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RJ Jariwalla, MW Roomi, B Gangapurkar, T Kalinovsky, A Niedzwiecki, and M Rath
Influenza is an acute viral disease of the respiratory tract that causes fever. It is one of the oldest and most common infections in the world, affecting one in five people (Jariwalla et al., 1). Annual influenza infections occur in epidemic proportions during winter and spring in the United States and result in significant economic costs, as well as morbidity and mortality. Vaccination is the primary prevention strategy for influenza, particularly for high-risk populations, although vaccines are often ineffective due to antigenic drift. Antigenic drift is a small change that occurs in the virus over time. This occurs when the virus mutates after the current year’s vaccine has been produced and cause the vaccine to be less effective in fighting the newly mutated strain of influenza (CDC).
Currently, two drugs marketed as Tamiflu and Relenza are approved for the treatment of influenza. In this study, authors have focused on developing strategies to inhibit viral infections and other diseases by naturally occurring nutrients, rather than prophylaxes. They test the efficacy of a unique nutrient mixture (NM), which contains ascorbic acid, green tea extract, lysine, proline, N-acetyle cysteine, selenium, and other micronutrients. This mixture was thought to have anti-carcinogenic and anti-atherogenic properties that combat influenza A virus subtype H1N1 multiplication in infected cells. Anti-carcinogenic agents are those that stop the accumulation and development of carginogens in the body (Merriam-Webster). Similarly, anti-atherogenic agents protect against the formation of abnormal deposits on arteries (The Pharmacy Review).
Methods and Materials
The first aspect of the authors’ methods and materials was the creation of the nutrient mixture with the ingredients described above. Next, cells that were infected with the virus as well as those that were not infected were cultured and incubated. At confluence, the cell plates were treated with various concentrations of the nutrient mixture (Jariwalla et al., 3). After the plates were treated, they were returned to the incubator for defined time intervals and assessed for viability and production of the viral agent.
Assays were conducted for viability and cytotoxicity. Cell viability was assessed by a test called the trypan blue due exclusion test. Cytotoxicity was determined by conducting a spectrophotometric measurement of total biomass by staining cellular proteins. This stain assessed the degree of cytotoxicity in terms of the change in absorbance relative to untreated control culture (Jariwalla et al., 3).
The effect of the nutrient mixture and ascorbic acid on influenza A nuclear protein was determined by examining two day old layers of Vero and MDCK cells that were washed and exposed to the human influenza A virus. Vero cells and MDCK cells are types of cell lines that are conducive to studying vaccine efficacy under controlled conditions (FDA). They were exposed in a serum-free medium at a temperature at which infection cells could multiply for one hour (Jariwalla et al., 4). These same cells were left with the infectious agent and were treated with various concentrations of the nutrient mixture or with ascorbic acid, and then were incubated further.
Study authors also assessed the influence of the virus pre-treatment with the nutrient mixture by pre-treating concentrations of the nutrient mixture and placing them on ice. This interaction was treated similarly to those of the Vero and MDCK cells. In this study, data were analyzed by conducting an independent sample 2-tailed t test.
Initially, the authors attempted to evaluate the effect of various concentrations of the nutrient mixture on the viability of Vero and MDCK cells that were infected with the virus. The nutrient mixture did not significantly affect the MDCK cells’ viability after 48 hours. Treating the Vero cells with low concentrations of the nutrient mixture had very little effect on cell viability, producing a 20% lowering of viable cell count (Jariwalla etl al., 5).
At higher concentrations of the nutrient mixture, there was an approximate 60-70% lowering in viability of Vero cells (Jariwalla et al., 6). Authors estimate that the effectiveness of the nutrient mixture is primarily due to the green tea extract on cell lines. Song and colleagues in a prior study have also reported this finding. Jariwalla and colleagues found that the sensitivity of cells to the nutrient mixture varies among different cell lines, which explains the increased sensitivity to toxicity of the nutrient mixture by Vero cells in contrast to MDCK cells (Jariwalla et al., 7).
Tests to investigate cytotoxicity and specificity of the antiviral effect of the nutrient mixture in Vero and MDCK cells concluded that the selectivity of the nutrient mixture had an inhibitory effect towards influenza rather than inhibiting host cells in the samples (Jariwalla et al., 8).
Green tea extract, as mentioned earlier, was a component of the nutrient mixture. This extract has been shown to inhibit influenza virus. Authors also evaluated the efficacy of ascorbic acid in the inhibition of influenza. This acid was found to have some inhibitory effects on the antigen production in influenza cultures that were treated, causing a 50% reduction in antigen (Jariwalla et al., 9). At higher concentrations, ascorbic acid was found to exert a dose-dependent suppression of the virus in MDCK cells. Green tea extract and ascorbic acid were tested individually and in combination.
The authors found that influenza virus can be controlled through a number of channels. Tamiflu and Relenza block viral uncoating through ion channels (Jariwalla et al., 11). However, these drugs have been shown to have toxic effects, which suggest the need for improved treatment methods. These results demonstrate that a nutrient mixture containing lysine, proline, ascorbic acid, green tea extract, selenium, and other micronutrients is a powerful inhibitor of influenza A growth and antigen production, as demonstrated through testing MDCK and Vero cell cultures (Jariwalla et al., 12). Inhibition happens when the nutrient mixture induces host cell toxicity and has a dose response relationship between antiviral efficacy and increasing concentrations of the mixture.
The antiviral effect of the mixture was enhanced when the virus was pre-treated with the mixture, as well as post-infection treatment of cells. The polyphenols present in green tea extract was thought to be the single most effective ingredient in the nutrient mixture at inhibiting the influenza A virus in this study, although the mixture’s other components were thought to have synergistic effects. In contrast to the toxic side effects of popular influenza medications, the nutrient mixture was found to be a safe therapeutic agent (Jariwalla et al., 13).
|This article was published on Sunday June 26, 2011.|
 Centers for Disease Control and Prevention, “How the Flu Virus Can Change: Drift and Shift”. http://www.cdc.gov/flu/about/viruses/change.htm.
 Food and Drug Administration, “History and Characterization of the Vero Cell Line”. http://www.fda.gov/ohrms/dockets/ac/00/backgrd/3616b1a.pdf.
 Jariwalla, RJ, Roomi, MW, Gangapurkar, B, Kalinovsky, T, Niedzwiecki, A, Rath, M. “Suppression of influenza A virus nuclear antigen production and neuraminidase activity by a nutrient mixture containing ascorbic acid, green tea extract and amino acids”. BioFactors 31(2007); 1-15.
 Merriam-Webster Medical Dictionary. “Anticarcinogenic”. http://mw1.merriam-webster.com/medical/anticarcinogenic.