Virucidal activity of cetyltrimethylammonium bromide below the critical micelle concentration

Abstract The virucidal activity of cetyltrimethylammonium bromide (CTAB) was investigated against a variety of different lipid-containing and non-lipid-containing bacterial viruses and 2 mammalian viruses. In all cases, the maximum virucidal effectiveness was obtained under conditions of low ionic strength and moderately basic pH. The virucidal effect is present well below the critical micelle concentration of CTAB, indicating that the initial interaction with viral surfaces is by monomers of CTAB and sodium dodecyl sulfate (SDS) exhibited no virucidal activity.     

pH modification of the effects of detergents on the stability of enteric viruses.

The effect of detergents on the stability of enteric viruses was found to be highly dependent on pH. This was demonstrated primarily with two ionic detergents, sodium dodecyl sulfate (an anionic detergent) and dodecyltrimethylammonium chloride (a cationic detergent). Both detergents were shown to be potent virucidal agents for reovirus, but the effects of sodium dodecyl sulfate were minimal near neutrality and much more pronounced at low than at high pH values. Dodecyltrimethylammonium chloride was extremely virucidal at high pH's but had little observable effect on reovirus stability at low pH values. In contrast, both detergents protected enteroviruses against heat at neutral and alkaline pH's. However, as was found with reovirus, sodium dodecyl sulfate was extremely virucidal at pH values below 5, even when the virus samples were incubated in ice. At different pH's the effects of detergents on the stabilities of coliphages T4, f1, and Q beta were qualitatively similar to those found with reovirus. Differences in viral stability in these experiments appeared to be due to the effects of pH on the ionic states of the viral capsid proteins.     

pH modification of the effects of detergents on the stability of enteric viruses.

The effect of detergents on the stability of enteric viruses was found to be highly dependent on pH. This was demonstrated primarily with two ionic detergents, sodium dodecyl sulfate (an anionic detergent) and dodecyltrimethylammonium chloride (a cationic detergent). Both detergents were shown to be potent virucidal agents for reovirus, but the effects of sodium dodecyl sulfate were minimal near neutrality and much more pronounced at low than at high pH values. Dodecyltrimethylammonium chloride was extremely virucidal at high pH's but had little observable effect on reovirus stability at low pH values. In contrast, both detergents protected enteroviruses against heat at neutral and alkaline pH's. However, as was found with reovirus, sodium dodecyl sulfate was extremely virucidal at pH values below 5, even when the virus samples were incubated in ice. At different pH's the effects of detergents on the stabilities of coliphages T4, f1, and Q beta were qualitatively similar to those found with reovirus. Differences in viral stability in these experiments appeared to be due to the effects of pH on the ionic states of the viral capsid proteins.     

Enzyme behaviour and molecular environment. The effects of ionic strength, detergents, linear polyanions and phospholipids on the pH profile of soluble cytochrome oxidase.

The activity vs. pH profile for the oxidation of ferrocytochrome c by purified cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was investigated as a function of ionic strength (from 10 to 200 mM) in the absence and in the presence of various perturbants: Tween 20, linear polyanions (RNA, heparin, polyglutamic acid) and phospholipids (asolectin, phosphatidylcholine, phosphatidic acid and cardiolipin). The activation induced by Tween 20 and "zero net charge" phospholipid liposomes was not pH dependent. On the other hand, linear polyanions and polyanionic liposomes strongly perturbed the pH profile, mostly at low ionic strength, by shifting the pH optimum about 1.7 pH units towards alkaline pH values. This effect was reversed by increasing ionic strength. These observations are interpreted in the light of polyelectrolyte theory. Since these results show striking with membrane-bound enzyme, it is concluded that in vivo cytochrome oxidase is located within polyanionic sites of the micochondrial membrane. The activation broght about by phospholipids may result from two posible processes: creation of a hydrophobic environment by the non-polar tails, preventing autoaggregation; and creation of a suitable polyelectrolytic environment by the polar heads (of non zero net charge), increasing the intrinsic reaction rate.     

