"Somewhere, something incredible is waiting to be known " - Carl Sagan University of Alberta
Antiviral pattern recognition receptors in the natural host of influenza, ducks (Anas platyrhynchos)
by
Megan Rani Winnifred Barber
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Physiology, Cell and Developmental Biology
Department of Biological Sciences
©Megan Rani Winnifred Barber Fall 2011 Edmonton, Alberta
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1*1 Canada Abstract
All strains of influenza replicate in the duck, the natural host of the virus. Ducks suffer little pathology from most influenza viruses, while the same strains are often fatal to chickens. The reasons why ducks and chickens have differing susceptibilities to influenza are not known. We speculated that innate immune mechanisms are particularly important for influenza defense to highly pathogenic avian influenza (HPAI) in ducks, due to the acute nature of this infection. We identified and characterized the known influenza detectors, toll like receptor 7
(TLR7) and retinoic-acid inducible gene-I (RIG-I) in ducks, to examine their early innate immune response to influenza. A comparison to chickens showed that ducks have a similar genomic organization of TLR7. However, the function of
TLR7 differs between ducks and chickens. Ducks, but not chickens, express TLR7 in the lung and always induce IFNa upon TLR7 triggering. Ducks have a functional RIG-I, while the gene appears to have been lost in chickens, affording them superior antiviral pattern recognition. Transferring duck RIG-I to chicken cells allows for recognition of RIG-I ligand, decreases low pathogenic avian influenza (LPAI) and HPAI virus titre, and induces antiviral gene transcription.
Thus, differences in the expression, function and even the repertoire of influenza- detecting pattern recognition receptors may all contribute to the relative resistance of ducks and susceptibility of chickens to influenza pathology. Understanding the immunological mechanisms mediating influenza resistance in ducks may provide insight into what constitutes a successful response to this virus. Acknowledgements
It is my pleasure to thank the individuals who made this thesis possible. First, I would like to thank my supervisor, Dr. Kathy Magor ("Boss"), to whom I am indebted for unwavering encouragement. She has allowed me the room to pursue my own ideas, but her door has always been open. It has been an honour to work with her and to be her first PhD student.
For their kind advice and support throughout the graduate school process, I would like to thank my committee members, Dr. Michele Barry and Dr. James Stafford. I would also like to extend my thanks to Dr. Warren Gallin and Dr. Vikram Misra for their involvement in my defence.
I am indebted to Dr. Robert Webster, who discovered the contribution of avian species to influenza, for welcoming me into his group at St. Jude Children's Research Hospital. Dr. Webster's continued mentorship, and the experience of pursuing influenza research in his world-class group, have been the highlights of my degree.
I am grateful for the generous support of a Natural Sciences and Engineering Research Council of Canada (NSERC) postgraduate scholarship (PGS-D) and a Canadian Poultry Research Council (CPRC) scholarship.
I have had the privilege of the entertaining company and scientific support of many colleagues who have become lifelong friends. I would like to thank members of Kathy Magor's lab, as well as Dr. Jerry Aldridge, Andrea Oko, and Daniela Villota.
I wish to thank my family for their support and for fostering a love of science. I am particularly grateful to Gil Barber, who is my everything. Lastly, I would like to thank my two trusty terriers, Ginger and Sprocket, who quite literally never left my side during the writing of this thesis. Table of contents
Examining committee
Abstract
Acknowledgements
Table of contents
List of figures
List of tables
List of abbreviations and symbols
Chapter I: Introduction 2
1.1 Influenza A virus biology 2
1.2 Evolution of influenza viruses - antigenic drift and shift 3
1.3 Natural ecology of influenza virus infection 4
1.4 Biology of LPAI infection in ducks 5
1.5 Adaptive immune mechanisms which favour LPAI perpetuation in ducks.. 6
1.6 Biology of HPAI infection in ducks 8
1.7 Innate immunity to influenza infection 9
1.7.1 Proinflammatory cytokines 12
1.7.2 Type I interferon and Interferon Stimulated Gene products 12
1.8 Pattern Recognition Receptors (PRRs) involved in influenza virus detection
14 1.8.1 TLRs 7 and 8 14
1.8.2 RIG-1 18
1.9 Influenza virulence factors 22
1.9.1 Counteracting host interferon with NS1 22
1.9.2 Beyond NS1 - other avian influenza virulence factors 23
1.10 Thesis rationale and objectives 26
Chapter II: Materials and methods 28
2.1 RNA collection from duck tissue and splenocytes for RT-PCR 28
2.2 Isolation of genomic and cDNA clones for duck TLR7 28
2.3 TLR bioinformatic analyses 29
2.4 RNA isolation, cDNA synthesis and TLR7 amplification from duck tissues
30
2.5 TLR agonist stimulation of isolated PBMCs and splenocytes 31
2.6 Identification and cloning of duck RIG-1 32
2.7Plasmids 33
2.8 In vitro transcription and RNAs 33
2.9 Cell culture, infections, and transfections 34
2.10 Immunofluorescence 35
2.11 Viruses and duck infections 36
2.12 Inoculation of embryonated eggs to grow and harvest influenza virus 36 2.13 MDCK plaque assays 37
2.14 Hemagglutination assays 37
2.15 Southern hybridization 37
2.16 Northern hybridization 38
2.17 Real-Time (qRT)-PCR 39
2.18 Microarray sample preparation, hybridization and analysis 41
Chapter III: Characterization of the duck TLR7/8 genomic loci 45
3.1 Introduction 45
3.2 Genomic organization of the duck TLR7/8 locus reveals that TLR8 is
disrupted 46
3.3 The structure of duck TLR7 is evolutionarily conserved 49
3.4 Structure of the rL/?7-hybridizing cDNA clone, 130K16 54
3.5 Tissue expression of duck TLR7 56
3.6 The TLR7 agonist, imiquimod, upregulates proinflammatory cytokines and
IFNa in duck splenocytes 57
3.7 Discussion 63
3.8 Author's contribution to data 66
Chapter IV: Identification of duck RIG-I and comparison to chicken 67
4.1 Introduction 68
4.2 RIG-I is present in ducks and apparently absent in chickens 69 4.3 Duck RIG-I detects in vitro transcribed RNA and activates the chicken
IFN(3 promoter 82
4.4 Transfected duck RIG-I detects influenza and induces an antiviral response
in chicken cells 86
4.5 RIG-Iis highly upregulated in ducks infected with VN1203 91
4.6 Discussion 94
4.7 Author's contribution to data 96
Chapter V: Functional characterization of duck RIG-I 97
5.1 Introduction 98
5.2 Duck RIG-I induces immune genes in response to BC500 100
5.3 Duck RIG-I induces immune genes in response to VN1203 105
5.4 Innate immune genes show increased expression in chicken cells expressing
duck RIG-I 114
5.5 Discussion 116
5.6 Author's contribution to data 120
Chapter VI: Conclusions and future directions 121
6.1 Summary 122
6.2 A model for innate defense to HPAI in ducks and chickens 127
6.3 Potential mechanisms for the loss of influenza-detecting PRRs in avian
species 130 6.4 Possible TLR7 and RIG-I driven strategies for influenza control in avian
species 132
6.5 Future directions 134
Chapter VII: Literature cited 136 List of tables
1.1 Innate immune receptors involved in host defense to influenza A infection... 11
5.1 GO terms for downregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with BC500 LPAI 102
5.2 GO terms for upregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with BC500 LPAI 103
5.3 A selection of immune genes upregulated more than twofold in RIG-I transfected DF-1 cells 15 hpi after infection with BC500 LPAI 104
5.4 GO terms for downregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with VN1203 HPAI 106
5.5 GO terms for upregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with VN1203 HPAI 108
5.6 A selection of immune genes upregulated more than twofold in RIG-I transfected DF-1 cells 15 hpi after infection with VN1203 HPAI Ill List of figures
1.1 Activation of TLR7 by influenza 16
1.2 Activation of RIG-I by influenza 21
3.1 Schematic diagram of the comparative genomic organization of the duck and chicken TLR7/8 loci 48
3.2 Amino acid alignment of duck, chicken, mouse and human TLR7 50
3.3 Schematic diagram of domain structures of duck, chicken, mouse, human and pufferfish TLR7 54
3.4 Structure of the rL/?7-hybridizing cDNA clone, 130K16 ....55
3.5 Analysis of duck TLR7 mRNA transcripts shows significant expression of duck TLR7 in the spleen, lung and bursa 57
3.6 IL-1/5 and IFNa mRNA expression in splenocytes stimulated with TLR7 and
TLR3 agonists 58
3.7 IL-ip, IL-6, IFNa, and TLR7 mRNA expression in splenocytes stimulated with TLR7 and TLR3 agonists 59
3.8 At 24 hours post-stimulation, IFNa is expressed in mock-treated splenocytes as well as those stimulated with TLR7 and TLR3 agonists 61
3.9 IL-lp, IL-6, IFNa, and TLR7 mRNA expression in PBMCs stimulated with
TLR7 and TLR3 agonists 62
4.1 Amino acid alignment of duck, zebra finch, and human RIG-I 70
4.2 Duck and chicken MDA5 are highly conserved 72 4.3 RIG-I is present in ducks and pigeons, but apparently absent in chickens 75
4.4 A single exon duck RIG-I probe fails to hybridize to human, mouse and chicken genomic DNA 76
4.5 RIG-I is present in ducks and pigeons, but apparently absent in chickens, while MDA5 is present in many avian species 78
4.6 RIG-I is present in pigeons, but apparently absent in other avian species 79
4.7 Pigeons are phylogenetically distant from ducks, compared to chickens 80
4.8 Hybridization of a single exon duck RIG-I probe to a northern blot containing
RNA from mock and H5N2-infected duck and chicken spleen, lung and intestine
RNA failed to detect RIG-I transcripts 82
4.9 Duck RIG-I rescues detection of 5'ppp RNA and induction of an antiviral response in DF-1 chicken embryonic fibroblasts 84
4.10 Supernatants from RIG-I transfected DF-1 cells reduce PR8 influenza replication in DF-1 chicken embryonic fibroblasts 88
4.11 RIG-I transfected DF-1 cells have significantly lower titres of HK213 90
4.12 RIG-I is dramatically upregulated in duck lung infected with VN1203 but not
BC500 93
5.1 Identification of duck RIG-I-responsive immune genes by microarray 112
5.2 Identification of duck RIG-I-responsive immune genes by microarray
(individual replicates) 113 6.1 A model for innate defense to influenza in duck and chicken epithelial cells 126
6.2 Simplified diagram of the airway epithelium, showing ciliated epithelial cells and resident plasmacytoid dendritic cells 129 List of abbreviations and symbols
a alpha
AI avian influenza
ANOVA analysis of variance
ATCC American type culture collection P beta
BC500 influenza A/mallard/British Columbia/500/2005
(H5N2) BLAST basic local alignment search tool BLASTn nucleotide BLAST BLASTp protein BLAST BLASTx translated nucleotide BLAST bp base pair BSA bovine serum albumin °C degrees Celcius CARD caspase activation and recruitment domain cDNA complementary deoxyribonucleic acid CIAP calf intestinal alkaline phosphatise CpG cytosine phosphate guanine CR1 chicken repeat 1 CT cycle threshold CTD c-terminal regulatory domain A change d days dATP deoxyadenosine triphosphate
DEPC diethylpyrocarbonate
DF-1 Doug Foster-1 cells (chicken embryonic fibroblasts, ATCC catalogue # CRL-12203)
DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate
DMEM Dulbecco's minimal essential media dsRNA double-stranded RNA
EDTA ethylenediaminetetraacetic acid eIF2a eukaryotic translation initiation factor 2A
EMEM Eagle's minimum essential media
EST expressed sequence tag
FBS fetal bovine serum
Fc fragment, crystallizable
GAPDH glyceraldehyde 3-phosphate dehydrogenase
GO gene ontology h hour(s)
HA hemagglutinin
HK213 influenza A/Hong Kong/213/97 (H5N1)
HPAI highly pathogenic avian influenza hpi hours post-infection
IFN interferon
IFNAR interferon-alpha/beta receptor Ig immunoglobulin
IL interleukin
IKK IKB kinase
ISG interferon stimulated gene
IPS-1 IFNp promoter stimulator 1
IRAK interleukin-1 -receptor-associated kinase
IRF interferon regulatory factor
JAK Janus kinase kb kilobase (s)
LB Luria-Bertani
LGP2 laboratory of genetics and physiology-2
LPAI low pathogenic avian influenza
LPS lipopolysaccharide
LRR leucine rich repeat
M molarity
Ml matrix 1
M2 matrix 2
MAVS mitochondrial antiviral signalling
MDA5 melanoma differentiation-associated gene-5 mDC myeloid dendritic cell
MDCK Madin-Darby canine kidney
MHC major histocompatibility complex mL millilitre MOI multiplicity of infection mRNA messenger ribonucleic acid
Mx myxovirus resistance protein
MyD88 myeloid differentiation primary response gene 88
NA neuraminidase
NCBI National Center for Biotechnology Information
NDV Newcastle disease virus
NEP nuclear export protein
NF-KB nuclear factor kappa-light-chain-enhancer of activated B cells ng nanogram
NLR NOD-like receptor
NOD nuclear oligomerization domain
NP nucleoprotein
NS1 nonstructural gene I
NTP nucleotide triphosphate
% percent
OAS 2'-5' oligoadenylate synthetase
OASL 2'-5' oligoadenylate synthetase-like
PA polymerase acidic protein
PB1 polymerase basic protein
PB1-F2 polymerase basic protein (frameshift)
PB2 polymerase basic protein 2
PBMC peripheral blood mononuclear cells PBS phosphate buffered saline
PCR polymerase chain reaction
PKR protein kinase R poly(I:C) polyriboinosinic:polyribocytidylic acid
PR8 influenza A/Puerto Rico/8/1934 (HlNl)
PRR pattern recognition receptor qRT-PCR quantitative polymerase chain reaction
RACE rapid amplification of cDNA ends
RIG-I retinoic acid inducible gene I
RLR RIG-I-like receptor
RNA ribonucleic acid
RNAi RNA interference
RT reverse transcription
SDS sodium dodecyl sulphate
SMART simple modular architecture research tool
SSPE saline-sodium phosphate-EDTA ssRNA single-stranded ribonucleic acid
STAT signal transducers and activators of transcription
TBK TANK binding kinase
Thl T helper 1
TIR toll/interleukin-1 receptor homology
TLR toll-like receptor
TNF tumor necrosis factor TPCK L-(tosylamido-2-phenyl) ethyl chloromethyl ketone
TRAF tumor necrosis factor receptor-associated factor
microgram(s) jiL microlitre (s)
\iM micromolar
UPL universal probe library
UTR untranslated region
VN1203 influenza A/Vietnam/1203/2004 (H5N1)
viral RNA Chapter I: Introduction
1 1.1 Influenza A virus biology
Influenza A viruses are enveloped, single-stranded negative-sense RNA viruses belonging to the Orthomyxoviridae family. The viral genome is composed of eight distinct ribonucleoproteins (RNPs), which encode 11 proteins. The viral
RNPs consist of viral RNA, a polymerase complex comprised of three subunits
(PA, PB1 and PB2), as well as nucleoprotein (NP) which surrounds the RNA.
Other structural proteins include the capsid matrix proteins (Ml and M2), and nuclear export protein (NEP), involved in the export of viral RNA (Tscherne and
Garcia-Sastre, 2011).
The virus surface contains the glycoproteins, hemagglutinin (H or HA) and neuraminidase (N or NA), which are used to classify the virus into antigenic subtypes. There are now 16 different H (H1-H16) and 9 different N (N1-N9) influenza A subtypes, and many different subtypes within each strain (Fouchier et al, 2005; Webster et al, 1992). Standard influenza nomenclature includes the virus genus (A, B or C), the species from which the virus was isolated (if other than human), the geographical location of isolation, number of isolate, and year of isolation, followed by the HA and NA subtype in parentheses (e.g. influenza
A/mallard/British Columbia/500/2005 (H5N2)).
A version of this chapter has been published. MacDonald (Barber) et al., 2007. Cytogenet Genome Res 117: 5913-5918.
2 HA is responsible for binding host cells through interactions with the terminal sialic acid residues that are present on many host cell glycoproteins. The species specificity of a given influenza A virus is partially determined by HA, because it shows specificity for sialic acids with distinct linkages. Human viruses preferentially bind sialic acids attached to the penultimate galactose by an a2,6 linkage and avian virus HAs preferentially bind an a2,3 linkage. Thus, human tracheal epithelial cells, the primary site of influenza replication, contain mainly a2,6 linkages between sialic acid and galactose (Couceiro et ai, 1993), while avian intestinal epithelial cells have predominantly a2,3 linkages (Ito et ai, 1998;
Ito et ah, 2000). After receptor binding, viral entry into the cell occurs by receptor-mediated endocytosis and subsequent HA fusion with the endosomal membrane. The viral genome is then released into the cytoplasm, followed by replication of the viral RNA in the nucleus. Viral assembly and budding occurs at the host cell membrane. The NA protein cleaves sialic acid from the host cell and nascent virions, which facilitates release of the virus from the cell membrane by preventing aggregation of virions (Bouvier and Palese, 2008).
1.2 Evolution of influenza viruses - antigenic drift and shift
Emergence of new strains of influenza occurs by two primary mechanisms. The viral RNA-dependent RNA polymerase does not have proofreading function, therefore like other RNA viruses, influenza is readily mutated. Rather than being genetically homogenous, a given influenza strain represents a quasispecies. This population of heterogenous genomes is dynamic -
3 constantly facing selection and undergoing evolution depending on the host environment. The high mutation rate allows for antigenic drift, whereby the HA and NA surface antigens gradually mutate. Eventually, an accumulation of mutations results in antigenic change significant enough to evade neutralizing antibodies (Bouvier and Palese, 2008).
The segmented RNA genome of influenza allows for the reassortment of genetic segments from different viral strains, known as antigenic shift. While antigenic drift is constant and gradual, antigenic shift is rare and unpredictable.
Antigenic shift results in the abrupt emergence of a novel influenza strain and is the mechanism responsible for the development of pandemic influenza viruses, strains to which humans are susceptible but immunologically naive (Bouvier and
Palese, 2008). Four influenza pandemics have arisen in the last century, in 1918,
1957, 1968 and 2009. In each case, they were caused by gene reassortment, resulting in novel H1N1, H2N2 and H3N2 subtypes (Tumpey et al, 2005;
Webster et al, 1992). A series of reassortment events led to the genesis of the
H5N1 strains currently circulating in Asia, which have devastated poultry flocks and caused human fatalities (Li et al, 2004).
1.3 Natural ecology of influenza virus infection
Influenza viruses cause infections in a wide variety of species, including humans, pigs, horses, sea mammals, dogs, and domestic poultry (Webster et al,
1992). Wild waterfowl, especially mallard ducks (Anas platyrhynchos), are the major natural reservoir of influenza A viruses and perpetuate all 16 HA and 9 NA
4 known subtypes of influenza. These avian viruses are the original source for all influenza pandemics (Webster et al, 1992). For the most part, influenza viruses cause asymptomatic infections in ducks. Presumably, lengthy coevolution between virus and host has led to an optimal evolutionary balance whereby the virus is perpetuated, while fitness costs to the host are limited (Kim et al, 2009).
Usually, influenza viruses have a restricted host range and replication is limited in other hosts (Beare and Webster, 1991; Hatta et al, 2002; Murphy et al,
1982). However, occasionally influenza viruses transmit to poultry or mammals where they have been shown to rapidly evolve (Ludwig et al, 1995). As a result of this process, strains may arise that are pathogenic to these non-natural hosts, as well as to the original duck reservoir. Only H5 and H7 subtypes are known to cause pathogenicity in ducks, and these subtypes can become highly pathogenic to both avian and mammalian species (Ludwig et al, 1995).
Influenza viruses are divided into low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), defined on the basis of their virulence in chickens. LPAI viruses cause only mild respiratory disease, depression and decreases in egg production, while HPAI infections have historically been called "fowl plague" for their ability to cause rapid, fatal disease
(Bosch et al, 1981; Bosch et al, 1979; Suarez and Schultz-Cherry, 2000).
Viruses that are highly pathogenic to chickens are usually non-pathogenic to ducks, the exception being some of the H5N1 strains currently circulating in Asia.
1.4 Biology of LPAI infection in ducks
5 LPAI is endemic in ducks, with up to 20% of juvenile mallards being infected during a given time (Hinshaw et ah, 1980b). Infectious virions can be isolated from lake water, allowing for fecal-oral transmission (Stallknecht et ah,
1990; Webster et ah, 1992). In ducks, avian influenza replicates primarily in the intestinal epithelial cells and virus is excreted in the feces, without causing apparent pathology (Webster et ah, 1978). Although the virus is cleared within a week, it may be that ducks can be reinfected with the same strain of virus, and they have limited serum antibody response (Kida et ah, 1980). Infection with one subtype does not protect against subsequent infection with other viral subtypes
(Austin and Hinshaw, 1984). Coinfection of ducks with more than one viral subtype (Sharp et ah, 1997), and reassortment between subtypes is common
(Hinshaw et ah, 1980a).
1.5 Adaptive immune mechanisms which favour LPAI perpetuation in ducks
The immunological mechanisms allowing ducks to host influenza are not known. However, elements of the duck cellular and humoral immune response may favour repeated influenza infections.
Influenza-specific IgA and IgY, an avian equivalent to IgG, have been demonstrated (Higgins et ah, 1987). However, ducks do not appear to mount a secondary antibody response to the virus (Kida et ah, 1980). The primary serum antibody, IgY, is made in two forms. The form that predominates later in the immune response is truncated and lacks the Fc region (Magor et ah, 1994; Magor et ah, 1992). While capable of neutralization, this truncated antibody presumably
6 lacks secondary effector mechanisms, including phagocytosis by professional antigen presenting cells. Such deficiencies in antigen uptake would decrease presentation on MHC II and limit the subsequent formation of memory responses.
Additionally, the immunoglobulin locus is unusually organized, with IgA being inverted in the opposite transcriptional orientation in the heavy chain locus. This means that class switching to IgA requires an inversion and cannot occur by normal excision of upstream genes, possibly making IgA expression unstable
(Magor etal., 1998).
Ability to initiate lasting cellular immune responses to influenza also depends on the diversity of Major Histocompatibility Class I (MHC I) alleles on the cell surface that are able to present viral peptides to circulating cytotoxic T cells. MHC I expression in ducks is very limited, with only one gene predominantly expressed (Mesa et al, 2004). On average, a given MHC allele is predicted to be able to present one or two peptides from a virus (Louzoun et al,
2006). Thus, the number of viral peptides able to be presented in ducks is extremely limited, and epitope mutation is likely a very effective strategy by which influenza could avoid a robust cellular immune response and T cell memory in its natural host.
Deficiencies in the duck adaptive immune system could be contributing to limited humoral and cellular responses and diminished viral memory, allowing ducks to become repeatedly infected by influenza and making them a very effective reservoir of the virus.
