Evaluation of a Virucidal Quantitative Carrier Test for Surface Disinfectants

Surface disinfectants are part of broader preventive strategies preventing the transmission of bacteria, fungi and viruses in medical institutions. To evaluate their virucidal efficacy, these products must be tested with appropriate model viruses with different physico-chemical properties under conditions representing practical application in hospitals.The aim of this study was to evaluate a quantitative carrier assay. Furthermore, different putative model viruses like adenovirus type 5 (AdV-5) and different animal parvoviruses were evaluated with respect to their tenacity and practicability in laboratory handling. To evaluate the robustness of the method, some of the viruses were tested in parallel in different laboratories in a multi-center study. Different biocides, which are common active ingredients of surface disinfectants, were used in the test. After drying on stainless steel discs as the carrier, model viruses were exposed to different concentrations of three alcohols, peracetic acid (PAA) or glutaraldehyde (GDA), with a fixed exposure time of 5 minutes. Residual virus was determined after treatment by endpoint titration.All parvoviruses exhibited a similar stability with respect to GDA, while AdV-5 was more susceptible. For PAA, the porcine parvovirus was more sensitive than the other parvoviruses, and again, AdV-5 presented a higher susceptibility than the parvoviruses. All parvoviruses were resistant to alcohols, while AdV-5 was only stable when treated with 2-propanol. The analysis of the results of the multi-center study showed a high reproducibility of this test system.In conclusion, two viruses with different physico-chemical properties can be recommended as appropriate model viruses for the evaluation of the virucidal efficacy of surface disinfectants: AdV-5, which has a high clinical impact, and murine parvovirus (MVM) with the highest practicability among the parvoviruses tested.     

Surface-Dried Viruses Can Resist Glucoprotamin-Based Disinfection

Touching of contaminated objects and surfaces is a well-known method of virus transmission. Once they are attached to the hands, viruses can easily get adsorbed and initiate infection. Hence, disinfection of frequently touched surfaces is of major importance to prevent virus spreading. Here we studied the antiviral activity of a glucoprotamin-containing disinfectant against influenza A virus and the model virus vaccinia virus (VACV) dried on inanimate surfaces. The efficacy of the surface disinfectant on stainless steel, polyvinyl chloride, and glass coupons was investigated in a quantitative carrier test. Vacuum-dried viruses were exposed to 0.25%, 0.5%, and 1% disinfectant for 5 min, 15 min, and 30 min without agitation, and residual infectivity was determined by endpoint titration. Although glucoprotamin was highly active against both viruses in suspension, limited antiviral activity against the surface-dried viruses was detected. Even after 30 min of exposure to 1% disinfectant, VACV was not completely inactivated. Furthermore, influenza A virus inactivation was strongly affected by the surface composition during the 5-min and 15-min treatments with 0.25% and 0.5% disinfectant. The results presented in this study highlight the relevance of practical tests to assess the antiviral activity of surface disinfectants. High virucidal activity in solution is not necessarily indicative of high antiviral activity against surface-dried viruses. In addition, we want to emphasize that the mere exposure of surfaces to disinfectants might not be sufficient for virus inactivation and mechanical action should be applied to bring attached viruses into contact with virucidal compounds.     

No influence of collagenous proteins of Achilles tendon, skin and cartilage on the virus-inactivating efficacy of peracetic acid–ethanol

The risk of transmitting human pathogenic viruses via allogeneic musculoskeletal tissue transplants is a problem requiring effective inactivation procedures. Virus safety of bone transplants was achieved using peracetic acid (PAA)-ethanol sterilisation. Proteins are known to have an adverse effect on the virus-inactivating capacity of PAA. Therefore we investigated virus inactivation by PAA in collagenous tissues. Achilles tendon, skin and cartilage were cut into small pieces, lyophilised and contaminated with pseudorabies virus (PRV) or porcine parvovirus (PPV). The inactivating capacity of PAA-ethanol was investigated by determining virus titres in the supernatant or the tissue pellet at different time-points. In all virus-contaminated tissue samples treatment for 10 min with PAA-ethanol resulted in titre reductions by a factor of >10(3). PRV was rapidly inactivated below the detection limit (< or =2.8 x 10(1) TCID(50)/ml). After 240 min a reduction by a factor of >10(4) was obtained for PPV in all samples, but a residual infectivity remained. Collagenous proteins of Achilles tendon, skin and cartilage had no adverse effect on the virus-inactivating capacity of PAA. PAA-ethanol used in the production process at the Charité tissue bank can therefore be recommended for treatment of non-osseous musculoskeletal tissues.     

