Characterization of the binding properties of the Avian Coronavirus

Transcription

University of Veterinary Medicine Hannover
Institute of Virology
Characterization of the binding properties of the
Avian Coronavirus spike protein
THESIS
Submitted in partial fulfillment of the requirements for the degree
-Doctor of Philosophy(PhD)
awarded by the University of Veterinary Medicine Hannover
by
Martina Hesse
(Fritzlar)
Hannover, Germany 2015
Supervisor:
Prof. Dr. Georg Herrler
Christine Winter, PhD
Supervision group:
Prof. Dr. Silke Rautenschlein, PhD
Prof. Dr. Wolfgang Garten
1st Evaluation:
University of Veterinary Medicine Hannover, Foundation
Prof. Dr. Silke Rautenschlein, PhD
Clinic for Poultry Institute
2nd Evaluation:
Prof. Dr. Heinz-Jürgen Thiel
Institute for Medical Virology
Justus Liebig University Giessen
Date of oral exam:
2015-05-07
2
____________________________________________________________________
To my loved ones
To my loved ones
h
Contents
................................................................................................................................... 0
List of figures ............................................................................................................. I
List of tables ............................................................................................................... II
List of Abbreviations .................................................................................................. III
Abstract .................................................................................................................... VII
Zusammenfassung .................................................................................................... IX
1
Introduction .......................................................................................................... 1
1.1
1.1.1
Epidemiology and diseases .................................................................... 1
1.1.2
History ..................................................................................................... 1
1.1.3
Taxonomy ............................................................................................... 2
1.1.4
Genome .................................................................................................. 3
1.1.5
Viral life cycle .......................................................................................... 4
1.1.6
Morphology ............................................................................................. 8
1.1.7
Attachment .............................................................................................. 9
1.2
The coronavirus spike protein ..................................................................... 10
1.2.1
Variability of the S1-subunit .................................................................. 11
1.2.2
The spike protein as host range determinant ........................................ 12
1.2.3
The spike protein as main immunogenic agent of coronaviruses.......... 13
1.2.4
The receptor-binding domains on the spike protein .............................. 14
1.3
2
Coronaviruses ............................................................................................... 1
IBV............................................................................................................... 14
1.3.1
Classification ......................................................................................... 14
1.3.2
Epidemiology ........................................................................................ 15
1.3.3
Pathogenesis and clinical signs ............................................................ 16
1.3.4
Control strategies .................................................................................. 16
Material .............................................................................................................. 19
2.1
4.1 Cell lines, primary cell cultures and tissue cultures ............................... 19
2.2
SPF eggs ..................................................................................................... 19
II
____________________________________________________________________
2.3
Chicken trachea and kidney ........................................................................ 19
2.4
Chicken erythrocytes ................................................................................... 19
2.5
Virus ............................................................................................................ 20
2.6
Bacteria ....................................................................................................... 20
2.7
Cell and tissue culture media ...................................................................... 20
2.7.1
DMEM (Dulbecco´s Minimal Essential Medium), pH 6.9....................... 20
2.7.2
EMEM (Eagle´s Minimal Essential Medium), pH 7.0............................. 20
2.7.3
Medium 199 Earle’s .............................................................................. 20
2.7.4
Medium 199 Hanks’ .............................................................................. 20
2.7.5
Fetale calve serum (FCS) ..................................................................... 20
2.7.6
Freezing Medium .................................................................................. 20
2.7.7
Trypsin/EDTA........................................................................................ 21
2.8
Bacteria media ............................................................................................ 21
2.8.1
Luria-Bertani (LB) media ....................................................................... 21
2.8.2
LB agar ................................................................................................. 21
2.9
Buffers and solutions ................................................................................... 21
2.9.1
Anode buffer I, pH 9.0 ........................................................................... 21
2.9.2
Anode buffer II, pH 7.4 .......................................................................... 22
2.9.3
Cathode buffer, pH 9.0.......................................................................... 22
2.9.4
DAPI staining solution ........................................................................... 22
2.9.5
Ethidium bromide staining solution ....................................................... 22
2.9.6
Mowiol ................................................................................................... 22
2.9.7
Paraformaldehyde (PFA), pH 7.4 .......................................................... 22
2.9.8 Phosphate buffered saline, supplemented with Calcium and magnesium
(PBSM), pH 7.5 .................................................................................................. 23
2.9.9
Phosphate buffered saline (PBS), pH 7.5 ............................................. 23
2.9.10
2.10
FACS-buffer....................................................................................... 23
Plasmids................................................................................................... 23
2.10.1
Expression plasmids .......................................................................... 23
2.10.2
cDNA Constructs ............................................................................... 24
2.10.3
Oligonucleotides ................................................................................ 25
2.11
3
Enzymes .................................................................................................. 26
2.11.1
Restriction enzymes .......................................................................... 26
2.11.2
Other enzymes .................................................................................. 26
2.12
Antibodies ................................................................................................ 26
2.13
Kits ........................................................................................................... 27
2.14
Chemicals ................................................................................................ 27
2.15
Other substances ..................................................................................... 29
2.16
Equipment ................................................................................................ 30
2.16.1
Agarose gel electrophoresis .............................................................. 30
2.16.2
Photometer ........................................................................................ 30
2.16.3
Bacteria culture .................................................................................. 30
2.16.4
Cell culture......................................................................................... 30
2.16.5
Centrifuges ........................................................................................ 30
2.16.6
Magnetic stirrer .................................................................................. 31
2.16.7
Microscope and image processing software ...................................... 31
2.16.8
PCR ................................................................................................... 31
2.16.9
Pipettes and pipette helpers .............................................................. 31
2.16.10
Reaction tubes and sterile filters ........................................................ 31
2.16.11
Centrifugal Filters .............................................................................. 32
2.16.12
