effect of the infection with porcine reproductive and respiratory syndrome virus on the regulation of

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Dissertations & Theses in Veterinary and Veterinary and Biomedical Sciences,
Biomedical Science Department of
Fall 8-2011
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Sakthivel Subramaniam
University of Nebraska-Lincoln, [email protected]
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Subramaniam, Sakthivel, "EFFECT OF THE INFECTION WITH PORCINE REPRODUCTIVE AND
RESPIRATORY SYNDROME VIRUS ON THE REGULATION OF CYTOKINES - TUMOR NECROSIS FACTOR-
ALPHA AND INTERLEUKIN-10" (2011). Dissertations & Theses in Veterinary and Biomedical Science. 7.
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EFFECT OF THE INFECTION WITH PORCINE REPRODUCTIVE AND
RESPIRATORY SYNDROME VIRUS ON THE REGULATION OF CYTOKINES -
TUMOR NECROSIS FACTOR-ALPHA AND INTERLEUKIN-10

by

Sakthivel Subramaniam

A DISSERTATION

Presented to the Faculty of
The Graduate College at the University of Nebraska
In partial Fulfillment of Requirements
For the Degree of Doctor of Philosophy

Major: Integrative Biomedical Sciences

Under the Supervision of Professor Fernando A. Osorio

Lincoln, Nebraska

August, 2011
EFFECT OF INFECTION WITH PORCINE REPRODUCTIVE AND RESPIRATORY
SYNDROME VIRUS ON THE REGULATION OF CYTOKINES - TUMOR
NECROSIS FACTOR-ALPHA AND INTERLEUKIN-10
Sakthivel Subramaniam, Ph.D.
University of Nebraska, 2011

Adviser: Fernando A. Osorio

Porcine reproductive and respiratory syndrome virus (PRRSV) causes late-term
abortion in sows and pneumonia in growing piglets. PRRSV evades the host immune
response by several mechanisms, including the modulation of cytokine secretions in
infected pigs, which is the subject of this dissertation. Particularly, PRRSV reduces the
secretion of the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α) but
increases the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). The
latter effect, however, is PRRSV strain-specific. In this dissertation, we have examined
mechanisms by which PRRSV regulates TNF-α and IL-10 expressions. The pathogenic
strain FL12, derived from a PRRSV infectious clone, consistently suppressed TNF-α but
failed to induce IL-10 in infected swine cells. In addition, no significant IL-10 production
takes place in pig tissues following infection with vFL12. Using a TNF-α promoter-
reporter gene construct, we demonstrated that the viral non-structural proteins, Nsp1α and
Nsp1β reduce the TNF-α promoter activity in transiently transfected cells, mainly by
inhibiting the cellular transcription factors Nuclear Factor-κB (NF-κB) and Specificity
Protein 1 (Sp1) respectively. Furthermore, screening of Nsp1α mutant constructs revealed
that five amino acid residues (Gly90, Asn91, Arg97, Arg100, and Arg124) are important
for inhibiting the TNF-α promoter activity. Nsp1α also reduced TNF-α protein levels in
an in vitro translation assay, and Gly90 was important for this activity. Screening of
Nsp1β mutant constructs showed that multiple amino acid stretches in all domains are
important for inhibiting the TNF-α promoter activity. We obtained two mutant vFL12
strains with substitutions at Nsp1αGly90 and Nsp1β70SMVRE74 positions. Both mutant
viruses increased TNF-α mRNA levels at early times after infection when compared to
parental vFL12 strain in infected macrophages. However, only Nsp1α mutant virus
induced higher TNF-α protein levels when compared to vFL12 in infected macrophage
cultures. Moreover, Nsp1β mutant virus was attenuated in infected pigs. In summary, we
have identified proteins that regulate the expression of TNF-α. These results may be
useful for designing novel prophylactics and therapeutics for PRRSV.
iv

Dedicated to my parents and teachers
v
ACKNOWLEDGEMENTS
I would like to express sincere gratification to my advisor, Dr. Fernando Osorio
for providing me the opportunity to pursue doctoral program under his guidance, for the
motivation to pursue the research topics, for constructive advices in research as well as
course works, for providing necessary needs to perform my studies. I also express sincere
thanks to my committee members, Dr. Asit Pattnaik, Dr. Clinton Jones, Dr. Jay Reddy
and Dr. Luwen Zhang for their valuable suggestions, advices to improve my skills
throughout research studies. I specially convey my thanks to faculty and staff of School
of Veterinary Medicine and Biomedical Sciences, Veterinary Diagnostic Center and
Animal Research Facility for their help in time, positive environment and support.
Particularly, I convey my special thanks to Judi at Veterinary Diagnostic Center who
allowed me to perform experiments in her lab.
I am also thankful for faculty, post-doctoral fellows and students at Nebraska
Center for Virology for their scientific interactions as well as discussions which helped
me to gain research knowledge and experience. I specially express sincere thanks to my
lab colleagues such as Kwon, Lalit, Hiep, Raje and former colleagues such as Marcelo,
Monica, for providing constructive suggestions in lab discussions as well as for their
positive interactions and help in the lab. I also thank students or research staffs from other
labs such as Debasis, Phat, Anshuman, Devis, Leticia, Pankaj, Chris, Chandir, Arun,
Nouhou for their good time, help in need and scientific interactions.
I also would like to express my sincere gratitude to my father and mother for their
love, sacrifices and encouragement. I convey special thanks to my wife, Priya for her
love, motivation and support.
vi
TABLE OF CONTENTS
TITLE i
ABSTRACT ii ii
DEDICATION iv iv
ACKNOWLEDGEMENTS v v
TABLE OF CONTENTS vi vi
CHAPTER I: Review of Literature 1

