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Appl Environ Microbiol. 2010 July; 76(13): 4318–4326.
Published online 2010 May 14. doi:  10.1128/AEM.02800-09
PMCID: PMC2897418

Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples[down-pointing small open triangle]

Abstract

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

Waterborne enteric viral illness is common worldwide (18). The enteric viruses that may be transmitted through water include enteroviruses, such as poliovirus, coxsackievirus, and echovirus; human caliciviruses, such as noroviruses (NoV) and sapoviruses; rotaviruses; hepatitis A virus (HAV); and adenoviruses. Enteroviruses can cause mild to severe and life-threatening illnesses ranging from mild gastroenteritis and upper respiratory tract infections to encephalitis, meningitis, and myocarditis (40). Noroviruses are the second most common cause of viral gastroenteritis next to rotaviruses worldwide (38). In recent years, a number of these enteric viruses have been the etiological agents of several waterborne outbreaks (1, 2, 10, 14, 21, 29, 32).

Currently, there are three primary approaches for detection of these viruses. The first approach is propagating the viruses in tissue culture and determining their cytopathic effects (CPE). While this approach yields information about the infectivity of a virus, it is expensive, labor-intensive, and time-consuming. It can take several weeks for detection of the CPE of some environmental strains using the Buffalo green monkey kidney (BGM) cell line that is often used for studies of the occurrence of enteric viruses in environmental water (12). In addition, the tissue culture method is not feasible for viruses which are not cytopathic in BGM cells, such as HAV and rotaviruses, and for noroviruses, which do not grow in established cell culture systems. Although noroviruses have been reported to grow in highly differentiated three-dimensional (3D) cell cultures (42), this system is labor-intensive and requires specialized equipment and extensive experience in the maintenance of 3D cell cultures.

The second approach for virus detection is PCR, which can be performed with and without reverse transcription for RNA and DNA viruses, respectively. PCR is rapid, sensitive, and specific and can be made quantitative by use of real-time quantitative PCR (qPCR) techniques. This approach, however, detects virus nucleic acids of both infectious and noninfectious viruses, which limits conclusions regarding the significance for public health. A third approach for virus detection is integrated cell culture PCR (ICC-PCR) (8, 35, 36). This approach combines the advantages of both tissue culture and PCR while overcoming some of the limitations of each of these methods. Viruses that replicate but do not produce cytopathic effects can potentially be detected, and this method can be performed in ways that detect only infectious virus; however, ICC-PCR does not currently detect the important norovirus group. ICC-PCR detects virus infectivity faster than cell culture alone, but this method is still labor-intensive, and it is at least 2 or more days before results can be obtained. Researchers have used this technique for detection of infectious adenoviruses (20) and other enteric viruses (8, 16, 19, 22, 35, 36).

From a public health protection perspective, the risk of exposure to enteric viruses and effective management strategies are determined better if there is information about the infectivity of the virus detected, which limits the value of studies using PCR to measure the occurrence of enteric viruses in recreational and drinking water. Virus detected by PCR may have been rendered noninfectious by natural die-off or by disinfection, and studies which have followed viral nucleic acids and infectivity in water environments have shown that nucleic acids last longer than infectivity (13). Therefore, for investigations of outbreaks and for monitoring recreational and drinking water, there is interest in a rapid approach that can distinguish between infectious and noninfectious virus particles (4, 37). Nuanualsuwan and Cliver (28) have demonstrated the feasibility of this type of approach for RNA viruses under certain conditions. These workers treated virus samples with protease and RNase prior to PCR. In theory, noninfectious viral capsid proteins should be more sensitive to cleavage by proteases, thus exposing the viral RNA to degradation by RNase. This approach was useful for viruses that were inactivated by treatment at 72°C, with chlorine, and with UV but not for viruses that were inactivated by treatment at 37°C (28). Baert et al. (4) also used the approach of Nuanualsuwan and Cliver to examine the infectivity of murine norovirus following heating at 80°C. They reported that heat treatment at 80°C for 150 s resulted in a 6.5-log10 reduction in infectivity but no decrease in the ability to detect the virus genome by qPCR. Treatment of noninfectious virus with proteinase K and RNase prior to qPCR resulted in only an additional 0.2-log10 reduction compared with that observed without this treatment. Thus, protease and RNase treatment may not be applicable to all types of RNA viruses.

