Figure 4.

Genetic Stability of nOPV2 in Cell Culture and Animal Model of Infection

(A) Virus adaptation to high temperature in Vero cells. At the top, schematic of experimental design. Sabin2 and nOPV2 were grown in Vero cells at 37°C to accelerate virus evolution. After 10 passages, viral genomes of the 10 passage viruses (P10) were analyzed by RNA-seq. At the bottom, Manhattan plot showing frequency of mutations at several locations (5′cre, domV, 2Ccre^mut, and HiFi/ Rec1) in nOPV2, compared with those in Sabin2. In the domV, the frequency of A481G (19.7%) in Sabin2 is compared with any mutation that increases the thermostability of domV in nOPV2, which is a “gatekeeper” structure involved in regain of virulence. Open circles represent frequencies of mutations observed at passage 8 and solid circles at passage 10.

(B) Genetic stability was further validated by comparing PD₅₀ of Sabin2 and nOPV2 before (P0) and after 10 passages (P10) of accelerated evolution at higher temperature as shown in (A). PD₅₀s of Sabin2 and nOPV2 (P0 and P10) were determined by intra-spinally inoculating Tg66 mice and calculated using the Spearman-Karber method.