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Journal of Virological Methods 168 (2010) 228–232 Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Protocols Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR Mariana Fittipaldi a , Nancy J. Pino Rodriguez a,b , Francesc Codony a,∗ , Bárbara Adrados a , Gustavo A. Peñuela b , Jordi Morató a a Laboratori de Microbiologia Sanitària i Mediambiental (MSM-Lab) – Aquasost. UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya, Violinista Vellsolá 37, 08222 Terrassa, Barcelona, Spain Grupo de Diagnóstico y Control de la Contaminación (GDCON), Sede de Investigación Universitaria, Universidad de Antioquia, Calle 62 # 52-59, Medellín, Colombia b a b s t r a c t Article history: Received 23 February 2010 Received in revised form 11 June 2010 Accepted 17 June 2010 Available online 25 June 2010 Keywords: Bacteriophage T4 Virus infectivity PMA Real-time PCR The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 ◦ C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 ◦ C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Viruses are some of the most significant pathogens for public health, recognized as leading causes of food-borne and water-borne disease (Bern et al., 1992; Mead et al., 1999). Detection of infectious viral particles in different types of samples is fundamental for an understanding of (i) the persistence capacity of viruses in the environment, (ii) the efficacy of disinfection treatments, and (iii) the evaluation of transmission risk to people. Many methods have been devised for detecting these viruses (Richards, 1999), and cell culture is employed currently to quantify the infectivity of certain viruses. However, culturing can take days to weeks to yield results, and many viruses important for public health are either difficult to culture or are non-culturable (Pecson et al., 2009). In addition, detection of infectious viruses in a sample will depend greatly on the assay conditions, such as the duration of exposure to host cells, volume of inocula, age of the cells, and ∗ Corresponding author at: Laboratori de Microbiologia Sanitària i Mediambiental (MSM-Lab), C/Violinista Vellsolà 37, EUOOT, TR8, 08222 Terrassa, Barcelona, Spain. Tel.: +34 93 7398328; fax: +34 93 7398328. E-mail address: [email protected] (F. Codony). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.06.011 the presence of inhibitory or toxic substances (Rodriguez et al., 2009). The advent of PCR and quantitative PCR (qPCR) has improved considerably the ability for detection of human viral pathogens in the environment (Abbaszadegan et al., 1993; Fong and Lipp, 2005; Gersberg et al., 2006; He and Jiang, 2005). In this sense, qPCR is a successful method for monitoring the viral contamination of water and food products (Beuret et al., 2002; Di Pinto et al., 2003; Lee and Kim, 2002). Although PCR-based methods have led to excellent sensitivity and specificity for virus detection and quantitation, a serious limitation is that the technique cannot discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard (Richards, 1999). Because of the persistence of DNA in the environment (Josephson et al., 1993; Masters et al., 1994), DNA-based assays cannot assess pathogenic risk because signals can originate from both infectious and non-infectious viruses (Pecson et al., 2009; Shin and Sobsey, 1998). To address this limitation, some new approaches have been developed recently (Rodriguez et al., 2009). Generally, additional pre-treatment steps have been employed, like immunocapture of the virus from the sample (Gilpatrick et al., 2000; Schwab et al., 1996); the use of cell attachment to remove viruses from the sample (Nuanualsuwan and Cliver, 2003); and enzymatic pre- M. Fittipaldi et al. / Journal of Virological Methods 168 (2010) 228–232 treatment (Nuanualsuwan and Cliver, 2002). Other strategies have included using longer amplicons (Allain et al., 2006; Duizer et al., 2004; Simonet and Gantzer, 2006) and the use of multiple PCR sites (Sobsey et al., 1998). Although enzymatic pre-treatment is one of the most promising techniques developed because it has become useful for measuring capsid integrity, none of these methods is applicable for all viruses because they do not quantify only infectious viruses. Furthermore, many fundamental questions regarding the relationship between virus inactivation and damage to the genome or damage to the capsid and the host attachment site remain unanswered. Most studies (Pecson et al., 2009; Rodriguez et al., 2009) agree in pointing out the importance of using qPCR coupled with other protocols for quantitation of infectious viral particles. In recent years, the use of qPCR and selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) has been evaluated effectively for different bacteria (Bae and Wuertz, 2009; Cawthorn and Witthuhn, 2008; Delgado Viscogliosi et al., 2009; Nocker et al., 2009; Pan and Breidt, 2007; Rudi et al., 2005; Soejima et al., 2007), spores (Rawsthorne