Ethidium monoazide (EMA) and propidium monoazide (PMA) are closely related DNA intercalating dyes with a photo-inducible azide group that covalently cross-links to DNA upon exposure to bright light (25). While studies with EMA have not been successful, PCR in conjunction with PMA has been used to distinguish between viable and nonviable bacteria (25, 26, 30) and fungi (45). PMA has also been used for detecting the viability of Giardia cysts (39) and Cryptosporidium oocysts (6). Recently, in studies similar to studies with bacteria, EMA did not distinguish between infectious and noninfectious avian influenza virus particles (15).

In this study we investigated the use of PMA to distinguish between infectious and noninfectious enteric RNA viruses. We developed a PMA-reverse transcriptase PCR (RT-PCR) assay, and here we report the results of a proof-of-concept study. Data obtained in this study suggest that pretreatment of viruses with PMA prior to RT-PCR is a reliable method for distinguishing between infectious and noninfectious viruses that were inactivated by treatment at 72°C or 37°C or by using hypochlorite. To our knowledge, this is the first report of application of this technique to enteric RNA viruses.

MATERIALS AND METHODS

Virus strains.

The enteric virus strains used in this study have positive-sense, single-stranded RNA. The specific viruses used in the study were poliovirus type 1, echovirus 7, coxsackievirus B5, and Norwalk virus (NV) (obtained from Gary Richards of the National Marine Fisheries Service). NV was extracted from stool samples as described by Parshionikar et al. (32).

Thermal inactivation of viruses.

Poliovirus, coxsackievirus B5, and echovirus 7 were inactivated thermally in phosphate-buffered saline (PBS) (pH 7.0) using water baths set at 19°C, 37°C, and 72°C. An initial concentration of 1,000 PFU/ml was used for each enterovirus to minimize multiple inactivation events, as recommended by Nuanualsuwan and Cliver (28). For inactivation at 19°C and 37°C, 50-ml conical tubes containing 1,000 PFU/ml of poliovirus, coxsackievirus B5, or echovirus 7 were placed in the appropriate water baths, and samples were monitored weekly for inactivation by performing plaque assays. For thermal inactivation at 72°C, 1.5-ml microcentrifuge tubes containing 500 μl of 1,000 PFU/ml virus were placed in water warmed by a heating block. The virus in each tube was incubated at 72°C for 5, 5.5, 6.0, or 6.5 min, quickly cooled by adding the inactivated virus to 4.5 ml of prechilled PBS (Sigma, St. Louis, MO), and then placed on ice. Inactivation of the virus, as measured by loss of infectivity at each time point, was analyzed by performing a plaque assay. For all inactivation temperatures, the lack of infectivity determined by the plaque assay was confirmed using the residual infectivity assay (see below). For enteroviruses, the terms infectious and noninfectious are used below to describe the native and inactivated states, respectively.

NV at a titer of 1,000 genomic copies/ml (see below) was inactivated thermally by incubating the virus in 50-ml conical tubes in 19°C and 37°C water baths for 4 weeks and 2 weeks, respectively. Since NV infectivity cannot be measured by the tissue culture method, the virus was assumed to have lost infectivity at the end of the heat treatment. Therefore, for Norwalk virus, the terms native and inactivated are used below.

Plaque assays.

Plaque assays were performed as described by Dahling and Wright (9). Briefly, stock cultures of the Buffalo green monkey kidney (BGM) cell line in 25-cm2 flasks were washed with Earle's balanced salts solution and then inoculated with 0.5 ml of a viral sample. Following a 60- to 90-min adsorption period, each infected BGM cell monolayer was overlaid with 10 ml of 1× minimum essential medium (MEM) overlay medium. The final concentrations of the ingredients of this medium are as follows: 1.5% agar, 2% calf serum, 2% dehydrated Bacto skim milk, 0.05% neutral red, 3% NaHCO3, 0.5% MgCl2, 0.05% tetracycline, 0.1% penicillin-streptomycin, and 0.02% amphotericin B (Fungizone). The flasks were then incubated at 37°C and checked daily for plaques for 1 week.

Residual infectivity assay.

To verify that all virus particles were inactivated, the residual infectivity test was carried out using a quantal assay. This assay is often more sensitive for virus detection than the plaque assay (41). The quantal assay was performed by adding 0.5 ml of a virus sample to a BGM cell monolayer on the inside surface of a cell culture tube. After the virus was allowed to adsorb for 60 to 90 min, 2 ml of Earle's balanced salts solution with 2% calf serum was added. The cell culture tubes were incubated at 37°C in a rotating apparatus and observed daily for cytopathic effects for 7 days.

