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Determining the virucidal effect of caprylic acid, monocaprylin, and sodium caprylate on avian influenza virus in tissue culture. Avian influenza virus is an important pathogen causing tremendous economic losses to the poultry industry. Further, zoonotic transmission of the virus to humans, and rapid human to human transmission highlight the public health significance of the virus. Moreover, resistance of avian influenza viruses to antiviral agents is an emerging, global public health problem. Since the virus is highly contagious, strict biosecurity measures can greatly help to contain the virus in a poultry farm. Contaminated food, water, and poultry manure represent three very important modes of transmission of the virus. Effective and safe antimicrobials that kill the virus in the aforementioned vehicles could greatly reduce spread of the virus. Further, since the gastrointestinal tract is one of the major target sites of the virus in birds, administration of a safe antiviral compound through feed and water could potentially kill the virus in live birds. Published literature, and previous research conducted revealed that caprylic acid, monocaprylin (monoglyceride of caprylic acid), and its sodium salt, sodium caprylate are highly effective in killing a variety of pathogens, including bacteria, and enveloped viruses. One major drawback of many antimicrobials is reduced or no antimicrobial activity in the presence of organic matter. However, research conducted in laboratory has revealed that caprylic acid and sodium caprylate are highly antimicrobial even in organic matter-rich media, including chicken cecal contents, bovine rumen fluid, and water containing bovine manure. Caprylic acid and monocaprylin can be added to poultry feed, whereas sodium caprylate, being stable and soluble in water, can be used as a potential antiviral agent in water. Delineating the antiviral properties of these molecules on the avian influenza virus will potentially help us to devise new antimicrobial approaches/tools to fight this fatal viral disease of the poultry industry. The proposed research is a first step in achieving this goal.
NON-TECHNICAL SUMMARY: Avian influenza is an important disease causing tremendous economic losses to the poultry industry. Further, transmission of the virus to humans highlights the public health significance of the disease. Since the virus is highly contagious, strict hygienic measures employed at the farm can greatly help to contain the virus. Contaminated food, water, and poultry manure are three important vehicles of the virus. Effective and safe antimicrobials that kill the virus in the aforementioned vehicles could greatly reduce spread of the virus. Caprylic acid is a natural fatty acid present in breast milk and coconut oil. From previously published research, it is known that caprylic acid and its other chemical forms, namely monocaprylin and sodium caprylate are highly effective in killing a variety of disease causing bacteria and viruses. Therefore, the proposed research aims to investigate the potential of caprylic acid and its molecules to kill avian influenza virus in a laboratory model. If the compounds are effective in killing the virus in the laboratory model, they could potentially be used to kill the virus in poultry feed and water. Moreover, since the virus resides the gastrointestinal tract of infected birds, supplementation of caprylic acid through feed and water could kill the virus in live birds. Determining the effect of these molecules on avian influenza virus may help us to devise new antimicrobial approaches/tools to fight this fatal viral disease affecting the poultry industry. The proposed research is a first step in achieving this goal.
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APPROACH: Madin-Darby canine kidney (MDCK) cells will be grown in minimal essential medium (MEM)supplemented with 5% fetal calf serum, 5 millimolar L-glutamine, sodium bicarbonate, 100 units of penicillin per milliliter, 100 micrograms of streptomycin sulfate per milliliter, and 100 micrograms of kanamycin sulfate per milliliter in a humidified atmosphere of 5% CO2. Caprylic acid, monocaprylin, and sodium caprylate will be obtained from Sigma. Two low pathogenic strains H5 and H9 and four isolates of low pathogenic H7/N5 viruses from NVSL, Ames, Iowa will be used in the study. The viruses will be propagated in the allantoic cavities of 10-day-old embryonated chicken eggs. The antiviral activity of caprylic acid, monocaprylin, and sodium caprylate will be determined by two different methods and further complemented with real time RT-PCR tests. A). the virucidal effect of the molecules on avian influenza virus will be determined by plaque reduction assay on MDCK cell. Briefly, six-well plates will be inoculated with the virus diluted in MEM to yield about 100 plaques per well. Cells will be incubated for 1 h at 37 C. After 3 days of incubation at 37 C, plaques will be visualized by staining with 0.1% crystal violet containing 10% formaldehyde. The percentage of inhibition of plaque formation compare to untreated controls will be calculated for each concentration of the three molecules. Three replicate experiments for each treatment will be performed to determine the concentration of each molecule that result in a 50% inhibition concentration (IC50) of the plaque number. B) Micro neutralization assay followed by a modified enzyme-linked immunoassay will be used to determine the antiviral effect of the three compounds. This assay detects the expression of viral nucleoprotein in infected cells. Briefly, MDCK cells will be seeded in 96-well culture plates at a density of 5000 cells per well in MEM containing 10% FBS, 100 Units per milliliter penicillin, 100 micrograms per mililiter streptomycin sulfate, and 100 micrograms per milliliter kanamycin sulfate. Cells will be incubated at 37C with 5% CO2 until 90% confluency is achieved. Cells will be then washed twice with serum-free MEM, and residual medium removed. Each micro titer plate will include uninfected control cultures, virus-infected control wells and virus-infected cultures to which antiviral compounds will be added. Plates will be incubated at 37 C for 30 minutes then allantoic fluid containing virus will be added to all wells except the wells with the cell control. After incubation for 18 h at 37C in a humidified atmosphere of 5% CO2, cells will be fixed by adding 100 microliter of cold acetone PBS mixture. The percent inhibition of virus replication by the antiviral compound will be calculated. The assay will be replicated three times. Real Time PCR will quantify viral RNA after and before treatment of the virus with the chemical compounds such as caprylic acid, monocaprylin, and sodium caprylate. The mean values, standard deviations, and significant differences will be analyzed by the Tukey-Kramer method.