Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Ozone inactivation of Cryptosporidium parvum in demand-free phosphate buffer determined by in vitro excystation and animal infectivity.

Inactivation of Cryptosporidium parvum oocysts by ozone was performed in ozone demand-free 0.05 M phosphate buffer (pH 6.9) in bench-scale batch reactors at 7 and 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times ranging from 5 to 15 min. The viability of the control and treated oocysts was determined by using in vitro excystation and infection in neonatal CD-1 mice. It was found that excystation consistently underestimated inactivation when compared with animal infectivity (P < or = 0.05). As inactivations increased, the difference between excystation and infectivity also increased. The inactivation kinetics of C. parvum by ozone deviated from the simple first-order Chick-Watson model and was better described by a nonlinear Hom model. The use of the Hom model for predicting inactivation resulted in a family of unique concentration and time values for each inactivation level rather than the simple CT product of the Chick-Watson model.     

Measurement of the inactivation kinetics of poliovirus by ozone in a fast-flow mixer.

Inactivation kinetics of poliovirus type 1 in ozone demand-free water was investigated by utilizing a fast-flow mixing apparatus. Ozonated water and a solution of ozone demand-free water containing a known quantity of poliovirus type 1 were introduced simultaneously into a mixing chamber, both at a constant rate. This mixture was then passed through a narrow tube of known length and diameter into a neutralizing solution. By altering the rate of introduction and/or tube length, different contact periods between ozone and virus could be determined with an accuracy of 0.01 s. Inactivation of the poliovirus occurred in two steps. During the first step, which lasted for 0.2 to 1.0 s, 95 to 99% of the virus was inactivated, depending on the ozone concentration (which ranged from 0.1 to 2.0 mg/liter). The second step apparently continued for several minutes; in this period the remainder of the virus was inactivated. An obvious dose-response relationship was demonstrated during the first step of the inactivation curve. The pH of the water slightly affected the viral inactivation rate, but these small differences seem to have no practical value.     

Measurement of the inactivation kinetics of poliovirus by ozone in a fast-flow mixer.

Inactivation kinetics of poliovirus type 1 in ozone demand-free water was investigated by utilizing a fast-flow mixing apparatus. Ozonated water and a solution of ozone demand-free water containing a known quantity of poliovirus type 1 were introduced simultaneously into a mixing chamber, both at a constant rate. This mixture was then passed through a narrow tube of known length and diameter into a neutralizing solution. By altering the rate of introduction and/or tube length, different contact periods between ozone and virus could be determined with an accuracy of 0.01 s. Inactivation of the poliovirus occurred in two steps. During the first step, which lasted for 0.2 to 1.0 s, 95 to 99% of the virus was inactivated, depending on the ozone concentration (which ranged from 0.1 to 2.0 mg/liter). The second step apparently continued for several minutes; in this period the remainder of the virus was inactivated. An obvious dose-response relationship was demonstrated during the first step of the inactivation curve. The pH of the water slightly affected the viral inactivation rate, but these small differences seem to have no practical value.     

Comparative biochemical studies of type 3 poliovirus

A study of the biochemistry of type 3 poliovirus strains which involves the examination of the virus-coded polypeptides in infected cells and the preparation of oligonucleotide maps is reported. The polypeptide patterns were shown to be a relatively stable property of virus strains and distinguished Sabin vaccine strains from wild strains of poliovirus type 3. This approach may be of value in deciding the origin (vaccine or nonvaccine) of field isolates of poliovirus. Oligonucleotide maps were found to be sensitive indicators of differences among strains and appear to form a basis for determining genetic relationships among strains. The nucleotide maps of two viruses isolated from human cases of paralytic poliomyelitis temporally associated with the administration of attenuated vaccine suggested a vaccine origin for the strain. In one case the nucleotide map was indistinguishable from that of the vaccine strain.     

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.     

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.     

Inactivation of coliphage MS-2 and poliovirus by copper, silver, and chlorine.