Hypochlorite inactivation of viruses.

Poliovirus, coxsackievirus B5, and echovirus 7 were diluted to obtain a concentration of 1,000 PFU/ml in 100 ml of prechilled demand-free phosphate buffer with a concentration of free chlorine of 0.5 mg/liter. This solution was placed in a 4°C refrigerator. Ten-milliliter samples were removed after 0.5, 1, 2, 5, 10, and 20 min and added to tubes containing 0.5% thiosulfate to neutralize any remaining free chlorine and to stop inactivation. Inactivation was examined at each time point by measuring the loss of infectivity with the plaque assay and the residual infectivity assay. The sample for the first time point at which infectivity was completely absent was used as the hypochlorite-inactivated virus sample in all subsequent analyses.

Propidium monoazide treatment.

PMA {phenanthridium, 3-amino-8-azido-5-[3-(diethylmethylammonio)propyl]-6-phyenol dichloride; Biotium, Inc., Hayward, CA} was reconstituted with 20% dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co., St. Louis, MO) to obtain a concentration of 1 mg/ml and was stored at −20°C. In a dark room, 25 μl of PMA was added to 100 μl of an infectious or native virus sample or a noninfectious or inactivated virus sample containing 100 PFU of enteroviruses or 100 genomic copies of NV in a 1.5-ml microcentrifuge tube, and the final concentration was adjusted to 100 or 200 μM with molecular-grade water. All sample tubes were then placed on a rocker, and the contents were mixed for 5 min. After mixing, the tubes were placed on their sides on ice to prevent overheating and exposed to a 650-W light at a distance of 20 cm for 3 min. Once the exposure to light was complete, the room lights were turned on, and the viral RNA was extracted (see below). The extracted RNA was used as a template in RT-PCR and quantitative RT-PCR experiments. RT-PCR assays performed with viruses that were pretreated with PMA are referred to below as PMA-RT-PCR assays. To determine whether PMA interferes with the ability of RT-PCR to detect viruses, controls consisting of viruses that were treated with PMA without exposure to light were included with each set of reactions. These controls are referred to below as PMA with no light. All PCR data in this paper are data that are representative of at least two experiments.

Viral RNA extraction.

Viral RNA was extracted using a QIAamp viral RNA minikit (Qiagen, Valencia, CA) according to the manufacturer's instructions. After the RNA was eluted into a 1.5-ml microcentrifuge tube, it was stored at −20°C, and it was subsequently thawed and analyzed by RT-PCR.

Conventional RT-PCR.

Primers used in this study are shown in Table Table1.1. Reverse transcription was performed using a 30-μl mixture. Ten microliters of an RNA sample was added to buffer containing 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, each deoxynucleoside triphosphate (dNTP) at a concentration of 20 nM, and 50 pmol of virus-specific cDNA sense primer. The reaction mixture was overlaid with 50 μl of mineral oil. Then 7.5 U of murine leukemia virus (MuLV) reverse transcriptase and 30 U of RNasin were added, and the reaction mixture was incubated at 43°C for 60 min and then at 95°C for 5 min. Each PCR was performed by adding 70 μl of a mixture containing 10 mM Tris (pH 8.3), 50 mM KCl, 3 mM MgCl2, 50 pmol of virus-specific RNA sense primer, and 5 U of Amplitaq Gold polymerase. Viral cDNA was amplified for 40 cycles, each of which consisted of 1 min at 95°C followed by 55°C for 1 min and 72°C for 1.5 min, and then it was kept at 4°C.

TABLE 1.
Sequences of primers and probes used for RT-PCR and qRT-PCR

Estimating the Norwalk virus genome copy number in stool samples.

The genome copy number for Norwalk virus was estimated using a Norwalk virus internal control (NORIC) as an internal standard in a TaqMan quantitative RT-PCR (qRT-PCR) assay. The development of NORIC has been described previously (31). NORIC was encapsidated with phage MS2 coat proteins by using the armored quant RNA technology of Ambion Inc. (33) and was supplied at a concentration of 1 × 1010 genome copies/ml. The armored NORIC was used to generate a standard curve for estimating the titer of Norwalk virus expressed in genome copies/ml (data not shown).

Quantitative RT-PCR.

