Magnesium-mediated Reversal of the Apparent Virucidal Effect of Ascorbic Acid or Congo Red Reactedin vitrowith the Human Immunodeficiency Virus

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The mechanism of in vitro inactivation of cell-free human immunodeficiency virus (CFHIV) with ascorbic acid (M) or Congo red (CR) was investigated with specific regard to the impact of an excess of magnesium ions on the viral inactivation. Quadruplicate reaction mixtures containing CFHIV were mixed with a virus-inactivating dose of 500 micrograms/ml ascorbic acid in RPMI medium devoid of fetal bovine serum and incubated for 3 h at 4 degrees C in two parallel sets of experiments. AA-free CFHIV and virion-free AA were included in each experiment as the positive and negative controls, respectively. After adding 10(6) MT2 cells to capture the surviving virons, the mixtures were incubated for 1 h at 37 degrees C. The cells from the first set were washed three times with Hanks balanced salt solution (HBSS) only, and those from the second set were washed with HBSS fortified with MgCl2 (1.0 mg/ml). Similarly, inactivation of CFHIV by increasing amounts of CR ranging between 12.5-100 micrograms/ml was also tested for the effect of MgCl2, except that (i) the assay was performed in subdued light, (ii) CFHIV-CR mixtures were incubated at 37 degrees C for 1 h in the dark and (iii) H9 cells were used instead of the MT-2 cells to capture the surviving virions in the test mixtures. The cells were cultured in RPMI with 20% FBS for 5 days at 37 degrees C. The absence of p24 antigen in the culture supernatant of MT2 or H9 cells indicated HIV inactivation by AA or CR, respectively. Remarkably, the cultured cells that were washed with HBSS + MgCl2 consistently expressed p24 antigen at levels comparable with those from the untreated virus control. Therefore, the apparent in vitro inactivation of CFHIV by either AA or CR was reversible as validated by washing of the cells with HBSS + MgCl2 following capture of the virions from CFHIV-AA or CFHIV-CR inactivation mixtures. These observations underscore the need for including extra magnesium ions as a control in validating various protocols used for assessing the in vitro virucidal activity of reverse transcriptase inhibitors, membrane binding dyes, or other candidate chemical agents.

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... This membrane-binding dye inactivates HIV in the presence of magnesium dichloride (Mg++ ions) only. This effect was found to be revesible as validated by washing of the cells by Hanks’ solution + MgCl2 following capture of the virions from cell-free HIV-Congo red inactivation mixture [18]. ...
... Vitamine C demonstrated a virucidal effect on HIV in the presence of Mg++ ions. Its virucidal properties are closed to those of Congo red [18]. ...
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Virucidal agents are chemical substances that attack and inactivate viral particles outside the cell (virions). In general this is accomplished by damaging their protein shells (capsid) or the substance penetrates the core itself, where it destroys the genetic material. Damage to the virion structure is also possible. These agents are used not only for traditional surface disinfection or sterilization of blood, blood products, and other medicinal products as well as in antiviral chemotherapy. They have also been used in recent times for inactivation of viruses in foodstuffs, detergents or cosmetics. Below is given an overview of the data currently available on the performance of these substances when used for the latter applications (cleaning and cosmetics). These include: hydrogen peroxide, hypochlorites, cupric and ferric ions, per-acids ethanol, parachlorometaxylenol in a sodium C14-16 olefin sulfonate, glutaraldehyde, quaternary ammonium salts, chlorhexidine and chlorhexidine gluconate, curdline sulphate, glycerol, lipids, azodicarbonamide, cicloxolone sodium, dichlorisocyanuric acid (sodium salt), benzalkonium salts, disulfate benzamides and benzisothiazolones, congo red, ascorbic acid, nonoxynol-9, para-aminobenzoic acid, bis(monosuccinamide) derivative of p,p’-bis(2-aminoethyl) diphenlyi-C60) (fullerene). merocyanine, benzoporphyrin derivative monoacid ring A, rose bengal, hypericin, hypocrellin A, anthraquinones extracted from plants, sulfonated anthraquinones and other anthraquinone derivatives gramicidine, gossypol, garlic (Allium sativum) extract and its components: ajoene, diallyl thiosulfinate (allicin), allyl methyl thioulfinate, methyl allyl thiosulfinate, extracts of ledium, motherworth, celandine, black currant, coaberry and bilberry, extract of Cordia salicifolia, steam distillate from Houttuynia cordata (Saururaceae) and its component, 5,6,7-trimethoxyflavone from Calicarpa japonica, isoscullarein (5,7,8,4’-tetrahydroxyflavone) from Scutellaria baikalensis and isoscutellarein-8-methylether, alkaloids and phytosteryl ester compounds.
