Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater.

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.     

Poliovirus retention in 75-cm soil cores after sewage and rainwater application.

The adsorption rate of a guanidine-resistant strain of poliovirus LSc 2ab was measured in Long Island soils with in situ field cores (10.1 by 75 cm). The test virus was chosen because it exhibited soil adsorption and elution characteristics of a number of non-polioviruses. After the inoculation of cores with seeded sewage effluent at a 1-cm/h infiltration rate, cores were extracted, fractionated, and analyzed for total plaque-forming units per each 5-cm fraction. The results showed that 77% of the viruses were adsorbed in the first 5 cm of soil. An additional 11% were found in the 5- to 10-cm fraction, and a total of 96% of the viruses were adsorbed by 25 cm. The remaining 4% were uniformly distributed over the next 50 cm of soil, with a minimum of 0.23% in each soil section. Few viruses (< 0.22%) were observed in core filtrates. Analysis of the viral distribution pattern in seeded cores, after an application of a single rinse of either sewage effluent or rainwater, indicated that large-scale viral mobilization was absent. However, localized areas of viral movement were noted in both of the rinsed cores, with the rainwater-rinsed cores exhibiting more expensive movement. All mobilized viruses were resorbed at lower core depths.     

Poliovirus retention in 75-cm soil cores after sewage and rainwater application

The adsorption rate of a guanidine-resistant strain of poliovirus LSc 2ab was measured in Long Island soils with in situ field cores (10.1 by 75 cm). The test virus was chosen because it exhibited soil adsorption and elution characteristics of a number of non-polioviruses. After the inoculation of cores with seeded sewage effluent at a 1-cm/h infiltration rate, cores were extracted, fractionated, and analyzed for total plaque-forming units per each 5-cm fraction. The results showed that 77% of the viruses were adsorbed in the first 5 cm of soil. An additional 11% were found in the 5- to 10-cm fraction, and a total of 96% of the viruses were adsorbed by 25 cm. The remaining 4% were uniformly distributed over the next 50 cm of soil, with a minimum of 0.23% in each soil section. Few viruses (< 0.22%) were observed in core filtrates. Analysis of the viral distribution pattern in seeded cores, after an application of a single rinse of either sewage effluent or rainwater, indicated that large-scale viral mobilization was absent. However, localized areas of viral movement were noted in both of the rinsed cores, with the rainwater-rinsed cores exhibiting more expensive movement. All mobilized viruses were resorbed at lower core depths.     

Isolation of apparently wild strains of poliovirus type 1 from sewage in the Ottawa area.

In the first 4 months of 1974, 140 gauze pad samples of sewage collected in the Ottawa area were analysed by the BS-C-1 cell system for the presence of viruses pathogenic for humans. Viruses were isolated from 111 (79%) of the samples. Of the 72 (65%) isolates identified by serology and electron microscopic examination, 56 (78%) were reoviruses and 16 (22%), enteroviruses. The enterovirus isolates included one coxsackievirus B4, one vaccine strain of poliovirus type 3, nine vaccine strains of poliovirus type 1 and five strains of poliovirus type 1 that proved by serodifferentiation and temperature marker tests to be different from vaccine strains. The fact that these strains were present in the community sewage in readily detectable concentrations at a time when immunity against polioviruses is declining in such communities is a cause for concern.     

Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland.

During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign.     

Viruses in sewage waters during and after a poliomyelitis outbreak and subsequent nationwide oral poliovirus vaccination campaign in Finland

During an outbreak of paralytic poliomyelitis in Finland in 1984 and 1985 the widespread circulation of the causative wild-type serotype 3 poliovirus in the population was documented by demonstrating the virus in sewage water specimens in 13 different locations in the greater Helsinki district and in 13 other cities or towns all over the country. After the nationwide campaign with oral poliovirus vaccine in 1985, poliovirus serotypes 2 and 3 were readily isolated from sewage waters for up to 2 months, whereas type 1 poliovirus seemed to disappear from the sewage more rapidly. All of these isolates were temperature sensitive and therefore most likely vaccine related. The efficacy of the vaccination campaign in regard to elimination of the epidemic type 3 strain was evaluated by a follow-up study on viruses in sewage waters continued for 12 months through the subsequent expected season of poliomyelitis. Several types of enteroviruses, including five vaccine-related poliovirus strains, were identified in the 72 virus-positive specimens out of 93 studied. No wild-type polioviruses were found, indicating the success of the campaign.     