Characterization of virucidal agents in activated sludge

A comprehensive study was carried out to determine the properties of agents responsible for loss of virus infectivity in mixed-liquor suspended solids (MLSS) of activated sludge. Initial experiments revealed that model enteric viruses (poliovirus-1 and rotavirus SA-11) were irreversibly inactivated in MLSS and released their RNA genomes. Enteric viruses belonging to other genera (echovirus-12, coxsackievirus A13, reovirus-3) were also shown to lose infectivity in MLSS. Although the virucidal activity decreased at reduced temperatures, MLSS still retained significant activity at 4 degrees C. The virucidal agents in MLSS were stable for months at 4 degrees C, but their activity decreased approximately 50% during 4 days of aeration at 26 degrees C. Primary effluent, the nutrient source for activated sludge, also contained virucidal activity. After centrifugation of MLSS, almost all virucidal activity was found in the particulate fraction because of inhibitory substances retained in the supernatant fraction. Decreasing or increasing the solids concentration of the particulate fraction did not increase the virucidal activity of the fraction. The effects of heat and antibiotics on the virucidal activity of MLSS, coupled with the finding that the activity can be produced in autoclaved primary effluent seeded with MLSS, strongly support the conclusion that microorganisms are responsible for this activity. Attempts to characterize the virucidal microbial components of MLSS indicated that treatments that resulted in the inactivation or removal of microorganisms also caused a loss of virucidal activity. Thus, it appears that the virucidal components of microorganisms are either short-lived or active only while bound to the organisms themselves.     

Characterization of virucidal agents in activated sludge.

A comprehensive study was carried out to determine the properties of agents responsible for loss of virus infectivity in mixed-liquor suspended solids (MLSS) of activated sludge. Initial experiments revealed that model enteric viruses (poliovirus-1 and rotavirus SA-11) were irreversibly inactivated in MLSS and released their RNA genomes. Enteric viruses belonging to other genera (echovirus-12, coxsackievirus A13, reovirus-3) were also shown to lose infectivity in MLSS. Although the virucidal activity decreased at reduced temperatures, MLSS still retained significant activity at 4 degrees C. The virucidal agents in MLSS were stable for months at 4 degrees C, but their activity decreased approximately 50% during 4 days of aeration at 26 degrees C. Primary effluent, the nutrient source for activated sludge, also contained virucidal activity. After centrifugation of MLSS, almost all virucidal activity was found in the particulate fraction because of inhibitory substances retained in the supernatant fraction. Decreasing or increasing the solids concentration of the particulate fraction did not increase the virucidal activity of the fraction. The effects of heat and antibiotics on the virucidal activity of MLSS, coupled with the finding that the activity can be produced in autoclaved primary effluent seeded with MLSS, strongly support the conclusion that microorganisms are responsible for this activity. Attempts to characterize the virucidal microbial components of MLSS indicated that treatments that resulted in the inactivation or removal of microorganisms also caused a loss of virucidal activity. Thus, it appears that the virucidal components of microorganisms are either short-lived or active only while bound to the organisms themselves.     

Identification of detergents as components of wastewater sludge that modify the thermal stability of reovirus and enteroviruses.

The agent in wastewater sludge previously shown to reduce the heat required to inactivate reovirus (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 34:681--688, 1977) was "separated" from other sludge components and analyzed by infrared spectroscopy. The infrared spectrum of this material was quite similar to the spectra of commercial anionic detergents, and subsequent analyses of the fractionated sludge samples revealed that anionic detergents in sludge were copurified with the virucidal activity. Further measurements on the virucidal activities of specific detergents revealed that ionic detergents reduce the heat required to inactivate reovirus, that cationic detergents are more active than anionic, and that nonionic detergents are inactive. Several detergents were also shown to protect poliovirus and other enteroviruses against inactivation by heat. These results indicate that ionic detergents are the major component in wastewater sludge that reduce the thermal stability of reovirus and, in addition, that detergents are able to protect enteroviruses against heat.     

Identification of detergents as components of wastewater sludge that modify the thermal stability of reovirus and enteroviruses.