7 1.6 Biology of HPAI infection in ducks
Transmission of endemic LPAI from wild birds to domestic poultry occasionally leads to reassortment and mutation of the virus into a HPAI strain, capable of causing significant pathology to poultry and mammals, and occasionally the natural duck reservoir. The HPAI H5N1 strains currently circulating in Asia arose in 1996, most probably from a low pathogenic H5 virus circulating in wild ducks or migratory waterfowl (Duan et al, 2007). A HPAI
H5N1 reassortant appeared in poultry and live bird markets in 1997, and was found to cause lethal infection in humans. A large number of H5N1 genotypes have arisen since 1997, potentially due to reassortment with LPAI strains in wild ducks. Unusually, some of these viruses have been pathogenic to wild birds, including ducks. However, while the viruses are universally lethal to domestic poultry, such as chickens, pathogenicity in ducks has varied. In Qinghai Lake,
China, most wild bird species died off due to four different H5N1 strains, but mallard ducks did not suffer pathology (Chen et al, 2005). Unlike LPAI strains,
HPAI appears to replicate in the lungs of ducks and virus titres can be higher in oropharyngeal swabs than cloacal swabs (Sturm-Ramirez et al, 2004). When
H5N1 viruses are pathogenic to ducks, the strains rapidly evolve to become less pathogenic (Hulse-Post et al, 2007; Hulse-Post et al, 2005).
Currently, numerous HPAI H5N1 strains are asymptomatic in ducks, causing them to spread the virus as "Trojan horses" of influenza (Hulse-Post et al, 2007; Kim et al, 2009; Sturm-Ramirez et al, 2005). These same viruses cause 100% mortality in chickens within hours. How ducks so successfully
8 respond to the same strain of influenza that is lethal in chickens is not known. Due to the rapid nature of a HPAI infection, we speculate that superior innate immunity to the virus may afford ducks increased protection, allowing for rapid detection and clearance of the virus. Deficiences in the adaptive immune response may allow ducks to perpetuate influenza by permitting a poor memory response to the virus. On the other hand, a successful innate immune response may limit pathology and decrease lethality from influenza, also contributing to the role of ducks as the reservoir for influenza.
1.7 Innate immunity to influenza infection
In response to viral manipulation of host cell machinery, the immune system has evolved means of detecting viral invasion and initiating antiviral defense. The outcome of infection with avian influenza viruses depends on early immune responses. The innate immune response involves rapid, non-specific, and germline-encoded detection of pathogens. Plants and invertebrate species primarily use RNA interference (RNAi) pathways for antiviral protection, whereby viral nucleic acids are specifically degraded. In vertebrates, antiviral defense depends on viral nucleic acid detection by pattern recognition receptors
(PRRs) and subsequent activation of the innate immune system.
PRRs are able to distinguish viral from host RNA. They can do this because of certain viral nucleic acid characteristics that identify them as foreign, or because of the unusual location of viral nucleic acids, such as in the endosome.
Numerous cellular antiviral proteins are triggered by dsRNA, which does not
9 normally accumulate in the cytoplasm, such as toll-like receptor (TLR) 3 and protein kinase R (PKR). While controversial, recent evidence suggests dsRNA accumulation to be minimal in influenza-infected cells (Weber et al, 2006).
Instead, genomic and antigenomic ssRNA with short dsRNA regions bearing a 5' triphosphate cap are an influenza signature because host RNAs are generally capped with a methylguanosine.
The mammalian immune system has evolved at least three classes of PRRs responsible for the detection of viral nucleic acids. These include the nucleotide oligomerization domain (NOD)-like receptors (NLRs), retinoic-acid inducible gene I (RIG-I) like receptors (RLRs), and the toll-like receptors (TLRs). The mammalian NLR family members are involved in the formation of inflammasomes, complexes of proteins which activate caspase-1 to induce the maturation of proinflammatory cytokines including IL-1(3, IL-18 and IL-33
(Kanneganti et al, 2007). Inflammasome activity is essential for adaptive immunity to influenza virus (Ichinohe et al., 2009). Specifically, the NLR molecule known as NLR family, pyrin domain containing 3 (NLRP3), has been associated with the innate immune response to influenza through the formation of the NLRP3 inflammasome. NLRP3 is activated by the influenza virus M2 ion channel protein (Ichinohe et al, 2010) and NLRP3 -/- mice have increased mortality and decreased cytokine production upon influenza infection (Allen et al, 2009; Thomas et al, 2009). However, NLR family members have yet to be identified and characterized in avian species.
In mammals, the cytosolic RIG-I receptor recognizes influenza genomic
10 RNA, while TLR7 and 8 detect incoming viral RNA in the late endosome. While
RIG-I is used in most cells, primarily plasmacytoid dendritic cells use the TLR pathway for influenza detection (Kato et al, 2005). Detection of influenza RNA by either receptor results in an antiviral state through the release of type I interferon (IFN) and proinflammatory mediators, and is critical for later adaptive immunity. Type I IFN instructs the adaptive immune system, serving to activate and mature dendritic cells (Proietti et al, 2002).
Other mammalian innate immune receptors with a demonstrated role in influenza defense include the natural killer cell activating receptors NKp44
(Arnon et al, 2001) and NKp46, which have been shown to be activated by influenza HA, leading to lysis of influenza-infected cells. The critical role of
Nkp46 has been demonstrated in mice which lack the NKp46 gene, Ncrl, which suffer lethal influenza infection (Gazit et al., 2006). The innate immune receptors with established roles in influenza defense are summarized in Table 1.1.
Table 1.1 Innate immune receptors involved in host defense to influenza A infection
Innate immune receptor Role
Natural Killer receptors • The activating receptors NKp44 and NKp46 recognize influenza HA, leading to lysis of infected cells. Nucleotide binding • Influenza triggers the NLRP3 inflammasome to activate oligomerization domain caspase 1, which leads to the subsequent maturation of (NOD)-like receptors pro-ILl (3 to mature IL-1 (3 (NLRs) Retinoic acid inducible • Recognizes influenza genomic RNA with 5' gene I (RIG-I) triphosphate ends within the cytoplasm of most cells to induce proinflammatory cytokine and interferon gene expression
11 Toll-like receptor 7 • Responsible for recognition of influenza genomic RNA (TLR7) within the endosome of immune cells, leading to proinflammatory cytokine and interferon gene expression Toll-like receptor 8 • Responsible for recognition of influenza genomic RNA (TLR8) within the endosome of immune cells, leading to proinflammatory cytokine and interferon gene expression
1.7.1 Proinflammatory cytokines
Influenza infection is accompanied by production of proinflammatory cytokines, such as tumor necrosis factor a (TNF-a), interleukin-ip (IL-ip), and interleukin-6 (IL-6), which promote lymphocyte infiltration and activation.
However, they may also exert negative effects on the host. Hypercytokinemia or
'cytokine storm' is an uncontrolled increase in proinflammatory cytokines leading to significant subsequent inflammation, and was hypothesized to be a major mechanism responsible for lethal influenza infection (de Jong et ah, 2006).
However, inhibiting proinflammatory cytokines in a mouse model through knockout or suppression with cytokine inhibitors does not protect against lethal
H5N1 infection (Salomon et ah, 2007).
1.7.2 Type I interferon and Interferon Stimulated Gene products
Type I IFNs (IFNa and IFNP) are a multigenic group of antiviral cytokines present in higher vertebrates. Type I IFNs were discovered in chicken cells in
1957 by Isaacs and Lindemann, due to their protective effects against influenza. It is still unclear whether or not different members of the Type IIFN family are induced differently, have distinct functions or are expressed by different cellular 12 subsets (Brideau-Andersen et al, 2007). After viruses are detected by the cell, signalling cascades activate transcription factors including IFN regulatory factors
(IRFs), nuclear factor KB (NFKB) and activating transcription factor (ATF)/c-jun
(Yoneyama and Fujita, 2009). When these transcription factors translocate to the nucleus, an 'enhanceosome' forms on the IFN promoter allowing for interaction with RNA polymerase, and the transcription of IFN (Carey, 1998). NFKB is also involved in proinflammatory cytokine expression. All the Type I IFNs act in an autocrine and paracrine fashion to induce transcription of interferon stimulated genes (ISGs), activate natural killer cells, differentiate cytotoxic T cells and to activate dendritic cells to prime the adaptive immune system (Randall and
Goodbourn, 2008). A successful innate immune response to influenza infection involves a robust, yet transient induction of IFN-stimulated antiviral genes.
All type I IFNs bind to the ubiquitously expressed IFNa/p receptor
(IFNAR). The pivotal role of this receptor is illustrated by IFNAR_/~ mice which are highly susceptible to viral infections (Hwang et al, 1995). After binding, signalling occurs through the Janus Kinase (JAK)/signal transducers and activators of transcription (STAT) signalling cascade, leading to activation of the transcription factor complex ISGF3, which is composed of STAT1, STAT2 and
IRF9 (Schindler et al, 2007). Formation of ISGF3 on ISG promoters leads to the expression of antiviral defense genes, such as protein kinase R (PKR), 2'-5' oligoadenylate synthetase (OAS) and myxovirus resistance protein (Mx)
(Yoneyama and Fujita, 2010). PKR is a dsRNA dependent serine/threonine kinase that binds dsRNA, leading to the phosphorylation of eukaryotic initiation factor
13 2a (eIF2a) and the subsequent inhibition of host and viral protein translation
(Garcia-Sastre and Biron, 2006). OAS also recognizes dsRNA, resulting in the production of 2'-5' oligoadenylates that activate RNase L. Activated RNase L degrades host and viral RNA, leading to transcription inhibition (Garcia-Sastre and Biron, 2006). Mx is a nuclear GTPase, which is proposed to interfere with influenza replication by directly inhibiting the viral polymerase complex within the nucleus (Engelhardt et ah, 2004).
Other genes stimulated by IFN include genes involved in antigen presentation, such as MHC I. IFN controls the expression of PRRs (such as RIG-
I) that are themselves responsible for IFN induction. This interferon-dependence serves to amplify the antiviral response in a positive feedback loop.
1.8 Pattern Recognition Receptors (PRRs) involved in influenza virus detection
1.8.1 TLRs 7 and 8
Toll was initially identified as a Drosophila protein involved in development and anti-fungal immunity (Lemaitre et ai, 1996). Mammalian homologues of Toll were then shown to be involved in innate immunity
(Medzhitov et ai, 1997). The Toll-Like Receptors (TLRs) are a family of transmembrane proteins possessing an ectodomain of leucine rich repeats (LRRs), which detect conserved microbial patterns. TLRs also have a conserved Toll/IL-I receptor (TIR) intracellular signalling domain. TLRs 7 and 8 are endosomal pattern recognition receptors that mediate innate immune activation to single-
14 stranded RNA (ssRNA) viruses such as influenza (Diebold et ah, 2004; Heil et al, 2004; Lund et al, 2004) that gain entry to the cell by receptor-mediated endocytosis. Maturation and acidification of the endosome can result in release of viral genomic ssRNA, which then serves as an agonist for TLRs 7/8. Triggering of TLR7/8 leads to the recruitment of other TIR-domain containing adaptor proteins such as MyD88, stimulating a signalling cascade that activates transcription factors such as nuclear factor KB (NF-KB) (Figure 1.1). The resulting upregulation of proinflammatory cytokines and type 1 IFN ultimately induces a
Thl-skewed, antiviral immune response. Half of the TLR7 ectodomain is proteolytically cleaved off within the endolysosome, and only this cleaved form can successfully recruit adaptor proteins, likely a mechanism to prevent self-RNA recognition by restricting receptor function to the endolysosomal compartment where self RNA is not normally present (Ewald et al, 2008).
15 LRR (leucine rich repeat) domain
TM (transmembrane) domain
TIR (toll/ll -1 receptor) domain
Figure 1.1 Activation of TLR7 by influenza. When influenza enters the cell by receptor mediated endocytosis, some release of the ssRNA genome may occur within the acidic endosome. These ssRNAs trigger TLR7/8, leading to recruitment of the TIR-domain containing adaptor protein, MyD88 and a subsequent complex of TRAF3, TRAF6, IRAKI/4, IKK-a and IRF7. Signalling through MyD88 activates the transcription factors IRF-7 and NF-KB, to induce IFNP and cytokine gene expression (Takeuchi and Akira, 2010). IRAK, IL-1R- associated kinase. TRAF, TNFR-associated factor. IKK, IKB kinase. IRF, interferon regulatory factor.
16 TLRs 7 and 8 are structurally and evolutionarily related as products of gene duplication, and are located in tandem in the genome. Despite their similarities, the specificity and function of TLRs 7 and 8 differs between vertebrate species. Human TLRs 7 and 8 respond to a different range of agonists and are also expressed in a distinct panel of cellular subsets, indicating they have at least partially unique function. In humans, compared to TLR8 agonists, TLR7- selective agonists better activate plasmacytoid dendritic cells (pDCs) and B cells to produce IFN and IFN-regulated chemokines. TLR8-selective agonists activate monocytes and myeloid dendritic cells, primarily inducing proinflammatory cytokines and chemokines (Gorden et al, 2005). Human TLR8 can detect the presence of single-stranded RNA sequences from influenza (Heil et al, 2004).
Mouse TLR8 does not respond to human TLR8 agonists, which are instead recognized by mouse TLR7 (Jurk et al, 2002). TLR7 knockout mice lose the ability to detect influenza (Lund et al., 2004). Although mouse TLR8 was previously thought to be non-functional, TLR7 and TLR9 knockout mice, have recently been shown to respond to a combination of imidazolquinoline and polyT oligodeoxynucleotides (Gorden et al., 2006).
In humans and mice, pDCs produce the vast majority of IFNa upon influenza infection, a process which is dependent on TLR7 and MyD88 (Diebold et al, 2004). Unlike other cell types, pDCs constitutively express IRF7. Viral cytosolic replication intermediates may be detected by TLR7 in pDCs by autophagy from the cytosol to lysosomes (Lee et al, 2007).
17 1.8.2 RIG-I
While TLR7 and 8 are necessary for influenza-induced IFN production by pDCs, most cell types ubiquitously express the RIG-I receptor which surveys the cytoplasm for virus. The cytoplasmic RLR family has three members, RIG-I itself, as well as MDA5 and LGP2. MDA5 is similar in structure to RIG-I, but recognizes a distinct subset of viruses. LGP2 lacks CARD domains and was originally suggested to play a regulatory role by inhibiting RIG-I and MDA5 signalling (Venkataraman et al, 2007). However, recent contradictory results demonstrate that LGP2 positively regulates RIG-I and MDA5 signalling through its ATPase domain (Satoh et al, 2010). The authors suggest LGP2 may modify viral RNA to facilitate detection by RIG-I and MDA5 (Satoh et al, 2010).
These receptors all share a DExD/H-box helicase domain, characterized by a DEAD motif (Asp-Glu-Ala-Asp). The RNA helicase domain has high homology to the ancient helicase, Dicer (Deddouche et al, 2008). Dicer is involved in viral defence through RNA interference in invertebrates, suggesting an evolutionary origin for RLRs in the RNAi pathway (Yoneyama and Fujita,
2010). The role of the helicase is unclear, as it has been shown to unwind dsRNA, but this ability does not affect IFN production (Takahasi et al, 2008).
Additionally, RIG-I and MDA5 have a C-terminal domain (CTD) which is involved in both agonist recognition (Takahasi et al, 2008) and repression of helicase activity in the absence of agonist.
For a long time, the exact identity of the natural agonist for RIG-I was unknown. In 2004, the receptor was shown to respond to the dsRNA homologue,
18 poly (I:C) or infection with a negative-stranded RNA virus, with dimerization of
IRF3 and activation of IFNp promoter activity (Yoneyama et al, 2004).
Subsequent studies showed that the receptor responded to in vitro transcribed
RNA but treating the RNA with calf alkaline intestinal phosphatase (CIAP) abrogated this response, suggesting that RNA bearing a 5' triphosphate triggers
RIG-I (Hornung et al, 2006). Pichlmair et al. showed that recognition of influenza genomic RNA was dependent upon at least one 5' phosphate (Pichlmair et al, 2006). Among other possible agonists, it was also suggested that host cell
RNA that has been cleaved by RNase L could trigger RIG-I (Malathi et al, 2007).
However, it now appears that RIG-I signalling is activated by 5' triphosphate
RNA containing short RNA (under 1 kb) with double-strand conformation, such as that derived from viral RNA with panhandle structures (Schlee et al, 2009;
Schmidt et al, 2009). Recently, it has been demonstrated that RIG-I agonists are full-length single-stranded (ss) RNA viral genomes with 5' triphosphates, exclusively (Rehwinkel et al, 2010). When an agonist is recognized by the CTD of RIG-I, a conformational change is triggered that allows for receptor dimerization and recruitment to the mitochondria for downstream signalling.
RIG-I and MDA5 possess two N-terminal caspase activation and recruitment domains (CARDs) that allow for interaction with the CARD-domain containing mitochondrial adaptor protein, IPS-1 (also known as MAVS, CARDIF and VISA) (Yoneyama and Fujita, 2008). Signalling downstream of IPS-1 results in the activation of interferon regulatory factors (IRFs) 3 and 7, as well as NF-KB, and subsequent production of proinflammatory cytokines and IFN (Figure 1.2).
19 RIG-I itself was originally identified as a cDNA clone that enhanced activity of an
IFN-driven promoter complex (Yoneyama et ai, 2004). RIG-I has since been found to be indispensable for the IFN response to many ssRNA viruses, including influenza and Newcastle Disease Viruse (NDV) as well as members of the paramyxovirus family and hepatitis C virus (Kato et ai, 2005). MDA5 detects long dsRNA (over 2kb) such as the synthetic dsRNA homologue polyinosinic:polycytidylic acid (poly (I:C)) and is essential for the detection of viruses which produce large amounts of dsRNA, such as picomaviruses (Kato et ai, 2005). A few RNA viruses, such as West Nile Virus are detected by both
RIG-I and MDA5 (Fredericksen et al, 2008).
20 Figure 1.2 Activation of RIG-I by influenza. Influenza-infected cells contain viral genomic RNA which is single-stranded RNA with short dsRNA regions, with a 5' triphosphate. RIG-I is normally present in an inactivated state whereby the tandem CARD domains are repressed from signalling by the CTD. When RIG-I is triggered by influenza genomic RNA, a conformational change and dimerization occurs. TRIM25 activates RIG-I via ubiquitination, an action which is prevented by the influenza NS1 protein. RIG-I is recruited to the mitochondrial adaptor protein, IPS-1. Signalling through IPS-1 activates the transcription factors IRF-3, IRF-7 and NF-KB, to induce IFNfi and cytokine gene expression. MDA5 is related to RIG-I and shares the same signalling pathway, but detects a distinct subset of viruses. CARD, caspase activation and recruitment domain. CTD, C- terminal domain. IKK, IKB kinase. IRF, interferon regulatory factor. TBK, TANK binding kinase. TRIM25, tripartite-motif protein 25.
21 Additionally, RIG-I directly activates the NLR-dependent inflammasome complex to mature proinflammatory cytokines (Poeck et al, 2010). There is also some suggestion that RIG-I may inhibit influenza replication directly and independent of IFN, because antibodies that neutralize DFNp do not completely abolish the antiviral activity of RIG-I (Kato et al, 2006).
RNA viruses have been shown to be more virulent and replicate to higher levels in mice lacking RIG-I (Kato et al, 2006). A hallmark of lethal influenza virus infection is interference with expression of RIG-I and downstream genes
(Kobasaera/., 2007).
1.9 Influenza virulence factors
1.9.1 Counteracting host interferon with NSl
Considering the central role of IFN in influenza defense, it is not surprising that the virus has evolved the means to counteract the IFN response at multiple levels. The major activity of the influenza NSl protein is to inhibit IFN and ISG products (Garcia-Sastre, 2001; Hale et al., 2008), which first became evident when NSl-deficient viruses were found to be unable to replicate in the presence of IFN (Garcia-Sastre et al, 1998). NSl blocks the cellular IFN response by several means, from evading viral recognition by PRRs to preventing IFN transcription (Garcia-Sastre, 2001). NSl complexes with TRIM25, preventing this cellular factor from activating RIG-I via ubiquitination (Gack et al, 2009). NSl can also bind to cellular factors responsible for cellular mRNA polyadenylation,
22 inhibiting the interferon response by selectively inhibiting transcription of host but not viral RNAs (Nemeroff et al, 1998).
The viral factors responsible for the extremely pathogenic nature of the pandemic 1918 HlNl strain have not been fully characterized. Hypercytokinemia has been frequently mentioned as a possible mechanism for the severity of infection with both 1918 HlNl and avian H5N1 strains. NSl proteins from H5N1
HPAI viruses are associated with dysregulation of the immune response and induction of high levels of proinflammatory cytokines. Several point mutations in
NS1 have been associated with enhanced virulence in chickens and mice, including D92E which leads to enhanced expression of proinflammatory cytokines and repression of the effect of NS 1 on IFN induction. A 5 amino acid deletion at position 80-94 also increases virulence and enhances resistance to
TNFa (Li et al, 2010; Long et al, 2008). Moltedo et al. demonstrated that influenza undergoes an almost "stealth" replication prior to the initiation of adaptive immunity. In humans infected with wildtype influenza, no evidence of an innate immune response was observed until two days after infection when a sudden inflammatory burst occurred, which was followed by dendritic cell migration to the lymph node. When NSl-deficient viruses were used, robust inflammation was detected almost immediately. Thus NS 1 affords the virus almost two days of replication prior to immune detection (Moltedo et al, 2009).
1.9.2 Beyond NSl - other avian influenza virulence factors
The pathogenicity of a given influenza strain is multigenic, and is also
23 specific to the host species infected. Numerous virulence determinants for avian influenza viruses have been determined, in the majority of influenza proteins
(recently reviewed by Tscherne and Garcia-Sastre, 2011).
A major determinant of virulence in many species is the presence of a stretch of multiple basic amino acids in the cleavage site of the HA gene. HA is expressed as a precursor, HAO, which must be subsequently cleaved into the HAl and HA2 to reveal the fusion peptide on HA2, which allows for viral fusion with the endosomal membrane (Horimoto and Kawaoka, 1994). HAO must be cleaved in nascent viruses in order for them to be infectious. In LPAI viruses, HAO is cleaved by trypsin-like enzymes and viral infection is limited to tissues expressing these enzymes. Acquisition of a multibasic cleavage site in HAO allows for cleavage by a wider range of host enzymes, which allows for systemic influenza infection (Kawaoka and Webster, 1988). This multibasic HAO cleavage site is required for lethal infection of H5 and H7 subtypes in chickens and mice (Hatta et al, 2001).
