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Introduction: Due to many genetic changes that have made it unfeasible to design efficient drugs, influenza virus has become one of the most important dangerous biologic agents. So, for its efficient treatment, researchers have shown an increased interest in novel drugs with natural origin and fewer side effects in compare with other conventional anti-influenza drugs. HESA-A is an herbal-marine compound consisting of rare elements and organic materials that have shown less cytotoxicity on normal cells. The influence of HESA-A on different biological agents and diseases has been investigated and its effects has been compared with other chemical drugs.
Materials and methods: HESA-A was prepared in normal saline as a stock solution (0.8 mg/ml, pH=7.4), sterilized and further diluted. Different concentrations of HESA-A were added to the cells and further incubated in different time points. Using MTT assay, percent cell survival was determined by ELISA reader at 540 nm. To study its potential antiviral activity, MDCK cells were treated with effective concentration (EC50) of HESA-A and 100 TCID50 of the virus sample during infection in different exposures. The viral titres were tested by hemagglutination assay (HA). To compare the viral genome load at different treatments, it was quantified by real-time PCR method using SYBR Green mix
Results: It was found that HESA-A has inhibitory effect on virus infection in cell culture by decreasing the titre of the virus.
Conclusion: The aim of this study was to evaluate and validate the anti-influenza effects of HESA-A on the load of influenza virus in cell culture. Besides, for rare reported side effects of HESA-A in comparison with other drugs, it seems that utilizing HESA-A as an anti-viral agent during upcoming epidemics can be priority to other chemical drugs.
Keywords: HESA-A, Influenza virus, Real-time PCR, MTT assay
Influenza virus is one of the most important causes of respiratory diseases worldwide and its genome is constantly evolving and new antigenic variants can cause different epidemics and pandemics. These mutations make it extremely difficult to develop effective vaccines and drugs. Therefore, it is needed to come back to traditional medication in combination with modern medicine to inhibit the viral activity or kill the virus (Vahabpour Roudsari, Shamsi Shahrabadi et al. 2007). HESA-A is an active natural biological compound with herbal and marine origin that has been patented in the Islamic Republic of Iran. General composition of HESA-A (X-ray results) is inorganic (50%), organic (45%) and aqueous (5%) fractions. Minerals are mixture of calcium carbonate, magnesium sulfate, potassium sulfate, sodium sulfate, magnesium phosphate, potassium phosphate and sodium phosphate, and elements such as strontium, titanium, manganese, nickel, mercury, copper, zinc, cadmium and cesium. It has shown antitumor, antimitotic, anticancer, antioxidant and anti-inflammation properties (Moallem, Ahmadi et al. 2007; Ahmadi, Barikbin et al. 2008; Ahmadi, Mohagheghi et al. 2008; Ahmadi, Mohagheghi et al. 2008) in different experiments. This project is involved MTT cytotoxicity assay and qPCR molecular technique to detect HESA-A anti-viral characteristic against influenza virus.
Material & methods
Influenza virus sample
Influenza A/New Jersey/8/76 (H1N1) vaccine strain obtained from ATCC (The Global Bioresource Center). It bared passage in Madin-Darby canine kidney (MDCK) cells in the presence of 1Âµg/ml of Trypsin_TPCK (Tosylamide, Phenylethyl Chloromethyl Keton-treated Trypsin) (Sigma Co.).
MDCK cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (ZenBio) contained 10% heat-inactivated Fetal Bovine Serum (FBS) (PAA, GmbH), 100 Units/ml Penicillin G and 100Âµg/ml Streptomycin (Sigma Co.) at 37Â°C in a humidified 5% CO2 incubator. During antiviral evaluations, the serum was removed and the medium was supplemented with 1Âµg/ml of Trypsin_TPCK.