Flow cytometry .................................................................................. 32
2.16.13
Safety cabinettes ............................................................................... 32
2.16.14
SDS-PAGE and Semi-dry Western-Blot ............................................ 32
2.16.15
Vortex ................................................................................................ 33
2.16.16
Scales ................................................................................................ 33
2.16.17
Homogenisator .................................................................................. 33
2.16.18
Water bath ......................................................................................... 33
2.16.19
Cryostat ............................................................................................. 33
Methods ............................................................................................................. 35
3.1
Cell and organ culture technique ................................................................. 35
3.1.1
Mycoplasma detection test ................................................................... 35
3.1.2
Cryoconservation .................................................................................. 35
IV
____________________________________________________________________
3.1.3
Preparation of primary embryonic chicken kidney cells ........................ 35
3.1.4
Preparation of primary chicken trachea cell cultures............................. 36
3.1.5
Preparation of tracheal organ culture .................................................... 37
3.2
Preparation of virus stocks .......................................................................... 37
3.3
Preparation of Cryosections ........................................................................ 37
3.4
Molecular Biology ........................................................................................ 38
3.4.1
Polymerase chain reaction .................................................................... 38
3.4.2
Agarose gel electrophoresis ................................................................. 40
3.4.3
DNA gel extraction ................................................................................ 40
3.4.4
Hybridization of DNA fragments ............................................................ 40
3.4.5
Restriction Enzyme Digest .................................................................... 41
3.4.6
PCR Purification.................................................................................... 41
3.4.7
Dephosphorylation ................................................................................ 42
3.4.8
Ligation ................................................................................................. 42
3.4.9
Measurement of DNA-concentration ..................................................... 42
3.4.10
Heat shock transformation of bacteria ............................................... 42
3.4.11
Colony-PCR ....................................................................................... 43
3.4.12
DNA preparation ................................................................................ 43
3.4.13
Sequence Analysis ............................................................................ 44
3.5
Transient transfection of mammalian cells .................................................. 44
3.6
Neuraminidase treatment ............................................................................ 44
3.7
Protease-treatment ...................................................................................... 45
3.7.1
Trypsin-treatment .................................................................................. 45
3.7.2
Pronase-treatment ................................................................................ 45
3.8
Infection of continuous cell lines with IBV .................................................... 45
3.9
Immunofluorescence ................................................................................... 46
3.9.1
Cell culture ............................................................................................ 46
3.9.2
Cryosections ......................................................................................... 46
3.9.3
Image analysis ...................................................................................... 47
3.10
FACS........................................................................................................ 47
3.11
Protein biochemistry ................................................................................. 47
4
3.11.1
Centrifugation of Cell culture supernatants for concentration ............ 47
3.11.2
Overlay binding assay ....................................................................... 48
3.11.3
SDS-PAGE ........................................................................................ 48
3.11.4
Western Blot ...................................................................................... 49
Results ............................................................................................................... 51
4.1
Binding properties of the IBV spike protein.................................................. 51
4.1.1
Structure and expression of chimeric soluble proteins .......................... 51
4.1.2
Detection of IBVS1Fc binding to different cells of chicken origin .......... 52
4.1.3
Analysis of IBVS1Fc binding to chicken primary cells in FACS analysis54
4.1.4
Binding of the IBV spike protein is sialic acid-dependent ...................... 56
4.1.5
The IBV spike is able to bind to permanent cell cultures....................... 58
4.1.6
The IBV strain B1648 does hardly infect permanent cell lines .............. 59
4.2
Analysis of the sialic acid binding domain within the IBV S1 ....................... 59
4.2.1 Deletion of 42 AA near the N-terminus of the spike protein results in a
reduced binding of the IBV spike protein............................................................ 59
4.2.2
The residual binding of the Del D mutant is not sialic acid dependent .. 61
4.2.3 Analysis of shorter amino acid sequences responsible for the sialic acid
binding capacity ................................................................................................. 64
4.2.4
4.3
5
Binding abilities of alanine mutants of the IBVS1Fc construct .............. 67
No evidence for a protein binding property of the IBV spike protein ............ 69
4.3.1
Overlay ................................................................................................. 69
4.3.2
Binding of different coronavirus spike proteins to protease-treated cells ..
.............................................................................................................. 70
Discussion.......................................................................................................... 73
5.1
Assessment of the binding characteristics of the construct IBVS1Fc .......... 73
5.2
Sialic acid dependence of IBVS1Fc binding ................................................ 76
5.3
Analysis of the sialic acid binding domain of IBVS1Fc ................................ 76
5.3.1
Assessment of mutant Del D-S1Fc ....................................................... 76
5.3.2 Identification of important sequences and single amino acids for
attachment within domain D ............................................................................... 78
5.4 Possibility of involvement of further main- or co-receptors in IBV host cell
attachment ............................................................................................................ 80
VI
____________________________________________________________________
5.5
Binding of the IBV spike to continuous cell lines ......................................... 81
5.6
Further factors possibly determining host range of IBV ............................... 82
5.7
Conclusion................................................................................................... 83
6
References......................................................................................................... 85
7
Appendix ............................................................................................................ 99
8
7.1
Amino acids ................................................................................................. 99
7.2
Sequences .................................................................................................. 99
7.2.1
B1648S1 nucleotide sequence ............................................................. 99
7.2.2
B1648S amino acid sequence ............................................................ 100
7.2.3
Nucleotide sequence of the linking sequence and of the Fc-tag ......... 100
7.2.4
Amino acid sequence of the linking part and the Fc-tag ..................... 101
7.2.5
Nucleotide sequences of different B1648S1 deletion mutants ............ 101
7.2.6
Amino acid sequences of different B1648S1 deletion mutants ........... 102
7.2.7
Alignment of the N-terminus of B1648S1, M41S1 and BeaudetteS1 .. 102
Acknowledgements .......................................................................................... 103
I
List of figures
List of figures
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16
Figure 17
Figure 18
Figure 19
Figure 20
Figure 21
Figure 22
Figure 23
Figure 24
Figure 25
The genome organization of IBV and the nested set of
subgenomic mRNAs produced during replication.
Coronavirus replication
Coronavirus virion
Schematic drawing of the IBV spike protein.