et al., 2009), and fungi (Vesper et al., 2008). EMA and PMA should penetrate only into membrane-compromised cells and once inside the cell, they bind irreversibly to the DNA by photoactivation. The presence of an azide group allows cross-linking of the dye to the DNA by exposure to strong visible light. The photolysis of EMA and PMA converts the azide group into a highly reactive nitrene radical, which can react with any organic molecule in its proximity, including the bound DNA. In its bound state, the DNA cannot be amplified theoretically by PCR (Nocker and Camper, 2009; Rudi et al., 2005). At the same time, when the cross-linking occurs, the light reacts the unbound excess dye with water molecules. The resulting hydroxylamine is no longer reactive, so the DNA extraction procedure does not modify the DNA from cells with intact membranes (Nocker et al., 2009). Thus, the approach relies on the integrity of the membrane to distinguish between viable and cells damaged irreversibly. As far as can be discerned, the utility of PMA for distinguishing between infectious and non-infectious viruses has not been confirmed. The qPCR method for quantitation of infectious viruses is based on the measure of genomic integrity. A PMA pre-treatment combined with qPCR (PMA-qPCR) could be useful for quantifying viruses that are inactivated because of damaged capsid integrity. It is equally probable, however, that such a PMA-qPCR method would have limitations because PMA can penetrate only into a virus with a damaged capsid, and not all non-infectious viruses have compromised capsids. As an example, a virus might lose the receptors that allow infection of the host, rendering it inactive, even while maintaining an intact capsid; PMA in this case would be unable to bind to its DNA. The purpose of this study was to determine if PMA pretreatment can be used with qPCR to quantify the infectivity of the bacteriophage T4, allowing discrimination of DNA from infectious and non-infectious bacteriophage T4 viruses. This phage was selected because it and its host, Escherichia coli, are well characterized and it is cultivated easily. 2. Materials and methods 2.1. Virus propagation Bacteriophage T4 and its E. coli host were purchased from the German Collection of Microorgansims and Cell Cultures (DSMZ, numbers 4505 and 613, respectively; Braunschweig, Germany). The viruses were inoculated initially into 250 ml of Tryptic Soy Broth (TSB, Merck, Darmstadt, Germany) containing log-phase E. 229 coli at roughly 106 colony forming units per milliliter (CFU/ml). The culture was kept 24 h at 37 ◦ C in an orbital shaker (Optic Ivymen System, Barcelona, Spain) at 120 rpm. After inoculation, 10% trichloromethane solution was added to the bacteria–virus suspension to facilitate cell lysis and viral particle release. The suspension was centrifuged 15 min at 4000 × g to separate the bacterial debris from the virus. The volume of supernatant containing the phage was adjusted using phage buffer (containing, per liter, 7 g Na2 HPO4 , 3 g KH2 PO4 , 5 g NaCl, 0.1 M MgSO4 10 ml, 0.1 M CaCl2 10 ml; Sigma Aldrich, Madrid, Spain) to obtain a stock virus solution. This suspension was kept at 4 ◦ C. Bacteriophage T4 was quantified using a double-layer agar technique, and infectious virus concentrations were measured in plaque-forming units per milliliter (PFU/ml). 2.2. Inactivating treatments Heat and proteolytic treatments were conducted to study the discrimination between infectious and non-infectious viruses. These modes of inactivation were chosen because they target predominantly the viral coat protein by physical and chemical routes respectively. In addition, heat is used commonly for food preparation and processing, and in emergencies with water (Nuanualsuwan and Cliver, 2002). Samples were taken in triplicate at 0, 5, 10, 20, and 30 min for inactivation rate analysis by cell culture, qPCR, and PMA-qPCR. Sample manipulation was carried out immediately following the inactivation treatment. The thermal inactivation temperature was 85 ◦ C. Aliquots (2 ml) of bacteriophage T4 stock solution were heated in a Thermomixer comfort (Eppendorf, Hamburg, Germany) at 85 ◦ C for the times specified above and then placed immediately at room temperature. Phage suspension without heating was used as a control. For proteolysis, 2-ml aliquots of bacteriophage T4 stock solution were mixed with Protease OB (Omega Bio-Tek, Norcross, GA, USA) at a 4 mg/ml concentration and incubated at 56 ◦ C for 30 min according to the manufacturer’s instructions. Samples were taken at 0, 5, 10, 20, and 30 min. Phage suspension without protease treatment was used as a control. A dilution series of the stock virus solution was prepared with phage buffer to obtain an initial bacteriophage T4 concentration of 1.2 × 1010 PFU/ml. Experiments with a high-temperature thermal inactivation temperature of 110 ◦ C were performed, as well. This high temperature was chosen to achieve complete destruction of the viral coat protein. Tubes containing 2 ml of phage suspension were heated at 110 ◦ C in a water bath for 15 min. The absence of infectious phages was confirmed by spread-plating 100-l aliquots of heat-treated viruses on plaques using a double-layer agar technique. Phage suspension without heat treatment was used as a control. 