Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests

Abstract

Objectives: A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans.

Methods: Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests.

Results: Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses.

Conclusions: Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC.

Figure 1

Indirect immunofluorescent antibody test for anti-HCoV-EMC IgG. (1A): positive; (1B): borderline; (1C): negative.

Figure 2

Structure-based protein sequence alignment of the S1 region of HCoV-EMC, SARS-CoV, HCoV-OC43 and HCoV-HKU1, constructed using PROMALS3D (http://prodata.swmed.edu/promals3d/). The receptor binding domain is highlighted. Identical and similar residues are shaded in black and grey respectively. Immunogenic regions predicted by Epitopia of at least 10 residues in length are highlighted by a black line. Only 1 representative sequence from each virus is used to improve clarity of presentation.

Figure 3

Structure-based protein sequence alignment of the S2 region of HCoV-EMC, SARS-CoV, HCoV-OC43 and HCoV-HKU1 constructed using PROMALS3D (http://prodata.swmed.edu/promals3d/). Identical and similar residues are shaded in black and grey respectively. Immunogenic regions predicted by Epitopia of at least 20 residues in length are highlighted by a black line. The heptad repeat regions are highlighted. Only 1 representative sequence from each virus is used to improve clarity of presentation.