The primers and probes used for qRT-PCR were designed using the Primer Express software (Applied Biosystems, CA) and are shown in Table Table1.1. qRT-PCR was performed with 10-fold serial dilutions of armored quant NORIC RNA and NV extracted from stool samples. A standard curve was obtained by plotting the threshold cycle (CT) value versus the log copy number. A qRT-PCR was also performed using RNA extracted from infectious or native viruses and noninfectious or inactivated viruses pretreated with PMA as described above. qRT-PCR was performed in two steps. First, reverse transcription was performed with a 25-μl mixture using a TaqMan core reagent kit obtained from Applied Biosystems Inc. The RT-PCR mixture contained 1.5 mM MgCl2, each dNTP at a concentration of 20 nM, 200 nM primer, 2.5 μl of 10× TaqMan buffer A, and 5 μl of the appropriate viral RNA or NORIC dilution. The reaction mixture was overlaid with 50 μl of mineral oil. Tubes containing NORIC were heated to 75°C for 5 min. Then 7.5 U of MuLV and 30 U of RNasin were added, and the reaction was performed using a program consisting of 43°C for 60 min, followed by 95°C for 5 min. Twenty-five microliters of a PCR mixture containing 5 mM MgCl2, 200 nM primer, 2.5 μl of 10× TaqMan buffer A, 20 nM probe, and 5 U of Amplitaq Gold polymerase was added to the cDNA. The cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min.

Calculation of virus inactivation.

The decrease in the poliovirus concentration caused by inactivation was calculated as follows: 2(average cycle threshold value of inactivated virus − average cycle threshold of native virus). The decrease in the poliovirus concentration caused by PMA treatment of noninfectious virus was calculated as follows: 2(average cycle threshold of inactivated virus pretreated with PMA − average cycle threshold of inactivated virus without PMA treatment).

Environmental water concentrates.

Environmental water concentrates were prepared as described by Fout et al. (11). Briefly, standard filter apparatuses (12) containing 1MDS positively charged, 10-in. cartridge filters were used to filter 200 liters of Ohio River water. The filters were eluted twice with 1.6 liters of 1.5% beef extract (Adams Scientific, West Warwick, RI) (pH 9.0), and the virus in each eluate was concentrated using 0.1% Celite at pH 4.0. The virus was eluted from the Celite with 0.15 M sodium phosphate (pH 9.0). The pH of the concentrate was adjusted to 7.0, and the concentrate was passed through a 0.2-μm-pore-size Acrodisc filter (Pall Gelman Laboratory, Ann Arbor, MI) and then treated for removal of inhibitors. Briefly, a portion of each concentrate was concentrated further by ultracentrifugation through a 30% sucrose layer and then treated with 0.01% dithiozone (diphenylthiocarbazone)-0.01 M 8-hydroxyquinoline-butanol-methanol-trichloroethane (0.1:0.9:1:0.25:0.25, vol/vol/vol/vol/vol). Stock solutions containing 0.01% dithiozone (Fisher Scientific, Pittsburgh, PA) and 0.01 M 8-hydroxyquinoline (Fisher Scientific) were prepared using chloroform. The aqueous layer was separated from the solvent layer by centrifugation, and then each sample was further concentrated using Microcon-100 filter units (Amicon, Inc., Beverly, MA) as described by the manufacturer. Tap water was processed similarly as a control. Dilutions (0, 10−1, 10−2, and 10−3) of Ohio River water concentrate after removal of inhibitors were seeded with 100 PFU of infectious and noninfectious poliovirus. PMA-RT-PCR was performed as described above using 15-μl portions of the dilutions listed above with 100 μM and 200 μM PMA separately. PMA-RT-PCR assay mixtures with seeded tap water samples were used as controls.

Dot blot hybridization.

Dot blot hybridization was performed as described by Fout et al. (11). Briefly, 15.75 μl of each RT-PCR product was denatured with 0.1 M NaOH and 0.4 M EDTA for 10 min. Ammonium acetate was added at a final concentration of 2 M for neutralization, and 50 μl of the mixture was spotted onto Magnagraph nylon membranes using a microsample filtration manifold (Schleicher & Schuell, Keene, NH). DNA on the membranes was UV cross-linked for 1 min. The membranes were prehybridized for 1 h at 51°C and then hybridized for 18 h at 51°C with individual samples labeled with digoxigenin-ddUTP (DIG Oligo 3-end labeling kit; Roche Molecular Biochemicals, Indianapolis, IN) as described by the manufacturer. The membranes were washed at 51°C in 0.25× SSC (prepared from 20× SSC from Fisher Scientific, Pittsburgh, PA) (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Following the stringency wash, the membranes were blocked and treated with anti-digoxigenin-alkaline phosphatase conjugate (Roche, Indianapolis, IN) as specified by the manufacturer. Hybridized probes were detected using the chemiluminescent substrate chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) (Tropix, Foster City, CA), and the blots were exposed to X-ray film (Eastman Kodak, Rochester, NY).