The viral polymerase, consisting of PA, PB1 and PB2 subunits, contributes to influenza virulence, likely by simply controlling the amount of vRNA produced. A comparison of two H5N1 viruses, A/duck/Fujian/01/2002 and A/duck/Guangxi/5 3/2002 found a correlation between polymerase activity and pathogenicity (Leung et al, 2010). Both viruses were isolated from apparently healthy ducks but they showed different virulence in mice. The determinants of virulence for the H5N1 viruses that acquired the ability to kill ducks are mostly uncharacterized, but substitutions in two different amino acids
24 (S223P and N383D) of the polymerase PA subunit have shown to contribute to a highly virulent phenotype (Song et al, 2011). The HPAI strain
A/Vietnam/1203/04 (H5N1) (VN1203) is an unusual virus in that it is highly pathogenic to ducks. A single amino acid substitution in the PA (T515A) protein has been reported to reduce pathogenicity in ducks, while PB1 Y436H reduces pathogenicity and transmissability in ducks (Hulse-Post et al, 2007). PB1-F2 is a small protein encoded by a frameshift of the PB1 gene that enhances polymerase activity by binding to PB1 (Mazur et al, 2008) and has proapoptotic function in immune cells (Chen et al, 2001; Zamarin et al, 2005). Several mutations in PB2 affect pathogenicity and host range (Hatta et al, 2001; Hatta et al, 2007; Mase et al, 2006; Puthavathana et al, 2005; Smith et al, 2006).
Besides the HA and polymerase proteins, influenza virulence is partially determined by its structural proteins. Mutations in the Ml (matrix) protein have been shown to contribute to the virulence of H5N1 in mice. Fan et al demonstrated that the differences in virulence of two H5N1 stains that were both lethal in chicken, but were of low and high pathogenicity for mice, were due to two point mutations in the Ml (matrix) protein (Fan et al, 2009). The structural viral NP protein coats and interacts with viral genomic RNA. The NP A184K mutation has been shown to induce immune gene expression and increase pathogenicity in chickens, possibly by increasing replication by changing the way
NP interacts with viral RNA (Wasilenko et al, 2009).
25 1.10 Thesis rationale and objectives
How ducks support influenza replication in the absence of overt disease is unknown. In the case of a LPAI infection, presumably ducks have an immune set point that controls viral replication but allows for frequent infection and thus persistence of the virus in the population. HPAI infections are also usually self- limiting in ducks, without causing significant immunopathology. These same
HPAI strains can cause severe disease with extreme morbidity and mortality in chickens, in as little as 18 hours. Due to the acute nature of this disease, it is predicted that innate immune mechanisms are particularly involved. Conceivably, ducks have rapid detection of influenza infection and initial clearance of the virus.
Understanding how ducks survive influenza infection could provide insight into what constitutes a successful immune response to the virus.
The aim of this project was to characterize influenza-detecting PRRs in ducks, with the aim of determining how they contribute to influenza defence in the natural reservoir of the virus. Comparisons were made to the chicken immune system to try to determine why there is differential susceptibility to influenza between ducks and chickens. The goal was to (1) determine the organization of the duck TLR7/8 locus and whether duck TLR7 is functional. We hypothesized that the TLR8 gene was intact in ducks but not chickens. The project then broadened to (2) assess for the presence of RIG-I in ducks and chickens and (3) characterize the antiviral function of duck RIG-I and its ability to complement the immune response in chicken cells.
26 Chapter II: Materials and methods
27 2.1 RNA collection from duck tissue and splenocytes for RT-PCR
White Pekin ducks (Anas platyrhynchos) were euthanized by lethal injection of Euthanol for collection of tissues that were used fresh, or snap frozen in liquid N2. Tissues used for isolation of RNA included spleen, duodenum, heart, kidney, liver, brain, lung and bursa. Blood was collected by wing puncture and peripheral blood mononuclear cells (PBMCs) were isolated from 10 mLs of whole blood via density-gradient centrifugation with Ficoll-paque PLUS (Amersham).
Splenocytes were dispersed by passage through a fine wire mesh followed by isolation using density-gradient centrifugation with Ficoll-paque PLUS.
2.2 Isolation of genomic and cDNA clones for duck TLR7
To identify duck TLR7, a fragment was amplified from duck spleen cDNA using primers in the N-terminal LRR (5'-TGT GGA GAT TGA CTT CAG
GTG CAA CTG C-3') and conserved regions of the TIR domain (5'-TGT GGA
GAT TGA CTT CAG GTG CAA CTG C-3'). Overlapping primers (5'-CTG AAT
GCT CTG GGA AAG GTT GTC-3') and (5'-AGG ACA GCC AGT CTT TGA
CAA CCT-3') within the partial TLR7 sequence were used to make a 32P labeled
'overgo' probe used to screen a genomic library from duck (Anas platyrhynchos)
(Moon and Magor, 2004) and two clones were isolated.
A version of this chapter has been published. MacDonald (Barber) et al., 2008. Mol Immunol 45: 2055-2061 and Barber et al, 2010. Proc Natl Acad Sci 13: 5913-5918.
28 Clone 124F14 was sequenced to an average depth of 5x with a minimum of bidirectional coverage, using the EZ::TN
(Applied Biosystems) on an ABI377 Sequencer (Applied Biosystems). Final assembly of the contig was confirmed by restriction mapping, and the complete sequence of 124F14 deposited in Genbank under the accession number
DQ888644.
A duck splenic cDNA library (Mesa et al, 2004) was arrayed on membranes (Xia et al, 2007) and screened using the TLR7 probe. Only one cDNA clone was identified, clone 130K16, which was sequenced by primer walking on both strands and deposited in Genbank under accession number
DQ888645. A continuous open reading frame could not be identified in this clone due to insertion of an alternate exon, 2a.
2.3 TLR bioinformatic analyses
Sequences were edited and assembled using VectorNTI Advance 10 software (Invitrogen). Augustus gene prediction (http://augustus.gobics.de)
(Stanke and Waack, 2003) and BLAST (www.ncbi.nlm.nih.gov/blast) (Altschul et al, 1990) were used to predict open reading frames. Repetitive elements were identified with RepeatMasker (http://repeatmasker.org) (Tarailo-Graovac and
29 Chen, 2009). Amino acid sequences were aligned using ClustalW
(www.ebi.ac.uk/clustalw) (Larkin et al, 2007) and edited with BOXSHADE
(http://bioweb.pasteur.fr/seqanal/interfaces/boxshade.html). SMART (Simple
Modular Architecture Research Tool - http://smart.embl-heidelberg.de) (Schultz et al, 1998) was used to predict the domain structure of duck TLR7.
2.4 RNA isolation, cDNA synthesis and TLR7 amplification from duck tissues
RNA was extracted using TRIZOL (Invitrogen) and quantified using a
NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies). For amplification of TLR7 transcripts from tissue samples, primers were designed spanning an intron, in the first (5'-CAA ATC TTT CAG CTG TGG AAG CAC A-
3') and third exons (5'-CCA CTC TCA CTG AAC CTT CAG AGG C-3') of
TLR7. First-strand cDNA was synthesized from 5 ug of RNA using the
Thermoscript RNase H- Reverse Transcriptase (Invitrogen) kit. The reverse transcription reactions were primed with an oligo(dT) primer, AdapT (5'-TCT
GAA TTC TCG AGT CGA CAT CT17-3'). cDNA was resuspended in 20 uL of
DEPC treated water and 1 uL used as template. TLR7 PCR cycling conditions were as follows: 5 minutes at 96°C, followed by 25 cycles of 96°C for 1 minute,
60°C for 30 seconds and 72°C for 30 seconds. Long terminal extension was performed at 72°C for 7 minutes. The product was subcloned into the TOPO 2.1 vector (Invitrogen) and sequenced for verification. The major TLR7 product amplified corresponded to ex on 2 joined directly to exon 3. No products
30 corresponding to the sequence of the cDNA clone carrying an alternate exon 2a were identified. Expression of glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) was used as a control for quality and quantity of cDNA. GAPDH primers, (5'-CCG TGT GCC AAC CCC CAA TGT CT-3') and (5'-GCC CAT
CAG CAG CAG CCT TCA TC-3'), spanned an intron. PCR conditions were performed as above, but for 20 cycles.
2.5 TLR agonist stimulation of isolated PBMCs and splenocytes
Isolated splenocytes (lymphoid cells isolated from the spleen) and PBMCs were resuspended and plated at 3 x 106 cells/mL in RPMI1640 supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, 0.25 fxg/mL fungizone
(P/S/F, Gibco/BRL), and 10% fetal calf serum (Gibco/BRL) and maintained at
37°C in 5% CO2. Cells were stimulated by addition of TLR agonists and incubated for 6 hours. TLR agonists (InvivoGen) were prepared in sterile endotoxin-free water or ethanol (loxoribine). Splenocytes and PBMCs were stimulated with a range of agonist concentrations and an optimal concentration for each agonist was chosen: 100 uM of loxoribine, 10 ug/mL imiquimod, 1 u.g/mL ssPolyU/Lyovec and 25 ug/mL poly (I:C). Splenocytes from two ducks and
PBMCs from three ducks were tested. Stimulation results were qualitatively similar for PBMC, however were more pronounced in splenocytes.
Each agonist was used to treat cells in triplicate for isolation of RNA.
RNA was extracted using TRIZOL (Invitrogen), followed by purification from the final aqueous phase using the RNeasy Mini Kit (Qiagen). First strand cDNA
31 synthesis was performed using the Superscript III kit (Invitrogen) using the
AdapT primer. Sequences for duck GAPDH DR765844, IL-1/3 AY426338, IL-6
AB191038, and IFNa AY879230 were retrieved from the NCBI database. Primers were chosen to span an intron to detect genomic contamination in the RNA, which was not seen. Initial PCR products were cloned and sequenced for verification. Approximately 25 ng of first strand cDNA was used as a template for cytokine and IFNa amplification. PCR cycling conditions for GAPDH and IFNa were as above, but with an annealing temperature of 60°C for 30s for 25 cycles.
Duck IL-ip (5'-CCC GTG TAC CGC TAC ACC CGC TCC-3') and (5'-GAT
GTC CCT CAT GAC GGC GGC CTC-3') was amplified using an annealing temperature of 62°C for 15s for 32 cycles. Duck IL-6 was amplified for 30 cycles with primers (5'-TGT GCG AGA ACA GCA TGG AGA TG-3') and (5'- GAA
TCT GGG ATG ACC ACT TCA TC-3'). TLR7 was amplified as above.
2.6 Identification and cloning of duck RIG-I
A PCR fragment of RIG-I was obtained from duck (Anas platyrhynchos) splenic cDNA using primers (5'-GAT CCC AGC A AT GAG AAT CCT AAA
CT-3') and (5'-CAA TGT CAA TGC CTT CAT CAG C-3'), based on a conserved region of the human RIG-I sequence. The complete cDNA sequence was obtained via 5' and 3' RACE using the SMART RACE cDNA Amplification Kit
(Clontech). The complete coding region of duck RIG-I was amplified using primers in the 5' UTR (5'-CGG CCG GCA GAG CCC AGC C-3') and 3' UTR
(5'-GTG TAG GAG AGT AAT AGA TGC ACT A-3') using Phusion High-
32 Fidelity DNA Polymerase (New England Biolabs), cloned into pCR 2.1-TOPO
(Invitrogen) and completely sequenced.
2.7 Plasmids
pcDNA-RIG was obtained by cloning duck RIG-I into the mammalian expression vector pcDNA 3.1 hygro (Invitrogen). Duck RIG-I was digested out of pCR 2.1-TOPO (Invitrogen) using Spel and Notl. RIG-I was then inserted between Nhel and Notl sites of pcDNA 3.1 hygro (Invitrogen). The chicken IFNP promoter luciferase reporter (pGL3-chIFNP) was constructed from White
Leghorn chicken genomic DNA using primers with incorporated Bglll and Mlul sites that amplified -158 to +14 of the chicken IFN-2 promoter, as previously done (Childs et al, 2007; Sick et al, 1998). The promoter fragment was then inserted between Bglll and Mlul sites of the pGL3-basic luciferase reporter vector
(Promega).
2.8 In vitro transcription and RNAs
The 21-mer RNA, 5'-pppGGGGCUGACCCUGAAGUUCCC-3'
(Hornung et al, 2006) was transcribed from annealed DNA oligonucleotides (5'-
TAA TAC GAC TCA CTA TAG GG-3') and (5'-GGG A AC TTC AGG GTC
AGC CCC TAT AGT GAG TCG TAT TA-3') containing a T7 promoter site, using the T7 Megashortscript kit (Ambion). In vitro transcription was carried out overnight, followed by DNasel digestion and precipitation. Calf intestinal alkaline phosphatase (CIAP) treatment was carried out for 3 h using 30 ug of in vitro
33 transcribed RNA, 30 U of CIAP (Invitrogen), lx CIAP buffer, and 200U
RNaseOut (Invitrogen). CIAP-treated 5'ppp RNA was purified by phenol- chloroform extraction and precipitation. Poly (I:C) (25 mg/mL) was obtained from InvivoGen.
2.9 Cell culture, infections, and transfections
UMNSAH/DF-1, a spontaneously immortalized chicken embryonic fibroblast cell line derived from East Lansing strain eggs (Schaefer-Klein et al,
1998) was maintained in DMEM plus 10% FBS. Cells (1.25 x 105) were seeded overnight in 24-well plates. Cells were co-transfected with 150 ng of pcDNA-RIG or empty pcDNA, 150 ng of pGL3-chIFNp, and 10 ng of the constitutive renilla luciferase reporter phRG-TK (Promega). Thirty hours after plasmid transfection, cells were transfected with 21-mer 5'ppp RNA (800 ng), CIAP-treated RNA (800 ng), or poly (I:C) (25 ug/mL). Plasmids and RNA agonists were transfected using
Lipofectamine 2000 (Invitrogen). Luciferase activity was measured using the
Dual-Luciferase Reporter Assay System (Promega) 15 h after challenge. For infection of transfected cells, DF-1 cells were maintained in DMEM plus 10%
FBS and 1.25 x 105 cells were seeded overnight in 24-well plates. Cells were transfected with 1 fj.g of pcDNA-RIG or empty pcDNA. Twenty four hours after transfection, cells were infected at a multiplicity of infection (MOI) of 1 with
H5N2 A/mallard/BC/500/05 or H5N1 A/Vietnam/1203/04 virus. L-(tosylamido-
2-phenyl) ethyl chloromethyl ketone-treated trypsin (Worthington Biochemicals) was used for infection with BC500 at a low concentration (0.1 ug/mL) in view of
34 the trypsin sensitivity of DF-1 cells (Lee et al, 2008). Exogenous trypsin was not added for infection with VN1203. Fifteen hours after infection, RNA was extracted from cells for quantitative RT-PCR (qRT-PCR) and influenza virus titer was determined by standard plaque assay on MDCK cells, which were maintained in EMEM + 10% FBS.
2.10 Immunofluorescence
DF-1 cells were maintained in DMEM + 10% FBS. 1.25 x 105 cells were seeded overnight in 24-well plates. Cells were transfected with 1 \ig of pcDNA-RIG or empty pcDNA. 48 hours post-transfection, cells were infected with 50 hemagglutinating units of A/PR/8/34 (H1N1) virus per mL. 15 hours post infection, 100 [xL of infected supernatant was used to infect fresh overnight cultures of 1.25 x 105 DF-1 cells. 3 hours post-infection, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 1% (v/v) Triton X-100 for 10 min, blocked with 4% BSA in PBS for 5 min, and incubated with 1:200 diluted
FITC-labelled anti-influenza NP (Argene) primary antibody in 4% BSA for 30 min. After incubation, cells were washed three times with PBS. Fluorescence microscopy was performed using a Zeiss inverted microscope (Zeiss) fitted with a
10 x Zeiss objective and Axiovision 4 imaging software (Zeiss). Images were collected at 1388 x 1040 pixel resolution and pseudo-coloured. CorelDRAW X3
(Corel) was used to render the images. Data shown is a representative of three independent experiments.
35 2.11 Viruses and duck infections
The H5N1 A/Vietnam/1203/04 HPAI was generated by reverse genetics
(Salomon et al., 2006) and H5N2 A/mallard/BC/500/05 LPAI was isolated by screening of environmental samples. The viruses were propagated in 10 day old embryonated chicken eggs and handled at St. Jude Children's Research Hospital, with VN1203 handled in biosafety level 3+ facilities approved by the United
States Department of Agriculture and Centers for Disease Control and Prevention.
Outbred White Pekin ducks (Anas platyrhynchos) were purchased from Ideal
Poultry or Metzer Farms, and all animal experiments were approved by the
Animal Care and Use Committee of St. Jude Research Hospital and performed in compliance with relevant institutional policies, National Institutes of Health regulations, and the Animal Welfare Act. A total of 106 of 50% egg infectious doses of BC500 and VN1203 were used to inoculate 6-week-old mallards via the natural route, in nares, eyes, and trachea. Mock infection was PBS only. Ducks were euthanized and tissues collected at day 1 and day 3 post infection (n = 3, except for BC500 day 3 samples, n = 2). Tracheal and cloacal swabs were collected to monitor viral shedding.
2.12 Inoculation of embryonated eggs to grow and harvest influenza virus
Chicken eggs were candled to ensure embryo viability and sterilized with
70% ethanol. A hole was drilled into the shell over the egg sac and 100 u.L of virus diluted in PBS and egg antibiotics (kanamicin, gentamicin, neomycin and polymixin B) was injected into the amniotic cavity. Eggs were incubated at 37°C
36 for 3 days to grow virus, prior to being chilled at 4°C to kill the embryos. Virus was then harvested from the allantoic fluid. Hemagglutination tests were performed to assess for presence of virus.
2.13 MDCK plaque assays
MDCK cells were plated overnight in 6-well plates. Serial 10-fold dilutions of virus samples were used to infect the confluent MDCK monolayers for 1 hour. Semisolid agar/EMEM was then applied to the cells. Three days post infection, plaques were visualized by staining with crystal violet/formaldehyde and counted.
2.14 Hemagglutination assays
Hemagglutination assays were carried out in 96 well round bottom plates.
A 0.5% suspension of chicken erythrocytes diluted in PBS was combined with serial two-fold dilutions of virus and incubated at room temperature for one hour.
Wells containing a homogenous layer of erythrocytes were scored as positive for hemagglutination, and wells were scored as negative if erythrocytes clumped together at the bottom of the well.
2.15 Southern hybridization
Genomic DNA was extracted from the blood of White Pekin duck (Anas platyrhynchos), White Leghorn chicken (Gallus gallus), pigeon (Columba livid),
Merriam's turkey (Meleagris gallopavo), pheasant (species unknown) and chukar
37 partridge (Alectoris chukar). Genomic DNA (10 ug) was digested to completion, separated on 0.8% agarose, and blotted to Nytran Supercharge (Schleicher &
Schuell). DNA was immobilized by UV cross-linking and baking for three hours at 80 °C. A multiple-exon 307-bp RIG-I probe in the helicase domain was amplified using the primers (5'-ACA GGT ATG ACC CTC CCA AGC CAG-3') and (5'-CAT CCC ATT TCT GGA TCT TTT CAA CAG-3') from a defined duck
RIG-I clone. A 171-bp probe predicted to contain a single exon was amplified from the same template using (5'-GTA TGA CCC TCC CAA GCC AGA AGG -
3') and (5'- CTC TGA CTT GGA TCA TTT TGG TCA CG-3'). A 150-bp duck
MDA5 probe was amplified with the following primers: (5'-GAG CAA AGG
GAA GTC ATT GAT AAA TTC C-3') and (5'-GGC CTG CAA CAT AGC AAT
TTC ATT-3') from a duck MDA5 clone. All probes were radiolabelled with a-32P dCTP by random priming (Prime It Random Primer Labeling Kit; Stratagene).
Blots were hybridized overnight at 42°C in 50% formamide, 5x Denhardt solution, 4x SSPE, 5% dextran sulfate, 1% SDS, and 100 ug/mL salmon sperm
DNA. Washes were carried out at low stringency in lx sodium chloride/sodium phosphate/EDTA and 0.1% SDS at 52°C. Film was exposed for three days. After development, the probe was stripped by pouring boiling stripping solution (O.lx
SSC, 0.1% SDS) over the blot then cooling to room temperature three times.
2.16 Northern hybridization
RNA was isolated from chicken and duck mock or virus-infected spleen, lung and intestine as described above and 10 jxg run on a 1.2% agarose, 0.6%
38 formaldehyde gel. The RNA gel was blotted onto a Nytran Supercharge nylon transfer membrane and UV cross-linked (UV Stratalinker 2400, Stratagene), followed by baking at 80°C for three hours. The blot was prehybridized at 42°C for two hours and hybridized overnight with GAPDH or RIG-I probes using the
50% formamide hybridization solution described above. The RIG-I probe was described above, and the GAPDH probe was prepared by amplifying a product from duck heart cDNA with the primers (5'-CCG TGT GCC AAC CCC CAA
TGT CT-3') and (5'- GCC CAT CAG CAG CAG CCT TCA TC-3'). Each fragment was random-primed with a-32P dCTP then purified using Sephadex G-
25 Fine (Sigma Chemical) size-exclusion chromatography. The northern blot was hybridized overnight at 42°C in 50% formamide. For the duck blot, three 15 minute high stringency washes (O.lx SSPE, 0.1% SDS) were carried out at 60°C.
For chicken blots, low stringency washes (lx SSPE, 0.1% SDS, 52°C) were used.
Blots were exposed to film at - 80°C for three days (GAPDH) or eight days (RIG-
I).
2.17 Real-Time (qRT)-PCR
RNA was extracted using TRIZOL (Invitrogen), followed by purification from the final aqueous phase using the RNeasy Mini Kit (Qiagen). First strand cDNA synthesis was performed using the Superscript III kit (Invitrogen) using
OligodT (Invitrogen) and random primers (Invitrogen). To quantify RIG-I gene expression from duck tissues, 50 ng of cDNA was amplified in a 10 uL reaction using the Applied Biosystems 7500 real-time PCR system (Applied Biosystems).
39 Duck primers and fluorogenic TaqMan FAM/TAMRA (6- carboxyfluorescein/6- carboxytetramethylrhodamine)-labeled hybridization probe mixes were obtained from Applied Biosystems and used with FastStart Universal Probe Master (Rox;
Roche Applied Science). Duck GAPDH was used as an endogenous control.
