The full length and the truncated fragments of the nucleocapsid were amplified by PCR. (a) Amplified PCR products of the nucleocapsid gene and the relative locations of the truncated fragments. Purified and expressed fragments of the nucleocapsid gene. The further diagnostic potential of the expressed recombinant protein was revealed by immunoblotting. The reactivities of the recombinant proteins with human SARS coronovirus-positive sera and animal coronavirus-positive sera were tested. In this study, in order to cast light on the possible diagnostic value of the N protein of SARS coronavirus, we expressed and purified full-length N and six truncated N proteins. Presumably, this recombinant protein could be made widely available. It would also decrease the risk of laboratory infections with the live virus. Thus, a reliable recombinant protein-based Western blot assay for the detection of antibodies against the SARS coronavirus that is not dependent on culturing of the SARS virus would be useful. However, it requires laboratories with biosafety level 3 (BSL-3) facilities, special equipment, and well-trained technicians. Currently, an immunofluorescence assay (IFA) is the “gold standard” for the detection of SARS coronavirus infection. Rapid viral diagnosis will become increasingly critical, both for the control of epidemics and for the management of patients with SARS coronavirus infections. It has been identified as a suitable candidate for diagnostic applications for animal coronaviruses. The N protein of coronaviruses (such as infectious bronchitis virus ) is highly conserved in each group, is immunogenic, and is abundantly expressed during infection. Based on previous findings, N was identified as the target gene for the development of a PCR for diagnosis ( 4, 6, 18). N also has a novel nuclear function, which could play a role in pathogenesis. The structural proteins (S, E, M, and N) function during host cell entry and virion morphogenesis and release.ĭuring virion assembly of the coronavirus, N binds to a defined packaging signal on the viral RNA, leading to the formation of a helical nucleocapsid. The genomic organization of the SARS coronavirus is typical of coronaviruses, with a characteristic gene order (replicase, spike, envelope, membrane, and nucleocapsid ). Phylogenetic analyses indicated that the SARS coronavirus is not closely related to any of the previously characterized coronaviruses and forms a distinct group within the genus ( 14).Ĭoronaviruses are the largest enveloped positive-stranded RNA viruses, with genome sizes ranging from 27 to 30 kb ( 8, 13). Several laboratories responded quickly by isolating a novel coronavirus ( 3, 5, 10, 12). It spread worldwide, affecting 8,360 individuals and resulting in 764 deaths by ( 9). Severe acute respiratory syndrome (SARS) is a newly emerging human disease, first identified in the Guangdong province of China in November 2002. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins.
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