A Novel Antiviral Drug to Treat Enterovirus 71

Table of Contents

Introduction

Methods
Transformation of E. coli with the genetic information from Enterovirus
Growth of E. coli colonies in the lab setting
2C protein purification
ATPase assays with drug testing:

Results
Transformation of E. coli with the genetic information from Enterovirus
Growth of the E. coli colonies in the lab setting
2C protein purification
ATPase assays with drug testing:

Discussion
Principle Results
Limitations

Acknowledgements

Multimedia Appendix

References

Abstract

Enterovirus 71, or EV 71, is responsible for causing Hand, Foot, and Mouth disease in humans. In particular, it is especially deadly when children and small infants are exposed. The objective of this research paper is to address the possibility of a novel antiviral drug that can be used once infection of EV 71 has occurred. The methods for this research include transformation of E. coli with the genetic information from Enterovirus 71, growth of the E. coli colonies in the lab setting, 2C protein purification, and ATPase assays with drug testing. Of the 364 drugs tested in the ATPase assay, a combination of two of them (Mitrofudil and N6- Benzyladenosine) indicated a stoppage in activity of ATPase, signaling no further activity of the enzyme and viral proliferation.

Keywords

Picornaviruses, Virology, ATPase, Antiviral, ATPase Assay, and Proteins.

Introduction

Human enterovirus 71 (EV71) is a small, non-enveloped, icosahedral virus that belongs to the human EV species A in the genus Enterovirus within the family Picornaviridae [1]. It is the virus that has the capability of infecting humans with Hand, Foot, and Mouth disease. There have been breakouts of EV 71 throughout the word, from the most prominent cases occurring in Eastern Asia. Viruses of this family need to reproduce actively in order to create and maintain a successful infection [2]. In order to do this, they rely on a protein known as 2C, which is responsible for viral replication and proliferation. Reproducing actively also requires the dephosphorylation of ATP to ADP and P through the enzyme ATPase [3]. The 2C protein acts as a platform to allow ATPase to breakdown ATP and fuel the creation of new viral particles. The aim of this study is to look at the drugs which can inhibit the activity of ATPase in regard to the 2C protein, thus preventing the viruses from reproducing.

Methods

Transformation of E. coli with the genetic information from Enterovirus 71

To begin, E. coli plates need to be grown out which have been transformed with the genetic information from EV 71 [4]. This is done by microwaving a plate agar bottle. The lid is loosened and the microwave is set for a time limit of 5 minutes. While heating, it is important to take out the bottle every 15 seconds to stir. After heating, tighten the lid of the bottle and let it cool. Turn on the Bunsen Burner and get a 50ml Falcon tube, pour 50ml of agar, 50 microliters of Ampicillin, and 50 microliters of Chloramphenicol. Make 2 plates, so pour out 25 ml of solution into one plate and 25 ml of solution into another. If E. coli samples are not already available, transformation needs to be done [5]. To transform, take a bucket of ice and get a sample of EV71 from fridge. Also, get purple PCR tubes which contain E. coli cell pellets. Label the PCR tubes. Turn on the hot water bath to 44 degrees C and hit the power button. Add 10 microliters of each EV 71 sample into the respective PCR tubes. Flick the tubes and place back in ice for 30 min. After 30 minutes, heat shock the two tubes by placing them into the heated water bath for 30 seconds. Label the Petri dishes. Add 450 microliters of SOL media to the small PCR tubes. Add 100 microliters of sample from the small PCR tube to the agar plate and 20 microliters for each of the four overnight tubes. Take the plate stick and put it in the flame. Repeat once more. Invert the stick and just streak it across the plate. Place the overnights and plates into shaker to incubate. Ensure everything is labeled (Multimedia Appendix 1).

