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.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

Inactivation of poliovirus in digested sludge.

The effect of anaerobically digested sludge on poliovirus during incubation at temperatures between 28 and 4 C was studied. Although virus was fully recoverable from sludge, its infectivity decreased in proportion to the time and temperature of incubation. The rate ranged from greater than 1 log per day at 28 C to about 1 log every 5 days at 4 C. The mechanism of inactivation was studied at the lower temperature where the sedimentation coefficients of most inactivated particles were not detectably modified. The ribonucleic acid (RNA) of these particles appeared to have been nicked and had an average sedimentation value about 70% that of RNA from infectious virus. Since the specific infectivity of RNA from particles recovered from sludge was directly proportional to that of the particles from which it was extracted, loss of infectivity was probably due to inactivation of RNA. Some breakdown was also found in the two largest viral proteins of inactivated particles. Thus, the mechanism of inactivation may be cleavage of viral proteins followed by nicking of encapsulated RNA. Because no virucidal activity was found in raw sludge, this component of digested sludge appears to be a product of the digestion process.     

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.     

Thermal and water source effects upon the stability of enteroviruses in surface freshwaters

The long-term survival of three human enterovirus serotypes, Coxsackievirus B3, echovirus 7, and poliovirus 1 was examined in samples of surface freshwater collected from five sites of physically different character. These were an artificial lake created by damming a creek, a small groundwater outlet pond, both a large- and a medium-sized river, and a small suburban creek. Survival was studied at temperatures of -20, 1, and 22 degrees C. The average amount of viral inactivation was 6.5-7.0 log10 units over 8 weeks at 22 degrees C, 4-5 log10 units over 12 weeks at 1 degree C, and 0.4-0.8 log10 units over 12 weeks at -20 degrees C. The effect of incubation temperature upon viral inactivation rate was statistically significant (p less than 0.00001). As determined by pairing tests, survival was also significantly related to both viral serotype and water source at each of the three incubation temperatures (p less than or equal to 0.05). Efforts were made to determine whether the rate of viral inactivation observed at the different incubation temperatures was related to characteristics inherent to the water that was collected from the different locations. The characteristics examined included physical and chemical parameters, indigenous bacterial counts, and the amount of bacterial growth that the waters would support (measured as the maximum number of generations which seeded bacteria could undergo after being placed into either pasteurized or sterile-filtered water samples). Analysis of viral inactivation rate versus these characteristics revealed three apparent effectors of viral persistence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infectivity and antigenicity reduction rates of human rotavirus strain Wa in fresh waters.

The rates of inactivation of human rotavirus type 2 (strain Wa) (HRV-Wa) and poliovirus type 1 (strain CHAT) were compared in polluted waters (creek water and secondary effluent before chlorination) and nonpolluted waters (lake water, groundwater, and chlorinated tap water). Viral infectivity titers were determined by plaque assays, while HRV-Wa antigenicity also was monitored by an enzyme-linked immunosorbent assay. Both viruses persisted longest in lake water and shortest in tap water. The actual inactivation times (i.e., times required for two-log10 reductions of initial viral titers) for the two viruses were significantly different in all waters except tap water. With the exception of the groundwater and secondary effluent results, the HRV-Wa inactivation times in the fresh waters tested were significantly different. Owing perhaps to aggregation, HRV-Wa appeared less susceptible to the effects of chlorine than previously reported for this virus and for the simian rotavirus SA11. HRV-Wa displayed prolonged survival in lake water and groundwater exceeding that previously reported for the SA11 virus. The HRV-Wa infectivity reduction rate (ki) was significantly correlated with the water pH (i.e., as pH increased, ki increased). The water pH may have influenced viral aggregation and thereby HRV-Wa susceptibility to other virucidal factors in the water. Enzyme-linked immunosorbent assay results showed similar inactivation patterns with the most significant reduction in HRV-Wa antigenicity occurring in polluted waters and tap water. In all waters, particularly tap water, infectivity declined at a faster rate than antigenicity. It is proposed that HRV-Wa can be used as a model for future studies of rotaviral persistence in the aquatic environment.     

Infectivity and antigenicity reduction rates of human rotavirus strain Wa in fresh waters

The rates of inactivation of human rotavirus type 2 (strain Wa) (HRV-Wa) and poliovirus type 1 (strain CHAT) were compared in polluted waters (creek water and secondary effluent before chlorination) and nonpolluted waters (lake water, groundwater, and chlorinated tap water). Viral infectivity titers were determined by plaque assays, while HRV-Wa antigenicity also was monitored by an enzyme-linked immunosorbent assay. Both viruses persisted longest in lake water and shortest in tap water. The actual inactivation times (i.e., times required for two-log10 reductions of initial viral titers) for the two viruses were significantly different in all waters except tap water. With the exception of the groundwater and secondary effluent results, the HRV-Wa inactivation times in the fresh waters tested were significantly different. Owing perhaps to aggregation, HRV-Wa appeared less susceptible to the effects of chlorine than previously reported for this virus and for the simian rotavirus SA11. HRV-Wa displayed prolonged survival in lake water and groundwater exceeding that previously reported for the SA11 virus. The HRV-Wa infectivity reduction rate (ki) was significantly correlated with the water pH (i.e., as pH increased, ki increased). The water pH may have influenced viral aggregation and thereby HRV-Wa susceptibility to other virucidal factors in the water. Enzyme-linked immunosorbent assay results showed similar inactivation patterns with the most significant reduction in HRV-Wa antigenicity occurring in polluted waters and tap water. In all waters, particularly tap water, infectivity declined at a faster rate than antigenicity. It is proposed that HRV-Wa can be used as a model for future studies of rotaviral persistence in the aquatic environment.     

