Round robin investigation of methods for the recovery of poliovirus from drinking water.

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration of Simian Rotavirus SA-11 from Tap Water by Membrane Filtratiòn and Organic Flocculation

Simian rotavirus SA-11 was concentrated from tap water by adsorption to and elution from microporous filters, followed by organic flocculation. Two types of filters were compared for their ability to concentrate the virus. Both Zeta Plus 60S and Cox AA type M-780 filters were efficient for virus adsorption, but the efficiency of virus elution was higher with Zeta Plus than with Cox filters. Optimum conditions for virus recovery from Zeta Plus filters included an input water pH of 6.5 to 7.5 and the use of 3% beef extract (pH 9.0) for elution. Under these conditions, an average of 62 to 100% of the virus was recovered in the concentrate. Organic flocculation was used as a second-step concentration method, with average recoveries of 47 to 69%. When the two methods were used to concentrate small numbers (7 to 75 PFU/liter) of input rotavirus, an average of 75 ± 40% recovery was achieved. With large volumes of input water, however, recovery was reduced to 16 ± 7%.     

Concentration of simian rotavirus SA-11 from tap water by membrane filtration and organic flocculation

Simian rotavirus SA-11 was concentrated from tap water by adsorption to and elution from microporous filters, followed by organic flocculation. Two types of filters were compared for their ability to concentrate the virus. Both Zeta Plus 60S and Cox AA type M-780 filters were efficient for virus adsorption, but the efficiency of virus elution was higher with Zeta Plus than with Cox filters. Optimum conditions for virus recovery from Zeta Plus filters included an input water pH of 6.5 to 7.5 and the use of 3% beef extract (pH 9.0) for elution. Under these conditions, an average of 62 to 100% of the virus was recovered in the concentrate. Organic flocculation was used as a second-step concentration method, with average recoveries of 47 to 69%. When the two methods were used to concentrate small numbers (7 to 75 PFU/liter) of input rotavirus, an average of 75 +/- 40% recovery was achieved. With large volumes of input water, however, recovery was reduced to 16 +/- 7%.     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Comparison of talc-Celite and polyelectrolyte 60 in virus recovery from sewage: development of technique and experiments with poliovirus (type 1, Sabin)-contaminated multilitre samples.

For virus recovery from sewage, a mixture of talc and Celite was tested as a possible inexpensive substitute for polyelectrolyte 60 (PE 60). After adjustment of pH to 6 and the addition of 45-60 plaque forming units (PFU)/ml of poliovirus type I (Sabin) to the sewage sample under test, 100 ml of it was passed through either a PE 60 (400 mg) or a talc (300 mg)-Celite (100 mg) layer; the layer-adsorbed virus was eluted with 10 ml of 10% fetal calf serum (FCS) in saline (pH 7.2). In these experiments, PE 60 layers recovered 73-80% (mean 76%) of the input virus. In comparison, virus recoveries with the talc-Celite layers were 65-70% (mean 68%). Passage of 5 litres of raw sewage (containing 50 to 1.26 X 10(5) PFU/100 ml of the poliovirus) through the talc (15 g)-Celite (5 g) layers and virus elution with 50 ml of 10% FCS in saline gave virus recoveries of 33-63% (mean 49%). Except for pH adjustment and prefiltration through two layers of gauze to remove large solids, no other sample pretreatment was found to be necessary. Application of this technique to recovery of indigenous viruses from field samples of raw sewage and effluents has been highly satisfactory.     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters.

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Biodiversity of soil microarthropods: the filtering of species

Decline of soil microarthropod biodiversity is described in the sequence: old forest stands, low-input grasslands, high-input grasslands (with and without the use of persistent pesticides). With this expected trend of declining biodiversity, the patterns of species losses are analysed with the use of classifications of life-history tactics, feeding guilds and tolerances to drought. Species intolerant to drought are absent in grasslands, but present in old forest stands. Also the fraction of thelytokously reproducing microarthropods is higher in old forest stands compared with low-input grasslands. The main difference between low-input and high-input grasslands is found in the feeding guild structure of the community. Low-input grasslands are dominated by (herbo-) fungivorous grazers, whereas high-input grasslands are dominated by fungivorous browsers. Application of DDT in high-input grasslands shows a high density of microarthropods with a high fraction of thelytokous reproduction, associated with a decrease in genetic variation in a thelytokously reproducing species. Patterns in species losses, the species filters, are explained and discussed. It is shown that the decline of biodiversity is not a randorn loss of species but follows and identifiable pattern.     

