The Antiviral Effect of Epigallocatechin Gallate-Stearate on Enterovirus-69" (2017)

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8-2017 The Antiviral Effect of Epigallocatechin Gallate- Stearate on Enterovirus-69 Hager Mohamed Montclair State University

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Enteroviruses are single stranded RNA viruses responsible for diseases from respiratory illness to hand-foot-and-mouth disease. As a rapidly evolving group, enteroviruses often gain genetic variability which leads to emergence of additional types, one of which is

Enterovirus 69 (EV-69). EV-69 has been associated with respiratory illness, possibly causing symptoms similar to those caused by EV-68. To challenge the ability of the virus to infect cells, this study examined the result of infection in lung cell lines A549 and

MRC-5 in response to Epigallocatechin gallate-stearate (EGCG-S) treatment of the virus before infection. EGCG-S, a modified green tea polyphenol, was assessed for its efficacy against EV-69 through examination of changes in cell viability, cytopathic effects, cell proliferation, and reactive oxygen species levels. Apoptosis in treated versus untreated virus infected controls was also assessed. The results of these assays indicate that

EGCG-S may be a suitable antiviral in synergy with another drug for inhibiting infection with EV-69 in susceptible cells.

THE ANTIVIRAL EFFECT OF EPIGALLOCATECHIN GALLATE-STEARATE ON

ENTEROVIRUS 69

A THESIS

Submitted in partial fulfillment of the requirements

For the degree of Master of Science

HAGER MOHAMED

Montclair State University

Montclair, NJ

2017

Acknowledgements

This research project was supported by the Bonnie Lustigman Research Award and completed with the guidance of Dr. Sandra Adams and Dr. Lee Lee, with whom it has been an honor working with. I would also like to thank Dr. Campanella for providing additional guidance as a member of my thesis committee, Dr. Anne Marie DiLorenzo for providing us with the MRC-5 cell line and ROS-Glo kit, and Dr. John Siekierka for allowing us to use his bioluminescence plate reader for the ToxGlo and ROS-Glo assays.

Finally, I would like to thank the faculty and students in the Montclair State University

Biology Department for their support and assistance with laboratory equipment throughout the research project.

Table of Contents

Abstract ………………………………………………………….………………....…….1

Acknowledgments………………………………………………….………….……...….4

List of Figures …………………………………………………...……………………….6

Introduction ……………………………………………………...……………………….7

Review of Literature……………………………………………….…………..….……....8

Methodology.……...………………………………………………...………….…...... 17

Results……………………………………………………………………..……………..22

Discussion…………………………………………………………….………………….32

Conclusion ...…………………………………………………………………………….35

Bibliography ……...………………………………………………………………….36-39

Supplemental Figures and Illustrations…...... 40

List of Figures

Figure 1: Genome of Enterovirus 68 consisting of a polyprotein with a region P1 encoding structural proteins VP1-4 and regions P2 and P3 coding for nonstructural proteins.……………………...…………………………………………………………....9

Figure 2: The Structure of EGCG (A) is modified to make it more stable by addition of an ester bond to make EGCG-S (B)……………………………………………………..14

Figure 3: MTS Cell Viability Assay. EGCG-S is not toxic to cells at up to 75µM…………………………………………………………………………….…...22-23

Figure 4: Morphology of Cells Infected with Treated and Untreated Enterovirus 69...……………………………………………………………………………….…..24-25

Figure 5: Trypan blue assay showing percentage of viable cells following infection of EGCG-S treated EV-69 in MRC-5 cells………………………….……………………..26

Figure 6: MTS Antiviral assay for treated EV-69 infected cultured lung cells……..27-28

Figure 7: Viral ToxGlo Assay showing increased cellular ATP levels in result of EGCG-S treatment.…………………………………………………………………….. 29

Figure 8: Fluorescence microscopy of DAPI stained EV-69 infected MRC-5 cells with and without 25-100µM EGCG-S treatment…………………………………………..…30

Figure 9: ROS-Glo H2O2 Antiviral Assay showing reduced ROS levels in MRC-5 cells infected with treated EV-69...……………………………………………….…………..31

Supplemental Figure 1: EV-69 infectious titer determined at 10-4 dilution in CPE assay showing cell death after 72 hrs……………………………………..……………………40

Introduction

Enterovirus 69 is a non-enveloped RNA virus that has been recently associated with respiratory disease, possibly exhibiting pathogenesis resembling that of Enterovirus

68 which has caused outbreaks of severe respiratory illness in immunocompromised individuals (Midgley et al., 2015). Enteroviruses are a rapidly evolving genus of the

Picornaviridae family with varied tissue tropism (Yang et al., 2009). This study will investigate the ability of EGCG-Stearate, a green tea catechin, to show increased inhibition of Enterovirus-69 infection in the lung cell lines A549 and MRC-5. EGCG-S is a modified formulation of EGCG, made to be more lipophilic and bioavailable (Zhong and Shahidi, 2011). As substantial research shows EGCG has broad spectrum activity against both enveloped and non-enveloped viruses with differing genomes, EGCG-S shows promise as a potential antiviral against EV-69 (Colpitts and Schang, 2014). This antiviral method can be efficacious even in the occurrence of EV-69 evolution, and current antiviral development would thus aid in preparation for possible future outbreaks caused by the rapidly increasing genetic variability in this and other enteroviruses.

