Daniel R. Dahling

A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater

ABSTRACT Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.

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.

Pilot-Scale Ozone Inactivation of Cryptosporidium and Other Microorganisms in Natural Water

Abstract A pilot-scale study was conducted to evaluate the inactivation by ozone against Cryptosporidium oocysts, Giardia cysts, poliovirus, and B. subtilis endospores spiked into Ohio River water. The indigenous Ohio River populations of total coliform bacteria, heterotrophic plate count bacteria and endospores of aerobic spore forming bacteria were also evaluated. Endospores were the only organisms found to be more resistant to ozone than Cryptosporidium oocysts. Endospores may serve as an indicator of microbial treatment efficiency. Cryptosporidium oocysts were more resistant than Giardia cysts or poliovirus. Although HPC bacteria were less resistant than Cryptosporidium oocysts, variability limits their usefulness as an indicator of treatment efficiency. Ozone inactivation data generated in a pilot-scale study employing natural surface waters were comparable to inactivation data derived from previously published bench-scale studies using laboratory waters. The ozone requirements for inactivation of Cryptosporidium oocysts may produce elevated levels of bromate and ozone byproducts.

Detection and enumeration of enteric viruses in cell culture

Abstract The use of cell cultures remains the most reliable and functional tool for the detection of viable human enteric viruses in environmental samples. There is, however, a definite need for uniformity in cell culture practices. Among the serious concerns repeatedly addressed for virus monitoring have been negative results arising, not from the absence of virus, but from the lack of sensitivity due to significant variations in detection methodology. Over the past 5 years, many cell culture procedures applied to environmental virology have been upgraded, refined, or replaced; thus, the continued use of insensitive methodology may hinder reliable virus monitoring programs. This report provides a singular updated resource, encompassing those changes which have been shown to make a difference in the sensitivity of the cell culture virus detection system.

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.

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.

Results of a survey of BGM cell culture practices

Abstract Ninety-eight laboratories in 16 countries were surveyed in 1979 to determine the uniformity of methods for the assay of human viruses in BGM cells. None of the 58 responding laboratories applied identical methodology. A number of these practices were sufficiently different to assure a significant variance in liter with the assay of standardized virus samples. The results of this survey indicate a definite need for implementing uniform cell culture practices for the enumeration and identification of viruses in the environment.

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.

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.

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.