Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia

TERAMICHI, Takurou; FUKUSHI, Shuetsu; HACHIYA, Yuma; MELAKU, Simenew Keskes; OGUMA, Keisuke; SENTSUI, Hiroshi

The neutralization test using VSV-MERS/GFP was performed as previously described [8] . A medium composed of Eagle's MEM supplemented with 5% FBS was used for virus and serum dilution. Serially diluted 0.05 ml of test sera were mixed with equal volumes of 3,000 FFU of VSV-MERS/GFP and incubated at 37°C for 1 hr. The mixture was inoculated to Vero cells seeded on a 96-well culture plate and incubated at 37°C for 24 hr in a CO 2 incubator. GFP-positive cells were then detected using a fluorescence microscope. The number of positive GFP cells was counted as described above. The neutralization titer was determined as the highest serum dilution showing ≤50% of the number of GFP-positive cells compared with the no serum control.