Wallace Snipes

Extreme Sensitivity of Enveloped Viruses, Including Herpes Simplex, to Long-Chain Unsaturated Monoglycerides and Alcohols

Unsaturated monoglycerides and alcohols of chain lengths of 16 or 18 carbons were found to be extremely potent inactivators of two enveloped viruses, herpes simplex virus type 2 and bacteriophage φ6. The lipid-containing bacteriophage PM2 was also inactivated by some of these amphiphilic molecules. Treatment of herpes simplex virus type 2 with these compounds at concentrations as low as 0.2 μM reduced virus survival to 50% in 30 min, making these agents the most potent inactivators of herpes simplex viruses discovered that are not cytotoxic to mammalian cells. Detailed characterizations of the effects of unsaturated monoglycerides and alcohols on bacteriophages φ6 and PM2 showed that the inactivated φ6 virion remained nearly intact but that PM2 was almost completely disrupted by the inactivating treatment. Some of the compounds inactivate the viruses even at low temperature (0°C). Excess amounts of diglycerides and phospholipids interfere with the inactivating abilities of some of the unsaturated monoglycerides and alcohols against φ6 and PM2. Our findings suggest that the unsaturated monoglycerides and some of the unsaturated alcohols should be further studied as potential antiviral agents, particularly for application to herpesvirus-infected areas of the skin and accessible epithelium.

Inactivation of the Enveloped Bacteriophage ø6 by Butylated Hydroxytoluene and Butylated Hydroxyanisole

Butylated hydroxytoluene (BHT) is a potent inactivator of the enveloped bacterial virus ø6 at concentrations as low as 3 × 10−5 M. The viral envelope is not removed by BHT treatment, in contrast to the effects of exposure to the detergent Triton X-100. BHT-treated viruses are morphologically indistinguishable from controls but are defective in their ability to attach to the host cell. Temperature at the time of exposure was found to be a crucial factor in the effectiveness of BHT against ø6. A precipitous drop in the degree of inactivation by 3 × 10−5 M BHT occurred when the temperature was lowered from 20 to 15 C. Calcium ions were found to potentiate the effect of BHT, particularly at lower temperatures where BHT alone was relatively ineffective. Barium and strontium, but not magnesium, were also effective in enhancing the activity of BHT. A structurally related molecule, butylated hydroxyanisole (BHA), was also found to inactivate ø6 virus, but higher concentrations were required than with BHT. Both BHT and BHA are commonly used as food additives, have apparent low toxicity to humans and other animals, and are potentially useful as antiviral agents.

Electron Spin Resonance Analysis of the Nitroxide Spin Label 2,2,6,6-Tetramethylpiperidone-N-Oxyl (Tempone) in Single Crystals of the Reduced Tempone Matrix

The nitroxide spin label Tempone (2,2,6,6-tetramethylpiperidone-N-oxyl) can be reduced with ascorbic acid to give a nonparamagnetic species. Single crystals of reduced Tempone serve as a suitable host matrix to orient trace quantities of Tempone for ESR analysis. In these crystals the majority of the Tempone molecules are well-oriented, but a smaller fraction of the molecules tumble freely to give an isotropic electron spin resonance (ESR) spectrum. ESR transitions for the oriented molecules are saturated at much lower microwave power levels than for the tumbling molecules. For the oriented molecules, an analysis of the anisotropy of the spectroscopic splitting factor (g) gives principal values of g(1) = 2.0094, g(2) = 2.0061, g(3) = 2.0021. The hyperfine coupling tensor is nearly axially symmetric, with principal values (in gauss) of A(1) = 6.5, A(2) = 6.7, A(3) = 33.0. Within experimental error, the principal axis systems for the g tensor and the hyperfine tensor are identical. Comparison of the average values of g and A with the isotropic values of these parameters for Tempone in solvents of different polarity suggests a method for choosing the most appropriate tensor elements to be used for spin label experiments in various solvent systems.

Radiation Damage to Artemia Cysts:Effects of Water Vapor.

