Potent Inhibition of Zika Virus Replication by Aurintricarboxylic Acid

Zika Virus is a virus that spreads mostly through mosquitoes. Normally a pregnant mother gets infected through a mosquito bite and passes it to their baby during pregnancy or during delivery. The virus is also known to spread through sexual contact. Blood transfusion has also been reported to be a way the virus can spread. The virus has been reported in South and Central America, United States, Africa, Pacific islands, parts of the Caribbean, and South Asia. Due to the outbreaks, researchers have been working on how to control the virus.

Zika virus (ZIKV) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the Western Hemisphere. Currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of ZIKV infections, and as of yet none are commercially available. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antimicrobial and antiviral activity. In this study, we evaluated ATA as a potential antiviral drug against ZIKV replication. The antiviral activity of ATA against ZIKV replication in vitro showed median inhibitory concentrations (IC₅₀) of 13.87 ± 1.09 μM and 33.33 ± 1.13 μM in Vero and A549 cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (CC₅₀) > 1,000 μM). Moreover, ATA protected both cell types from ZIKV-induced cytopathic effect (CPE) and apoptosis in a time- and concentration-dependent manner. In addition, pre-treatment of Vero cells with ATA for up to 72 h also resulted in effective suppression of ZIKV replication with similar IC₅₀. Importantly, the inhibitory effect of ATA on ZIKV infection was effective against strains of the African and Asian/American lineages, indicating that this inhibitory effect was not strain dependent. Overall, these results demonstrate that ATA has potent inhibitory activity against ZIKV replication and may be considered as a potential anti-ZIKV therapy for future clinical evaluation.”

To understand what the Zika virus is all about, it is important to understand where it comes from. This is because the treatment or possible cure will start by looking at how the virus survives and what can be used to inhibit its spread.

Zika virus (ZIKV) belongs to the genus Flavivirus within the Flaviviridae family. ZIKV is an enveloped positive sense single-stranded RNA virus with a genome size of ∼10.7 kb that encodes a single polyprotein, which is post-translationally processed by cellular and viral proteases into three structural (capsid, C; pre-membrane, prM; and envelope, E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (Tripathi et al., 2017; Avila-Perez et al., 2018).

Zika virus was initially isolated from Uganda in 1947 and viral infections only occurred sporadically in Africa and Asia until 2007. ZIKV appeared explosively as the first large-scale outbreak occurred in the Yap island in 2007 and French Polynesia in 2013 (Weaver et al., 2016). Most recently, in 2015, the first local transmission of ZIKV was found in territories of Latin America and the Caribbean, resulting in up to 1.3 million of ZIKV infection suspected cases (Tang et al., 2016; Tripathi et al., 2017).

There are several vaccines and antiviral drugs currently under development for the prevention or treatment of ZIKV infection (Abbink et al., 2016; Larocca et al., 2016; Shan et al., 2017; Fink et al., 2018). DNA-based (Abbink et al., 2016; Larocca et al., 2016), inactivated (Abbink et al., 2016; Larocca et al., 2016; Shan et al., 2017), live-attenuated and mRNA (Richner et al., 2017) vaccines have been proposed for the prophylactic treatment of ZIKV infections. On the other hand, arbidol (ARB) (Fink et al., 2018; Haviernik et al., 2018), bortezomib, mycophenolic acid, daptomycin (Barrows et al., 2016), obatoclax, saliphenylhalamide, gemcitabine (Kuivanen et al., 2017), emetine (Yang et al., 2018), and sofosbuvir (Bullard-Feibelman et al., 2017) have been proposed for the therapeutic treatment of ZIKV infection. Despite these tremendous efforts, there is currently no Food and Drug Administration (FDA)-approved vaccines and/or anti-viral drugs available for the treatment of ZIKV infection. Since vaccination takes at least 2 weeks to several months to show protective effects against ZIKV infection, vaccination is probably not the most appropriate prophylactic method for those who are traveling to areas where ZIKV is epidemic, endemic, or have already been infected. Moreover, vaccination may cause an important issue, such as antibody-dependent enhancement (ADE) (Bardina et al., 2017; Priyamvada et al., 2017). ADE, which has been extensively described in DENV (Priyamvada et al., 2017), is a phenomenon where preexisting antibodies facilitate binding and infection during subsequent exposure to infectious viruses, instead of neutralizing them, resulting in exacerbation of clinical signs (Bardina et al., 2017; Priyamvada et al., 2017). Because of the structural similarities between DENV and ZIKV, DENV immunity–linked ADE of ZIKV infection has also been reported (Bardina et al., 2017; Priyamvada et al., 2017). Since vaccination for ZIKV could lead to DENV ADE, antivirals could represent a better choice for the control of ZIKV infection.