Detection of soluble chimeric proteins derived from
coronaviruses IBV-B1648 and SARS-CoV
Binding of IBVS1Fc to chicken tissues.
Binding of IBVS1Fc to primary CEKC.
Quantification of the binding capacity of IBVS1Fc to
CEKC in FACS analysis.
tracheal cells in FACS-Analysis.
chicken erythrocytes in FACS Analysis.
Reduction of IBVS1Fc binding to cryosections of chicken
kidneys after neuraminidase treatment.
Reduction of IBVS1Fc binding to neuraminidase-treated
CEKC in FACS-analysis.
Binding of IBVS1Fc to permanent cell lines.
Infection of permanent cell line with B1648.
Schematic drawing the B1648 deletion mutant Del DS1Fc.
Detection of soluble chimeric proteins derived from IBVB1648 deletion mutant Del D.
Quantification of the binding capacity to CEKC of the
deletion mutant Del D-S1Fc compared to the parental
protein in FACS analysis.
Quantification of the reduction of IBVS1Fc binding to
CEKC after Neuraminidase-treatment in FACS-analysis.
Binding of IBVS1Fc and Del D-S1Fc to neuraminidasetreated and untreated CEKC cell cultures.
Binding of IBVS1Fc and Del D-S1Fc to neuraminidasetreated and untreated cryosections of chicken trachea.
Schematic drawing of IBVS1Fc deletion mutants.
Detection of different IBVS1Fc deletion mutants
Quantification of the reduction of different IBVS1Fc
deletion mutants’ binding to CEKC in FACS-analysis.
Quantification of the reduction of three further IBVS1Fc
deletion mutants’ binding to CEKC in FACS-analysis.
Alanine exchange mutants of the IBVS1Fc chimeric
4
7
8
51
52
53
54
55
55
56
57
57
58
59
60
60
61
62
63
64
65
66
66
67
67
List of tables
II
____________________________________________________________________
Figure 26
Figure 27
Figure 28
Figure 29
Figure 30
protein.
Detection of seven alanine exchange mutants in
supernatants of transfected BHK21 cells.
Quantification of the reduction of different alanine
exchange mutants’ binding capacities to CEKC in
FACS-analysis.
Search for cellular receptors with soluble proteins.
Quantification of the reduction of different coronavirus
spike proteins’ binding capacity to target cells after
trypsin-treatment in FACS-analysis.
Quantification of the reduction of different coronavirus
spike proteins’ binding capacity to target cells after
pronase-treatment in FACS-analysis.
68
69
70
71
72
List of tables
Table 1
Table 2
Table 3
Immortalised cell lines
Primary cell and tissue cultures
Antibodies
10
10
26
III
of figures
List ofList
Abbreviations
List of Abbreviations
A
Ampere
AA
Amino acid
AvCoV
Avian Coronavirus
ACE2
Angiotensin Converting Enzyme 2
APN
Aminopeptidase N
Bp
Basepairs
BCA
Bicinchoninic acid
BHK
Baby hamster kidney
BSA
Bovine serum albumine
cDNA
Complementary DNA
CEKC
Chicken kidney cells
CO2
Carbon dioxide
CoV
Coronavirus
C-terminal
COOH terminus
Cy3
Indocarbocyanine
DAPI
4’,6’-Diamidino-2-phenylindole
DEPC
Diethylpyrocarbonat
DMEM
Dulbecco’s modified Eagle medium
DNA
Desoxyribonucleic acid
dNTP
Desoxynucleotide
Dpi
days post infection
DTSSP
3,3’-dithiobis[sulfosuccinimidylpropionat]
et al.
et alii (and others)
E. coli
Escherichia coli
EDTA
Ethylenediaminetetraacetic acid
EMEM
Eagle´s Modified Essential Medium
ER
Endoplamatic reticulum
ERGIC
Endoplasmatic reticulum Golgi
intermediate compartment
IV
____________________________________________________________________
FACS
Fluorescence activated cell sorting
FCoV
Feline Coronavirus
FCS
Fetal Calf Serum
FFU
Focus-forming units
Fig.
Figure
FITC
Fluoresceine isothiocyanate
g
Gramm or gravitational force
h
hour
HCoV
Human Coronavirus
HI
Hemagglutination inhibition, -ing
Hpi
hours post infection
HRP
Horse raddish peroxidase
IBV
Infectious Bronchitis Virus
IgG
Immunoglobulin G
IF
Immunofluorescence
Kb
Kilobases
kDa
Kilodalton
L
Liter
LB
Luria Bertani
M
Molarity, - molar
mA
Milliampere
MHV
Mouse hepatitis virus
MCS
Multiple cloning site
MDCK
Madin-Darby canine kidney
MERS
Middle East respiratory syndrome
Mg
Milligramm
MHV
Murine Hepatitis Virus
Min
Minute
mL
Milliliter
MOI
Multiplicity of infection
MW
Molecular weight
V
List ofList
Abbreviations
of figures
N
Nano
NA
Neuraminidase
N-terminal
NH2 terminus
ORF
Open reading frame
PBS
Phosphate buffered saline
PCLS
Precision-cut lung slices
PEI
Polyethylenimine
Pen/Strep
Penicillin and Streptomycin
PFA
Paraformaldehyde
pH
potentia Hydrogenii
TOC
Tracheal organ culture
TSR
transcription regulating sequences
S
Spike protein
S1
N-terminal subunit of the spike protein
SARS
Severe acute respiratory syndrome
SPF
Specific pathogen free
ST
Swine testis
U
Unit
V
Volt
VN
Virus neutralization, -ing
°C
degree Celsius
α
anti (antibodies) or alpha (sialic acids)
µ
micro (gram or liter for example)
VI
____________________________________________________________________
VII
List of Abstract
figures
Abstract
Characterization of the binding properties of the avian coronavirus
spike protein
Martina Hesse
The avian coronavirus (AvCoV) infectious bronchitis virus (IBV) is a pathogen
affecting the respiratory and the genito-urinary tract of birds, mainly of chickens
(gallus gallus) and other galliforme birds. For all viruses the attachment to host cells
is the first and an important step in the viral life cycle. The binding ability of
coronaviruses resides in the S1-subunit of their spike protein and in the case of IBV
host cell binding has been demonstrated to be dependent on the presence of sialic
acids on the cellular surface.
