Download UV Inactivation of Coxsackievirus, Echovirus, and Poliovirus

UV Inactivation of Coxsackievirus, Echovirus, and Poliovirus
Quantified Using an Integrated Cell Culture-qRTPCR Assay
Q-469
Brooke Mayer,
Mayer, Hodon Ryu,
Ryu, Dan Gerrity,
Gerrity, and Morteza Abbaszadegan
For copies of poster: http://wqc.asu.edu
Department of Civil and Environmental Engineering, National Science
Science Foundation Water Quality Center, Arizona State University, Tempe,
Tempe, AZ
Contact Information: Brooke Mayer, Ph.D.; EE-mail: [email protected];
[email protected]; Phone: (480) 277277-7200
Motivation
The simultaneous promulgation of the Environmental Protection Agency
Agency’’s (EPA) Disinfectant and Disinfection Byproduct Rule and Enhanced
Enhanced Surface Water Treatment Rule emphasized the importance of balancing
balancing the risks posed by microbial and chemical contamination of drinking water. Ultraviolet (UV)
disinfection produces no known disinfection byproducts, and as such,
such, has generated interest as an alternative disinfectant for drinking
drinking water treatment. Accordingly, it is important to understand
understand the efficacy of UV disinfection for microbial inactivation, particularly emerging pathogens such as those on the
EPA’
EPA’s Contaminant Candidate List (CCL). The traditional infectivity assays required for such studies are timetime- and materialmaterial-intensive, which limits understanding of UV efficacy. In particular,
particular, a better understanding of the effect of UV on emerging microbes,
microbes, such as coxsackievirus and echovirus (which are
currently included on the CCL), is needed. This study demonstrates
demonstrates the applicability of integrated cell cultureculture-quantitative RTPCR (ICC(ICC-qRTPCR) for the simultaneous quantification of infectious coxsackieviruses,
coxsackieviruses, echoviruses, and polioviruses and validates its use in UV disinfection studies.
Results
Background
ICCICC-qRTPCR Method Development and Optimization
Type 1, Strain
LscLsc-2ab
Host: BGM cells
Optimization of Number of Washes for ICCICC-qRTPCR
1.E+05
1.E+03
1.E+02
1.E+01
Significant removal of nonnon-infectious viruses
using 3 postpost-infection washes
0
4
6
Coxsackievirus
Standard Infectivity Assay for Enteroviruses : InIn-Vitro Cell Culture:
1.E+02
1.E+01
8
0
24
1
3
Echovirus
Echovirus: Log(Virus Concentration) =
2
0.7224 * Log(ICC-QPCR) - 0.50; R = 0.8326
1.E+04
Poliovirus: Log(Virus Concentration) =
2
1.2675 * Log(ICC-QPCR) - 5.15; R = 0.8742
1.E+03
1.E+02
1.E+01
1.E+00
1.E+00
5
1.E+01
1.E+02
1.E+03
Number of Washes
Coxsackievirus
Poliovirus
Echovirus
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
ICC-QPCR Quantity (TCID50/ml)
Poliovirus
Coxsackievirus Echovirus Poliovirus
Incubation Time Optimization experiments were performed to determine the optimal incubation time for quantification of the mixed enteroviruses. Serial dilutions (106 through 101
TCID50/ml) were applied to cells and harvested after different incubation
incubation times (0, 4, 6, 8, and 24 hours post inoculation). A time of 24 hours post inoculation was identified as optimal based on
consistent and significant replication of viruses at different initial
initial concentrations. Samples with high virus concentrations (low
(low inactivation) should be diluted to avoid cell capacity limitations.
limitations.
1.0E+06
1.0E+05
ICC-qRTPCR Quantity
100 10-1 10-2 10-3 10-4 (-)
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.0E+07
1.E+01
Inactivated
Coxsackievirus
1.0E+05
1.0E+03
1.0E+02
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+01
Inactivated
1.0E+06
Echovirus
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
Inactivated
Poliovirus
1.0E+05
1.0E+04
Quantity
1.E+01
1.0E+04
ICC-qRTPCR Quantity
• Depends on visual differentiation between infected host cells demonstrating
demonstrating cytopathic (CPE) effects and
healthy, uninfected cells.
• For the TCID50 assay, a single sample may require 24 wells.
• For the TCID50 assay, cells are observed for up to 14 days and statistical methods
methods are used to estimate the
concentration of the original sample based on the number of positive
positive and negative replicates.
• Method is timetime- and costcost-intensive, and somewhat subjective.
l)
1.E+03
Post Infection Incubation Time (hr)
Use standard curves to quantify
unknown samples
Coxsackievirus: Log(Virus Concentration) =
2
0.968 * Log(ICC-QPCR) - 1.70; R = 0.7057
1.E+04
1.E+00
1.E+00
ICCICC-qPCR Standard Curves for 24 h.p.i.
h.p.i.
1.E+05
5 0 /m
No significant replication of nonnon-infectious viruses
1.E+04
IC C -Q P C R Q u a n tity (T C ID 5 0 /m l)
Poliovirus
Echovirus
Type 12
Host: BGM cells
Coxsackievirus
Type B6
Host: BGM cells
NonNon-Infectious Virus ICCICC-qRTPCR Control
IC C -Q P C R Q u a n tity (T C ID 5 0 /m l)
• Coxsackievirus B6, Echovirus type 12, and poliovirus type 1 were simultaneously quantified in this study.
• As members of the enterovirus genus, they are small (24–
(24–30 nm), nonnon-enveloped, positive strand RNA viruses.
