Fig 2: EEEV-infected NHP brains were fixed in glut/PFA and homogenized alongside positive control tissue samples. Centricon filtration was used to remove formalin, and supernatants were used to perform the first cell passage on ATCC Vero 76 cells for 72 h. A second passage was also performed for 72 h. CPE was observed and recorded daily. Column controls consisted of diluted virus run through a centricon filtration system, and the safety test control was performed by adding diluted virus directly to cell culture. Plaque assay was performed on glut/PFA fixed samples and control samples to determine EEEV titers. Stock virus was diluted and used as a plaque assay positive control. The limit of detection for the assay was 1 PFU/mL. (A) CPE and plaque assay data for replicate experiment 1. (B) CPE and plaque assay data for replicate experiment 2. (C) CPE and plaque assay data for replicate experiment 3.

Fig 3: EEEV-infected NHP brains were fixed in 10% formalin and homogenized alongside positive control tissue samples. Centricon filtration was used to remove formalin, and supernatants were used to perform the first cell passage on ATCC Vero 76 cells for 72 h. A second passage was also performed for 72 h. CPE was observed and recorded daily. Column controls consisted of diluted virus run through a centricon filtration system, and the safety test control was performed by adding diluted virus directly to cell culture. Plaque assay was performed on formalin-fixed samples and control samples to determine EEEV titers. Stock virus was diluted and used as a plaque assay positive control. The limit of detection for the assay was 1 PFU/mL. (A) CPE and plaque assay data for replicate experiment 1. (B) CPE and plaque assay data for replicate experiment 2. (C) CPE and plaque assay data for replicate experiment 3.

Fig 4: EEEV-infected NHP brains were fixed in glut/PFA and homogenized alongside positive control tissue samples. Centricon filtration was used to remove formalin, and supernatants were used to perform the first cell passage on ATCC Vero 76 cells for 72 h. A second passage was also performed for 72 h. CPE was observed and recorded daily. Column controls consisted of diluted virus run through a centricon filtration system, and the safety test control was performed by adding diluted virus directly to cell culture. Plaque assay was performed on glut/PFA fixed samples and control samples to determine EEEV titers. Stock virus was diluted and used as a plaque assay positive control. The limit of detection for the assay was 1 PFU/mL. (A) CPE and plaque assay data for replicate experiment 1. (B) CPE and plaque assay data for replicate experiment 2. (C) CPE and plaque assay data for replicate experiment 3.

Fig 5: EEEV-infected NHP brains were fixed in glut/PFA and homogenized alongside positive control tissue samples. Centricon filtration was used to remove formalin, and supernatants were used to perform the first cell passage on ATCC Vero 76 cells for 72 h. A second passage was also performed for 72 h. CPE was observed and recorded daily. Column controls consisted of diluted virus run through a centricon filtration system, and the safety test control was performed by adding diluted virus directly to cell culture. Plaque assay was performed on glut/PFA fixed samples and control samples to determine EEEV titers. Stock virus was diluted and used as a plaque assay positive control. The limit of detection for the assay was 1 PFU/mL. (A) CPE and plaque assay data for replicate experiment 1. (B) CPE and plaque assay data for replicate experiment 2. (C) CPE and plaque assay data for replicate experiment 3.