Review of Literature

Enterovirus 69 is a single-stranded, non-enveloped RNA virus classified into

Human-enterovirus B (HEV-B), one of twelve species of enteroviruses. They are primarily distinguished from other enterovirus species due to sharing a greater sequence similarity in a region encoding for a capsid protein (Tang et al., 2014). Enteroviruses are a diverse genus in the Picornaviridae family, with tissue tropism varying from cells in the gastrointestinal system to those in the respiratory tract. Accordingly, the cell receptors to which they attach were predominantly found to be sialic acid containing glycans (Yang et al., 2009; Alexander et al., 2002). EV-69 has recently been associated with respiratory disease, similar to the better characterized Enterovirus 68, in which infection has caused respiratory failure or pneumonia in children and immunocompromised individuals (Yin-

Murphy and Almond, 1996). EV-68 has been associated with sporadic outbreaks predominantly in the U.S. and several countries in South Asia including Thailand and

Japan (Midgley et al., 2015; Huang et al., 2015). The largest outbreaks of respiratory disease in the U.S. and Canada occurred in 2014 (Peci et al., 2015). Moreover, enteroviruses have been extensively associated with viral meningitis, having accounted for approximately 75,000 cases in the U.S. every year (Desmond et al., 2006).

Even within the same species, the pathogenesis of enteroviruses can greatly vary.

Other members of the same HEV-B species, which include coxsackie B and A9 viruses and echoviruses, cause diseases such as myocarditis, pericardial effusion, pancreatic inflammation, hepatitis, and encephalitis (Mena et al., 2000; Danthanarayana et al.,

2015). Infection with EV-68 as with many enteroviruses has been described to generally cause mild respiratory or cold-like symptoms in healthy individuals but may cause severe

8 respiratory disease requiring hospitalization in children and the elderly. In recent outbreaks in the U.S., children particularly between one and four years old have been affected (Huang et al., 2015).

The RNA genome of enteroviruses is about 7,500 bases long and encodes a polyprotein with a region coding for nonstructural proteins (P2 and P3) and one coding for structural, capsid proteins (P1) (Fig. 1) (Chau et al., 2007). Upon cleavage by viral proteases, the P1 region of the polyprotein yields four peptides encoding for the proteins

VP1-VP4, and the P2 and P3 regions yield seven peptides encoding for proteins required for viral replication including VPg, replicase, and proteins that manipulate the cell, causing lysis (Chau et al., 2007).

Figure 1. Genome of Enterovirus 68 consisting of a polyprotein with a region P1 encoding structural proteins VP1-4 and regions P2 and P3 coding for nonstructural proteins. Adapted from W. Huang, et al., (2015)

Members of the Picornaviridae family follow the same general infectious cycle, beginning with the virus becoming endocytosed upon receptor binding and subsequent uncoating which releases the RNA into the cytoplasm (Seitsonen et al., 2012). Following this, cap-independent translation of the genome to a polyprotein occurs directly and is

9 initiated by an Internal Ribosome Entry Site (IRES), located on the 5’ untranslated region

(UTR). This process is facilitated by the capsid proteins and the seven nonstructural proteins resulting from translation of the genome are then critical in facilitating the viral

RNA replication (van der Schaar et. al., 2013). Replication also occurs through an RNA- dependent-RNA polymerase (RdRp) and the protein VpG serving as a primer (Kaku et. al., 2002).

Infection of a cell with EV-69 is currently not well understood beyond the general life cycle of all Picornaviruses. While the receptors are currently unidentified, research on other enteroviruses gives insight into its likely varied tropism and clinical manifestations.

As with many nonenveloped enteroviruses including EV-71 and EV-68, EV-69 likely binds to sialic acid containing glycans which are numerous on many cell types (Baggen et. al., 2016). Like EV-68, it is possible that EV-69 infects both the upper and the lower respiratory tract, then may reach other tissues from this initial point of infection by a distinct mechanism. It may also bind to multiple sialylated or even non sialylated receptors and infect erythrocytes through sialylated receptors, as well as dendritic cells through binding of the lectin receptor CD209 (Baggen et al., 2016; Chen et al., 2012).

Some enteroviruses have been found to bind to integrins, intercellular adhesion molecule

1 (ICAM-1) and CD55, in addition to sialic acid receptors (Nilsson et al., 2008). The tropism of EV-68 has not been limited to the respiratory tract, however, as the virus has been shown to invade neural cells of the central nervous system (Michos et al., 2007).

This process is hypothesized to possibly occur either via retrograde transport through neuromuscular junctions of axons, usually exacerbated by muscular injury, or through transport by leukocytes after they are infected (Ohka et al., 2004; Espinoza et al., 2013).

EV-68 has also been isolated from patients during outbreaks in North America of acute flaccid paralysis (AFP), which resembled paralysis caused by a polio infection

(Greninger et al., 2015). More case studies are needed to further elucidate this causation, however. A distinct characteristic of EV-68 is that unlike many enteroviruses which are named for infecting the gastrointestinal tract, EV-68 exhibits poor stability in stomach acid, thus EV-69 is likely to be a respiratory virus with these properties. This also suggests that primary transmission of EV-69 may be, as with EV-68 through contact with respiratory secretions; inhalation of aerosols, or introduction into the airways by direct contact with fomites (Oberste et al., 2004).