... In addition , it was recorded that Albedo had the highest antioxidant activity reflecting its higher flavonoid and total phenolics content and results showed that Albedo was the main source of glycosylated flavanones and flavedo of 299 metoxylated flavones (Wang et al., 2008 andXu et al., 2008). Vitamin C as OAlb E content proved to exert virucidal activity as was proved by Abd elgaied, et al., Rawal, et al., (1996), who Institute of Environmental Studies and Research -Ain Shams University Vol.32, No.1, March, 2016 196 mentioned that Rabies and HIV viruses could be completely inactivated with ascorbic acid . Regarding the antiviral activity, present data recoded considered the use of HAV and RVF as viral models was in agreement with Su,& D'Souza, (2011), despite our use of a safe concentrations for both OAlbE and GSE were effective to reduce the infectivity titers of HAV in a dose-dependent manner. ...
... The dye also blocked neuraminidase activity of myxoviruses (Newcastle disease virus and fowl plaque virus), their multiplication and release from infected cells (Becht and Drzeniek, 1968). Finally, it was shown that CR inactivates human immunodeficiency virus (HIV) (Balzarini et al., 1986), although this effect was reversed by addition of magnesium (Rawal and Vyas, 1996). In conclusion, anti-HIV-1 effect of CR might be mediated by inhibition of reverse transcriptase, HIV-1 proteinase , and HIV replication (Mohan et al., 1990; Brinkworth and Fairlie, 1992). ...
Congo red is a commonly used histological dye for amyloid detection. The specificity of this staining results from Congo red's affinity for binding to fibril proteins enriched in beta-sheet conformation. Unexpectedly, recent investigations indicate that the dye also possesses the capacity to interfere with processes of protein misfolding and aggregation, stabilizing native protein monomers or partially folded intermediates, while reducing concentration of more toxic protein oligomers. Inhibitory effects of Congo red upon amyloid toxicity may also range from blockade of channel formation and interference with glycosaminoglycans binding or immune functions, to the modulation of gene expression. Particularly, Congo red exhibits ameliorative effect in models of neurodegenerative disorders, such as Alzheimer's, Parkinson's, Huntington's and prion diseases. Another interesting application of Congo red analogues is the development of imaging probes. Based on their small molecular size and penetrability through blood-brain barrier, Congo red congeners can be used for both antemortem and in vivo visualization and quantification of brain amyloids. Therefore, understanding mechanisms involved in dye-amyloidal fibril binding and inhibition of aggregation will provide instructive guides for the design of future compounds, potentially useful for monitoring and treating neurodegenerative diseases.
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The concept of reducing cell-associated blood-borne viruses (BBVs) by filtration of the vector leukocytes from blood collected for transfusion has led to the development of high efficiency filters. Improved filtration technology demands newer methodology to accurately estimate the residual cells. We have developed an experimental model based on the hemocytometer counts and the polymerase chain reaction (PCR), performed on the lymphocytes derived from the units of red cell mass inoculated with marker cells (H9) persistently carrying cell-associated human immunodeficiency virus DNA (CA-HIV). We measured the efficiency of 6 units of a prototype filter using our model and found an estimated mean of less than 4 residual cells per milliliter in the filtered blood. This represents a mean 5.84 log10 reduction of normal PBMC and CA-HIV in pre- and post-filtration aliquots and exemplifies the application of our model for evaluating a new generation of blood filters. Our model illustrates that a biological tracer (ie, DNA) is a better measure of the efficacy of a leukocyte filter than the hemocytometric enumeration of pre- and post-filtration PBMC concentrates.
