Montelukast, an Anti-asthmatic Drug, Inhibits Zika Virus Infection by Disrupting Viral Integrity

Abstract

The association of Zika virus (ZIKV) infection and severe complications including neurological sequelae especially fetal microcephaly has aroused global attentions since its outbreak in 2015. Currently, there are no vaccines or therapeutic drugs clinically approved for treatments of ZIKV infection, however. And the drugs used for treating ZIKV in pregnant women require a higher safety profile. Here, we identified an anti-asthmatic drug, montelukast, which is of safety profile for pregnant women and exhibited antiviral efficacy against ZIKV infection in vitro and in vivo. And we showed that montelukast could disrupt the integrity of the virions to release the viral genomic RNA, hence irreversibly inhibiting viral infectivity. In consideration of the neuro-protective activity that montelukast possessed, which was previously reported, it is promising that montelukast could be used for patients with ZIKV infection, particularly for pregnant women.

Keywords: Zika virus; flavivirus; montelukast; viral inactivator; viral integrity.

FIGURE 1

The chemical structure of montelukast and its inhibitory activity against Zika virus (ZIKV) strains from Asian and African lineages in two host cells. (A) Chemical structure of montelukast. Dose-dependent inhibition of ZIKV strain SZ01 (B), MR766 (C), and FLR (D) infection by montelukast in BHK-21 cells and inhibition of ZIKV strain SZ01 (E), MR766 (F), and FLR (G) infection in Vero E6 cells. Curcumin, an anti-ZIKV drug, was included as positive control. All experiments were carried out in triplicate, and the error bars stand for standard deviation (SD). The 50% inhibitory concentration (IC50) is presented as means ± SD and summarized in Table 2.

FIGURE 2

The cytotoxicity of montelukast in different cell lines. BHK-21 (A), Vero E6 (B), or U-251 MG (C) cells were treated with serially diluted montelukast. Then cells viability were determined 2 days later by Cell Counting Kit-8 (CCK-8) kit according to the instruction manual. All experiments were carried out in triplicate, and the error bars stand for standard deviation (SD). The 50% cytotoxicity concentration (CC50) is presented as means ± SD and summarized in Table 3.

FIGURE 3

Antiviral activity of montelukast against other two flaviviruses [dengue virus (DENV)-2 and yellow fever virus (YFV) 17D] in two host cells. Dose-dependent inhibition of DENV-2 (A) and YFV 17D (B) by montelukast in BHK-21 and Vero E6 cells. All experiments were carried out in triplicate, and the error bars stand for standard deviation (SD). The 50% inhibitory concentration (IC50) is presented as means ± SD and summarized in Table 2.

FIGURE 4

Montelukast inhibited flavivirus infection at the early stage of virus life cycle. Time of addition experiment of montelukast against Zika virus (ZIKV) SZ01 (A), dengue virus (DENV)-2 (B), and yellow fever virus (YFV) 17D (C). Montelukast hardly inhibited ZIKV SZ01 (D), DENV-2 (E), and YFV 17D (F) infection at post-entry stage. NITD008, an adenosine analog, inhibiting flaviviruses RNA replication by terminating viral RNA synthesis was included as control. All experiments were carried out in triplicate, and the error bars stand for standard deviation (SD). The data are presented as means ± SD. NS, not significant. Student’s two-tailed t-test.

FIGURE 5

Montelukast blocked flaviviruses adsorption, not internalization. Virus adsorption assay of montelukast against Zika virus (ZIKV) SZ01 (A), dengue virus (DENV)-2 (B), and yellow fever virus (YFV) 17D (C); and curcumin, an inhibitor known to block virus adsorption, was included as control. Virus internalization assay of montelukast against ZIKV SZ01 (D), DENV-2 (E), and YFV 17D (F), and chloroquine, an inhibitor known to block virus internalization, was included as control. All experiments were carried out in triplicate, and the error bars stand for standard deviation (SD). The data are presented as means ± SD. NS, not significant; ***P < 0.001; ****P < 0.0001, Student’s two-tailed t-test.

FIGURE 6

Montelukast irreversibly disrupted viral infectivity. Infectivity inhibition reversibility assay of montelukast against Zika virus (ZIKV) SZ01 (A), dengue virus (DENV)-2 (B), and yellow fever virus (YFV) 17D (C). All experiments were carried out in triplicate, and the error bars stand for standard deviation (SD). The data are presented as means ± SD. NS, not significant; ****P < 0.0001, Student’s two-tailed t-test.

FIGURE 7

Degradation of released genomic RNA of flaviviruses mediated by montelukast treatment in an RNase digestion assay. The release and degradation of genomic RNA of Zika virus (ZIKV) SZ01 (A), dengue virus (DENV)-2 (B), yellow fever virus (YFV) 17D (C), and purified virions of ZIKV SZ01 (D), DENV-2 (E), and YFV 17D (F) were detected by using their respective primers targeting different regions in the viral genome. All experiments were carried out in triplicate, and the error bars stand for standard deviation (SD). The data are presented as means ± SD.

FIGURE 8

Protection against vertical transmission of Zika virus (ZIKV) in montelukast-treated pregnant C57BL/6 mice. (A) Viremia of pregnant C57BL/6 mice (n = 12 in each group). (B) Viral RNA loads in the placentas (n = 24 in each group). Two embryos of each pregnant mouse were randomly collected, and the viral RNA load in each placenta was determined. (C) Viral RNA loads in the fetal heads (n = 24 in each group). The viral RNA load in the fetal head of each collected embryo was determined. The bars reflect median values. The horizontal dotted lines represent limits of detection. **P < 0.01; ***P < 0.001, Mann–Whitney test.

FIGURE 9

Protective activity of montelukast against lethal Zika virus (ZIKV) infection in type I interferon receptor-deficient A129 mice. (A) Survival of ZIKV-infected A129 mice. ***P < 0.001, log-rank (Mantel–Cox) test. (B) Viral RNA loads in sera of ZIKV-infected A129 mice. Whiskers: 5th–95th percentile. ***P < 0.001, Mann–Whitney test.