Detection of low levels of enteric viruses in metropolitan and airplane sewage.

To detect less prevalent viruses, such as wild-type polioviruses in sewage from a highly immunized community, a method was developed to efficiently recover viruses and remove PCR inhibitors. The method consisted of initial separation of solids from liquid, followed by solvent extractions, polyethylene glycol precipitations, Sephadex G-200 chromatography, and guanidinium isothiocyanate (GIT) extraction. To elute viruses from the separated solids, 0.5 M threonine (pH 7.5) was as efficient as 3% beef extract but conferred no PCR inhibition. In samples that were concentrated approximately 1,000-fold, 21% of the initially seeded viruses were recovered. When poliovirus type 3 (PV3) Sabin strain at low levels and PV1 LSc strain at high levels were seeded in raw sewage, PV3 was specifically detected in the final sample concentrates at sensitivities of 14 PFU by direct PCR and 0.7 PFU by GIT extraction-PCR. While applying the method to international airplane sewage, which contains high levels of solids as well as commercial sanitizers, 44% (7 of 16) of the samples were found to harbor enteroviruses by both cell culture infectivity and pan-enterovirus PCR analyses. Nucleotide sequencing of the PCR products revealed that multiple enterovirus genotypes were amplified from each final sewage concentrate, whereas the fewer virus genotypes detected by cell culture infectivity were probably the better growing strains. By this method, we demonstrated that air travel may contribute to the intercontinental dissemination of enteric pathogens.     

Polioviruses and other enteroviruses in urban sewage from Laval (Canada): presence of non-vaccinal strains of poliovirus.

During a 13-month period (August 1, 1977-August 31, 1978) 55 samples of 2-4 L of raw sewage from an urban residential area were collected and studied for the presence of human enteroviruses. Viruses were recovered from 47 (90.5%) of the samples. Of the 201 viral isolates propagated, 180 (89.5%) were identified by the serum neutralization test as poliovirus types 1,2, or 3; 16 (8%) were identified as coxsackie B viruses; and 5 (2.5%) could not be identified by the methods used. While all polioviruses, types 2 and 3 isolates, were vaccine-like (rct-/d-) or intermediate strains, 14 poliovires, type 1 out of 55 selected isolates, were found to be non-vaccine-like (rct+/d+), 8 were rct-/d-), and 33 were intermediate strains. Out of nine samples submitted to the serodifferentiation (McBride) test, six poliovires, type 1 rct+/d+ and three rct+/d-, were related to wild strains.     

Poliovirus retention in soil columns after application of chemical- and polyelectrolyte-conditioned dewatered sludges

The transport of poliovirus type 1 (strain LSc) was studied in Red Bay sandy loam columns that were treated with chemical- or polyelectrolyte-conditioned dewatered sludges and then leached with natural rainwater under saturated flow conditions. Poliovirus was concentrated in the alum and ferric chloride sludges that were produced following the flocculation of virus-seeded raw sewage. Virtually complete inactivation of the virus was observed following the flocculation of raw sewage or the stabilization of alum and ferric chloride sludges with lime at pH 11.5. Poliovirus was also concentrated in polyelectrolyte-conditioned dewatered sludge that was produced from virus-seeded, anaerobically digested sludge. Despite the saturated flow conditions for a sustained period, no viruses were detected in the leachates of the soil columns that were treated with these chemical and chemically treated sludges. Since the viruses were mostly associated with the solids in these sludge samples, it is believed that they were immobilized along with the sludge solids in the top portion of the soil columns.     

Poliovirus retention in soil columns after application of chemical- and polyelectrolyte-conditioned dewatered sludges.

The transport of poliovirus type 1 (strain LSc) was studied in Red Bay sandy loam columns that were treated with chemical- or polyelectrolyte-conditioned dewatered sludges and then leached with natural rainwater under saturated flow conditions. Poliovirus was concentrated in the alum and ferric chloride sludges that were produced following the flocculation of virus-seeded raw sewage. Virtually complete inactivation of the virus was observed following the flocculation of raw sewage or the stabilization of alum and ferric chloride sludges with lime at pH 11.5. Poliovirus was also concentrated in polyelectrolyte-conditioned dewatered sludge that was produced from virus-seeded, anaerobically digested sludge. Despite the saturated flow conditions for a sustained period, no viruses were detected in the leachates of the soil columns that were treated with these chemical and chemically treated sludges. Since the viruses were mostly associated with the solids in these sludge samples, it is believed that they were immobilized along with the sludge solids in the top portion of the soil columns.     

