expression as above. The recombinant protein229E-NP and OC43-NP were analyzed by Western blot with immune serum from rabbits and cultural supernatant of hybridoma.Recombinant plasmid pFastBac HTC-229E-NP and pFastBac HTC-OC43were constructed.229E-NP and OC43-NP solution were obtained with the relative molecular mass in the range of40-55kDa and the relative molecular mass in the range of50-70kDa, respectively. However pure protein solution could not be obtained since they can not be purified well by Ni-NTA. The Western blot showed that the recombinant protein229E-NP and OC43-NP can reacted with immune serum of229E-NP and OC43-NP from rabbits, cultural supernatant of229E-NP and OC43-NP, respectively. That meant229E-NP and OC43-NP can be expressed in the Bac-to-BacBaculovirus Expression System, and obtained antigenic specificity.2. Development of a generic nest PCR for detection of human coronavirusesWe developed a generic nest PCR for detection of human coronaviruses with primers that supplied by Wuhan Institute of Virology of the Chinese Academy of Sciences, and then sequence the T-A clones of PCR products to analyze coronavirus type. The primers targeted the RNA-dependent RNA polymerase gene (RdRp gene) of coronavirus. The specificity of the method was evaluated by detecting11non-coronavirus respiratory viruses icluding Influenza virus A (IVA) and its subtypes (HI N1-09, H3N2), Influenza virus B (IVB), Parainfluenza virus type1-4(PIV1, PIV2, PIV3, PIV4), Respiratory syncytial virus (RSV), Rhinovirus (RhV), Adenovirus (ADV). Its LOD was evaluated by detecting T-A cloning plasmid of human coronaviruses which were10fold serial dilution. The nest PCR was applied to384respiratory specimens from patients with respiratory syndrome and fever of emergency, outpatient and inpatient in Zhujiang hospital in2013, and compared with Real-time RT-PCR.A generic nest PCR method for detection of human coronaviruses was developed with limit of detection (LOD) of5.84×103copies/μ.L,8.36×103copies/μL,6.06×l03copies/μL and6.18×102copies/μL to hCoV-229E, hCoV-OC43, hCoV-NL63and hCoV-HKUl, respectively and specificity to coronaviruses. According to the preliminary evaluation of the nest PCR, the positive rates of the Real-time RT-PCR to total human coronaviruses, hCoV-229E, hCoV-OC43, hCoV-NL63, hCoV-HKU1were4.95%(19/384),2.34%(9/384),1.82%(7/384),0(0/384),0.78%(3/384), respectively; the positive rates of the nest PCR to total human coronaviruses, hCoV-229E, hCoV-OC43, hCoV-NL63, hCoV-HKU1were3.65%(14/384),1.56%(6/384),1.82%(7/384),0(0/384),0.26%(1/384), respectively and no novel human coronavirus. The positive rates analyzed by SPSS were no statistical differences between the nest PCR and Real-Time RT-PCR. By reference to the results of Real-Time RT-PCR, the diagnostic sensitivity of the nest PCR to total human coronaviruses, hCoV-229E, hCoV-OC43and hCoV-HKUl were73.7%,66.7%,100%,33.3%, respectively; and its specificity and positive predictive value of diagnosis to total human coronaviruses, hCoV-229E, hCoV-OC43and hCoV-HKU1were100%.Conclusion1. We successful expressed SARS-NP,229E-NP and OC43-NP in Bac-to-Bac Baculovirus Expression System, obtained recombinant proteins with antigenicity. SARS-NP was preliminarily deemed that it can not react with serum from patients infected with hCoV-229E and hCoV-OC43. The deficiency to229E-NP and OC43-NP was that the pure proteins could not be obtained.2. We developed a generic nest PCR for pan-coronaviruses which was preliminarilyconsidered to be applied to clinical diagnosis. Our future studies will focus on developing antibody-capture ELISAs with the three recombinant proteins and further evaluation of the nest PCR to discover a novel human coronavirus. |
