Recombinant Receptor Binding Domain Protein Induces Partial Protective Immunity in Rhesus Macaques Against Middle East Respiratory Syndrome Coronavirus Challenge()

Lan, Jiaming; Yao, Yanfeng; Deng, Yao; Chen, Hong; Lu, Guangwen; Wang, Wen; Bao, Linlin; Deng, Wei; Wei, Qiang; Gao, George F.; Qin, Chuan; Tan, Wenjie

Even though the qRT-PCR is considered as a highly sensitive assay for detection the viral nucleic acid, an assay of viral isolation is absolute necessity to detect the real viral replication. To this end, all tissues were homogenised to a final 10% suspension in DMEM and clarified by low-speed centrifugation at 4500 g for 30 min at 4°C. Swab samples were immersed in 1-mL DMEM, vortexed, and clarified by low-speed centrifugation at 5000 g for 10 min at 4°C. Virus titres were determined in Vero cell monolayers grown in 96-well plates as described previously (Yao et al., 2013) . In brief, 1.5 × 10 4 /well of Vero cells were seeded in a 96-well plate and incubated overnight at 37°C in a CO 2 incubator. One-hundred microliters of 10-fold serially diluted suspension was then added to each well in quadruplicate. The virus was allowed to adsorb to the cells at 37°C for 1 h. After adsorption, viral inocula were removed, and 100 μL of DMEM (2% FBS) was added to each well. Plates were incubated in a CO 2 incubator at 37°C for 3 days, following which CPEs were observed microscopically at 40× magnification. The virus titres expressed as the TCID 50 of each specimen were calculated by the Reed-Muench method.