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Growth of E. coli colonies in the lab setting

Next, take out the four overnights with the bacteria/virus with the 2XYT LB media, at 20 ml volume. Take 4 big growing jar/flask filled with agar that has been pre prepped. Add 1 ml of ampicillin and 1 ml of chloramphenicol to the big flask. Take the overnight and pour into the big flask. Put the aluminum foil lid back on and place the flask into the shaker. Push the big button to change the temperature to 37 degrees Celsius. Incubate for 2 hrs. Do the nanodrop by taking out 1 ml of sample and 1 ml of original growth media. Use the nanodrop machine to set the original growth media as a blank to compare with the new sample reading. Look for a 0.5 OD value from the reader [6]. This signifies that E. coli growth is sufficient. Then add 1 ml of IPTG to the big flask. Put the flask back into the shaker and change the temperature into 30 degrees Celsius. Incubate it for an additional 6 hours. Pour the liquid into 2 small plastic centrifuge containers. Balance out the masses. Put them into the centrifuge for 15 minutes. Pour out the supernatant and store the cell pellet at -80 degrees Celsius.

2C protein purification

Get 50 ml of Buffer A into 50 ml blue-top Falcon tube. Add 1 protease inhibitor tablet, and dissolve. Put Buffer A back in the fridge. Label it Buffer A + protease inhibitor. 10 ml of the solution into each pellet. Mix thoroughly. Take two Olderidge centrifuge tubes. Put them on ice, take the top off of one of them. Need the silver cap, put the lid on. Get some tissue paper. Pour both into one container. Suck up the remaining with the pipet.

Do 7ml for each blast. Put it in so that it is parallel and push down. Align the locking mechanism by matching the dots. Put it in the metal stick. Take it out once done and push off the liquid.

Put finger on anti-foam device and pour into tube. Keep it on ice. After this, get a small beaker and balance out with the other tube. Balance out the two tubes, one of them with water. Change speed on centrifuge to 25,000 xG and spin for 30 minutes [7].

Then, proceed to AKTA start, system control. Plug into computer. Press connect. Check Buffer levels. For nodes, do A to A, B to B. Check to see if waste is low. Change to a Nickel column (Multimedia Appendix 2).

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Press manual 1, change to 5 ml/min. Press blue button to make sample value green. Switch it off when you get to the 10 ml mark. Turn it back to Buffer B at that point for another 10ml. Switch to Buffer A and give 10 ml. Switch to weak value and give it 20 ml. Fill up the rotor with empty tubes in the meantime. Press stop once 20 ml of weak value are added. (square button). Push the tube over to (1), the starting position.

To filter the samples, take the tubes out of centrifuge. Bring ice box with you. Need a 100 ml bottle. Put it in ice. Set up the filter. Put in a filter paper. Put on the lid for the filter. Attach it to the vacuum for filtration. Pour a little at a time (do not pour the pellet). Replace the filter. Need a clear 50 ml Falcon tube. Pour the extract into the tube. Then, set up the Method run. Change one of the leads to point to the lysed cell extract. Press on the sample value to make it green. Change the volume to account for how much you have.

For comparison with an established ladder through SDS-PAGE, get 2x loading dye and precision-plus protein unstained standards. Do pellet for ladder, supernatant, T2, T6, T12, T13, T14, T16, T17, and T18 (Multimedia Appendix 3).

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Get a PCR tube, yellow micropipette, 10 microliters, and put 10 microliters of loading dye into each. Put the sample into each PCR tube as well (10 microliters). Label the tubes. Put PCR tubes into Thermal Cycler, Home Button, incubate (98 degrees C for 5 min), and run. Use the non-native gel. Put the covering to the gasket side. Match the colors to the master position. Use the SDA buffer. Pour into the gel to fill it up. Take p1000 micropipette and special gel micropipette tips and rest the buffer between the two [8]. Need 5 microliters of ladder, and 10 microliters of sample into each column in the gel. Move through the gel asymmetric when placing samples. Fill up the wells, run at 150V, and check the mA value.

To read the SDS-PAGE, remove the covers and take the gel out of the covering. Place it into the gel reader tray (pour distilled water to rid the gel of bubbles). Press green button and open the gel reading computer software. Label the gel based on the positions of each respective sample. Toss the gel into the trash once the file has been saved under your name with all the info.