The enhanced virulence of enteroviruses that allows them to invade other tissue types, as with EV-68 becoming neurotrophic, has been attributed to polymorphisms in regions of the polyprotein (Greninger et al., 2015). The variation in pathogenesis observed in the enterovirus genus is partly due to the high genetic variability in the species of viruses and their ability to undergo genetic recombination even between different serotypes. Their error-prone RdRp and the lack of proofreading during the RNA replication also accounts for the significant variability in the sequences among the serotypes (Lindberg et al., 2003; Woodman et al., 2016). Recombination occurring between two enteroviruses in a host cell, as opposed to the proposed accumulated mutations in the genome, has moreover been associated with the diversity observed in the

5’ UTR sequences among species of enteroviruses. The twelve species currently identified were categorized based on sequence similarity in certain parts of the RNA genome. Generally, enteroviruses in the same species have been found to possess greater than 75% similarity in a segment of the genome encoding the structural protein, VP1, in

11 particular (Tang et al., 2014). However, within the species, studies have found there is generally a greater variability in the region P1 which codes for all the capsid proteins, than in the P2 and P3 regions coding for the non-structural proteins involved in viral replication. This variance may be attributed to greater fitness or may have evolved independently as a mechanism to escape immune surveillance (Lindberg et al., 2003).

Increasing pathogenesis among enteroviruses has been attributed to enhanced ability to evade innate immunity through varying mechanisms, which may occur in result of recombination. Chen et al. (2016) suggested, for example, EV-71 evades the innate immune response partly by interfering with the ubiquitination of the pattern recognition receptor, RIG-I, which senses viral RNA products of replication and initiates a cascade to stimulate the production of interferons (IFNs). In particular, two nonstructural proteases,

2A and 3C, of EV-71 were shown to be involved in this interference of IFN-stimulated gene expression, by binding or cleaving a variety of signaling molecules, including

MDA5 in addition to RIG-I, which were also responsible for initiating the necessary cascade to activate these genes (Chen et al., 2016). The numerous methods of immune surveillance as well as modulation in viral infections allows for several possibilities in which enteroviruses can evolve, as did EV-71, differing mechanisms of ensuring replication and shutting down the host cell. These mechanisms would be largely mediated by the nonstructural viral proteins.

van der Schaar et al. (2013) examined the methods of inhibiting these nonstructural proteins involved in replication, and the variability in the pathogenesis of enteroviruses presents a challenge in developing an antiviral which may work across different species and serotypes in the genus. Some developments are targeting the general

12 host factors, such as the kinase PI4KIIIβ which have been shown to be required for viral replication, potentially creating a broad spectrum drug. However, such drugs are anticipated to risk exhibiting significant toxicity to the host since they may necessitate damage to the host factors utilized (van der Schaar et al., 2013). On the other hand, drugs which target viral replication proteins, particularly in RNA viruses that mutate frequently, have raised the concern of the viruses evolving resistance to the drug, necessitating a combinatorial therapy and other drug targets identified to strengthen treatment (Tang et al., 2014).

A more feasible drug target appears to be the structural proteins of the enteroviruses which are involved in the early stages of the infectious cycle. The capsid binding antiviral, Pleconaril, has been shown in clinical trials to reduce disease symptoms in subjects with enteroviral meningitis as well as respiratory associated disease symptoms. However, following these clinical trials, as reported by Desmond et al.

(2006), the drug was rejected by the Food and Drug Administration due to side effects reported in the subjects and the possibility of a low success rate associated with drug- drug interactions and poor host metabolism of the compound. Effects of such compounds suggest a treatment using natural or nature derived compounds can be more effective while also less toxic to the host. Overall, the treatments which currently exist for enteroviral diseases involve medications to help alleviate symptoms of the various infections, as no current antiviral is approved for use (Desmond et al., 2006).

One nature derived antiviral currently being investigated as a potential long term treatment of diseases caused by enteroviruses is epigallocatechin-3-gallate (EGCG), the predominant catechin in green tea derived from the plant Camellia sinensis. Ho et al.

(2009) evaluated the potential of EGCG to act as an antiviral against Enterovirus-71, and found it can significantly reduce viral titers by as much as 95% while not exhibiting toxicity on cells treated without the virus. Clinical tests using EGCG treatment for diseases, including infections with Herpes simplex virus 1 (HSV-1), have shown that symptoms can be reduced or eliminated upon application of a formulation with EGCG.

Moreover, the antiviral effect was observed both in the early and late stages of the infection. Since EGCG is hydrophilic, recent pharmaceutical developments have made it more stable and bioavailable with enhanced lipophilicity (Zhong and Shahidi, 2011). The structure of EGCG was modified by esterification to produce a lyophilized EGCG-acyl ester derivative containing stearic acid, also termed EGCG-Stearate (Fig. 2) (EGCG-S)

(Zhong and Shahidi, 2011). Due to the enhanced solubility, EGCG-S is more potent and can be used in formulations in medicine (Zhao et al., 2013). This form of the catechin may overcome the challenges of poor host cell absorption and susceptibility to in situ metabolic changes (Colpitts and Schang, 2014).