Water vapor altered the form and greatly increased the rate of decay of the electron-spin resonance pattern of long-lived free radicals obtained upon gamma irradiation of Artemia salina cysts ( brine shrimp eggs). These results, combined with data on radiation survival, indicate that the water vapor protects the cysts from radiation damage, or heals the damage. They also indicate that water protects the cysts from the effect of oxygen by neutralizing the radiation-induced free radicals before they can interact with oxygen to produce irreversible damage.

Effect of Isoniazid on the Protoplasmic Viscosity in Mycobacterium tuberculosis

The effect of isoniazid on the protoplasmic viscosity in the H37Ra strain of Mycobacterium tuberculosis was determined by using electron spin resonance spectroscopy and a small spin label tempone (2,2,6,6-tetramethylpiperidone-N-oxyl radical). Isoniazid (0.5 μg/ml) caused the internal cellular viscosity to increase gradually over the first 15 h of exposure from a rotational correlation time value (Tc) of 2.4 × 10−10 to 3.4 × 1010 s and then decrease linearly to the control level after 27 h. These results could be interpreted to mean that isoniazid allows a continued and normal synthesis of the protoplasmic components while the rate of increase in the cell volume is reduced. A degradative process may begin after the initial 15-h exposure time, which would cause the reduction in the internal viscosity.

Inactivation of the Lipid-Containing Bacteriophage PM2 by Butylated Hydroxytoluene

Several factors have been investigated which are of significance in the inactivation of PM2, a lipid-containing bacterial virus, by butylated hydroxytoluene (BHT). Studies of the time dependence of inactivation during exposure to BHT showed that virus killing occurs rapidly, with the majority of the effect taking place in the first 5 min. The degree of inactivation is dependent upon the initial virus titer, the solvent from which BHT is added, and the presence of a variety of protective agents, including surfactants, bovine serum albumin, and bacterial cells. Sucrose gradient analysis of 32P-labeled, BHT-treated virus was used to determine the degree to which the virion is disrupted by BHT. These experiments show that the 32P-labeled molecules are converted into very slowly sedimentable material by BHT treatment, indicating complete destruction of the virus particle. Images

Temperature sensitivity of the assembly process of the enveloped bacteriophage ϕ6

Abstract 1. 1. Bacteriophage ϕ6 can produce plaques at temperatures up to about 30 °C. Above this temperature (31 or 32 °C), infected cells do not lyse. Temperature shift and pulse experiments ( 25 → 31 ° C , 31 → 25 ° C, and 31 → 25 → 31 ° C ) indicate that this temperature sensitivity for production of infectious virus particles occurs very late in infection and is reversible. 2. 2. The use of spin-labeled hydrocarbons to probe the membranes of host cells and virus indicates the occurrence of a “phase transition” in both cellular and viral mambrances at about 30 °C. 3. 3. The phospholipid composition of infected cultures (at 25 or 31 °C) very late in infection is intermediate between that of uninfected cells and purified virus. 4. 4. These results are discussed in relation to the assembly of ϕ6 membrane components.

The antiviral effectiveness of butylated hydroxytoluene on herpes cutaneous infections in hairless mice

Abstract Hairless mice, cutaneously infected with herpes simplex virus type 1 (HSV-1), were treated topically with butylated hydroxytoluene (BHT). The effectiveness of BHT in shortening the duration of infections was assayed under three conditions. In the first experiments, mice undergoing primary infections with no prior immunity to HSV-1 were utilized. These animals tended to develop deep lesions that were not typical of recurrent HSV-1 infections in humans. A second set of experiments utilized mice that had recovered from a primary infection and that were immunosuppressed by γ irradiation. Immunosuppression was essential for the full development of lesions upon reinfection. The lesions in these animals remained more localized with less tendency to spread into deep tissues. A third set of experiments utilized animals that were subcutaneously inoculated with human serum γ-globulin 24 hr prior to infection. Lesions on these animals also remained localized and did not penetrate into deep tissues. Under all three conditions, BHT was found to be effective in reducing the clearance time of HSV-1 cutaneous lesions when applied topically to the infected area.