Inactivation of enteroviruses by ascorbic acid and sodium bisulfite.

Poliovirus type 1, coxsackievirus type A9, and echovirus type 7 were inactivated by sodium bisulfite and ascorbic acid. Inactivation rates depended upon concentration, temperature, and pH. RNA infectivity was lost during inactivation; the capsid was also altered by these inactivating agents, as determined by enzyme sensitivity assays and by tests of adsorption to cells. Structural modifications of the virus particles were not identical, suggesting that the mechanism of inactivation by ascorbic acid differs from that of sodium bisulfite.     

Inactivation of enteroviruses by ascorbic acid and sodium bisulfite.

Poliovirus type 1, coxsackievirus type A9, and echovirus type 7 were inactivated by sodium bisulfite and ascorbic acid. Inactivation rates depended upon concentration, temperature, and pH. RNA infectivity was lost during inactivation; the capsid was also altered by these inactivating agents, as determined by enzyme sensitivity assays and by tests of adsorption to cells. Structural modifications of the virus particles were not identical, suggesting that the mechanism of inactivation by ascorbic acid differs from that of sodium bisulfite.     

Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.     

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation.

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.     

Inactivation of enteric viruses in wastewater sludge through dewatering by evaporation.

The effect of dewatering on the inactivation rates of enteric viruses in sludge was determined. For this study, water was evaporated from seeded raw sludge at 21 degrees C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.     

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.     

TnBP?Triton X-45 Treatment of Plasma for Transfusion Efficiently Inactivates Hepatitis C Virus

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products.     

Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water.

The potential for human immunodeficiency virus (HIV) to enter domestic sewers via contaminated body fluids such as blood has spurred interest in the survival of this virus in water and wastewater. This study focused on establishing the inactivation of HIV and productively infected lymphocytes in dechlorinated tap water. In addition, HIV survival was compared with that of poliovirus. Results indicated that either free HIV or cell-associated HIV was rapidly inactivated, with a 90% loss of infectivity within 1 to 2 h at 25 degrees C and a 99.9% loss by 8 h. In comparison, poliovirus showed no loss of infectivity over 24 h. The presence of human serum in tap water slowed the rate of HIV inactivation through 8 h but did not stabilize the virus through 24 h. In addition, blood from stage IV AIDS patients was introduced into tap water, and the recovery of HIV was monitored by using both an infectivity assay and polymerase chain reaction amplification of viral sequences. Virally infected cells were no longer detectable after 5 min in dechlorinated tap water, while little diminution in amplifiable sequences was observed over 2 h. Thus, detection of viral sequences by polymerase chain reaction technology should not be equated with risk of exposure to infectious HIV.     

Virion conformational forms and the complex inactivation kinetics of echovirus by chlorine in water

Aberrant inactivation kinetics were observed when monodispersed echovirus type 1 (Farouk) was inactivated with chlorine. An initial 1- to 2-log10-unit decrease in titer was followed by lag period, during which the titer stayed the same or increased, and this was followed by a final decline in titer. First-order kinetics were obtained with poliovirus type 1 under the same conditions. Isoelectric focusing studies of echovirus before chlorine treatment showed that the virus distributed into two pH-dependent and interconvertible isoelectric forms. After chlorine treatment all remaining virus infectivity was associated with a third pH-independent isoelectric form. The complex inactivation kinetics appeared to be due to shifts between these conformational forms during inactivation in certain ionic environments. Under certain conditions the conformational shifts resulted in substantial resistance of monodispersed echovirus to chlorine.     

Virion conformational forms and the complex inactivation kinetics of echovirus by chlorine in water.

Aberrant inactivation kinetics were observed when monodispersed echovirus type 1 (Farouk) was inactivated with chlorine. An initial 1- to 2-log10-unit decrease in titer was followed by lag period, during which the titer stayed the same or increased, and this was followed by a final decline in titer. First-order kinetics were obtained with poliovirus type 1 under the same conditions. Isoelectric focusing studies of echovirus before chlorine treatment showed that the virus distributed into two pH-dependent and interconvertible isoelectric forms. After chlorine treatment all remaining virus infectivity was associated with a third pH-independent isoelectric form. The complex inactivation kinetics appeared to be due to shifts between these conformational forms during inactivation in certain ionic environments. Under certain conditions the conformational shifts resulted in substantial resistance of monodispersed echovirus to chlorine.