A B . A .

Figure 2. The Structure of EGCG (A) is modified to make it more stable by addition of an ester bond to make EGCG-S (B). Adapted from Colpitts and Schang, (2014) and Hsu (2014)

The inhibition of infection by EGCG-S may be attributed to the catechin’s ability to interfere with the first step to infecting susceptible cells which is attachment to the cell

14 receptors, as found in a study by Colpitts and Schang (2014) examining its effect on both enveloped and non-enveloped viruses. The unrelated viruses tested bind to either heparan sulfate or sialic acid containing receptors on susceptible cells. Inhibition of infection with these viruses observed after pretreatment with EGCG suggested it can act as a receptor mimetic due to possessing one moiety analogous to the structure of heparan sulfate and another to sialic acid (Colpitts and Schang, 2014). A study by Mukoyama et al. (1991) supported that it may prevent the adsorption of specifically enteroviruses to cells, although this excluded poliovirus due to its receptor being a distinct immunoglobulin receptor. EGCG also inhibits virulent, respiratory viruses such as Influenza A which supports the potential of the polyphenol to inhibit the similar pathogenesis of the distinct respiratory enteroviruses, including EV-69 (Colpitts and Schang, 2014). Calland et al.

(2012) examined the effect of EGCG treatment on infection with Hepatitis C virus

(HCV) throughout each stage from attachment to release, and found that EGCG did not inhibit infection at other stages once viral entry into the cells was allowed before treatment with the catechin. Instead, RNA replication and assembly were observed to continue in result. Furthermore, as numerous enterovirus genotypes exist, the possibility of significantly differing antiviral potency dependent on this variable has been overturned by a study by Ciesek et al., (2011) demonstrating similar inhibitory effects of viral entry in viruses with varying genomes of the same species.

However, although some research has supported an antiviral effect of EGCG on enteroviruses such as EV-71, other research has suggested it does not inhibit infection on other enteroviruses. Isaacs et al. (2011) reported that EGCG had no effect on the titers of enteroviruses coxsackie B4, coxsackie A9, and echovirus 6 following infection, while it

15 greatly reduced those of the enveloped HSV-1. Due to the lack of consistency regarding both the antiviral effect of EGCG-S on all groups of viruses and the extent of its antiviral activity, substantial research is needed to evaluate the efficacy of this treatment particularly on non-enveloped, RNA viruses. Furthermore, the effect of this green tea polyphenol on EV-69 has not yet been reported.

Materials and Methods

Cell Culture Maintenance

The adherent human lung epithelial A549 cells (CCL-185) and the fetal lung fibroblast IRR-MRC-5 cells (ATCC 55-X) (American Type Culture Collection (ATCC)

Manassas, VA) were maintained in T25 flasks at 37°C in a 5% CO2 incubator. The A549 cell line and MRC-5 cell line were propagated in F12K and DMEM media, respectively, both of which have been supplemented with 10% Fetal Bovine Serum and 1% gentamicin.

Virus Propagation

Cells were sub-cultured into a 25T flask and incubated 24 hrs to reach 70-80% confluency. 100µL of virus (Enterovirus type 69 (American Type Culture Collection

(ATCC® VR-1077™) Manassas, VA) was used to infect the confluent cell monolayer for one hr after which 5mL of media was supplemented. A549 cells were checked after 48-

72 hrs and MRC-5 after 24 hrs for cytopathic effect (CPE). Virus was harvested when at least 90% of cells showed CPE, through removal of the media and centrifugation for 5 min to eliminate cellular debris. The supernatant containing virus was stored in cryogenic tubes at -80°C and used for the following experiments.

EGCG-S Preparation

EGCG-ester from green tea extract in powder form (US Patent 20120172423) was obtained from Stephen Hsu at Camellix, LLC (Georgia Health Sciences University

Center of Innovation for Life Sciences, August, GA). It was dissolved in 1ml of DMSO to a final stock concentration of 5mM and stored at 4°C. Treatment concentrations were

17 prepared from this stock by dissolving in F12K or DMEM to make 25µM, 50µM, 75µM, and 100µM.

MTS Cell Viability Assay

The CellTiter 96Aqueous One Solution Cell Proliferation Assay (MTS) kit (Promega

Corp., Madison, WI) quantifies viable cells through detection of cellular respiration using the insoluble formazan product as an indicator. This formazan product is derived from the

MTS substrate (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt) which becomes converted by the mitochondrial dehydrogenase enzymes present in viable cells. The following procedure is based on the protocol for the product G3580. A549 and MRC-5 cells were plated from a 100% confluent flask into a 96 well plate and incubated for 24 hrs at 37°C under 5% CO2 prior to performing the assay. Cells were then treated at 70-80% confluency with 100µL of

25µM, 50µM, 75µM, and 100µM of EGCG-S for one hr. The unadsorbed EGCG-S was then aspirated and cells were supplemented with medium and incubated for 24 hrs at

37°C under 5% CO2. After incubation, 20µL of MTS reagent was added to each well and the plate was incubated for one hr. Absorbance was read at 490nm using a 96 well plate reader spectrophotometer.