ATPase assays with drug testing:

Wrap a 100ml bottle in foil. Dissolve 45mg of Malachite green in 100ml of Milli-Q H2O. Mix overnight using the blue stirrer. Store at 4 degrees C for 1 week minimum. For 180ml of 4.2% ammonium molybdate in 4N HCL. Dissolve 7.56 g of ammonium molybdate in 120 ml of Milli-Q H2O and add 60ml of 12N HCL. Dilute the 2C protein sample in Buffer B of TALON. Calculate the dilution to make the fraction reach a final concentration of 20 micromolar [9]. In a 50ml Falcon tube, mix 12.5ml ammonium molybdate stock with 37.5ml of malachite green. Put on a spin rotor for 25 minutes. In a new 50 ml Falcon tube add 1ml of 1% Tween stock. Set up the assay in a clear 384 well plate. The final volume should be 50 microliters before adding in the color reagent. Add the antiviral drugs and compounds across the well plates. Make sure the micropipette does not touch the interior of the wells to prevent cross contamination. Next, add in 25 microliters of ATPase to each of the wells. Once this is done, add 25 microliters of the color reagent to each of the wells. Bring the ATPase assay to the computer for image processing (Multimedia Appendix 4).

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Results

Transformation of E. coli with the genetic information from Enterovirus 71

The results from the transformation showed that EV 71 genetic information was successfully transferred to the E. coli colonies. This result was obtained by physically transforming the E. coli through the method described above.

Growth of the E. coli colonies in the lab setting

The results from the growth of the E. coli showed that they initially grew at an exponential rate without being hindered by resources or space. However, as time progressed, the rate of growth became logarithmic and slowly ceased. The growth of the bacteria colonies in the lab setting followed a normal growth rate pattern among E. coli.

2C protein purification

The results from the 2C protein purification showed that the protocol for extracting the protein was successful. The AKTA computer system was utilized to detect changes in conductivity at various time intervals as the sample was analyzed [10]. These changes in conductivity were subjected to SDS-PAGE and compared to a standardized 2C protein ladder. The extracted protein and the ladder matched in terms of kDa length and band formation [11].

ATPase assays with drug testing:

The ATPase assays with drug testing revealed that a combination of two different antiviral compounds (Mitrofudil and N6- Benzyladenosine) was able to cease ATPase activity. This was qualitatively measured by the fluorescence exhibited by each well based on the presence of the color reagent. A yellow fluorescence indicates that there is no free phosphate available in the well, meaning that ATP was not being dephosphorylated [12]. Four subsequent wells next to one another had to display this phenomenon to ensure that it was indeed the drug halting the breakdown of ATP [13]. Mitrofudil and N6- Benzyladenosine together were able to prevent ATPase activity. A green fluorescence indicates that there is free phosphate available as a direct result of ATPase breaking down ATP to ADP and P [14]. This shows that the other antiviral compounds would not inhibit ATPase activity and still allow new viral particle formation for this particular strain [15].

Discussion

Principle Results

The principle results from this experiment are that a conjunction of two antiviral compounds are best at blocking ATPase rather than each compound on its own [16]. Coloration of wells when both drugs were combined appeared to be of a greater shade of yellow than when each compound was tested individually. This suggests that the mechanism of blocking ATPase is two-fold in EV 71 [17].

Limitations

It is important to note that more trials are needed to show that these two antiviral compounds are best suited for EV 71 treatment. These drugs have also not yet been tested on actual patients with Hand, Foot, and Mouth disease.

Acknowledgements

This research would not have been possible without the assistance of Dr. Simon White and the equipment in his laboratory. These discoveries were all made in the Biophysics Building at the University of Connecticut, Storrs campus. This work was also partially funded by a grant awarded to the White Lab for advancements in virology.

Conflicts of Interest:

No conflicts of interest to report.

Multimedia Appendix

Multimedia Appendix 1: EV 71 genome incorporated into transformed E. coli.

Multimedia Appendix 2: AKTA computer processing technique with Nickel column to detect the 2C protein.

Multimedia Appendix 3: SDS-PAGE showing successful 2C protein elution.

Multimedia Appendix 4: ATPase assay and drug testing. Notice that the colors are not just green and yellow. Over time, the colors will fade as the samples are oxidized.

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