Analysis of the sialic acid binding activity of the hemagglutinins of influenza viruses and
its role in host tropism
THESIS
Submitted in partial fulfillment of the requirements for the degree
-Doctor rerum naturalium- Dr. rer. nat.
at the University of Veterinary Medicine Hannover
by
Anne-Kathrin Sauer (Hannover)
Hannover 2013
Advisory Committee: Prof. Dr. Georg Herrler Prof. Dr. Silke Rautenschlein Prof. Dr. Rita Gerady-Schahn
1st Evaluation Prof. Dr. Georg Herrler Institute of Virology,
University of Veterinary Medicine Hannover, Foundation Prof. Dr. Silke Rautenschlein
Clinic for Poultry,
University of Veterinary Medicine Hannover, Foundation Prof. Dr. Rita Gerardy-Schahn
Institute for Cellular Chemistry, Hannover Medical School
2nd Evaluation Prof. Dr. Wolfgang Garten Institute for Virology,
Philipps University Marburg
Date of oral exam: 26. April 2013
This work was financed by the Federal Ministry of Education and Research (BMBF):
FluResearchNet
List of Abbreviations V
List of Figures VIII
List of Tables X
Abstract XI
Zusammenfassung XIII
1 Introduction 1
1.1 Pathology . . . 2
1.2 Taxonomy . . . 4
1.3 Virus structure . . . 4
1.4 Viral life cycle . . . 8
1.5 The hemagglutinin . . . 10
1.5.1 The H9 subtype . . . 12
1.5.2 The H7 subtype . . . 13
1.5.3 The H5 subtype . . . 14
1.5.4 The H1 subtype . . . 14
1.5.5 The H17 subtype . . . 16
1.6 Sialic acids . . . 16
1.7 Aim of the study . . . 21
2 Material 22 2.1 Cell lines . . . 22
2.2 Bacteria . . . 22
2.3 Fertilized eggs . . . 22
2.4 Porcine tissues . . . 23
2.5 Plasmids . . . 23
2.5.1 pCG1 . . . 23
I
2.5.2 pCGFc . . . 23
2.5.3 pCGT6his . . . 24
2.6 cDNA . . . 24
2.7 Viruses . . . 26
2.8 Media . . . 27
2.8.1 EMEM (Eagle’s minimal essential medium), pH 7.0 . . . 27
2.8.2 DMEM (Dulbecco’s minimal essential medium), pH 6.9 . . . 27
2.8.3 HAM’s F12, pH 7.0 - 7.5 . . . 27
2.8.4 CLEC213 Medium, pH 6.9 . . . 28
2.8.5 Freezing medium . . . 28
2.8.6 LB-Medium, pH 7.7 . . . 28
2.9 Buffers and solutions . . . 28
2.10 Synthetic Oligonucleotides . . . 32
2.11 Enzymes . . . 34
2.11.1 Restriction enzymes . . . 34
2.11.2 Other enzymes . . . 34
2.12 Antibodies . . . 34
2.13 Lectins . . . 36
2.14 Excitation and emission wavelentgh of fluorescent dyes . . . 36
2.15 Kits . . . 36
2.16 Other substances . . . 37
2.17 Chemicals . . . 37
2.18 Equipment . . . 39
3 Methods 41 3.1 Cell Culture . . . 41
3.1.1 DAPI test . . . 41
3.1.2 Cryoconservation . . . 41
3.1.3 Transfection of HEK293T cells via calcium phosphate precipitation 42 3.1.4 Preparation of primary chicken kidney cells (pCKC) and tissue sections . . . 42
3.2 Molecular biology . . . 43
3.2.1 Polymerase chain reaction (PCR) . . . 43
3.2.2 PCR purification . . . 45
3.2.3 Restriction . . . 45
3.2.4 Agarose gel electrophoresis . . . 45
3.2.5 DNA extraction from gel . . . 46
3.2.6 Ligation . . . 46
3.2.7 Transformation . . . 46
3.2.8 Colony PCR . . . 47
3.2.9 Plasmid preparation . . . 47
3.2.10 Determination of DNA concentration . . . 48
3.2.11 Sequencing . . . 48
3.3 Protein biochemistry . . . 48
3.3.1 Preparation of soluble glycoproteins . . . 48
3.3.2 Fast protein liquid chromatography, FPLC . . . 49
3.3.3 Quantification of soluble proteins . . . 50
3.3.4 Immunofluorescence . . . 51
3.3.5 Quantification of HA binding by Flow Cytometry . . . 52
3.3.6 SDS PAGE . . . 53
3.3.7 Western Blot . . . 53
3.3.8 Overlay Assay . . . 54
3.4 Virological methods . . . 55
3.4.1 Propagation of virus in embryonated chicken eggs . . . 55
3.4.2 Immunoplaque assay . . . 55
3.4.3 Virus binding assay . . . 56
4 Results 57 4.1 Expression and purification of soluble HAs . . . 57
4.2 Binding of soluble HAs to different cell lines . . . 60
4.2.1 Binding of H7Fc and H9Fc to different cell lines . . . 61
4.2.2 Binding of other solHAs to cells . . . 62
4.2.3 Lectin staining of cell lines . . . 69
4.3 Neuraminidase pretreatment of cells . . . 71
4.3.1 Quantification via flow cytometry . . . 72
4.3.2 NA effect on the HA binding to different cell lines . . . 72
4.4 Virusbinding to cells . . . 78
4.5 Binding to respiratory tissue sections . . . 79
4.6 Glycan array . . . 84
4.7 Investigations of the binding properties of H17 . . . 88
4.7.1 Cleavability of H17 HA0 . . . 89
4.7.2 Binding to cell lines . . . 90
4.7.3 Binding to permanent cell lines from bats . . . 92
4.8 Search for potential interactions partners of the influenza HA . . . 97
4.9 Sequence alignment of HA subtypes . . . 97
5 Discussion 101 5.1 Expression of soluble HAs . . . 102
5.2 Binding of solHAs to different cell lines . . . 103
5.2.1 Neuraminidase pretreatment . . . 107
5.2.2 Virus binding . . . 107
5.3 Binding to respiratory tissue sections . . . 108
5.4 Binding properties of bat H17 subtype . . . 112
5.5 Other applications for solHAs and outlook . . . 115
6 References 118 7 Sequences 129 7.1 Sequence of H7Fc . . . 129
7.2 Sequence of H7T6his . . . 131
7.3 Sequence of H9Fc . . . 133
7.4 Sequence of H9T6his . . . 135
7.5 Sequence of H5Fc . . . 137
7.6 Sequence of H1_2009Fc . . . 139
7.7 Sequence of H1_1918Fc . . . 141
7.8 Sequence of H1_WFc . . . 143
7.9 Sequence of H17Fc . . . 146
8 Abbreviations of amino acids 149
9 Affidavit 150
10 Acknowledgments 151
aa Amino acid
APS Ammonium peroxide disulfate
BHK Baby hamster kidney
bp Base pair
BSA Bovine serum albumine
BSL3 Biosafety level 3
ca. circa
cDNA complementary DNA
cRNA cellular RNA
CO2 Carbon dioxide
C-terminal COOH terminus of proteins
Cy3 Indocarbocyanine
dH2O pure water
DAPI 4’,6’-Diamidino-2-phenylindol
DMEM Dulbeccos Modified Eagle Medium
DNA Desoxy ribonucleic acid
dNTP Desoxy nucleoside triphosphate
DTT Dithiothreitol
E. coli Escherichia coli
ER Endoplasmatic Retikulum
et al. et alii (and others)
FACS Fluorescence activated cell sorting
FCS Fetale calves serum
fig. figure
FITC Fluorescine isothiocyanate
Gal Galactose
Glc Glukose
HA Hemagglutinin
HAT Human airway trypsin-like protease
V
HRP Horse raddish peroxidase
IF Immunofluorescence
Ig Immunglobulin
kb Kilo base pairs
kDa Kilodalton
LB Luria Bertani; luria broth
M Molarity; -molar
MAA Maackia amurensisagglutinin
MDCK Madine-Darby canine kidney
mRNA messenger RNA
MW Molecular weight / mass
NA Neuraminidase
N-terminal NH2-terminal of a protein
NEP Nuclear export protein
Neu5Ac N-acetyl-neuraminic acid
Neu5Gc N-glycolyl-neuraminic acid
NP Nucleoprotein
PBS Phosphate buffered saline
PBSM PBS without calcium and magnesium
PCR Polymerase chain reaction
PE Phycoerythrin
PFA Paraformaldehyde
Pfu Pyrococcus furiosus
pfu plaque forming units
pH Potentia Hydrogenii
rcf Relative centrifugal force
RNA Ribo nucleic acid
rpm Rounds per minute
RT Room temperatur
SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
Sia Siali acid
SNA Sambuccus nigraagglutinin
SN Supernatant
TAE Tris, Acetate, EDTA
Taq Therus aquaticus
TEMED N,N,N’,N’-Tetramethylethylendiamin
Tris Tris(hydroxymethyl)aminomethan
TRITC Tetramethyl Rhodamine
U unit [µmol/min]
WB Western blot
α anti (antibodies) or alpha (sialic acids)
µ mikro (gramm or litre for example)
1.1 Schematic drawing of an Influenza A virus particle . . . 5
1.2 Single cell replication cycle of Influenza A viruses . . . 8
4.1 Western blot of Fc-tagged solHA proteins and FcATG under non reducin conditions . . . 59
4.2 Western blot of H9Fc, H7Fc and FcATG . . . 60
4.3 Western Blot of H7T6his and H9T6his . . . 60
4.4 Binding test on different cell lines with H9Fc . . . 63
4.5 Binding test on different cell lines with H7Fc . . . 64
4.6 Binding test on different cell lines with FcATG . . . 65
4.7 Binding of his-tagged H7 and H9. . . 66
4.8 Binding test on different cell lines with 100 pmol solHAs . . . 68
4.9 Binding test on different cell lines with 100 pmol solHAs (cont.) . . . 69
4.10 Lectin staining of different cell lines . . . 71