Cytopathic Effects in Cells with EGCG-S treated Virus

Cells were plated in a 6-well plate and incubated for 24 hrs until 70-80% confluency. Virus was treated with 25µM, 50µM, 75µM, and 100µM EGCG-S prior to infection. Cells in each well were then infected with 50µL EGCG-S treated and untreated virus, respectively, for one hr after which unabsorbed virus was aspirated and cells were replenished with media. The plate was incubated at 37°C under 5% CO2 and cells were

18 examined at 400X using an inverted microscope and images were taken at 48 and 72 hrs post infection (hpi).

Trypan Blue Assay

The number of viable cells remaining after infection with EGCG-S treated or untreated virus were counted and Trypan blue solution (Thermo Fisher Scientific) was used to stain and detect nonviable or dead cells. The procedure was done using a modified protocol of the Thermo Fisher Scientific Trypan Blue Exclusion protocol.

MRC-5 cells were seeded into 6 well plates and allowed to reach 80% confluency before infecting with treated or untreated virus for one hr. After infection, wells were aspirated and 3mL of media was supplemented into each well. Cells were incubated for 24 hrs at

37°C and 5% CO2. After incubation, media was aspirated and 250 µL of trypsin EDTA was added to each well for 5 minutes to allow all cells to detach. Media (2mL) was then added to each respective well to inactivate the trypsin and the contents of each well were transferred to separate conical tubes to be centrifuged for 5 minutes. The pellet was resuspended in 0.5 mL of media. Trypan blue (500µL) was then added to 500µL of cell slurry in each sample and subsequently loaded onto a hemocytometer for counting.

Viable and dead cells were calculated as a percentage of the total cell count.

MTS Antiviral Cell Proliferation Assay

Cells were plated in a 96-well plate and incubated for 24 hrs at 37°C in a humidified CO2 atmosphere until 80-90% confluent. Virus was treated with 25µM,

50µM, 75µM, and 100µM EGCG-S for one hr prior to infection. Cells were then infected with 100µL EGCG-S treated and untreated virus, respectively, for one hr after which unadsorbed virus was aspirated and cells were replenished with media. The plate was

19 incubated for 24 hrs at 37°C in a humidified CO2 atmosphere, at which point 20µL of the

MTS reagent was added and absorbance was read at 490nm using a 96 well plate reader spectrophotometer following an hr of incubation with the reagent.

Antiviral ToxGlo Assay

A 96-well plate was plated with MRC-5 cells and incubated for 24 hrs at 37°C in a humidified CO2 atmosphere until 80-90% confluent. Virus was treated with 25µM,

50µM, 75µM, and 100µM EGCG-S for one hr prior to infection. Media was aspirated from wells and cells were then infected with 100µL EGCG-S treated and untreated virus for one hr. After infection, unadsorbed virus was aspirated and cells were replenished with media. The Viral ToxGlo™ assay kit (Promega Corp., Madison, WI) was used to detect viable cells by measuring ATP levels in test conditions. The following procedure was based on the protocol for product G8941. The plate was incubated for 24 hrs at 37°C in a humidified CO2 atmosphere, at which point 10µL of the ATP detection reagent was added to each well and the plate was incubated for one hr. Relative luminescence units

(RLU) was then read using a Biotek Synergy™ 2 luminometer.

Fluorescence Microscopy

MRC-5 cells were plated on coverslips within 24-well plates and incubated until

80% confluent. The infectivity assay using EGCG-S treated and untreated virus was conducted as previously described, and cells were incubated at 37°C in a humidified CO2 atmosphere for 5 hrs. Based on the counterstaining protocol from Thermo Fisher

Scientific, cells were then stained with 150 µL of 300 nM DAPI (4, 6-diamidino-2- phenylindole) stain (Thermo Fisher Scientific) for 5 mins in a 37˚C incubator in the dark.

After incubation, 4% paraformaldehyde solution was added to fix cells for 20 minutes at

20 room temperature, then rinsed with PBS (Dulbecco’s Phosphate Buffered Saline). A solution of 90% glycerin and 10% PBS was added to glass slides for fixing the mounted coverslips. Images of cells on the coverslips were taken with a digital camera using a

Zeiss Axiovision fluorescence microscope at a magnification of 400×.

Antiviral ROS-Glo H2O2 Assay

The ROS-Glo™ H2O2 assay kit (Promega Corp., Madison, WI) was used to assess oxidative stress induced by viral infection by measuring the levels of H2O2, a reactive oxygen species (ROS). MRC-5 cells were plated in a 96-well plate and allowed to reach 80% confluency, then infected with EGCG-S treated or untreated virus for one hr as described in the previous infectivity assays. The lytic assay protocol for product

G8820 was followed. Cells were then incubated at 37°C in a humidified CO2 atmosphere for 7 hrs to allow infection, after which 20uL of H2O2 substrate solution was added to each well and cells were incubated for an additional 5 hrs, for a total of 12 hrs of infection. After incubation, 100µL of the ROS-Glo™ detection solution reaction was added to each well and incubated for 20 mins. ROS levels were measured in RLU and read using a Biotek Synergy™ 2 luminometer.