Arbuscular mycorrhizal colonization of lime in different agroecosystems of the dry tropics

 Tecomán, in the Mexican state of Colima, had the world's greatest production of lime Citrus aurantifolia Swingle. Typical farming systems in the area include: (a) high-input monoculture, (b) a high-input system in which lime trees grow together with coconut palms, (c) a low-input system called "Family Farms" or "Family Gardens". In the Family Gardens, cultural practices are minimal and other fruit trees (about 16 species) coexist with the lime trees. This traditional minimal input system makes use of locally available resources and they are structurally very diverse. Arbuscular mycorrhizae may be crucial for sustainable production in Family Gardens. Root colonization and spore populations of fungi were scored at 2-week intervals in the three agroecosystems during a 6-month period. First samples were taken after the application of chemical fertilizer and irrigation in the high-input systems. Root colonization of lime was much higher and consistent in the low-input plots than in conventionally farmed plots, with colonization levels of 50–62% that remained the same throughout the sampling time; the high-input systems showed a high variation and lower level of colonization, 36% and 27% in associated and monoculture systems, respectively. Spore abundance was higher in the high-input systems but showed constant variation. The results suggest a strong effect of agroecosystem on mycorrhizal colonization of lime roots. Accepted: 12 September 1995     

Development and application of different methods for the detection of Toxoplasma gondii in water

Two methods, centrifugation and flocculation, were evaluated to determine their efficiencies of recovery of Toxoplasma gondii oocysts from contaminated water samples. Demineralized and tap water replicates were inoculated with high numbers of sporulated or unsporulated T. gondii oocysts (1 x 10(5) and 1 x 10(4) oocysts). The strain, age, and concentration of the seeded oocysts were recorded. Oocysts were recovered either by centrifugation of the contaminated samples at various g values or by flocculation with two coagulants, Fe(2)(SO(4))(3) and Al(2)(SO(4))(3). The recovery rates were determined with the final pellets by phase-contrast microscopy. Sporulated oocysts were recovered more effectively by flocculation with Al(2)(SO(4))(3) (96.5% +/- 21.7%) than by flocculation with Fe(2)(SO(4))(3) (93.1% +/- 8.1%) or by centrifugation at 2,073 x g (82.5% +/- 6.8%). For the unsporulated oocysts, flocculation with Fe(2)(SO(4))(3) was more successful (100.3% +/- 26.9%) than flocculation with Al(2)(SO(4))(3) (90.4% +/- 19.1%) or centrifugation at 2,565 x g (97.2% +/- 12.5%). The infectivity of the sporulated oocysts recovered by centrifugation was confirmed by seroconversion of all inoculated mice 77 days postinfection. These data suggest that sporulated Toxoplasma oocysts purified by methods commonly used for waterborne pathogens retain their infectivity after mechanical treatment and are able to induce infections in mammals. This is the first step in developing a systematic approach for the detection of Toxoplasma oocysts in water.     

Survival of duck plague virus in water from Lake Andes National Wildlife Refuge, South Dakota

An isolant of duck plague herpesvirus from the Lake Andes Refuge outbreak was seeded in raw and filter-decontaminated water from two locations on the refuge, held at 4 C, and assayed for infectivity intermittently over a period of 2 mo. From an initial level of about 10(5) PFU per ml, infectivity in the filtered samples uniformly dropped to about 10(4) PFU per ml. Infectivity in the raw samples declined much more rapidly; infectious virus remaining at the end of 2 mo (ca. 10(1) PFU per ml) was only about 0.01% of that originally seeded.     

Virus detection in soils: a comparison of four recovery methods.