Virus safety of avital bone tissue transplants: evaluation of sterilization steps of spongiosa cuboids using a peracetic acid-methanol mixture.

The aim of this study was to validate the virus-inactivating/eliminating capacity of the manufacturing process of spongiosa cuboids. Both the sterilization step with peracetic acid (PAA)/ethanol and the defatting step of bones with chloroform/methanol (2:1, v/v) were investigated. Relevant enveloped, non-enveloped, and model viruses belonging to different virus families were included in the investigation: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), poliovirus (PV-1), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine virus diarrhoea virus (BVDV). Treatment of virus-spiked spongiosa cuboids for 4 hours at room temperature (RT) with 1% PAA/24% ethanol (PES) efficiently inactivated most viruses. Titres were reduced by more than 4 log(10)with the exception of HAV. The defatting step with chloroform/methanol reduced HAV titres by a factor of >/=7.0 log(10). From these results it can be concluded that the treatment of spongiosa cuboids with (i) chloroform/methanol and (ii) 1% PAA/24% ethanol solution leads to a virus-safe medicinal product.     

Virucidal effect of sodium p-chloromercuribenzoate on influenza viruses attributable to inhibition of virus particle-associated RNA-dependent RNA polymerase.

Sodium p-chloromercuribenzoate (PCMB) caused a noticeable reduction of infectivity of prototype strains of type A and Lee strain of type B influenza viruses at concentrations of 100 and 200 mug/ml, respectively, after an incubation at 37 C for 60 min. The virucidal effect on A/AA/2/60 (H2N2) strain was dependent on the concentration of the drug and temperature as well as on the time of incubation. The reagent exerted this effect at a concentration which induced little change in the hemagglutinating and neuraminidase activities of the virus. PCMB inhibited by 50% the virus particle-associated RNA polymerase activity of all prototype strains of type A influenza virus at about 2 mug/ml and that of Lee strain of type B influenza virus at 8.5 mug/ml. Other sulfhydryl reagent such as phenylmercuric nitrate also exhibited virucidal effect on A/AA/2/60 virus which paralleled their inhibition of the virus particle-associated RNA polymerase activity. From these results it was considered likely that the virucidal action of PCMB on influenza viruses was attributable to inhibition of the virus particle-associated RNA polymerase activity.     

Virucidal Effect of Sodium p-Chloromercuribenzoate on Influenza Viruses Attributable to Inhibition of Virus Particle-Associated RNA-Dependent RNA Polymerase

Sodium p-chloromercuribenzoate (PCMB) caused a noticeable reduction of infectivity of prototype strains of type A and Lee strain of type B influenza viruses at concentrations of 100 and 200 μg/ml, respectively, after an incubation at 37 C for 60 min. The virucidal effect on A/AA/2/60 (H₂N₂) strain was dependent on the concentration of the drug and temperature as well as on the time of incubation. The reagent exerted this effect at a concentration which induced little change in the hemagglutinating and neuraminidase activities of the virus. PCMB inhibited by 50% the virus particle-associated RNA polymerase activity of all prototype strains of type A influenza virus at about 2 μg/ml and that of Lee strain of type B influenza virus at 8.5 μg/ml. Other sulfhydryl reagent such as phenylmercuric nitrate also exhibited virucidal effect on A/AA/2/60 virus which paralleled their inhibition of the virus particle-associated RNA polymerase activity. From these results it was considered likely that the virucidal action of PCMB on influenza viruses was attributable to inhibition of the virus particle-associated RNA polymerase activity.     

Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat.