Primer and probe sequences were as follows: duck RIG-I primers (5'-GTG TAT
GGA GGA AAA CCC TAT TTC TTA ACT-3') and (5'-GGA GGG TCA TAC
CTG TTG TTT GAT-3'), and probe (5'-TTC CGC GCC CCA TCA A-3'). Duck
GAPDH primers were (5'-GCC TCT TGC ACC ACC AAC T-3') and (5'-GGC
ATG GAC AGT GGT CAT AAG AC-3') and probe (5'-CAC AAT GCC AAA
GTT G-3'). Changes in gene expression post infection were expressed as a ratio of the level observed in a mock-infected animal. RT-PCR was performed for RIG-I and GAPDH in a single-plex format, with the following cycling conditions: 95°C for 10 min for activation, followed by 40 cycles at 95 °C for 15 s and 60°C for 1 min. Quadruplicate cycle threshold CT values were analyzed with SDS software
(Applied Biosystems) using the comparative CT (AACT) method. To quantify gene expression from transfected DF-1 cells, 20 ng of cDNA was amplified in a
10 JJ,L reaction using the Applied Biosystems 7500 real-time PCR system
(Applied Biosystems). Chicken 28s RNA (endogenous control) and IFNfi probes and primers (Peters et al., 2003) were obtained from Applied Biosystems and used with TaqMan Fast Universal PCR Master Mix (Applied Biosystems). For
Influenza A matrix gene expression, primers were designed according to CDC recommendations. InfA forward primer was (5'-GAC CRA TCC TGT CAC CTC
TGA C-3'); MA reverse was (5'-AGGGCA TTY TGG ACA AAK CGT CTA-
40 3'); and MA probe (5'-TGC AGT CCT CGC TCA CTG GGC ACG-3') (Centers for Disease Control and Prevention). The primers and probes for chicken Mxl
(accession number NM_204609) and PKR (accession number NM_204487.1) were designed with the Roche online Universal Probe Library (UPL) system
Assay Design Center. The primers for Mxl were (5'-GTT TCG GAC ATG GGG
AGT AA-3') and (5'-GCA TAC GAT TTC TTC AAC TTT GG -3') (UPL probe
80). The primers for PKR were (5'-TGC TTG ACT GGA AAG GCT ACT-3') and
(5'-TCA GTC AAG AAT AAA CCA TGT GTG-3') (UPL probe 29). Cycling conditions were 95 °C for 20 s for activation, followed by 40 cycles at 95 °C for 3 s and 60°C for 30 s. Changes in gene expression were expressed as a ratio of the level observed with cells transfected with RIG-I or vector only.
2.18 Microarray sample preparation, hybridization and analysis
Cloning of duck RIG-I into pcDNA, cell transfections and infections were performed as described above. Briefly, DF-1 cells were transfected with 1 u,g of pcDNA-RIG or empty pcDNA. Twenty-four hours after transfection, cells were infected at a multiplicity of infection (MOI) of 1 with H5N2
A/mallard/BC/500/05 or reverse genetics H5N1 A/Vietnam/1203/04 virus. Fifteen hours after infection, RNA was extracted from cells for microarray analysis. The microarray (Agilent-015068, Chicken Gene Expression 4x44K), consisting of
42,034 60-mer in situ synthesized oligonucleotides, was designed and manufactured by Agilent Technologies (Santa Clara, CA). Labelled cRNA was prepared from 500 ng of total RNA using the Agilent labelling protocol, and
41 microarray hybridization was performed at 65 °C for 18 hours in Agilent's microarray hybridization chambers, followed by wash procedures according to the manufacturer's recommended protocols. The testing samples (Cy5) were co- hybridized with uninfected, untransfected reference DF1 samples (Cy3). Three sets of replicates, including duck RIG-I transfected samples along with vector- only transfected samples, were performed following BC500 infection; while two sets of replicate were performed following VN1203 infection. The microarray was scanned in an Agilent scanner at 3 urn resolution, and the array data was extracted with Agilent feature extraction software (version 10.5.1.1) using the
GE2_105_Jan09 protocol. Reproducibility and reliability of each single microarray was assessed using Quality Control report data. Expression ratios compared with the reference control were calculated and log2 transformed.
Lowess normalization on background-subtracted signal intensity was performed to correct the intensity bias, and additional filtering steps, including valid measurement in at least 50% of the tested samples, were applied to further improve the quality of the data set. The relative differential gene expression between RIG-I transfected and vector-only transfected samples was reported as the difference of log2 ratios between these two groups, and a p-value using t-test was calculated for each gene as statistical measurement of the replicates. The selection of the genes for Gene Ontology term analysis (DAVID, http://david.abcc.ncifcrf.gov [42]) was based on a change of at least twofold and a probability of >95% (P < 0.05) on differential expression in at least two experiments. The data discussed in this publication have been deposited in NCBI's
42 Gene Expression Omnibus [43] and are accessible through GEO Series accession number GSE29596
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSEGSE29596).The selection of the genes for Gene Ontology term analysis (DAVID, http://david.abcc.ncifcrf.gov (Huang et al, 2009)) was based on a change of at least twofold and a probability of > 95% (p < 0.05) on differential expression in at least two experiments.
43 Chapter III: Characterization of the duck TLR7/8 genomic loci 3.1 Introduction
Toll like receptors (TLRs) mediate proinflammatory signaling in response to conserved molecular patterns (Barton, 2007; Uematsu and Akira, 2007). TLR7 and TLR8 respond to U-rich single-stranded RNA (ssRNA), and are candidates for the detection of influenza (Lund et al, 2004). When viruses enter the cell via receptor-mediated endocytosis, acidification may release the genome, which then triggers TLR7/8. The endosomal location allows discrimination against self- nucleic acids that could otherwise initiate the same immune responses (Marshak-
Rothstein and Rifkin, 2007). These receptors are type I transmembrane proteins, possessing extracellular tandem leucine-rich repeats, and a toll-interleukin-1 receptor (TIR) intracellular domain, responsible for initiating an inflammatory response. Diebold et al, 2004 have demonstrated that pDC recognition of influenza is TLR7- and MyD88- dependent.
The TLR7 and 8 genes lie adjacent to each other in the genomes of mammals and fish (Meijer et al, 2004; Roach et al, 2005), and are clearly products of an ancient gene duplication, being structurally and functionally related
(Heil et al, 2003). Analysis of the chicken TLR7/8 genomic locus revealed that chickens possess an intact TLR7 gene, but that TLR8 is only present in a series of small fragments which are disrupted by a retroviral, CRl-type insertion element
(Philbin^aZ.,2005).
A version of this chapter has been published. MacDonald (Barber) et al, 2008. Mol Immunol 45: 2055-2061 45 Using primers in a TLR8 fragment and the CR1 insertion, the authors amplified a fragment confirming this disruption existed in all galliform birds (chickens and their relatives). The hemi-nested PCR failed to amplify a fragment in anseriform birds, including ducks. These results were taken to suggest that there was a galliform-specific disruption of the TLR8 gene. The suggestion that ducks might possess TLR8, while chickens have dispensed with this receptor, was intriguing in light of the different susceptibility to influenza of ducks and chickens. To determine if ducks have TLR8, we sequenced the duck TLR7/8 genomic locus.
Results
3.2 Genomic organization of the duck TLR7/8 locus reveals that TLR8 is disrupted
Genes encoding TLRs 7 and 8 are adjacent to each other in the genomes of fish and mammals. Chickens have lost TLR8. To determine whether ducks possess an intact TLR8, we examined the TLR7/8 genomic locus of ducks. We fully sequenced the 44 kb clone 124F14, which included TLR7 and approximately
15 kb of sequence downstream. The duck TLR7/8 locus shows a well-conserved organization compared to the chicken with TLR7 downstream of phosphoribosyl pyrophosphate synthetase II (PRPS2) (Figure 3.1). Both species possess an intact
TLR7 gene and only fragments of a TLR8-like gene. Open reading frames in
124F14 (38 598 bp) were revealed by nucleotide and protein BLAST searches,
AUGUSTUS gene prediction software, as well as by mapping the duck TLR7
46 cDNA onto the genomic sequence. The 5' end of 124F14 contains three exons (5,
6 and 7) of the PRPS2 gene, which is directly upstream of TLR7 in other species examined to date. Approximately 13 kb downstream of PRPS2, a 3138 bp exon was identified, possessing high homology to the major coding exon (3) of TLR7.
TLR7 is completely encoded within a single exon (exon '3' in chicken, human and mouse), except for the initiating methionine (located within exon '2' in chicken, human and mouse). Approximately 11 kb further downstream, three fragments (411, 488 and 147 bp) with homology to TLR8 (Sus scrofa E value 3e-
15) were identified by BLASTX search. Repeatmasker analysis revealed that
124F14 contains several retroviral insertion elements, including a 1.4 kb CR1 element present between fragments of the TLR8 gene, similar to chicken. Six other CRl-type retroviral insertion elements were also interspersed throughout
124F14.The failure of the PCR to amplify the TLR8 disruption in ducks (Philbin et ai, 2005) is due to differences in the sequence under the primers. While the sequence under the forward primer in the TLR8 fragment had only two mismatches, sequences matching the two reverse primers in the CR1 element could not be identified in duck, accounting for the failure of the PCR.
47 kb 0 10 20 30 40 50 60 I I
DUCK (124F14) U* 1 5 6 7 2 3 PRPS2 TLR7 CR 1 & TLR8
CHICKEN f 7 2 3 PRPS2 TLR7 CR1& TLR8 TMB4SX
Figure 3.1 Schematic diagram of the comparative genomic organization of the duck and chicken TLR7/8 loci. In both ducks and chickens, TLR7 is intact, while only fragments of TLR8 are present, disrupted by a CRl-type retroviral insertion element. PRPS2, phosphoribosyl pyrophosphate synthetase 2. TMB4SX, thymosin beta 4 X-linked.
48 3.3 The structure of duck TLR7 is evolutionarily conserved
To compare the amino acid sequence of duck TLR7 to known TLR7s we
generated an alignment of duck TLR7 with that of other vertebrates (Figure 3.2).
Significant conservation was revealed between all TLR7s. The translated amino
acid sequence of duck TLR7 reveals a predicted protein of 1047 amino acids, with
features of all TLRs, including an ectodomain composed of tandem leucine rich
repeats (LRRs), a transmembrane domain, and a conserved intracellular TIR
signaling domain. Duck TLR7 contained variants of the conserved boxes within
the TIR region, important for signaling (box 1 and 2): FDAFISY and GYKCC-
RD-PG, and localization (box 3): a W surrounded by basic residues (Slack et al,
2000). The conserved proline found at position 712 in mouse TLR4, essential for
signaling through TIR (Underhill et al, 1999) is also present in duck TLR7. The
duck receptor exhibited 66% amino acid identity to human TLR7 while only 44%
amino acid identity to human TLR8 (BLASTp), confirming its identity as a bona fide TLR7 orthologue.
49 DuTLR7 0 CHTLR7 0 M0TLR7 0 HUTLR7 0
DUTLR7 96 ChTLR7 96 MOTX.R7 100 HUTLR7 100 CfilPJ
DuTLR7 ChTLR7 MOTLR7 HUTLR7
DuTLR7 295 gflRHlgS ChTLR7 295 HHSIJ|SL MoTLR7 300 aB3VHPTl HUTLR7 300 a321JVQP]
DuTLR7 395 ChTLR7 395 MoTLR7 400 HuTLR7 400 fi
DuTLR7 495 ChTLR7 4 95 MoTLR7 498 HuTLR7 497
DuTLR7 595 ChTLR7 595 MOTLR7 598 HuTLR7 597
DuTLR7 695 CbTLR7 695 MoTLR7 698 HuTLR7 697
DuTLR7 7 95 ChTLR7 7 95 MoTLR7 7 98 HuTLR7 797
DuTLR7 895 ChTLR7 895 HoTLR7 898 HTKNSJ HuTLR7 897
DuTLR7 995 ChTLR7 995 MoTLR7 998 BuTLR7 997
• Signal Peptide • LRR • i Transmembrane Domain
• TTR dornam
50 Figure 3.2 Amino acid alignment of duck, chicken, mouse and human TLR7. Alignment was performed using the CLUSTAL W program and edited with BOXSHADE. Black shading indicates amino acid identity, grey shading indicates similarity (50% threshold). Underlined structural domains are predicted by SMART (Simple Modular Architecture Research Tool) and refer to duck TLR7. Boxes labeled 1, 2 and 3 refer to conserved regions of TIR known to be essential for signaling (Box 1: FDAFISY, Box 2: GYKCC-RD-PG, Box 3: W surrounded by basic residues). Asterisks indicate the conserved cysteines in the TLR9 family motif. LRR, leucine rich repeat. TIR, toll-ILl receptor signaling domain. TLR7 sequences are shown for duck (Du), chicken (Ch), mouse (Mo) and human (Hu).
51 The amino acid identity of chicken and duck TLR7 was only 85%. The differences were largely in the LRR domains, since conservation of the LRR region between ducks and chickens was 82% as compared to 93% amino acid identity in the conserved TIR domain. A loop excluded from the LRR regions, proposed to be the cap structure separating the LRR region into two horseshoe domains in the TLR7, 8, and 9 family, corresponding to amino acids 435-497 of human TLR7 (Matsushima et al, 2007) showed the greatest interspecies variation. This region is followed by LRR domains in which mutations cause loss of function, D543A in human TLR8 (Gibbard et al, 2006) and D535A in TLR9
(Rutz et al, 2004), and mouse L499P in TLR9 (Tabeta et al, 2004). Residues corresponding to both the aspartate and leucine are present in the duck sequence,
L517 and D553. There are numerous non-conservative amino acid differences between the duck and chicken sequences.
To compare the secondary structure of duck TLR7 to that of other species, we identified the domain structure using the SMART program (Figure 3.3).
Secondary structure analysis of duck TLR7 revealed that it possesses a signal peptide, followed by 18 leucine rich repeats, including cysteine rich N-terminal and C-terminal LRRs. The assignment of the repeats, important in agonist- binding, varied between species. Ducks possess 16 internal LRR repeats, while chickens have only 15 detected by this program. Domains with high interspecies conservation included the N-terminal and C-terminal LRRs, as well as the transmembrane and TIR domains. LRR domains with insertions, thus not detected by this program, have been implicated in agonist-binding (Bell et al, 2003). A 16-
52 residue insertion unique to and highly conserved within the TLR7, 8 and 9 family is also seen in the duck sequence. It has four conserved cysteines in the pattern
CxRCxxxxx CxxC. The motif is also seen in the CpG binding protein, and is thought to co-ordinate a metal ion involved in nucleic acid binding (Lee et al.,
2001). This region in human TLR8 is critical for signaling presence of imidazoquinoline and resquimod, and mutation of any of the cysteine residues results in loss of function (Gibbard et al, 2006).
53 Amino Acids 1 200 i_ i i DuTLR7 of j— - -j -....-MCIO- ""•0-lHHHF-j OK* MOTLR7D[] . _ . 0K> HuTLR7 of 1—1 U U 1 U L_ - _ . _ OIO PuTLR7 of j— Ml- - — -MDK* D Signal Peptide 0 N-terminal LRR 1 1 C-terminal LRR 1 Transmembrane Domain LRR, Leucine Rich Repea< o TIR Domain
Figure 3.3 Schematic diagram of domain structures of duck, chicken, mouse, human and pufferfish TLR7. Domains were predicted by SMART. Genbank accession numbers are: DuTLR7 (DQ888644), ChTLR7 (NM001011688), MoTLR7 (NM133211), HuTLR7 (NM016562) and PuTLR7 (AAW69375).
3.4 Structure of the TZJ? 7-hybridizing cDNA clone, 130K16
To determine the sequence of the TLR7 transcript, we isolated and sequenced a rL/?7-hybridizing cDNA clone, 130K16 (3578 bp) (Figure 3.4).
While we could identify the major exon of TLR7, 13OK 16 lacked a putative in- frame initiating methionine. The only methionine upstream of the exon 3
54 (encoding the majority of the receptor) for TLR7 that was not followed by a stop codon, was out of frame with the remainder of the protein.
kb 12 3 4 5 6 7 8 9 ! 1 i | 1 1 I 1 1
2 2a 3 -> RT-PCR Product |j (Exon 2-3) I | y / Figure 3.4 Structure of the rLfl7-hybridizing cDNA clone, 130K16. Duck TLR7 is expressed in alternatively spliced isoforms. Exon 2 of the cDNA clone 130K16 contains the 5' UTR and terminates with the translational start codon for TLR7. Exon 2a cannot be translated in frame, while the last exon (3) encodes the majority of the receptor. RT-PCR using primers annealing to the second and third exons amplifies only a product without exon 2a. Untranslated regions are indicated in black, the alternate exon indicated in grey. Mapping the sequence of the 7X/?7-hybridizing cDNA clone, 130K16, back to the genomic sequence revealed elements of 4 separate exons. The first contained a partial 5' UTR, terminating with the translational start codon, similar to exon 2 of humans, mouse and chicken. The entire major coding exon 3 was also contained within 130K16 - although there were 2 nucleotide mismatches between exon 3 in the genomic clone sequence and exon 3 in 130K16, the translated amino acid sequence was identical. However an additional 212 bp exon is present between exon 2 and 3. This region is largely non-coding in each reading 55 frame - any putative translational start codons are later followed by stop codons. BLAST and SMART searching of other vertebrate sequences in the database revealed no significant matches to this exon (2a). Alternatively spliced isoforms of TLR7 have been identified previously in chickens, as well as mice and humans (Philbin et al, 2005), although the significance of these variants is unclear. The 3' end of 124F14 also includes partial UTR sequence, some of which is present within exon 3, and some further sequence which falls in an additional exon. We have not attempted to identify the equivalent of the mammalian exon 1 (containing only 5' UTR in humans and mice) in ducks. To examine whether the exon 2a-containing clone 130K16 represented a significant isoform of TLR7, RT-PCR was perfomed using a forward primer designed in the first coding exon of TLR7 (exon 2), and a reverse primer in the last exon (exon 3) (Figure 3.4). RT-PCR with these primers on splenic cDNA revealed only one 166 bp product, which corresponded to the second and third exons only, without the additional exon 2a present in 130K16. Thus, 130K16 probably represents a rare and non-coding TLR7 transcript. 3.5 Tissue expression of duck TLR 7 Expression of duck TLR7 transcripts was assessed by RT-PCR on various tissues, using primers in the second and third exons of the gene (Figure 3.5). TLR7 expression is highest in the lymphoid tissues of spleen and bursa, and also very high in lung. Low expression was found in the duodenum, kidney and liver, and minimal expression is seen in heart and brain. GAPDH was amplified as a control for cDNA quantity and quality. 56 Figure 3.5 Analysis of duck TLR7 mRNA transcripts shows significant expression of duck TLR7 in the spleen, lung and bursa. Gene expression was measured with RT-PCR, using a forward primer in TLR7 exon 2 and reverse primer in exon 3. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as a control for cDNA quantity and quality. 3.6 The TLR7 agonist, imiquimod, upregulates proinflammatory cytokines and IFNa in duck splenocytes To assess the ability of duck TLR7 to respond to mammalian TLR7/8 agonists, we first stimulated freshly isolated duck splenocytes with multiple concentrations of TLR agonists and measured gene expression of IL-lfi and IFNa to determine if agonists were in an acceptable concentration range to induce cytokine gene expression (Figure 3.6). We then treated splenocytes with several TLR agonists for 6 hours and measured gene expression oflL-lfi, IL-6 and IFNa and TLR7 (Figure 3.7). The agonists used included loxoribine (a guanosine analogue), imiquimod (an imidazoquinoline amine analogue), and ssPolyU/ Lyovec which is a single-stranded poly-uridine oligonucleotide. The TLR3 agonist, polyriboinosinic polyribocytidylic acid (poly (I:C)), a synthetic analog of dsRNA, was also used as a control for proinflammatory cytokine and IFNa 57 expression. Expression of IL-lfi, 1L-6 and IFNa and TLR7 was measured by RT- PCR. At 6 hours, IL-lfi was slightly upregulated in all samples, with highest expression occurring in the imiquimod-treated samples. IL-6 was upregulated in imiquimod-treated, and to a lesser extent, poly (LC)-treated samples. IFNa expression was also notably increased in the imiquimod and poly (I:C)-treated samples. Figure 3.6 IL-ip and IFNa mRNA expression in splenocytes stimulated with TLR7 and TLR3 agonists. Freshly isolated splenocytes were stimulated for 6 hours in tissue culture medium alone, or with TLR7/8 agonists (loxoribine, imiquimod, ssPolyU) or the TLR3 agonist, poly (I:C). RNA was extracted and expression of IL-lfi and IFNa was determined by RT-PCR, using GAPDH expression as a control for cDNA quantity and quality. Splenocytes were treated with 50 or 150 JJM of loxoribine, 1, 5 or 10 ug/mL imiquimod, 1, 10 or 25 ug/mL ssPolyU/Lyovec and 20, 25 or 30 ug/mL poly (I:C). 58 .g 3? i .5 C *^i ** ll-lp IL-6 IFNa TLR7 GAPDH Figure 3.7 IL-lfi, IL-6, IFNa, and TLR7 mRNA expression in splenocytes stimulated with TLR7 and TLR3 agonists. Freshly isolated splenocytes were stimulated for 6 hours in tissue culture medium alone, or with TLR7/8 agonists (loxoribine, imiquimod, ssPolyU) or the TLR3 agonist, poly (I:C). RNA was extracted and expression of IL-lfi, IL-6 and IFNa was determined by RT-PCR, using GAPDH expression as a control for cDNA quantity and quality. Splenocytes were treated with 100 uM of loxoribine, 10 ug/mL imiquimod, 1 u.g/mL ssPolyU/Lyovec and 25 ug/mL poly (I:C), with three biological replicates performed. The treatment of splenocytes with TLR7 agonists or poly (I:C), did not significantly affect the regulation of the TLR7 transcript (Figure 3.7). This is in contrast to IFN-induction of TLR7 gene expression in isolated human primary macrophages (Miettinen et al., 2001). However, duck TLR7is already highly expressed in spleen and lung, suggesting TLR7 is poised for activation of innate immune responses in a cell population that is resident in these tissues in ducks. Stimulation of splenocytes with the TLR7/8 agonists, imiquimod, loxoribine and ssPolyU, induced upregulation of IFNa at the 24 hour time-point, 59 however expression of IFN was also high in the mock treated samples (possibly due to culture conditions), making the effect of TLR agonists difficult to ascertain (Figure 3.8). All agonists were also tested on isolated PBMCs, which showed qualitatively similar results, with upregulation of proinflammatory cytokine and IFN a genes (Figure 3.9). 60 •••:_ ."tV'VT*!** / - i - •*• *- IFNa •_-_-_ Sfct'.'-:. •.,!"•" ?:A K,;i GAPDH tl/L: .