key: cord-1019840-7q9h92le authors: Gong, Mei‐jiao; Li, Shi‐fang; Xie, Yin‐li; Zhao, Fu‐rong; Shao, Jun‐jun; Zhang, Yong‐guang; Wang, Wen‐hui; Chang, Hui‐yun title: Inhibitory effects of homoharringtonine on foot and mouth disease virus in vitro date: 2019-05-29 journal: J Med Virol DOI: 10.1002/jmv.25494 sha: e228e1640e98017ea60d8df614187ce67430b175 doc_id: 1019840 cord_uid: 7q9h92le Foot‐and‐mouth disease (FMD) is a highly contagious disease that affects cloven‐hoof animals including cattle, swine, sheep, goats, and lots of wild species. Effectively control measures are urged needed. Here, we showed that homoharringtonine treatment exhibited a strong inhibitory effect against two different strains of FMDVs (O/MYA98/BY/2010 and A/GD/MM/2013) in swine kidney (IBRS‐2) cells. Further experiments demonstrated that homoharringtonine did not affect virus attachment or entry. Using time‐of‐addition assays, we found that the antiviral activity of homoharringtonine occurred primarily during the early stage of infection. These results demonstrated that homoharringtonine might be an effective anti‐FMDV drug. Further studies are required to explore the antiviral activity of homoharringtonine against FMDV replication in vivo. Foot-and-mouth disease (FMD) is one of the most contagious disease of the cloven-hoofed animals and has a great potential for causing severe economic losses in susceptible farms. 1 Generally, the clinical symptoms of infected animals, such as cattle, pigs, sheep, and goats, were characterized by vesicles in the feet, in and around the mouth, and on the mammary gland in females. 2 There are seven serotypes of FMDV, namely, O, A, C, Asia 1, SAT1, SAT2, and SAT3, and infections with any serotype do not confer immunity against others. 3 Vaccination is currently the preferred method of preventing FMDV infection. However, these vaccines provide complete clinical protection of the animals at least 7 days after vaccination. 4 In addition, these emergency vaccines are serotype and subtype specific, and as a result, their using often requires specific diagnostic tests to distinguish between vaccinated and unvaccinated animals. 5 An alternative strategy that can be used is to use an immunomodulatory or antiviral molecules to induce immediate and serotype nonspecific protection of the animal under FMDV infection. 6 Viral proteins are needed for lots of virus to form new particles. This process relies on the cellular translational machinery, thus, inhibition of protein translation process may become a new antiviral therapeutic strategy to control infection. To date, a number of compounds including silvestrol, 7 hippuristanol, 8 rocaglamide, 9 elisabatin, and allolaurintenol 10 have been identified to have a specific effect on inhibiting the translation process. Homoharringtonine is known to inhibit the first cycle of the elongation phase of eukaryotic translation. 11, 12 Homoharringtonine was used in China for the treatment of chronic myeloid leukemia (CML) and other types of tumor diseases for the past 30 years. 13 Besides that, homoharringtonine was reported to exhibit antiviral activity, such as hepatitis B virus (HBV), bovine viral diarrhea virus (BVDV), chikungunya virus, mouse hepatitis coronavirus, varicella-zoster virus, vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), porcine epidemic diarrhea virus (PEDV), type 1 herpes simplex virus (HSV-1), and pseudorabies virus (PRV). [14] [15] [16] [17] [18] All of these characteristics make homoharringtonine an attractive candidate for development as an antiviral therapeutic, but its activity against FMDV remains unexplored. In the current study, the inhibitory effect of homoharringtonine on FMDV replication in IBRS-2 cells was demonstrated. Also, this study investigated potential mechanisms by which the homoharringtonine could exert its antiviral effects and provided preliminary evidence for its possible use as an antiviral agent during the early stage of viral infection. Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5- ODuntreated is the absorbance obtained with untreated cells; ODtreated represents the absorbance value of treated cells. After 24 hours post infection, the cell lysates were subjected to three freeze-thaw cycles in preparation for determining the FMDV 2B mRNA and VP1 protein levels by qPCR and Western blot analysis, respectively. The collected cells were lysed using 100 µL of Pierce radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, UK) and incubated on ice for half hours. After centrifugation at 12 000 × g for 30 minutes, the supernatants were collected and used to determine To determine the inhibitory effects of homoharringtonine on FMDV infection, viral 2B mRNA was measured by q-PCR as previously described with modifications. 