The growth of PRRSV was significantly inhibited in Marc–145 treated with SR
To analyze whether SR shares the antiviral activity against PRRSV, the following experiments were carried out. First, the cytotoxicity of SR to MARC–145 cells were tested using MTT Cell Proliferation and Cytotoxicity Assay Kit. In comparison to mock and SR treatment at different concentrations had no obvious impact on the viability of MARC–145 cells by measuring the absorbance of cell cultures at 570 nm (Fig.1B). And then, we investigated the inhibitory effect of SR on PRRSV (Fig.1C).
In order to explore the antiviral function of SR, we infected Marc–145 with PRRSV DJY and SR mixture, cultured at 37 ℃ for two hours, washed the cells with PBS, removed the non-adsorbed virus particles, then added 2% fresh medium containing, and measured the relative expression of ORF7 mRNA by qRT-PCR at 48hpi. The results showed that compared with mock, SR significantly and continuously inhabit the replication of PRRSV (Fig.1D). And in the presence of different concentrations of SR (0, 10, 20, 40, 60, 80 and 100 μM), compared with PRRSV infected Marc–145 cells without SR treatment, the virus titer was decreased, which also showed dose-dependent inhibition (Fig.1E).
The proliferation of PRRSV were inhibited in Marc–145 treated with SR
Marc–145 cells were pretreated with 100μM SR or PBS for 24 hours, and then infected with PRRSV DJY at the dose of MOI = 1. The virus titers of Marc–145 cells pretreated with SR were measured at 12, 24, 36 and 48 hpi, respectively. Compared with PBS group, the virus titer of Marc–145 cells pretreated with SR decreased to some extent (Fig. 2A). These results showed that PRRSV DJY proliferation in SR pretreated cells was affected, but in the later stage, the inhibition decreased gradually with the increase of time.
We examined whether SR can inactivate PRRSV infection. PRRSV DJY (MOI = 0.1) was mixed with gradient SR, and the mixture was incubated at room temperature for 2 hours, then infected with Marc–145 of 96 well plate, and the virus titer was determined after 48dpi. As shown in Figure 2B, compared with PBS treatment, SR treatment with concentration of 100μM, 10μM and 1μM had no significant effect on PRRSV titer, indicating that SR could not directly inactivate PRRSV infectivity.
PRRSV attachment and entry were significantly inhibited by SR
In order to further determine the precise steps of SR inhibition in the virus life cycle, we first evaluated the effect of SR on PRRSV adsorption. The precooled Marc–145 cells were inoculated with a mixture of PRRSV DJY and SR (100 μM) of different MOI (MOI = 10,1,0.1,0.01), and then placed at 4 ℃ for another 2 hours. The cells were washed with PBS to remove the non-adsorbed virus particles, then incubated at 37 ℃, and the virus titer was measured at 48 hpi. As shown in the Figure 3A, compared with PBS treatment, SR treatment significantly inhibited the PRRSV of each infection dose, indicating that SR may inhibit the adsorption of PRRSV to the cell surface, but the effect was not obvious.
In order to further explore the effect of SR on PRRSV entry, PRRSV DJY was allowed to adsorb on precooled Marc–145 cells at 4 ℃ under different MOI. SR (100 μM) was then added to the virus adsorbed cells and the cells were placed at 37 ℃. After incubation for 3 hours, replace the medium containing SR with fresh medium, and the cells were further cultured at 37 ℃ for 48 hours to determine the virus titer. The results showed that the virus titer of Marc–145 cells treated with SR decreased about 5 times under 10 to 0.01 MOI virus compared with PBS (Fig.3B). In conclusion, these results suggest that SR inhibits PRRSV from entering Marc–145 cells.
SR had significant effect on genome synthesis of PRRSV
In order to solve the problem of whether SR inhibit PRRSV genome synthesis, we analyzed the effect of SR on PRRSV genome synthesis. Marc–145 cells were infected with PRRSV DJY (MOI = 1) at 37 ℃ for 2 hours, and then treated with SR (100 μM) or PBS. Samples were taken at the designated time points (12h, 24h, 36h) to prepare total RNA and analyze the PRRSV genome by real-time RT-PCR. The results showed that the relative level of GP7 in Marc–145 cells treated with SR was significantly reduced compared with PBS at all the time, and showed a time gradient. At 12 hours after treatment, the relative level of PRRSV had been reduced by 10 times (Fig.3C), indicating that SR could significantly inhibit the synthesis of PRRSV genome.
In order to further study whether SR can inhibit the release of PRRSV, Marc–145 cells were infected with PRRSV DJY (MOI = 1). At 24 hpi, the inoculum was replaced with fresh medium containing SR. The virus titers in supernatant were measured 15 minutes, 30 minutes, 45 minutes and 60 minutes after medium replacement. As shown in Figure 3D, in the supernatant, compared with PBS treatment, the PRRSV titer of cells treated with SR did not decrease significantly at different time points, indicating that SR had no effect on the release of PRRSV virus particles.