Antiviral Activity of Benzavir-2 against Emerging Flaviviruses

Gwon, Yong-Dae; Strand, Mårten; Lindqvist, Richard; Nilsson, Emma; Saleeb, Michael; Elofsson, Mikael; Överby, Anna K.; Evander, Magnus

Plasmids containing a full-length infectious clone (icDNA) of the Brazilian ZIKV isolate, containing the ZsGreen reporter (pCCI-SP6-ZIKV-ZsGreen) were kindly provided by Dr. A. Merits (Tartu University, Tartu, Estonia), and the strategy previously described for ZIKV icDNA rescue was used [21] . Briefly, 5 µg of pCCI-SP6-ZIKV-ZsGreen were linearized using AgeI restriction enzyme prior to in vitro transcription. The resulting DNA fragments were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany) and eluted with 6 µL water. The mMESSAGE mMACHINE SP6 transcription kit (Thermo Scientific, USA) was used to synthesize in vitro transcribed RNA from 3 µL of cDNA in a 20 µL reaction volume. The capped RNA transcripts were transfected into 4 × 10 5 Vero B4 cells [22] using the TransIT ® -mRNA Transfection Kit (Mirus Bio, Madison, WI, USA). Transfected cells were monitored daily and, when significant, the cytopathogenic effect and the ZsGreen signal was observed, the stocks of rescued viruses (rZIKV-ZsGreen), passage number 0 (p0 stocks) were harvested, centrifuged at 1500 rpm for 5 min to remove cellular debris, and aliquoted with 20% fetal bovine serum (FBS). Aliquots were stored at −80 • C until use.