RESULTS

PMA-RT-PCR.

Coxsackievirus B5, echovirus 7, poliovirus, and Norwalk virus were inactivated thermally and by using hypochlorite. The infectious or native viruses and noninfectious or inactivated viruses were then treated with PMA, RNA was extracted, and RT-PCR was performed. Figure Figure11 shows the results obtained when poliovirus was rendered noninfectious (data not shown) by treatment at 72°C. The presence of PMA, either with or without exposure to light, had no effect on detection of infectious poliovirus (Fig. (Fig.1,1, compare lane 3 with lanes 1 and 2). The 195-bp poliovirus amplicon was produced when noninfectious virus was assayed without PMA treatment (lane 8). When noninfectious virus was assayed with PMA treatment, the expected amplicon was observed when light treatment was not used, but not it was not observed when light treatment was used (lanes 7 and 6). This indicates that PMA can covalently bind to viral RNA, but only when it is exposed to light. The results for inactivation of coxsackievirus B5 and echovirus 7 by treatment at 72°C were identical to those shown in Fig. Fig.11 (data not shown). Figure Figure22 shows a summary of the results of experiments performed with enteroviruses rendered noninfectious by treatment at 37°C and by treatment with hypochlorite. The results shown in Fig. Fig.22 and the associated control results (not shown) were identical to the results shown in Fig. Fig.11.

FIG. 1.
Analysis of poliovirus inactivated at 72°C. Infectious or native virus (NPV) (lanes 1 to 3) and noninfectious or inactivated virus (IPV) (lanes 6 to 8) was treated with PMA (lanes 1, 2, 6, and 7) with cross-linking by light (L) (lanes 1 and 6) ...
FIG. 2.
Composite image of individual gels showing results for enteroviruses inactivated at 37°C (panel A, lanes 1 to 6) or by hypochlorite (panel B, lanes 7 to 12). Infectious or native poliovirus (NPV) (lanes 1 and 7), coxsackievirus (NCXB5) (lanes ...

Since it was not possible to determine the exact time at which NV was inactivated by exposure to 72°C, several time points (0.5 to 10 min) were examined by using PMA-RT-PCR. Figure Figure33 shows that treatment times as short as 0.5 and 1 min at 72°C were sufficient to prevent detection of Norwalk virus when it was pretreated with PMA. When Norwalk virus was inactivated at 37°C for 1 month, the PMA-RT-PCR results were negative, while the results for native Norwalk virus were positive (Fig. (Fig.4).4). However, the amount of amplicon for the inactivated virus that was not treated with PMA was reduced, and no product was seen for inactivated virus that was treated with PMA in the absence of light.

FIG. 3.
Analysis of Norwalk virus inactivated at 72°C. Native virus (NNV) (lanes 1 to 3), virus inactivated for 0.5 min (INV-0.5 Min.) (lanes 7 to 9), and virus inactivated for 1.0 min (INV-1.0 Min.) (lanes 10 to 12) were treated with PMA (lanes 1, 2, ...
FIG. 4.
Analysis of Norwalk virus inactivated at 37°C. Native virus (NNV) (lanes 1 to 3) and inactivated virus (INV) (lanes 6 to 8) were treated with PMA (lanes 1, 2, 6, and 7) with cross-linking by light (L) (lanes 1 and 6) or without light (NL) (lanes ...

The PMA-RT-PCR results for all enteroviruses rendered noninfectious by treatment at 19°C and for Norwalk virus inactivated by treatment at 19°C were positive, like the results for their native counterparts (data not shown). Table Table22 shows a summary of the results of PMA-RT-PCR analyses of infectious or native viruses and noninfectious or inactivated viruses.

TABLE 2.
Summary of PMA-RT-PCR results for enteric viruses used in this study

PMA-qRT-PCR.

PMA-qRT-PCR was performed with infectious and noninfectious poliovirus. Poliovirus primers were designed for the highly conserved 5′ untranslated region (5′ UTR), and they generated a 144-bp amplicon. For Norwalk virus, two primer-probe combinations were used to target two different amplicons (59 and 96 bp). The same virus RNA samples that were used in the conventional PMA-RT-PCR were used in the PMA-qRT-PCR.

