Enterovirus inactivation in soil.

The inactivation of radioactively labeled poliovirus type 1 and coxsackievirus B 1 in soils saturated with surface water, groundwater, and septic tank liquor was directly proportional to temperature. Virus persistence was also related to soil type and the liquid amendment in which viruses were suspended. At 37 degrees C, no infectivity was recovered from saturated soil after 12 days; at 4 degrees C, viruses persisted for at least 180 days. No infectivity was recovered from dried soil regardless of temperature, soil type, or liquid amendment. Additional experiments showed that evaporation of soil water was largely responsible for the decreased recovery of infectivity from drying soil. Increased rates of virus inactivation at low soil moisture levels were also demonstrated.     

Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water.

The survival in mineral water of hepatitis A virus (HAV) and poliovirus type 1 was compared, under controlled experimental conditions, at 4 degrees C and room temperature. Viral infectivity titers were determined by cell culture titration, while HAV antigenicity was monitored by radioimmunoassay-endpoint titration. Both viruses persisted longest at 4 degrees C. At this temperature, after 1 year of exposure, the inactivation of either HAV or poliovirus type 1 was not important. At room temperature, poliovirus type 1 was not detected after 300 days, whereas HAV was still infectious. For both temperatures, the computed regression coefficients of best-fit lines for inactivation rates for the two viruses were significantly different. The survival of HAV was also studied at 4 degrees C and room temperature in mineral water with 5- and 50-micrograms/ml protein concentrations (i.e., purity of the virus suspension) for 120 days. As shown by a comparison of the regression coefficients for the inactivation rates, the stability of HAV in mineral water depends on protein concentration and temperature. Radioimmunoassay-endpoint titration results showed inactivation patterns similar to those of cell culture titration, with the most significant reduction in HAV antigenicity at room temperature. At the two temperatures, the infectivity of HAV declined at a faster rate than the antigenicity.     

Die-off of Cryptosporidium parvum in soil and wastewater effluents

AIMS: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. METHODS AND RESULTS: The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. CONCLUSIONS: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.     

Persistence of human rhinovirus infectivity under diverse environmental conditions

The persistence of human rhinovirus type 2 and type 14 infectivity was studied under various laboratory conditions designed to mimic those commonly found in the environment. The effects of temperature, ionic strength, protein content, and evaporation were compared. Both viruses were stable (less than 0.3-log decrease in titer) at 6 and 23 degrees c for 24 h in the liquid state regardless of salt or protein additives; a titer decrease of less than 1.0 log was noted at 37 degrees C. However, evaporation at 37 degrees C reduced virus infectivity by 3.2 to 4.5 logs in buffered water, an effect which could be significantly lessened by the addition of bovine serum albumin in saline (2.0- to 2.9-log decrease in titer). These studies support and extend observations by others that the human rhinoviruses retain sufficient infectivity after drying on hard surfaces to permit their transmission to susceptible persons upon contact.     

Persistence of human rhinovirus infectivity under diverse environmental conditions.

The persistence of human rhinovirus type 2 and type 14 infectivity was studied under various laboratory conditions designed to mimic those commonly found in the environment. The effects of temperature, ionic strength, protein content, and evaporation were compared. Both viruses were stable (less than 0.3-log decrease in titer) at 6 and 23 degrees c for 24 h in the liquid state regardless of salt or protein additives; a titer decrease of less than 1.0 log was noted at 37 degrees C. However, evaporation at 37 degrees C reduced virus infectivity by 3.2 to 4.5 logs in buffered water, an effect which could be significantly lessened by the addition of bovine serum albumin in saline (2.0- to 2.9-log decrease in titer). These studies support and extend observations by others that the human rhinoviruses retain sufficient infectivity after drying on hard surfaces to permit their transmission to susceptible persons upon contact.     

Capsid functions of inactivated human picornaviruses and feline calicivirus

The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72 degrees C), and physiological temperature (37 degrees C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37 degrees C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72 degrees C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37 degrees C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72 degrees C inactivation is the capsid and that the target of thermal inactivation (37 degrees C versus 72 degrees C) is temperature dependent.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Inactivation of polioviruses and coxsackieviruses in surface water.

Inactivation rates of polioviruses 1 and 3 and coxsackieviruses A-13 and B-1 were determined in situ in the Rio Grande in southern New Mexico, using membrane dialysis chambers. Inactivation of the viruses was exponential, and the rates of inactivation were apparently affected principally by the water temperature. Stability of the viruses in river water differed, with poliovirus 1 and coxsackie B-1 being most stable. Typically 1-log reductions of infectivity at water temperatures ranging between 23 and 27 degrees C required 25 h for poliovirus 1, 19 h for poliovirus 3, and 7 h for coxsackie virus A-13. At water temperatures of 4 to 8 degrees C, the log reduction times for poliovirus 1 and coxsackievirus B-1 were 46 and 58 h, respectively. Results obtained with labeled poliovirus 1 and coxsackievirus B-1 and with infectious ribonucleic acid indicate that inactivation was due to damage to viral ribonucleic acid. Virus-inactivation rates were also affected by heat sterilization of river water, indicating the presence of a heat-labile or volatile inactivating factor. The inactivating factor in Rio Grande water was apparently present at a constant concentration over a 1-year period.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.     