The agent in wastewater sludge previously shown to reduce the heat required to inactivate reovirus (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 34:681--688, 1977) was "separated" from other sludge components and analyzed by infrared spectroscopy. The infrared spectrum of this material was quite similar to the spectra of commercial anionic detergents, and subsequent analyses of the fractionated sludge samples revealed that anionic detergents in sludge were copurified with the virucidal activity. Further measurements on the virucidal activities of specific detergents revealed that ionic detergents reduce the heat required to inactivate reovirus, that cationic detergents are more active than anionic, and that nonionic detergents are inactive. Several detergents were also shown to protect poliovirus and other enteroviruses against inactivation by heat. These results indicate that ionic detergents are the major component in wastewater sludge that reduce the thermal stability of reovirus and, in addition, that detergents are able to protect enteroviruses against heat.     

Differential Effects of Ionic Strength,Divalent Cations and pH on the Pore-forming Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Insecticidal Toxins

The combined effects of ionic strength, divalent cations, pH and toxin concentration on the pore-forming activity of Cry1Ac and Cry1Ca were studied using membrane potential measurements in isolated midguts of Manduca sexta and a brush border membrane vesicle osmotic swelling assay. The effects of ionic strength and divalent cations were more pronounced at pH 10.5 than at pH 7.5. At the higher pH, lowering ionic strength in isolated midguts enhanced Cry1Ac activity but decreased considerably that of Cry1Ca. In vesicles, Cry1Ac had a stronger pore-forming ability than Cry1Ca at a relatively low ionic strength. Increasing ionic strength, however, decreased the rate of pore formation of Cry1Ac relative to that of Cry1Ca. The activity of Cry1Ca, which was small at the higher pH, was greatly increased by adding calcium or by increasing ionic strength. EDTA inhibited Cry1Ac activity at pH 10.5, but not at pH 7.5, indicating that trace amounts of divalent cations are necessary for Cry1Ac activity at the higher pH. These results, which clearly demonstrate a strong effect of ionic strength, divalent cations and pH on the pore-forming activity of Cry1Ac and Cry1Ca, stress the importance of electrostatic interactions in the mechanism of pore formation by B. thuringiensis toxins.     

Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine.

Some of the kinetic properties of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) purified from hog lung have been determined using hippurylglycylglycine as substrate. The effects of pH and ionic environment on enzyme activity are complex and interdependent. At 0.1 M NaCl, the pH-activity curve shows an abrupt decrease in V/Km as the pH rises from 6 to 6.5, implying that ionization of a group in the enzyme with a pK in this range aids in binding of the substrate. Chloride is required for enzyme activity; there are two phases in the effect of NaCl. At both pH 6 AND 8, THE FIRST PHASE (UP TO 0.1 M NaCl) is activation. The second phase (above 0.1 M) at pH 6 is inhibition, while at pH 8 there is further activation which appears to be dependent upon ionic strength rather than a specific Cl-effect. Activation by cobalt and inhibition by EDTA are somewhat more effective at pH 6 than at pH 8. The nonapeptide inhibitor less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is nearly equipotent at both pH 6 and 8, but Arg-Pro-Pro is more inhibitory at pH 8 than at pH 6.     

Biological and molecular characteristics of the premalignant mouse mammary gland

We tested the ability of saturated n-monocarboxylic acids ranging from eight to 12 carbons in length to self-assemble into vesicles, and determined the minimal concentrations and chain lengths necessary to form stable bilayer membranes. Under defined conditions of pH and concentrations exceeding 150 mM, an unbranched monocarboxylic acid as short as eight carbons in length (n-octanoic acid) assembled into vesicular structures. Nonanoic acid (85 mM) formed stable vesicles at pH 7.0, the pK of the acid in bilayers, and was chosen for further testing. At pH 6 and below, the vesicles were unstable and the acid was present as droplets. At pH ranges of 8 and above clear solutions of micelles formed. However, addition of small amounts of an alcohol (nonanol) markedly stabilized the bilayers, and vesicles were present at significantly lower concentrations (approximately 20 mM) at pH ranges up to 11. The formation of vesicles near the pK(a) of the acids can be explained by the formation of stable RCOO(-)...HOOCR hydrogen bond networks in the presence of both ionized and neutral acid functions. Similarly, the effects of alcohols at high pH suggests the formation of stable RCOO(-)...HOR hydrogen bond networks when neutral RCOOH groups are absent. The vesicles provided a selective permeability barrier, as indicated by osmotic activity and ionic dye capture, and could encapsulate macromolecules such as DNA and a protein. When catalase was encapsulated in vesicles of decanoic acid and decanol, the enzyme was protected from degradation by protease, and could act as a catalyst for its substrate, hydrogen peroxide, which readily diffused across the membrane. We conclude that membranous vesicles produced by mixed short chain monocarboxylic acids and alcohols are useful models for testing the limits of stabilizing hydrophobic effects in membranes and for prebiotic membrane formation.     

