A method to remove environmental inhibitors prior to the detection of waterborne enteric viruses by reverse transcription-polymerase chain reaction.

A method was developed to remove environmental inhibitors from sample concentrates prior to detection of human enteric viruses using the reverse transcription-polymerase chain reaction (RT-PCR). Environmental inhibitors, concentrated along with viruses during water sample processing, are removed by the method through a series of steps that includes dialysis, solvent extraction, ultrafiltration and glass purification. The method was tested by spiking sodium phosphate with poliovirus type 1 with or without humic or fulvic acids and then measuring virus recovery by plaque assay and RT-PCR. Results of the study indicated that (i) 90% of the spiked virus could be recovered from samples at the end of the ultrafiltration step, (ii) virus was detected in the final eluate of samples containing as much as 0.5 mg of humic acid or 5.0 mg of fulvic acid, and (iii) as little as 0.06 plaque forming units (PFU) was detectable per RT-PCR reaction. These results indicate that the described purification method along with RT-PCR is a feasible approach for detecting waterborne human enteric viruses in the presence of interfering substances.

Detection of viruses in environmental samples: suitability of commercial rotavirus and adenovirus test kits.

Commercially marketed kits are now available for rapid viral assay of clinical specimens. This study was conducted to determine the suitability of these kits for use in environmental testing. Eight rotavirus kits and one enteric adenovirus kit were screened for sensitivity using simian rotavirus SA11, human rotavirus Wa, and adenovirus 41. The most sensitive rotavirus kit and the adenovirus kit were selected for further evaluation using virus-seeded and unseeded sewage samples. The selected rotavirus kit proved capable of detecting virus at the 10(1) PFU/ml level. The enteric adenovirus kit was similarly sensitive, detecting virus at the 10(1) TCID50/ml level. Neither kit was adversely affected by the presence of sewage. Kit assay revealed 3 of 30 unseeded sewage samples to be positive for rotavirus. Adenovirus positive samples were not detected among the 30 samples. These results were confirmed using electron microscopy. It was concluded that sensitive commercial kits could provide a reasonable alternative to cell culture for the presumptive testing of environmental samples.

Comparison of fortified calf serum, serum substitutes and fetal calf serum with or without extenders for propagation of cell cultures for virus plaque assays.

Two studies comparing sewage-isolated and laboratory stock viruses were conducted to determine if alternative forms of serum or serum extenders could be used in place of fetal bovine serum without a significant loss of viral titer. In the first study, BGM cells were grown in standard MEM-L15 medium which was supplemented with Nuserum, Sigma serum replacement (CPSR-1), HyClone defined iron supplemented calf bovine serum, fetal bovine serum (FBS) or FBS supplemented with either SerXtend or Mito serum extenders. Comparison of virus titers showed that CPSR-1 gave the best overall results and was comparable to FBS. Of the serum extenders, only SerXtend improved virus recovery from sewage samples. In the second study, all sera were tested with and without SerXtend. In these experiments, SerXtend enhanced virus sensitivity of the BGM cell line grown in the HyClone serum but reduced the sensitivity of those cultured in Sigma serum. In both series, the growth of BGM cells was monitored for 12 weeks and all test products were shown to support long-term cell growth.

Factors affecting virus plaque confirmation procedures.

Direct plaque counts obtained by using the monolayer cell culture assay technique reliably confirmed the number of viruses isolated. Analysis revealed some significant differences in the false-positive rate, depending on the test method used or virus samples evaluated. Plaques from laboratory stock viruses showed a higher confirmation rate than sewage plaque isolates. Test results with laboratory stock viruses suggested that confirmation rates may be affected by virus types present in the sample. Plaques picked by the stab and scrape method and immediately passed into cell culture tubes produced the most reliable counts as compared to those picked by the stab only method or those stored at -70 degrees C in Earle's balanced salt solution with or without fetal calf serum. Plaque confirmation using this method was 90% or better. Although the term 'false positive plaque' has been applied to a particular plaque that was not confirmed, five of ten plaques picked by the stab and scrape method in one series of experiments were confirmed when repicked by the same method from original plaque bottles, indicating that a substantial number of unconfirmed plaques may be caused by plaque transfer techniques.