• Although poliovirus is perhaps the most wellwell-studied of all viruses, less is known regarding the occurrence,
health effects, treatment, etc. of other enteroviruses, which led
led to the inclusion of coxsackievirus and
echovirus on the EPA’
EPA’s Contaminant Candidate List.
• Enterovirus infections are common (annually 30–
30–50 million in the U.S.), but are often manifested subclinically.
subclinically.
However, a range of clinical outcomes is possible, including colds
colds and fevers, handhand-footfoot-andand-mouth disease,
conjunctivitis, meningitis, and poliomyelitis.
In it ia l V iru s C o n c e n t ra t io n ( T C ID
Study Viruses
1.0E+03
1.0E+04
1.0E+03
1.0E+02
1.0E+02
1.0E+01
1.0E+01
NonNon-Infected BGM Cells
2424-Well Tray Per Sample
Infected BGM Cells Showing CPE
Plaque Assay Infection
1.0E+01
1.0E+00
1.0E+00
0
4
6
8
1.0E+00
0
24
4
6
8
0
24
4
6
8
24
Post-Inoculation Incubation Time (hr)
Post-Inoculation Incubation Time (hr)
Post-Inoculation Incubation Time (hr)
Quantitative RealReal-Time qPCR using Taqman Probes:
UV Disinfection Quantified Simultaneously using ICCICC-qRTPCR
1.0E+05
1.0E+07
3.5
Coxsackievirus ICC-qPCR Standard Curve
Log (Spiking Level) = 0.82 Log (ICC-qPCR Quantity) + 0.56
R2 = 0.9428
3.0
1.0E+06
Probe Cleavage and Fluorescence
• Reverse transcription at 48oC for 30 min; RT inactivation and initial denaturation at 95oC for 10 min;
40 cycles of denaturation at 95oC for 15 sec, annealing and extension at 60oC for 30 sec.
1.0E+04
1.0E+03
1.0E+03
1.0E+02
Echovirus ICC-qPCR Standard Curve
Log (Spiking Level) = 0.69 Log (ICC-qPCR Quantity) + 0.54
R2 = 0.9493
1.0E+02
1.0E+01
1.0E+01
Experimental Approach
1.0E+00
Log Inactivation [-log(N t/N o)]
Primer Extension
1.0E+05
Spiking Level (TCID 50/mL)
Primer and Probe Annealing
ICC-qPCR Quantity [TCID50/mL]
1.0E+04
Poliovirus ICC-qPCR Standard Curve
Log (Spiking Level) = 1.10 Log (ICC-qPCR Quantity) - 3.15
R2 = 0.9842
1.0E+2
TCID 50/mL
1.0E+3
TCID50 /mL
1.0E+4
TCID 50/mL
1.0E+5
TCID 50/mL
Spiking Level (based on CPE) [TCID50/mL]
1.0E+6
TCID 50/mL
1.0E+6
TCID50/mL
(Inactivated)
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
2.5
2.0
1.5
1.0
0.5
1.0E+05
1.0E+06
1.0E+07
0.0
0
ICC-qPCR Coxsackievirus Quantity (TCID50/mL)
5
10
15
20
25
30
2
Coxsackievirus
Echovirus
Poliovirus
ICCICC-qRTPCR Validation at 24
h.p.i.
h.p.i. for UV Experiment
Coxsackievirus
Echovirus
Poliovirus
Power (Coxsackievirus)
Power (Echovirus)
Power (Poliovirus)
ICCICC-qRTPCR Standard Curve
for UV Experiment
UV Dose (mJ/cm )
UV DoseDose-Response Curves
Black lines and symbols represent experimental data;
colors signify reports from the literature.
Summary
Mixed Virus
Sample
Collimated Beam
UV Reactor
InIn-vitro Cell Culture
Well Harvest
Purification
RealReal-time Quantitative
RTPCR (qRTPCR)
•
•
•
•
The integrated cell cultureculture-quantitative RTPCR (ICC(ICC-qPCR) assay was successfully applied for the simultaneous quantification
quantification of coxsackievirus, echovirus, and poliovirus.
The assay provides significant time and material savings and offers
offers a practical alternative to traditional methods for the quantification
quantification of infectious viruses.
Assay optimization demonstrated that 24 h.p.i.
h.p.i. and 3 postpost-infection washes was optimal for the simultaneous quantification of coxsackievirus, echovirus, and poliovirus.
ThreeThree-log inactivation was achieved at UV doses of 30–
30–44, 28–
28–42, and 28–
28–29 mJ/cm2 for coxsackievirus B6, echovirus 12, and poliovirus 1, respectively,
respectively, which is similar to values reported using
conventional methods.
Acknowledgements
This research was partially funded by the National Science Foundation
Foundation Water Quality Center (NSF WQC) at Arizona State University.
University. It was performed while on appointment as a U.S. Department of
of Homeland Security (DHS) Fellow under the DHS Scholarship and Fellowship
Fellowship Program. The DHS program is
administered by the Oak Ridge Institute for Science and Education
Education (ORISE) for DHS through an interagency agreement with the U.S Department of Energy (DOE). ORISE is managed by Oak Ridge Associated
Associated Universities under DOE contract number DEDE-AC05AC05-06OR23100. All opinions expressed here are the
authors’
ICC-qPCR assay in disinfection studies, please visit Poster QQ-471: BenchBench-Scale and PilotPilot-Scale Photocatalytic Inactivation of Viruses with Titanium Dioxide
Dioxide Nanoparticles.
authors’ and do not necessarily reflect the policies and views of ASU, NSF
NSF WQC, DHS, ORISE, or DOE. For additional information regarding the use of this ICC-