Poliovirus rendered noninfectious by treatment at 37°C and poliovirus treated with PMA and light had undetectable CT values (>40) when PMA-qRT-PCR was performed (Table (Table3).3). This result was in agreement with the conventional RT-PCR results (Fig. (Fig.11 and and2).2). PMA-qRT-PCR showed that the overall relative virus concentration decreased about 13-fold during inactivation (Table (Table3).3). PMA pretreatment of noninfectious poliovirus resulted in an undetectable CT value, and there was at least 6-fold decrease in addition to that due to inactivation alone.

TABLE 3.
Detection of poliovirus and Norwalk virus by PMA-qRT-PCR

Like the concentration of poliovirus, the overall relative concentration of Norwalk virus inactivated by treatment at 37°C decreased about 11-fold (Table (Table3).3). Unlike the results for poliovirus, however, PMA treatment resulted in only a small additional decrease in the virus titer (about 1.4-fold).

A higher PMA concentration (200 μM) was tested next to determine whether this concentration reduced the detection of inactivated NV virus by qRT-PCR. Once again, there was little difference in the CT values for native and inactivated NV (data not shown). A primer set designed for a different and slightly larger region of the RNA polymerase gene was also tested, and there was no difference in the CT values for native and inactivated NV (data not shown).

Limit of detection.

In order to determine the limit of detection of the assay, PMA-RT-PCR was performed with 100 PFU, 10 PFU, 1 PFU, 0.1 PFU, 0.01 PFU, and 0.001 PFU of infectious and noninfectious poliovirus (based on the original native titer). The detection limit of this assay was 0.1 PFU (data not shown).

PMA-RT-PCR assay with mixed samples (infectious virus and noninfectious virus).

It is thought that the populations of contaminating enteric viruses in water samples usually are mixtures of infectious and noninfectious viruses. Therefore, it is important that the PMA-RT-PCR assay is able to distinguish infectious viruses from a mixed pool of infectious and noninfectious viruses. To determine whether the assay can do this, 10 PFU of infectious poliovirus and 10 PFU of noninfectious poliovirus were mixed, and the PMA-RT-PCR assay was performed. As shown in Fig. Fig.5,5, the PMA-RT-PCR results for the mixture of infectious virus and noninfectious virus were positive, indicating that the presence of noninfectious virus did not interfere with the detection of infectious poliovirus.

FIG. 5.
Analysis of poliovirus inactivated at 37°C. Infectious or native virus (NPV) (lanes 1 to 3), noninfectious or inactivated virus (IPV) (lanes 6 to 8), or a mixture of infectious and noninfectious viruses (Mix) (lanes 9 to 11) were treated with ...

PMA-RT-PCR with seeded environmental water samples.

To test the effect of environmental water matrices on the PMA-RT-PCR assay, 0, 10−1, 10−2, and 10−3 dilutions of Ohio River water concentrates were seeded with 100 PFU of infectious and noninfectious poliovirus. Plaque assays with the Ohio River water concentrate showed that it did not contain infectious enteroviruses (data not shown).

This seeded sample was treated with 100 μM and 200 μM PMA. Figure Figure66 shows that the matrix did not interfere with detection of poliovirus seeded into the sample or with the ability of PMA at a concentration of 200 μM to prevent detection of inactivated virus. The results of tests with 100 μM PMA were the same, except that many more nonspecific bands were observed, as has been reported previously for environmental matrices examined with the primer set used (11). All results were confirmed by dot blot hybridization (data not shown).

FIG. 6.
Analysis of undiluted Ohio River water concentrates (lanes 1 and 5) and concentrates diluted 1:10 (lanes 2, 6, and 9), 1:100 (lanes 3, 7, and 10), or 1:1,000 (lanes 4, 8, and 11) that were seeded with infectious or native poliovirus (NPV) (lanes 1 to ...

DISCUSSION

Routine water quality monitoring for human enteric viruses, gathering of virus occurrence data, and the interpretation of PCR data are hampered by the limitations of the current enteric virus detection techniques in spite of the improvements made in recent years. Methods that yield information regarding infectivity are of particular interest for detection of enteric viruses. Generally, for public health protection, the usefulness of a method is determined by its applicability to all culturable and nonculturable environmental strains, its detection of viral infectivity, and the rapidity with which results are obtained.