In the present study chimeric soluble S1-subunits of the IBV spike protein connected
to human IgG-Fc-tags are prepared and used as tools to analyze the binding
properties of the S1-domain of IBV in more detail. These chimeric proteins are
expressed in continuous cell lines, concentrated by centrifugation through filter
columns and can easily be detected with fluorescence labeled anti-human IgGantibodies. Out of the huge variety of different IBV strains, the spike protein of the
B1648 strain, a field isolate from Belgium, was chosen for this study. It has not
acquired additional binding affinities by passages in non-avian cells like the
Beaudette strain and it is pathogenic in vivo. In this work, two questions were
addressed regarding the IBV spike binding characteristics. First, it was examined
where the sialic acid binding site is located within the S1 subunit of the spike protein.
Second, it was analyzed, if the IBV spike protein harbors any binding properties apart
from sugar binding.
For localization of the sugar binding domain, stretches of amino acids in the
sequence of the S1 domain were deleted to see if the binding ability is impaired by
these changes. The importance of 42 amino acids located near the N-terminus of the
molecule for binding was demonstrated. In a second step, this range was narrowed
Abstract
VIII
____________________________________________________________________
down to seven amino acids. The residual binding, that all deletion mutants exhibited,
was not sialic acid-dependent.
To analyze if a protein receptor is involved in the IBV attachment, the soluble spike
proteins were used to search for interacting cellular surface proteins and protease
treatment of cells was applied to analyze the role of proteins in viral binding. The
former approach did not result in the identification of specific interactions. The latter
revealed that spike protein binding to protease-treated cells was not reduced, arguing
against the involvement of a specific protein receptor in host cell binding of IBV.
Moreover, the ability of the soluble spike protein to bind to continuous cell lines was
tested. Since spike protein binding is viewed as a main factor determining the host
range, it was unexpected to see that the construct was able to bind to cells, which
were hardly susceptible to infection by the corresponding IB virus strain B1648.
IX
Zusammenfassung
List of figures
Zusammenfassung
Charakterisierung der Bindungseigenschaften des Spike Proteins
des Aviären Coronavirus
Martina Hesse
Das aviäre Coronavirus (AvCoV) Infektiöses Bronchitis-Virus ist ein Erreger, der den
Respirations- und den Urogenitaltrakt von Vögeln, hauptsächlich vom Huhn (gallus
gallus) und anderen Galliformen, befällt. Für alle Viren ist die Bindung an Wirtszellen
der erste und ein wichtiger Schritt im Replikationszyklus. Für die Bindungsfähigkeit
der Coronaviren ist die S1-Untereinheit des Spike-Proteins verantwortlich. Im Fall
von IBV hängt die Bindung an Wirtszellen von der Präsenz von Sialinsäuren auf der
Zelloberfläche ab.
In der vorliegenden Studie wurden chimäre lösliche S1-Untereinheiten, versehen mit
einem humanen IgG-Fc-Marker-Peptid, hergestellt und als Werkzeuge zur
detaillierteren Untersuchung der Bindungseigenschaften der S1-Domäne von IBV,
benutzt. Diese chimären Proteine können in permanenten Zelllinien exprimiert, durch
Zentrifugation ankonzentriert und mithilfe Fluoreszenz-markierter anti-human-IgGAntikörper leicht nachgewiesen werden. Aus den vielen verschiedenen IBVStämmen wurde für diese Untersuchungen der B1648-Stamm für die Erzeugung der
löslichen Proteine ausgewählt. Dabei handelt es sich um einen Feldstamm, der in
Belgien isoliert wurde. Dieser Stamm weist keine zusätzliche, durch Passage in
nichtaviären Zellen gewonnene, Bindungseigenschaft auf, wie beispielsweise der
Beaudette-Stamm. Außerdem verhält sich dieser Stamm in vivo pathogen. In dieser
Arbeit haben wir uns zwei Fragen bezüglich der Bindungseigenschaften des IBV
Spike-Proteins gewidmet. Einerseits wurde untersucht, wo auf dem Spike-Protein die
rezeptorbindende Domäne lokalisiert ist. Andererseits wurde analysiert, ob das IBV
Spike-Protein weitere Bindungseigenschaften, außer der Fähigkeit an Zucker zu
binden, besitzt.
Zur
Lokalisationsbestimmung
der
zuckerbindenden
Domäne
wurden
einige
Aminosäuren in der Sequenz der S1-Untereinheit deletiert, um so festzustellen, ob
Zusammenfassung
X
____________________________________________________________________
durch diese Deletionen die Bindungsfähigkeit des Proteins herabgesetzt wird. Dabei
konnte die Bedeutung von 42 Aminosäuren, die nahe am N-Terminus des Proteins
lokalisiert sind, für die Bindung gezeigt werden. In einem zweiten Schritt wurde
dieser Bereich auf sieben besonders wichtige Aminosäuren eingegrenzt. Die dabei
verbleibende Restbindung, die bei allen Mutanten auftrat, war nicht abhängig von
Sialinsäuren.
Um zu untersuchen, ob ein Proteinrezeptor an der Bindung von IBV beteiligt ist,
wurden
die
löslichen
Spike-Proteine
zur
Ermittlung
von
Interaktionen
mit
Zelloberflächenproteinen eingesetzt. Protease-Behandlung von IBV-empfänglichen
Zellen wurde angewandt, um generell eine Beteiligung von Zelloberflächenproteinen
bei der Virusbindung zu untersuchen. Der erste Ansatz führte nicht zur Identifizierung
spezifischer Interaktionspartner. Der zweite Ansatz konnte zeigen, dass die Bindung
der Spike-Proteine an proteasebehandelte Zellen nicht herabgesetzt war, was gegen
die Beteiligung eines spezifischen Proteinrezeptors spricht.