To determine the efficacy of a clean-in-place system for the inactivation of viruses present in human plasma, the effect of 0.1 M sodium hydroxide at 60 degrees C on viral infectivity was investigated. Inactivation of the following model and relevant viruses were followed as a function of time: human hepatitis A virus (HAV), canine parvovirus (CPV; a model for human parvovirus B-19) pseudorabies virus (PRV, a model for hepatitis B virus), and bovine viral diarrhoea virus (BVDV, a model for hepatitis C virus and human immunodeficiency virus). Infectivity of CPV was determined by a novel in situ EIA method which will prove useful for studies to validate parvovirus inactivation or removal. Infectivity of BVDV, PRV and CPV were shown to be reproducibly inactivated below the limit of detection by 0.1 M NaOH at 60 degrees C within 30 s. HAV was inactivated to below the limit of detection within 2 min. Treatment with heat alone also resulted in some log reduction for all viruses tested except for CPV which remained unaffected after heating at 60 degrees C for 16 min. Treatment of HAV with hydroxide alone (up to 1.0 m) at 15 degrees C did not lead to rapid inactivation. Collectively, these data suggest that 0.1 M NaOH at 60 degrees C for two min should be sufficient to inactivate viruses present in process residues.     

Virus safety of a porcine-derived medical device: evaluation of a viral inactivation method

The goal of this study was to evaluate the efficacy of a virus-inactivating process for use during the preparation of porcine-derived extracellular matrix biomaterials for human clinical implantation. Porcine small intestine, the source material for the tissue-engineered, small intestinal submucosa (SIS) biomaterial, was evaluated. Relevant enveloped, non-enveloped, and model viruses representative of different virus families were included in the investigation: porcine parvovirus (PPV), porcine reovirus, murine leukemia retrovirus (LRV), and porcine pseudorabies (herpes) virus (PRV). Samples of small intestine were deliberately inoculated with approximately 1 x 10(7) plaque-forming units (PFU) of virus which were thereafter exposed to a 0.18% peracetic acid/4.8% aqueous ethanol mixture for time periods ranging from 5 minutes to 2 hours. Enveloped viruses were more easily inactivated than non-enveloped viruses, but material processed for 30 minutes or longer inactivated all of the viruses. D(10) values were calculated and used to extrapolate the extent of inactivation after 2 hours. Viral titers were reduced by more than 14.0 log(10) PPV, 21.0 log(10) reovirus, 40.0 log(10) PRV, and 27.0 log(10) LRV, meeting international standards for viral sterility. These results demonstrate that treatment of porcine small intestine with a peracetic acid/ethanol solution leads to a virus-free, non-crosslinked biomaterial safe for xenotransplantation into humans.     

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.     

Mechanism of inactivation of enteric viruses in fresh water.

Fresh water obtained from nine sources was shown to cause inactivation of poliovirus. Further testing with four of these water samples showed that enteric viruses from different genera were consistently inactivated in these freshwater samples. Studies on the cause of inactivation were conducted with echovirus type 12 as the model virus. The results revealed that the virucidal agents in the waters tested could not be separated from microorganisms. Any treatment that removed or inactivated microorganisms caused loss of virucidal activity. Microbial growth in a sterilized creek water seeded with a small amount of stream water resulted in concomitant production of virucidal activity. When individual bacterial isolates obtained from a stream were grown in this sterilized creek water, most (22 of 27) produced a large amount of virucidal activity, although the amount varied from one isolate to the next. Active and inactive isolates were represented by both gram-positive and gram-negative organisms. Examination of echoviruses inactivated in stream water revealed that loss of infectivity first correlated with a slight decrease in the sedimentation coefficient of virus particles. The cause appeared to be cleavage of viral proteins, most notably, VP-4 and, to a lesser extent, VP-1. Viral RNA associated with particles was also cleaved but the rate was slower than loss of infectivity. These results suggest that proteolytic bacterial enzymes inactivate echovirus particles in fresh water by cleavage of viral proteins, thus exposing the viral RNA to nuclease digestion.     

Mechanism of inactivation of enteric viruses in fresh water

Fresh water obtained from nine sources was shown to cause inactivation of poliovirus. Further testing with four of these water samples showed that enteric viruses from different genera were consistently inactivated in these freshwater samples. Studies on the cause of inactivation were conducted with echovirus type 12 as the model virus. The results revealed that the virucidal agents in the waters tested could not be separated from microorganisms. Any treatment that removed or inactivated microorganisms caused loss of virucidal activity. Microbial growth in a sterilized creek water seeded with a small amount of stream water resulted in concomitant production of virucidal activity. When individual bacterial isolates obtained from a stream were grown in this sterilized creek water, most (22 of 27) produced a large amount of virucidal activity, although the amount varied from one isolate to the next. Active and inactive isolates were represented by both gram-positive and gram-negative organisms. Examination of echoviruses inactivated in stream water revealed that loss of infectivity first correlated with a slight decrease in the sedimentation coefficient of virus particles. The cause appeared to be cleavage of viral proteins, most notably, VP-4 and, to a lesser extent, VP-1. Viral RNA associated with particles was also cleaved but the rate was slower than loss of infectivity. These results suggest that proteolytic bacterial enzymes inactivate echovirus particles in fresh water by cleavage of viral proteins, thus exposing the viral RNA to nuclease digestion.     