Isolation of enterovirus and reovirus from sewage and treated effluents in selected Puerto Rican communities.

Sewage treatment plant effluents were surveyed for viral contributions to gastroenteritis outbreaks in Puerto Rico. Of the 15 sewage treatment plants studied, all discharged their effluents upstream from water treatment plant intakes. No base-line data on the degree of viral challenge to these sewage treatment plants or the subsequent reduction of viruses before discharge existed. Enterovirus counts were generally much higher than those found in the continental United States. At four plants, viruses in the incoming sewage exceeded 100,000 PFU/liter, and one of these, a trickling filter plant, was discharging 24,000 PFU/liter to receiving waters. Virus identification showed that more than 80% of the enterovirus isolates were coxsackievirus B5. These overwhelming viral numbers pointed to defects in the sewage treatment processes. Without reasonable barriers to protect receiving waters, several of the downstream communities were using raw waters that posed extraordinary demands on the ability of their water treatment plants to supply virologically safe drinking water.

A comparison of recovery of virus from wastewaters by beef extract-Celite, ferric chloride, and filter concentration procedures.

An improved concentration method using sample volumes as large as 1500 ml has been developed to monitor for viruses in wastewaters. Non-precipitating dry beef extract powder is added to wastewater samples to give a 3% concentration and mixed until dissolved. This is followed by the addition of Celite as a virus adsorbent. By manipulating pH, viruses are eluted from the Celite in small volumes of phosphate buffer. This procedure was further tested without the aid of the Celite additives using a precipitating beef extract powder and substituting FeCl3 as an alternate reagent for the Celite. Comparison testing was also made with the currently recommended cartridge and disc filter procedures. In all cases, the non-precipitating beef extract-Celite method gave higher recovery rates in highly polluted waters.

Optimization of suspended cell method and comparison with cell monolayer technique for virus assays.

The suspended cell technique for enumeration of viruses from environmental samples was evaluated in regards to the number of cells necessary per bottle or flask, contact time with virus prior to overlay, and number of viruses per sample for maximum enumeration, using 40-cm2 (6 oz) glass bottles or 25-cm2 plastic culture flasks. Optimum cell numbers were determined to be 4 x 10(7) cells for the 40-cm2 bottles and 2.0 x 10(7) cells for the 25-cm2 flasks. The optimum exposure time of virus to cells at 37 degrees C was 60 min with significantly higher recoveries with shaken mixtures as opposed to static mixtures. Upon comparing the suspended cell technique with that of the cell monolayer system, using two sets of environmental sewage sample concentrates, 62 of the 67 samples yielded an average of 5-8 times greater viral recoveries with the suspended cell procedure. Based on our data we feel that all environmental samples should be tested using the suspended cell procedure.

Comparison of the in-line injector and fluid proportioner used to condition water samples for virus monitoring.

An in-line injector system is described and compared with the fluid proportioner for the injection of chemicals into water systems during filtration for viruses. Data on flow rates and virus recoveries of this system indicate that it is a suitable alternative to the fluid proportioner and other systems currently in use.

Recovery of viruses from water by a modified flocculation procedure for second-step concentration.

A reduction in virus recovery efficiencies stemming from a change in the commercial processing of powdered beef extract was reversed by the addition of Celite analytical filter aid. Supplementing beef extract with this silicate is recommended as a modification to the organic flocculation procedure for second-step concentration in monitoring for waterborne viruses. Considerable differences in virus recovery were found among lots of beef extract and Celite preparations; this indicates that the performance of each lot of these substances should be checked before use.

Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment.

An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.

Use of bituminous coal as an alternative technique for field concentration of waterborne viruses.