4.11 Binding of solHAs and lectins to neuraminidase treated MDCKII cells . . . 73
4.12 Quantification of the neuraminidase effect on the binding of solHAs and lectins on MDCKII cells . . . 74
4.13 Binding of H5Fc and H9Fc on different cell lines with and without prior pre- treatment with 200 mU NA fromClostridium perfringes . . . 76
4.14 Binding of FcATG and lectins on different cell lines with and without prior pretreatment with 200 mU NA fromClostridium perfringes . . . 77
4.15 Binding of H7N7 and H9N2 viruses to different cell lines with 5x105 ffu/ml. . 78
4.16 Binding of H7Fc, H9Fc and FcATG to the respiratory epithelium of chicken, turkey and swine trachea as well as the porcine bronchus. . . 80
4.17 Binding of solHAs to the respiratory epithelium of chicken, turkey and swine trachea as well as the porcine bronchus. . . 82
4.18 Lectin staining of the respiratory epithelium of chicken, turkey and swine trachea as well as the porcine bronchus. . . 84
4.19 Glycan array analysis of the solHAs H9Fc and H7Fc . . . 86
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4.20 Glycan array analysis of the lectins MAAII and SNA . . . 87
4.21 Sialosides used in the glycan arrays. . . 88
4.22 Coomassie Gel of trypsin treated and untreated solHAFc . . . 89
4.23 Binding of H17Fc on different cell lines with and without prior pretreatment with 200 mU NA from Chlostridium perfringes . . . 91
4.24 Binding of solHAs on different bat cell lines with and without prior pretreat- ment with 200 mU NA fromChlostridium perfringes . . . 93
4.25 Binding of different solHAs to CpKd cells. . . 94
4.26 Flow cytometry of solHA binding to MDCKII cells with and without pretreat- ment with NA from C. perfringens. . . 95
4.27 Enzymatic treatment of cells and solHA binding. . . 96
4.28 Overlay assay . . . 98
4.29 Overlay assay . . . 99
4.30 Sequence alignment of the RBS of all used HA subtypes. . . 100
4.31 Sequence alignment of the RBS of H1 subtypes. . . 100
2.1 Adherent, continuous cell lines . . . 22
2.2 fertilized eggs . . . 22
2.3 cDNA of hemagglutinins, avian origin . . . 25
2.4 cDNA of hemagglutinins, porcine origin . . . 25
2.5 cDNA of hemagglutinins, human origin . . . 26
2.6 cDNA of hemagglutinins, bat origin . . . 26
2.7 Strains of avian influenza viruses used in this work . . . 27
2.9 Synthetic oligonucleotides . . . 32
2.10 Antibodies . . . 35
2.11 Lectins; all lectins were used diluted in 1%BSA, 1:200 . . . 36
2.12 Excitation and emission wavelength of fluorescent dyes . . . 36
3.1 Standard protocol forPfu PCR . . . 44
3.2 PCR conditions for standardPfu PCR with a touchdown temperature style 44 3.3 Standard protocol for ligation . . . 46
3.4 Standard protocol for colony PCR , reaction for one colony . . . 47
3.5 PCR conditions for colony PCR . . . 47
4.1 List of soluble HAs used in this work . . . 58
4.2 Scoring of solHA binding . . . 62
4.3 Scoring of solHA binding . . . 69
4.4 Scoring of lectin staining . . . 70
4.5 Scoring of solHA binding on tissue sections . . . 83
8.1 Abbreviations of amino acids . . . 149
X
Analysis of the sialic acid binding activity of the hemagglutinins of influenza viruses and its role in host tropism
Anne-Kathrin Sauer
Influenza A viruses belong to the familyOrthomyxoviridae. They are single-stranded RNA viruses with a segmented genome of negative orientation. The influenza hemag- glutinin (HA) is the viral envelope glycoprotein responsible for binding of the virion to the host cell and fusion of the viral with the cellular membrane. It is synthesized as an uncleaved precursor and introduced into the host cell secretory pathway where it is posttranslationally modified and forms trimers. The receptor determinants for influenza A viruses are sialic acids, terminal sugars of glycan chains present on glycolipids or glycoproteins. Different virus strains (e.g. avian and human strains) have different affinities for either α2,3- or α2,6-linked sialic acids. To distinguish between these two types of receptor determinants, the plant lectins MAA (from Maackia amurensis) and SNA (from Sambucus nigra) were used in many studies. Due to the huge diversity of oligosaccharide structures on the one hand and the different hemagglutinin subtypes on the other hand, those two plant lectins are not sufficient to characterize the binding properties of influenza hemagglutinins.
Therefore, soluble hemagglutinins (solHAs) were generated that can be used as lectins for detection of those sialoglycoconjugates that are interaction partners for influenza
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viruses. Fusion of the ectodomain to the Fc-component of a human IgG and to a mod- ified GCN4 leucin zipper motif as trimerization domain leads to chimeric proteins that are secreted into cell culture supernatant after transfection of HEK293T cells. These soluble hemagglutinins of different avian, human and porcine viruses as well as that of newly discovered bat influenza virus were analyzed for the binding to cells and tissue sections by confocal laser scanning microscopy and the results were compared with pictures obtained by staining with plant lectins.
Binding of solHAs was reduced when sialic acids were released from the cell surface by prior neuraminidase pretreatment of cells. The binding patterns obtained with H7Fc and H9Fc was comparable to those observed with the respective intact virions, i.e.
H7N7 and an H9N2 strain. SolHAs of different subtypes bound with different efficien- cies to continuous cell lines from different species. Although most cell lines expressed both sialic acid linkage types in similar intensities, the binding of HA subtypes differed.
To investigate the binding to the respiratory epithelium of potential hosts, binding tests were perfomed with tracheal sections from chicken, turkey and swine as well as with sections from the pig lung. Again, this assay showed different efficiency of the different HA subtypes that were not predictable by lectin staining. The affinity to sialoglycans was further analysed by applying the glycan array technology. This showed a clear dif- ference between the glycoconjugates detected by the two plant lectins and by solHAs.
These data showed that plant lectins are not the optimal tool to investigate the com- plex issue of receptor binding of influenza viruses. Using solHAs we have been able to detect even small differences in receptor specificity and to show an approach that finally may even allow the identification of specific glycoprotein or glycolipid receptors for influenza viruses.
Analyse der Sialinsäure-Bindungsaktivität der Hämagglutinine von Influenzaviren und deren Bedeutung für den Wirtstropismus
Anne-Kathrin Sauer
Influenza A Viren gehören der Familie Orthomyxoviridae an. Ihr einzelsträngiges RNA-Genom ist segmentiert und weist eine negative Orientierung auf. Das virale Glykoprotein Hämagglutinin (HA) ist verantwortlich für Rezeptorbindung und für die Fusion der viralen mit der zellulären Membran. Die Rezeptordeterminanten für Influen- zaviren sind Sialinsäuren, die eine terminale Position in Zuckerketten von Glykolipiden und Glykoproteinen einnehmen. Verschiedene Influenzavirus-Stämme (z.B. aviären und human Ursprungs) besitzen unterschiedliche Affinitäten zu Sialinsäuren, die in α2,3 oder α2,6 Konformation mit einer Galactose verbunden sind. Um diese beiden Rezeptor-Determinanten zu unterscheiden werden in vielen Studien die Pflanzenlek- tine MAA (von Maackia amurensis) und SNA (von Sambucus nigra) verwendet. Da aber einerseits eine große Vielfalt an Zuckerstrukturen existieren und andererseits eine Reihe unterschiedlicher HA-Subtypen beschrieben wurden, wird die Nutzung von nur zwei Pflanzenlektinen dieser Diversität nicht gerecht und kann die natürlichen Zusam- menhänge nur unzureichend wiedergeben.