Aurintricarboxylic acid (ATA), a polyanionic aromatic compound, has been shown to have inhibitory properties against several bacteria and viruses including, among others, Yersinia pestis (Liang et al., 2003), Cryptosporidium parvum (Klein et al., 2008), human immunodeficient virus (HIV) (Mitra et al., 1996; De Clercq, 2005), hepatitis C virus (HCV) (Chen et al., 2009; Mukherjee et al., 2012; Shadrick et al., 2013), Vaccinia virus (Myskiw et al., 2007), influenza virus (Hung et al., 2009), Enterovirus 71 (Hung et al., 2010) and severe acute respiratory syndrome coronaviruses (SARS-CoV) (He et al., 2004). Mechanistic studies have suggested that ATA has the ability to modulate various cellular enzymes such as activators of the Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) families (Rui et al., 1998), inhibitors of nucleases (Shadrick et al., 2013), glucose-6-phosphate dehydrogenase (Bina-Stein and Tritton, 1976), and topoisomerase II proteins (Catchpoole and Stewart, 1994; Benchokroun et al., 1995) as well as the enzymatic activity of the Vaccinia virus AH1L phosphatase (Smee et al., 2010). However, to date, the ability of ATA to inhibit ZIKV infection has not been evaluated. Herein, we investigated ATA as a plausible prophylactic and therapeutic candidate against ZIKV infection. Our results demonstrate that ATA has a potent and effective antiviral activity against ZIKV in pre- and post-infection settings, including broadly antiviral activity against strains of the African and American/Asian lineages with no toxicity up to 1,000 μM in cultured cells. These data support the feasibility of implementing ATA for the treatment of ZIKV infection.

Materials and Methods

Cell Lines and Viruses

African green monkey kidney epithelial Vero (ATCC CCL-81) and human adenocarcinoma alveolar basal epithelial A549 (ATCC CCL-185) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Inc.) supplemented with 5% fetal bovine serum (FBS) and 1% PSG (100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine) at 37°C in a 5% CO2 atmosphere.

Paraiba/2015 ZIKV isolate was kindly provided by Stephen Dewhurst (Department of Microbiology and Immunology, University of Rochester). Uganda/1947 (MR_766 strain, Catalog No. NR-50065) and Nigeria/1968 (IbH 30656 strain, Catalog No. NR-50066) ZIKV isolates were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). Puerto Rico/2015 (PRVABC59 strain) and French Polynesia/2013 ZIKV isolates were kindly provided from the Centers for Disease Control and Prevention (CDC). Virus stocks were propagated in Vero cells and titrated by plaque assay as previously described (Marquez-Jurado et al., 2018).

Compounds

Aurintricarboxylic acid (Catalog No. A1895) and Arbidol (ARB, Catalog No. SLM0860) were purchased from Sigma-Aldrich, MO, United States. Both compounds were prepared at 100 mM stock solution dissolved in dimethyl sulfoxide (DMSO) and kept at -20°C until experimental use. Each drug was diluted into infectious media (DMEM 2% FBS, 1% PSG) for the described experiments, where the maximum DMSO concentration was 0.1%.

Cell Viability Assay

Cell viability in Vero and A549 cells was measured using the CellTiter 96 Non-Radioactive Cell Proliferation assay (Promega) following the manufacturer’s instructions. Briefly, confluent Vero or A549 cells (96-well plate format, 5 × 104 cells/well, triplicates) were treated with 100 μl of DMEM containing serially diluted (twofold dilutions, starting concentration of 1,000 μM) chemicals or 0.1% DMSO (vehicle control). Plates were incubated at 37°C in a 5% CO2 atmosphere for 36 or 72 h. Samples were treated with 15 μl of Dye Solution and incubated at 37°C in a 5% CO2 atmosphere for 4 h. Next, cells were treated with 100 μl of Solubilization Solution/Stop Mix and absorbance at 570 nm was measured using a Vmax kinetic microplate reader (Molecular Devices, Waltham, MA, United States). Viability of compound-treated cells was calculated as a percentage relative to values obtained with DMSO-treated cells. Non-linear regression curves and the median cytotoxic concentration (CC50) were calculated using GraphPad Prism software version 8.0.