The inactivation of foot and mouth disease, Aujeszky's disease and classical swine fever viruses in pig slurry

The aim of the study was to investigate the decontamination of pig slurry containing exotic viruses of pigs, foot AND mouth disease virus (FMDV), Aujeszky's disease virus (ADV) AND classical swine fever virus (CSFV). Laboratory-scale decontamination experiments showed that FMDV, ADV and CSFV were heat inactivated in slurry within 3 min at 67 degrees C, 3 min at 62 degrees C and 3 min at 60 degrees C and in Glasgow Eagles medium within 5 min at 67 degrees C, 4 min at 65 degrees C and 2 min at 65 degrees C, respectively. At pilot scale, FMDV was heat inactivated at 66 degrees C in water and 61 degrees C in slurry, ADV at 61 degrees C in water or slurry and CSFV at 62 degrees C in water and 50 degrees C in slurry. Treatment of pig slurry for the inactivation of exotic viruses may be achieved through the use of a thermal pilot plant operating in continuous mode. The work demonstrates the suitability of thermal treatment in ensuring the safety of pig slurry following a disease outbreak.     

Elimination of Mycoplasma Contaminants from Virus Stocks by Treatment with Nonionic Detergents

Five nonionic detergents (Tweens 20, 40, 60, and 80, and Triton WR-1339) were tested for their ability to inactivate four Mycoplasma species which are common contaminants of animal cell cultures. Tween 20 was found to be the most effective, in that a concentration of 2.5 mg/ml completely inactivated cultures of M. hominis, M. hyorhinis, and Acholeplasma laidlawii within 1 hr and a culture of M. orale within 3 hr. The other detergents exhibited various degree of activity against the different mycoplasmas, with Triton WR-1339 being the least effective. The virucidal activity of the detergents was determined for six viruses. All four Tween compounds were highly virucidal for herpes simplex virus. Tween 20 also exhibited virucidal effects against vesicular stomatitis virus, California encephalitis virus, and Newcastle disease virus, and Tween 80 was found to be active against California encephalitis and Newcastle disease viruses. Detergent treatment procedures were effective in two instances in eliminating mycoplasma contaminants from virus preparations while the preparations retained most of the viral infectivity. The limitations of this technique for routine use are discussed.     

Polyhexamethylene biguanide exposure leads to viral aggregation

Aims: This study reports the activity of two biguanides against MS2 bacteriophage used as a surrogate virus for nonenveloped mammalian viruses and provides an explanation as to their apparent limited efficacy. Methods and Results: When tested in a standard suspension test, two polyhexamethylene biguanides (PHMB), VANTOCIL? TG and COSMOCIL? CQ, reduced the viability of MS2 by only 1–2 log₁₀ PFU ml^?1. Exposure time up to 30 min did not affect the activity of the biguanides, although both PHMB were shown to strongly interact with MS2 proteins. Conclusions: Inactivation kinetics and change in virus hydrophobicity suggested that PHMB induces the formation of viral aggregates. This hypothesis was supported using dynamic light scattering that showed an increase in viral aggregates sizes (up to 500 nm) in a concentration‐dependent manner. Significance and Impact of the Study: It has been reported that viral aggregation is responsible for virus survival to the biocide exposure. Here, this might be the case, because the virucidal activity of the biguanides was modest and viral aggregation important. The formation of viral aggregates during virus exposure to PHMB was unlikely to overestimate the virucidal potential of the biguanides.     

