key: cord-0033673-miupbeuy authors: Yuan, Jun-fa; Li, Yan; Zhang, Hua-jun; Zhou, Peng; Ke, Zhen-hua; Zhang, Yun-zhi; Shi, Zheng-li title: Indirect Enzyme-Linked Immunosorbent Assay based on the nucleocapsid protein of SARS-like coronaviruses date: 2009-04-14 journal: Virol Sin DOI: 10.1007/s12250-009-3037-0 sha: b5042f7a9e88b4f64c47b297deaf0bc24f0be591 doc_id: 33673 cord_uid: miupbeuy The nucleocapsid protein (N) is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus (SL-CoV) has a high similarity with that of SARS-CoV. In this study, the SL-CoV N protein was expressed in Escherichia coli, purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay (indirect ELISA) was developed for detection of SARS- or SL-CoV infections in bat populations. The detection of 573 bat sera with this indirect ELISA demonstrated that SL-CoVs consistently circulate in Rhinilophus species, further supporting the proposal that bats are natural reservoirs of SL-CoVs. This method uses 1–2 μl of serum sample and can be used for preliminary screening of infections by SARS- or SL-CoV with a small amount of serum sample. to the phylogenetic analysis, these SARS-and SL-CoVs are grouped into a subgroup of CoV group 2, named group 2b ( 3, 4, 8) . The SARS-CoV N protein is the most immunogenic protein and stimulates high-level of antibody response in the host and is an ideal viral antigen for detection of virus infection. SL-CoVs are very similar to SARSCoV in genome organization and gene products. In particular, the N proteins share 95%-97% identity ( 4) . Thus the N protein of SARS or SL-CoV can be used as antigen for detection of infections by group 2b viruses. In this study, SL-CoV N protein was expressed in Escherichia coli, purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay (indirect ELISA) was developed for detection of SARS or SL-CoV infections. This method uses 1-2 µL of serum sample and so can be used for testing of serum sample with limited quantity. The human cell lines 293T and HeLa were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% heated-inactivated fetal calf serum (Gibco, USA or Sijiqi, China). HeLa cell lines that stably express human ACE2 protein were established in a previous study (6) . Rabbit serum against inactivated SARS-CoV was kindly offered by Dr. Lin-Fa Wang at the Australian Animal Health Laboratory, CSIRO Livestock Industries. AP-conjugated goat anti-rabbit IgG were purchased from PTGLab (Chicago, IL). Horseradish-peroxidaseconjugated protein G was obtained from PIERCE (Rockford, IL). Bat sera were collected from the field as described previously (4) . The viral cDNA was prepared from SL-CoV positive bat fecal sample as described previously and used as template (4) . Two primers which target the SL-CoV N gene, To establish the baseline for the tests, sera from healthy bats were tested in this assay and the mean absorbance value (Nc) was calculated. The cut-off value was determined as 2.1×Nc. The sample was determined as positive when the absorbance value at 450 was greater than 2.1×Nc. To evaluate the specificity of the indirect ELISA, sera from healthy bat, mouse and rabbit were tested according to the protocol described above. Sera were diluted at 1/100, 1/500 and 1/1000 respectively. The mean absorbance value for each dilution was calculated. Using the protocol described above, we tested for the presence of IgG antibody against the bat SL-CoV N protein in bat serum samples. Rabbit serum against inactivated SARS-CoV and healthy bat serum was used as positive and negative control, respectively. The construction of a codon-optimized full-length spike protein (S) gene of SARS-CoV BJ01 was prepa- (7). Due to a lack of permissive cell lines for bat SL-CoV, the sera neutralization assay to this virus was not conducted. The N protein was successfully expressed in soluble solution in E. coli and purified. The size of the recombinant N protein was consistent with the expected size of 49.6 kDa (Fig. 1a) . Western-blot analysis confirmed that the expressed protein cross reacted with the rabbit serum against SARS-CoV (Fig. 1b) . Different concentrations of the recombinant N protein (1, 2, 10 µg/mL) were used as coated antigen to optimize the amount of coated antigen. The results showed that a concentration of 1 µg/mL is optimal for plate coating (data not shown). The results tested with sera from healthy bat, mouse and rabbit demonstrated that the purified SL-CoV N protein has no cross reactivity when the sera were diluted at 1:100 (data not shown). A total of 573 bat sera, collected during 2005-2008, were tested for presence of SL-CoV infections. As shown in Table 1 Double-antigen sandwich ELISA for detection of antibodies to SARSassociated coronavirus in human serum Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats Bats are natural reservoirs of SARS-like coronaviruses Severe acute respiratory syndrome Identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features