In 2002, severe acute respiratory
syndrome-associated coronavirus (SARS-CoV) emerged in humans, causing a
global epidemic. By phylogenetic analysis, SARS-CoV is distinct from
known 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 an enzyme-linked
immunosorbent assay (ELISA) and Western blot analysis using specific
antisera to animal CoVs and SARS-CoV and SARS patient convalescent-phase
or negative sera. Moderate two-way cross-reactivity between SARS-CoV and
porcine CoVs (transmissible gastroenteritis CoV [TGEV] and porcine
respiratory CoV [PRCV]) was mediated through the N but not the spike
protein, whereas weaker cross-reactivity occurred with feline (feline
infectious peritonitis virus) and canine CoVs. Using Escherichia
coli-expressed recombinant SARS-CoV N protein and fragments, the
cross-reactive region was localized between amino acids (aa) 120 to 208.
The N-protein fragments comprising aa 360 to 412 and aa 1 to 213 reacted
specifically with SARS convalescent-phase sera but not with negative
human sera in ELISA; the fragment comprising aa 1 to 213 cross-reacted
with antisera to animal CoVs, whereas the fragment comprising aa 360 to
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
HCoV-NL63 did not cross-react with SARS-CoV. One-way cross-reactivity of
HCoV-NL63 with group 1 CoVs was localized to aa 1 to 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 level of amino acid
identity between SARS-CoV and porcine CoV nucleoproteins, because
sequence comparisons indicated that SARS-CoV N protein has amino acid
identity similar to that of infectious bronchitis virus N protein and
shares a higher level of 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.
