Chimeric SR-PLP-rSCV grew less efficient in presence of type I interferon (IFN).

Virus growth was compared in (a) type I IFN-deficient (Vero) primate cells, and (b) IFN-competent (MA-104) primate cells. For virus growth kinetics, cells were infected with rSCV and SR-PLP-rSCV (MOI 0.01). Supernatants were taken at 0, 8, 14, 24 and 48 hpi, and viral replication was determined by a plaque titration assay. Growth experiments in Vero and MA-104 cells were done in triplicate and repeated twice. Error bars indicate the standard deviations of the means. Infectious particle production of rSCV and SR-PLP-rSCV was compared using SPSS Version 23.0.0.0 and a general linear regression model. Growth of both viruses did not significantly differ in Vero cells (p = 0.929 and R-square = 0.670). In MA-104 cells growth of the viruses significantly differed (p = 0.038 and R-square = 0.612). (c) Vero cells were treated with 100 IU/ml of recombinant pan-species IFN-α. At 16 h post IFN treatment, cells were infected with rSCV and SR-PLP-rSCV (MOI 0.01), respectively. Supernatants were taken at 24 hpi, and viral replication was determined by a plaque titration assay. The experiment was performed in triplicate and repeated twice. Error bars indicate the standard deviations of the means. One representative experiment is shown. (d) Virus growth was compared in IFN-competent human cells (Calu-3) as described above. The experiment was performed in triplicate. (e) To determine IFN-β expression, Calu-3 cells were infected with rSCV, SR-PLP-rSCV or IFN-inducing RVFV Cl 13 (control of IFN-β expression) at an MOI of 1. Total mRNA was extracted from cell lysates at 24 hpi. IFN-β expression was determined using quantitative real-time PCR analysis. The mean fold change in IFN-β expression was calculated using TATA-binding protein (TBP) expression as a reference gene and the 2−ΔΔCt analysis method [55]. The experiment was done in quadruplicates. Statistical significance between the indicated groups was determined using a two-sided t test. (f) Human airway epithelial cells (HAE) were infected with rSCV and SR-PLP-rSCV with an absolute infectious dose of 40,000 PFU. At 0, 48, 72 and 96 hpi samples were taken and viral replication was determined by a plaque titration assay. The experiment was done in duplicate and repeated three times independently. Statistical significance in (d-f) was determined using a two-sided t test.