## 4 Discussion  
Use of inactivated vaccines is an effective way to protect against a viral infections [1]. Inactivated viral vaccines produced using Vero cells are the most used product in the market. However, safety concerns posed by its residual non-human DNA need to be addressed. This has also led to such vaccine products being extremely demanding for DNA residual removal in quality control [39]. The demand for safer and more protective vaccines has given rise to a strategy to cultivate virus using human diploid cells such as MRC-5 cells and gradually replace Vero cells [40]. However, our results yielded a low RABV production in MRC-5 cells (Figure 1A). [PERSON] et al. [41] reported similar results: incubation with the challenge virus standard (CVS) RABV strain in a MRC-5 cell line at 24 hpi resulted in a low infectivity of approximately 30%, which resulted in low yields. Due to the low RABV production, human diploid cell-based inactivated RABV vaccines are expensive, limiting their mainstream production in the market. In 1977, [PERSON] et al. [42] increased the infectivity of human diploid cells by screening for the appropriate viral strains. [PERSON] et al. [43] reported that the inhibition of IFN-B gene expression increased RABV infection. However, neither of these studies improved the yield of RABV. The cell's innate immunity against viral infection involves the upregulated expression of antiviral proteins that are mediated by the type I IFN signaling pathway [44; 45]. The IFN-induced antiviral response controls most viral infections and is indispensable for controlling viral infections in vertebrates [29]. The RABV P-protein targets both IFN induction and signaling [46]. The globular C-terminal domain of the P-protein (P-CTD) contains sites required for binding and activating STAT1, which, in turn, binds to downstream DNA, blocking the effects of IFN [47]. In 2002, [PERSON] et al. elucidated the practical applications of IFN-non-responsive cells in vaccine development and manufacture [48]. In this study, we found that RABV infection activated a strong IFN response in MRC-5 cells but almost none in Vero cells (Figure 1) [11]. This result is supported by several studies [49; 50; 51], suggesting that inhibition of the IFN response could result in better RABV production in MRC-5 cells. We successfully enhanced RABV production by treatment with IFN inhibitors. The RABV production in MRC-5 cells treated with IFN inhibitors, TPCA-1 or Ruxolitinib, at 168 hpi increased yields by 10- and 13-fold, respectively, comparable to that in Vero cells (Figure 2). The receptor of IFN-\(\alpha\) and IFN-\(\beta\), IFNAR1 is the key node for IFN antiviral activity. In our study, the utilization of IFNAR1 mAbs, \(\alpha/\beta\)-IFNAR1 mAb, and \(\alpha\)-IFNAR1 mAb also significantly enhanced RABV production in MRC-5 cells (Figure 3). Additionally, we successfully established a stable MRC-5\({}^{\text{IFNAR1}-}\) cell line via GenCRISPR(tm) gene editing technology. The IFN and OAS1 mRNA expression were inhibited, resulting in increased RABV production by 6-fold in the MRC-5\({}^{\text{IFNAR1}-}\) cells (Figure 4). We showed that the MRC-5\({}^{\text{IFNAR1}-}\) engineered cells could be used for inactivated RABV vaccine production. Additionally, the MRC-5\({}^{\text{IFNAR1}-}\) cell line could be applied in the production of other viral vaccines as well as a detection cell line for viruses with low sensitivity to human diploid cells. A marketed human diploid cell RABV vaccine produced by Chengdu Kanghua Biological Products Co. in China has a near 100% efficacy rate when used appropriately and greatly reduces the carcinogenic risk due to human residues [52]. However, due to its lower yield, this vaccine is significantly more expensive than Vero cell RABV vaccines. Nevertheless, in 2020, the use of this rabies vaccine expanded 10-fold compared to that in 2010.