—i«iW*6?i«S s-M" Figure 3.8 At 24 hours post-stimulation, ZfTVa is expressed in mock-treated splenocytes as well as those stimulated with TLR7 and TLR3 agonists. Freshly isolated splenocytes were stimulated for 24 hours in tissue culture medium alone, or with TLR7/8 agonists (loxoribine, imiquimod, ssPolyU) or the TLR3 agonist, poly (I:C). RNA was extracted and expression of IFNa was measured by RT-PCR, using GAPDH expression as a control for cDNA quantity and quality. Splenocytes were treated with 100 uM of loxoribine, 10 ug/mL imiquimod, 1 |ag/mL ssPolyU/Lyovec and 25 ug/mL poly (I:C). 61 Figure 3.9 IL-lp, IL-6, IFNa, and TLR7 mRNA expression in PBMCs stimulated with TLR7 and TLR3 agonists. Freshly isolated PCMCs were stimulated for 6 hours in tissue culture medium alone, or with TLR7/8 agonists (loxoribine, imiquimod, ssPolyU) or the TLR3 agonist, poly (I:C). RNA was extracted and expression of IL-lfi, IL-6 and IFNa was measured by RT-PCR, using GAPDH expression as a control for cDNA quantity and quality. Splenocytes were treated with 100 uM of loxoribine, 10 ug/mL imiquimod, 1 ug/mL ssPolyU/Lyovec and 25 ug/mL poly (I:C). 62 3.7 Discussion The duck TLR7/8 locus was sequenced and, as seen in chicken, this was comprised of an intact TLR7 gene and a disrupted TLR8 gene with a CRl-type retroviral insertion element. Re verse-transcription PCR confirmed that the TLR7 transcript was encoded by just two exons and is abundantly expressed in spleen, lung and bursa. Isolated splenocytes upregulate proinflammatory cytokine and IFNa genes in response to TLR7 agonists, suggesting this pathway is functional in ducks. The common mechanism of inactivation of duck and chicken TLR8, by insertion of a CRl element, indicates that TLR8 was rendered non-functional in a common ancestor of ducks and chickens. Given the early divergence of ducks within the avian lineage, this may be true of all birds. Prior failure of PCR to amplify the TLR8 disruption in ducks (Philbin et al, 2005) was identified as inability of the reverse primers to bind the CRl element. In mammals, both TLR7 and TLR8 are implicated in detection of single- stranded viruses. Although they respond to some of the same agonists, human TLR7 and TLR8 have different agonist specificity, tissue expression patterns, and are expressed in different cellular subsets (Gorden et al, 2006; Heil et al, 2003; Hornung et al, 2002). Human TLR7 is expressed predominantly in pDCs, and TLR function is required for the production of massive amounts of IFN in response to a viral infection. Human TLR8 is expressed on monocytes and myeloid-derived mDCs, responsible for the priming of antigen specific immune responses (Ito et al, 2005; Kadowaki et al, 2001). Human TLR8 responds to the 63 synthetic agonist R848, while mouse TLR8 does not, suggesting they do not share identical specificity (Jurk et al, 2002). Mouse TLR7, but not TLR8 can detect the presence of ssRNA, and indeed, TLR7 knockout mice are susceptible to influenza (Lund et al, 2004). The complicated overlap and differentiation between the function of TLR7/8 in mouse and humans has led to the suggestion that mouse TLR8 and human TLR7 are evolving to become non-functional (Crozat and Beutler, 2004). In humans, inflammatory responses initiated by TLR7 agonists can be down modulated by antagonistic interaction involving TLR8 and TLR9 receptors (Ghosh et al, 2007; Wang et al, 2006). It has been suggested that mouse TLR8, which is widely expressed but poorly responsive to agonist stimulation, may function primarily to limit the extent of the inflammatory response (Ghosh et al, 2007). In birds, in the absence of TLR8 and possibly TLR9, this mechanism of modulation of inflammatory signals cannot operate. TLR9 has not been found in chickens (Iqbal et al, 2005; Yilmaz et al., 2005) although they can respond to CpG (He et al, 2007), which has been found to occur through TLR21 (Brownlie et al, 2009; Keestra et al, 2008) Duck TLR7 was highly expressed in lymphoid tissues such as the spleen and bursa. In addition, high expression is seen in the lung of ducks, which is distinct from the expression pattern of chickens. In chicken, the highest TLR7 expression was seen in lymphoid tissues (spleen, bursa and caecal tonsil), with lower expression in gut associated tissue samples of chicken (Philbin et al, 2005). The duck TLR7 expression pattern is comparable to that for human, which is 64 highest in spleen, with significant expression in lung (Chuang and Ulevitch, 2000; Nishimura and Naito, 2005). High pulmonary expression of TLR7 could be significant in the context of highly pathogenic H5N1 avian influenza, which is primarily a lung infection (Pantin-Jackwood and Swayne, 2007). We postulate that the observed difference in TLR7 expression may be due to differences in the organization of lymphoid tissue in the lung of ducks and chickens. Ducks have high expression of several immune genes in lung (our unpublished results) suggesting that lung is a significant lymphoid tissue in ducks. Alternatively, the high TLR7 expression in duck lung could be due to the presence of resident cells expressing the TLR7 receptor, which are absent in the chicken. In chickens, TLR7 is highly expressed in B cells, although transcript is detectable in primary macrophages and heterophils, the macrophage-like cell line HD11, and DT40 B cells (Philbin et ai, 2005). Unfortunately duck cell lines are not available to examine for TLR7 expression. In humans, lung expression of TLR7 is due primarily to plasmacytoid dendritic cells, the major IFN producers (Demedts et ai, 2006; Diebold et ai, 2004; Kadowaki et ai, 2001). It is tempting to speculate that the pulmonary expression of TLR7 seen in ducks (and not chickens) could be due to the presence of pDCs, although not yet identified in birds. Triggering of duck splenocytes with mammalian TLR7 agonists resulted in upregulation of transcripts encoding proinflammatory cytokines and IFNa. In contrast, IFN was not upregulated in chicken splenocytes and cell lines in response to TLR7 agonists, although proinflammatory cytokines were upregulated 65 (Philbin et al, 2005). Schwarz et al. (2007) (Schwarz, 2007) showed isolated chicken splenocytes responded to the TLR7 agonist, R848, by activating an Mx promoter in an indicator cell line, presumably through IFN. However, transfection of a TLR7 construct in HEK293 cells, failed to activate an NF-KB reporter construct, although TLR3 and human TLR7 did. Recent results suggest that ability to upregulate IFN may be dependent on chicken genotype. Chicken heterophils from the commercial poultry line B, but not line A, can respond to TLR7 agonists such as loxoribine with upregulation of IFNa expression (Kogut et al, 2006). This data taken together suggests that chicken TLR7 may exist in functional and non-functional alleles, or a downstream mediator in the pathway is dysfunctional in certain cell or poultry lines. The critical differences between ducks and chickens in response to influenza remains to be identified. If TLR7 is involved in detection of influenza in birds, then the distinctions between ducks and chicken in tissue distribution or function of the receptor may be important. A difference in the TLR7/8 genomic loci is not involved. In ducks, TLR7 is expressed in respiratory and lymphoid tissues, and triggering of TLR7 results in rapid upregulation of proinflammatory cytokines and IFNa mRNA, critical mediators of viral defense. 3.8 Author's contribution to data I performed all experiments in this chapter, except for the isolation of the genomic and cDNA clones from our library (Debra Moon and Jianguo Xia). 66 Chapter IV: Identification of duck RIG-I and comparison to chicken 4.1 Introduction All strains of influenza A virus are perpetuated in the duck reservoir (Webster et al,. 1992). Although rare, when influenza viruses cross over from the avian reservoir into humans they may evolve into pandemic strains. After emerging in 1996 the HPAIH5N1 viruses have evolved into multiple clades and subclades and have spread to Europe and Africa, probably by wild birds (Duan et al, 2008). Although H5N1 viruses do not frequently infect humans they are often highly pathogenic and cause death in otherwise healthy individuals. Of 445 laboratory-confirmed human H5N1 infections the mortality rate is 60% (Barrat et al, 2005). In 2002, H5N1 viruses began killing wild waterfowl (Ellis et al, 2004; Sturm-Ramirez et al, 2004), including ducks, which is highly unusual and a disruption of the normal ecology (Webster et al, 1992). Typically ducks do not show signs of disease upon infection with influenza. H5N1 viruses may rapidly adapt, losing their pathogenicity in ducks (Hulse-Post et al, 2007; Kim et al, 2009; Sturm-Ramirez et al, 2005), suggesting that virus evolution helps maintain the balance between virus and host. Many strains of highly pathogenic H5N1 now cause asymptomatic infection in ducks, making them a 'Trojan horse' in the spread of these influenza viruses (Hulse-Post et al, 2005; Kim et al, 2009; Webster et al, 1992). A version of this chapter has been published. Barber et al, 2010. Proc Natl Acad Sci. 13: 5913-5918. 68 In contrast, many of these same H5N1 viruses cause 100% mortality in chickens within hours or days. We therefore reasoned that superior innate immunity might protect the duck during this critical period. The molecular basis of the natural resistance of ducks to influenza infection is unresolved. A successful innate immune response to influenza infection involves a robust, yet transient induction of interferon-stimulated antiviral genes. RIG-I is a cytoplasmic RNA sensor (Yoneyama et al, 2004) and triggering by influenza virus leads to production of IFN|3 and expression of downstream IFN stimulated antiviral genes (Loo et al., 2008). A hallmark of lethal influenza virus infection is interference with expression of RIG-I and downstream genes (Kobasa et ah, 2007). Further, RNA viruses have been shown to be more virulent and replicate to higher levels in mice lacking RIG-I (Kato et al, 2005). Considering this, we speculated that the outcome of influenza infection in birds may also be determined by RIG-I and the downstream subset of IFN- stimulated genes. Results 4.2 RIG-I is present in ducks and apparently absent in chickens Given its role in antiviral defence in mammals, we searched for avian homologues of RIG-I. We identified a duck (Anas platyrhynchos) RIG-I homologue with 53% amino acid identity to human and 78% identity to zebra finch RIG-I (Figure 4.1). Remarkably, we are unable to identify a chicken homologue of RIG-I, even though we used a variety of approaches to identify one. Searches of the chicken {Gallus gallus) genome (2004) with the duck or finch 69 RIG-I sequence do not reveal a match. In addition, a phylogenetic analysis of RIG-like receptors also noted that RIG-I was absent in chickens (Zou et al., 2009). Duck Finch Human Duck 100 Finch 100 L Human 101 %fc% Duck 19S lBJB13?Cli3»Si Finch 199 HKHIMJWHFMTIMM ^_^^ Hunan 199 DLEDKHBTspigjFg|g|pp|jg8-ij Duck 299 j Finch 299 I Human 296 FgNOlS Duck 399 N-gjs: Finch 398 OSHJKS! Human 396 GGgsGPHJiVBI;; Duck 797 3118 Finch 797 WgTjpE; Human 796 a-PSUVPDXEl Duck 897 I Finch 897 '• Human 894 • >._ CARD "—^DEXD box • •••i Belicase ••••»» C-teroinal Regulatory Figure 4.1 Amino acid alignment of duck, zebra finch, and human RIG-I. Alignment of duck RIG-I (accession number EU363349), zebra finch (accession numberXM_002194524), and human RIG-I (accession number AF038963) was performed using the ClustalW program and edited with Boxshade. Black shading indicates amino acid identity and gray shading indicates similarity (50% threshold). Plus signs indicate human residues involved in polyubiquitination and asterisks indicate residues involved in agonist binding. The ATP binding motif is boxed. 70 However, the related Melanoma Differentiation Associated gene-5 (MDA5) is present in the chicken genome (Sarkar et al, 2008). RIG-I and MDA5 initiate signalling cascades that converge on the same pathway at IPS-1 and lead to induction of IFN|3 and expression of downstream interferon-stimulated antiviral genes. MDA5 is a detector of long double-stranded RNA, polyinosinic:polycytidylic acid (poly I:C), and picornaviruses (Kato et al, 2006). To ensure that the helicase we isolated was RIG-I and distinct from MDA5, we amplified a large fragment of duck MDA5. The fragment of MDA5 shared 91% amino acid identity to chicken MDA5 (Figure 4.2), yet only 33% identity to duck RIG-I. We also attempted to amplify a duck LGP2, the third known member of the RLR family, but three sets of cross-species primers failed to amplify the gene. 71 Duck ASEgGKVIVLVNKVPLVEQHLRKEFNPFLKRWYQVlGLSGDS ^Gjj Chicken 620 E5333 ASEMGKVIVLVNKVPLVEOHLRKEFNPFLKRWYQVIGLSGDS sm Duck CHHTQKEGVYNNIMRRY HASQLKNQVK.JSPFKKTV1A Chicken 719 HASOLKNOVKEPFKKTVIA Duck AKEEKRKERVCAEHLKKYNDALQINDTIRMVDAYNHLNNFYKE SKFSjIGg Chicken 819 •AKEEKRKERVCAEHLKKYNDALOINDTIRMVDAYNHLNNFYKE dRRSJAES 1 v^,„ 919 mammmsmmimmwMKmimmMaaBmxmsm Duck iTKPMTQNEQREVIDKF ILLIATTVAEEGLDIKECNIVIRYGLVTNEIAM Chicken 101915 3TKPMTONEOREVIDKF • Partial DEXD box i Partial Helxcase Figure 4.2 Duck and chicken MDA5 are highly conserved. Alignment of partial duck MDA5 (accession number GU936632) and chicken MDA5 (accession number XM422031) was performed using the Clustal W program and edited with BOXSHADE. Black shading indicates amino acid identity and grey shading indicates similarity (50% threshold). 72 We identified the RIG-I syntenic region on the chicken Z chromosome where acontinase I is encoded, a gene flanking the mammalian RIG-I homologue. A local BLAST search of the adjacent 4 Mb reveals no match to RIG-I, although there are sequence ambiguities in this region. Confirming the syntenic region is conserved in other birds, we identified a RIG-I homologue in the recently released draft genome for zebra finch {Taeniopygia guttata) on chromosome Z and flanked by acontinase I. A search of the finch expressed sequence tag (EST) database revealed two RIG-I transcripts among the 92,000 sequences. In contrast, no RIG-I sequences are present among the 600,000 ESTs from chicken. Notably, MDA5 transcripts are present. Thus, RIG-I appears to be absent from the chicken genome sequence derived from the Red Jungle Fowl, which resembles the ancestral chicken, as well as the modern chicken lines represented in the EST sequences. Combined, these data suggest that chickens may have lost RIG-I prior to their domestication. Avian RIG-I proteins have features in common with mammalian RIG-I. Duck RIG-I is 933 amino acids, and zebra finch RIG-I is 927 amino acids. Domain prediction reveals the expected tandem N-terminal CARD domains, a helicase domain, and a DExD/H box helicase domain, consistent with the mammalian structure (Takahasi et al, 2008; Yoneyama et ah, 2004). RIG-I is a agonist-dependent ATPase, and the Walker A ATP-binding motif is conserved. The hydrophobic core and the four lysine residues implicated in agonist-binding, K858/861/888/907 (Takahasi et al, 2008), are completely conserved within the 73 C-terminal regulatory domain. However, residues T55 and K172, critical for attachment and polyubiquination by TRIM25 needed for IPS-1 binding and signal induction (Gack et al., 2008; Gack et al, 2007) are not conserved, suggesting this pathway does not function or involves different residues in birds. To provide further evidence for the absence of RIG-I in chickens, we hybridized a Southern blot of genomic DNA from ducks and chickens with a probe amplified from the helicase region of duck RIG-I (Figure 4.3a). The probe spanned a complete exon and part of two other exons, and had over 70% nucleotide identity between the duck sequence, horse (Equus caballus), platypus {Ornithorhynchus anatinus), human {Homo sapiens), mouse (Mus musculus), Rhesus monkey (Macaca mulatto) and chimp (Pan troglodytes). While we observed a polymorphic pattern of hybridization to duck DNA, we did not detect cross-hybridization to chicken DNA except for faint hybridization in Chicken 1 for the Xbal digest, which we thought could be cross-hybridization to a different gene. 74 JF% Duck Chicken Duck Chicken Hindlll Xbai Kb 23 9.4 6.6 4.4 * J* • §ii • mttm • RIG-I Duck Pigeon Dock Chicken Pigeon KB Chicken Kb P N S P N S p N s PUS P N S P N S • ! 4.4 2.3 2.0 • RIG-I MDAS Figure 4.3 RIG-I is present in ducks and pigeons, but apparently absent in chickens. (A) Hybridization of a multiple exon duck RIG-I probe to Hindlll and Xbal-digested genomic DNA from four White Pekin ducks and two White Leghorn chickens. (B) Hybridization of a single exon duck RIG-I probe to PstI, Ndel and Sad digested genomic DNA from duck, chicken and pigeon. (C) Hybridization of a single exon duck MDA5 probe to same blot. We then limited the probe to spanning only one exon to refine our result. To determine if our duck probe could hybridize to other species we initially hybridized the probe to a Southern blot containing genomic DNA prepared from a human (RAMOS) and mouse (18-81) cell line, as well as chicken (Figure 4.4). 75 c c c J —» 4.4 'v-,"»CtfV5 irt Figure 4.4 A single exon duck RIG-I probe fails to hybridize to human, mouse and chicken genomic DNA. Hybridization of a single exon duck probe to a Southern blot containing Hindlll-digested genomic DNA from chickens, a human (RAMOS) and mouse (18-81) cell line. A positive control lane containing duck genomic DNA hybridized to the same probe was exposed on a separate film. The probe failed to hybridize to this Southern blot, so we restricted our attempts to show that our probe was capable of hybridizing to other avian species. The duck RIG-I probe cross-hybridizes with RIG-I from the more phylogenetically distant pigeon (Figure 4.3b, Figure 4.5a, Figure 4.7), providing evidence that the probe 76 can recognize RIG-I from other avian species. The duck RIG-I probe hybridized to pigeon DNA in only the PstI digestion, although this may be because the Ndel and Spel digestions produced hybridizing fragments of DNA too small to be visualized. It is noteworthy that pigeons are remarkably resistant to influenza viruses, including HPAIH5N1 (Perkins and Swayne, 2002). In comparison, strong cross-hybridization of a duck MDA5 probe with DNA from pigeon and chicken (Figure 4.3c) suggests it has diverged considerably less than RIG-I. The duck MDA5 probe also cross-hybridized with three other species including turkey, pheasant and partridge (Figure 4.5b). It is difficult to draw conclusions as to the presence of RIG-I in these species, as hybridization to RIG-I in these species occurred only on some blots and was possibly at the limit of detection (Figure 4.5a, Figure 4.6). Demonstrating that our duck RIG-I probe hybridizes to pigeon but not to chicken genomic DNA further supports the absence of RIG-I in chickens. However, we cannot rule out that chicken RIG-I has diverged to an extent that it is not detectable through bioinformatics or hybridization approaches, which may preclude function in any case. 77 r-4 CD O o -E Kb TO Pheasan t Pigeo n Duc k 1 (J (J Turke y CL 23 4. •* 9 4 •&4 6.6 <# J^4 4 &\G- Kb 23 9.4 -- ^^n 6.6 ^^K^ 6 ^i^^T ^PHW^ ^H^ %*# O 44 MDAS Figure 4.5 RIG-I is present in ducks and pigeons, but apparently absent in chickens, while MDA5 is present in many avian species. (A) Hybridization of a single exon duck RIG-I probe to Hindlll and digested genomic DNA chicken from turkey, pheasant, partridge, pigeon and duck. (B) Hybridization of a single exon duck MDA5 probe to same blot. 78 t~ i 44 Figure 4.6 RIG-I is present in pigeons, but apparently absent in other avian species. Hybridization of a single exon duck RIG-I probe to Hindlll and digested genomic DNA chicken from pigeon, chicken, turkey and partridge. 79 Paleognathae (emus, ostriches and relatives) Neoaves (most modern birds, including pigeon) IIIIIIIIII Galliformes (Chickens, turkey, quail, and relatives) Neognathae Galloanserae Anseriformes (Ducks, geese and relatives) Figure 4.7 Pigeons are phylogenetically distant from ducks, compared to chickens. Ducks are a member of the anseriformes and share a common ancestor with chickens that presumably possessed RIG-I. Pigeons are a member of neoaves and strong Southern blot hybridization to duck RIG-I suggests that a common ancestor of the galloanserae and neoaves possessed RIG-I and that absence of hybridization of chicken genomic DNA to duck RIG-I is indicative of loss or disruption of the gene in chickens. Finally, we attempted northern blotting to determine whether chickens expressed a transcript hybridizing to duck RIG-I (Figure 4.8a). We hybridized a blot containing RNA from mock and H5N2 infected ducks and chickens to the duck RIG-I probe, but were unable to detect hybridization in either ducks or chickens. To determine if our RNA was intact and our procedure sound, we hybridized the same blot to a duck GAPDH probe (Figure 4.8b). This resulted in 80 hybridization to all of our samples, thus our approach was not sensitive enough to detect RIG-I transcripts. One previous group used a northern blot approach to assess RIG-I expression in human epithelial cells, however they used poly A+ RNA only and hybridization was faint even in LPS treated cells that were expected to have high expression of RIG-I (Imaizumi et al, 2002). 81 cv £\l ^ 2 2 ij !3 £ o X X H"I (1f) (!) 8%% E r- CV -* ft "> o"I Xf X £ A 1 $ B 'v*> -A m *>* 4# f f f Figure 4.8 (A) Hybridization of a single exon duck RIG-I probe to a northern blot containing RNA from mock and H5N2 infected duck and chicken spleen, lung and intestine RNA failed to detect RIG-I transcripts. (B) Hybridization of a duck GAPDH probe to a northern blot containing RNA from mock and H5N2- infected duck and chicken spleen, lung and intestine RNA. Transcripts were identified in each sample. 