A filter system that sandwiches a bituminous coal preparation between two prefilters was comparable to those presently used to recover human viruses from large volumes of water. This filter was effective over a pH range of 3.0 to 7.0. Poliovirus type 1 recoveries from 100-liter seeded samples of Cincinnati tap water did not vary significantly when compared with those of identical samples processed through Filterite and Millipore filters. In studies with raw domestic sewage, virus recoveries were nearly identical from comparable samples filtered through coal and Millipore disk filters. Thus, the availability of coal makes this filter system an inexpensive analytical tool, especially in developing nations, for virus concentration.

Comparison of commercial beef extracts and similar materials for recovering viruses from environmental samples.

Microbiological- and food-grade beef extracts, protein hydrolytic, enzymatic and autolytic digestion products, and whole protein materials were examined for their potential effectiveness for eluting adsorbed enteroviruses from membrane filters with observed efficiencies ranging from less than 1 to 69%. Concentration of enteroviruses from solutions of these protein and protein-derived products by organic flocculation ranged in efficiency from 2 to 125%. Both elution and concentration were dependent upon virus type, as well as nature, source, and production lot of the material being tested. Determining the efficiency of virus concentration was complicated by virus aggregation and apparent virus inactivation by low pH. Effectiveness of concentrating viruses by organic flocculation from solutions prepared with the various test materials seemed independent of the amount of precipitate produced during the flocculation procedure. Quality assurance tests were proposed by which solutions prepared from beef extracts, whole protein, and protein-derived materials could be evaluated for use in eluting adsorbed viruses from membrane filters and for concentrating viruses by organic flocculation. Food-grade beef extract seemed equal to microbiological-grade beef extract in terms of both virus elution and concentration. Several of the nonbeef extract materials evaluated were as effective as beef extract for virus concentration, but were less effective for virus elution.

Processing and transport of environmental virus samples.

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.

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)

Method for recovering viruses from river water solids.

Small numbers of virions (poliovirus 1) that had been adsorbed to river water solids were eluted by mixing the solids for 30 min with a 10% solution of beef extract that contained sufficient Na2HPO4 to bring the molarity of the salt to 0.05 and sufficient citric acid to bring the pH to 7. The virions were recovered by inoculating the beef extract onto cell cultures. With this method, 39 to 63% of the poliovirions that had been adsorbed onto the river water solids were recovered.

Survival of enteric viruses under natural conditions in a subarctic river.

The survival of enteric viruses was studied in the vicinity of Fairbanks, Alaska at selected stations along a 317-km section of the Tanana River. This section was located downstream from all known domestic wastewater sources and was effectively sealed by a total ice cover. The mean flow time through the region was 7.1 days, during which initial viral population showed a relative survival rate of 34%. The tracing of native viruses at such great distances in the complete absence of other point and nonpoint viral sources has not been previously reported. Of the two methods of virus concentration used, viral recoveries from the disk adsorption virus elution procedure were far greater than those achieved with the Aquella system employed at that time. The fact the ratio of enteric viruses to fecal indicator bacteria was not constant clearly inferred that these bacteria were not an effectual measure of virus concentration. The persistence of fecal coliforms and fecal streptococci, however, attested to the microbiological health risk involved.

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.

Recovery of small quantities of viruses from clean waters on cellulose nitrate membrane filters.

A method is described for quantitatively recovering small amounts of viruses from large volumes of buffered, distilled water. Development of the method was motivated by the anticipated need for testing large volumes of renovated sewage for viruses. The method consists of adsorbing viruses onto cellulose nitrate membrane filters (0.45 mum pore size) from water containing sufficient Na(2)HPO(4) to produce a molarity of 0.05 and sufficient citric acid to produce a pH of 7, and eluting the adsorbed viruses in 3% beef extract under extended sonic treatment. Complete recovery of poliovirus 1, echovirus 7, and coxsackievirus B3 resulted when less than 100 plaque-forming units were added to 1-liter quantities of water. Recoveries of reovirus 1 were almost as good. Preliminary studies indicate that good recoveries can be made from 25-gal quantities of water. The method described is efficient in waters of high quality and may be useful for recovering viruses in renovated, and perhaps in tap waters, but not in waters containing certain organic matter unless that matter is first removed.