*2.9. Detection of SUDV Neutralizing Antibodies by a Pseudotyped Virus*

A pseudotyped virus neutralization assay was performed to test the neutralzing antibodies. Recombinant lentiviral vectors that express SUDV glycoprotein and carry a luciferase reporter gene were produced as described previously [31], with some modifications. Briefly, 239T cells were seeded in 60-mm culture dishes (Corning, NY, USA), and 24 h later, the 80% subconfluent cells were co-transfected with 3 μg of pcDNA 4.0-GP (the glycoprotein expression vector) and 3 μg of pNL4-3.Luc. RE vector by LipofectamineTM 3000 Transfection Reagent (Thermo Scientific, Carlsbad, CA, USA). After 2 days, the supernatant containing the pseudotyped viruses was harvested, and the titer was determined in Huh7 cells as previously described [32]. For testing of neutralizing antibodies, 2-fold serially diluted serum samples were mixed with 100 TCID50 of pseudotyped viruses for 0.5 h at 37 ◦C and were then added to Huh7 cells. After 4 h, the inoculum was removed and replaced with fresh media. Then, cells were lysed at 48 h with cell lysis buffer, that was followed by the addition of 100 μL of luciferase substrate (Promega, Madison, WI, USA) to determine luciferase activity. The luciferase activity of the samples was measured with the Infinite M200 Microplate Spectrophotometer (Tecan, Mannedorf, Switzerland). The percent inhibition rate was calculated as previous showed [1]. The experiments were independently repeated three times.
