Development of a plaque assay based on Avicel-crystal violet staining for detection of Middle East respiratory syndrome coronavirus infection and neutralizing antibody

Abstract

Objective To establish a plaque assay for detection of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Methods Vero and Huh7.5 cells were infected with MERS-CoV (EMC/2012) strains for different days (two or three days) and covered with different overlays (agarose or Avicel). The optimal host cells, time of infection and overlay were screened out by analyzing plague formation to establish the plaque assay for detection of MERS-CoV infection. Neutralizing antibodies against MERS-CoV were detected by the established assay. Results Clear plaques were observed on Vero cells infected with MERS-CoV at 3 days post-infection (p.i.), while only tip-size plaques were observed at 2 days p. i.. Clearer and bigger sizes of plaques were observed under the overlay of Avicel than those under the agarose. The Avicel-based plaque assay was suitable for detecting neutralizing antibodies against MERS-CoV. Conclusion We develop a modified plaque assay for detecting MERS-CoV based on Avicel-crystal violet staining. It is easier to operate the modified plaque assay in biological safety level 3 (BSL-3) laboratories. This study paves a way for quantification of MERS-CoV and detection of neutralizing antibodies against MERS-CoV. Key words: Middle East respiratory syndrome coronavirus (MERS-CoV); Biological safety level 3 (BSL-3) laboratory; Plaque assay; Vero cell; Avicel; Neutralization test