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. 2006 Sep;80(18):8920-8.
doi: 10.1128/JVI.00649-06.

Extremely low exposure of a community to severe acute respiratory syndrome coronavirus: false seropositivity due to use of bacterially derived antigens

D T M Leung  1 , W W C van Maren, F K L Chan, W S Chan, A W I Lo, C H Ma, F C H Tam, K F To, P K S Chan, J J Y Sung, P L Lim
Affiliations

Extremely low exposure of a community to severe acute respiratory syndrome coronavirus: false seropositivity due to use of bacterially derived antigens

D T M Leung et al. J Virol. 2006 Sep.

Abstract

Estimates of seropositivity to a new infectious agent in a community are useful to public health. For severe acute respiratory syndrome (SARS), the figures are conflicting. Herein, we screened 12,000 people in a community stricken by SARS 10 months previously and found 53 individuals (0.44%) who had immunoglobulin G antibodies to the SARS coronavirus (SARS-CoV) nucleocapsid (N) produced in bacteria. However, only seven of these (group 1) had sera which also reacted with the native N antigen expressed in SARS-CoV-infected Vero cells, N-transfected 293T cells, and tissues of infected SARS patients. Of these, six individuals had had SARS previously. The remaining person, as well as the 46 other individuals (group 2), were healthy and had no history of SARS. Group 1 antibodies recognized epitopes located slightly differently in N from those of group 2 antibodies, and a mouse hybridoma antibody resembling the former type was generated. Unusually, group 2 antibodies appeared to recognize cross-reactive bacterial epitopes that presumably were posttranslationally modified in eukaryotes and hence were probably not induced by SARS-CoV or related coronaviruses but rather by bacteria. The N antigen is thus highly unique. The extremely low rate (0.008%) of asymptomatic SARS infection found attests to the high virulence of the SARS-CoV virus.

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Figures

FIG. 1.
FIG. 1.
Serological identification of community subjects exposed to SARS-CoV. A) Map of SARS-CoV N protein showing the various fragments (Na, Nb, Nb1, and Nb2) produced as GST fusion proteins in bacteria used for serological detection. Numbers denote amino acid numbering of protein. B) Purity of the recombinant fusion proteins (rNa-GST and rNb-GST) isolated by affinity chromatography, separated on 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and stained by Coomassie blue. Starting material (crude lysate) and molecular weight markers are also shown. C) ELISA results of community subjects screened for IgG antibodies to a combination of rNa-GST and rNb-GST. Comparative data of SARS patients using sera obtained during acute-convalescence phase are shown (OD values >3.5 are plotted as 3.5). The shaded bar denotes the cutoff used to differentiate between elevated and normal levels of antibodies. ▵, CS individuals; ▿, CSN individual; •, CN individuals.
FIG. 2.
FIG. 2.
Western blot results showing specificity of serum antibody activities of representative individuals from community groups (CS, CSN, and CN) against SARS-CoV crude, native antigens or a recombinant GST-fused antigen (rNb). Numbers refer to identification of individuals used throughout the text. A) Results for crude, native antigens separated on 10% gel. The reactivity of a SARS patient's convalescence serum is included for comparison. B) Specific reactivity of two individual CN sera to the viral protein moiety rather than the GST carrier shown in gel where the two components of the fusion protein (rNb-GST) were separated on 14% gel following thrombin digestion. The leftmost panel shows locations of the dye-stained components.
FIG. 3.
FIG. 3.
More-detailed characterization of antibody activities in representative community group subjects, based on reactivity to various rNb subunits. A) ELISA results for individual subjects examined for type (IgM, IgA, or IgG) and specificity (crude native antigens or recombinant rNa or rNb) of antibodies present. Shown are nine sera obtained from SARS patients during acute-convalescence phase, 6 CS sera, 1 CSN serum, 8 CN sera, and 5 sera from healthy subjects. The shaded area denotes negative reactivity, based on a value of mean plus 2 SD for healthy (non-SARS) individuals. B) WB results for selected individuals showing reactivity of sera for rNb1-GST or rNb2-GST in 14% gel.
FIG. 4.
FIG. 4.
Specificity of rNa- or rNb-purified antibodies from CS or CN individuals and related mouse MAbs. A) ELISA results showing absence of cross-reactivity between rNa and rNb used as the detecting antigen (coat) probed with rNa- or rNb-purified antibodies or with mAb13. B) IFA results showing reactivity of CS-purified rNb-specific antibodies and mAb13 with SARS CoV-infected Vero cells (×400) and the lung and intestinal tissues (×600) of SARS patients, and the lack of such reactivity by CN-purified rNb-specific antibodies. Inset shows Vero cells stained with hematoxylin. For the human tissues, green denotes reagent antibody fluorescence, blue (or purple) denotes DNA (nuclear) staining, and red (or orange) denotes nonspecific staining of red blood cells or the cytoplasm of other cells.
FIG. 5.
FIG. 5.
Specificity of mAb13 and the prevalence of mAb13-like antibodies in human sera. A) WB results showing reactivity of MAb for rNb and the native N antigen but not rNa. B) Inhibition ELISA determining the presence of mAb13-like antibodies in serum of various groups of individuals. Antigen used, rNb-GST; indicator MAb used, biotinylated mAb13. Results indicate % inhibition of buffer control. Shaded area denotes negative reactivity, based on mean plus 2 SD for healthy subjects.
FIG. 6.
FIG. 6.
Examination of CN sera for reactivity with 293T cells transiently transfected with rNb. IFA results (×600) show reactivity of a CS serum and mAb13 but not the CN serum. Also shown are transfected cells stained with hematoxylin.
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