Among nine eluents tested, 0.5% (w/v) isoelectric casein at pH 9.0 and 0.5% (w/v) non-fat dry milk (pH 9.0) were the most efficient in eluting poliovirus type 1 (Sabin) from Eustis fine sand. However, no significant difference was found between the overall (elution followed by concentration) virus recoveries by non-fat dry milk, isoelectric casein, beef extract, and glycine-EDTA methods. High overall recovery (75%) of low input (200 PFU) of viruses from 100 g of soil was achieved by the isoelectric casein method. It was found that the recovery efficiency of this method was not significantly affected by the soil type, following examination of four Florida soils. The mean overall recovery for the four soils was 50%. For other enteroviruses, the overall recovery for coxsackie B3 was 88% but was significantly lower (23%) for echovirus 4. Examination of the efficiency of the casein method under field conditions showed that it was possible to recovery low poliovirus numbers from soil (0.9-1.3 PFU/g soil).     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples.

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Molecular filtration for recovery of waterborne viruses of fish.

The effectiveness of tangential flow filtration (TFF) for the recovery of infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) from large volumes of water was evaluated. In laboratory studies, virus recovery from IHNV-seeded water following concentration by TFF was approximately 13%. However, the addition of 0.1 and 1% fetal bovine serum to deionized water stabilized the virus, increasing virus recoveries to 95%. The addition of 0.03 and 0.3% beef extract resulted in IHNV recoveries of 80 and 61%, respectively. Similar results were obtained with IPNV-seeded water. Field studies using the TFF procedure were conducted with water from areas where IHNV is endemic. IHNV was detected in effluent from an adult steelhead trout (Salmo gairdneri) holding pond at an estimated concentration of 1 PFU/5 ml of water. It was also detected at levels of 1 PFU/50 ml in water from a 2-m-diameter circular tank containing IHNV-infected steelhead trout fry. IHNV isolated in samples taken from the Metolius River was detected by TFF at estimated levels of 1 PFU/3 liters.     

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency’s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.     

Predictors of outcome of dream work for East Asian volunteer clients: Dream factors, attachment anxiety, Asian values, and therapist input.

Eighty-eight East Asian volunteers were paired with 6 East Asian therapists who provided low or high input in single-dream sessions. Volunteer clients with poor initial functioning on the target problem associated with their dreams and high self-efficacy for working with dreams profited more from dream sessions than did their counterparts. Although no main effects were found for therapist input, volunteer clients who scored higher on attachment anxiety had better outcome in the low-input condition, whereas clients who scored lower on attachment anxiety had better outcome in the high-input condition. Volunteer clients with lower Asian values evaluated low-input sessions more positively, whereas volunteer clients with higher Asian values evaluated high-input sessions more positively. Implications for dream work and future research are suggested. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mosquitoes inoculate high doses of West Nile virus as they probe and feed on live hosts

West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (10(1.2)-10(4.3) PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 10(4.3) PFU and 10(5.0) PFU for Culex tarsalis, 10(5.9) PFU and 10(6.1) PFU for Cx. pipiens, 10(4.7) PFU and 10(4.7) PFU for Aedes japonicus, and 10(3.6) PFU and 10(3.4) PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus ( approximately 10(2) PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.     

Molecular filtration for recovery of waterborne viruses of fish

The effectiveness of tangential flow filtration (TFF) for the recovery of infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) from large volumes of water was evaluated. In laboratory studies, virus recovery from IHNV-seeded water following concentration by TFF was approximately 13%. However, the addition of 0.1 and 1% fetal bovine serum to deionized water stabilized the virus, increasing virus recoveries to 95%. The addition of 0.03 and 0.3% beef extract resulted in IHNV recoveries of 80 and 61%, respectively. Similar results were obtained with IPNV-seeded water. Field studies using the TFF procedure were conducted with water from areas where IHNV is endemic. IHNV was detected in effluent from an adult steelhead trout (Salmo gairdneri) holding pond at an estimated concentration of 1 PFU/5 ml of water. It was also detected at levels of 1 PFU/50 ml in water from a 2-m-diameter circular tank containing IHNV-infected steelhead trout fry. IHNV isolated in samples taken from the Metolius River was detected by TFF at estimated levels of 1 PFU/3 liters.