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8/11/2011
Demonstration of
nucleic acid
repair in viruses
after UV
disinfection using
coliphages as
surrogates
Christian McGuire REU – University of
Arkansas
Motivation
• Improve water treatment
technology
• Develop understanding of
high levels of resistance for
certain waterborne pathogens
Acknowledgments
• Connie Chan SMART – Columbia
University
• Mentors:
• Karl Linden Ph.D – University of Colorado,
Boulder
• Roberto Rodriguez Ph.D – University of
Colorado, Boulder
• Sarah Bounty – University of Colorado,
Boulder
• And REU program
Background
• Low pressure mercury vapor lamp (monochromatic
light)
• 254 nm wavelength
• Medium pressure mercury vapor lamp (polychromatic
light)
UV!
• 200-600 nm wavelengths
Zimmer, J. L. et al. 2002. Appl. Environ. Microbiol.
68(7):3293-3299
Background ~ UV damage and
repair
• Germicidal wavelength : 200-300 nm
• Peak DNA absorption at 260 nm
• Photolyase: photo-repairing enzyme
• Activated at wavelengths 350-450 nm
Objectives
• Determine possible reactivation of
viruses utilizing host photorepair
mechanisms
• Determine If MP lamps
(polychromatic) are more effective
than LP lamps (monochromatic) at
inactivating phages.
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Experimental process
Phage and Host
• Phage
Phase 1
Phase 2
Determine dose
response for
each of the
three bacterial
host under the
LP UV lamp
(Famp, CN13,
& LT2).
Confirm
photo-repair
ability and
parameters
of bacteria
after
exposure to
LP UV lamp.
Phase 3
Determine
dose
response for
each phage
(T1, PRD-1,
PHIX174)
under both
LP and MP
UV lamps.
Phase 4
Demonstrate
reactivation
of virus using
host repair
mechanisms
after
exposure to
LP and MP
UV lamps.
– PRD-1: doublestranded DNA
• Bacteria
– LT2 (salmonella)
– T1: doublestranded DNA
– phiX174: single
stranded DNA
`
– CN13 (E.Coli)
Bacteria dose-response
Results
Low pressure
UV
collimated
beam system
Photo repair shop
Bacteria Photorepair
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Maximum photoreactivation of the
bacterial host after UV exposure
2
1.8
Log Photoreactivation
1.6
1.4
1.2
1
0.8
0.6
Phage Dose-response
0.4
0.2
0
Salmonella (LT2) 3 hrs
Ecoli (Famp) 2hr
E coli (CN13) 1 hr
Spot plating
• Saved time and equipment
• 5 dilutions per plate opposed to three plates per
dilution
Titering of phage
Plating of LT2
Comparison to Double agar layer method:
1st trial: DAM ~ 3.3 x 104 2nd trial: DAM ~ 5.1 x 109
spot -titer ~ 4 x 104
Spot - titer ~ 4.8 x 109
Phage Repair Experiment
PRD-1
Initial Concentration
Concentration after LP exposure ~ 40 mj/cm2
Phage repair using host photorepair mechanism
Concentration after 6 hours of light exposure
Concentration after 6 hours of dark
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Phage repair
Phage photoreactivation
• 6 hours under photo-repair lamp
3
ss DNA
Log Photoreactivation
2.5
ds DNA
2
1.5
1
0.5
phiX174 – LP system
PRD-1 – LP system
0
PhiX174-LP
UV dose-response of phage PRD-1 with
and without photoreactivation
6
Log (10) Inactivation
5
T1-LP
PRD-1-LP
PRD-1-MP
Conclusion
• Phage with double-stranded DNA were able to
repair using bacterial host repair-mechanisms,
but phage with single stranded DNA were not
LP
4
MP
3
LP - Light
• More experiments need to be run to determine if
MP lamps are more effective at inactivating
PRD-1, T1, and phiX174
2
MP - Light
1
0
0
20
40
60
Dose (mJ/cm2)
80
100
120
Future work
• Develop dose response curves
for Photoreactivation of phage
• Further investigate efficacy of
Medium pressure lamps as
opposed to Low pressure lamps.
Questions?
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References
Rocky Mountain Laboratories, NIAID, NIH
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