Two-way antigenic cross-reactivity between SARS-coronavirus (CoV) and group 1 animal CoVs is mediated through an antigenic site in the N-terminal region of the SARS-CoV nucleoprotein

Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691-4096; Istanbul University, Faculty of Veterinary Medicine, Department of Virology, Avcilar, 34320, Istanbul, Turkey. Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases, Division of Viral Diseases, 1600 Clifton Road, Atlanta, GA 30333; Center for Infectious Disease Research and Vaccinology, Veterinary Science Department, South Dakota State University, Brookings, SD 57007; Department of Medicine, Medical School, University of Massachusetts, 364 Plantation Street, Worcester, MA 01605

^* To whom correspondence should be addressed. Email: saif.2{at}osu.edu.

	Abstract

In 2002, SARS-associated coronavirus (SARS-CoV) emerged in humans causing a global epidemic. By phylogenetic analysis, SARS-CoV is distinct from known coronaviruses (CoVs) and most closely related to group 2 CoVs. However, no antigenic cross-reactivity between SARS-CoV and known CoVs was conclusively and consistently demonstrated except for group 1 animal CoVs. We analyzed this cross-reactivity by ELISA and Western blot using specific antisera to animal CoVs and SARS-CoV and SARS patient convalescent or negative sera. Moderate two-way cross-reactivity between SARS-CoV and porcine CoVs (TGEV and PRCV) was mediated through the N, but not the spike protein, whereas weaker cross-reactivity occurred with feline (FIPV) and canine CoVs. Using E.coli-expressed recombinant SARS-CoV N-protein and fragments, the cross-reactive region was localized between amino acids (aa) 120-208. The N-protein fragments aa360-412 and aa1-213 reacted specifically with SARS-convalescent sera, but not with negative human sera in ELISA; fragment aa1-213 cross-reacted with antisera to animal CoVs, whereas fragment aa360-412 did not cross-react and could be a potential candidate for SARS diagnosis. Particularly noteworthy, a single substitution at aa 120 of PRCV N-protein diminished the cross-reactivity. We also demonstrated that the cross-reactivity is not universal for all group 1 CoVs, because human CoV-NL63 did not cross-react with SARS-CoV. One-way cross-reactivity of human CoV-NL63 with group 1 CoVs was localized to aa1-39 and at least one other antigenic site in the N-protein C-terminus, differing from the cross-reactive region identified in SARS-CoV N-protein. The observed cross-reactivity is not a consequence of a higher amino acid identity between SARS-CoV and porcine CoV nucleoproteins because sequence comparisons indicated that SARS-CoV N-protein has similar amino acid identity with infectious bronchitis virus (IBV) N-protein and shares higher identity with bovine CoV N-protein within the cross-reactive region. The TGEV and SARS-CoV N-proteins are RNA chaperons with long disordered regions. We speculate that during natural infection, antibodies target similar short antigenic sites within the N proteins of SARS-CoV and porcine group 1 CoVs that are exposed to an immune response. Identification of the cross-reactive and non-cross-reactive N-protein regions allows development of SARS-CoV specific antibody assays for screening animal and human sera.