HESA-A was received from Dr. Ahmadi, university of Tehran. In brief, it was dissolved in normal saline (pH 1.5), shacked for 30 minutes and filtered. Prior to its use, this stock solution (0.8 mg/ml, pH 7.4) was sterilized using 0.22Âµm syringe filter and diluted (Ahmadi 2009) to final concentrations of 0.4, 0.2, 0.1, 0.05, 0.025 &0.0125 mg/ml.
MDCK cells (4Ã-104cells/well) in 96-well micro-plate incubated for 24hrs at 37oC. Two-fold serial dilutions of HESA-A prepared by DMEM were added to the semi-confluent cells and incubated in different time points to obtain EC50. Colorimetric MTT assay was performed according to Mehrbod et al (Mehrbod, Motamed et al. 2009). MTT is reduced to purple formazan by NADH pathway. Insoluble formazan needs an organic solvent to be soluble before measuring the absorbance. Briefly, 100Âµl of 1x MTT was added to each well after removing medium of the confluent cells. Following incubation at 37Â°C with 5% CO2 for 3hrs and discarding the solution, 100Î¼l of acidic isopropanol was added and mixed thoroughly to release the color from the cells. The absorbance of the color in the solution was analyzed using ELISA reader machine (EL 800) at 540 nm. The 50% cytotoxic concentration (CC50), effective concentration (EC50) and viability of the cells were defined by this method.
HESA-A inhibitory effect on the virus
The cells were infected with 0.5 multiplicity of infection (MOI) of influenza virus (100 TCID50) in different exposures with EC50 of HESA-A. Following 1h incubation of pre-penetration, post-penetration and co-penetration exposures at 37Â°C, unabsorbed viruses were washed with phosphate buffer saline (PBS) and TPCK-containing medium was added (100Î¼l/well). After 48 h incubation at 37Â°C, viabilities of infected and non-infected cells were evaluated by MTT method. Then the virus titer was determined by HA assay and the viral genome load were evaluated by quantitative real-time PCR using SYBR green mix.
Percent protection of HESA-A was calculated using MTT results by Microsoft office Excell 2010 by means of viability of mock-infected and infected cells after 48hrs exposure (Shigeta, Shuichi et al. 1997).
To evaluate presence of the virus in cell culture, serial dilutions of the culture media were added to 96-well U-shape micro-plate. Chicken red blood cells (cRBCs) (0.5%) were added to each well. Following incubation at least for 1h at room temperature, the data calculating in Spss, Anova showed the meaningful differences among the results (Mehrbod, Motamed et al. 2009).
RNA Extraction and cDNA synthesis
Viral genomic RNA was extracted from virus exposed cell culture using Viral Nucleic Acid Extraction kit II (Geneaid, Taiwan). Briefly, 200Âµl cell culture media was used and RNA was bound to glass fibers fixed in a column and finally was isolated and eluted in 50Âµl RNase-free water. The cDNA synthesis was carried out by RevertAid H Minus First Strand cDNA kit (Fermentas, Malaysia). Ten microliter RNA sample along with random hexamer primers were incubated at 65Â°C for 5 min, added to a mixture of buffer, Ribolock RNase inhibitor, dNTP mix and MMUL-V as instructed in the kit. The cDNA products were stored at -20Â°C for long time use.
The primers (HPLC purified) for this study were designed by kind assistance of Dr. Cheah, First Base Co. Malaysia. Primers designed for this study amplified NP genes of influenza A H1N1/ New Jersey/8/76. They were:
NP-A-For: 5Â´_CAG ACC AAA TGA AAA CCC AGC_3Â´ nt; 973-992
NP-A-Rev: 5Â´_AAT CTG AAC CCC TCT TGT GG_3Â´ nt; 1101-1120
The primers corresponded to the influenza A H1N1/ New Jersey/8/76 sequence obtained from GenBank accession number CY039994.
Quantitative Real-time PCR
Maxima SYBR Green/Fluorescin qPCR (Fermentas, Malaysia) in real-time PCR was used on 5Âµl cDNA and 1 Âµl of each primer. Thermal cycling was run on a thermal Cycler C1000 (BIO RAD CFX96) instrument under the following conditions: 3 min at 95Â°C; 40 cycles of 15 sec at 95Â°C, 30 sec at 60Â°C; and 20 sec at 72 Â°C.