In this study, we describe development of a novel PMA-RT-PCR assay for enteric RNA viruses that is based on pretreatment of viruses with propidium monoazide prior to RT-PCR. This assay is based on penetration of PMA through damaged or compromised capsids of inactivated and noninfectious viruses and its covalent binding to viral RNA upon exposure to visible light, which makes the RNA unavailable for amplification by RT-PCR. This assay allows differentiation between infectious and noninfectious viral particles. Also, because this assay is PCR based, it is rapid and potentially applicable to all culturable and nonculturable viruses. To our knowledge, this is the first study that used propidium monoazide in conjunction with RT-PCR to successfully distinguish between infectious and noninfectious enteric RNA viruses under the defined conditions described below.

To develop this assay, poliovirus 1, coxsackievirus B5, and echovirus 7 were selected as representative viruses that can be easily cultured. Norwalk virus (genogroup I.1) was used as a representative of viruses that cannot be grown in cell culture but which often cause waterborne outbreaks. Although culturable surrogates of human norovirus could have been chosen, the commonly used feline calicivirus is not an enteric virus and thus is not an appropriate surrogate for human noroviruses in drinking water (3, 7, 44), and murine norovirus was not available for this study. With the aim of causing differential capsid damage, we used three temperatures for inactivation of viruses; 72°C was chosen because it caused the most capsid damage, while 37°C was chosen because it caused more subtle capsid damage. In addition, these temperatures were used in a protease-RNase study of Nuanualsuwan and Cliver (27, 28). A temperature of 19°C was because it represents the average ambient surface water temperature in the United States (U.S. Environmental Protection Agency ICR Aux 1 database). Inactivation at 19°C was expected to result in the subtlest form of capsid damage. Inactivation by hypochlorite was used because it caused damage resembling capsid damage resulting from conventional drinking water treatment.

For enteroviruses inactivated at 72°C, at 37°C, and by hypochlorite, the PMA-RT-PCR assay (with a PMA concentration of 100 μM) was able to distinguish between infectious and noninfectious viruses (Fig. (Fig.11 and and2).2). However, the PMA-RT-PCR assay was not able to distinguish between infectious enteroviruses and noninfectious enteroviruses that were inactivated at 19°C (Table (Table2).2). This indicated that although these viruses lost infectivity, as shown by tissue culture assays, their capsids did not allow penetration of PMA. Poliovirus has been demonstrated to be a dynamic virus that is capable of undergoing conformational alterations at physiological temperatures (23). Because changes in capsid protein conformation can result in loss of infectivity as measured by tissue culture assays without compromising capsid integrity (28), future studies should also include HAV inactivated at 19°C to test binding to homologous receptors to better understand the mechanism of capsid damage in viruses inactivated at 19°C. Increased incubation time with PMA, changes in pH, and addition of proteases with PMA should also be tested to see if it is possible to identify conditions that distinguish between infectious viruses and noninfectious viruses that are inactivated at 19°C and to determine what the positive PMA-RT-PCR results for viruses rendered noninfectious by treatment at 19°C actually mean. Future studies should also address the effect of PMA on UV-exposed enteric viruses. In bacteria, the loss of viability after a short exposure to UV could not be monitored with PMA as UV light causes DNA damage without directly affecting membrane permeability (26). It is possible that the same effect will be observed with UV-treated enteric viruses, but pretreatment with a protease or some other capsid-disrupting aid in conjunction with PMA may be warranted (28).

The results for Norwalk virus were similar to the results for enteroviruses in that PMA-RT-PCR was able to distinguish between native virus and virus inactivated at 37°C and 72°C (Fig. (Fig.33 and and4)4) but it was not able to distinguish between native virus and virus inactivated at 19°C (Table (Table2).2). Note that in the case of Norwalk virus complete inactivation and thus loss of infectivity were only assumed to occur as complete inactivation and loss of infectivity could not be confirmed by tissue culture. Research with murine norovirus, which is a culturable surrogate for human noroviruses, is necessary to confirm the data presented here.

Besides being rapid, qRT-PCR reduces the level of false-positive results by reducing post-PCR handling of amplicons. In order to test whether PMA-qRT-PCR performs like PMA-RT-PCR, PMA-qRT-PCR was performed with infectious poliovirus and poliovirus rendered noninfectious by treatment at 37°C. The lack of detection of CT values for inactivated virus indicated that there was agreement between the PMA-RT-PCR and PMA-qRT-PCR results. However, the PMA-qRT-PCR assay demonstrated that the overall virus concentration decreased 13-fold after treatment at 37°C. This overall decrease would be expected if inactivated virus were degraded and thus more susceptible to contaminating nucleases during the inactivation period or during sample processing.