Efficacy of certain disinfectants against infectious pancreatic necrosis virus

The virucidal properties of iodophor, chlorine (sodium hypochlorite), formalin, thimerosal (organic mercurial compound), malachite green, and acriflavine were tested on infectious pancreatic necrosis virus (IPNV). Iodine and chlorine showed good activity, but efficacy depended on the concentration of virus, the presence of organic matter (calf serum), and water _pH. Water hardness (0-300 mg 1⁻¹ as CaCO₃) did not affect virucidal activity. In a 5 min exposure, 4 mg 1⁻¹ available iodine inactivated 10^3.9 TCID₅₀ m1⁻¹ IPNV but 16 mg 1⁻¹ iodine were needed for inactivation of 10^6.3TCID₅₀m1⁻¹. The addition of 0-5% calf serum significantly reduced the iodine concentration and the virucidal activity. In comparison, 4 mg 1⁻¹ chlorine were needed to inactivate 10⁴⁶ TCID₅₀ m1⁻¹ IPNV in 5 min. However, the addition of 0-07 % serum greatly reduced the chlorine concentration and extended the virucidal contact time to 30 min or more. IPNV at 10^6.3 TCID₆₀ m1⁻¹ was not inactivated by exposures for 60 min to 0-2% formalin, 10 min to 0-2% thimerosal, 60 min to 5 mg 1⁻¹ malachite green, or 20 min to 500 mg 1⁻¹ acriflavine. However, acriflavine at 0-5 mg 1⁻¹ in cell culture media prevented the development of cytopathology caused by IPNV and may be useful in the treatment of the disease.     

Insights into virus inactivation by polysorbate 80 in the absence of solvent

Triton X-100 has long been used either alone or in combination with solvent to inactivate enveloped viruses in biopharmaceutical manufacturing. However, European Chemicals Agency (ECHA) officially placed Triton X-100 on the Annex XIV authorization list in 2017 because 4-(1,1,3,3-tetramethylbutyl) phenol, a degradation product of Triton X-100, is of harmful endocrine disrupting activities. As a result, any use of Triton X-100 in the European Economic Area would require an ECHA issued authorization after the sunset date of January 4, 2021. In search of possible replacements for Triton X-100, we discovered that polysorbate 80 (PS80) in absence of any solvents was able to effectively inactive enveloped viruses such as xenotropic murine leukemia virus and pseudorabies virus with comparable efficacy as measured by log reduction factors. Interestingly, PS80 did not show any virucidal activities in phosphate buffered saline (PBS) while achieving robust virus inactivation in cell-free Chinese hamster ovary (CHO) bioreactor harvests. This intriguing observation led us to speculate that virus inactivation by PS80 involved components in the cell-free CHO bioreactor harvests that were absent in PBS. Specifically, we hypothesized that esterase and/or lipases in the cell-free bioreactor harvests hydrolyzed PS80 to yield oleic acid, a known potent virucidal agent, which in turn inactivated viruses. This theory was confirmed using purified recombinant lysosomal phospholipase A2 isomer (rLPLA2) in PBS. Subsequent characterization work has indicated that virus inactivation by PS80 is effective and robust within temperature and concentration ranges comparable to those of Triton X-100. Similar to Triton X-100, virus inactivation by PS80 is dually dependent on treatment time and temperature. Unlike Triton X-100, PS80 inactivation does not correlate with concentrations in a simple manner. Additionally, we have demonstrated that PS20 exhibits similar virus inactivation activities as PS80. Based on the findings described in the current work, we believe that PS80 is potentially a viable replacement for Triton X-100 and can be used in manufacturing processes for wide spectrum of biopharmaceuticals to achieve desirable virus clearance. Finally, the advantages and disadvantages of using PS80 for virus inactivation are discussed in the contexts of GMP manufacturing.     

Halophilic Bacteria Susceptibility to Peracetic Acid Vapor and Ethylene Oxide

Extremely to slightly halophilic bacteria were tested for susceptibility to two sterilizing agents, peracetic acid (PAA) and ethylene oxide (ETO). PAA susceptibility was explored by two methods: an agar plate (constant pH of 7.2) and a filter strip (constant incubation period of 37 days); 100% susceptibility was obtained by both methods. The dosage (0.5 ml/min) was applied to a filter pad in a petri dish cover. Glove box experiments with ETO (input 1.5 lb. [ca. 680.4 g]/24 hr, the only constant) yielded 100% susceptibility for all halophiles tested. These experiments demonstrated the efficacy of two lethal agents for extreme halophiles, PAA and ETO. Variation in pH did not affect susceptibility.     

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.