Different quantities in viral load in PCR products in different treatments were interpreted using analysis of variance (ANOVA) Spss 18.0.
Viabilities of MDCK cells were determined after different time exposures to HESA_A different concentrations by MTT method reading optical densities at 540 nm. The results shown in figure 1 were clear that HESA_A had no lethal effect on the cells at concentration up to 0.05mg/ml. EC50 of this compound was calculated from MTT results by two-way Anova test at 0.025mg/ml that had no obvious cytopathic effect on the cell as in control while reducing CPE of the virus.
Figure1: the graph shows MTT results of different concentrations of HEA (from right to left: 0.8, 0.4, 0.2, 0.1, 0.05, 0.025mg/ml and negative control) in different time exposures to cells (24, 48 &72 hr). Cytotoxicity of different concentrations of HESA on MDCK cells as meanÂ±SD is as follow respectively: 0.27Â±0.43, 0.39Â±0.09, 0.49Â±0.02, 0.57Â±0.004, 0.67Â±0.001, 0.74Â±0.00 and 0.79Â±0.00. With P<0.05, the CC50 is 0.05mg/ml and EC50 with no significant difference with negative control is 0.025mg/ml (column number 2). Results are averages of four independent experiments.
HESA-A inhibitory effect on the virus
In this experiment, there has been some increase in optical densities measured after running MTT assay in different exposures in compare with virus sample that is the result of HESA_A effect on virus activity. But as it is obvious from the figure2 and table1, the meaningful raise in OD is related to co-penetration exposure.
Figure2: this graph illustrates MTT results of different exposure manners of the virus and HESA_A that are averages of at least 4 independent tests (meanÂ±SD).
:ÙSignificantly different from value obtained for co-penetration treatment compared to virus untreated sample (p<0.05).
Table1: OD at 540nm for different exposure manners of HESA_A and virus
Different exposures showed increase in OD, but there was just co-penetration treatment (Ù) that showed significant increase with p<0.05 (meanÂ±SD).
Percent protection results
The viabilities of cells were evaluated by determination of formazan absorbance at 540 nm after 48 hrs exposure. Untreated cells were considered as negative control. The percent protection was calculated using this formula:
Percent protection = [(ODT)V-(ODC)V] / [(ODC)M-(ODC)V] Ã- 100
That (ODT)V, (ODC)V and (ODC)M imply absorbance of the sample treated, the virus-infected control (no compound) and negative control (no virus and no compound), respectively (Shigeta, Shuichi et al. 1997). The results are shown in table2.
Table2: MTT results for percent protection
Percentage averages (%)
Values are percentage averages of four independent experiments.
It was found that co-penetration-treated sample compared to the other treated and untreated samples was more protective on the cell viability against the virus CPE.
Hemagglutination assay results
Antiviral activity of HESA_A against influenza virus cell culture in different sets of experiments was assessed by hemagglutination assay. Its inhibitory effect was shown by HA titre reduction in figure3 and table3.
Figure3: this illustration clearly clarifies the inhibitory effect of the HESA_A on HA titer of influenza virus at different exposures. Data are averages of 4 independent tests.
:ÙSignificantly different from value obtained for virus untreated sample compared to combination treatments (p<0.05).
Table3: HA results for antiviral activity of HESA_A against influenza virus A
Values are averages of four independent HA examinations.
*: Significantly different from values obtained for HESA_A_treated samples compared to untreated sample (p<0.05).
Quantitative Real-time PCR
The effect of HESA_A on the viral genome load was shown by increase in cycle threshold (figure4). Quantitative analysis on PCR products on NP gene of influenza virus A in pre & co-penetration exposures for 1 hour showed statistically meaningful decrease in genome content in direct exposure of HESA_A to the virus. Data are shown in figure5.