The efficacy of electrolytically generated copper and silver ions (400 and 40 micrograms/L, respectively) was evaluated separately and in combination with free chlorine (0.2 and 0.3 mg/L) for the inactivation of coliphage MS-2 and poliovirus type 1 in water at pH 7.3. The inactivation rate was calculated as log10 reduction/min: k = -(log10 Ct/C0)/t. The inactivation of both viruses was at least 100 times slower in water containing 400 and 40 micrograms/L copper and silver, respectively (k = 0.023 and 0.0006 for MS-2 and poliovirus, respectively), compared with water containing 0.3 mg/L free chlorine (k = 4.88 and 0.036). Significant increases in the inactivation rates of both viruses were observed in test systems containing 400 and 40 micrograms/L copper and silver, respectively, with 0.3 mg/L free chlorine when compared with the water systems containing either metals or free chlorine alone. Poliovirus was approximately 10 times more resistant to the disinfectants than coliphage MS-2. This observation suggests either a synergistic or an additive effect between the metals and chlorine for inactivation of enteric viruses. Use of copper and silver ions in water systems currently used in swimming pools and spas may provide an alternative to high levels of chlorination.     

Hypochlorite inactivation kinetics of Listeria monocytogenes in phosphate buffer

The inactivation kinetics of Listeria monocytogenes in a phosphate buffer (PB) was determined at different hypochlorite concentrations, pH values and temperatures. D-values, using a linear regression, of L. monocytogenes in PB (pH 6.5) were 23.54, 17.40, 14.24 and 12.00s at 5, 10, 50 and 100 mg l(-1) hypochlorite, respectively, at 30 degrees C. The k-values ranged from 0.098 to 0.192s(-1) and 0.007 to 0.018s(-1) for hypochlorite concentrations (from 5 to 100 mg l(-1)) in PB (pH 6.5) and PB containing 0.1% peptone (pH 6.5), respectively, at 30 degrees C. D-values of L. monocytogenes exposed to hypochlorite were decreased with decreasing pH of PB (pH from 8.5 to 4.5). Hypochlorite showed higher antimicrobial activity at higher temperature. Not only the effect of hypochlorite concentration on the inactivation of L. monocytogenes but also other parameters like temperature, pH and suspending solutions effect the inactivation rates.     

Mechanism of poliovirus inactivation by ammonia.

Poliovirus inactivation by ammonia causes a slight reduction in the sedimentation coefficients of viral particles, but has no detectable effect on either the electrophoretic pattern of viral capsid proteins or the isoelectric points of inactivated particles. These virions still attach to cells, but are unable to repress host translation or stimulate the synthesis of detectable amounts of viral RNA. Although ammonia has no detectable effect on naked poliovirus RNA, it causes cleavage of this RNA when still within viral particles. Therefore, the RNA genome appears to be the only component of poliovirus significantly affected by ammonia.     

Mechanism of poliovirus inactivation by bromine chloride.

The mechanism of poliovirus inactivation by BrCl was determined by exposing poliovirus to various concentrations of BrCl and correlating the loss of virus infectivity with structural changes of the virus. Concentrations of 0.3 to 5 mg of BrCl per liter resulted in 95% to total inactivation of poliovirus. However, the inactivated virus retained structural integrity, as determined by buoyant density measurements of poliovirus labeled with radioactivity. However, at concentrations of 10 to 20 mg of BrCl per liter, total inactivation of poliovirus was associated with the degradation of the structural integrity of the virus. Since infectious ribonucleic acid at similar concentrations could be recovered from untreated poliovirus and poliovirus treated with 0.3 mg of BrCl per liter, it was concluded that BrCl as HOBr or bromamines inactivates poliovirus by reacting with the protein coat of the virus. Moreover, this inactivating reaction does not result in the degradation of the structure of the virion, nor does it affect the biological activity of the internal ribonucleic acid of the virus.     

Effect of the method of preparing monochloramine upon inactivation of MS2 coliphage, Escherichia coli, and Klebsiella pneumoniae.