Daher wurden in dieser Arbeit lösliche Hämagglutinine (engl. soluble hemagglutinins, solHAs) erzeugt, deren eigene Sialinsäure-bindende Eigenschaften genutzt wurden
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um die Bindung an Sialoglykokonjugate zu untersuchen, die Interaktionspartner für Influenzaviren darstellen. Die Verknüpfung der HA Ektodomäne mit der konstanten Region einens humanen IgG-Moleküls (-Fc) oder an eine modifizierte GCN4 Leucin- zipper Sequenz, die als Trimerisierungsdomäne diente führte zu löslichen Proteinen, die über ihren jeweiliges Anhängsel (engl. tag) einfach zu reinigen und nachzuweisen waren.
SolHAs von verschiedenen Subtypen aviären, humanen und porzinen Ursprungs und sogar eines aus einem neu entdeckten Fledermaus Influenzavirus wurden in dieser Arbeit in Bezug auf ihre Bindung an Zellen und Gewebe untersucht. Die Ergebniss wurden verglichen mit Bildern, die durch Bindungsversuche mit Pflanzenlektinen er- halten wurden.
Die Bindung der solHAs an Zellen war reduziert, wenn die Sialinsäuren durch vorherige Neuraminidase-Bahndlung von der Zelloberfläche entfernt wurden. Das Bindungsmuster der löslichen H7Fc und H9Fc Proteine entsprach dem, das mit intakten Viruspartikeln der betreffenden Subtypen erhalten wurden. Obwohl die meisten in dieser Arbeit untersuchten Zelllinien Sialinsäure in beiden Verknüpfungstypen exprimierten unter- schied sich die Bindung der verschiedenen HA-Subtypen teils stark. Um die Rezep- torverteilung in primären Gewebe zu untersuchen, wurden Bindungstests mit Cryoschnit- ten von Trachea aus Huhn, Pute und Schwein und Lungenschnitten vom Schwein durchgeführt. Auch hierbei wurde deutlich, dass die verschiedenen HAs eine unter- schiedliche Bindungseffizienz aufwiesen, die durch Lektinfärbung nicht vorherzusagen war. Die Affinität zu verschiedenen Sialoglykanen wurde weiterhin mittels der Glycan Array Technologie untersucht. Diese Daten zeigen deutlich, dass die Pflanzenlektine und die HAs unterschiedliche Glykokonjugate erkennen.
Die in dieser Arbeit dargestellten Daten zeigen, dass Lektine kein optimales Hilfs- mittel sind, um die komplexen Zusammenhänge der Rezeptorbindung und Rezep- torverteilung bei Influenzaviren zu untersuchen. Durch die Benutzung von löslichen
HAs konnten wir sogar kleinere Unterschiede in der Rezeptorbindung verschiedener HA Subytpen aufzeigen. Mit diesem Ansatz könnte es in Zukunft sogar mögliche sein, definierte Glykoprotein- oder Glykolipid-Rezeptoren für Influenzaviren zu identifizieren.
Influenza viruses have attracted public interest since they have been identified more than 80 years ago. Seasonal epidemics occurring especially in the cold season lead to 3 to 5 million severe cases with 250.000 to 500.000 deaths (WHO [2009]). The risk group for influenza infections are especially the elderly, infants and immunocompro- mised people where case severity is highest. Prevalence is highest in school aged children (J. K. Taubenberger [2008]). Current seasonal epidemics are caused by in- fluenza A viruses (IAV) of the H1N1 and H3N2 subtypes as well as by influenza B viruses. The high antigenic variation of the viruses surface glycoproteins via antigenic drift (the gradual accumulation of mutations, that allow the virus to escape immunity) makes it necessary to develop new vaccines for every season.
The natural reservoir for IAV are aquatic and shore birds (Anseriformes like ducks and geese; Charadriiformes like gulls). But it is possible for the virus to cross species bar- riers to other avian species, like domestic poultry or to mammals (e.g. horses, seals, pigs, humans) (Webster & Bean [1992], Wright & Webster [2001]).
Every now and then, when an influenza A virus strain enters the human population and meets immunogenic naive hosts, this can lead to a pandemic outbreak. The last one was in 2009, when a triple reassortant virus spread from pigs to humans. The first cases were found in Mexico from where the virus spread around the world and caused
>18.000 deaths worldwide and 43 million to 89 million infections within a year (Medina
& García-Sastre [2011]).
1
The most severe influenza pandemic was the "Spanish flu" from 1918 to 1919 which caused the deaths of presumably more than 40 million people around the world. This virus of the H1N1 subtype probably spread from birds to humans. The 1918 H1N1 virus circulated among the human population until the next pandemic, the "Asian flu", occurred in 1957. At that time a strain of the H2N2 subtype replaced the descendent of the H1N1 "Spanish flu". In 1968 an H3N2 virus led to the next pandemic, the "Hong Kong flu". Both strains resulted from the reassortment of viral genes derived in part from avian influenza viruses. In 1977, an H1N1 virus "re-emerged", maybe from a lab- oratory (J. Taubenberger & Morens [2006], Baigent & McCauley [2003]). None of the following pandemics have been as severe as the "Spanish flu".
It is believed that direct transmission of avian IAV to the human population is inefficient but may be facilitated by the involvement of an intermediate host like the pig (Neumann
& Kawaoka [2006]). Around the turn of the millennium, humans were directly infected by a highly pathogenic avian H5N1 strain. Since 1997 to date an overall of 610 cases with 360 deaths have been reported worldwide (WHO [2012], report 17.12.2012). So far the virus does not spread from humans to humans but H5N1 infections in wild birds, poultry stocks and transmission to humans is closely monitored.
Similar zoonotic events recently also occurred with other avian IAV strains. Cases of human infections by H9 and H7 virus subtypes occur sporadically and raise con- cern over the possibility of new pandemic outbreaks (Horimoto & Kawaoka [2005], J. K. Taubenberger & Kash [2010]).
1.1 Pathology
IAV infection in humans results in sudden onset of fever, cough, headache and inflam- mation of the upper and lower respiratory tract. Symptoms persist seven to ten days,
weakness and fatigue for weeks. Complications may include hemorrhagic bronchitis, pneumonia, acute respiratory distress syndrome and lead to death. Effective coun- termeasures are prevention by vaccination or hygiene and the application of antiviral drugs (J. K. Taubenberger [2008]).
In waterfowl, IAV infections are asymptomatic although virus can be found in many or- gans as well as in the blood. In aquatic and shore birds, influenza viruses are mainly found in the intestine and are transmitted oral-fecally (Horimoto & Kawaoka [2005],- J. K. Taubenberger & Kash [2010]).
In poultry (order Galliformes: turkey, chicken, quail, etc), two forms of IAV are dis- tinguished: low pathogenic and highly pathogenic avian influenza viruses. LPAI only cause localized infections in respiratory or intestinal tract with mild or no symptoms.
HPAI infections on the other hand lead to loss in egg production, lethargy, sinusitis, diarrhea, nervous system disorders and death. Infected flocks are culled to prevent spreading (Horimoto & Kawaoka [2005]). The major difference between LPAI and HPAI refers to cleavage site of the viral surface protein hemagglutinin (see section 1.5).
In pigs, the signs of disease consist of coughing, fever, labored breathing, conjunc- tivitis and pneumonia. Several lineages of porcine influenza viruses are known. In the 1930’s the first isolation of IAV was from swine (Shope [1931]). This "classical"
swine H1N1 virus causes enzootic disease in pigs. Since 1998 there are also triple reassortants containing gene segments from swine H1N1, human H3N2 and avian IAV infecting pigs and spreading like the classical H1N1 viruses in North America and worldwide. By adaptation of an avian IAV to swine in the 1970’s a Eurasian strain devel- oped (J. K. Taubenberger & Kash [2010]). Swine have been shown to be susceptible for avian and human influenza strains and are therefore considered the aforementioned intermediate host or "mixing vessel" for the generation of IAV that may infect humans.
1.2 Taxonomy
Influenza A viruses are classified in the genus Influenzavirus A within the family Or- thomyxoviridae and are enveloped viruses with a single stranded segmented RNA genome of negative orientation. Other genera of this viral family are theInfluenzavirus B andC,ThogotovirusandIsavirus. Influenza B and C viruses only infect humans and swine or seals respectively. Influenza A viruses on the other hand infect a variety of mammalian and avian species. Influenza C infections are usually mild or asymptomatic whereas influenza A and B can cause severe illness.
