Article

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

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Abstract

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). ...
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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.