19 Briefly, the collected cells were subjected to RNA extraction with the TRIzol reagent (Invitrogen). The concentration required to reduce virus-induced cytopathogenicity by 50% of the control value (EC 50 ) was calculated by Graphpad Prism 7 (GraphPad Software, Inc., La Jolla, CA). Selectivity indices (SI) were was derived as SI = CC 50 /EC 50 . The statistical significance was analyzed with Student t tests, and values of P < 0.05 were considered significant. Data are presented as means ± SD. The virus binding assay and virus entry assay were carried out as described previously. 20 Usually, at 4°C, the viruses can attach to cell receptors but do not enter the cell until the temperature rise to 37°C . According to these features, this study was performed to evaluate whether homoharringtonine suppresses FMDV attachment and entry. Figure 6A and 6B indicated that homoharringtonine did not exhibit any significant inhibiting capacity against FMDV at binding and entry stages. To further determine at which time after infection We observed that viral attachment and entry were not affected by the treatment of homoharringtonine. Time-of-addition studies were also performed to identify the window in the FMDV replication cycle when homoharringtonine exhibited its antiviral effect. Our study revealed F I G U R E 5 Evaluation of the effects of homoharringtonine by IFA. After the cells infected with FMDV O/MYA98/BY/2010 at an MOI of 1 for 1 hour, IBRS-2 cells were treated with the indicated concentrations of homoharringtonine at 37°C for 12 hours or 0.025% DMSO (mock). IFA was performed and fluorescence was observed using a fluorescent microscope. DMSO, dimethyl sulfoxide; IFA, immunofluorescence assay F I G U R E 6 Homoharringtonine has no effect on virus attachment and entry. A, Relative viral RNA levels of cells treated with homoharringtonine (6.2, 12.5, and 25 μM) at the viral attachment stage. B, Relative viral RNA levels of cells treated with homoharringtonine (6.2, 12.5, and 25 μM) at the viral entry stage. Differences in suppression, compared with the DMSO group, were assessed with an analysis of variance followed by Student t tests. Data were expressed as mean ± SD from three independent experiments that homoharringtonine remained potent when added at post infection from 0 hours to 8 hours post infection, indicating that the inhibitory effect of homoharringtonine on FMDV replication occurred primarily at the early stages. Similar result was also obtained from the study of Dong et al, 17 in their study, they found that homoharringtonine play its roles in the viral replication cycle after viral entry. Although homoharringtonine shows broad antiviral activity against lots of virus in cell cultures, such as VSV, NDV, PEDV, HSV-1, and PRV etc, its action was reported to be restricted to some RNA viruses, for example, Dong et al, 17 reported that homoharringtonine treatment only moderately inhibited the infection by avian influenza virus. In this study, we found that homoharringtonine exerts antiviral activity against FMDV, a porcine Picornaviridae, including two different strains of FMDV. Therefore, this study broadened our understanding of the characteristics of its antiviral activity. Further study is needed to investigate the inhibitory effect of homoharringtonine on FMDV infections in vivo. In conclusion, we demonstrated that FMDV infection was inhibited in a dose and time dependent manner by homoharringtonine treatment. Furthermore, homoharringtonine specifically targeted the virus at the early phases of viral replication. These results indicated that homoharringtonine could become a promising candidate for further development as an FMDV inhibitor. Further research will be required to explore whether homoharringtonine has an antiviral effect in vivo. 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A final concentration of 12.5μM homoharringtonine was added at specified time points that represent the time points during adsorption (0 hours), and post infection (2, 4, 8, and 16 hours). 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