Schließlich wurde auch die Fähigkeit der löslichen Proteine, an permanente Zelllinien
zu binden, untersucht. Da das Bindungsvermögen der Spike-Proteine als ein
Hauptfaktor für die Festlegung des Wirtsspektrums angesehen wird, überraschte es,
dass die Konstrukte in der Lage waren, an Zellen zu binden, die in unseren
Experimenten kaum durch das korrespondierende Virus B1648 infiziert werden
konnten.
1
Introduction
1 Introduction
1.1 Coronaviruses
1.1.1 Epidemiology and diseases
Coronaviruses infect a wide range of vertebrates including mammals, reptiles, birds
and fish and cause a broad spectrum of different diseases. Humans may also be
affected. Remarkable, however, is the fact that over the last years coronaviruses
have demonstrated their potential to cross species barriers (VIJGEN et al. 2005; JIN
et al. 2007; ALEKSEEV et al. 2008; CHU et al. 2011).
Coronaviruses are commonly associated with digestive symptoms such as diarrhea
or respiratory signs like the common cold. Severe respiratory diseases can be
induced by the severe acute respiratory syndrome-coronavirus- (SARS-CoV) or the
middle east respiratory syndrome-coronavirus- (MERS-CoV)-infection in humans. In
birds, mainly chickens, the avian coronavirus (AvCoV), also known as infectious
bronchitis virus (IBV), is also associated with respiratory symptoms but in addition it
may affect the kidneys and the oviduct. More rarely, hepatitis, peritonitis and
neurological disease may be observed upon infection with coronaviruses. The course
of disease can be either acute or persistent.
1.1.2 History
The infectious bronchitis virus (IBV) was described in 1932 as the first coronavirus. It
was then recognized as the cause of a respiratory disease in chickens, that could be
transmitted via contact exposure, bronchial exudates and via cloaca (SCHALK a.
HAWN 1931; HUDSON 1932).
Despite the discovery of several related viruses which are pathogenic for humans,
coronaviruses have been regarded to be mainly of veterinary interest throughout the
20th century (BELOUZARD et al. 2012). All human coronaviruses that were known
during that time were associated with only mild respiratory diseases (VAN DER
HOEK 2007). In contrast to this, coronaviruses have been known to be the cause of
Introduction
2
____________________________________________________________________
much more severe problems in veterinary contexts. IBV for instance has caused
tremendous economic losses for the poultry industry for decades (CAVANAGH
2007). TGEV, the transmissible gastroenteritis virus, was responsible for mortality
rates in piglets of up to 100% before it was replaced by a naturally occurring variant
that only induces mild respiratory disease (PENSAERT et al. 1986). With the
appearance of the severe acute respiratory syndrome (SARS) in a small scale
epidemic in 2002 and 2003, which was caused by the SARS coronavirus (SARSCoV), the general interest in coronavirus research was strongly increased (PEIRIS et
al. 2003; CHERRY a. KROGSTAD 2004; ZHONG a. WONG 2004; BELOUZARD et
al. 2012). The SARS-CoV infected about 8000 people and killed approximately 10%
of the diseased persons, before its spread could be stopped by aggressive public
health intervention strategies. It was shown that SARS originated from an animal
reservoir and crossed the species barrier, indicating the potential threat of animal
coronaviruses for the human population (CHERRY a. KROGSTAD 2004; LI et al.
2005b; BELOUZARD et al. 2012). This notion was strengthened by the appearance
of the middle east respiratory syndrome in 2012 in Saudi-Arabia, another severe
respiratory disease caused by a coronavirus (BERMINGHAM et al. 2012; ZAKI et al.
2012).
The increase in coronavirus research after the SARS-outbreak in 2002/2003 led to a
large increase of knowledge regarding coronavirus phylogeny. A first common
ancestor of coronaviruses, which might have infected either bats or birds, evolved
into coronaviruses infecting bats and birds. These viruses were the ancestors for
alpha and beta coronaviruses, and in case of the avian virus for gamma and delta
coronaviruses. (WOO et al. 2012).
1.1.3 Taxonomy
The discovery of IBV in 1932 was followed over the next decades by the discovery of
several related viruses and led to the recognition of the new viral family
Coronaviridae by the international committee on the taxonomy of viruses in 1975
(INTERNATIONAL COMMITTEE ON THE TAXONOMY OF VIRUSES 1975), first
based on a characteristic morphology (TYRRELL et al. 1975; SIDDELL et al. 1983).
3
Introduction
In 1996 the genera Coronavirus and Torovirus within the family Coronaviridae were
established and the families Arteri- and Coronaviridae were classified within the order
Nidovirales (PRINGLE 1996). Today, the viral family Coronaviridae belongs to the
order Nidovirales
together with
the families
Arteriviridae, Roniviridae
and
Mesoniviridae. Two subfamilies, the Coronavirinae and the Torovirinae are
distinguished within this coronavirus family. Since in 2008 the nomenclature of
Coronaviridae was revised, the former genus Coronavirus has been replaced by the
three genera Alpha-, Beta- and Gammacoronavirus, each of them including several
species (DE GROOT 2008). In the same year the new species AvCoV was created.
Two years later, the genus Deltacoronavirus with three species affecting different bird
species was established (DE GROOT 2010).
1.1.4 Genome
The genome organization of coronaviruses has been reviewed by de Vries et al.
(1997). Coronaviruses possess a single-stranded RNA genome of positive
orientation. It is the largest viral RNA genome currently known. The genome is not
segmented and comprises between 27000 and 32000 nucleotides. At the 5’ end a
cap structure is present and the genome possesses between 6 and 10 open reading
frames (ORFs). The first ORFs, ORF 1a and 1b, are overlapping. They code for the
proteins of the replicase/transcription complex. Subsequently ORFs coding for
varying nonstructural proteins follow. The 3’ part of the genome comprises the genes
for the structural proteins in the conserved order (HE)-S-E-M-N (see Fig. 1). Between
the genes for the structural proteins, genes for accessory proteins are interspersed.
