Sampling for airborne influenza virus using RNA preservation buffer

85 Pages·2017·1.74 MB·English

University of Iowa
Iowa Research Online
Theses and Dissertations
Summer 2014
Sampling for airborne influenza virus using RNA
preservation buffer : a new approach
Elanie Michelle Girlando
University of Iowa
Copyright 2014 Elanie Michelle Girlando
This thesis is available at Iowa Research Online: https://ir.uiowa.edu/etd/1324
Recommended Citation
Girlando, Elanie Michelle. "Sampling for airborne influenza virus using RNA preservation buffer : a new approach." MS (Master of
Science) thesis, University of Iowa, 2014.
https://doi.org/10.17077/etd.lg6lfxwf
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of theOccupational Health and Industrial Hygiene Commons
SAMPLING FOR AIRBORNE INFLUENZA VIRUS
USING RNA PRESERVATION BUFFER:
A NEW APPROACH
by
Elanie Michelle Girlando
A thesis submitted in partial fulfillment
of the requirements for the Master of
Science degree in Occupational and Environmental Health
in the Graduate College of
The University of Iowa
August 2014
Thesis Supervisor: Assistant Professor Matthew W. Nonnenmann
Copyright by
ELANIE MICHELLE GIRLANDO
2014
All Rights Reserved
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
MASTER'S THESIS
_______________
This is to certify that the Master's thesis of
Elanie Michelle Girlando
has been approved by the Examining Committee
for the thesis requirement for the Master of Science
degree in Occupational and Environmental Health at the August 2014
graduation.
Thesis Committee: ___________________________________
Matthew W. Nonnenmann, Thesis Supervisor
___________________________________
Thomas Peters
___________________________________
Lucy Desjardin
ACKNOWLEDGMENTS
I would like to thank my advisor, Matt, for working hard to get funding for this
research and for being willing to work outside of his comfort zone to support my research
interests. I couldn’t have done this without your support and direction during my thesis
work and certainly wouldn’t have completed this work without your unending supply of
patience when I had none.
I would also like to thank the team at NIOSH, Drs. John Noti and Bill Lindsley
for all of your guidance and direction in this research, for always being available for
questions, and for supplying us with the NIOSH Biosamplers used in this experiment. To
our partners at the State Hygienic Lab at the University of Iowa, Drs. Lucy Desjardin and
Travis Henry, thank you for your collaboration with us on this project. Lucy, you were a
wonderful source of knowledge on a topic of research we knew very little about, and kept
us from straying down the wrong path several times. Travis, you were the best mentor I
could ask for in my microbiology work. Thanks for taking the time to teach me how to do
things the right way. We look forward to collaborating with both NIOSH and the State
Hygienic Lab on future projects.
I also want to thank my family for always being there for me and unconditionally
supporting every life decision I have made. To my parents, thank you for helping me get
to where I am today, for being an example of strength and determination, and for
instilling in me a sense of adventure and love for learning. To my sister, thanks for being
my friend and confidant, and for being a wonderful example to me all of my life of
caring, compassion, and intelligence. Seeing you accomplish your goals and dreams by
hard work inspired me to do the same.

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To my friends, you have supported me every step of the way, and I am eternally
thankful for you. Thank you for sharing in the good times and bad, and for always being
there for me. To Mary and Erin Dunn, thank you for being a source of guidance and
stability to me through many important and difficult moments in life. To my boyfriend
Chris, thank you for pulling me back from the ledge on more than one occasion, for being
sweet and caring when I needed it, and for making me laugh when I wanted to cry. I
appreciate you all more than you know.
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ABSTRACT
Characterizing airborne influenza virus exposure is important for infection
prevention and exposure control in health care and public settings. Detecting airborne
influenza virus is important in assessing infection risk. The virus must also be protected
from deterioration during aerosol sampling and long term storage. RNA preservation
buffers (RNAPBs) may stabilize influenza virus after sampling and during storage.
Bioaerosol samplers are used to collect airborne influenza virus, and many different types
of samplers are available.
The objectives of this experiment were to: 1) compare influenza virus
concentrations across bioaerosol samplers; 2) compare the efficacy of RNAPB over
Hanks Balanced Salt Solution (HBSS) as a sample collection media; and 3) determine
whether RNAPB stabilizes viral particles stored over time. In this experiment we
aerosolized active influenza virus (H1N1) in a bioaerosol chamber and compared
sampling efficiencies using two different samplers: the SKC Biosampler and NIOSH
Biosampler, and two different medias: Hanks Balanced Salt Solution (HBSS) and an
RNAPB. Ten 15-minute experimental trials were completed. We also compared HBSS
and RNAPB in terms of the maintenance of virus RNA integrity during storage at room
temperatures. Samples were stored at room temperature for 1, 4, 7, and 14 days. Virus
concentrations were measured and compared at each time point.
Significant differences were found between sampler and media type – the SKC
Biosampler collected a higher concentration of virus than the NIOSH Biosampler, and
HBSS collected a higher concentration of virus than RNAPB. In storage at room
temperature conditions, RNAPB maintained virus in concentrations significantly greater

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than in HBSS. The results of this experiment indicates that the SKC Biosampler should
be used to characterize airborne influenza and that RNAPB should not be used as a
sampling media but can be used to preserve samples if needed.
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TABLE OF CONTENTS
LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
CHAPTER
I. LITERATURE REVIEW .................................................................................1
The Significance of Influenza ...........................................................................1
Modes of Influenza Virus Transmission ...........................................................3
Present Difficulties in the Detection of Airborne Influenza Virus ...................4
Sampling Techniques for Airborne Influenza Virus ........................................5
Selection of the Liquid Sampling Media for Use With Liquid Impinger
Samplers ...........................................................................................................8
RNA Preservation Buffers ................................................................................9
Analysis of Influenza Virus Samplers ............................................................11
Objectives .......................................................................................................12
II. THE USE OF HBSS AND RNAPB IN SKC AND NIOSH BIOSAMPLERS .......14
Introduction .....................................................................................................14
Methods ..........................................................................................................16
Virus Aerosol Sampling ..........................................................................19
Virus Isolation and RT qPCR Extraction................................................20
Total Virus Particle Collection ...............................................................21
Effect of Media Type on Virus Stability in Room Temperature
Storage ....................................................................................................23
Results.............................................................................................................25
Total Virus Particle Collection ...............................................................25
Effect of Media Type on Virus Stability in Room Temperature
Storage ....................................................................................................25
Discussion .......................................................................................................33
Limitations ..............................................................................................36
Conclusions.....................................................................................................39
III. CONCLUSION .....................................................................................................41
APPENDIX A: SOP BIOAEROSOL CHAMBER ...........................................................43
APPENDIX B: STANFORD UNIVERSITY RNAPB RECIPE .......................................50
APPENDIX C: CALCULATIONS ...................................................................................51
APPENDIX D: STANDARD CURVE .............................................................................54
APPENDIX E: RAW DATA .............................................................................................55
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APPENDIX F: ANOVA TABLE ......................................................................................62
APPENDIX G: FLOW RATE VARIANCE ....................................................................63
REFERENCES .................................................................................................................64

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