The PMA-qRT-PCR results obtained with native and inactivated Norwalk virus (Table (Table3)3) were not consistent with the PMA-RT-PCR results (Fig. (Fig.3).3). With two different real-time primer-probe sets and even when the PMA concentration was doubled, PMA-qRT-PCR could not be used to distinguish between native and inactivated Norwalk virus. As observed for inactivation of poliovirus, inactivation of Norwalk virus at 37°C resulted in a ~11-fold decrease in the overall virus concentration (Table (Table3).3). However, PMA treatment resulted in only an additional ~1.4-fold decrease, suggesting that PMA-qRT-PCR cannot distinguish infectious Norwalk virus from noninfectious Norwalk virus with the primer sets used.

Based on binding of ethidium bromide to DNA, PMA binds to DNA by intercalating between the nucleotide heterocyclic bases with little or no sequence preference and with a stoichiometry of one dye molecule per 4 or 5 bp (24, 25, 48). Because intercalation is required for covalent binding to DNA via the photo-inducible azide moiety, it is likely that the secondary structure of the RNA genome plays a role in the observed differences between poliovirus and Norwalk virus PMA-qRT-PCR results. The qRT-PCR primer set used for enteroviruses is targeted to the 5′ UTR. This region has a strong secondary structure (5, 34). The qRT-PCR primer sets used for Norwalk virus are targeted to the RNA polymerase region. When the secondary structures of the different primer set regions were examined, the targeted enterovirus region had a minimum free energy of −45.8 kcal/mol (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi), while the energies for the two qRT-PCR Norwalk virus primer sets used were −11 and −20.4 kcal/mol. Whether these secondary structures exist in intact viral particles is unknown, but because of its high free energy, encapsidated poliovirus RNA is more likely to retain its secondary structure. This can explain the consistency of the poliovirus PMA-RT-PCR and PMA-qRT-PCR results as regions targeted by both primer sets have high free energy. The presence of the secondary structure in intact virus particles can also explain the difference between PMA-qRT-PCR results (Table (Table3)3) and PMA-RT-PCR results (Fig. (Fig.4)4) for Norwalk virus, as the minimum free energy of the region targeted by the PMA-RT-PCR primers is −90 kcal/mol.

The stable secondary structures may facilitate covalent binding of PMA to viral RNA, rendering the RNA undetectable by RT-PCR. Future research should include further optimization of the PMA-qRT-PCR for Norwalk virus by development of a real-time RT-PCR primer set to amplify a region with stronger secondary structure. However, in light of the low infectious dose of enteric viruses (43, 47), until a PMA-qRT-PCR assay for Norwalk virus is developed, the qualitative, presence-absence PMA-RT-PCR assay will provide valuable information concerning the risk of virus exposure.

To test whether environmental matrices interfered with the ability of the PMA-RT-PCR assay to detect only infectious virus, the assay was used with seeded Ohio River water. PMA-RT-PCR was able to distinguish between infectious and noninfectious poliovirus. In initial experiments using 100 μM PMA, nonspecific bands were observed, as has been reported previously for environmental matrices when the conventional PCR assay was used (11). A very valuable observation was that most of the nonspecific bands could be removed by increasing the PMA concentration to 200 μM (Fig. (Fig.6).6). This is in agreement with the findings of other workers, who have reported that PMA is useful for removing nonspecific PCR products common in environmental water samples (17, 46).

We concluded that the PMA-RT-PCR assay that includes pretreatment of enteric viruses with propidium monoazide prior to the RT-PCR allows rapid differentiation between infectious and noninfectious enteric viruses when virus particles are inactivated by heating at 72°C or 37°C or by using hypochlorite. This assay can potentially be used with all nonculturable and difficult-to-culture environmental strains inactivated under these conditions. Although the assay cannot be used at this time to measure waterborne viruses that are inactivated at 19°C, it should be useful for studies of the efficacy of chlorine disinfection by reducing the need for cell culture studies and for studies testing the efficacy of pasteurization for virus die-off. With further development, this assay has the potential to provide much needed information on viral infectivity to assess the risk of virus exposure (and thereby provide better risk management strategies) for water from conventional drinking water plants that use chlorine as the disinfectant.

Acknowledgments

This research was supported through appointment of Ian Laseke to the Postgraduate Research Program at the Technical Support Center, Office of Ground Water and Drinking Water. This appointment was administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S Environmental Protection Agency.

We thank Jennifer Cashdollar and Gretchen Sullivan for providing BGM cells used in this study.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 May 2010.

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