KCl potentiation of the virucidal effectiveness of free chlorine at pH 9.0.

In studies at 5 degrees C and pH 9.0, poliovirus 1 was inactivated about 15 times more rapidly by free chlorine (FC) in purified water in the presence of 1,262 mg of KCl per liter (approximately 0.0169 M) than in the absence of KCl. In the presence of 526 mg of KCl per liter, the virus was inactivated about seven times more rapidly by FC than in the absence of KCl. At a level of 21 mg/liter, KCl did not significantly potentiate the virucidal activity of FC in purified water. Although poliovirus 1 was inactivated almost three times more rapidly by FC in borate-buffered purified water than in purified water, the presence of the buffer did not alter the extent of potentiation by KCl. Most of FC exists as OCl- at pH 9.0. Tap water has been shown to markedly potentiate the polivirucidal effectiveness of FC at pH 9.0. For the same degree of virucidal potentiation of FC at this pH, a considerably greater quantity of KCl was required in purified water than the total salt content that appeared to be present in the tap water.     

Inactivation of human and simian rotaviruses by chlorine dioxide

The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.     

Inactivation of human and simian rotaviruses by chlorine dioxide.

The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.     

Virucidal Effect of Cold Atmospheric Gaseous Plasma on Feline Calicivirus,a Surrogate for Human Norovirus

Minimal food-processing methods are not effective against foodborne viruses, such as human norovirus (NV). It is important, therefore, to explore novel nonthermal technologies for decontamination of foods eaten fresh, minimally processed and ready-to-eat foods, and food contact surfaces. We studied the in vitro virucidal activity of cold atmospheric gaseous plasma (CGP) against feline calicivirus (FCV), a surrogate of NV. Factors affecting the virucidal activity of CGP (a so-called radio frequency atmospheric pressure plasma jet) were the plasma generation power, the exposure time and distance, the plasma feed gas mixture, and the virus suspension medium. Exposure to 2.5-W argon (Ar) plasma caused a 5.55 log₁₀ unit reduction in the FCV titer within 120 s. The reduction in the virus titer increased with increasing exposure time and decreasing exposure distance. Of the four plasma gas mixtures studied (Ar, Ar plus 1% O₂, Ar plus 1% dry air, and Ar plus 0.27% water), Ar plus 1% O₂ plasma treatment had the highest virucidal effect: more than 6.0 log₁₀ units of the virus after 15 s of exposure. The lowest virus reduction was observed with Ar plus 0.27% water plasma treatment (5 log₁₀ unit reduction after 120 s). The highest reduction in titer was observed when the virus was suspended in distilled water. Changes in temperature and pH and formation of H₂O₂ were not responsible for the virucidal effect of plasma. The oxidation of viral capsid proteins by plasma-produced reactive oxygen and nitrogen species in the solution was thought to be responsible for the virucidal effect. In conclusion, CGP exhibits virucidal activity in vitro and has the potential to combat viral contamination in foods and on food preparation surfaces.     

Identification of the virucidal agent in wastewater sludge.

Anaerobically digested sludge contains an agent that causes irreversible inactivation of poliovirus. It has now been shown that the agent responsible for this activity is ammonia. The effect of ammonia on poliovirus appears to be typical for picornaviruses, but reovirus, an enteric virus of another group, is quite resistant to this compound. Because ammonia is not virucidal in its charged state, it expresses significant activity only at pH values greater than 8. Therefore, increasing the pH of sludge should cause rapid inactivation of indigenous picornaviruses.     

Identification of the virucidal agent in wastewater sludge.