Infectivity of RNA from inactivated poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

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 caliciviruses

The viruses most commonly associated with food- and waterborne outbreaks of gastroenteritis are the noroviruses. The lack of a culture method for noroviruses warrants the use of cultivable model viruses to gain more insight on their transmission routes and inactivation methods. We studied the inactivation of the reported enteric canine calicivirus no. 48 (CaCV) and the respiratory feline calicivirus F9 (FeCV) and correlated inactivation to reduction in PCR units of FeCV, CaCV, and a norovirus. Inactivation of suspended viruses was temperature and time dependent in the range from 0 to 100 degrees C. UV-B radiation from 0 to 150 mJ/cm(2) caused dose-dependent inactivation, with a 3 D (D = 1 log(10)) reduction in infectivity at 34 mJ/cm(2) for both viruses. Inactivation by 70% ethanol was inefficient, with only 3 D reduction after 30 min. Sodium hypochlorite solutions were only effective at >300 ppm. FeCV showed a higher stability at pH <3 and pH >7 than CaCV. For all treatments, detection of viral RNA underestimated the reduction in viral infectivity. Norovirus was never more sensitive than the animal caliciviruses and profoundly more resistant to low and high pH. Overall, both animal viruses showed similar inactivation profiles when exposed to heat or UV-B radiation or when incubated in ethanol or hypochlorite. The low stability of CaCV at low pH suggests that this is not a typical enteric (calici-) virus. The incomplete inactivation by ethanol and the high hypochlorite concentration needed for sufficient virus inactivation point to a concern for decontamination of fomites and surfaces contaminated with noroviruses and virus-safe water.     

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.     

Physicochemical stability and inactivation of human and simian rotaviruses

The effects of various physical and chemical treatments on the stability of a human serotype 1 rotavirus and simian agent 11 (SA11) were compared by using a fluorescence focus assay. The infectivity of both strains was retained after storage at room temperature for 14 days, 4 degree C for 22 days, and -20 degree C for 32 days; lyophilization; and treatment at pH 3 to 11. Both viruses were inactivated at pH 12, as was the human virus at pH 2, although this pH resulted in only partial inactivation of SA11. The human virus also appeared to be more sensitive than SA11 to the action of ether and chloroform. The infectivity of both viruses was lost after UV irradiation for 15 min and after treatment with 8% formaldehyde for 5 min, 70% (vol/vol) ethanol for 30 min, and 2% lysol, 2% phenol, and 1% H2O2 for 1 h each.     

Chlorine inactivation of adenovirus type 40 and feline calicivirus

Ct values, the concentration of free chlorine multiplied by time of contact with virus, were determined for free-chlorine inactivation experiments carried out with chloroform-extracted (dispersed) and non-chloroform-extracted (aggregated) feline calicivirus (FCV), adenovirus type 40 (AD40), and polio virus type 1 (PV-1). Experiments were carried out with high and low pH and temperature conditions. Ct values were calculated directly from bench-scale free-chlorine inactivation experiments and from application of the efficiency factor Hom model. For each experimental condition, Ct values were higher at pH 8 than at pH 6, higher at 5 degrees C than at 15 degrees C, and higher for dispersed AD40 (dAD40) than for dispersed FCV (dFCV). dFCV and dAD40 were more sensitive to free chlorine than dispersed PV-1 (dPV-1). Cts for 2 log inactivation of aggregated FCV (aFCV) and aggregated PV-1 (aPV-1) were 31.0 and 2.8 orders of magnitude higher than those calculated from experiments carried out with dispersed virus. Cts for 2 log inactivation of dFCV and dAD40 in treated groundwater at 15 degrees C were 1.2 and 13.7 times greater than in buffered-demand-free (BDF) water experiments at 5 degrees C. Ct values listed in the U.S. Environmental Protection Agency (EPA) Guidance Manual were close to, or lower than, Ct values generated for experiments conducted with dispersed and aggregated viruses suspended in BDF water and for dispersed viruses suspended in treated groundwater. Since the state of viruses in water is most likely to be aggregated and associated with organic or inorganic matter, reevaluation of the EPA Guidance Manual Ct values is necessary, since they would not be useful for ensuring inactivation of viruses in these states. Under the tested conditions, dAD40, dFCV, aFCV, dPV-1, and aPV-1 particles would be inactivated by commonly used free chlorine concentrations (1 mg/liter) and contact times (60 to 237 min) applied for drinking water treatment in the United States.     

Physicochemical stability and inactivation of human and simian rotaviruses.

The effects of various physical and chemical treatments on the stability of a human serotype 1 rotavirus and simian agent 11 (SA11) were compared by using a fluorescence focus assay. The infectivity of both strains was retained after storage at room temperature for 14 days, 4 degree C for 22 days, and -20 degree C for 32 days; lyophilization; and treatment at pH 3 to 11. Both viruses were inactivated at pH 12, as was the human virus at pH 2, although this pH resulted in only partial inactivation of SA11. The human virus also appeared to be more sensitive than SA11 to the action of ether and chloroform. The infectivity of both viruses was lost after UV irradiation for 15 min and after treatment with 8% formaldehyde for 5 min, 70% (vol/vol) ethanol for 30 min, and 2% lysol, 2% phenol, and 1% H2O2 for 1 h each.     

Survival of enteric viruses adsorbed on electropositive filters.

Three viruses (poliovirus type 1, rotavirus SA-11, and bacteriophage f2) adsorbed on electropositive microporous filters survived at least 5 weeks at 4 degrees C. Poliovirus type 1 and bacteriophage f2 also survived at least 6 weeks at -20 degrees C. Rotavirus SA-11 was not recovered after 1 week at -20 degrees C. The stability of viruses adsorbed on electropositive filters may enable extensive monitoring of viruses in water.