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. 2005 Jul;12(7):848-54.
doi: 10.1128/CDLI.12.7.848-854.2005.

Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay

Fuxun Yu  1 , Mai Quynh Le, Shingo Inoue, Hong Thi Cam Thai, Futoshi Hasebe, Maria Del Carmen Parquet, Kouichi Morita
Affiliations

Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay

Fuxun Yu et al. Clin Diagn Lab Immunol. 2005 Jul.

Abstract

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N' protein and (N)Delta(121) protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N' protein-based ELISA showed a highly nonspecific reaction. The (N)Delta(121) protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N' protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV (N)Delta(121) protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the (N)Delta(121) protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.

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Figures

FIG. 1.
FIG. 1.
Recombinant plasmids containing the N′ and NΔ121 genes were transformed into E. coli strain XL1-Blue and induced with IPTG. E. coli cell lysates were analyzed in a 10% SDS-PAGE gel and revealed with Coomassie brilliant blue staining. Lane M, protein marker (SDS-7B; Sigma, St. Louis, Mo.); lanes 1 and 4, supernatant of sonicated E. coli cell lysate after centrifugation; lanes 2 and 5, pellet of sonicated E. coli cell lysate; lanes 3 and 6, purified recombinant protein.
FIG. 2.
FIG. 2.
Western blot analysis of purified N′ and NΔ121 proteins. The prestained protein marker and purified recombinant proteins were separated by SDS-PAGE and transferred to a PVDF membrane. Each membrane was incubated with diluted serum, followed by horseradish peroxidase-conjugated anti-rabbit IgG, anti-mouse IgG, or anti-human IgG (1:1,000 dilution), and detected by DAB staining. (A) Reactivity of recombinant proteins to rabbit anti-SARS-CoV serum. (B) Reactivity of recombinant proteins to mouse antihistidine serum. (C) Reactivity of recombinant proteins to SARS patient serum. Lanes M, protein marker (SDS-7B); lanes 1, purified SARS N′ protein; lanes 2, purified SARS NΔ121 protein.
FIG. 3.
FIG. 3.
Western blot analysis of sera that reacted with the N′ protein but not with the NΔ121 protein. Purified recombinant N′ and NΔ121 proteins were separated by SDS-PAGE and transferred to a PVDF membrane separately. The membranes were cut into strips and incubated with diluted serum (1:100 dilution) from seven healthy volunteers (I, II, III, IV, V, VI, and VII), followed by horseradish peroxidase-conjugated anti-human IgG (1:1,000 dilution), and detected by DAB staining. These sera showed high titers of antibodies to the N′ protein by the N′ protein-based ELISA but were negative by the NΔ121 protein-based ELISA. P, SARS patient serum used as a positive control. The odd-numbered lanes represent reactions with the N′ protein, and the even-numbered lanes represent reactions with the NΔ121 protein.
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