The name of the order Nidovirales refers to the unique genome organization and
expression shared by all members and is a reference to the Latin word nidus
meaning “nest”. All members of this order produce a 3' co-terminal nested set of
subgenomic mRNAs during replication (see 1.1.5 Viral life cycle).
Introduction
4
____________________________________________________________________
Fig. 1: The genome organization of IBV (Panel A) and the nested set of
subgenomic mRNAs produced during replication (Panel B). Only mRNAs for
translation of structural proteins are depicted; Leader sequence indicated by grey
ellipse; TRS=transcription regulating sequence, indicated by black circle;
UTR=untranslated region, indicated by black star, poly A-tail indicated by AA(A)n
1.1.5 Viral life cycle
After attachment of enveloped viral particles to host cells, the fusion of the viral
envelope with cellular membranes is initiated which involves a conformational
change of the attachment protein. In case of some coronaviruses, fusion seems to be
mediated by endosomal proteases (SIMMONS et al. 2005; HUANG et al. 2006; QIU
et al. 2006). After this fusion event, the nucleocapsid is released into the cytoplasm
where the RNA replication and the synthesis of viral proteins take place. The
translation begins at ORF 1a and may be continued after a ribosomal frameshift with
the ORF 1ab (WEISS et al. 1994; NAMY et al. 2006). The resulting polyproteins pp1a
and pp1ab undergo cotranslational proteolytic processing and then form the
membrane-bound replication/transcription complexes. They are responsible for the
production of negative strand RNAs that serve as template for the further replication
steps, for the replication of progeny genomes and for the production of subgenomic
5
Introduction
mRNAs. Subgenomic mRNAs have the same 3’end and the same “leader” sequence
derived from the 5’end of the genome. They are a result of discontinuous
transcription, a process regulated by so-called transcription regulating sequences
(TRS) (D. SAWICKI et al. 2001; S. G. SAWICKI a. SAWICKI 2005) located at the 3’
end of the genome and at the beginning of the ORFs encoding the structural and
accessory proteins. That means that the transcription starts in front of one of these
ORFs and continues from there through the subsequent ORFs to a poly-A-tail at the
3’end of the genome. At last, the leader sequence is added. The minus strands with
subgenomic length that are thus produced, serve as template for the final positive
strand subgenomic mRNAs (S. G. SAWICKI a. SAWICKI 2005), which as a
consequence of discontinuous transcription comprise different numbers of ORFs
(see Fig. 1) (DE VRIES et al. 1997). From each of the subgenomic mRNAs only the
first ORF is translated (S. G. SAWICKI et al. 2007). Assembly of the translated
structural proteins with the encapsidated genomes occurs in the ER-Golgi
intermediate compartment (ERGIC). Viral particles bud into the ERGIC-lumen and
are then transported in vesicles along the secretory pathway to the cellular surface
(TOOZE et al. 1984; DUNPHY a. ROTHMAN 1985; RISCO et al. 1998).
A peculiar feature of coronaviruses is the relatively low error rate during replication
compared to other RNA-viruses due to the presence of proofreading enzymes in this
group (Graham and Baric). For the SARS-CoV MINSKAIA et al. (2006) identified an
exoribonuclease (ExoN) encoded within the ORF1 nsp14 that acts on both ssRNAs
and dsRNAs in a 3′→5′ direction. Nsp14 shows homologies to cellular exonucleases.
Viruses containing mutations within the active site of the enzyme were correlated
with reduced viable viral progeny, reduced genome replication and defects in
synthesis of subgenomic RNAs (MINSKAIA et al. 2006) The proofreading function of
nsp14 has further been evidenced by ECKERLE et al. (2007) who could
demonstrate, that MHV mutant viruses with a defective nsp14 are 15-fold more errorprone than wild type virus. This corresponds to error rates reported for RNAdependent RNA-polymerases from other RNA virus families. From these data BARR
and FEARNS (2010) concluded that nsp14 is responsible for removal and repair of
misincorporated nucleotides. Additionally, an endoribonuclease has been identified,
Introduction
6
____________________________________________________________________
the SARS-CoV nsp15 (BHARDWAJ et al. 2004) and active sites of this ribonuclease
are superimposable with an orthologous enzyme found in MHV (KANG et al. 2007). It
is assumed that for viruses it is necessary to keep the error rate below a certain
threshold, which still allows the production of sufficient viable progeny virus. That is
especially true for coronaviruses due to their extremely large genomes compared to
other RNA-viruses (BARR a. FEARNS 2010). This is supported by the fact that related
viral families like the Arteriviridae lack some of the proteins which confer increased
transcription fidelity (MINSKAIA et al. 2006). Nevertheless an error rate below the
assumed threshold seems to facilitate efficient replication in the complex
environment of an animal host. In fact, any given RNA-virus population consists not
of a single genotype but of different so called “quasispecies” (VIGNUZZI et al. 2006).
In situations of selective pressure, it may even be favorable to increase the mutation
rate further to render adaptation to new situations possible. Additionally, there have
been indications that coronaviruses appear to have the ability to induce directed
mutation rather than just increase the error rate stochastically (GRAHAM a. BARIC
2010).
7
Introduction
Fig. 2:"Coronavirus replication" by Crenim at English Wikipedia - Own work.