Determination of Viral Titer

MRC-5 cells were plated onto 6-well plates and allowed to reach 100% confluency. Cells were then infected with 500uL of EV-69 stock as a positive control and at dilutions of 10-3 to 10-6 and observed for cytopathic effect (CPE) 12 hrs later. The infectious virus titer, found to be 10-4 infectious units/mL, was determined at the highest dilution which began to exhibit visible CPE after 72 hrs of infection (Supplementary

Fig. 1).

Results

EGCG-S treatment concentrations at up to 75µM do not negatively affect lung fibroblast and epithelial cell proliferation

The effect of EGCG-S alone on cell viability was determined through the MTS assay, in which the viable cell number is directly related to the amount of NAD(P)H- dependent dehydrogenases present to convert the MTS substrate into the formazan product detected. At 70-80% confluency, A549 cells were treated with 25-100µM

EGCG-S dissolved in media. Cell viability was calculated as a percentage of the average of absorbance values for wells with untreated cells, designated as the negative control.

A549 cells maintained at least a 94.3% cell viability relative to the untreated control 24 hrs after treatment with EGCG-S at concentrations between 25µM and 75µM, and MRC-

5 cells maintained at least a 92.7% cell viability (Fig. 3). A decrease was observed for

100µM which resulted in an 84.9% cell viability in A549 cells and a 76.4% cell viability in MRC-5 cells.

A. 120

100

% % Cell Viability 20

0 25μM EGCG-S 50μM EGCG-S 75μM EGCG-S 100μM EGCG-S Treatment Concentration

Negative Control Treated Cells

B. 120 100 80 60 40

20 % % Cell Viability 0 25µM EGCG-S 50µM EGCG-S 75µM EGCG-S 100µM EGCG-S Treatment Concentration Negative Control Treated Cells

Figure 3. MTS Cell Viability Assay. EGCG-S is not toxic to cells at up to 75µM in A549 (A) and (B) MRC-5 cells.

Cytopathic effects in EV-69 infected cells were reduced upon EGCG-S treatment

Treatment of EV-69 with EGCG-S resulted in more cell proliferation for both

MRC-5 and A549 cells at all concentrations in comparison to cell growth in the untreated virus infected cells, which was used as the positive control (Fig. 4). Significant decrease in cell viability was characterized as more than 90% of cells exhibiting rounding and lifting for A549 cells, and by the presence of primarily cellular debris as a result of prominent lysis in MRC-5 cells after 48 hrs. The differences observed in the two cell lines reflect a much shorter infectious cycle observed in MRC-5 cells. In both cell lines more extensive proliferation than the untreated virus positive control was observed after

72 hpi in result of 50µM and 75µM treatment.

Figure 4. Morphology of Cells Infected with Treated and Untreated Enterovirus 69. Treatment of EV-69 with EGCG-S resulted in reduced CPE in (A) A549 Cells and (B) MRC-5 Cells.

Infection with EGCG-S treated EV-69 increased the viable cell count

Cell viability was assessed by using the trypan blue assay and counting the stained dead and non-stained live cells using a hemocytometer. The number of viable cells was observed at 24 hrs following infection with virus treated with 25-100µM of

EGCG-S, and compared to that of the positive control with untreated virus. At

25 concentrations 50µM and 75µM, cells remained viable relative to the number quantified for the negative control with uninfected cells (Fig. 4). EGCG-S exhibited some protective effect at 25µM and to a much lesser extent at 100µM, with a 56.87% and 44.69% cell viability, respectively, as compared to 23.55% observed for the positive control.

100 90 80 70 60 50 40 30

% Viable Cells Viable % 20 10 0 25µM EGCG-S 50µM EGCG-S 75µM EGCGS 100µM EGCG-S Treatment Concentrations Positive Control Treated Virus Negative Control Figure 5. Trypan blue assay showing percentage of viable cells following infection of EGCG-S treated EV-69 in MRC-5 cells.

EV-69 Treatment with EGCG-S increased cellular respiration during infection

The MTS antiviral assay was used to measure cell viability in infected cells in response to EGCG-S treatment of the virus. The activity of mitochondrial dehydrogenases in metabolically active cells converts the MTS substrate to a formazan dye product which can be quantified by measuring absorbance at 490nm. Concentrations of 25µM-100µM of EGCG-S were tested and the percent cell viability was calculated through comparison to the negative control with uninfected cells. Infected cells exhibit a lower absorbance, as observed for the positive control with untreated virus, and a high inhibition of infection by the tea is characterized as increased absorbance values relative to the negative control. Inhibition of infection was also calculated as a percentage of the

26 negative control which would exhibit 100% inhibition of infection, using the formula in

Figure 6. Treatment with EGCG-S at 50µM and 75µM resulted in a two-fold or greater increase, respectively, in cell viability in comparison to the positive control in A549 cells, and almost a two-fold increase in MRC-5 cells (Fig. 6A and B). However, the highest percent of inhibition, also observed at 50µM and 75uM EGCG-S, were 59.4% and 66.0% for A549, and 27.1% and 29.5% MRC-5 cells, respectively (Figure 6C and D).