Figure4: Quantification curves relating cycle number and maxima Sybr Green fluorescence signals obtained in Thermal Cycler during real time PCR amplification of influenza A (H1N1) New Jersey NP gene.
(H+V: co-penetration, Hâ€¦V: pre-penetration, V: Virus inoculation, Vâ€¦H: post-penetration, NC: Negative Control, HESA: HESA treatment)
Figure5: these results which are averages of 4 independent repeats, show obvious increase in pre & co-penetration exposures cycle threshold in compared with virus untreated sample.
QPCR data obtained in Thermal Cycler using sybr green mix during real time PCR amplification of influenza A H1N1 NP gene. Cycle threshold of fluorescence obtained from viral RNA amplification after exposure to HESA_A (MeanÂ±SD) which are averages of four independent examinations are as follow: Virus sample: 12.76Â±0.81, Post-penetration: 14.44Â±0.22, Pre-penetration: 18.90Â±0.39*, Co-penetration: 22.09Â±0.73* and Negative Control: 35.66Â±2.61*
*: Significantly different from values obtained for pre & co-penetration exposure samples compared to untreated sample (p<0.05).
Discussion and conclusion
For hundreds of years humans have struggled with seasonal inï¬‚uenza epidemics. This virus still causes severe respiratory disease that remains a leading source of annual morbidities and mortalities (Lamb and Takeda 2001; Fedson 2008). So, it is required to identify and develop novel anti-influenza compounds preferably with natural origin for prevention and treatment of potential influenza pandemics. Existing therapeutic antiviral agents have limited clinical efficiency and many toxic side effects, but antiviral compounds of natural origin are more easily available and mostly nontoxic (Vahabpour Roudsari, Shamsi Shahrabadi et al. 2007).
HESA-A which is a natural compound with herbal-marine origin, contains inorganic, organic and aqueous fractions with a wide range of useful effects demonstrated in different experiments (Moallem, Ahmadi et al. 2007; Ahmadi, Balali-Mood et al. 2008; Ahmadi, Mohagheghi et al. 2008; Tafreshi, Ahmadi et al. 2008; Ahmadi 2009; Y., N. et al. 2010).
In this study, we studied the antiviral activity of HESA_A and evaluated interactions between HESA_A and influenza virus A/H1N1. To test our hypothesis for effect of this natural compound, we explored its in vitro antiviral activity against influenza A virus.
This compound was not toxic on MDCK cells up to 0.05mg/ml concentration. As calculated from MTT results by two-way Anova test, EC50 of this compound was obtained 0.025mg/ml with no clear cytopathic effect on the cell as in control.
From MTT results and percent protection calculation in different exposures of HESA_A and virus that show the optical densities for living cells, it was shown that co-penetration and pre-penetration exposures with 83.77% and 69.61% protection ,respectively were more effective (p<0.05) in decreasing the virus activity in compared to the other exposure. Meanwhile, HA data showed significant drop in HA titre in all combination treatments as compared to positive control virus-treated sample. From these results it can be estimated that HESA_A may interfere with viral membrane fusion by inhibition of penetration or adsorption through interfering with HA glycoprotein.
After these detection methods to confirm the antiviral effect of this herbal-marine drug on influenza virus load in MDCK cell culture, quantitative real time PCR assay was applied. A significant increase in cycle threshold was shown once HESA_A was applied in pre-penetration and especially co-penetration treatments.
It can be suggested that HESA_A could be a good nominee as a natural medication to prevent and also remediation of this viral infection.
Research on HESA_A interactions with different bio-systems has been started recently. Knowing the exact effect of HESA_A on the virus life cycle or even the cellular structure to prevent cellular damage is in progress nowadays and provides the incentive for further research especially on cytokine dysregulation that makes the disease by this infection more severe (Frost, Petersen et al. 2007) and also is one of the most characteristicss of HESA_A which is under research by the authors.