Effect of soil permeability on virus removal through soil columns.

Laboratory experiments were performed on four different soils, using 100 cm long columns, to determine the extent of virus movement when wastewater percolated through the soils at various hydraulic flow rates. Unchlorinated secondary sewage effluent seeded with either poliovirus type 1 (strain LSc) or echovirus type 1 (isolate V239) was continuously applied to soil columns for 3 to 4 days at constant flow rates. Water samples were extracted daily from ceramic samplers at various depths of the column for the virus assay. The effectiveness of virus removal from wastewater varied greatly among the different soil types but appeared to be largely related to hydraulic flow rates. At a flow rate of 33 cm/day, Anthony sandy loam removed 99% of seeded poliovirus within the first 7 cm of the column. At flow rates of 300 cm/day and above, Rubicon sand gave the poorest removal of viruses; less than 90% of the seeded viruses were removed by passage of effluent through the entire length of the soil column. By linear regression analyses, the rate of virus removal in soil columns was found to be negatively correlated with the flow of the percolating sewage effluent. There was no significant difference in rate of removal between poliovirus and echovirus in soil columns 87 cm long. The rate of virus removal in the upper 17 cm of the soil column was found to be significantly greater than in the lower depths of the soil column. This study suggests that the flow rate of water through the soil may be the most important factor in predicting the potential of virus movement into the groundwater. Furthermore, the length of the soil column is critical in obtaining useful data to predict virus movement into groundwater.     

Effect of soil permeability on virus removal through soil columns

Laboratory experiments were performed on four different soils, using 100 cm long columns, to determine the extent of virus movement when wastewater percolated through the soils at various hydraulic flow rates. Unchlorinated secondary sewage effluent seeded with either poliovirus type 1 (strain LSc) or echovirus type 1 (isolate V239) was continuously applied to soil columns for 3 to 4 days at constant flow rates. Water samples were extracted daily from ceramic samplers at various depths of the column for the virus assay. The effectiveness of virus removal from wastewater varied greatly among the different soil types but appeared to be largely related to hydraulic flow rates. At a flow rate of 33 cm/day, Anthony sandy loam removed 99% of seeded poliovirus within the first 7 cm of the column. At flow rates of 300 cm/day and above, Rubicon sand gave the poorest removal of viruses; less than 90% of the seeded viruses were removed by passage of effluent through the entire length of the soil column. By linear regression analyses, the rate of virus removal in soil columns was found to be negatively correlated with the flow of the percolating sewage effluent. There was no significant difference in rate of removal between poliovirus and echovirus in soil columns 87 cm long. The rate of virus removal in the upper 17 cm of the soil column was found to be significantly greater than in the lower depths of the soil column. This study suggests that the flow rate of water through the soil may be the most important factor in predicting the potential of virus movement into the groundwater. Furthermore, the length of the soil column is critical in obtaining useful data to predict virus movement into groundwater.     

Influence of pH, salinity, and organic matter on the adsorption of enteric viruses to estuarine sediment.

This study was designed to determine the degree of adsorption of enteric viruses to marine sediment and factors controlling this association. Adsorption and elution characteristics of several enteroviruses and one rotavirus to estuarine sediments were studied under varying conditions of pH, salinity, and presence of soluble organics. Greater than 99% of the added poliovirus type 1 (LSc), coxsackievirus type B3 (Nancy), echovirus type 7 (Wallace), and rotavirus (SA-11) adsorbed to sediment. Echovirus 1 (Farouk) and a recent isolate typed as coxsackievirus B4 adsorbed significantly less than poliovirus 1 under similar conditions of varying salinity and pH. The presence of soluble organic matter, in the form of secondary sewage effluent or humic acid, did not affect these patterns of adsorption. Only echovirus 1 (Farouk) desorbed when the pH or salinity was altered and then only to a small extent. Three recent isolates of echovirus 1 and echovirus 29 (strain JV-10) also demonstrated varying amounts of adsorption to sediment. These data indicate that enteric viruses can become readily associated with sediment in the estuarine environment and that this association may play a major role in their hydrotransportation and survival.     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater.

