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The standard method of enterovirus assay and detection involves the use of animal cell culture. The presence of virus is indicated by the destruction of cells, referred to as the cytopathogenic effect (CPE), and requires from 3 to >14 days (8). Often secondary passage of the environmental samples onto a fresh monolayer of cells is necessary before CPE is produced, but secondary passage is not routinely conducted (3, 10). Integrated cell culture-PCR (ICC-PCR) combines the speed and sensitivity of PCR with a cultural viability assay to form a molecular detection method capable of isolating low levels of infectious virus (8). This study determined the efficacy of ICC-PCR to evaluate the chlorine contact time necessary to ensure that all target viruses were inactivated.

Poliovirus type 1 (strain Lsc-2ab) was grown in buffalo green monkey kidney (BGM) cells (passages 135 to 195) at 37°C until CPE occurred. Poliovirus was isolated by freeze-thawing and centrifugation as previously described (8). The titers of virus stocks were determined at 2.3 × 10⁷ PFU/ml using conventional plaque assay methodology (4). All glassware was exposed to >5% sodium hypochlorite overnight, rinsed with high-performance liquid chromatography (HPLC) grade water, and exposed to UV light for at least 1 h to eliminate chlorine demand. Virus stock and sodium hypochlorite were added to 50 ml of chlorine demand-free HPLC grade water for a final concentration of 2.3 × 10² PFU/ml and 0.5 mg/liter, respectively. Chlorine levels were measured at room temperature and pH 5.5 using a spectrophotometer (model 2000; Hach, Loveland, Colo.). Three replicate studies indicated that chlorine concentrations remained constant for longer than the duration of the experiments, 10 min.

BGM continuous cell culture flasks (25 cm²) were incubated with 1 ml of sample at 37°C for time intervals of 0, 1, 2, 3, 4, and 14 days before freezing at 80°C for PCR analysis. For conventional cell culture assays, flasks were incubated up to 14 days and observed daily for CPE. Secondary BGM assays were performed with lysate from the first passage, but the flasks were not frozen until CPE, or another 2-week incubation, occurred. Prior to PCR analysis, samples were centrifuged (model 5415C; Sorvall, Newtown, Conn.) for 15 min at 735 × g and 5°C to remove cellular debris.

Target sequences were reverse transcribed as previously described (8). Cell culture lysate template (5 µl) was added to reverse transcription reagents and extracted by heat (5 min at 98°C), followed by a single cycle of 24°C for 10 min, 44°C for 60 min, 99°C for 5 min, and 5°C for 5 min. For cDNA amplification, 1.5 µl of 25 mM MgCl₂, 3.5 µl of 10× buffer II (Perkin-Elmer Cetus Corporation, Norwalk, Conn.), 0.25 µl of upstream primer at 50 pmol/reaction mixture, 0.25 µl of downstream primer (2) at 50 pmol/reaction mixture, 28.75 µl of HPLC grade water, and 0.25 µl of AmpliTaq Gold Polymerase (Perkin-Elmer Cetus Corporation) were added per 50-µl reaction mixture. Samples were amplified by single PCR as follows: (i) 95°C for 5 min; (ii) 25 cycles, with 1 cycle consisting of 99°C for 1 min, 60°C for 45 s, and 72°C for 1 min; and (iii) a single 10-min extension step at 72°C. For seminested PCR, 5 µl of the single PCR mixture volume was added to 50-µl reaction mixtures of fresh reagents with the upstream external primer and a downstream primer internal to the sequence amplified in single PCR (7). Amplification conditions were identical to those used in single PCR for an additional 20 cycles.

PCR products (15 µl) were mixed with 3 µl of Ficoll loading buffer (20% Ficoll, 1% sodium dodecyl sulfate, 0.25% bromophenol blue, and 0.1 M EDTA [pH 8.0]) and subjected to 1.5% low-electroendosmosis agarose gel electrophoresis at 100 V for 1.75 h (Fisher Scientific, Fair Lawn, N.J.). The gel was stained with SyberGold for 45 min and evaluated with an AlphaImager 2000 (Alpha Innotech Corporation, San Leandro, Calif.) for the presence of PCR and seminested PCR product bands at 195- and 106-bp locations, respectively. Positive seminested PCR products were prepared for dye termination sequencing (University of Arizona's Laboratory of Molecular Systematics and Evolution sequencing facility) using a QIAquick-Gel Extraction Kit per the manufacturer's suggestions (QIAGEN, Inc., Valencia, Calif.).

Previous studies indicate that poliovirus inactivation with free chlorine occurs after 2 min of exposure to 0.5 mg/liter, yielding a required C_t value (concentration of disinfectant × time of exposure) of 1.0 (1, 5). Using first-passage cell culture as the method for detection of infectious virus, the results of this study agree with previous studies (Table 1). However, ICC-PCR provided a more rapid and effective detection method, since analysis of cell culture lysate from first-passage samples demonstrated that infectious poliovirus was still present even after 6 min of exposure to 0.5 mg of free chlorine per liter. A second passage in continuous cell culture confirmed that not only was poliovirus present but it was also cytopathogenic. Furthermore, ICC-PCR-positive results required only 2 days of incubation in cell culture versus up to 14 or more days with conventional cell culture. Ultimately, both methodologies yielded the same result, but cell culture alone required a second assay to accurately establish necessary contact time. At 8 min of exposure to 0.5 mg of free chlorine per liter, most samples were completely disinfected, as determined by two passages in cell culture and ICC-PCR analysis. Clearly, 8 min was close to the threshold value for complete disinfection, since a second passage in cell culture showed that only two of the six replicates harbored infectious poliovirus. ICC-PCR results from the second-passage lysates confirmed these results. Only after 10 min of exposure to 0.5 mg of free chlorine per liter was the poliovirus totally inactivated in all samples, yielding a required C_t value of 5.0. DNA sequencing analysis of the PCR product indicated that the observed virus was poliovirus type 1 (data not shown).

In conclusion, ICC-PCR provides a more sensitive and rapid method for detection of infectious poliovirus following chlorine disinfection compared to conventional cell culture. This new application of ICC-PCR proved to be more effective at determining the potency of chlorine disinfection, minimizing the chance for false-negative results with single-passage cell culture alone. The results of this study suggest that previous research to determine the resistance of viruses to chlorine may have underestimated the resistance of at least poliovirus populations, as previous studies involved only direct cell culture assay of the virus with no secondary passage and no detection via ICC-PCR (3, 10). This has serious implications since the determination of inactivation rates of waterborne virus is crucial to the drinking water industry. The amount of chlorine that must be applied is determined by the contact time required to inactivate a given percentage of a pathogen. The results of this study suggest that current contact values could be in error of up to 400% (C_t values of 1 versus 5) (1, 11) and that a reevaluation of the contact time necessary for the true disinfection of all infectious viruses present may be needed. This could help to explain why infectious viruses have been detected in drinking water after receiving what was believed to be adequate disinfection (6, 9). Additional studies are needed to determine the exact mechanism of resistance and if other types of enteric viruses or disinfectants present similar phenomena.