The full length strain nomenclature of influenza virus strains indicates the genus (In- fluenzavirus A, B, C), the species of the host (if other than man) from which the strain was isolated, the country or region of discovery, a laboratory number to distinguish different isolates, and the year of isolation, followed by the hemagglutinin (HA) and neuraminidase (NA) subtypes: e.g. A/chicken/Emirates/R66/2002 (H9N2). According to the HA and NA serotypes, influenza A subtypes are termed for example H1N1 or H7N7. So far 10 NA and 17 HA subtypes are known. The latest, H17N10, was discov- ered in 2012 (Tong et al. [2012]) in a virus isolated from bats in Guatemala.
All HA and NA subtypes can be found in aquatic birds (exception H17N10, which has only been isolated from bats). In waterfowl influenza A viruses cause no or mild dis- ease. Aquatic birds are probably the natural reservoir for influenza A viruses and spread from this host either directly or via adaptation processes to other host species like domestic poultry or mammals (J. K. Taubenberger & Kash [2010]).
1.3 Virus structure
Influenza viruses are enveloped viruses with a segmented RNA genome of negative orientation with a total of about 13 kb. The viral particles can be spherical with a diam- eter of about 80 to 130 nm in size, but filamentous forms - especially in fresh isolates -
Figure 1.1: Schematic drawing of an Influenza A virus particle, adapted from Flint et al. [2004]
have also been reported (Elton et al. [2006]). The 8 genomic segments code for a total of 11 proteins: 9 structural and 2 non-structural.
The RNA segments are associated with the nucleoprotein NP and the polymerase pro- teins PA, PB1 and PB2, which together form the viral ribonucleoprotein (vRNP). The inner surface of the viral envelope is coated by the matrix protein M1. Integrated inside the viral envelope are the ion channel protein M2 as well as the viral glycoproteins hemagglutinin and neuraminidase.
The vRNPs consist of the single-stranded vRNA covered by NP proteins, forming a panhandle structure, and the heterotrimeric polymerase complex. The RNA hairpin results from double-stranded stretches formed by the homologous 5’ and 3’ ends of the vRNA. This duplex is also the binding site for the polymerase complex (Portela &
Digard [2002], Elton et al. [2006]). The RNA-dependent polymerase complex consists of the PB1 (polymerase and endonuclease activity), PB2 (cap snatching) and PA (func- tion unknown) protein.
The matrix protein M1 is he most abundant protein in the virion. It interacts with newly synthesized vRNPs, is important for nuclear export as well as assembly and budding of new virus particles (see also section 1.4)(Barman et al. [2001]).
The ion channel protein M2 is encoded from a spliced mRNA of the M1 segment. The tetrameric protein is activated by the low pH of the endosomes enabling acidification of the lumen of the virus vesicles and thus supporting the uncoating of the virus (Horimoto
& Kawaoka [2001]).
The neuraminidase is a receptor destroying enzyme, cleaving sialic acids from sialo- glycoconjugates, allowing nascent virions to be released from the host cell. During viral replication the enzyme also cleaves sialic acids from HA and NA proteins. It is a type II membrane protein, which forms tetramers and has a mushroom-like shape.
To date there are 10 NA subtypes known. The latest, N10, as mentioned above, was isolated from bats and differs from the other 9 subtypes. It is more distantly related to the nine other NA subtypes. Although it shares structural features with the so far described NAs, it lacks certain amino acids in the sialic acid binding and cleavage sites and has indeed no sialidase activity (Zhu et al. [2012], Li et al. [2012], García-Sastre [2012]).
The NA is the target of current anti-influenza drugs (zanamivir and oseltamivir) which interact with the catalytic domain of the enzyme. The occurrence of resistant strains makes the development of new antiviral compounds an urgent task. Like the HA, the NA is subject to selective pressure and both are major targets of the adaptive immunity (Flint et al. [2004], Gamblin & Skehel [2010], Webster & Bean [1992]). The counterpart, responsible for receptor binding is the hemagglutinin which will be described in detail in section 1.5. Its receptor specificity is important for the adaptation to new hosts and for crossing species barriers. The HA is also responsible for fusion of the viral and the host membrane during virus entry. As HA and NA activity go hand in hand in the viral infection, both proteins have to evolutionary co-adapt.
The non-structural protein NS1 plays many roles in the viral life cycle. Some of those are regulating vRNA synthesis, influencing viral mRNA splicing and translation and an- tagonizing host innate immunity, namely the interferon production, an antiviral mecha- nism of the cell (Hale et al. [2008]). Therefore the NS1 is also an important virulence factor of influenza virus strains.
Through alternative splicing from the NS1 segment, the nuclear export protein is syn- thesized, which as the name indicates facilitates the export of new vRNPs from the nucleus (see section 1.4).
From an alternative open reading frame of the PB1 segment, the small protein PB1-F2 is produced. PB1-F2 is a proapoptotic protein, contributing to viral pathogenicity. It can only be found in some strains, but its presence has been shown to favor secondary bacterial infections. Likewise the introduction of the PB1-F2 of the 1918 pandemic strain into the apathogenic PR8 strain led to increased pathogenicity with and without bacterial infections (Conenello & Palese [2007]).
1.4 Viral life cycle
Figure 1.2: Single cell replication cycle of Influenza A viruses, references see text
The viral replication cycle begins with the binding of the HA to its cellular sialic acid receptors. Upon attachment the virus particle is endocytosed via clathrin-dependent or independent endocytosis (Sieczkarski & Whittaker [2002]). It is transported to the late endosomes where the low pH leads to a conformational change of the HA. The HA1 subunit de-trimerizes but remains connected to the HA2 subunit by disulfide bonds (Harrison [2008]). Re-folding of the HA2 subunits, which now form a long helix, con- nects the viral and the cellular membrane via the fusion peptide in an intermediate hemifusion form. When this intermediates collapses both membranes are brought into contact, leading to the opening of a fusion pore (Skehel & Wiley [2000], Harrison [2008],
Jiang et al. [2010]).
The decreasing pH of the endosome results in an activation of the M2 ion channel and as a consequence in the acidification of the viral core. Under the acidic conditions the vRNPs dissociate from the M1 protein releasing the genome through the fusion pore into the cytoplasm (Nayak et al. [2004], Samji [2009]).
The vRNPs are translocated into the nucleus via the nuclear localisation signals of the proteins associated with the viral RNA: PA, PB1, PB2 and NP. To synthesize viral pro- teins, the negative sense viral RNA has to be transcribed into mRNA. For this purpose the PB2 protein cleaves the 5’ methylated cap together with 10 to 15 nucleotides of nascent cellular mRNAs, that serve as primer for viral transcription. This process has been termed "cap snatching" (Samji [2009]).
Viral mRNAs also possess a 3’ poly-A tail produced by a uridine repeat at the 5’ end of the template vRNP molecules which results in a stuttering process, amplifying and complementing the 5 to 6 uridine residues (Flint et al. [2004]). The freshly transcribed, capped and polyadenylated viral mRNAs are translocated into to cytoplasm. Here the M1, NS1 and NEP mRNAs are translated and proteins translocated back into the nucleus. The mRNAs for HA, NA and M2 are translated at the rER and the proteins translocated into the lumen of the ER. Here HA and NA are subject to glycosylation and oligomerization and transported along the secretory pathway, from the ER, via ERGIC and Golgi to the plasma membrane. The freshly translated NP, PA, PB1 and PB2 are transported back to the nucleus to participate in the synthesis of new full-length vRNA.
The replication of viral RNA does not need primers and results in an un-capped cRNA that does not carry a polyA tail and serves as a template for new negative-sense vR- NAs. The exact mechanism by which switch from mRNA synthesis to the synthesis of full-length vRNAs is triggered is yet unclear, but it appears that an association of the positive sense cRNA with NP plays an important role in this process. NP-cRNA inter- action might result in the transcription into negative sense vRNA, either by a soluble
polymerase complex in trans in contrast to mRNA synthesis which is carried out by the polymerase complex associated with the vRNPs (incis) (for detailed review of this topic see Resa-Infante et al. [2011] and Portela & Digard [2002]).
The newly synthesized vRNPs interact with the M1 protein, which can bind N-terminally to the nuclear export protein NEP, thereby masking the nuclear localization signal. The NEP facilitates the export of the vRNP-M1-NEP complex into the cytoplasm using prob- ably the CRM1-dependent export pathway (Samji [2009]).
Maturation of influenza viruses occurs by a budding process at the apical site of po- larized cells. For this purpose, the M1-vRNP complexes have to be transported to the plasma membrane by an so far unknown mechanism. HA, NA and M2, all of which are transported along the secretory pathway are also sorted to the apical membrane and accumulate in TX-100 resistant membrane microdomains, so-called lipid rafts (Nayak et al. [2004] and Takeda et al. [2003]). The M1 protein probably acts as a bridge be- tween HA/NA/envelope and the vRNPs. Due to the segmented genome of influenza viruses, all eight segments have to be packaged into the virion to become infectious particles. Recent studies showed that this step in the replication cycle most likely occurs via a specific packaging process (Naim & Roth [1993], Nayak et al. [2009]).
