Blebbistatin as a novel antiviral agent targeting equid herpesvirus type 8

Abstract

Introduction: Equid herpesvirus type 8 (EqHV-8) poses a significant threat to equine health, leading to miscarriages and respiratory diseases in horses and donkeys, and results in substantial economic losses in the donkey industry. Currently, there are no effective drugs or vaccines available for EqHV-8 infection control.

Methods: In this study, we investigated the in vitro and in vivo antiviral efficacy of Blebbistatin, a myosin II ATPase inhibitor, against EqHV-8.

Results: Our results demonstrated that Blebbistatin significantly inhibited EqHV-8 infection in Rabbit kidney (RK-13) and Madin-Darby Bovine Kidney (MDBK) cells in a concentration-dependent manner. Notably, Blebbistatin was found to disrupt EqHV-8 infection at the entry stage by modulating myosin II ATPase activity. Moreover, in vivo experiments revealed that Blebbistatin effectively reduced EqHV-8 replication and mitigated lung pathology in a mouse model.

Conclusion: Collectively, these findings suggest that Blebbistatin holds considerable potential as an antiviral agent for the control of EqHV-8 infection, presenting a novel approach to addressing this veterinary challenge.

Keywords: EqHV-8; animal model; antiviral activity; blebbistatin; myosin II ATPase inhibitor.

Figure 1

The chemical structure and cytotoxicity of Blebbistatin. (A) The chemical structure of Blebbistatin. (B) The cytotoxicity of Blebbistatin at different concentrations was determined in RK-13 and MDBK cells by CCK-8 kit and was expressed as relative cell viability by comparing with the viable cells in the absence of Blebbistatin (set up as 100%). These data shown are representatives from three independent experiments. *p < 0.05; **p < 0.01, ns: no significant.

Figure 2

Blebbistatin inhibited EqHV-8 replication in susceptible cell. RK-13 (A,B) or MDBK (C,D) cells were pretreated with Blebbistatin at different concentrations (0, 2.5, 5, 10, and 20 μM) for 1 h, then infected with EqHV-8 SDLC66 (0.1 MOI) for 1 h at 37°C, then these cells were collected to detect gD expression by western blot at 24 hpi. Meanwhile, those cellular supernatants were harvested to assess virus titer using TCID_50. Data were presented as the means of normalized data ± standard deviations (error bars) based on at least three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001, ns: no significant.

Figure 3

Blebbistatin show antiviral activity against other EqHV-8 strains. RK-13 cells were pretreated with Blebbistatin at different concentrations (0, 5, 10, or 20 μM) or DMSO for 1 h at 37°C, respectively, infected with EqHV-8 SDLC66, SD2020113, or Donkey/Shandong/10/2021 at 0.1MOI for 1 h in the presence of Blebbistatin at indicated concentrations. These cells were fixed with paraformaldehyde and stained using mouse anti-EHV-8 positive serum to detect EHV-8 proteins at 36 hpi (red), and the nucleocapsid was counterstained with DAPI (blue). Images were captured using Leica microsystems (DMi8, Germany). Scale bar, 100 μm. The mock-infected cells were served as negative control. Additionally, the progeny viral titer in cellular supernatants was analyzed by TCID₅₀. Data were presented as the means of normalized data ± standard deviations (error bars) based on at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Blebbistatin inhibits EqHV-8 infection mainly at initial stage. Time-of-addition schematic (A). The RK-13 cells were infected with EqHV-8 SDLC66 (MOI = 0.1) and treated with Blebbistatin at different time points, including before infection (Pre-treatment), during infection (Co-treatment), after infection (Post-treatment), and All-stage treatment. The RK-13 cells were treated with Blebbistatin (20 μM) as Pre-treatment, Co-treatment, Post-treatment and All-stage treatment for 24 hpi, and infected with EqHV-8 SDLC66 (MOI = 0.1), the cells were collected to detect gD expression for western blot (B), meanwhile, the cell supernatants were also harvested to progeny virus generation by qPCR (C). α-Tubulin acts as loading control, the data represent as mean ± SD. from three independent experiments, **p < 0.01; ***p < 0.001, ns: no significant.

Figure 5

Blebbistatin suppress EqHV-8 replication at adsorption and internalization stage. The RK-13 cells were incubated with a mixture of Blebbistatin (20 μM) or DMSO and EqHV-8 (1 MOI) for 1 h at 4°C, and washed by PBS and incubated with 3% FBS MEM at 37°C for 24 h before the gD expression was tested using qPCR (A). RK-13 cells were pretreated with Blebbistatin (20 μM) for 1 h and then incubated with EqHV-8 (MOI = 1) at 4°C for 1 h. The cells were washed with PBS and incubated with Blebbistatin (20 μM) or DMSO for another 1 h at 37°C, then washed by citrate buffer. The gD expression was detected using qPCR at 24 hpi. (B) **p < 0.01; ***p < 0.001; ns: not significant.

Figure 6

Blebbistatin inhibits EqHV-8 replication in vivo (A) The pattern diagram of the animal experiments. (B) The titration of EqHV-8 in lung from different group. RK-13 cells were used to titrate in lung from EqHV-8 infected or Blebbistatin treated EqHV-8 infected mice using the Reed–Muench method. The results are presented as the mean ± SD (error bars). *p < 0.05. (C) Representative images of hematoxylin and eosin (H&E) in the lungs derived from mice in indicated groups. Bar, 100 μm.