Generation and Transduction Efficiency of MERS-CoV Pseudotypes

We employed a previously described protocol for the generation of VSV pseudotype particles (VSVpp) that is based on a replication-deficient VSV vector that lacks the genetic information for VSV-G but instead contains the genetic information for eGFP and firefly luciferase (fLuc) as reporters of transduction efficiency (VSV*ΔG-fLuc, kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern/Switzerland) [37 (link),40 (link)]. In brief, 293T cells transfected with expression vectors for MERS-CoV S, VSV-G (positive control) or empty expression vector (negative control) were inoculated with VSV*ΔG-fLuc for 1 h before being washed with PBS and further incubated for 16 h with culture medium that was supplemented with anti-VSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) (except for cells expressing VSV-G). The produced VSVpp were inoculated onto BHK-21 cells expressing WT or mutant DPP4, or no DPP4 (empty expression vector, negative control) and incubated for 16–18 h before fLuc activity in cell lysates was quantified as an indicator for transduction efficiency using the Beetle-Juice kit (PJK) and a plate luminometer (Hidex) [41 (link)].