ucalgary_2013_Chaudhuri_Sibapriya.pdf

UNIVERSITY OF CALGARY
Investigating the changes in phagosomal function in Porcine Reproductive and Respiratory
Syndrome Virus infection
by
Sibapriya Chaudhuri
A THESIS
SUBMITTED TO THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCES
DEPARTMENT OF VETERINARY MEDICAL SCIENCES
CALGARY, ALBERTA
SEPTEMBER, 2013
© Sibapriya Chaudhuri 2013

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Abstract
Porcine Reproductive and Respiratory Syndrome is one of the most economically
devastating diseases of the swine industry and affects all swine producing countries worldwide.
The disease is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The
virus is macrophage-tropic and infects tissue macrophages in the host animal. However,
nothing is known about how the phagosome lumenal microenvironment in the macrophages
may be modified in PRRSV infection. Such knowledge is crucial for understanding how
microbicidal and antigen presentation functions of the macrophage may be compromised
during PRRSV infection. The field of PRRSV biology also suffers from the dearth of existence of a
good in vitro system for studying the virus biology in a methodical and reductionist fashion. This
thesis establishes an in vitro system for studying PRRSV infection. Using this newly established
in vitro system of PRRSV infection in porcine bone marrow derived macrophages, this study
investigates how the phagosome microenvironment changes in porcine macrophages in PRRSV
infection. This study also investigates how phagosome lumenal properties are modified in
porcine alveolar macrophages isolated from PRRSV infected pregnant gilts.

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Acknowledgements
I would like to thank my supervisor Dr. Robin Yates for his guidance and support for this
study. Working under the supervision of a scientist as enthusiastic and motivated as him, has
been a great experience. Not only was his door always open for discussions, but he was also
always enthusiastic to troubleshoot, teach or work at the bench together.
I would like to thank Dr. Yan Shi, Dr Faizal Careem and Dr. Derek Mckay for investing
their time to be on my supervisory committee.
I would like to thank Dr. Neil McKenna for helping me carry out some of the ex vivo
experiments, proof-reading my thesis, and for all his invaluable suggestions for troubleshooting
experiments. He is a great co-worker who always greatly contributes to making working in lab
super fun for all lab members. I would also like to thank him for his patience when I was slow to
learn, and for having faith in my abilities.
I would like to thank everyone in the Yates lab - Dale Balce for his invaluable suggestions
on phagosomal assays, Jason Lemmon for valuable tips on improving writing skills. I would like
to thank Euan Allan, Jason Abboud, Pankaj Tailor, Paymon, Bernard Euanchak, Raymond Lam,
Samuel Lee for being great people to work with.
I would like to thank our collaborator Dr. John Hardings for sending us exvivo samples.
Finally, I would like to thank my family and friends for their moral support, particularly
my parents for having faith in my abilities and my sister who made me see “the other side of
the coin”. Moving half way across the globe can be challenging. I would like to thank all who
made this transition easy – particularly Meenakshi Rawat, Demyana Boles and my flatmate
Liane Babes.

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Dedication
Dedicated to my parents - who inspired me to dream big and believed in me at all times, even
when I did not believe in myself.

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TABLE OF CONTENTS
CHAPTER 1 : INTRODUCTION
1
1.1: Phagocytosis
2
1.2: Professional phagocytes
3
1.3: The macrophage
4
1.3.1: Alveolar macrophages
9
1.4: Phagosome formation
10
1.5: Phagosome maturation
14
1.6: Microbicidal effectors of the phagolysosome
19
1.7: Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)
23
1.7.1: Clinical symptoms:
25
1.8: PRRSV genome organization and structural proteins:
25
1.9: PRRSV cell tropism:
28
1.10: Entry of PRRSV into the host cell: PRRSV receptors
30
1.11: Immune responses of the host animal to PRRSV
35
1.12: Hypothesis and Specific Aims:
38
1.12.1: HYPOTHESIS:
38
1.12.2: Specific Aim I
38
1.12.3: Specific Aim II
39
1.12.4: Specific Aim III
39
CHAPTER 2: METHODS
44
2.1: Cell lines used
45
2.1.1: MARC 145 cell line
45
2.1.2: L929 cell line
45
2.2: Virus strain used
46
2.3: Growing PRRSV isolate NVSL 97-7895 in MARC 145 cells
46
2.4: Titration of virus by plaque assay
47
2.5: Cultivation of L929 supernatant
50
2.6: In vitro differentiation of porcine bone marrow cells into porcine bone marrow
derived macrophages
51
2.7: Freezing and thawing of porcine bone marrow cells
55
2.8: Infection of pBMMØ
55
2.9: Maintenance and infection of pregnant gilts:
56
2.10: Isolation of porcine alveolar macrophages (PAM) from BAL samples by density
gradient centrifugation
57
2.11: Assays for studying phagosomal lumenal properties
60