Clustering of the M1 protein at the budding site might lead to bending of the membrane resulting in the initiation of the budding process. Budding and membrane fission are completed by cellular proteins and the process is not completely solved to date (for review see Nayak et al. [2009]). The viral NA releases sialic acids from the cell surface allowing the virus to spread from infected to uninfected cells (Samji [2009]).
1.5 The hemagglutinin
The influenza HA is one of the most intensively studied glycoproteins in respect to structure, function and intracellular trafficking. The type I integral membrane protein is
translated as a precursor protein HA0 at the rER and translocated into the lumen of the ER where it is folded and forms trimers along its way to the ER-Golgi-intermediate compartment (ERGIC) (Tatu et al. [1995]). During transport along the secretory path- way the HA0 gets glycosylated at four to eleven potential N-glycosylation sites and it is cleaved by host cell proteases into the subunits HA1 and HA2 which remains con- nected via disulfide bonds. The HA1 subunit, comprising 324 aa, carries the sialic acid binding domain and forms the globular head; the HA2 (222 aa) forms the stalk and car- ries the fusion peptide at the N-terminus which is buried in a negatively charged cavity at neutral pH. Upon acidification in the endosomes, the fusion peptide is exposed and inserted into the cellular membrane, which is the first step in membrane fusion (See section 1.4; Skehel & Wiley [2000], Harrison [2008]). Thus, the proteolytic activation of the HA is an essential step in order to get infectious virus.
HA0 cleavage is an important determinant of the pathogenicity in influenza viruses. In most HA subtypes, the cleavage site consists of a single arginine residue which can be cleaved on the cell surface or on released virus particles. Such cleavage sites are accessible to a set of proteases expressed only in a limited number of tissues, e.g.
the respiratory and intestinal epithelium. Those proteases are called "trypsin-like" pro- teases and include for example tryptases, blood-clotting factor Xa (e.g. in chicken eggs) in in vitro situations. TMPSSR2 (transmembrane protease serine 2) and HAT (human airway trypsin-like protease) were shown to cleave human HAs in vivo (Klenk [2008]).
Infections by such avian influenza viruses are local and usually not life-threatening.
Cleavage sites of highly pathogenic avian influenza viruses (HPAI) consist of several basic amino acids with a consensus motif R-X-R/K-R that can be cleaved by ubiquitous proteases like furin. Infection with these viruses therefore can occur in a variety of tis- sues resulting in a more acute and systemic course of disease (Horimoto & Kawaoka [2001], Klenk [2008]). HPAI viruses are only found among the H5 and H7 subtypes.
A major task of the HA protein is the recognition of and the binding to cellular re-
ceptors. The receptor determinants of influenza viruses are sialic acids i.e. N-acetyl- neuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc) or modifications of these two types of sialic acid. Sialic acids are usually located at the outermost position of oligosaccharides on glycoproteins or glycolipids. Sialic acids connected to galactose by anα2,3- or anα2,6-linkage have been shown to be recognized by influenza viruses.
The receptor binding site (RBS) of the HA is located at the tip of the molecule and con- sists of residues 190 -198 forming an α-helix and two loops formed by residues 133 - 138 and 220 - 229. The amino acids responsible for receptor recognition of the HA of some subtypes are well characterized. The receptor determinant sialic acid forms hydrogen bonds with amino acids (aa) 135, 136, 137 as well as 183, 190, 98 and is connected via van der Waals forces with aa 153 and 194. Essential for receptor speci- ficity are the amino acid positions 226 and 228. A leucine (L) can be found in viruses withα2,6-linkage specificity and glutamine (Q) inα2,3-specific viruses (Skehel & Wiley [2000]). A serine (S) at position 228 is found in human viruses and a glycine (G) in the HA of avian viruses (Vines et al. [1998]).
1.5.1 The H9 subtype
H9N2 viruses were found in many countries in Asia, Europe, Middle East and Africa and can be isolated from many avian species, as well as from pigs and even humans (Dong et al. [2011]).The H9 HA from H9N2 srains is of particular interest as several cases of transmission of H9N2 strains to humans have been reported since 1997 (Butt et al. [2010]). Many field isolates have acquired human virus-like receptor specificity with a leucine at HA aa position 226 (H3 numbering) (M. Matrosovich et al. [2001], Choi et al. [2004]) although this appears not to be a prerequisite for H9N2 viruses to infect humans as also the "avian" glutamine has been found in H9N2 isolates from humans (Butt et al. [2010]). H9N2 isolates from 2009 also showed a different proteolytic
cleavage site. Chicken-adapted H9N2 viruses possess an R-S-S-R motif, associated with low pathogenicity. 2009 isolates from humans on the other hand showed a R-S- N-R motif, which before was only found in avian isolates from Israel (Perk et al. [2006]) and India (Tosh et al. [2008]). Whether this correlates with higher pathogenicity is not yet known (Butt et al. [2010]).
As H9N2 viruses are highly prevalent in Asia and readily infect humans they are a reservoir from which a future pandemic virus may evolve. These viruses have been shown to undergo reassortment with other avian viruses in this region, e.g. stains of the H5N1 lineage, further increasing the pandemic risk (Medina & García-Sastre [2011]).
1.5.2 The H7 subtype
Influenza viruses of the H7 subtype also have been recently associated with direct transmission from avian species to humans. In 2003, a H7N7 HPAI outbreak in com- mercial poultry farms in the Netherlands resulted in the infection of 89 humans. The course of disease was mild but human-to-human transmission occurred in three cases (Fouchier et al. [2004], Koopmans et al. [2004]).
In solid phase binding assays avian H7 isolates showed preference for α2,3-linked Neu5Ac, which is in accordance with earlier investigations of the receptor-binding ac- tivity of H7 viruses. Many North American and Eurasian isolates also showed weak binding of human-like sialoglycans, showing the potential for avian-to-human transmis- sion (A. S. Gambaryan et al. [2012]). In the same study, equine isolates of H7 were shown to have adapted to Neu5Gc , which is abundant in horses.
1.5.3 The H5 subtype
H5N1 HPAI outbreaks occur regularly in domestic birds in Asia, Europe and Africa.
Transmission to humans happened so far only after direct contact with infected ani- mals and did not spread from humans to humans. The possibility of the virus to infect humans without prior adaptation steps is the reason for concerns that H5N1 virus may cause a future pandemic. H5N1 isolates from chicken show less specificity for α2,3- linked sialic acids than do their relatives from ducks but nevertheless do not show specificity toα2,6-linked sialic acids (M. Matrosovich et al. [1999]). H5N1 isolates from chicken and humans showed an additional glycosylation site at the tip of the HA and a deletion in the NA stalk region (M. Matrosovich et al. [1999], Horimoto & Kawaoka [2005]). To date ten clades of H5N1 viruses have been described (Medina & García- Sastre [2011]).
Experimentally inserted amino acid changes Q196R, Q226L and G228S have been shown to confer α2,6-linkage specificity (L.-M. Chen et al. [2012]) and experimentally adaptation in ferrets showed that it is possible for H5 viruses to acquire transmissibility via droplets (Imai et al. [2012],Herfst et al. [2012]).
1.5.4 The H1 subtype
The HA of the pandemic 2009 strain (swine origin influenza virus, SOIV) is derived from a North American swine H1N2 strain, which itself is a triple reassortant virus with the HA from the classical swine lineage, a descendant of the 1918 H1N1 strain (J. K. Taubenberger & Kash [2010]). The classical swine lineage in pigs was sub- ject to little antigenic variation due to the short life span of pigs whereas the human H1N1 strains had to undergo substantial antigenic drift. Thus the human population had mainly antibodies against the drifted, seasonal human H1 which are not cross- protective against the swine origin H1 (Garten et al. [2009]). SOIV H1 has been shown
to bind toα2,3- andα2,6-linked sialic acids, which is a possible explanation why infec- tions with this virus led to severe cases of viral pneumonia (Medina & García-Sastre [2011], L.-M. Chen et al. [2011]). This is believed to be associated with infection of the lower respiratory tract of humans, where α2,3-linked sialic acids are expressed (van Riel et al. [2007], Medina & García-Sastre [2011]).
As the 2009 pandemic H1 is a descendant of the "Spanish flu" HA, the original 1918 H1 can confer cross reactive antibodies in mice (Baigent & McCauley [2003], J. K. Tauben- berger & Kash [2010], Medina & García-Sastre [2011]). Sequences of the 1918 HA are divergent. Three of five show a G225D mutation (glycine G to aspartic acid D; G be- ing the conserved avian form), two retain the G225. All five show a change at aa 190 from E (glutamic acid) to D. HAs with both 190 and 225 changes showed α2,6-linkage specificity whereas variants with only the 190 change bound both sialic acid linkage types (Reid et al. [2003], J. K. Taubenberger et al. [2008], Stevens et al. [2006]).
