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Journal of Virological Methods 168 (2010) 228–232
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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,
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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.
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