Microplaque Reduction Assay and Immunostaining

Confluent monolayers (96-plate format, 5 × 104 cells/well, triplicates) of Vero cells were infected with 25 plaque forming units (PFU)/well of Paraiba/2015, Uganda/1947, Nigeria/1968, Puerto Rico/2015, and French Polynesia/2013 ZIKV strains at 37°C in infection media. After 1 h of adsorption, virus inoculum was removed and cells were washed three times with infection media before adding fresh infection media containing 1% microcrystalline cellulose (Avicel, Sigma-Aldrich) and the indicated concentration of compounds, or 0.1% DMSO as vehicle control. In case of pre-treatment experiments, the cell monolayers were treated with the indicated concentration of compound, or 0.1% DMSO, for the indicated times before ZIKV infection. Infected cells were incubated at 37°C for 36–60 h, depending on virus strains. For immunostaining, cells were fixed with 4% paraformaldehyde for 1 h, washed three times with phosphate buffered saline (PBS) and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. Then, the plates were blocked with 1.25% bovine serum albumin (BSA) in PBS (blocking solution) for 1 h at room temperature, followed by incubation with 1 μg/ml of the pan-flavivirus envelop (E) protein monoclonal antibody 4G2 (ATCC, Catalog No. VR-1852) diluted in blocking solution for 1 h at 37°C. After incubation with the primary antibody, cells were washed three times with PBS and developed with the Vectastain ABC kit and the DAB Peroxidase Substrate kit (Vector Laboratory, Inc., CA, United States) according to the manufacturers’ instructions. Stained plaques were analyzed using the CTL ImmunoSpot plate reader and counting software (Cellular Technology Limited, Cleveland, OH, United States). Virus titers were calculated as PFU/ml (Nogales et al., 2015). Non-linear regression curves and the median inhibitory concentration (IC50) were determined as described above.

Zika Virus Growth Kinetics

Confluent monolayers (24-well plate format, 2.5 × 105 cells/well, triplicates) of Vero or A549 cells were infected (multiplicity of infection, MOI, 0.1) with Paraiba/2015 diluted in infection media for 1 h at room temperature. After viral absorption, cells were incubated with infection media containing the indicated concentrations (250, 25, 2.5, and 0 μM) of ATA. At 12, 24, 48, and 72 h post-infection (h p.i.), tissue culture supernatants were collected and titrated on Vero cells by immunostaining as described previously (Marquez-Jurado et al., 2018).

Apoptosis Assay

Levels of apoptosis were measured using the Caspase-Glo® 3/7 Assay (Promega, WI, United States) following the manufacturer’s instruction. Briefly, Vero and A549 cells (24-well plate format, 2.5 × 105 cells/well, triplicates) were infected with ZIKV Paraiba/2015 (MOI of 0.1) and, at the indicated times post-infection, cells and tissue culture supernatants were collected and centrifuged. Twenty five microliters of supernatants were mixed with 25 μl of Caspase-3/7 reagent using a plate shaker, incubated at room temperature for 1 h, and luminescence at 570 nm was measured using a SpectraMax iD5 (Molecular Devices, Waltham, MA, United States) following the manufacturer’s instructions.

Statistical Analysis

Two-way ANOVA was used to evaluate significant differences. Data are expressed as the mean ± standard deviation (SD) of at least three independent experiments in triplicates using Microsoft Excel software. Value were considered statistically significant when ∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001. All data were analyzed with Prism software version 8.00 (GraphPad Software, CA, United States). CC50 and IC50 were determined using sigmoidal dose response curves (GraphPad Software, CA, United States). The selective index (SI) of each compound was calculated by dividing the CC50 with the IC50.

Results

Analysis of ATA Toxicity in Vero and A549 Cells

Before examining the inhibitory effect of ATA (Figure 1) against ZIKV infection, we first determined the CC50 of ATA on Vero and A549 cells (Figure 2). For this, we treated both cell lines with serial (twofold) dilutions of ATA and measured cell viability at 36 and 72 h post-treatment. As an internal control for these studies, we used ARB, a drug that has been previously described to have antiviral activity against ZIKV in Vero (Haviernik et al., 2018) and A549 (Fink et al., 2018) cells. We did not observe any toxicity with ATA in Vero (Figure 2A) or A549 (Figure 2B) cells at 36 or 72 h post-treatment, even at the highest concentration tested (1,000 μM), while ARB showed CC50 values of 74.71 ± 1.09 or 59.37 ± 1.10 μM in Vero (Figure 2C) and 114.6 ± 1.08 or 91.0 ± 1.08 μM in A549 (Figure 2D) cells (Table 1) at 36 or 72 h post-treatment, respectively.”

Beyond the treatment and efforts, it is important that pregnant mothers protect themselves and their children. This can be through using treated mosquito nets, wearing long-sleeve tops and long pants to prevent mosquito bites, and use of condoms to prevent sexually transmitted Zika virus.

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