Porcine influenza was first clinically detected in 1918 in association with the 1918 pan- demic. It is not clear which came first, infection of pigs or humans (J. K. Taubenberger
& Kash [2010]). The classical swine lineage of influenza viruses derived from the 1918 strain and remained antigenically stable in pigs (Medina & García-Sastre [2011]). From 1998 different lineages of triple reassortant viruses with genes from humans, birds and pigs have emerged (Zhou et al. [1999]). In Europe, a novel lineage arose in the 1970’s by adaptation of an avian strain. Several strains are currently co-circulating in the swine population and completely human strains were isolated in 2004 and 2005 in North America. The swine influenza virus population consists of two lineages, a North American and a Eurasian (Nelson et al. [2011]).
The possibility of pigs being co-infected with both avian and human IAV strains makes them a potential mixing vessel for reassortment and intermediate host for adaption to human-like sialic acids.
1.5.5 The H17 subtype
The H17 is the most recently described HA. The viral RNA was found in bats in Guatemala. Of 316 captured bats only three rectal swab samples from little yellow shouldered bats (Sturnira lilium) were positive in a pan-influenza RT-PCR. The HA se- quence, obtained by next generation sequencing of the samples, shows conserved sequence motifs, like the receptor binding domain, but also changes in positions re- sponsible for receptor specificity. It is related to known HA subtypes, but more distantly than the subtypes H1 to H16 are related to each other. The NA protein of this virus is very divergent from known NAs and less related to them than the known nine NAs are to influenza B neuraminidases (Tong et al. [2012]). In a recent publication (Zhu et al. [2013]) the authors showed that H17, expressed in a baculovirus system is not de- graded by trypsin digestion at pH 4 like other HAs. Glycan arrays with this H17 showed no binding to sialoglycans.
The N10 lacks conserved amino acids responsible for the cleavage of sialic acids and shows no sialidase activity (Zhu et al. [2012], Li et al. [2012]) and is therefore termed by Zhu et al. [2013] a NA-like (NAL) protein. As the function of HA and NA, of recep- tor binding and release, have to be in balance and to co-adapt in order to maintain a evolutionary "fit" virus these findings raise the question whether the H17 hemagglutinin recognizes sialic acids of either linkage type. This of course raises the next question:
if sialic acids are not the receptor determinants for this bat influenza virus, what is the receptor?
1.6 Sialic acids
The term sialic acid describes a large group of 9-carbon monosaccharides located on the outermost position of glycoproteins and glycolipids of higher animals and in some bacteria. The group comprises 50 members, the most common one being N-acetyl-
neuraminic acid (Neu5Ac). The carbon 1 carries a carboxyl group that is responsible for the negative charge of the molecule. Hydroxylation of the N-acetyl group of Neu5Ac results in N-glycolyl-neuraminic acid (Neu5Gc). Another derivative of Neu5Ac is KDN (2-keto-3-deoxy-nonulosonic acid) which is obtained when the 5-amino group is re- placed by a hydroxyl group. Further possible modifications are acetylation, methylation or the addition of phoshates or sulphates. If the exact chemical structure is not speci- fied, the respective molecules are termed sialic acids, abbreviated Sia (A. Varki [1997]).
The addition of Sias to glycan chains is catalyzed by enzymes called sialyltransferases.
Those are localized in the Golgi as part of the complex glycosylation machinery. Each sialyltransferase transfers a sialic acid residue to a specific sugar in a certain orien- tation. Most common are the α2,3- and α2,6-linkages to an underlying galactose, N-acetylglucosamine or N-acetylgalactosamine molecule. Also possible is the attach- ment to another sialic acid in α2,8 linkage (Nicholls et al. [2008], Kleineidam et al.
[1997]).
The expression of sialic acids is tissue-dependent and developmentally regulated. In humans only Neu5Ac is expressed, Neu5Gc can only be found in certain tumors. In other species like pigs Neu5Gc is quite abundant. Due to their negative charge Sias repel each other, giving mucus its viscosity or preventing thrombocytes from clump- ing. They play an important role in cellular and molecular recognition. The immune system employs Sias for self/non-self recognition. In cancer sialylation patterns are often changed, it has been shown for example that the ST6Gal1 sialyltransferase is upregulated in carcinomas of the colon, cervix, breast and others (N. Varki & Varki [2007]). Also erythrocytes carry sialic acids and therefore participate to the glycosyla- tion pattern resulting in different blood types (Traving & Schauer [1998]). In the form of polysialic acids, Sias play an important role in neural development and plasticity (A. Varki & Schauer [2009]).
However, sialic acids are not only recognized by cells but also by different pathogens.
The invasion of red blood cells by Plasmodium falciparium, the causative agent of malaria, depends on the expression of certain Sias. The bacteriumHelicobacter pylori binds to gastric mucins and bacterial toxins like cholera toxin also recognize sialic acids (Traving & Schauer [1998]).
In 1941 George Hirst discovered that influenza viruses are able to agglutinate chicken red blood cells and later Gottschalk showed that this effect is prevented by removal of Sias (Nicholls et al. [2008]). In the next decades many efforts were undertaken to elu- cidate the receptor binding of influenza viruses. So it is known that influenza C viruses and some coronaviruses use 9-O-acetylated sialic acids as receptors (Herrler et al.
[1985], Schultze & Herrler [1992]).
General methods to investigate the distribution of sialic acids on cells and tissues is the histological or immunofluorescent staining with plant lectins. It is possible to discrim- inate between α2,3- and α2,6-linked sialic acids by using lectins derived from Sam- bucus nigra (SNA, α2,6-linkage) and Maackia amurensis (MAA, α2,3-linkage). Stud- ies using these lectins often led to different results because of the two isoforms of MAA, for example when addressing the question whether ciliated cells in the human lung expressed α2,3- or α2,6-linked sialic acids (Nicholls et al. [2008]). MAAII has a more restricted binding pattern and recognizes Sia-α2,3-Gal-β1,2(Siaα2,6)-GalNAc whereas MAAI also binds to other sialylated glycans (Sia-α2,3-Gal-β1,4-GlcNAc/Glc and even to non-sialic acid residues (Nicholls et al. [2007]). Furthermore lectins differ in their affinity to inner components of carbohydrate chains, which is also known to af- fect the binding of different HAs (A. Gambaryan et al. [2005]). Thus lectins might bind to sialoglycans that do not serve as receptors for influenza viruses and some glycans recognized by influenza viruses might not be detected by lectin staining.
Several studies in the last years investigated the presence of receptor type sialic acids on the epithelium of the human respiratory tract to see whether humans would be sus- ceptible for infection with avian influenza viruses. These reports (M. N. Matrosovich et
al. [2004], Shinya et al. [2006], van Riel et al. [2006]) all came to different conclusion with respect to the cells in the human respiratory tract that are targeted by human or avian viruses and with respect to the type of sialic acids that is expressed on those cells (for review see Nicholls et al. [2008] and Hong [2009]). Therefore, Nicholls et al. [2007]
suggest to use both MAA isoforms as they could show using glycan array technology that both MAAI and H5N1 bound to certain sialoglycans that might not be detected by MAAII.
Similar studies showed, that pigs express both α2,3- and α2,6-linked sialic acids and, therefore, may serve as a mixing vessel for genetic reassortment between avian viruses, which favor α2,3-linked Sias, and human IAV, that preferentially bindα2,6-linked ones.
After several reports of direct bird-to-human transmission and studies that demon- strated the presence of both linkage types in the human lung, this notion had to be revised (Horimoto & Kawaoka [2005]). Recent detailed studies of the porcine respi- ratory epithelium have shown that indeedα2,6-linked sialic acids are the predominant sialic acid species and that pigs in this respect resemble humans and not birds (Bate- man et al. [2010]). Other studies using lectin staining have shown, that the respiratory epithelia of chicken, turkey and quail express both sialic acid linkage types and there- fore may allow the adaptation to "human type" receptor Sias (Kimble et al. [2010], Kuchipudi et al. [2009], Imai et al. [2012]).
To investigate receptor binding properties of different influenza strains methods like solid phase binding assays or hemagglutination assays with erythrocytes from differ- ent species or resialylated red blood cells were used. Avian strains were shown to bind preferentially α2,3-linked sialic acids. Human viruses on the other hand preferentially boundα2,6-linked sialic acids. New methods to investigate receptor binding profiles of different subtypes are glycan arrays, where different sialoglycans are printed on glass slides and the respective viruses are bound. The general preferences of human and avian strains for α2,6- or α2,3 respectively has not been refuted by these studies but
they add more detail to assessment of HA binding as they take underlying glycans into account. Still, these arrays, can not show on what cells and tissues these sugars are expressed (Blixt et al. [2004], J. K. Taubenberger [2008], Liao et al. [2010]) and so this question remains. First attempts have been undertaken by analyzing primary porcine or human cells with mass spectrometry with respect to the glycans present (Chandrasekaran et al. [2008] and Bateman et al. [2010]).