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.     

Enteric viruses in renovated water in Manitoba

Using various concentration procedures (including the gauze pad method, ultrafiltration, and adsorption-elution), enteric viruses, mainly vaccine strains of polioviruses, were isolated from 61.8% of sewage, 20.5% of effluents, 3.0% of river water, and 6.7% of drinking water samples collected from a waterway system in Manitoba. The presence of polioviruses in the drinking water of the town utilizing this waterway was not correlated with an increase in the number of infectious diseases reported.     

2010年山东省环境污水中脊髓灰质炎病毒的监测及其基因特征分析

本研究在山东省开展了脊髓灰质炎病毒(Poliovirus,PV)的外环境监测,从济南、临沂两地采集污水标本,浓缩处理后进行病毒分离,对分离到的PV采用中和试验进行血清定型,并对其VP1及3D区进行序列测定,分析其基因突变和重组情况。2010年,共采集污水标本32份,PV阳性10份,阳性率31.3%;分离到18株PV(PV1型3株,PV2型9株,PV3型6株),均为疫苗相关株,VP1完整编码区核苷酸变异数在0~4个之间,在3株PV2型病毒和4株PV3型病毒的基因组中发现重组;对VP1区影响神经毒力的减毒位点分析发现,PV1型病毒中有1株在nt 2 749发生突变(A→G),PV2型病毒中有1株在nt2 908发生A→G突变,3株在nt2 909发生U→C突变,6株PV3型病毒全部在nt2 493发生C→U突变。环境污水中可以分离到PV,其基因重组率和主要减毒位点的回复突变率较高,未发现脊灰野毒株和疫苗衍生株脊灰病毒(Vaccine-derived poliovirus,VDPV)。     

Efficiency of beef extract for the recovery of poliovirus from wastewater effluents.

The efficiency of poliovirus elution from fiber glass cartridge filters (K27), epoxy-fiber glass-asbestos filters (M780), and pleated cartridge filters was assessed by using 3% beef extract (pH 9.0) or 0.1 M glycine (pH 11.5). Poliovirus type I, strain LSc, was seeded into 20- to 25-gallon (ca. 75.6- to 95.6-liter) samples of treated sewage effluent and concentrated by using a filter adsorption-elution technique. Virus elution was accomplished by using either two 600-ml portions of 3% beef extract (pH 9.0), or two 1-liter portions of 0.1 M glycine (pH 11.5). In all experiments, beef extract elution followed by organic flocculation was found to be superior, yielding a mean recovery efficiency of 85%, with recoveries ranging from 68 to 100%. Elution with 0.1 M glycine (pH 11.5) followed by inorganic flocculation resulted in a mean recovery efficiency of 36%. The variable range of recoveries with beef extract could not be significantly improved by varying the type of beef extract or by extending the elution time to 30 min. Second-step reconcentration of 1-liter seeded sewage effluent and renovated wastewater samples indicated that organic flocculation was a more efficient method for virus recovery than inorganic flocculation. Beef extract concentrations of less than 3% were found to be efficient in the recovery of poliovirus from renovated wastewater.     

Development of a quantitative method for the detection of enteroviruses in soil.

A method is described for efficiently concentrating enteroviruses from soil. Viruses were eluted from soil by mechanical agitation in high pH glycine buffer containing ethylenediaminetetraacetic acid. The eluted viruses were concentrated on a floc that formed de novo upon adjustment of the soil eluate to 0.06 M aluminum chloride and pH 3.5. Viruses not pelleted with the floc were concentrated by adsorption to and elution from membrane filters. This method yielded an average efficiency of 66% recovery from loamy sand soil for four enteroviruses. Virus recovery from soil was consistently high, with samples ranging in size from 25 to 500 g. The method was used successfully to isolate naturally occurring viruses from soil beneath a wastewater land treatment site. Recovery of enteroviruses by this method form different types of soil was dependent on percentage of clay, surface area, and cation exchange capacity. Recovery was not dependent on soil saturation pH or on percentage of organic matter. This method should prove useful for studying enterovirus migration and survival during the land application of domestic sewage.