Iodine is a nonmetallic essential nutrient with a potent broad range of microbicide actions against almost all of the important health-related microorganisms, including bacteria, fungi, viruses and protozoa. Although a high content of iodine species with free molecular form (I2) and hypoiodous acid (HOI) in aqueous solution has powerful microbicidal effects but can cause volatility, stinging and cytotoxicity [30–32]. To overcome these problems, iodine was combined with neutral carrier polymers to increase iodine solubility and to keep low the release of iodine as a solubilizing agent and to act as an iodine reservoir [30, 33]. The most popular carrier in current use is povidone [32, 33], which has no microbicidal activity . Since povidone slowly and continuously releases free iodine into solution, these properties help to maintain antimicrobial capacity for a long period and to decrease toxicity.
By using the cell counting kit-8 assay, we found that the IC50 cytotoxicity of MDCK cells following 24-h exposure to PVP-I was 2.4 ± 0.2 mg/ml. Based on morphological criteria , cell shrinkage, rounding and detachment from the surface of the culture plate after treatment with 3.1 mg/ml of PVP-I suggested that the cells were undergoing apoptosis. Therefore, we used low concentrations of PVP-I that did not cause any toxicity to host MDCK cells in order to investigate its anti-influenza virus activity.
Our results confirmed that PVP-I is a potent inhibitor of influenza virus production in MDCK cells. We indicated that PVP-I inhibits the viral replication in a dose dependent manner and is more active against human viruses (H1N1, H3N2) than avian viruses (H1N1, H5N3, H9N2). PVP-I appeared to inhibit binding of human A/Memphis/1/71 (H3N2) virus to specific sialoglycopolymers but not that of avian A/DK/HK/313/78 (H5N3) virus. Hemagglutination of erythrocytes induced by human viruses was inhibited by PVP-I, while hemagglutination inhibition of avian viruses required higher PVP-I concentrations. Differences in hemagglutination inhibitory activity of PVP-I against various viruses may be associated with the different structure of HA protein of each virus type. Unlike the α2,3 and α2,6 sialoconjugated protein fetuin , which reduces HA binding activity of both avian and human influenza viruses via competition for binding with sialyloligosaccharide receptor substrates to the viruses , blockage of viral HA attachment to receptor substrates by PVP-I may result from alteration of viral HA protein structure by reaction of free iodine with basic -NH groups, phenolic groups, and -SH groups of amino acid residues . Although avian viruses appear to be less sensitive than human viruses to PVP-I, based on results of the erythrocyte agglutination assay, which reflects viral attachment to host cells, agglutination of avian A/DK/HK/313/78 virus (~400 HAU) was completely inhibited after a second exposure to 5 mg/ml of PVP-I. This is in agreement with the finding that titers of a highly pathogenic avian virus (H5N1) and three low pathogenic avian viruses (H5N3, H7N7 and H9N2) cultivated in embryonated eggs become undetectable by incubation with a commercial PVP-I product for 10 seconds before inoculation . These results suggest that gargling with PVP-I could prevent human infection not only by human influenza viruses that bind to sialyl α2,6 Gal receptors in the upper part of human trachea but also by avian viruses that bind to sialyl α2,3 Gal receptors that exist deep in the human respiratory tract . This could consequently minimize the risk of avian virus mutation, either by adaptation or reassortment, to recognize the human host predominately carrying α2,6-linked sialic acids.
PVP-I inhibited sialidase activity as a mixed-type inhibitor, indicating that free iodine is capable of binding to either free sialidase or sialidase complexed with its substrate, but iodine binding to free sialidase is more efficient than that to sialidase-substrate complex as Ki was 16-fold lower than Kis. This may be explained by the distribution of lysine, arginine, histidine, cysteine and tyrosine residues throughout the sequence of the NA molecule, which are reactive with iodine . Although the Ki value for iodine was higher than the Km value, indicating that affinity of the MUNA substrate for sialidase is higher than iodine, the activity of sialidase to hydrolyze MUNA (Vmax/Km = 0.12) was reduced in the presence of PVP-I (75 μg/ml) (Vmax/Km = 0.02), comparable to that in the presence of OC (4 nM). The reduction in sialidase activity should result in a decrease in influenza replication.
There have been a number studies on the development of harmless carriers (such as cyclodextrin) that slowly release free iodine at a concentration retaining antimicrobial activity without a cytotoxic effect against mammalian cells for use of iodine in therapeutic applications [28, 39]. Intravenous administration of iodine-lithium-α-dextrin has successfully prevented lethal infection of Staphylococcus aureus in rats .