Glaser et al. [2007] showed, that infection of mice lacking aα2,6 sialyltransferase (thus without abundance of α2,6-linked Sias) with human influenza viruses led to equally high titers as in wildtype mice. Also influenza viruses were still able to bind to desialy- lated MDCK cells (Stray et al. [2000]).
The topology of the glycan also appears to be important and might play a role in recep- tor binding. One is the narrow cone-like topology in which sialic acid and the following sugars span like a cone into the HA RBS. This topology is adopted byα2,3 linked sialic acids and short α2,6 glycans. The amino acids associated withα2,3 specificity (E190 and Q226) are involved in this conformation. The degree of branching after the first trisaccharide plays a crucial role in the second possible, umbrella like, topology. The amino acids involved in the binding to these glycans are not conserved among-human adapted H1 and H3 viruses. The broader umbrella-like topology is unique to longα2,6 terminated glycans (Chandrasekaran et al. [2008], Viswanathan et al. [2010]).
Future studies will continue to clarify the relationship between sialic acid linkage, gly- can structure and receptor specificity as well as the distribution of influenza receptors and will determine what the prerequisites for an avian HA to bind to human cells are.
1.7 Aim of the study
The aim of this thesis is embedded in a long-term project that investigates the role of sialic acids in adaptation of influenza viruses to new hosts. During adaptation to new hosts, the receptor specificity of the viral surface protein hemagglutinin changes and allows binding to different receptor types of different host species. To analyse the distribution of receptor type sialic acids, the plant lectins MAA and SNA are utilized to distinguish between α2,3-linked and α2,6-linked sialic acids. But these two lectins have a specificity for certain glycans and those glycans are not necessarily the same as those detected by influenza HAs or they may be recognized with different affinity.
Direct infection of humans with avian H5, H7 or H9 strains led to a rethinking about the previous notion of avian viruses binding toα2,3-linked sialic acids and human viruses binding to α2,6-linked sialic acids. Now we know that sugars beyond the terminal Neu5Ac-Gal disaccharide are involved in receptor specificity. Modern technologies like glycan arrays allow to investigate which glycan chains are detected by different HA subtypes and mass spectrometry can show what glycans are present in a certain sus- ceptible cell or tissue. But still in most cases MAA and SNA are used for direct staining of influenza receptor determinants on host cells.
To circumvent these problems and to take into account the complexity of glycan struc- tures and the variety among HA subtypes, we aimed at generating soluble hemagglu- tinins that would allow to determine the presence or absence of receptors for influenza A viruses on various cells and tissues. In this way, the receptor specificity of each HA subtype can be used to probe for receptors for the respective virus that might not be detected when plant lectins are used. This approach will help in the future to under- stand the role of sialic acids in adaptation processes to new host as the binding to receptor type sialic acids is a first crucial step in the viral replication cycle.
2.1 Cell lines
Table 2.1: Adherent, continuous cell lines
Cell line Species Source Properties Growth medium
A549 Human respiratory epithelium non-polar DMEM + HAM’s F12 1:2 + 5%FCS
Calu-3 Human respiratory epithelium polar EMEM + 5% FCS
HEK 293T Human Kidney non-polar DMEM + 5%FCS
CLEC213 Chicken respiratory epithelium non-polar CLEC213 Medium
MDCKII Dog kidney epithelium polar DMEM + 5% FCS
NPTr Pig tracheal epithelium polar EMEM + 5% FCS
2.2 Bacteria
Chemically competent E. coli MRF XL-1 blue obtained from Stratagene and HB101 from Gregor Meyers, FLI, Tübingen were used for heat shock transformation of plasmid DNA (see section 3.2.7).
2.3 Fertilized eggs
Table 2.2: fertilized eggs
SPF chicken eggs Lohmann, Cuxhaven
turkey eggs Moorgut Kartzfehn von Kameke, Bösel
22
2.4 Porcine tissues
Porcine trachea and lung explants were obtained from three month old crossbred pigs housed in the Clinics for Swine and Small Ruminants and Forensic Medicine at the University of Veterinary Medicine, Hannover.
2.5 Plasmids
2.5.1 pCG1
The pCG1 plasmid was obtained from R. Cattaneo (Mayo Clinic College of Medicine, Rochester, Minnesota, USA). Between the promotor of the cytomegalovirus (CMV) and the multiple cloning site (MCS) there is an intron inserted derived from the rabbit β-tubulin gene. The plasmid contains an ampicillin resistance gene for selection in bacteria. This plasmid was used to express the soluble influenza hemagglutinins in eukaryotic cell lines.
2.5.2 pCGFc
The pCGFc vector is derived from the pCG1 plasmid. In the MCS of the original plas- mid the Fc fragment of human immunoglobulin G is inserted via an SphI restriction site. The pCGFc plasmid was used to generate soluble hemagglutinins fused to a Fc- tag to enable easy detection of the chimeric protein. A derivative of this vector is the pCGFcATG vector which expresses the Fc alone because of the inserted start codon ATG in front of the Fc gene.
2.5.3 pCGT6his
The pCGT6his vector is derived from the pCG1 plasmid. A modified GCN4 leucin zip- per trimerization domain was cloned into the MCS via a SphI restriction. This trimer- ization domain was used to stabilize soluble hemagglutinin trimers.
An amino acid exchange from valine or asparagine or leucine to isoleucines results in increased trimer formation of the coiled-coiled α-helices (Harbury et al. [1993]). This GCN4-pII mutant (in the following referred to as "trimerization domain" (TD)) was used by X. Yang, Farzan, et al. [2000] to stabilize the human immunodeficiency virus enve- lope glycoprotein in membrane-bound (X. Yang, Farzan, et al. [2000]) and soluble form (X. Yang, Florin, et al. [2000]).
Via subsequent PCR the his-tag has been added to the trimerization domain for easy detection and for purification.
2.6 cDNA
The cDNA for the hemagglutinin of A/turkey/Turkey/1/2005 (H5N1) HPAI and A/chicken/- Netherlands/621557/2003 (H7N7) HPAI was provided by Ben Peters (Wageningen Uni- versity and Research Center, Leylstadt, The Netherlands). The cDNA for the HA of A/chicken/Emirates/R66/2002 (H9N2) was provided by Jürgen Stech (Friedrich-Löffler- Institute, Greifswald, Insel Riems) and the cDNA for A/swine/Wisconsin/1/67 (H1N1) by Thorsten Wolff (Robert Koch Institute, Berlin, Germany). The cDNAs of the HAs of human HAs A/South Carolina/1/18 (H1N1) and A/California/04/2009 (H1N1) were provided by Thorsten Wolff (Robert Koch Institute, Berlin,Germany).
The fragments encoding the ectodomain of the HA were amplified and inserted into the MCS of the pCGFc and pCGT6his vector upstream of the human Fc-tag.
The soluble consructs of the the human H1 subtypes have been cloned and initially tested by Meike Erdt.