Anaerobically digested sludge contains an agent that causes irreversible inactivation of poliovirus. It has now been shown that the agent responsible for this activity is ammonia. The effect of ammonia on poliovirus appears to be typical for picornaviruses, but reovirus, an enteric virus of another group, is quite resistant to this compound. Because ammonia is not virucidal in its charged state, it expresses significant activity only at pH values greater than 8. Therefore, increasing the pH of sludge should cause rapid inactivation of indigenous picornaviruses.     

Influence of ionic liquid cosolvent on transgalactosylation reactions catalyzed by thermostable beta-glycosylhydrolase CelB from Pyrococcus Furiosus

The synthesis of glycosides by enzymatic transglycosylation is a kinetically controlled reaction performed in the context of a non-favorable thermodynamic equilibrium. An unreactive organic cosolvent which increases the selectivity of the enzyme for glycosyl transfer to the acceptor nucleophile compared with water (Ksel) could improve maximum product yield. Here we report on the effect of the ionic liquid 1,3-dimethylimidazoliummethylsulfate on hydrolase and transferase activities of the hyperthermostable beta-glycosidase CelB from the archaeon Pyrococcus furiosus. CelB retained full catalytic efficiency for lactose hydrolysis at 80 degrees C in a 50% (by vol.) solution of ionic liquid in sodium citrate buffer, pH 5.5. It was inactive but not irreversibly denatured at 70% ionic liquid. Using lactose (0.15 M) as galactosyl donor, values of Ksel for a representative series of eight acceptor alcohols were determined in kinetic assays at 80 degrees C and found to increase between 1.3-fold (D-xylose) and 3.1-fold (glycerol) in 45% ionic liquid. Enhancement of Ksel was dependent on ionic liquid concentration and higher than expected from the decrease in water activity caused by the cosolvent. Experimental molar ratios of D-glucose and D-galactose produced during enzymatic conversion of lactose (75-150 mM) in the presence of D-xylose (0.5 M) or glycerol (0.5 M) showed excellent agreement with predictions based on Ksel values and confirm a significant, yet moderate effect of 45% ionic liquid on increasing the yield of D-galactoside product, by < or = 10%.     

Effect of alpha2 macroglobulin on some kinetic parameters of trypsin.

1. Complex formation of trypsin with alpha2 macroglobulin results in marked changes of the Michaelis-Menten constant, pH optimum and sensitivity to ionic strength in a system using N-carbobenzoxy-glycylglycyl-L-arginine-2-naphthylamide as substrate. 2. In contrasts to the inhibition (50%) observed when alpha2 macroglobulin-bound trypsin is assayed under conditions optimal for the free enzyme, there is minimal reduction of activity when determinations are performed at a substrate concentration and pH optimal for the bound enzyme. 3. The changes in substrate concentration and ionic environment required for maximum activity of alpha2 macroglobulin-bound trypsin are similar to those observed with enzymes embedded in polyelectrolyte matrices and may reflect alterations in the microenvironment of the enzyme resulting from conformational changes of the macromolecule during interaction with trypsin. 4. Enzymatic activity of trypsin towards casein is greatly reduced by alpha2 macroglobulin, even under assay conditions optimal for the bound enzyme, confirming previous findings that access to the active center for high-molecular weight substrates is sterically hindered by alpha2 macroglobulin.     

Virucidal activity of the new disinfectant monopercitric acid

AIMS: The virucidal efficacy of monopercitric acid (MPCA) was evaluated against the enveloped vaccinia virus as well as the nonenveloped adenovirus type 2 and poliovirus type 1. The results were compared with that obtained with peracetic acid (PAA). METHODS AND RESULTS: In the virucidal suspension test without and with protein burden, all viruses were inactivated by 0.5% MPCA within 0.5 min or by 0.1% MPCA within 5 min as measured by a >10(4)-fold reduction in virus titres. For MPCA, there was a better virucidal efficacy than for PAA which inactivated all viruses included in the test within 15-30 min at a concentration of 0.2%. SIGNIFICANCE AND IMPACT OF THE STUDY: The high virucidal activity, short exposure times, and nontoxic by-products seem to make MPCA suitable as disinfectant for medical use and should warrant further investigation.