2.3. PMA treatment PMA (Biotium, Inc., Hayward, CA, USA) was dissolved in 20% dimethylsulfoxide (Sigma Aldrich, Madrid, Spain) to obtain a 2-mM stock solution. Given a scarcity of data regarding viruses and PMA in the literature, the PMA concentration used in this study was chosen based on references related to other microorganisms and considering that there was sufficient PMA for the DNA-PMA binding (Bae and Wuertz, 2009; Brescia et al., 2009; Chang et al., 2010; Varma et al., 2009). The PMA solution was added at a final concentration of 100 M to a 200-l aliquot of either heat-treated (85 ◦ C) or phages inactivated enzymatically, heat (110 ◦ C)-inactivated virus, or infectious virus without inactivating treatment, following an incubation period of 5 min in the dark at room temperature with occasional mixing to allow PMA penetration. After incubation in the dark, 230 M. Fittipaldi et al. / Journal of Virological Methods 168 (2010) 228–232 Fig. 1. Bacteriophage T4 quantitation over time by plate count, qPCR, and PMA-PCR. (A) Heat treatment (85 ◦ C). (B) Protease treatment. The qPCR approach overestimates infectious virus quantitation, and the PMA-qPCR approach is not sufficiently effective to distinguish between infectious and non-infectious viruses. Error bars show standard deviations from three independent assays. samples were exposed to light for 15 min using a photoactivation system (Led-Active Blue, Ingenia Biosystems, Barcelona, Spain). 2.4. Real-time PCR The quantitation of bacteriophage T4 was conducted using a LightCycler 1.5 PCR system (Roche, Barcelona, Spain). Genomic DNA from samples was extracted using the E.Z.N.A. DNA extraction kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. Primers specific to bacteriophage T4 (Thermo Scientific Biopolymers, Waltham, MA, USA), T4F (5 -AAGCGAAAGAAGTCGGTGAA-3 ), and T4R (5 CGCTGTCATAGCAGCTTCAG-3 ) were used to amplify a 163-bp region of the gp18 tail protein (Gerriets et al., 2008). The qPCR amplification was performed in a total volume of 20 l comprising 10 l of FastStart SYBR Green Master (Roche, Barcelona, Spain), 0.5 M forward and reverse primers, and 10 l of DNA template or PCR-grade water (Eppendorf, Hamburg, Germany) for negative control. The experimental LightCycler protocol consisted of one step of 10 min at 95 ◦ C for Taq polymerase activation followed by 45 cycles of PCR amplification. Each PCR amplification cycle consisted of 15 s of denaturation at 95 ◦ C, 60 s of annealing at 60 ◦ C, and 60 s of elongation at 72 ◦ C. A standard curve was constructed by making serial 10-fold dilutions of the initial virus stock dilution using sterile buffer to obtain the set of dilutions for the curve. DNA was then extracted, and qPCR amplification was carried out in triplicate. after 20 min, very strong DNA amplification signals were obtained by the detection of infectious viruses using qPCR and PMA-qPCR. PMA-qPCR and qPCR resulted consistently in higher levels of survival compared to plate counts, which indicates that inactivated viruses can be detected still by molecular methods. Similar results were found for samples inactivated by proteolysis. Fig. 1B shows the proteolytic inactivation curves by plaque count, qPCR, and PMA-qPCR. In this case, after only 10 min, the initial infectivity of 1.2 × 1010 ± 4.5 PFU/ml of bacteriophage T4 was detectable no longer by culture while inactivated viral particles were detectable by molecular methods. In addition, assays were conducted to examine the efficiency of PMA pre-treatment in minimizing detection signals from noninfectious viruses inactivated by heat at 110 ◦ C for 15 min. The results showed that PMA pre-treatment resulted in a significant decrease in the detection of non-infectious virus by qPCR. The PMApre-treated non-infectious virus concentration was 7.63 log lower than the PMA-pre-treated infectious virus concentration (Fig. 2). The phage infectivity was evaluated by spread-plating 100 l aliquots of heat-treated viruses on plaques using a double-layer agar technique. The results were all negative. The concentration of 3. Results 3.1. Standard curve A linear regression analysis was performed by plotting the cycle threshold (Ct) values against the logarithm of the PFU/mL of bacteriophage T4. The experimental points aligned in a straight line with a correlation coefficient (r) = −0.993 (R2 = 0.9867). The quantitation limit was estimated to be 1 × 102 PFU/ml of sample. 