Meclizine Inhibits Pseudorabies Virus Replication by Interfering With Virus Entry and Release

Abstract

Pseudorabies virus (PRV) is a pathogen that causes substantial economic losses to the swine industry. With the emergence and widespread of PRV variants since 2011 in China, current commercial vaccines cannot provide complete protection against PRV infection. Therefore, antiviral drugs may work as an alternative way to control and prevent PRV. In this study, the inhibitory effects and underlying molecular mechanisms of meclizine against PRV were studied. Meclizine displayed a significant inhibitory effect against PRV when it was added before, simultaneously with, or after virus infection. The inhibitory effect of meclizine occurred during viral entry and cell-to-cell spreading but not at viral attachment into PK-15 cells. Meclizine also inhibited viral particle release at the late stage of infection. The antiviral effect of meclizine was tested in mice, and the results showed that meclizine reduced the severity of clinical symptoms and the viral loads in tissues, and delayed the death, after PRV challenge. The above results indicated that meclizine had an inhibitory effect on PRV. Our findings will contribute to the development of potential therapeutic drugs against PRV infection.

Keywords: antiviral activity; meclizine; pseudorabies virus; virus entry; virus release.

Figure 1

Meclizine inhibited PRV infection and replication in PK-15 cells. (A) Potential cytotoxicity of meclizine against PK-15 cells was detected with the Enhanced Cell Counting Kit-8. PK-15 cells were seeded in 96-well plates and then exposed to different concentrations of meclizine or DMSO. After 24 h of incubation, 10 μl CCK-8 solution was added for another 3 h. The absorbance was measured at 450 nm. (B–D) PK-15 cells were infected with PRV variant strain (PRV-W) and Bartha K61 strain (PRV-V) at MOI of 1 in the presence of different concentrations of meclizine, DMSO (control) for 24 h at 37°C with 5% CO₂. The supernatant and cell samples were harvested. Supernatants were subjected to virus titration. The expression levels of gB protein were analyzed by immunoblotting (C). The copy numbers of PRV-W and PRV-V DNA were quantified by real-time PCR (D). Data were presented as means±SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

Meclizine decreased the expression levels of PRV gB protein detected by IFA. PK-15 cells were infected with PRV-W (A) and PRV-V (B) at MOI of 1 in the presence of different concentrations of meclizine or DMSO (control groups) for 24 h at 37°C with 5% CO₂. Cells were detected by IFA. Scale bars = 200 μm. Typical figures were presented from three independent experiments.

Figure 3

Meclizine inhibited PRV infection during the different stages of infection. (A) Schematic diagram of meclizine administration. PK-15 cells were infected with different PRV strains (PRV-W and PRV-V) at MOI of 1 for 1 h, and cells were treated with 150 μM meclizine at different time points. Meclizine treatment was performed either before (pre), simultaneously with (co), or after PRV infection (post), respectively. At 24 h, cell samples were harvested. (B) The copy numbers of PRV DNA were quantified by qPCR. (C–F) The expression levels of gB protein and GAPDH were analyzed by immunoblotting. The analysis of WB band gray value was visualized by Image J software. Data were presented as means±SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4

Meclizine inhibited PRV infection by interfering with the entry, release and cell-to-cell spreading but not attachment into PK-15 cells. PK-15 cells were seeded in 6-well plates and cultured overnight. When the cells reached approximately 70–80% confluence before carrying out antiviral assays. (A) Virus attachment assay. (B,C) Virus entry assay. (D,E) Virus release assay. (F,G) The cell-to-cell spreading assay of meclizine about PRV-W. The results of PRV-V were showed in Supplementary Figure S1. Scale bars = 200 μm. Data were presented as means±SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

Meclizine inhibits PRV infection in mice. (A) Schematic diagram of mice experiment and mortality of mice in different groups. (B) Survival profile of mice after PRV challenge in different groups. (C) Brain, lung, and liver samples of mice were collected for pathological examination. Typical figures were presented from three independent experiments.

Figure 6

Meclizine treatment decreased the viral loads in tissues after viral challenge. 0.1 g of brain (A), lung (B), and liver (C) samples were taken for DNA extraction. The viral loads in tissues were analyzed by qPCR. *, p < 0.05; Data were presented as means ± SD from three independent experiments.