Since its emergence in 2012, more than 900 laboratory-confirmed cases of Middle East respiratory syndrome (MERS) have been reported with a fatality rate of more than 30%. However, no antigen detection assay for commercial use is available for diagnosis. In this study, the full-length nucleocapsid protein (NP) gene of MERS coronavirus (MERS-CoV) was cloned and expressed in Escherichia coli. A MERS-CoV NP capture enzyme-linked immunosorbent assay (ELISA) using two MERS-CoV-NP-specific monoclonal antibodies (MAbs) generated was developed. The ELISA was evaluated using 129 nasopharyngeal aspirates (NPAs) positive for various respiratory viruses and simulated positive NPAs by adding serial dilutions of MERS-CoV. Using a cutoff OD of 0.19, all 129 NPAs positive for respiratory viruses showed very low OD, with a specificity of 100%. For the two simulated MERS-CoV-positive NPAs with serial dilutions of live MERS-CoV, all samples with ≥10 50% tissue culture infective dose (TCID50)/0.1 mL showed positive results. For the 10 additional NPAs with 20 and 200 TCID50/0.1 mL of live MERS-CoV added, all were positive. A highly sensitive and specific MAbs-based antigen capture ELISA has been developed for MERS. This sensitive and specific antigen capture ELISA should be useful for detection of MERS-CoV in human and dromedaries and in field studies.

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The nucleoprotein (NP) of influenza A virus plays a critical role in the formation of viral ribonucleoprotein (vRNP) complex. However, it remains unclear which key residues in NP are associated with the assembly of vRNP and contribute to virus replication. Here, a highly conserved aspartic acid at residue 88 (D88) of NP was identified by molecular docking of NP with the Fv region of a broad-spectrum anti-NP mAb 19C10 and further demonstrated to be an important residue contributes to the RNP activity, virus growth in MDCK cells and replication in lungs of infected mice by comparing recombinant wild-type A/WSN/1933 virus to the mutant virus that contains an alanine instead of aspartic acid at NP residue 88. D88 was also predicted to interact with PB2 by molecular docking and further verified by immunoprecipitation. These findings provide new information for understanding the interaction between NP and other polymerase subunits in virus replication.