Table 2.3: cDNA of hemagglutinins, avian origin
Name Discription Size (bp)/ aa
H5 Full length HA of 1704/568
A/turkey/Turkey/1/2005 (H5N1) HPAI
solH5 ectodomain HA of 1599/523
A/turkey/Turkey/1/2005 (H5N1) HPAI
H5Fc Fc-tagged ectodomain of HA 2367/789
A/turkey/Turkey/1/2005 (H5N1) HPAI
H5T6his T6his-tagged ectodomain of HA 1743/581
A/turkey/Turkey/1/2005 (H5N1) HPAI
H7 Full length HA of 1715/562
A/chicken/Netherlands/621557/2003 (H7N7) HPAI
solH7 ectodomain HA of 1575/525
A/chicken/Netherlands/621557/2003 (H7N7) HPAI
H7Fc Fc-tagged ectodomain of HA 2346/782
A/chicken/Netherlands/621557/2003 (H7N7) HPAI
H7T6his T6his-tagged ectodomain of HA 1722/574
A/chicken/Netherlands/621557/2003 (H7N7) HPAI
H9 Full length HA of 1683/561
A/chicken/Emirates/R66/2002 (H9N2)
solH9 ectodomain of HA of 1545/515
A/chicken/Emirates/R66/2002 (H9N2)
H9Fc Fc-tagged ectodomain of HA 2325/775
A/chicken/Emirates/R66/2002 (H9N2)
H9T6his T6his-tagged ectodomain of HA 1701/567
A/chicken/Emirates/R66/2002 (H9N2)
Table 2.4: cDNA of hemagglutinins, porcine origin
Name Discription Size (bp)/ aa
H1W Full length HA of 1746/582
A/swine/Wisconsin/1/67 (H1N1)
solH1W ectodomain HA of 1587/529
A/swine/Wisconsin/1/67 (H1N1)
H1WFc Fc-tagged ectodomain of HA 2367/789
A/swine/Wisconsin/1/67 (H1N1)
H1WT6his T6his-tagged ectodomain of HA 2340/780 A/swine/Wisconsin/1/67 (H1N1)
Table 2.5: cDNA of hemagglutinins, human origin
Name Discription Size (bp)/ aa
H1_2009 Full length HA of 1701/567
A/California/04/2009 (H1N1)
solH1_2009 ectodomain HA of 1587/529
A/California/04/2009 (H1N1)
H1_2009Fc Fc-tagged ectodomain of HA 2352/784 A/California/04/2009 (H1N1)
H1_2009T6his T6his-tagged ectodomain of HA 2728/576 A/California/04/2009 (H1N1)
H1_1918 Full length HA of 1701/567
A/South Carolina/1/18 (H1N1)
solH1_1918 ectodomain HA of 1590/530
A/South Carolina/1/18 (H1N1)
H1_1918Fc Fc-tagged ectodomain of HA 2331/777 A/South Carolina/1/18 (H1N1)
H1_1918T6his T6his-tagged ectodomain of HA 1728/576 A/South Carolina/1/18 (H1N1)
Table 2.6: cDNA of hemagglutinins, bat origin
Name Discription Size (bp)/ aa
H17 Full length HA of 1695/565
A/lit. yel. shouldered bat/Guat./153/09 (H17N10)
solH17 ectodomain HA of 1584/528
A/lit. yel. shouldered bat/Guat./153/09 (H17N10)
H17Fc Fc-tagged ectodomain of HA 2364/788
A/lit. yel. shouldered bat/Guat./153/09 (H17N10)
H17T6his T6his-tagged ectodomain of HA 1740/580
A/lit. yel. shouldered bat/Guat./153/09 (H17N10)
2.7 Viruses
The viruses were propagated in 10 days old embryonated chicken eggs to achieve high infectivity titers.
Table 2.7: Strains of avian influenza viruses used in this work
Name Provided by
A/duck/Potsdam/15/80 (H7N7) LPAI Clininc for Poultry, University of Veteri- nary Medicine Hannover
A/chicken/Emirates/R66/2002 (H9N2) LPAI
Friedrich-Löffler-Institut, Greifswald, In- sel Riems
2.8 Media
2.8.1 EMEM (Eagle’s minimal essential medium), pH 7.0
EMEM powder (GIBCO BRL) 9.6 g/l
NaHCO3 2.2 g/l
Penicillin G 0.06 g/l
Streptomycin-Sulfate 0.05 g/l
Non-essential aminoacids
2.8.2 DMEM (Dulbecco’s minimal essential medium), pH 6.9
DMEM Powder 13.53 g/l
NaHCO3 2.2 g/l
2.8.3 HAM’s F12, pH 7.0 - 7.5
Ham’s F12 with L-Glutamine PAA, Pasching, Austria
ready to use
2.8.4 CLEC213 Medium, pH 6.9
DMEM + HAM’s F12 1:2
FCS 10%
BSA 3%
Insulin, Transferrin, Selenium 1%
Pen/Strep 1%
EGF 0,25%
2.8.5 Freezing medium
DMEM / EMEM
+ FCS 10%
Glycerol (sterile) 10%
2.8.6 LB-Medium, pH 7.7
Tryptone 10 g/l
Yeast extract 5 g/l
Sodium chloride 10 g/l
2.9 Buffers and solutions
Anode buffer I 1M Tris 300 ml
pH 9.0 (with HCl) Ethanol 200 ml
Aqua bidest. ad 1 l
Anode buffer II 1M Tris 25 ml
pH 7.4 (with HCl) Ethanol 200 ml
Aqua bidest. ad 1 l
Cathode buffer 1M Tris 25 ml
pH 9.4 (with HCl) Aminocaproic acid 5.25 g
Ethanol 200 ml
Aqua bidest. ad 1 l
Crystal violet 37% Formalin 270 ml
Aqua bidest. 730 ml
crystal violet 1 g
DAPI staining solution DAPI 10µg
Ethanol ad 10 ml
Ethidium bromide Ethidium bromide 1 g
staining solution TAE buffer ad 100 ml
Mowiol Mowiol 2.5 g
Glycerol 6 g
Aqua bidest. 6 ml
DABCO 2.5%
PBS NaCl 8 g
pH 7.5 KCl 0,2 g
Na2HPO4 1.15 g
KH2PO4 0.12 g
MgCl2 · 6 H2O 0.1 g CaCl2 · 2 H2O 0.132 g
Aqua bidest. ad 1 l
PBSM NaCl 8 g
pH 7.5 KCl 6 0.2 g
Na2HPO4 1.15 g
KH2PO4 0.2 g
Aqua bidest. ad 1 l
PBSM 0.1% Tween PBSM 2 l
Tween 20 2 ml
10x SDS running buffer SDS 10 g
pH 8.4 Tris 30 g
Glycine 144 g
Aqua bidest. ad 1 l
2x SDS sample buffer 0.5 M Tris/HCl, pH 6.8 10 ml
10% SDS 20 ml
Glycine 10 ml
2% Bromophenol blue 1 ml
Aqua bidest. ad 50 ml
Separating gel (8%) H2O 2.3 ml
for SDS PAGE Acrylamide solution 30% 1.3 ml
1M Tris/HCl, pH 8.8 1.3 ml 10% SDS (in H2O) 50µl 10% APS (in H2O) 50µl
TEMED 8µl
Stacking gel H2O 3.4 ml
for SDS PAGE Acrylamide 30% 0.83 ml
1M Tris/HCl, pH 6.8 0.63 ml 10% SDS (in H2O) 50µl 10% APS (in H2O) 50µl
TEMED 50µl
TAE buffer Tris 40 mM
pH 8.0 Sodium acetate 20 mM
EDTA 2 mM
TBE buffer Tris 10 mM
pH 8.0 Boric acid 89 mM
EDTA 2 mM
Trypsin 0.125% NaCl 8.0 g
pH 7.0 KCl 0.20 g
Na2HPO4 · 12 H2O 2.31 g KH2PO4 · 2 H2O 0.20 g
CaCl2 0.13 g
MgSO4 · 7 H2O 0.10 g
Trypsin 1.25 g
Versen (EDTA) 1.25 g
Streptomycin 0.05 g
Penicillin 0.06 g
Aqua bidest. ad 1 l
2x HBS Buffer NaCl 280 mM (1.63 g)
Na2HPO−4 · 7H20 1.5 mM (0.2 g) HEPES 50 mM (1.2 g)
dH2O 100 ml
CaCl2 CaCl2 · 2H2O 2.5 M (18.375 g)
dH2O 50 ml
his-tag Binding Buffer 20 mM Sodium-Phosphate (Na3PO4 7.6024 g
0.5 M NaCl 29.22 g
30 mM Imidazol 2.04 g
pH 7.4 dH2O 1 l
his-tag Elution Buffer 20 mM Sodium-Phosphate (Na3PO4 3.8012 g
0.5 M NaCl 14.61 g
500 mM Imidazol 17.02 g
pH 7.4 dH2O 500 ml
his-tag Stripping Buffer 20 mM Sodium-Phosphate (Na3PO4 3.8012 g
0.5 M NaCl 14.61 g
50 mM EDTA 7.306 g
pH 7.4 dH2O 500 ml
Elution Buffer, Protein A sodium citrate 0.1 M (14.705 g)
dH2O 500 ml
Neutralization Buffer, Protein A Tris 0,1 M, pH 9
2.10 Synthetic Oligonucleotides
Oligonucleotides (see Table 2.10) were synthesized by Sigma-Aldrich Chemie GmbH, Steinheim and used for PCR in a 10µM concentration.
Table 2.9: Synthetic oligonucleotides
No. Name Sequence 5’→3’
1 Turkey-HA-fo TTT TTT AAT TAA ATG GAG AAA ATA GTG CTT CTT CTT GCA ATA
2 H5_PacI_AS AAA TTA ATT AAA GTT CCT ATT GAT TCC A 3 Turkey-Bp480-S AGA AAT GTG GTA TGG CTT AT
4 Turkey-Bp980-S AGT CCT TGC TAC TGG GCT CA 5 H7N7-HA-Bp480 CTC CTG TCA AAC ACA GAC AA 6 H7N7-HA-Bp980 AGG AAT GAA GAA TGT TCC CG
8 H9_PacI_S TTT TTA ATT AAA TGG AGA CAA TAT CAC TG
9 H9_PacI_AS AAA ATT AAT TAA GTA AGT TCC CTC AGA TTC CAA 10 H9stop_PacI_AS AAA ATT AAT TAA TTA GTA AGT TCC CTC AGA TTC
CAA
11 H9_Bp800s TCT TTC AGG AGG GAG CCA TG
