Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169-422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50-150 as the RNA-binding domain and the C-terminal aa 169-422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-nucleocapsid protein (NP) antibodies produced were mapped to aa 1-20, aa 150-170 and aa 390-410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15-20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.

Previous studies have reported that a prokaryotic-expressed recombinant nucleocapsid protein (NP) is a suitable reagent for the epidemiological screening of coronavirus infection. In this study, soluble recombinant human coronavirus OC43 (HCoV-OC43) NP was produced to examine the antigenicity of the HCoV-OC43 NP of betacoronavirus. Using the purified recombinant NP as an antigen, a polyclonal antibody from rabbit serum with specificity for HCoV-OC43 NP was generated; this antibody reacts specifically with HCoV-OC43 NP and does not cross-react with other human CoV NPs (including those of SARS-CoV and HCoV-229E) by Western blot. Sera from 26 young adults, 17 middle-aged and elderly patients with respiratory infection, and 15 cord blood samples were also tested. Strong reactivity to the NPs of HCoV-OC43 was observed in 96%, 82%, and 93% of the serum samples from the young adults, respiratory patients, and cord blood samples, respectively. To identify the immunoreactivities of the three structural regions of the NP that are recognised by the rabbit polyclonal antibody and human serum, the antigenicities of three protein fragments, including the N-terminal domain (aa 1-173), the central-linker region (aa 174-300), and the C-terminal domain (aa 301-448), were evaluated by Western blot. The rabbit polyclonal antibody demonstrated greater immunoreactivity to the central-linker region and the C-terminal domain than to the N-terminal domain. Three different patterns for the immunoreactivities of the three structural regions of HCoV-OC43 NP were observed in human serum, suggesting variability in the immune responses that occur during HCoV-OC43 infection in humans. The central-linker region of the NP appeared to be the most highly immunoreactive region for all three patterns observed. The goal of this study was to offer insight into the design of diagnostic tools for HCoV infection.

No abstract

The SARS-CoV spike protein, a glycoprotein essential for viral entry, is a primary target for vaccine and drug development. Two peptides denoted HR-N(SN50) and HR-C(SC40), corresponding to the Leu/Ile/Val-rich heptad-repeat regions from the N-terminal and C-terminal segments of the SARS-CoV spike S2 sequence, respectively, were synthesized and predicted to form trimeric assembly of hairpin-like structures. The polyclonal antibodies produced by recombinant S2 protein were tested for antigenicity of the two heptad repeats. We report here the first crystallographic study of the SARS spike HR-N/HR-C complex. The crystal belongs to the triclinic space group P1 and the data-set collected to 2.98 A resolution showed noncrystallographic pseudo-222 and 3-fold symmetries. Based on these data, comparative modeling of the SARS-CoV fusion core was performed. The immunological and structural information presented herein may provide a more detailed understanding of the viral fusion mechanism as well as the development of effective therapy against SARS-CoV infection.

The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein.