Introduction: Due to many familial alterations that have made it impracticable to plan efficient drugs, grippe virus has become one of the most of import unsafe biologic agents. So, for its efficient intervention, research workers have shown an increased involvement in fresh drugs with natural beginning and fewer side effects in comparison with other conventional anti-influenza drugs. HESA-A is an herbal-marine compound consisting of rare elements and organic stuffs 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 farther diluted. Different concentrations of HESA-A were added to the cells and farther incubated in different clip points. Using MTT check, per centum cell endurance was determined by ELISA reader at 540 nanometer. To analyze its possible antiviral activity, MDCK cells were treated with effectual concentration ( EC50 ) of HESA-A and 100 TCID50 of the virus sample during infection in different exposures. The viral titers were tested by haemagglutination check ( HA ) . To compare the viral genome burden at different interventions, it was quantified by real-time PCR method utilizing SYBR Green mix
Consequences: It was found that HESA-A has repressive consequence on virus infection in cell civilization by diminishing the titer of the virus.
Decision: The purpose of this survey was to measure and formalize the anti-influenza effects of HESA-A on the burden of influenza virus in cell civilization. Besides, for rare reported side effects of HESA-A in comparing with other drugs, it seems that using HESA-A as an anti-viral agent during upcoming epidemics can be precedence to other chemical drugs.
Keywords: HESA-A, Influenza virus, Real-time PCR, MTT assay
Influenza virus is one of the most of import causes of respiratory diseases worldwide and its genome is invariably germinating and new antigenic discrepancies can do different epidemics and pandemics. These mutants make it highly hard to develop effectual vaccinums and drugs. Therefore, it is needed to come back to traditional medicine in combination with modern medical specialty to suppress 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 beginning that has been patented in the Islamic Republic of Iran. General composing of HESA-A ( X-ray consequences ) is inorganic ( 50 % ) , organic ( 45 % ) and aqueous ( 5 % ) fractions. Minerals are mixture of Ca carbonate, Mg sulphate, K sulphate, Na sulphate, Mg phosphate, K phosphate and Na phosphate, and elements such as Sr, Ti, manganese, nickel, quicksilver, Cu, Zn, Cd and caesium. It has shown antitumor, antimitotic, anticancer, antioxidant and anti-inflammation belongingss ( Moallem, Ahmadi et Al. 2007 ; Ahmadi, Barikbin et Al. 2008 ; Ahmadi, Mohagheghi et Al. 2008 ; Ahmadi, Mohagheghi et Al. 2008 ) in different experiments. This undertaking is involved MTT cytotoxicity check and qPCR molecular technique to observe HESA-A anti-viral feature against influenza virus.
Material & A ; methods
Influenza virus sample
Influenza A/New Jersey/8/76 ( H1N1 ) vaccinum strain obtained from ATCC ( The Global Bioresource Center ) . It bared transition in Madin-Darby eyetooth kidney ( MDCK ) cells in the presence of 1Aµg/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 100Aµg/ml Streptomycin ( Sigma Co. ) at 37A°C in a humidified 5 % CO2 brooder. During antiviral ratings, the serum was removed and the medium was supplemented with 1Aµg/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 proceedingss and filtered. Prior to its usage, this stock solution ( 0.8 mg/ml, pH 7.4 ) was sterilized utilizing 0.22Aµm syringe filter and diluted ( Ahmadi 2009 ) to concluding concentrations of 0.4, 0.2, 0.1, 0.05, 0.025 & A ; 0.0125 mg/ml.
MDCK cells ( 4A-104cells/well ) in 96-well micro-plate incubated for 24hrs at 37oC. Double consecutive dilutions of HESA-A prepared by DMEM were added to the semi-confluent cells and incubated in different clip points to obtain EC50. Colorimetric MTT check was performed harmonizing to Mehrbod et Al ( Mehrbod, Motamed et Al. 2009 ) . MTT is reduced to purple formazan by NADH tract. Insoluble formazan needs an organic dissolver to be soluble before mensurating the optical density. Briefly, 100Aµl of 1x MTT was added to each well after taking medium of the feeder cells. Following incubation at 37A°C with 5 % CO2 for 3hrs and flinging the solution, 100I?l of acidic isopropyl alcohol was added and assorted exhaustively to let go of the colour from the cells. The optical density of the colour in the solution was analyzed utilizing ELISA reader machine ( EL 800 ) at 540 nanometer. The 50 % cytotoxic concentration ( CC50 ) , effectual concentration ( EC50 ) and viability of the cells were defined by this method.
HESA-A inhibitory consequence on the virus
The cells were infected with 0.5 multiplicity of infection ( MOI ) of grippe virus ( 100 TCID50 ) in different exposures with EC50 of HESA-A. Following 1h incubation of pre-penetration, post-penetration and co-penetration exposures at 37A°C, unabsorbed viruses were washed with phosphate buffer saline ( PBS ) and TPCK-containing medium was added ( 100I?l/well ) . After 48 h incubation at 37A°C, viabilities of septic and non-infected cells were evaluated by MTT method. Then the virus titre was determined by HA check and the viral genome burden were evaluated by quantitative real-time PCR utilizing SYBR green mix.
Percent protection of HESA-A was calculated utilizing MTT consequences by Microsoft office Excell 2010 by agencies of viability of mock-infected and septic cells after 48hrs exposure ( Shigeta, Shuichi et Al. 1997 ) .
To measure presence of the virus in cell civilization, consecutive dilutions of the civilization media were added to 96-well U-shape micro-plate. Chicken ruddy blood cells ( cRBCs ) ( 0.5 % ) were added to each well. Following incubation at least for 1h at room temperature, the informations calculating in Spss, Anova showed the meaningful differences among the consequences ( Mehrbod, Motamed et Al. 2009 ) .
RNA Extraction and complementary DNA synthesis
Viral genomic RNA was extracted from virus exposed cell civilization utilizing Viral Nucleic Acid Extraction kit II ( Geneaid, Taiwan ) . Briefly, 200Aµl cell civilization media was used and RNA was bound to glaze fibres fixed in a column and eventually was isolated and eluted in 50Aµl RNase-free H2O. The complementary DNA synthesis was carried out by RevertAid H Minus First Strand complementary DNA kit ( Fermentas, Malaysia ) . Ten microliter RNA sample along with random hexamer primers were incubated at 65A°C for 5 min, added to a mixture of buffer, Ribolock RNase inhibitor, dNTP mix and MMUL-V as instructed in the kit. The complementary DNA merchandises were stored at -20A°C for long clip usage.
The primers ( HPLC purified ) for this survey were designed by sort aid of Dr. Cheah, First Base Co. Malaysia. Primers designed for this survey amplified NP cistrons of grippe A H1N1/ New Jersey/8/76. They were:
NP-A-For: 5A?_CAG ACC AAA TGA AAA CCC AGC_3A? nt ; 973-992
NP-A-Rev: 5A?_AAT CTG AAC CCC TCT TGT GG_3A? nt ; 1101-1120
The primers corresponded to the grippe A H1N1/ New Jersey/8/76 sequence obtained from GenBank accession figure CY039994.
Quantitative Real-time PCR
Maxima SYBR Green/Fluorescin qPCR ( Fermentas, Malaysia ) in real-time PCR was used on 5Aµl complementary DNA and 1 Aµl of each primer. Thermal cycling was run on a thermic Cycler C1000 ( BIO RAD CFX96 ) instrument under the undermentioned conditions: 3 min at 95A°C ; 40 rhythms of 15 sec at 95A°C, 30 sec at 60A°C ; and 20 sec at 72 A°C.
Different measures in viral burden in PCR merchandises in different interventions were interpreted utilizing analysis of discrepancy ( ANOVA ) Spss 18.0.
Viabilities of MDCK cells were determined after different clip exposures to HESA_A different concentrations by MTT method reading optical densenesss at 540 nanometer. The consequences shown in figure 1 were clear that HESA_A had no deadly consequence on the cells at concentration up to 0.05mg/ml. EC50 of this compound was calculated from MTT consequences by bipartisan Anova trial at 0.025mg/ml that had no obvious cytopathic consequence on the cell as in control while cut downing CPE of the virus.
Figure1: the graph shows MTT consequences of different concentrations of HEA ( from right to go forth: 0.8, 0.4, 0.2, 0.1, 0.05, 0.025mg/ml and negative control ) in different clip exposures to cells ( 24, 48 & A ; 72 hour ) . Cytotoxicity of different concentrations of HESA on MDCK cells as meanA±SD is as follow severally: 0.27A±0.43, 0.39A±0.09, 0.49A±0.02, 0.57A±0.004, 0.67A±0.001, 0.74A±0.00 and 0.79A±0.00. With P & lt ; 0.05, the CC50 is 0.05mg/ml and EC50 with no important difference with negative control is 0.025mg/ml ( column figure 2 ) . Consequences are norms of four independent experiments.
HESA-A inhibitory consequence on the virus
In this experiment, there has been some addition in optical densenesss measured after running MTT assay in different exposures in comparison with virus sample that is the consequence of HESA_A consequence on virus activity. But as it is obvious from the figure2 and table1, the meaningful rise in OD is related to co-penetration exposure.
Figure2: this graph illustrates MTT consequences of different exposure manners of the virus and HESA_A that are norms of at least 4 independent trials ( meanA±SD ) .
: USignificantly different from value obtained for co-penetration intervention compared to virus untreated sample ( p & lt ; 0.05 ) .
Table1: Doctor of optometry at 540nm for different exposure manners of HESA_A and virus
Different exposures showed addition in OD, but at that place was merely co-penetration intervention ( U ) that showed important addition with p & lt ; 0.05 ( meanA±SD ) .
Percentage protection consequences
The viabilities of cells were evaluated by finding of formazan optical density at 540 nanometers after 48 hour exposure. Untreated cells were considered as negative control. The per centum protection was calculated utilizing this expression:
Percentage protection = [ ( ODT ) V- ( ODC ) V ] / [ ( ODC ) M- ( ODC ) V ] A- 100
That ( ODT ) V, ( ODC ) V and ( ODC ) M imply optical density of the sample treated, the virus-infected control ( no compound ) and negative control ( no virus and no compound ) , severally ( Shigeta, Shuichi et Al. 1997 ) . The consequences are shown in table2.