3.2. Thermal and proteolytic inactivation Bacteriophage T4 thermal inactivation assays were conducted at 85 ◦ C. The detection limit of the cell culture technique was 1 PFU/ml. Fig. 1A shows the thermal inactivation curves by plaque count, qPCR, and PMA-qPCR. After 20 min, the initial infectivity of 1.2 × 1010 ± 4.5 PFU/ml of bacteriophage T4 was detectable no longer by culture, which implies a ∼10 log reduction. However, Fig. 2. PMA pre-treatment did block qPCR amplification of heat-treated bacteriophage T4 DNA when the bacteriophage T4 was inactivated by heat at 110 ◦ C. The virus concentration was measured by qPCR with or without PMA pre-treatment. The plate count of heat-treated viruses was negative. Error bars show standard deviations from three independent assays. M. Fittipaldi et al. / Journal of Virological Methods 168 (2010) 228–232 heat-treated phages was a little lower (1.39 log) than the concentration of untreated phages by qPCR; in addition, the concentration of PMA-pre-treated infectious viruses was only slightly lower (0.66 log) than the infectious virus concentration obtained by qPCR (Fig. 2). 4. Discussion Real-time PCR is an excellent tool for environmental virology and has been used successfully to determine the concentrations of viral genomes in the environment (Abbaszadegan et al., 1999; Choi and Jiang, 2005). However, these assays do not provide information about the infectious risk because this technique does not discriminate between infectious viruses and non-infectious viruses that convey no health threat. Currently, the combination of PCR and cell culture offers the best approach to assess viral infectivity. Nevertheless, there are difficulties in achieving a cell culture model for the detection of important water-borne pathogens, leaving the use of direct PCR as the most feasible technique. Therefore, the ability of PCR to assess viral infectivity would enhance greatly its application for the monitoring of water and food quality and for treatment processes (Rodriguez et al., 2009). In this study, a PMA pre-treatment was used to assess the potential of the PMA-qPCR method to discriminate between infectious and non-infectious viruses. Infectious viral particles of most enteric viruses consist of two major components, the capsid and the genome (Strauss and Strauss, 2002), and their infectivity requires the functional integrity of both components. Because the genomes of all viruses comprise essentially the same nucleic acids (either RNA or DNA), it appears that the stability of some viruses lies primarily in the capsid structure. At the protein-structure level, conformational changes in the capsid that diminish viral stability or affect attachment to a cell receptor jeopardize directly viral infectivity (Nuanualsuwan and Cliver, 2003); therefore, critical damage to the capsid is not required necessarily to inactivate viruses. Indeed, Nuanualsuwan and Cliver (2003) found that the capsids of inactivated human picoviruses and feline calcivirus at 37 ◦ C could still protect the viral genome from RNase pre-treatment. Also, many viruses (such us the influenza virus) have viral envelopes covering their capsids that are used to help viruses enter host cells, and most enveloped viruses are dependent on the envelope for their infectivity (Mahy, 1998). Thus, some inactivation mechanisms may damage the viral envelope, inactivating the virion but leaving its capsid uncompromised. These previous findings regarding capsid integrity could explain some of the current results. The findings from the thermal and proteolytic virus inactivation assays in this study demonstrated that bacteriophage T4 is inactivated relatively rapidly, at 20 and 10 min, respectively, as detected by culture. However, positive and similar results were obtained by qPCR for each sample time of inactivation, either by heat at 85 ◦ C or enzymatic treatment. Consequently, the qPCR alone, which targeted genomic integrity, did not distinguish infectious from non-infectious viruses. Earlier studies have described similar results using the same or different inactivation methods and viruses (Baert et al., 2008; Gassilloud et al., 2003; Graiver et al., 2009; Lee et al., 2008; Safaa et al., 2008; Simonet and Gantzer, 2006). These outcomes could arise from the fact that the main mechanism of virus inactivation under the conditions used in this study (heat at 85 ◦ C and enzymatic treatments) did not involve DNA degradation, at least at the primer location. However, the phage concentration did not change significantly when PMA pretreatment followed by PCR was performed with the tests involving heat at 85 ◦ C or proteolytic inactivation. These findings indicate that moderate heating (85 ◦ C) and protease treatment may cause a loss of infectivity in bacteriophage T4, as cell culture results showed, but that these treatments do not appear to cause critical damage to the 231 viral capsid. It is also possible that more damage to the viral capsid may occur with heat treatment than with enzymatic treatment. Graiver et al. (2009) found that EMA pre-treatment titers were significantly higher than the measured cell culture titers when EMA pre-treatment followed by PCR was carried out to determine the survival of influenza