4.3 Duck RIG-I detects in vitro transcribed RNA and activates the chicken IFNP promoter The apparent absence of RIG-I in chickens led us to investigate whether the DF-1 chicken cell line (chicken embryonic fibroblasts) can respond to a RIG-I agonist. RIG-I signalling is activated by 5' triphosphate RNA (5' ppp RNA) containing short RNA with double-strand conformation, such as that derived from 82 viral RNA with panhandle structures (Schlee et al, 2009; Schmidt et al, 2009), and from in vitro transcribed products (Hornung et al, 2006). With this in mind, we challenged DF-1 cells with an in vitro transcribed 21mer 5'ppp RNA shown to activate mammalian RIG-I (Hornung et al, 2006). Transfection of this agonist failed to activate a chicken IFN(3 promoter-luciferase reporter (Figure 4.9a). Poly (I:C), known to drive the chicken IFNP promoter in this cell line (Childs et al, 2007), showed a 2.4 fold increase in IFNp promoter activity compared to unstimulated cells. Poly (I:C) triggers the cytoplasmic receptor MDA5 and the endosomal dsRNA receptor, TLR3. However, the response to poly (I:C) in DF-1 cells is largely through MDA5, as viral inhibition of MDA5 signalling nearly abrogates IFNp promoter activity (Childs et al, 2007). Thus DF-1 cells respond to poly (I:C) mostly through MDA5, demonstrating that they possess the downstream components of the shared RIG-I/MDA5 pathway. One duck cell line is commercially available (duck embryonic fibroblasts, American Type Culture Collection (ATCC) number CCL-141) and we attempted to propagate this cell line in order to test the ability of duck cells to respond to RIG-I ligand. We has one successful result using the luciferase assay that suggested a ~ three fold induction of the chicken IFNP promoter upon poly (I:C) transfection and a ~ five fold induction upon 5' triphosphate ligand transfection in the duck CCL-141 cells. Unfortunately, we were unable to confirm this result as all attempts to culture this cell line past the first few passages failed, in both our hands and our collaborators at St. Jude Children's Hospital failed. 83 prutaL , i i i J O © © IFN-I'. Fold Index 1 1 1 -t KJ W ^ J I JL I £rnpt» vector mock H Empty vecloi 5'pppRNA RIfi-3 mock R1G4 5'pppRNA RIG-1CI<\PRNA RIG-3 poly (fcQ 00 pfu/mL Fold expression [RlG-l vector) ffl o « « 5 o ~* *J « C © © influenza A matrix Figure 4.9 Duck RIG-I rescues detection of 5fppp RNA and induction of an antiviral response in DF-1 chicken embryonic fibroblast cells. (A) IFNp promoter activity in RIG-I or empty vector transfected DF-1 cells following 15 hours of agonist stimulation compared to mock-treated cells, shown as mean fold induction (+/- s.d.). Results are representative of 3 independent experiments and were analysed using a single-factor ANOVA and Tukey's posthoc test (different letters P<0.05). (B) RIG-I transfected DF-1 cells respond to BC500 infection (MOl 1) with increased expression of chicken IFNp and the interferon stimulated genes Mxl and PKR, and decreased influenza matrix gene expression, relative to empty vector transfected cells. RNA was extracted from cells for qRT-PCR 15 hours post-infection, and fold difference in gene expression calculated for RIG-I or vector-only transfected DF-1 cells. Results are representative of 3 independent experiments and error bars show RQMin/Max at a 95% confidence level and represent standard error (n=3). (C and D) RIG-I transfected DF-1 cells had significantly lower influenza virus titres compared to empty vector transfected cells for (C) BC500 or (D) VN1203. Both infections were performed 24 hours post-transfection at a MOl of 1. After 15 hours, titre was determined by plaque assay from triplicate wells and results were analyzed with the two-tailed Student's t test (n=3, P=0.002). CIAP, calf intestinal alkaline phosphatase. 85 We next investigated whether duck RIG-I could confer recognition of a RIG-I agonist and signal through these downstream components. DF-1 cells were transfected with duck RIG-I prior to stimulation with 5'ppp RNA. DF-1 cells expressing duck RIG-I responded to 5'ppp RNA with a 2 fold induction of the IFN(3 promoter as compared to mock-transfected cells. However, phosphatase removal of the 5' triphosphate abrogated the response similar to the previous report in mammalian cells (Hornung et ai, 2006). Thus duck RIG-I is functional and can induce IFN(3 promoter activity in the DF-1 chicken cell line. While the two-fold induction of the IFNP promoter is modest, this is consistent with the upregulation by poly (I:C) stimulation downstream of chicken MDA5. Given the evolutionary divergence of chickens and ducks, it is remarkable that the duck RIG-I presumably even binds to chicken IPS-I to connect to downstream signalling components. 4.4 Transfected duck RIG-I detects influenza and induces an antiviral response in chicken cells Initially, we attempted to determine if RIG-I could induce an antiviral response in the chicken DF1 cells by transfecting the cells with duck RIG-I or vector only, followed by infection with A/PR/8/34 (HlNl) influenza. To visualize the viral titre we transferred cell supernatants to DF1 cell monolayers, allowed infections to proceed for three hours, and performed immunofluorescence with a FITC-labelled anti-influenza NP antibody. Reduced NP staining and fewer cells showing cytoplasmic NP accumulation (Figure 4.10) was initially seen for 86 supernatants from RIG-I transfected cells. The supernatants from all cells transfected with duck RIG-I could reduce influenza replication in chicken cells, although these preliminary experiments were not quantified and we had not performed an isotype control. When continuing these experiments at St. Jude Children's Research hospital, we realized that the conditions of the experiment were suboptimal as we were using 7.5% Bovine Serum Albumin (Gibco) instead of 7.5% Bovine Serum Albumin (Sigma) during viral infections, which contained trypsin inhibitors that which reduced the amount of infectious virus in the sample and also disguised the fact that the concentration of TPCK-trypsin being used was toxic to the DF-1 cells. 87 Figure 4.10 Supematants from RIG-I transfected DF-1 cells reduce PR8 influenza replication in DF-1 chicken embryonic fibroblasts. DF-1 cells were transfected with empty vector or duck RIG-I. 24 hours post-transfection, cells were infected with A/PR/8/34 (H1N1) influenza. 15 hours later, influenza supematants were applied to a fresh DF-1 monolayer, and virus was visualized by immunofluorescence with a FITC-labelled anti-influenza nucleoprotein (NP) monoclonal antibody. Anti-influenza NP immunofluorescence on DF-1 cells treated with supematants from influenza infected (A) empty vector-transfected and (B) RIG-I-transfected DF-1 cells. 88 We went on to refine this experiment with different reagents and a lower TPCK-trypsin concentration (0.1 ug/mL) at St. Jude Children's Hospital. To determine if duck RIG-I could detect influenza virus and induce an antiviral response in the chicken DF-1 cells, we transfected the cells with duck RIG-I or vector only, followed by infection with LPAI or HPAI. We chose H5N2 A/mallard/BC/500/2005 (BC500), a LPAI isolated from wild ducks that causes no pathology in its natural host and H5N1 A/Vietnam/1203/04 (VN1203) and reverse genetics H5N1 A/Hong Kong/213/03 (HK213), HPAI isolates from fatal human infections and known to be lethal to ducks and chickens (Hulse-Post et al., 2007). After a 15 hour infection with BC500, there was increased expression of IFNfi as well as the antiviral interferon-stimulated genes Mxl and PKR, known to be RIG-I responsive in mouse fibroblasts (Loo et al, 2008) (Figure 4.9b). Although IFNfi and PKR were only slightly upregulated, the interferon-stimulated gene Mxl was induced approximately 30 fold. Influenza A matrix gene expression was significantly reduced in RIG-I transfected DF-1 cells as compared to vector- transfected control cells (Figure 4.9b). Furthermore, the interferon response initiated by duck RIG-I resulted in significant reduction in viral titre for both BC500 (Figure 4.9c) and VN1203 (Figure 4.9d) infection, indicating that duck RIG-I is capable of reducing influenza replication in chicken cells. Similar results were seen for another HPAI human isolate, HK213 (Figure 4.11). 89 Figure 4.11 RIG-I transfected DF-1 cells have significantly lower titres of HK213. Infection was performed 24 hours post-transfection at a MOI of 1. After 15 hours, titre was determined by plaque assay from triplicate wells and results were analyzed with the two-tailed Student's t test (n=3, P=0.002). 90 4.5 RIG-I is highly upregulated in ducks infected with VN1203 To determine whether RIG-I contributes to the antiviral response to influenza infection in ducks, we measured the expression of RIG-I in 6-week-old White Pekin duck tissues following infections with BC500 (H5N2) or VN1203 (H5N1). Infection with VN1203 induced significant upregulation of RIG-I gene expression in the infected lung (Figure 4.12a). RIG-I expression was induced over 200-fold by dl PI, whereas by d3 PI, RIG-I was only modestly expressed, suggesting the induction is early and transient (Figure 4.12b). In comparison, infection with BC500 induced only slight upregulation of RIG-I in lung tissue. Because LPAI strains predominately replicate within the intestinal epithelium of ducks (Webster et ai, 1978), we also assessed RIG-I expression in BC500 infected duck intestine (Figure 4.12c,d). RIG-I expression was not significantly induced by BC500 infection in duck intestine on dl or d3 PI. Duck RIG-I is thus expressed early during an innate immune response to a highly pathogenic influenza virus. However, it is not clear why VN1203 infection results in tremendous upregulation of duck RIG-I, while BC500 does not. Considering that RIG-I expression is itself regulated by interferon, high expression in the lungs following VN1203 infection may reflect interferon levels. Additionally, influenza viruses vary in their ability to actively inhibit interferon induction in birds and mammals, a function which is dependent on the viral protein NS1 (Egorov et ai, 1998; Garcia-Sastre et al„ 1998; Li et ai, 2006) that directly targets the RIG-I pathway preventing activation of IFNp1 (Mibayashi et ai, 2007). One possibility is that the NS1 protein of BC500 interferes with viral activation of the interferon 91 pathway more efficiently than the NS1 of VN1203. Although VN1203 is potentially lethal in ducks (Hulse-Post et al, 2007; Kim et al, 2009; Sturm- Ramirez et al, 2005), none of the ducks showed severe symptoms or died within the three days of the experiment. Recent data, published after our experiments, has shown that our infections may have been sublethal because the reverse genetics VN1203 strain that we used inadvertently contained amino acid mutations in the PB1-F2 open reading frame, which result in reduced lethality in ducks (Marjuki et al, 2010). 92 B < •Z 250- E T 200- o a. = 150" O '35 tg 100- a (§ 50- o Mock BC500 VN1203 Mock BC500 VN1203 D < < 15- Z 4-| z a. E £ 6 3i 6 10- 5 .2 2- c .2 "to to £ 5- a xo u Figure 4.12 RIG-I is dramatically upregulated in duck lung infected with VN1203 but not BC500. Lung and intestinal RNA was extracted dl or d3 PI and analyzed by qRT-PCR for RIG-I expression as compared to mock-infected animals. (A) Fold expression of RIG-I mRNA in duck lung dl PI with VN1203 or BC500. (B) Fold expression of RIG-I mRNA in duck lung d3 PI. (C) Fold expression of RIG-I mRNA in duck intestine dl PI. (D) Fold expression of RIG-I mRNA in duck intestine d3 PI. Error bars show RQMin/Max at a 95% confidence level and represent standard error (n=4). Separate bars represent individual animals. 93 4.6 Discussion This study establishes the presence of RIG-I in ducks, the natural reservoir of influenza viruses, but apparent absence in chickens. This may reflect the differential susceptibility of ducks and chickens to influenza-induced pathology. We found that RIG-I is expressed during the innate immune response to influenza infection in ducks, providing evidence for the antiviral relevance of RIG-I in the natural host of the virus. Furthermore, RIG-I contributes to the innate immune response to VN1203 infection and is highly upregulated early in an infection in ducks. In contrast, RIG-I appears to be absent in chickens and a chicken embryonic fibroblast cell line fails to respond to 5' ppp RNA, a function that can be conferred on the cells by transfection with duck RIG-I. Expression of duck RIG-I augments the antiviral interferon response and reduces influenza replication in chicken cells. Despite the lack of RIG-I, chicken cells do produce interferons. Indeed, interferons were initially discovered in chicken cells treated with heat-inactivated influenza virus (Isaacs and Lindenmann, 1957). While other pathways can produce IFNa, IFNP production upon influenza infection is largely dependent on RIG-I. Embryonic fibroblasts from a RIG-I knockout mouse fail to induce IFN-fi and a subset of genes involved in innate immunity following influenza infection (Loo et al, 2008). siRNA knockdown of RIG-I (Opitz et al, 2007) or introduction of a dominant negative RIG-I (Siren et al, 2006) has been demonstrated to significantly reduce the influenza-induced IFNP production in human cell lines. Additionally, IFNp_/~ mice show reduced survival and enhanced 94 influenza viral titres in the lung (Koerner et al, 2007). Thus, IFNP appears to be protective during an influenza infection and cannot be compensated for by IFNa. Chicken embryonic fibroblasts infected with influenza produce interferon and inhibit an IFN-sensitive vesicular stomatitis virus (VSV)-GFP (Li et al, 2006), however, this response is 80% IFNa (Schwarz et al, 2004). Infection of primary chicken embryo fibroblasts with H5N1 strains induced low Mxl and IFNa gene expression, while IFNfi was not significantly induced (Sarmento et al, 2008b). Similar to influenza infection, Newcastle Disease Virus (NDV), also detected by RIG-I, causes substantially more pathology in chickens than ducks. In accord with our findings, others have found that NDV infection does not induce I FN ft promoter activity in chicken embryonic fibroblasts (Sick et al, 1998), also consistent with lack of RIG-I. Here we provide evidence for the antiviral function of RIG-I in ducks, and apparent absence in chickens. RIG-I independent pathways, such as TLR7, also contribute to influenza detection and interferon production in both chickens (Philbin et al, 2005) and ducks (MacDonald et al., 2008). However, chickens lack a key regulator of the antiviral response thus it is not surprising that they suffer remarkable pathology from HPAI infection compared to ducks. Chickens lacking RIG-I would be without a first line of defence at the lung epithelial cell layer during influenza infection, and antiviral genes downstream of RIG-I may not be expressed (Loo et ai, 2008). Considering that RIG-I may also inhibit influenza replication directly and independent of interferon (Yoneyama et al., 2004), loss of this undefined pathway may also contribute to unchecked virus 95 replication in chickens. RIG-I loss-of-function mutants in humans (Pothlichet et al, 2009; Shigemoto et al, 2009) may also be predicted to cause increased viral pathogenicity. The presence of a functional RIG-I in ducks eliciting an early antiviral response may contribute to survival of what may otherwise be a lethal influenza infection. 4.7 Author's contribution to data I performed all experiments in this chapter, except the in vivo duck and chicken influenza infections (performed by Dr. Magor). For HPAI DF-1 experiments, I set up the experiment and performed the transfections and qRT- PCR, however Dr. Jerry Aldridge infected the DF-1 cells with VN1203, as I did not have Biosafety Level 3+ clearance. 96 Chapter V: Functional characterization of duck RIG-I 97 5.1 Introduction Influenza A viruses cause largely asymptomatic infection in ducks, the natural reservoir (Webster et al, 1992). However, when influenza crosses the species barrier into chickens it causes symptomatic infection. Both the H5 and H7 subtypes can suddenly evolve from low pathogenic to highly pathogenic avian influenza (HPAI) within chickens (Pasick et al, 2005; Swayne and Suarez, 2000). Thus, chickens are an important intermediate in the genesis of novel influenza strains, whereas this is not the case for ducks. Adaptation of avian influenza viruses to humans can result in pandemic influenza strains. Thus, controlling infection in chickens is integral to preventing the emergence of HPAI strains with pandemic potential. Recent H5N1 HPAI strains are of concern because of their pronounced pathogenicity and transmissibility among chickens, as well as for their ability to infect humans. All H5N1 HPAI strains are lethal to chickens but only certain strains can kill ducks. Typically, ducks are able to limit H5N1- induced pathology (Sturm-Ramirez et al, 2005; Swayne and Pantin-Jackwood, 2006). Because of the acute onset of the infection, we predict that innate immune mechanisms are crucial in this response. An immune response to HPAI that allows for survival of the host likely involves a robust response in the cells that are initially infected. The initial cells infected with HPAI are respiratory tract epithelial cells (Swayne, 2007; Swayne and Pantin-Jackwood, 2006). A version of this chapter has been submitted for publication. Barber et al. 2011. BMC Genomics. 98 Both epithelial and fibroblast cells rely on viral detection by the RIG-I pattern recognition receptor. We have previously shown that ducks have an intact and functional RIG-I while chickens appear to lack the gene for the receptor (Barber et a/., 2010). Chickens cannot respond to RIG-I ligand but transferring the duck receptor to chicken cells reconstitutes this recognition. Demonstrating that signalling downstream of RIG-I is intact in these cells, we showed that induction of the immune genes Mxl, IFNfi and PKR upon influenza infection was significantly higher in chicken DF-1 cells transfected with duck RIG-I (Barber et al, 2010). Additionally, the presence of duck RIG-I in DF-1 cells reduces the titre of both LPAI and HPAI (Barber et al, 2010). However the genes that contribute to this antiviral effect are not known. To investigate the genes that are altered downstream of RIG-I upon influenza infection, we used a microarray approach. We used 44k Agilent chicken microarrays to compare responses to a LPAI and HPAI virus following transfection with RIG-I or vector-only in a chicken embryonic fibroblast (DF-1) cell line. These microarrays showed significant induction of many innate antiviral genes by influenza infection in chicken cells carrying duck RIG-I. Thus, duck RIG-I is functional in chicken cells and elicits a robust immune response to influenza. The global transcriptional profile reveals important mediators of the RIG-I dependent antiviral response. Results 99 5.2 Duck RIG-I induces immune genes in response to BC500 To investigate differences in DF-1 gene expression with and without duck RIG-I, we used an Agilent chicken gene expression microarray consisting of 42,034 60-mer oligonucleotides, which represents the entire chicken genome. We transfected DF-1 cells with pcDNA 3.1 or pcDNA 3.1 containing duck RIG-I. Twenty-four hours after transfection, we infected the cells with a low pathogenic avian influenza (LPAI) H5N2 A/mallard/British Columbia/500/2005 (BC500), or the highly pathogenic avian influenza (HPAI) H5N1 A/Viet Nam/1203/2004 (VN1203) at a multiplicity of infection (MOI) of 1. RNA was extracted for microarray analysis at 15 hours post infection, across triplicate (LPAI samples) or duplicate (HPAI samples) biological replicates. Overall analysis of the microarray data revealed that 1379 genes were altered in expression by greater than twofold upon infection with BC500 in RIG-I transfected cells, as compared to vector-only transfected cells. There were statistically significant changes in the transcription of 395 (P < 0.05) genes. Of the RIG-I responsive genes altered more than twofold during BC500 infection, 206 were statistically significant between biological replicates (P < 0.05). Gene ontology (GO) analysis revealed that genes with altered expression are involved in many functional classes, including the immune response (Table 5.1, Table 5.2). Several genes appear in multiple hierarchical GO classes. Of the differentially expressed genes (greater than twofold), 21 were associated with the immune response, with differential expression of 6 of them being statistically significant across replicates (Table 5.3). All of the differentially expressed immune genes 100 were upregulated by RIG-I, consistent with the role of the gene in eliciting a type I interferon response upon influenza infection. No downregulated immune genes were identified. The results of this microarray were consistent with previous qRT- PCR results in the same RNA samples which indicated significant induction of Mxl, PKR and IFNfi (Barber et al., 2010). 