1. Introduction 1

2. Pathogenesis of PRRS 2
2.1. Clinical signs
2.2. Mortality and morbidity
2.3. Transmission and sites of replication
2.4. Lesions

3. Molecular biology of PRRSV 4
3.1. Structure of virion
3.2. Viral entry and uncoating
3.3. Viral replication, transcription, and assembly

4. Cell tropism of PRRSV 6
4.4.1. Macrophages
4.4.2. Dendritic cells
4.4.3. Host factors required for PRRSV infection

5. Innate immune responses during PRRSV infection 8
5.1. Type I Interferon responses against PRRSV infection
5.2. TNF-α response against PRRSV infection
5.3. Modulation of macrophages survival and innate function by PRRSV

6. Adaptive immune responses during PRRSV infection 11
6.1. Humoral immune responses against PRRSV
6.2. Cell-mediated immune responses against PRRSV

7. Cytokine responses during PRRSV infection 15
7.1. Modulation of pro-inflammatory cytokines by PRRSV
7.2. Anti-viral cytokines that inhibit PRRSV replication
7.3. Modulation of anti-inflammatory cytokines during PRRSV infection
vii
7.4. Modulation of T cell cytokines by PRRSV

8. TNF-α and its modulation by viruses 18
8.1. Biological functions of TNF-α
8.2. Mechanism and Regulation of TNF-α induction
8.3. Modulation of TNF-α by viruses

9. IL-10 and its modulation by viruses 19
9.1. Biological functions of IL-10
9.2. Modulation of IL-10 by viruses

10. PRRSV vaccine development 20

11. Objective 21

CHAPTER II
Porcine reproductive and respiratory syndrome virus non-structural protein 1
suppresses tumor necrosis factor-alpha promoter activation by inhibiting NF-κB
and Sp1 22

Table
1. Primers used for PCR amplification 40

Figures
1. PRRSV down modulates TNF-α response during in vitro infection in PBMC-
derived macrophages 41
2. PRRSV Nsp1α and Nsp1β down modulate the activity of TNF-α promoter at
the proximal region 43
3. Transcription factors affected by Nsp1α and Nsp1β at the proximal region (-
194) of TNF-α promoter 45
4. Nsp1α inhibits NF-κB activation 48
5. Nsp1β suppresses the trans-activation of Sp1 49
6. Proposed mechanisms behind PRRSV-mediated suppression of TNF-α
promoter 52

CHAPTER III
Identification of amino acids in non-structural proteins 1α and 1β of porcine
reproductive and respiratory syndrome virus that contribute to tumor necrosis
factor-alpha down regulation 53

Tables
1a. Primers used in site-directed mutagenesis 78
1b. Primers used in semi-quantitative RT-PCR 79
2. Recovery of PRRSV mutant viruses by reverse genetics 79
viii
Figures
1. PRRSV Nsp1α and Nsp1β suppress TNF-α promoter activity independent of
their cysteine protease functions 80
2. Characterization of Nsp1α amino acid residues necessary for reducing TNF-α
promoter activity 83
3. All domains of Nsp1β contribute to TNF-α promoter suppression 85
4. Nsp1β70-5A mutant protein is unable to suppress Sp1-dependent transcription 86
5. Nsp1α suppresses TNF-α protein expression and mutation at Gly90 position
partially relieves the suppression 88
6. Nsp1 mutant viruses have reduced growth when compared to wild type vFL12
virus 91
7. Effect of mutations in Nsp1 proteins on TNF-α as well as TNF-α inducible
chemokines during PRRSV infection in vitro 92
8. Comparison of viremia, TNF-α serum levels, and antibody responses between
pigs infected with vFL12 wild type and pigs infected with mutant viruses. 95

CHAPTER IV
A virulent strain of porcine reproductive and respiratory syndrome virus does not
up-regulate interleukin-10 levels in vitro or in vivo. 97

Figures
1. Phenotypic characterization of monocyte-derived macrophages and monocyte-
derived dendritic cells 112
2. PRRSV-vFL12 strain does not up-regulate IL-10 during in vitro infection of
monocyte-derived macrophages 114
3. PRRSV-vFL12 up-regulates IL-10 mRNAs but does not induce IL-10 protein
secretion in infected monocyte-derived dendritic cells 116
4. Confocal immunofluorecence staining of PRRSV-vFL12 infected and mock-
infected control tonsils for swine IL-10 and PRRSV nucleocapsid antigen 118

CONCLUSIONS 120

BIBLIOGRAPHY 124
1
CHAPTER I
Literature Review
1. Introduction
Porcine reproductive and respiratory syndrome (PRRS) is a disease affecting
swine, which is characterized by reproductive failure in pregnant sows and pneumonia in
growing pigs. The causative agent, porcine reproductive and respiratory syndrome virus
(PRRSV) belongs to the family Arteriviridae classified under the order Nidovirales (193).
The virus is classified into two genotypes, European (genotype I) and North American
(Genotype II) based on genetic relatedness (111). Within each genotype, several
genetically distinct strains are circulating in the swine populations temporally and
geographically (153, 162).
Since its identification in the late 1980s (77), the control of PRRS in the field was
unsuccessful due to the lack of effective vaccines. At present, the virus is endemic
throughout the world and is an economically important pathogen of swine. Current live
attenuated vaccine strains do not provide complete protection against heterologous strains
and may revert back to virulence (91, 128). Even with the availability of reverse genetics
systems for PRRSV (113, 114, 126, 178), the knowledge about the basis of attenuation,
means to increase heterologous protection, means to avoid immune evasion mechanisms
are needed to develop safe, effective, live attenuated PRRSV vaccines. In this dissertation,
we aimed to understand and explore means to attenuate PRRSV by knocking down
cytokine evasion mechanisms, which could lead to better vaccine development in the
future.