(Experiemental well − positive control)

(Negative control − positive control)

Equation for percent inhibition of infection

A. A549 120

100

% % Cell Viability 20

0 0.5% DMSO 25µM EGCG-S 50µM ECGG-S 75µM EGCG-S 100µM EGCG-S Treatment Concentration Positive Control Treated Virus Negative Control

B. MRC-5

120

100 80

% Cell Viability 20 0 0.5% DMSO 25µM EGCG-S 50µM EGCG-S 75µM EGCG-S 100µM EGCG-S Treatment Concentration Positive Control Treated Virus Negative Control

C. A549 100 90 80 70 60 50 40 30 % % Inhibiition 20 10 0 Negative 0.5% 25µM 50µM 75µM 100µM Control DMSO EGCG-S ECGG-S EGCG-S EGCG-S Treatment Concentration

D. MRC-5 100 90 80 70 60 50 40 30

% Inhibition 20 10 0 Negative 0.5% 25µM 50µM 75µM 100µM Control DMSO EGCG-S EGCG-S EGCG-S EGCG-S

Treatment Concentration Figure 6. MTS Antiviral assay for treated EV-69 infected cultured lung cells. Treatment increased viability of infected cells (A and B) but did not result in a high percent inhibition of infection (C and D).

EGCG-S treatment inhibits apoptosis during EV-69 infection

The infectivity of A549 cells by EV-69 declined over time. Due to the cells no longer being infected by EV-69, experimenting with A549 cells was discontinued and all subsequent assays were completed using MRC-5 cells, beginning with the antiviral

ToxGlo assay. To verify the increase in cell viability observed in the MTS antiviral assay, cells were assessed for ATP levels after infection with EGCG-S treated or untreated virus using the Viral ToxGlo Assay. The Viral ToxGlo assay quantifies ATP as a direct

28 measure of cell viability. The luminescent signal, measured as RLU, is proportional to the amount of ATP in the sample. Treatment with 50µM and 75µM EGCG-S resulted in up to a two-fold increase in cellular ATP levels as compared to the positive control with untreated virus (Fig. 7). However, this increase was not similar to the untreated cells negative control, which exhibited 3.5-fold increase in ATP levels compared to the positive control. These results further demonstrate that EGCG-S treatment exhibits some protective effect on MRC-5 cells from EV-69 infection.

120500

100500

80500

60500 RLU 40500

20500

500 0.5% DMSO 25µM EGCG-S 50µM EGCG-S 75µM EGCGS 100µM EGCG-S Treatment Concentrations

Positive control Treated Virus Negative control

Figure 7. Viral ToxGlo Assay showing increased cellular ATP levels in result of EGCG-S treatment.

Nuclear Integrity is maintained in infected cells upon EGCG-S treatment

Since nuclear breakdown is a characteristic indicator of nonviable cells in result of a chronic viral infection, the effect of EGCG-S treatment on viruses prior to infection was assessed using DAPI, which at high concentrations is permeable and stains nuclei of viable cells. The stain was added after infection to each MRC-5 cell sample corresponding to the treatment condition, and cells were imaged using a fluorescent

29 microscope (Fig. 8). A maintenance of nuclear integrity most closely resembling the uninfected, negative control, was observed at 75µM EGCG-S, and a similar protective effect was observed at 50µM treatment. In contrast, excessive nuclear breakdown and accordingly lack of fluorescence was observed for the untreated virus infected positive control and similarly for treatment of the virus with 100µM EGCG-S.

Figure 8. Fluorescence microscopy of DAPI stained EV-69 infected MRC-5 cells with and without 25-100µM EGCG-S treatment.

EGCG-S treatment reduces oxidative stress induced by EV-69 infection

Infection with EV-69 leads to increased ROS (reactive oxygen species) levels in addition to decreased cellular ATP and increased mitochondrial distress. To verify the inhibitory effect of treatment with 50µM and 75µM EGCG-S observed previously, treated MRC-5 cells were also assessed for their ROS levels after infection. The levels of

ROS produced in treated cells was compared with the untreated virus positive control and

30 uninfected cells negative control. Both 50µM and 75µM EGCG-S treatment resulted in a

68.5% and 39.6% decrease, respectively, in ROS levels (Fig. 9). These levels, particularly at 50µM were more comparable to those observed for uninfected cells which exhibited 60.9% less ROS levels than the positive control.

90000 80000 70000 60000 50000

RLU 40000 30000 20000 10000 0 50µM EGCG-S 75µM EGCGS Treatment Concentration Positive Control Treated Cells Negative Control

Figure 9. ROS-Glo H2O2 Antiviral Assay showing reduced ROS levels in MRC-5 cells infected with treated EV-69.

Discussion

Neurological complications have resulted from infection with several species of enteroviruses that differ in cell tropism and species such as EV-68 have recently emerged as a public health threat globally (Greninger et al., 2015, Huang et al., 2015). Although the tropism varies among enteroviruses, their RNA genome enables the species to undergo rapid mutations which may continue to pose a challenge for new drug development (Lindberg et al., 2003). In addition, considering their rapid infectious cycle, there is a need for a drug which may work quickly or at the beginning stages of the infection. This is further complicated by the viruses lacking an envelope, a property enabling them to resist damage by the acidic environment in the stomach and other barriers to infection in the gastrointestinal tract (Oberste et al., 2004). Consequently, there is a lack of current treatment for enteroviral infections.

