## 3 Results
### RABV Production Was Higher in MRC-5f\({}^{\text{IFNAR1}-}\) Cell Line
The target sequence was amplified by PCR with IFNARI gRNA MRC-5F\({}^{\text{IFNAR1}-}\)-1,2,3 primers in Table 4 and then was sequenced. The deficiency of IFNARI in three MRC-5F\({}^{\text{IFNAR1}-}\) cell lines were confirmed by sequencing trace analysis. The gRNA target area is marked in gray. Mutated bases are marked in red, and—means deletion. (**B**) Three MRC-5F\({}^{\text{IFNAR1}-}\) cell lines, MRC-5F\({}^{\text{IFNAR1}-}\) cell lines, MRC-5\({}^{\text{NC}}\), and MRC-5 cells were cultured in 6-well plates to 80% confluence. Total RNA from cells was extracted, and the expression of IFNAR1 mRNA was detected by qRT-PCR. (**C**) Three MRC-5F\({}^{\text{IFNAR1}-}\) cell lines and MRC-5 cells were cultured in 6-well plates to 80% confluence. The cells were infected with the PM RABV strain at an MOI of 0.05. The total RNA from cells was extracted at 24 hpi, and the expression of IFN-\(\beta\) and OAS1 mRNA was detected by qRT-PCR. (**D**) Viral RNA in the culture supernatant were detected by qRT-PCR at 96 and 168 hpi. \({}^{\text{+}}\): Viral infection; NC: non-targeting control; \({}^{\text{+}}p<0.05\); \({}^{\text{**}}p<0.01\); \({}^{\text{**}}p<0.001\); \({}^{\text{**}}p<0.0001\); ns \(p\geq 0.05\) compared with MRC-5\({}^{\text{+}}\) at the indicated gene or time. The MRC-5 and MRC-5\({}^{\text{IFNAR1}-}\) cells were passaged in 1500 square centimeter cell spinner flasks (Figure 5A). The MRC-5 cells were treated with 4 uM TPCA-1, 4 uM ruxolitinib, or both 2 uM TPCA-1 and Ruxolitinib 2 h before RABV infection. Subsequently, MRC-5 cells were infected with the PM RABV strain at a MOI of 0.05. The viral titer in the culture supernatant was determined by FFU assays at 96, 168, and 240 hpi. The viral production was increased by 8.3-, 5.2-, and 12.3-fold by TPCA-1, Ruxolitinib, or both TPCA-1 and Ruxolitinib, treatment, respectively, at 168 hpi (Figure 5C). The MRC-5\({}^{\text{IFNAR1}-}\)2 cells were infected with the PM RABV strain at a MOI of 0.05. The viral titer in the culture supernatant was determined by FFU assays at 168 hpi. The viral production was increased 4-fold compared with that using MRC-5 cells (Figure 5D). Compared with IFN inhibitors, the MRC-5\({}^{\text{IFNAR1}-}\) cell line did not perform efficiently and showed a substantial decrease at 240 hpi. Cell growth status of each experimental group was recorded at 168 hpi. Cell growth status showed that the IFN inhibitor and IFNAR1 silencing had no significant effect on the normal growth of MRC-5 cells. The difference in cytopathy shows the promoting effect of IFN inhibitor and IFNAR1 silencing on viral infection (Figure 5B). More than 80% cell death in the MRC-5\({}^{\text{IFNAR1}-}\) cell group was detected at 240 hpi. This may be related to the higher population of MRC-5\({}^{\text{IFNAR1}-}\) cells used. Compared with Vero cells, no significant difference in RABV production was observed in TPCA-1 or Ruxolitinib-treated MRC-5 cells. Addition of IFN inhibitors or the use of INFAR1-defective cell lines are potential solutions for the production of RABV vaccines in MRC-5 cells. Figure 5: The IFN inhibitor or MRC-5\({}^{\text{IFNAR1}-}\) cell lines enhanced rabies virus (RABV) production in pilot-scale experiments. (**A**) The pilot production platform consists of 30 spinner flasks with a surface area of 1500 cm\({}^{2}\) and a constant temperature flask-rotating machine. The MRC-5\({}^{\text{IFNAR1}-}\) cells and MRC-5 cells treated with both 2 uM TPCA-1 and Ruxolitinib 2 h before RABV infection were infected with the PM RABV strains at an MOI of 0.05 in 1500 square centimeter spinner flasks, respectively. Vero cells were infected with the aG RABV strains at a MOI of 0.001. (**B**) Representative cell status was recorded at 168 hpi. (**C**) The MRC-5 cells were treated with 4 uM TPCA-1, 4 uM Ruxolitinib, or both 2 uM TPCA-1 and Ruxolitinib 2 h before RABV infection. Viral titers in the culture supernatant were detected by focus-forming unit (FFU) assays at 96, 168, and 240 hpi. (**D**) The MRC-5 or MRC-5\({}^{\text{IFNAR1}-}\) cells were infected with RABV.