Licensed
under
CC
BY-SA
3.0
via
Wikimedia
Commons
http://commons.wikimedia.org/wiki/File:Coronavirus_replication.png#mediaviewer/Fil
e:Coronavirus_replication.png
1) Attachment 2) uptake of the virion and uncoating 3) synthesis of the viral RNA
polymerase, followed by synthesis of the negative RNA strand 4) replication of
genome and transcription of subgenomic mRNAs 5) assembly of N-protein with
genomic RNA, integration of M-, S-, and eventually HE-protein into the ERmembrane, budding into the ER-lumen 6) transport to the cellular surface
Introduction
8
____________________________________________________________________
1.1.6 Morphology
Fig. 3:„Coronavirus virion“ by Belouzard, et al http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397359/. Licensed under CC BY 3.0
via Wikimedia Commons http://commons.wikimedia.org/wiki/File:Coronavirus_virion.jpg#mediaviewer/File:Coro
navirus_virion.jpg
The virions are enveloped, pleomorphic particles with a size ranging from 80 to 200
nm. From the surface club-shaped projections protrude that in electron microscopy
remind of the solar corona and therefore gave the virus family its name (TYRRELL et
al. 1975; SIDDELL et al. 1983) These projections are the spike (S) proteins, which
are responsible for virus attachment to host cells and for fusion of the viral envelope
with the host cell membrane. Besides the spike protein, coronavirus particles feature
regularly three further structural proteins, namely the nucleocapsid- (N), the
membrane- (M) and the envelope- (E) protein. The N protein is associated with the
viral RNA and creates the helical symmetry of the nucleocapsid. It also plays an
important role in transcription processes (VERHEIJE et al. 2010; ZUNIGA et al.
2010). The M protein is a membrane protein that interacts both with the spike protein
and the nucleocapsid protein (OPSTELTEN et al. 1995b, a). It is thought to
orchestrate particle formation. In cryo-electron tomography, the viral membrane of
MHV revealed an unusual thickness (about twice of the norm) and made it possible
to distinguish an extra shell from the lipid bilayer (BARCENA et al. 2009). It was
proposed that this extra shell is formed by the C-terminal ends of the M-protein. For
TGEV, too, it has been suggested that the M-protein together with the N-protein
9
Introduction
forms a spherical core shell within the virus particle (RISCO et al. 1996). The function
of the E-protein could so far not be entirely clarified. However, since the E-protein
together with only the M-protein is able to form virus-like particles it seems to play a
role in viral assembly (VENNEMA et al. 1996). In addition to these four structural
proteins some betacoronaviruses also possess the hemagglutinin-esterase, the HEprotein. Like the neuraminidase of influenza A viruses, it is a receptor-destroying
enzyme (HERRLER et al. 1991).
1.1.7 Attachment
Although in rare cases coronaviruses seem to circumvent receptor binding entirely,
possibly by usage of highly fusogenic spike proteins (MCROY a. BARIC 2008;
MIURA et al. 2008), host cell attachment is usually the first and an important step in
the viral life cycle. In case of coronaviruses it is mediated by the spike protein. The
receptor varies not only within different coronavirus genera but also from strain to
strain (GRAHAM a. BARIC 2010).
While many coronaviruses bind to protein receptors, several have additionally the
ability to bind different sialic acids expressed on cellular surfaces or present in mucus
on epithelial surfaces. For some species, however, no protein receptor has been
identified yet and it seems possible that they solely rely on glycans as receptor
determinants.
For many alpha- and betacoronoaviruses binding to a single protein receptor seems
to be sufficient for host cell attachment. But for instance the beta-coronavirus SARSCoV binds to a cellular protein receptor, the angiotensin-converting enzyme (ACE-2)
(LI et al. 2003) and additionally may use different alternative proteins for attachment
including DC-SIGN, DC-SIGNR and LSECTin as has been summarized by GRAHAM
and BARIC (2010). Aminopeptidase N (APN) has been demonstrated to serve as
receptor for some alphacoronaviruses including HCoV-229E, FCoV, CCoV and
TGEV (DELMAS et al. 1992; TRESNAN a. HOLMES 1998). The latter however also
possesses the ability to bind sialic acids (SCHULTZE et al. 1993; SCHULTZE et al.
1995). It seems that this additional binding activity helps the virus to attach under
unfavorable environmental conditions, for instance in the gut (SCHWEGMANN-
Introduction
10
____________________________________________________________________
WESSELS et al. 2011). The virus uses the additional binding capacity to bind to sialic
acids present in the mucus layer that covers the gut epithelium and may therefore get
the chance to reach the APN-expressing cells. A mutant, which has lost the sialic
acid binding activity, is still able to infect respiratory tissues, but no longer the
intestine.
In contrast to that, some viruses bind only to sialic acids. Especially some
betacoronaviruses, for instance HCoV-OC43, resemble influenza C viruses with
respect to their binding behavior and they possess the hemagglutinin-esterase (HE),
a receptor-destroying enzyme. Whereas these viruses bind to 9-O-acetylated sialic
acids for host cell attachment, the HE-protein has also an acetylesterase activity to
prevent virions from getting trapped in sialic acid rich mucus and helps to release
newly formed viral particles from the cellular surface (HERRLER et al. 1991). A
specific protein receptor is not known for this group of sialic acid binding
betacoronaviruses (KREMPL et al. 1995).
The gammacoronavirus IBV also uses sugars, α2,3-linked sialic acids, for attachment
to host cells. A protein receptor of this virus has not been identified yet (WINTER et
al. 2006; WINTER et al. 2008; ABD EL RAHMAN et al. 2009). Unlike the mentioned
betacoronaviruses IBV does not possess a receptor-destroying enzyme. Therefore,
the question arises how virus particles detach from the cellular surface once they
have passed through the viral life cycle. It has been proposed that this detachment
may be possible due to a lower affinity of IBV to its receptor determinant and a more
narrow
spectrum
of
sialic
acids
recognized
by
this
virus
compared
to
betacoronaviruses or influenza viruses (WINTER et al. 2006; WICKRAMASINGHE et
al. 2011).
1.2 The coronavirus spike protein
The coronavirus spike protein constitutes a class 1 viral fusion protein (BOSCH et al.