Table2: MTT consequences for per centum protection
Percentage norms ( % )
Valuess are per centum norms 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 consequences
Antiviral activity of HESA_A against influenza virus cell civilization in different sets of experiments was assessed by haemagglutination check. Its repressive consequence was shown by HA titer decrease in figure3 and table3.
Figure3: this illustration clearly clarifies the repressive consequence of the HESA_A on HA titre of influenza virus at different exposures. Datas are norms of 4 independent trials.
: USignificantly different from value obtained for virus untreated sample compared to combination interventions ( P & lt ; 0.05 ) .
Table3: HA consequences for antiviral activity of HESA_A against influenza virus Angstrom
Valuess are norms of four independent HA scrutinies.
* : Significantly different from values obtained for HESA_A_treated samples compared to untreated sample ( p & lt ; 0.05 ) .
Quantitative Real-time PCR
The consequence of HESA_A on the viral genome burden was shown by addition in rhythm threshold ( figure4 ) . Quantitative analysis on PCR merchandises on NP cistron of influenza virus A in pre & A ; co-penetration exposures for 1 hr showed statistically meaningful lessening in genome content in direct exposure of HESA_A to the virus. Datas are shown in figure5.
Figure4: Quantification curves associating rhythm figure and maxima Sybr Green fluorescence signals obtained in Thermal Cycler during existent clip PCR elaboration of grippe A ( H1N1 ) New Jersey NP cistron.
( H+V: co-penetration, Haˆ¦V: pre-penetration, V: Virus vaccination, Vaˆ¦H: post-penetration, NC: Negative Control, HESA: HESA intervention )
Figure5: these consequences which are norms of 4 independent repetitions, show obvious addition in pre & amp ; co-penetration exposures rhythm threshold in compared with virus untreated sample.
QPCR informations obtained in Thermal Cycler utilizing sybr green mix during existent clip PCR elaboration of grippe A H1N1 NP cistron. Cycle threshold of fluorescence obtained from viral RNA elaboration after exposure to HESA_A ( MeanA±SD ) which are norms of four independent scrutinies are as follow: Virus sample: 12.76A±0.81, Post-penetration: 14.44A±0.22, Pre-penetration: 18.90A±0.39* , Co-penetration: 22.09A±0.73* and Negative Control: 35.66A±2.61*
* : Significantly different from values obtained for pre & A ; co-penetration exposure samples compared to untreated sample ( p & lt ; 0.05 ) .
Discussion and decision
For 100s of old ages worlds have struggled with seasonal ini¬‚uenza epidemics. This virus still causes terrible respiratory disease that remains a taking beginning of one-year morbidities and mortalities ( Lamb and Takeda 2001 ; Fedson 2008 ) . So, it is required to place and develop fresh anti-influenza compounds sooner with natural beginning for bar and intervention of possible grippe pandemics. Existing curative antiviral agents have limited clinical efficiency and many toxic side effects, but antiviral compounds of natural beginning are more easy available and largely atoxic ( Vahabpour Roudsari, Shamsi Shahrabadi et Al. 2007 ) .
HESA-A which is a natural compound with herbal-marine beginning, contains inorganic, organic and aqueous fractions with a broad scope of utile 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 survey, we studied the antiviral activity of HESA_A and evaluated interactions between HESA_A and influenza virus A/H1N1. To prove our hypothesis for consequence of this natural compound, we explored its in vitro antiviral activity against grippe A virus.
This compound was non toxic on MDCK cells up to 0.05mg/ml concentration. As calculated from MTT consequences by bipartisan Anova trial, EC50 of this compound was obtained 0.025mg/ml with no clear cytopathic consequence on the cell as in control.
From MTT consequences and percent protection computation in different exposures of HESA_A and virus that show the optical densenesss for life cells, it was shown that co-penetration and pre-penetration exposures with 83.77 % and 69.61 % protection, severally were more effectual ( p & lt ; 0.05 ) in diminishing the virus activity in compared to the other exposure. Meanwhile, HA information showed important bead in HA titer in all combination interventions as compared to positive control virus-treated sample. From these consequences it can be estimated that HESA_A may interfere with viral membrane merger by suppression of incursion or surface assimilation through interfering with HA glycoprotein.
After these sensing methods to corroborate the antiviral consequence of this herbal-marine drug on grippe virus burden in MDCK cell civilization, quantitative existent clip PCR check was applied. A important addition in rhythm threshold was shown one time HESA_A was applied in pre-penetration and particularly co-penetration interventions.
It can be suggested that HESA_A could be a good campaigner as a natural medicine to prevent and besides redress of this viral infection.
Research on HESA_A interactions with different bio-systems has been started late. Knowing the exact consequence of HESA_A on the virus life rhythm or even the cellular construction to forestall cellular harm is in advancement presents and provides the inducement for farther research particularly on cytokine dysregulation that makes the disease by this infection more terrible ( Frost, Petersen et Al. 2007 ) and besides is one of the most feature of HESA_A which is under research by the writers.