Severe acute respiratory syndrome virus (SARS-CoV) was the causative agent resulting in the SARS outbreak in 2002-2003 China (5). The identification of SARS-CoV in civet cats and other wild animals in live animal markets revealed that this novel pathogen emerged as a result of an interspecies transmission between animals and hunman (2). In our previous work, we have detected a high sero-prevalence of SARS-CoV infection in horseshoe bats by using a sandwich ELISA kit (Beijing Wantai Biological Pharmacy Enterprise Co., Ltd, China) based on the recombinant nucleocapsid protein (N) of SARS-CoV and an indirect ELISA using inactivated viruses as coated antigen (4). The identification of several different CoVs which are similar to SARS-CoV, called SARS-like CoVs (SL-CoVs), suggests that bats are natural reservoirs of SARS-CoV. According 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 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).
Figure 1. SDS-PAGE (A) and western-blot analysis (B) of the SL-CoV recombinant N protein expressed in E. coli. Lane 1, Supernatant of sonicated cells transformed with pET28b-N; lane 2, Purified recombinant protein; lane 3, Purified recombinant protein probed with serum against SARS-CoV; M, Low molecular protein marker.
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, most of positive samples were detected in the Rhinolophus species, with a higher prevalence in R. sinicus. These results are inconsistent with previous reports published by ourselves and other teams (3, 4). Furthermore, we have detected SL-CoV infections in Myotis, suggesting that Myotis species are also susceptible to SARS-or SL-CoV.
Table 1. Detection of SL-CoV antibody
The positive sera for the SL-CoV N protein were tested for activity to neutralize the HIV/BJ01. The results showed that none of these positive sera could neutralize SARS-CoV.