101 Table 5.1 GO terms for downregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with BC500 LPAI. Gene Ontology Term P-value Genes GO 0043169-cation binding 0 00971 TSHZ3, F10, GBGT1, ESR1, CPXM2, MGP, LOC419888, CDH4, CDH5, MTMR3, TRIM2, FBLN1, CHID1, GATA5, Z1C4, PRDM10, LOC424261, ST18, ADAMTS3, LOC422310 GO 0043167~ion binding 0 0104 TSHZ3, F10, GBGT1, ESR1, CPXM2, MGP, WC419888, CDH4, CDH5, MTMR3, TR1M2, FBLN1, CHID1, GATA5, ZIC4, PRDM10, LOC424261, ST18, ADAMTS3, LOC4223W GO 0046872-metal ion binding 0 0190 TSHZ3, F10, GBGTl, ESR1, CPXM2 MGP, LOC419888, CDH4, CDH5, MTMR3, TRIM2, FBLN1, GATA5, ZIC4, PRDM10, LOC424261, ST18 ADAMTS3, LOC422310 GO 0007186~G protein coupled receptor protein 0 0327 GABRG1, LPHN3, FZD10, S1PR1, signaling pathway CGNRH-R, FZD5 GO 0022610~biological adhesion 0 0378 OPCML, CPXM2, FCGBP, SOX9, CDH4, CDH5 GO 0007155-cell adhesion 0 0378 OPCML, CPXM2 FCGBP, SOX9 CDH4, CDH5 GO 0031224-intnnsic to membrane 0 0417 RAMP3, GABRG1, PALM, OPCML, ATP4B, GBGTl, PRTG, FZD5, CDH4, CDH5, FZD10, S1PR1, FUT4, LOC424261, CGNRH-R GO 0008270-zinc ion binding 0 0418 TSHZ3, MTMR3, TRIM2, GA TA5, ZIC4, PRDM10, ESR1, CPXM2, ST18, LOC419888, ADAMTS3, LOC422310 GO 0007166~cell surface receptor linked signal 0 0421 GABRG1, LPHN3, FZD10, S1PR1, PTPRA, transduction LOC424261, CGNRH-R, FZD5, IRS 1 GO 0046914~transition metal ion binding 0 0763 TSHZ3, GBGTl, ESR1, CPXM2, LOC419888, MTMR3, TR1M2, GATA5, ZIC4, PRDM10, ST18, ADAMTS3, LOC422310 GO 0043565-sequence-specific DNA binding 0 0903 TSHZ3, GATA5, MEOX2, ESR1, LOC419888, SOX9 GO 0003700-transcnption factor activity 0 0904 TSHZ3, GATA5, MEOX2, ESR1, ST18, LOC419888, SOX9 102 Table 5.2 GO terms for upregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with BC500 LPAI. Gene Ontology Term P-value Genes GO 0006955-immune response 1 05E-04 OASL, TNFSF10, CCL20, TLR15, 1RF7, IRF8, TNFSF15, CCL19, TLR3 GO 0007186~G-protein coupled receptor protein 0 0438 GNAT1, LOC396318, P2RY5, GPR92, signaling pathway MTNR1B, RGS6, BDKRB2 GO 0046489-phosphoinositide biosynthetic process 0 0575 PIGN, PIGA GO 0006506-GPI anchor biosynthetic process 0 0575 PIGN, PIGA GO 0006505-GPI anchor metabolic process 0 0575 PIGN, PIGA GO 0006952-defense response 0 0799 NFKB1Z, TLR15, TLR3, GAL12 GO 0042107~cytokme metabolic process 0 0851 IRF7,TNFSF15 GO 0006497-protein amino acid lipidation 0 0986 PIGN, PIGA GO 0008270-zinc ion binding 0 00772 LOC4I6147, ZDHHC4, ZC3HAV1, LMX1B, ADAM23, ZBTB6, ZNFX1, DTX3L, ZBTBI1, MMP3, CSRP3, PHF2, PLEKHF2, PARP12, CI40RF131, PRDM10, RC3H2, CD4, ZNF804A GO 0003950-NAD+ ADP-nbosyltransferase activity 0 00780 PARP12, ZC3HAV1, PARP14 GO 0008502-melatonin receptor activity 0 0454 LOC396318, MTNR1B GO 0046914~transition metal ion binding 0 0574 WC416147, ZDHHC4, ZC3HAV1, LMX1B, ADAM23, ZBTB6, ZNFX1, DTX3L, ZBTBII, MMP3, CSRP3, PHF2, PLEKHF2, PARP12, C140RF131, PRDM10, RC3H2, CD4, ZNF804A GO 0016763-transferase activity, transferring pentosyl 0 0638 PARP12, ZC3HAVI, PARP14 groups 103 Table 5.3 A selection of immune genes upregulated more than twofold in RIG-I transfected DF-1 cells 15 hpi after infection with BC500 LPAI. Statistically significant genes (P < 0.05) are bolded. Gene Name Accession Fold P-value Number Change CCL19 chemokine (C-C motif) ligand 19 BX929857 9 25 0 0899 CCL20 chemokine (C-C motif) ligand 20 AB101005 3 34 0 398 DHX58 (LGP2) probable ATP dependent RNA helicase DHX58 AM070728 3 67 0 0724 E1F2AK2 eukaryotic translation initiation factor 2-alpha kinase 2 AB125660 3 22 0 0269 (PKHj GAL12 beta defensm 12 AY534898 2 48 0 0678 IFIH1 (MDA5) interferon-induced helicase C domain-containing CR385175 7 33 0 0461 protein 1 IFIT5 interferon-induced protein with tetratricopeptide XM_421662 27 5 0 0200 repeats 5 IFITM5 interferon induced transmembrane protein 5 XM_420924 231 0 0686 IRF1 interferon regulatory factor 1 L39766 3 98 0 0364 1RF7 interferon regulatory factor 7 U20338 281 0 264 IRFS interferon regulatory factor 8 L39767 2 15 0 0805 ISG12 2 interferon stimulated gene 12 2 protein-like NMJXH001296 8 23 0 0660 MX1 myxovirus (influenza virus) resistance 1 AB088533 32 6 0 0096 NFKBIZ nuclear factor of kappa light polypeptide gene enhancer AJ721113 2 36 0 406 in B-cells inhibitor, zeta OASL 2 -5'-oligoadenylate synthetase-like NM_205041 3 81 0 0563 TLR15 toll-like receptor 15 DQ267901 2 76 0 557 TLR3 toll like receptor 3 AY633575 2 37 0 457 TNFSF10 tumor necrosis factor (ligand) superfamily, member 10 AJ720191 2 16 0 507 TNFSF15 tumor necrosis factor (ligand) superfamily, member 15 BX930081 2 79 0 101 ZC3HAV1 zinc finger CCCH type, antiviral 1 CR524013 3 73 0 0910 ZNFX1 zinc finger, NFXl-type containing 1 XM_417395 7 96 0 0448 104 5.3 Duck RIG-I induces immune genes in response to VN1203 After infection with the HPAI virus, 2400 genes were altered in expression by more than twofold. In all, 1422 genes were differentially expressed to a statistically significant level (P < 0.05) in RIG-I transfected chicken cells, as compared to vector-only transfected cells. Of the RIG-I responsive genes altered more than twofold, 377 genes were statistically significant at a P value of < 0.05. GO analysis revealed that RIG-I detection of influenza induced the expression of a wide array of genes in different functional groups, including those associated with transcriptional and translational regulation, and the immune response (Table 5.4, Table 5.5). Twenty eight genes were identified as immune genes and they were all induced by RIG-I, with upregulation of 21 of these genes being statistically significant between biological replicates (Table 5.6). An increased number of statistically significant upregulated immune genes are seen in the VN1203-infected samples compared to BC500-infected. This likely reflects less variability among the two VN1203 replicates compared to the three BC500 replicates. All experiments were independently performed transient transfections of RIG-I or vector and variability between individual experiments is due to many factors including transfection efficiency, cell viability and quality of RNA isolated. . Figure 5.1 shows a heatmap of the overall immune gene expression patterns averaged across replicates, while Figure 5.2 presents the same genes showing variation in individual experimental replicates. 105 Table 5.4 GO terms for downregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with VN1203 HPAI. Gene Ontology Term P-value Genes GO 0003677-DNA binding 7 05E 04 TSHZ3, SOX3, TBX20, HOXD13, TBP, PAX3, HDX, YBX1, GATA2, GF11B, DNTT, NKX2 1, SOX18, ATOH7, SCX, NKX2-5 DMBX1, DNMT3A, TCF7, GEN1 ESR1 ARNTL, HES6, FMN1, ASCL1, TBX19 GO 0030528-transcnption regulator activity 0 00282 DMBX1, TSHZ3, TCF7, TBX20, ESR1 HOXD13, TBP, PAX3 HES6, ARNTL HDX, TAL2, ASCL1, GATA2, NCOA3, NKX2-1, ATOH7, SCX, NKX2 5 TBX19 GO 0045449-regulation of transcription 0 00341 DMBX1, TSHZ3, DNMT3A, TCF7, SOX3, TBX20, ESR1, HOXD13, TBP, PAX3, HES6, ARNTL, HDX, YBX1, TAL2, ASCL1, GATA2, GFI1B, NCOA3, NKX2 1 ATOH7, SCX, NKX2 5, TBX19 GO 0006350-transcnption 0 00882 DNMT3A, SOX3, TBX20, ESR1, HOXD13, TBP, PAX3, ARNTL YBX1, GATA2, GFI1B, ATOH7, SCX, TBX19 GO 0048610~reproductive cellular process 0 0149 SOX3, NKX2 1, GDF9, FCGBP, VMOl GO 0010843-promoter binding 0 0176 ESR1, NKX2 1, NKX2-5 GO 0007610~behavior 0 0180 CRHR1, CCK, CSF3R, NKX2 1 SEMA3A, ESPN, AMPH GO 0006355-regulation of transcription, DNA- 0 0217 DMBX1, TSHZ3, TCF7, TBX20, ESR1, dependent HOXD13, TBP, PAX3, HES6, ARNTL HDX, YBX1, GATA2, NCOA3, NKX2-1, NKX2 5, TBX19 GO 0051252-regulation of RNA metabolic process 0 0252 DMBX1, TSHZ3 TCF7, TBX20, ESR1, HOXD13, TBP, PAX3, HES6, ARNTL HDX, YBX1, GATA2, NCOA3, NKX2-1, NKX2-5, TBX19 GO 0016502-nucleotide receptor activity 0 0252 GPR174, ADORA3, P2RX1 GO 0001614-punnergic nucleotide receptor activity 0 0252 GPR174, ADORA3, P2RX1 GO 0003700-transcnption factor activity 0 0380 DMBX1, TSHZ3, TCF7, TBX20 ESR1, HOXD13, PAX3, ARNTL, HDX, GATA2, NKX2-1, NKX2-5, TBX19 GO 0000904-cell morphogenesis involved in 0 0472 CCK, NKX2-1, GDF9, SEMA3A, B3GNT2 differentiation GO 0042923-neuropeptide binding 0 0770 PRLHR, NPFFR1, NPY7R GO 0008188~neuropeptide receptor activity 0 0770 PRLHR, NPFFR1, NPY7R GO 0003006-reproductive developmental process 0 0859 TCF7, SOX3, HOXD13, NKX2 1, GDF9 GO 0032504-multicelluIar organism reproduction 0 0889 P2RX1, SOX3, NKX2-1, GDF9, VMOl GO 0048609-reproductive process in a multicellular 0 0889 P2RX1, SOX3, NKX2 1, GDF9, VMOl organism GO 0007409-axonogenesis 0 0894 CCK, NKX2 1, SEMA3A, B3GNT2 GO 0048812~neuron projection morphogenesis 0 0975 CCK, NKX2-1, SEMA3A, B3GNT2 106 Gene Ontology Term P value Genes GO 0030878-thyroid gland development 0 0983 NKX2 1, NKX2 5 GO 0031224-intnnsic to membrane 0 0987 GPR174, PRLHR, ADORA3, SLC15AI, ATP4B, CDIC, CNTFR, DLLl, CACNG3, TECPRl, VIPR2, CDH6, GJA3, PROMI, CRHR1, TMEM56, P2RX1, B3GNT2, NPY7R, NPFFR1 GO 0008134~transcnption factor binding 0 0994 GATA2, NCOA3, TBP, ARNTL, NKX2 5 107 Table 5.5 GO terms for upregulated genes in RIG-I transfected DF-1 cells compared to empty-vector transfected DF-1 cells, 15 hpi with VN1203 HPAI. Gene Ontology Term P-value Genes GO 0009615~response to virus 2 14E-05 1FNB, MYD88, IRF7, 1FNG, TLR3 GO 0006955-immune response 1 90E 04 OASL, MYD88, CCL20, IRF7,1RF8, 1FNG, ZAP70, CCL19, TLR3, IL10 GO 0005576-extracellular region 2 39E-04 FU, WNT10A, LPL, NOG, LOC396260, UTS2D, CCU9, MMP3, GDNF, IL10, IFNB, GPC5, APOB, TNFRSF11B, CCL20 IFNG, ASIP, THPO GO 0005125-cytokine activity 5 48E-04 IFNB, CCL20, IFNG, CCL19, IL10, THPO GO 0003950-NAD+ ADP-nbosyltransferase activity 8 11E-04 PARP9, PARPI2, ZC3HAV1, PARP14 GO 0045080-positive regulation of chemokine 0 00131 MYD88, IFNG, TLR3 biosynthetic process GO 0045073-regulation of chemokine biosynthetic 0 00131 MYD88, IFNG, TLR3 process GO 0006952-defense response 0 00218 IFNB, LSPI, LIPA, MYD88, IFNG, TLR3, IL10 GO 0032642-regulation of chemokine production 0 00321 MYD88, IFNG, TLR3 GO 0010033-response to organic substance 0 00378 MYD88, IFNG, CHRNB4, TLR3, EIF2AK2, IRG1, ASIP, IL10 GO 0044421 -extracellular region part 0 00390 IFNB, GPC5, LPL, WNTI0A, NOG, TNFRSFI1B, APOB, CCL20, IFNG, MMP3, IL10 GO 0042035 -regulation of cytokine biosynthetic 0 00815 MYD88, IFNG, TLR3, IL10 process GO 0048878-chemical homeostasis 0 0117 LPL, APOB, EPASI, CHRNB4, PTCH1, CALBI, RHAG GO 0016763-transferase activity, transferring pentosyl 0 0125 PARP9, PARP12, ZC3HAV1, PARP14 groups IFNB, LPL, NOG, APOB, CCL20, IFNG, GO 0005615-extracellular space 0 0128 IL10 P2RX7, SCN1A, KCNK9, GABRB3, GO 0005216~ion channel activity 0 0137 KCNN2, SCN2A, CHRNB4, GLRA4, SCN5A NOG, MYD88, TLR3, IL10 GO 0051098-regulation of binding 0 0140 GO 0022838-substrate specific channel activity 0 0141 P2RX7, SCN1A, KCNK9, GABRB3, KCNN2, SCN2A, CHRNB4, GLRA4, SCN5A GO 0015267-channel activity 0 0145 P2RX7, SCN1A, KCNK9, GABRB3, KCNN2, SCN2A, CHRNB4, GLRA4, SCN5A GO 0022803-passive transmembrane transporter 0 0145 P2RX7, SCN1A, KCNK9, GABRB3, activity KCNN2, SCN2A, CHRNB4, GLRA4, SCN5A GO 0051091-positive regulation of transcription factor 00156 MYD88, TLR3, IL10 activity GO 0032675-regulation of interleukin-6 production 0 0180 IFNG, TLR3, IL10 GO 0031226-intnnsic to plasma membrane 0 0188 SCN1A, MDGAI, NTRK1, RORI, CHRNB4, PTCH1, SCN5A GO 0042592-homeostatic process 0 0190 LPL, APOB, LIPA, EPASI, IFNG, CHRNB4, PTCH1, CALBI, RHAG 108 Gene Ontology Term P value Genes GO 0001816-cytokine production 0 0233 LWA, MYD88, IRF7 GO 0043388-positive regulation of DNA binding 0 0233 MYD88, TLR3, ILW GO 0051099-positive regulation of binding 0 0262 MYD88, TLR3, ILW GO 0009617~response to bacterium 0 0278 MYD88, IFNG, IRG1, ILW GO 0051241-negative regulation of multicellular 0 0294 NOG, IFNG, PTCH1, ILW organismal process GO 0045348-positive regulation of MHC class H 0 0298 IFNG, IL10 biosynthetic process GO 0045346-regulation of MHC class II biosynthetic 0 0298 IFNG, IL10 process GO 0045211-postsynaptic membrane 0 0300 GABRB3, CHRNB4 GLRA4, CALB1 GO 0042108-positive regulation of cytokine 0 0323 MYD88, IFNG, TLR3 biosynthetic process GO 0051090-regulation of transcription factor activity 0 0323 MYD88, TLR3, IL10 GO 0001817-regulation of cytokine production 0 0366 MYD88, IFNG, TLR3, IL10 GO 0002237-response to molecule of bacterial origin 0 0389 MYD88, IRG1, IL10 GO 0042627-chylomicron 0 0396 LPL, APOB GO 0001819-positive regulation of cytokine production 0 0423 MYD88, IFNG, TLR3 GO 0046464-acylglycerol catabolic process 0 0444 LPL, APOB GO 0050707-regulation of cytokine secretion 0 0444 IFNG, IL10 GO 0046503-glycerolipid catabolic process 0 0444 LPL, APOB GO 0032606-type I interferon production 0 0444 MYD88, IRF7 GO 0050715~positive regulation of cytokine secretion 0 0444 IFNG, IL10 GO 0044269-glycerol ether catabolic process 0 0444 LPL, APOB GO 0045351 -type I interferon biosynthetic process 0 0444 MYD88, IRF7 GO 0046461 -neutral lipid catabolic process 0 0444 LPL, APOB GO 0019433-tnglycende catabolic process 0 0444 LPL, APOB GO 0008201 -heparin binding 0 0450 LPL, APOB, PTCHI GO 0010557-positive regulation of macromolecule 0 0512 MYD88, EPAS1, MAFB, IFNG, TLR3, biosynthetic process GDNF, TLX1, IL10 GO 0001518~voltage-gated sodium channel complex 0 0525 SCN1A, SCN5A GO 0005887-integral to plasma membrane 0 0570 SCN1A, NTRK1, ROR1, CHRNB4, PTCHI, SCN5A GO 0051101-regulation of DNA binding 0 0573 MYD88, TLR3, ILW GO 0005248-voltage-gated sodium channel activity 0 0580 SCN1A, SCN5A GO 0031328-positive regulation of cellular biosynthetic 0 0585 MYD88, EPAS1, MAFB, IFNG, TLR3, process GDNF, TLX1, ILW GO 0030903-notochord development 0 0587 NOG, GNOT1 GO 0009891-positive regulation of biosynthetic process 0 0596 MYD88, EPAS1, MAFB, IFNG, TLR3, GDNF, TLX1, ILW GO 0034702-ion channel complex 0 0638 SCN1A, GABRB3, CHRNB4, SCN5A GO 0034706-sodium channel complex 0 0652 SCN1A, SCN5A GO 0034361-very-low density lipoprotein particle 0 0652 LPL, APOB GO 0034385-tnglyceride rich lipoprotein particle 0 0652 LPL, APOB 109 Gene Ontology Term P value Genes GO 0044456-synapse part 0 0661 GABRB3, CHRNB4, GLRA4, CALB1 GO 0035295-tube development 0 0723 NOG, LIPA, EPAS1, PTCH1, GDNF GO 0032760-positive regulation of tumor necrosis 0 0728 MYD88, TLR3 factor production GO 0032770-positive regulation of monooxygenase 0 0728 IFNG, GDNF activity GO 0042089-cytokine biosynthetic process 0 0728 MYD88, 1RF7 GO 0042107-cytokine metabolic process 0 0868 MYD88,1RF7 GO 0051043-regulation of membrane protein 0 0868 IFNG, IL10 ectodomam proteolysis GO 0051353-positive regulation of oxidoreductase 0 0868 IFNG, GDNF activity GO 0050714~positive regulation of protein secretion 0 0868 IFNG, IL10 GO 0045072-regulation of interferon gamma 0 0868 TLR3.IL10 biosynthetic process GO 0050810~regulation of steroid biosynthetic process 0 0868 APOB, IFNG GO 0045893-positive regulation of transcription, DNA- dependent - 0 0879 EPAS1, MAFB, IFNG, GDNF, TLX1, IL10 GO 0051254-positive regulation of RNA metabolic process 0 0916 EPAS1, MAFB, IFNG, GDNF, TLX1, IL10 GO 0009953-dorsal/ventral pattern formation 0 0917 NOG PTCH1, GNOT1 GO 0005539-glycosaminoglycan binding 0 0945 LPL, APOB, PTCH1 GO 0044057-regulation of system process 0 0961 EPAS1, IFNG, CHRNB4, GDNF 110 Table 5.6 A selection of immune genes upregulated more than twofold in RIG-I transfected DF-1 cells 15 hpi after infection with VN1203 HPAI. Statistically significant genes (P<0.05) are bolded. Gene Name Accession Fold P-value Number Change CCL19 chemokine (C-C motif) ligand 19 BX929857 7 48 0 0201 CCL20 chemokine (C-C motif) ligand 20 AB101005 4 33 0 00970 EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase 2 AB125660 2 44 0 0698 (PKR) EPAS1 endothelial PAS domain protein 1 AF129813 2 88 0 376 GDNF glial cell derived neurotrophic factor AF176017 2 20 0 378 IFIH1 (MDA5) interferon-induced helicase C domain-containing CR385175 187 0 0159 protein 1 IFITS interferon-induced protein with tetratricopeptide XM_421662 13 8 00138 repeats 5 IFITM5 interferon induced transmembrane protein 5 XM_420924 4 39 0 0456 IFNB interferon beta AY831397 4 21 0 00862 IFNG interferon, gamma AY705909 5 66 0 0309 IL10 interleukin 10 AJ621254 2 38 0 245 IRF1 interferon regulatory factor 1 L39766 291 0 0474 IRF7 interferon regulatory factor 7 U20338 3 12 0 0314 IRF8 interferon regulatory factor 8 L39767 3 70 0 00949 IRG1 immunoresponsive 1 homolog (mouse) AJ720739 5 44 0 00689 ISG12-2 ISG12-2 protein-like NM_001001296 14 8 0 00209 LIPA lipase A, lysosomal acid, cholesterol esterase XM_421661 2 73 0 0140 LSP1 lymphocyte-specific protein 1 AB101641 4 36 0 171 MAFB v-maf musculoaponeurotic fibrosarcoma oncogene NM_001030852 281 0 0282 homolog B MX1 myxovirus (influenza virus) resistance 1 AB088533 20 8 0 0209 MYD88 myeloid differentiation primary response gene (88) CR389938 2 34 0 00948 OASL 2'-5'-oligoadenylate synthetase-like NM_205041 4 43 0 0226 THPO thrombopoietin AY613434 2 06 0 124 TLR3 toll-like receptor 3 AY633575 3 23 0 0137 TLX1 t-cell leukemia homeobox 1 AF071874 2 44 00156 ZAP70 zeta-chain-associated protein kinase 70 XM_418206 2 11 0 120 ZC3HA VI zinc finger CCCH-type, antiviral 1 CR524013 6 43 00113 ZNFX1 zinc finger, NFXl-type containing 1 XM_417395 451 0 0145 111 •(_fl r CCL19 i.j'^yHMHRII CCL20 • i%;» - IFIH1(MDA5) r IFIT5 - IFITM5 • IFNB(IFNP) IFNGOFNy) IRF1 H IRF7 1*1 IRF8 IRG1 ISG12-2 im mm MX1 ^•^H MYD88 OASL TLR3 ZC3HAV1 ZNFX1 RIO-I(BCSOO ) • RIG- I (VN1203 ) ' Empt y vecto r (BC500 ) • Empt y vecto r (VN1203 ) • Colours: Log2R to Refeiraice -4 9 Figure 5.1 Identification of duck RIG-I-responsive immune genes by microarray. Chicken embryonic fibroblasts (DF-1 cells) were either transfected with empty vector (pcDNA 3.1) or duck RIG-I. 24 hours post-transfection, the transfected DF-1 cells were infected with LPAI virus A/BC/500/2003 (BC500) or HPAI virus A/Viet Nam/2004 (VN1203) at a multiplicity of infection (MOI) of 1. At 15 hpi, cellular RNA was extracted for microarray analysis. Heat map shows the differential expression of a bioset of RIG-I-responsive genes following LPAI or HPAI infection, averaged across replicates. Samples were normalized to a reference sample (untransfected, uninfected DF-1 cell RNA). 112 cai9 CCL20 IFIH1 (MDA5) IFIT5 IFITM5 IFNB(IFNP) IFNG(IFNy) IRF1 IRF7 IRF8 IRG1 ISG12-2 MX1 MYD88 OASL F 1 K§3jS: ' 1 TIK3 ;&•> ••" ;<;•'• ZC3HAV1 ZNFX1 ^ M£W* OOOC3C5OC0C0C0C0 oooooooooo ioir>ir>ir>ir>iooicMoiCM OOOOOO'-'-'-'^' IUIDIDIDlllfllzzzz OI CO x— CM CO T— CM CM Hi ii S. S O (D «> Colours. Log2R to Reference -4 9 Figure 5.2 Identification of duck RIG-I-responsive immune genes by microarray in individual replicates. Chicken embryonic fibroblasts (DF-1 cells) were either transfected with empty vector (pcDNA 3.1) or duck RIG-I. 24 hours post-transfection, the transfected DF-1 cells were infected with LPAI virus A/BC/500/2003 (BC500) or HPAI virus A/Viet Nam/2004 (VN1203) at a multiplicity of infection (MOI) of 1. At 15 hpi, cellular RNA was extracted for microarray analysis. Heat map shows the differential expression of a bioset of RIG-I-responsive genes following LPAI or HPAI infection, in each individual replicate sample. Samples were normalized to a reference sample (untransfected, uninfected DF-1 cell RNA) 113 5.4 Innate immune genes show increased expression in chicken cells expressing duck RIG-I Many immune genes were induced by infection with both BC500 and VN1203 and their expression is increased in chicken cells expressing duck RIG-I, rather than vector-only (Figure 5.1 and 5.2). Of these, several genes have known roles in influenza defense or are involved in interferon signal transduction. Signalling through RIG-I leads to translocation of IRF7, which drives expression of IFNp, leading to increased induction of interferon responsive genes by positive feedback. Both IRF7 and IFNJ3 are induced by influenza infection in chicken cells transfected with duck RIG-I. Three genes with established roles in influenza defense are Myxovirus resistance protein (Mx), oligoadenylate synthetase (OAS), and Protein Kinase R (PKR), and induction of all of three genes by influenza was significantly augmented by the presence of duck RIG-I. DF-1 cells expressing duck RIG-I demonstrated a 33-fold increase in Mxl expression in response to infection with BC500, and 21-fold by VN1203. The interferon-inducible Mx proteins confer antiviral function in transfected cells and transgenic animals (Engelhardt et al, 2004; Pavlovic et ai, 1995; Pavlovic et al., 1990). In mice, Mxl is interferon- induced and protects against lethal infection by both the 1918 pandemic H1N1 strain and VN1203 (Tumpey et al., 2007). Chickens have a single, polymorphic, interferon-stimulated Mxl gene (Schumacher et al., 1994). Although previous studies questioned the efficacy of some chicken Mx alleles (Ko et al, 2002; Ko et 114 al., 2004b; Schumacher et al, 1994), recent data showed antiviral effects against two different HPAI strains (Ewald et al., 2011). The pronounced induction of Mxl is likely one of mechanisms by which duck RIG-I contributes to the antiviral response. OAS and PKR are induced by interferon and have known antiviral function. OAS synthesizes 2'-5'-oligoadenylates which activate RNaseL to degrade host and viral RNA, resulting in transcriptional shutdown (Garcia-Sastre and Biron, 2006). The related OAS-like gene (OASL) was induced approximately 4-fold by both viruses in the presence of duck RIG-I, compared to vector-only transfected cells. Protein kinase R (PKR) (EIF2AK2) was induced in chicken cells transfected with duck RIG-I after both BC500 and VN1203 infection, at a P value < 0.05 for the BC500 but not VN1203 infection. Chicken PKR is a polymorphic protein with demonstrated antiviral function against VSV infection (Ko et al., 2004a). However, recent data shows PKR is required for IFNa/p induction in response to several viruses, but not influenza (Schulz et al, 2010). Although their contribution to influenza defense is not well-defined in chickens, OASL and PKR are classical antiviral effectors and both are induced downstream of duck RIG-I upon influenza infection. In both BC500 and VN1203 infections, interferon-stimulated genes were significantly induced. IFIT5, a member of the IFN-induced proteins with tetratricopeptide repeats (IFIT) family, was induced in cells carrying duck RIG-I by 28-fold upon infection with BC500 or 14-fold by VN1203. IFIT family members are implicated in antiviral activity, likely through interference with 115 protein translation (Zhang et al, 2007). ISG12-2 was induced in RIG-I transfected compared to vector-only transfected DF-1 cells by 8-fold in response to BC500 and 15-fold by VN1203. ISG12-2 is induced in chicken lung and tracheal organ culture after influenza infection and is predicted to have antiviral function through an unknown mechanism (Reemers et al, 2009; Reemers et al, 2010b). Human ISG12a has been implicated in sensitizing cells for apoptosis by mitochondrial membrane destabilization (Rosebeck and Leaman, 2008). Apoptotic cell death has been proposed as a mechanism to limit influenza spread, and this effect is much greater in duck than chicken cells (Kuchipudi etal.,2011). RIG-I may be inducing apoptosis upon influenza infection through the pronounced expression of ISG12-2. CCL19 expression is interferon-dependent (Pietila et al, 2007) and was induced by 7-fold by HPAI infection in chicken cells carrying duck RIG-I compared to vector-only, while CCL20 was induced by 4-fold. Both CCL19 (Forster et al, 2008) and CCL20 (Le Borgne et al, 2006) are involved in recruitment of dendritic cells and naive lymphocytes to initiate adaptive immune responses. 5.5 Discussion Through a microarray approach, we have characterized the transcriptional response induced by influenza in chicken cells in the absence or presence of duck RIG-I. We demonstrate that the induction of key innate immune genes by both BC500 and VN1203 was augmented by the presence of duck RIG-I in chicken 116 cells. Many of the immune genes that were induced in cells carrying duck RIG-I correspond to those reported as RIG-I responsive in a microarray comparison of wildtype and RIG-I"/_ mouse fibroblasts, including IFNfi, IRFl, IRF7, MDA5, and Mxl (Loo et al, 2008). Thus, duck RIG-I regulates a similar subset of genes as those defined as the RIG-I bioset in the mouse. RIG-I stimulates the production of IFNp\ which is essential for protection against influenza (Koerner et al, 2007) because it orchestrates the production of antiviral interferon stimulated genes (ISGs). Thus, the RIG-I bioset of genes defines a set of candidate antiviral effectors, which can be mined for those with direct antiviral function. We expect that some of these effectors are responsible for our previous observation that duck RIG-I reduces influenza virus titre in chicken DF-1 cells (Barber et al, 2010). Interestingly, most of the immune genes that were significantly induced downstream of duck RIG-I in both the BC500 and VN1203 infections were induced to lower levels in the VN1203-infected samples (IFIT5, IRFl, Mxl and ZNFXl). This is despite VN1203 replicating to slightly higher levels in DF-1 cells compared to BC500-infected cells (Barber et al, 2010). One possible explanation is that VN1203 replicated faster than BC500, and the peak of interferon was earlier for this infection. A second possibility is that the NS1 protein of VN 1203 is more immunosuppressive than the NS1 of BC500. The influenza NS1 protein is known to modulate the interferon-mediated antiviral response (Chan et al, 2005; Garcia-Sastre et al, 1998; Zielecki et al, 2010) and RIG-I is a known target of NS1 (Gack et al, 2009; Opitz et al, 2007; Rehwinkel et al, 2010). 