virus in reverse-osmosis water at 37 ◦ C. A possible reason for this behaviour is that EMA did not inhibit RT-PCR amplification because inactivation mechanisms other than capsid compromise were at work. When a higher inactivation temperature (110 ◦ C) was applied, a clear discrimination of infectious and non-infectious viruses was observed with PMA-qPCR (Fig. 2). A slight difference between qPCR and PMA-qPCR for concentrations of infectious virus was observed, which may indicate that some viruses could be non-infectious before the heat treatment or that PMA could have a slightly antivirucidal effect. However, this effect appeared to be weak and therefore not an impediment to the use of PMA pre-treatment. The obtained concentrations using qPCR alone were similar for infectious and non-infectious viruses. An amplification signal that was only slightly lower than the amplification signal of untreated phages was observed when heat-treated phages were amplified by qPCR, indicating that little genome damaged had occurred; however, the non-infectious virus concentration reported based on PMA-qPCR fell dramatically. These results suggest that PMA could penetrate the capsid and bind to DNA as a result of the denaturation of the viral capsid protein at high temperatures. Although the data from the PMA-qPCR method were in better agreement with the cell culture data than the qPCR for this case, the PMA-qPCR method resulted in a positive signal amplification. This outcome suggests that PMA-qPCR could yield false positives. Similar results have been observed with bacteria in previous reports (Bae and Wuertz, 2009; Chang et al., 2010; Kralik et al., 2010). Many factors could cause bias in the application of PMA-qPCR; for example, the presence of a high number of dead cells is one possible reason for false-positive detection signals (Wang et al., 2009). Nevertheless, optimization of the PMA method can help reduce false-positive results. Although experiments should be done that focus on factors that can affect viability results when PMA pre-treatment is applied, without doubt the PMA approach is an important step forward in the quantitation of viable microorganisms by DNA detection-based methods and allows for better characterization of microbial dynamics in most of areas of microbiology, including environmental and clinical microbiology and quality control. In conclusion, PMA pre-treatment could allow differentiation of non-infectious from infectious viruses in cases of significant damage to the integrity of the viral capsid. On the other hand, the PMA approach is not sufficiently efficient for distinguishing noninfectious viruses when moderate heat and protease treatment are used; in these cases, an intact capsid may protect the viral nucleic acid from PMA and a positive qPCR will result even for non-infectious viruses. In such cases, a different complementary treatment will be necessary to distinguish between infectious and non-infectious viruses using molecular methods. Despite the use of a single virus strain in this study, it is very probable that using different viruses under similar experimental conditions would provide results equivalent to those reported in this study. However, other factors suitable for optimization, such as the concentration of PMA, incubation time, the light source, distance from the light source, and light exposure time may lead to a more effective PMA pre-treatment for viruses. Extreme conditions of heat, chlorine, or acidity are required to inactivate enteric viruses rapidly and efficiently in food or water (Croci et al., 1999; Ma et al., 1994; Wang et al., 1995). In these cases, the inactivating agent is likely to attack the viral capsid protein, and the PMA approach could prove to be useful. In this sense, the PMAqPCR approach can be a good method for assessing damage to the 232 M. Fittipaldi et al. / Journal of Virological Methods 168 (2010) 228–232 capsid and provide a more reliable understanding of the factors that contribute to viral inactivation. Acknowledgements This research was supported by pre-doctoral scholarships, FI and FPI, from the Comissionat per a Universitats i Recerca del Departament d’Innovació, Universitats i Empresa de la Generalitat de Catalunya i del Fons Social Europeu, and Ministry of Education and Science of Spain, respectively. Financial support was provided by Alfa Network TECSPAR (RED ALFA II-0543-FI-FAFCD; sustainable technologies for potabilization and wastewater treatment), and by grant CTM2008-06676-C05-02/TECNO from the Ministry of Science and Innovation of Spain to Jordi Morató. References Abbaszadegan, M., Huber, M.S., Gerba, C.P., Pepper, I.L., 1993. Detection of enteroviruses in groundwater with the polymerase chain reaction. Appl. Environ. Microbiol. 59, 1318–1324. Abbaszadegan, M., Stewart, P., LeChevallier, M., 1999. A strategy for detection of viruses in groundwater by PCR. Appl. Environ. Microbiol. 65, 444–449. 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