Neutralised ascorbic acid is found to exert a strong bactericidal action on Pseudomonas aeruginosa suspended in isotonic phosphate buffer at pH 7.1. Both the bactericidal and bacteriostatic action of ascorbic acid are antagonised by magnesium ions. In the absence of complex formation between magnesium and ascorbic acid it is concluded that ascorbic acid acts by competing with the magnesium binding sites in the cell wall, cell membrane or ribosomes. Using the chequer-board titration method the synergistic action of ascorbic acid and erythromycin is determined; such a potentiation of erythromycin is also adversely affected by magnesium ions. P. aeruginosa cells, washed and suspended in isotonic phosphate buffer containing ascorbic acid, became increasingly susceptible to the action of polymyxin, erythromycin, chloramphenicol, neomycin and tetracycline. It is suggested that ascorbic acid alters the cell surface to render it increasingly permeable to these antibiotics.
Most chemicals with potential virucidal activity are extremely cytotoxic even at very small concentrations, thus introducing a number of technical problems and uncertainties in the evaluation of the net virucidal effect. In the present study, an attempt was made to confirm the reported virucidal activity of certain well-known chemicals and a number of new compounds were investigated. The results suggest that HIV inactivation is dependent on the viral concentration, the time of incubation in presence of the putative disinfectant and the degree of virucidal activity of the latter. The data illustrate methodological problems arising from residual cytotoxicity of the chemical which may mask or mimic the presence of a true virucidal activity and lead to erroneous conclusions. Alcohol, the most commonly used disinfectant, was found to be ineffective for high viral concentrations, whilst sodium hypochlorite was the most efficient.
In vitro inactivation of cell-free human immunodeficiency virus (CFHIV) was investigated by mixing replication-competent virions with aliquots of a culture medium (RPMI) containing increasing amounts (62.5-500 micrograms/ml) of ascorbic acid (AA) at pH7. Similarly, mixtures of CFHIV and 500 micrograms/ml AA in whole blood (WB) and leukocyte depleted blood (LDB) were made; control mixtures containing either CFHIV or AA alone in each experiment were included. After holding the mixtures for 3 h at 4 degrees C, the tubes containing WB and LDB mixtures were centrifuged to remove the blood cells. The respective supernatants, including the control aliquots, were layered over 0.5 x 10(6) MT2 cells in quadruplicate wells in microtitre plates. After 1 h of incubation at 37 degrees C in an atmosphere of 5.0% carbon dioxide to permit contact of viable virions, the fluid in each well was replaced with RPMI containing 20% fetal bovine serum (FBS). The incubation was then continued at 37 degrees C for 5 days. On the basis of (1) absence of syncytia formation, (2) 100% viability of MT2 cells as compared with the cell controls, (3) absence of p24 antigen in the culture supernates, and (4) absence of HIV DNA in MT2 cells, we conclude that 500 micrograms/ml AA, in (a) RPMI, (b) WB, or (c) LDB, inactivated CFHIV in vitro. Furthermore, we determined that addition of 500 micrograms/ml AA to platelet concentrates did not adversely affect the platelet function tests during 5 days of storage at room temperature. These data warrant further work to evaluate the mechanism of CFHIV inactivation by treatment of blood products with AA.
The mechanism of the antiviral activity of hypericin was characterized and compared with that of rose bengal. Both compounds inactivate enveloped (but not unenveloped) viruses upon illumination by visible light. Human immunodeficiency and vesicular stomatitis viruses were photodynamically inactivated by both dyes at nanomolar concentrations. Photodynamic inactivation of fusion (hemolysis) by vesicular stomatitis, influenza, and Sendai viruses was induced by both dyes under similar conditions (e.g., I50 = 20-50 nM for vesicular stomatitis virus), suggesting that loss of infectivity resulted from inactivation of fusion. Syncytium formation, between cells activated to express human immunodeficiency virus gp120 on their surfaces and CD4+ cells, was inhibited by illumination in the presence of 1 microM hypericin. Hypericin and rose bengal thus exert similar virucidal effects. Both presumably act by the same mechanism--namely, the inactivation of the viral fusion function by singlet oxygen produced upon illumination. The implications of this photodynamic antiviral action for the potential therapeutic usefulness of both hypericin and rose bengal are discussed.
Solvent/de-tergent-treated plasma: a virus-inactivated substitute for fresh plasma
  • B Horowitz
  • R Bonomo
  • Prince
  • Am
Horowitz B, Bonomo R, Prince AM et al. Solvent/de-tergent-treated plasma: a virus-inactivated substitute for fresh plasma. Blood 1992; 79: 826–831.