117 Our experiment does not address which genes would be upregulated by influenza infection in untransfected chicken cells. However, others have used gene expression profiling by microarray to examine the early transcriptional responses to influenza A viruses in chickens (Reemers et al, 2009; Reemers et al, 2010a; Sarmento et al, 2008a; Sarmento et al, 2008b). An examination of the genes expressed in primary chicken embryonic fibroblasts infected with two H5N1 HPAI strains, A/Ck/Hong Kong/220/97 and A/Egret/Hong Kong/757.2/02 at four hpi showed that of 191 genes that demonstrated a twofold induction, 10 were associated with the immune response (Sarmento et al, 2008a). None of these genes appear to be associated with the innate immune response, and the genes induced in RIG-I transfected chicken cells are notably absent. Chickens certainly have an interferon response to influenza, and are able to induce an inflammatory response upon infection with HPAI (Karpala et al, 201 la). Chickens also have a strong innate immune response to other RNA viruses such as Newcastle Disease Virus (NDV) (Rue et al, 2011). Furthermore, influenza-infected chicken trachea and tracheal organ cultures express numerous innate immune genes (Reemers et al, 2009). Thus, it may be that chickens have a robust inflammatory response to influenza, but that loss of RIG-I prevents this response in epithelial and fibroblast cells, which are the primary target of HPAI strains. A recent study strongly supports this hypothesis. Type I IFN, as measured by an IFN bioassay, was induced by H5N1 HPAI infection in chicken lung, plasma and spleen. However, infection with HPAI or LPAI isolates completely failed to induce Type 1 IFN in primary cultures of chicken embryonic fibroblasts 118 or DF-1 cells. In contrast, infection with HPAI consistently induced high levels of Type I interferon in chicken splenocytes (spleen PBMCs) (Moulin et al, 2011). Thus, while chickens do produce large amounts of interferon in response to HPAI infection, it seems to be arising from leukocytes, likely through the TLR7 pathway. Lack of protection by RIG-I in chicken epithelial cells is likely detrimental in an acute influenza infection, given that they are the initial cells infected with influenza viruses. The possibility of augmenting influenza defense in chickens through transgenesis is compelling. Very recently, Lyall and colleagues successfully used such an approach by creating a transgenic chicken expressing a short-hairpin RNA that inhibits the influenza polymerase. While the chickens still succumbed to infection, importantly, transmission of influenza to uninfected chickens was reduced (Lyall et al., 2011). Because duck RIG-I can induce many known antiviral mediators in chicken cells, a transgenic chicken expressing duck RIG-I could theoretically translate to increased protection to influenza in vivo. Chickens expressing duck RIG-I in tracheal epithelial cells would be expected to control influenza virus replication more efficiently. There are possible drawbacks to creating a transgenic chicken with RIG-I, such as creating a chicken that can replicate HPAI strains without symptoms. However, a superior innate immune response may limit viral replication and thus reduce the emergence of highly pathogenic strains. Our data suggests that RIG-I transgenic chickens would show increased resistance to influenza infections through the induction of important antiviral genes. Here we compare global gene expression by microarray for 119 chicken DF-1 cells infected with low or highly pathogenic strains of influenza in the presence or absence of duck RIG-I. Overall, the microarray results demonstrate that duck RIG-I can function to elicit an interferon-driven, antiviral response in chicken embryonic fibroblasts. Additionally, this experiment provides proof-of-principle that incorporation of duck RIG-I into chickens by transgenesis would augment innate immune gene expression upon influenza infection. 5.6 Author's contribution to data I designed this experiment with advice from the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital (Michael Wang and Granger Ridout). The Hartwell Center prepared labelled cDNA and performed the microarray hybridization and data extraction with the transfected/infected samples I prepared. 120 Chapter VI: Conclusions and future directions 121 6.1 Summary Wild birds, especially ducks (Anas platyrhynchos), serve as the reservoir for all strains of influenza (Webster et al, 1992). Ducks propagate most influenza strains without apparent pathology. Occasionally, avian influenza strains emerge that are highly pathogenic to domestic poultry species such as chickens. HPAI viruses are devastating to many species, but generally still cause limited pathology in the original duck reservoir. How ducks, but not domestic poultry, survive HPAI is unknown. We speculated that innate immune mechanisms are particularly important in HPAI defense in ducks due to the acute nature of this infection. For a successful antiviral response, pattern recognition receptors (PRRs) must first detect influenza and trigger subsequent immune responses. We examined toll like receptor 7 (TLR7) and retinoic-acid inducible gene-I (RIG-1) in ducks, to examine their early innate immune response to influenza. TLR7 is an endosomal PRR found in specialized immune cells such as plasmacytoid dendritic cells (pDCs) (Diebold et al, 2004). Upon triggering, TLR7 signals for the release of large amounts of type I interferon (IFN) and proinflammatory cytokines which induce dendritic cell maturation and contribute to an antiviral state in neighbouring cells (Steinman and Hemmi, 2006). TLR8 is a highly related TLR that arose through TLR7 gene duplication, which also contributes to influenza detection in mammals. Original reports suggested that the TLR8 gene was disrupted in chickens but possibly intact in ducks, potentially contributing to differential influenza detection between the two species (Philbin et al, 2005). We 122 fully sequenced a genomic clone containing the TLR7/8 locus in ducks and found that like chickens, TLR8 is only present in small fragments and is additionally disrupted by a retroviral insertion element. While the genomic organization of TLR7/TLR8 is thus similar between ducks and chickens, we demonstrated that duck TLR7 can induce IFNa while some chicken cell lines cannot (Philbin et al., 2005). Additionally, we demonstrated that duck TLR7 is significantly expressed in the lung which is not the case in chicken and may be important in light of the respiratory nature of HPAI infections. In mammals, RIG-I is essential for the response to influenza in the primary site of replication of HPAI, respiratory epithelial cells. We have also shown that ducks, but not chickens, have a functional RIG-I that is significantly induced in the duck lung during highly pathogenic (HPAI) infection (Figure 6.1). Transfection of the chicken embryonic fibroblast cell line, DF-1, with duck RIG-I rescued the ability of chicken cells to detect an in vitro transcribed RIG-I ligand, induced transcription of the known antiviral mediators IFNp, Mxl and PKR, and reduced the titre of both LPAI and HPAI influenza strains. To better understand the transcriptional response elicited by RIG-I in response to influenza, we performed whole genome microarrays on duck RIG-I transfected chicken embryonic fibroblast (DF-1) cells. A strong transcriptional response was observed with the induction of many genes involved in innate antiviral and proinflammatory responses. While surviving HPAI infection is a polygenic trait, we have identified several distinctions in the pattern recognition receptors that recognize influenza between ducks and chickens which appear to provide ducks 123 with superior innate immune detection of influenza. Characterizing the immunological mechanisms mediating influenza resistance in the natural duck reservoir may elucidate what constitutes a successful response to this virus, providing insight into the pathogenesis of the virus. 124 • Duck Epithelial Cells Influenza A virus 5-ppp \/X)0O(/\S RIG-I MDA5 Antiviral program Limited pathology Chicken Epithelial Cells Xg| Influenza A virus 3fiBC.]fflfc 5-ppp VAXXX/V Mitochondrion MDA5 5-ppp \y\50©O\/ 5-ppp \/\300C/\/ 5-ppp V/NJOOOY/ NFKB Increased pathology 125 Figure 6.1 A model for innate defense to influenza in duck and chicken epithelial cells. RIG-I detection of influenza viral RNA triggers the interferon response in duck epithelial cells, while chickens apparently lack RIG-I. MDA5 and RIG-I, pattern-recognition receptors, initiate signalling pathways that converge at the activation of the transcription factors interferon (IFN)-regulatory factor 3 (IRF3), IRF7 and/or nuclear factor-KB; this leads to the expression of IFNp. Hypothetically, in duck epithelial cells, influenza infection stimulates IFNP production and an antiviral program that reduces viral replication. In chicken epithelial cells lacking RIG-I, there is a delayed or weak antiviral program and influenza rapidly replicates to cause a lethal infection, ppp, 5' triphosphate 126 6.2 A model for innate defense to HPAI in ducks and chickens Ducks and chickens have differential susceptibility to influenza pathology, and these studies provide the first evidence for immunological mechanisms that may mediate this distinction. The first cells to be infected by influenza are the respiratory epithelial cells (Figure 6.2). Our prediction is that ducks rapidly detect influenza in this cell type with RIG-I to induce an antiviral state, but that chickens do not have this crucial influenza sensor. Since RIG-I is the first line of protection in the respiratory epithelium, chickens may have a few rounds of replication occur before resident immune cells such as plasmacytoid dendritic cells are triggered to produce IFNa through the TLR7 pathway. Both ducks and chickens have a functional TLR7 receptor, but TLR7 appears to be consitutively expressed in duck but not chicken lung. This may also lead to delayed induction of IFN and viral control in the influenza-infected chicken lung. Most likely, ducks have more immediate detection of influenza infection, allowing for a more rapid immune response, and some RIG-I responsive genes which are known to be antiviral may be produced in ducks but not chickens. Since undertaking our studies, the findings of several other groups have supported our results. Primary cultures of chicken embryonic fibroblasts and DF-1 cells have been shown to fail to induce type 1 IFN upon influenza infection, while high amounts of IFN are produced in whole lung, plasma and spleen (Moulin et ai, 2011). Very recently, it has been shown that chicken cells fail to generate a response to 5' triphosphorylated dsRNA and that chicken MDA5 cannot compensate for the lack of RIG-I (Karpala et ah, 201 lb). Additionally, it has been 127 observed that duck embryonic fibroblasts rapidly undergo apoptosis, DNA fragmentation and activation of caspase 3/7 upon H5N1 HPAI infection (Kuchipudi et al, 2011). Chicken cells undergo reduced apoptosis upon the same infection. Perhaps the rapid cell death in duck cells is a mechanism contributing to successful host defense to H5N1 (Kuchipudi et al, 2011). While the authors do not determine a mechanism responsible for this differential apoptosis, it is interesting to note that IRF-3 activation downstream of RLR signalling not only leads to the induction of innate immune genes, but also the induction of apoptosis through interaction with the pro-apoptotic protein, Bax (Chattopadhyay et al, 2010). Blocking RIG-I signalling with a dominant-negative RIG-I mutant protects Sendai virus infected cells from apoptosis (Chattopadhyay et al, 2010). Thus, perhaps the decreased apoptosis in chicken compared to duck cells derives from the loss of RIG-I, and is one of the mechanisms by which loss of this gene enhances influenza pathology. All of the above recent studies are consistent with the loss of RIG-I in chickens and its presence in ducks. 128 ciliated epithelial cells Figure 6.2 Simplified diagram of the airway epithelium, showing ciliated epithelial cells and resident plasmacytoid dendritic cells. Influenza viruses primarily infect respiratory ciliated epithelial cells, which rely on RIG-I for the influenza detection. As a second line of defense, resident immune cells such as pDCs also recognize influenza with TLR7, triggering production of large amounts oflFNa. 129 6.3 Potential mechanisms for the loss of influenza-detecting PRRs in avian species Given the important and evolutionarily conserved role of PRRs in pathogen recognition, it is intriguing why ducks and chickens may have lost potential influenza detectors. It bears mentioning that avian species harbour many other RNA viruses besides influenza, including coronaviruses, West Nile Virus, and Newcastle Disease Virus, which may all exert pressure on PRR repertoire and function. The significance of the apparent loss of TLR8 (in ducks and chickens) and RIG-I (in chickens) appears great, but a few different possibilities for PRR loss can be envisioned. Selective forces have affected the diversity of avian immune genes in many ways. There is evidence of pathogen-driven selection in chicken immune genes, which usually have lower sequence conservation than other gene categories (International Chicken Genome Sequencing Consortium, 2004). Immune genes are usually one of the most positively selected gene groups (Yang, 2005) and Mx is under positive selection in both ducks and chickens (Hou et al, 2007). Chickens are highly inbred and during the process of domestication, maladaptive immune traits may have become fixed due to linkage with breeding traits. However, in the case of TLR8 and RIG-I loss, presumably negative selection occurred. It has been shown that the absence of TLR8 in chickens is the result of gene loss (Temperley et al, 2008), presumably in a common ancestor of ducks and chickens. RIG-I is also missing from the chicken genome which is derived from Red Jungle Fowl, the ancestor of modern commercial chickens (Fumihito et 130 al, 1994), so it is not likely that RIG-I was lost during domestication either. Phylogenetic analyses have suggested that the RIG-I like helicases arose in invertebrates and that the gene has been lost in chickens (Sarkar et al, 2008; Zou et al, 2009). It is possible that TLR8 simply was not functional, as has been suggested for mouse (Heil et al, 2004), and there was no selective pressure to retain the gene in a functional form. Another possibility is that TLR8 (in a common ancestor of ducks and chickens) or RIG-I (in the case of chickens) may have become detrimental and undergone negative selection. Negative selection could be exerted on either receptor if they were to recognize self-antigen and induce autoimmunity. There is substantial evidence that TLRs can recognize self- antigens, which can lead to autoimmune disease (reviewed by (Fischer and Ehlers, 2008). However, it is not known if avian species suffer autoimmune conditions. Another theoretical possibility is that a pathogen may have subverted TLR8 or RIG-I for its own benefit, leading to selective pressure to inactivate either gene. An example of viral subversion of a PRR is found in TLR3, whose absence actually increases host survival from several viral infections. TLR3- induced proinflammatory cytokines allow WNV to cross the blood-brain barrier (Wang et al, 2004). TLR3 function has also been positively associated with influenza virus induced pneumonia (Le Goffic et al, 2006) and morbidity from vaccinia infection (Hutchens et al, 2008). Thus, viral subversion of a PRR may create selection to disable the gene. 131 6.4 Possible TLR7 and RIG-I driven strategies for influenza control in avian species While influenza viruses are in evolutionary stasis in their reservoir, when they jump to chickens they may evolve into strains which are highly pathogenic to chickens, and possibly also the original reservoir. This is not only an agricultural challenge, but transmission of avian influenza to domestic poultry creates a scenario for the genesis of novel influenza strains with pandemic potential. The reassortment of LPAI strains from ducks with H5N1 HPAI strains has produced many genotypes of the virus (Peiris et al., 2007), although human-to-human transmission has not yet evolved. In terms of strategies for influenza control, the virus is not a good candidate for eradication because it is a zoonotic disease (Kim et al, 2009). Culling wild birds is not a viable solution for control of pandemic influenza strain emergence (Kim et al, 2009). Instead, control of HPAI should occur in the domestic poultry population, and a long term goal is to introduce natural disease resistance genes from the reservoir species into domestic poultry (Forrest and Webster, 2010; Kim et al, 2009). Currently, agricultural vaccines are employed to protect chickens against influenza however they do not achieve sterile immunity, even against the same viral subtypes (Swayne and Kapczynski, 2008). Antigenic shift and drift also complicate vaccine strain selection. Furthermore, vaccinating domestic poultry creates additional selective pressure for the virus to mutate. In Mexico, vaccinations against H5N2 induced protection, but this was followed by rapid evolution into multiple viral lineages due to antigenic drift (Lee et al, 2004). The 132 identification and characterization of a functional duck TLR7 and RIG-I may allow for ligands of these PRRs to be included in duck vaccine strategies to improve vaccine efficacy. In mice, incorporation of a RIG-I agonist into a HA DNA vaccine improved humoral immunity as measured by an increase in HA- specific antibodies (Luke et al., 2011). The use of transgenesis as a means to create an influenza-resistant chicken has been suggested (Hunter et al. 2005). Very recently, Lyall et al. created a chicken transgenic for a short hairpin RNA that blocks influenza polymerase, which blocked HPAI transmission between chickens (Lyall et al., 2011). Duck RIG-I induces known antiviral mediators in chicken cells and it is theoretically possible that this could translate to in vivo antiviral function. The possibility of augmenting influenza defense in chickens through the creation of a transgenic chicken expressing duck RIG-I is intriguing. Potentially, RIG-I presence in a transgenic chicken could also increase the efficacy of avian influenza vaccination by providing an additional receptor to recognize a live vaccine and potentiate the response. To this end, we have filed a United States patent for the creation of RIG-I transgenic chickens. The most obvious downfall of such a transgenic chicken is the possibility that the chickens could become a "Trojan horse" of influenza, able to acquire and transmit HPAI influenza strains without disease symptoms, while still retaining the ability to pass the strains to mammalian species. 133 6.5 Future directions Overall, we have characterized some of the PRRs that are likely involved in influenza detection in its natural host. Many basic questions remain unanswered. Plasmacytoid dendritic cells have not been identified in avian species and it is not known which cells express TLR7 in the avian lung. Additionally, whether TLR7 signalling in avian species initiates adaptive immunity is not known. PRRs are important in initiating dendritic cell maturation upon infection. Ducks have limitations in their ability to develop a secondary immune response to influenza (Kida et al., 1980), and perhaps dendritic cell maturation is not being effectively initiated by TLR7 signalling. The presence of a functionally antiviral RIG-I in ducks underscores the importance of this receptor in influenza detection. However, we have yet to discover which cell types in the duck express RIG-I, and have not defined the pathways leading to increased IFN following RIG-I signalling. As the duck genome becomes available, identification of signaling components will become more possible. Due to the lack of a defined duck cell line, all functional work characterizing duck RIG-I has been performed in chicken cells. However, duck and chickens are separated by 90 million years of evolution and it is entirely possible that duck RIG-I signals for different downstream responses in duck than chicken cells. Whether RIG-I contributes differently to the control of enteric LPAI infection than pulmonary HPAI influenza detection remains unknown. While LPAI and the duck immune response have had many millennia to evolve 134 together, HPAI is really an accidental infection in ducks and is not the normal occurrence. While ducks have superior immunity to HPAI, this is perhaps because the fundaments of successful control of influenza infection are in place from constant exposure to LPAI viruses. We do not yet know whether the presence of RIG-I in other avian species correlates to influenza susceptibility, or whether RIG-I has equivalent function in ducks as it does in difficult-to-infect avian species such as pigeons. It also remains to be seen if duck RIG-I has the same detection and/or signalling abilities as mammalian RIG-I. Given that duck RIG-I is only 53% conserved to human RIG-I at the amino acid level, it is quite possible that it has different functionality. A corollary to this is whether duck RIG-I interacts with the viral NS1 protein in the same way as mammalian RIG-I. NS1 can be divided into two alleles, A and B. A alleles are both avian and mammalian and B alleles are avian only. After an avian NS1 is introduced into mammals it is rapidly selected against (Hayman et al, 2007). Perhaps this selection relates to its interaction with RIG-I, and RIG-I-NS1 interactions happen in a species-specific manner. Understanding how influenza infection is controlled by the natural reservoir of the virus is fundamental to understanding influenza pathogenesis and may suggest what constitutes a successful innate immune response to influenza. 135 Chapter VII: Literature cited 136 1. 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