This study investigated whether EGCG-S would exhibit an antiviral effect on

EV-69 in a similar manner as observed in Ho et al., 2009 on EV-71. Our results indicate that EGCG-S inhibited infection, as evinced by an increase in cell proliferation and cellular respiration, accompanied by a decrease in ROS produced by the cells (Fig. 9).

The increase in cell viability however was limited by up to two-fold at concentrations

50µM and 75µM, with the greatest percent viability being at 70% in A549 cells. In the cell viability assay conducted by Ho et al., (2009), a five-fold increase was observed for the tested 25µM concentration of EGCG compared to an untreated EV-71 infected control, but the percent viability was similarly less than 70% in the Vero cells. This considerable decrease from the percent viability for uninfected cells may indicate EGCG

32 or EGCG-S treatment alone may not be substantial to prevent infection efficiently particularly with a highly virulent strain.

MRC-5 cells appeared to be more susceptible to infection with EV-69. While

EGCG-S treatment similarly inhibited lysis, MRC-5 cells exhibited less growth as compared to A549 cells after infection with treated virus (Fig. 3). Treatment at 50μM and 75μM, however, increased nuclear and membrane integrity during infection, with a viable cell count resembling more that of the uninfected negative control (Figs. 4 and 8).

A decrease in viral infectivity was also evident as at least a 39.6% decrease in ROS levels resulted after 75μM of treatment (Fig. 9). The MTS antiviral assay results suggested the percent of inhibition at 75μM is minimal, at 29.5% as compared to the 66.5% inhibition observed in A549 cells. Accordingly, cellular ATP levels were two-fold greater after treatment while the uninfected control was 3.5-fold greater. As viral infection leads to increased production of reactive oxygen species and excessive production of ROS may lead to cell death, the inhibitory effect of EGCG-S observed may be attributed to its antioxidant potential (Ho et al., 2009).

The mechanism for the antiviral effect observed for EGCG has been debated.

Many studies have reported its ability to inhibit infection by interfering with attachment of the virus to the host cells (Colpitts and Schang, 2014; Isaacs et al., 2011). However some suggest it is instead due to the antioxidant capacity and manipulating the cellular redox to normal levels (Ho et al., 2009). Alternatively, viral replication may be affected by EGCG inhibiting necessary viral proteins such as viral proteases (Weber et al., 2003).

Several factors in the EV-69 infectious cycle need to be further established prior to investigating the mechanism of action for potential antivirals. Attachment of the virus

33 to cells, for example, is not fully characterized as also for other enterovirus species such as EV-71. Su et al., (2012) reported that EV-71 was unable to attach to susceptible cells upon neuraminidase treatment to remove sialic acids on the cells. Other studies have demonstrated the efficiency in EGCG in preventing attachment to cells by interfering with binding, possibly by acting as a sialic acid or heparan sulfate mimetic (Colpitts and

Schang, 2014). Thus, if this mechanism may only apply to certain cell receptors, the activity of EGCG may be negligible for viruses that are able to infect by binding to different receptors, as is the case with poliovirus, also a picornavirus, which establishes infection by binding to an immunoglobulin like receptor (Colpitts and Schang, 2014). It is possible that while EV-69 may predominantly bind to sialic acid containing glycans to attach to cells, it may also utilize an alternate receptor in their absence and thus evade the receptor mimetic antiviral mechanism of EGCG-S proposed by Colpitts and Schang,

(2014).

The mechanism of viral inhibition by EGCG-S based on the results of the current study thus far cannot be concluded, and may be due to any of or a combination of these mechanisms. Future studies will investigate and compare the antiviral effect of EGCG-S treatment in attachment assays with its effect after the virus has infected the cell by evaluating the resulting viral titers as well resulting viral RNA. Moreover, other polyphenols such as black tea theaflavins may exert a more protective effect as found in the study by Isaacs et al., 2011 on coxsackieviruses. Due to the minimal inhibitory effect observed, EGCG-S may be a more efficient antiviral if used synergistically with another compound for treatment of EV-69 infections.

Conclusion

The current study demonstrated that EGCG-S may increase cell viability and reduce ROS levels during EV-69 infection, but it is not sufficient enough on its own as an antiviral due to a low percent of inhibition of infection. Considering it is a nature derived compound, EGCG-S may be a more benign option for synergistic treatment avoiding the challenges of side effects observed for trials of other enteroviral drugs in development.

As outbreaks have recently emerged with EV-68 and EV-71, there is a need to develop many antiviral options, moreover with activity accounting for a more rapid infectious cycle and other mutations arising which confer resistance to previously investigated drugs. Polyphenols that have exhibited antiviral activity against several, unrelated viruses in past studies are thus an attractive target for treatment against several enteroviral infections.

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Supplemental Figures and Illustrations

Supplemental Figure 1. EV-69 infectious titer determined at 10-4 dilution in CPE assay showing cell death after 72 hrs.