Monochloramine prepared in situ by first adding chlorine to a suspension of microorganisms, followed by subsequent addition of ammonia, inactivated the MS2 coliphage more rapidly than did exposure of phage to monochloramine prepared either by adding chlorine to ammonia or by adding chlorine and ammonia simultaneously. The rapid viral inactivation was apparently due to the exposure of MS2 to free chlorine before the addition of ammonia. The average 99% CT value of MS2 when exposed to free chlorine was 1.3 and 1.1 at 5 and 15 degrees C, respectively. The average 99% CT values of MS2 briefly exposed to the combined action of free chlorine followed by the addition of ammonia to form monochloramine in situ were 19.3 and 1.5 at 5 and 15 degrees C, respectively. No 99% CT values were calculated for the inactivation of MS2 with preformed monochloramine because less than 1 log (90%) of inactivation occurred during a 4-h contact time. Inactivation of MS2 by monochloramine was more rapid at 15 than at 5 degrees C and when the chlorine to nitrogen weight ratio was 5:1 compared with 3:1. Monochloramine was a more efficient inactivating agent for the coliforms Escherichia coli and Klebsiella pneumoniae than it was for the MS2 coliphage.     

Comparative study on the inactivation of MS2 and M13 bacteriophages using energetic femtosecond lasers

Femtosecond (fs) laser irradiation techniques are emerging tools for inactivating viruses that do not involve ionizing radiation. In this work, the inactivation of two bacteriophages representing protective capsids with different geometric constraints, that is, the near‐spherical MS2 (with a diameter of 27 nm) and the filamentous M13 (with a length of 880 nm) is compared using energetic visible and near‐infrared fs laser pulses with various energies, pulse durations, and exposure times. Intriguingly, the results show that inactivation using 400 nm lasers is substantially more efficient for MS2 compared to M13. In contrast, using 800 nm lasers, M13 was slightly more efficiently inactivated. For both viruses, the genome was exposed to a harmful environment upon fs‐laser irradiation. However, in addition to the protection of the genome, the metastable capsids differ in many properties required for stepwise cell entry that may explain their dissimilar behavior after (partial) disassembly. For MS2, the dominant mechanism of fs‐laser inactivation was the aggregation of the viral capsid proteins, whereas aggregation did not affect M13 inactivation, suggesting that the dominant mechanism of M13 inactivation was related to breaking of secondary protein links.     

Mechanism of enteroviral inactivation by ozone.

The mechanism of enteroviral inactivation by ozone was investigated with poliovirus 1 (Mahoney) as the model virus. Ozone was observed to alter two of the four polypeptide chains present in the viral protein coat of poliovirus 1. However, the alteration of the protein coat did not significantly impair virus adsorption or alter the integrity of the virus particle. Damage to the viral RNA after exposure to ozone was demonstrated by velocity sedimentation analysis. It was concluded that the damage to the viral nucleic acid is the major cause of poliovirus 1 inactivation by ozone.     

Restricted fragmentation of poliovirus type 1, 2 and 3 RNAs by ribonuclease III.

Cleavage of the genome RNAs of poliovirus type 1, 2 and 3 with the ribonuclease III of Escherichia coli has been investigated with the following results: (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack “primary cleavage sites”; (2) lowering the salt concentration to 0.1 m or below allows RNase III to cleave the RNAs at “secondary sites”. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)- linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.     

Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics

Of thirteen ribosome-directed antibiotics surveyed for their inhibitory effect on viral protein synthesis in Escherichia coli infected with coliphage MS2, three antibiotics, kasugamycin, tetracycline and chloramphenicol, were found to exert differential inhibition, with synthesis of maturation protein being more sensitive than coat protein synthesis. Differential effects of kasugamycin and tetracycline were also observed in vitro. Such differential inhibition might reflect the presence of cistron-speciflc ribosomes or the induction of functional ribosomal heterogeneity by these antibiotic ligands.