2003). The protein is heavily glycosylated (CAVANAGH 1983a; BINNS et al. 1985). It
consists of a signal peptide, the ectodomain, the transmembrane region and the
cytoplasmic tail. The signal peptide directs the spike protein to the endoplasmic
reticulum (ER), where it is cleaved off (BINNS et al. 1985), while the hydrophobic
11
Introduction
transmembrane region later anchors the spike protein in the virus membrane
(CAVANAGH 1983c). Within the ectodomain of the spike protein, two subunits can
be distinguished, of which the S1-subunit forms the N-terminal part and the S2subunit the C-terminal part (CAVANAGH 1983b; JACKWOOD et al. 2001; YAMADA
a. LIU 2009). In some coronaviruses, including IBV, the subunits are cleaved by host
cell proteases (JACKWOOD et al. 2001; DE HAAN et al. 2004; YAMADA a. LIU
2009). The S1-subunit appears as the bulbous head of the protein and is responsible
for attachment to host cells (KOCH et al. 1990; LAUDE et al. 1995; TAGUCHI 1995;
TAGUCHI et al. 1995) whereas the shaft of the protein represents the S2-subunit and
mediates the fusion process of the viral envelope with the host cell membranes (LUO
a. WEISS 1998). Heptad repeat regions in the S2-subunit are involved in
oligomerization of the protein monomers (DE GROOT et al. 1987) resulting in the
homotrimeric form of the spike which is present in virus particles (DELMAS a. LAUDE
1990). This process takes place in the ER (CASAIS et al. 2003) where the spike is
assembled into viral membranes through noncovalent interactions with the M-protein
(GODEKE et al. 2000).
1.2.1 Variability of the S1-subunit
One characteristic feature of IBV is the high sequence variability of the S1-subunit of
the spike protein. This is not only true for spike proteins of different genera or species
but in case of IBV even for different strains of the same virus. IBV strains of different
serotypes show a variation in the amino acid sequence of S1- proteins of about 2025%, in some cases even up to 50% (reviewed by CAVANAGH CAVANAGH (2007)).
Interestingly, this seems not to be true for the tertiary structure of different spike
proteins. LI (2012) compared the structures of the alphacoronavirus NL63-CoV and
that of the betacoronavirus SARS-CoV. It could be shown that in spike proteins of
both viruses the different secondary structural elements are connected in the same
order.
In contrast to the S1-subunit, the S2-subunit is conserved even among different
coronavirus genera (XU et al. 2004a; ZHENG et al. 2006).
Introduction
12
____________________________________________________________________
A reason for the high mutation frequency in coronaviruses is the non-processive
nature of the viral RNA-polymerase, which leads to genetic drift. However, GRAHAM
and BARIC (2010) state that the error rate of the polymerase in coronaviruses is
lower compared to other RNA-viruses. This has been attributed to the fact that
Coronaviridae in contrast to other RNA-viruses possess RNA-processing and editing
enzymes, that eliminate incorrectly synthesized RNA-sequences (BHARDWAJ et al.
2004; BARR a. FEARNS 2010), (see chapter 1.1.5). Another reason for the variability
of the spike protein gene is the occurrence of recombination, the incorporation of
foreign genetic material is also possible (KOTTIER et al. 1995).
1.2.2 The spike protein as host range determinant
It has been suggested that the spike protein is the key determinant of the host range
of coronaviruses (KUO et al. 2000; SCHICKLI et al. 2004; THACKRAY a. HOLMES
2004; THACKRAY et al. 2005; DE HAAN et al. 2006; WICKRAMASINGHE et al.
2011). The reason is the observation that the substitution of the spike ectodomain in
a coronavirus results in viruses with the tropsim of the donor spike (KUO et al. 2000;
CASAIS et al. 2003; HAIJEMA et al. 2003; DE HAAN et al. 2006).
Both, the S1 subunit and the S2 subunit play a role in host range determination.
WICKRAMASINGHE et al. (2011) have tested the binding abilities of soluble S1subunits connected to a Strep-tag to different cells and tissues. They concluded that
the binding abilities of the IBV-M41, IBV-Beaudette and IBV-B1648 spike protein
reflect the host and tissue tropism of the corresponding viruses. GRAHAM and
BARIC (2010) report that changes in the receptor binding domain of SARS-CoV,
which is located in the S1-subunit, play an important role in the determination of the
tropism to civets or humans. SANCHEZ et al. (1999) and SCHICKLI et al. (2004)
have shown that alteration of host range is influenced by mutations in the S1-subunit.
Others, however, have reported that alterations in the S2-subunit may affect the
tropism of a coronavirus and thus host range can also be fusion-dependent (MCROY
a. BARIC 2008; YAMADA et al. 2009). This finding is in accordance with the fact that
also other viruses apart from the coronavirus family, which possess class I fusion
13
Introduction
proteins, show a change in host range after mutations in the fusion subunit
(AMBERG et al. 2006; KELETA et al. 2008).
Apart from host cell attachment and the fusion with host cell membranes, also the
cleavage of the spike protein, which leads to its activation and which is mediated by
host cell proteases, may play a role for the tropism of IBV. The extended host range
of the Vero cell-adapted IBV-strain Beaudette has been associated not only with an
additional binding activity for heparan sulfate, but also with the acquisition of an
additional cleavage site located in the S2-subunit and therefore designated as S2’
(MILLET a. WHITTAKER 2014).
1.2.3 The spike protein as main immunogenic agent of coronaviruses
The coronavirus spike protein has been identified as the main inducer of protective
immune responses after infection (CAVANAGH a. DAVIS 1986; CAVANAGH et al.
1986; SONG et al. 1998; JOHNSON et al. 2003). This protection however, might not
solely be based on an antibody response, since several studies could show a lack of
correlation between serum titers of virus neutralizing (VN) and hemagglutination
inhibiting (HI) antibodies produced after application of an inactivated virus and the
hereby induced protection against challenge (RAGGI a. LEE 1965; CAVANAGH et
al. 1986).
CAVANAGH a. DAVIS (1986) could show that virus particles from which the S1subunit had been removed by urea-treatment did neither result in protection against
challenge nor in the production of VN- or HI-antibodies. In contrast, four
intramuscular injections of monomeric S1-proteins did lead to the production of both
VN- and HI-antibodies. In another study it was shown that vaccination with a
recombinant baculovirus expressing the S1-subunit of the IBV-strain KM91 confers
protection against challenge and induces an increase of HI-titers (SONG et al. 1998).
These results are also supported by findings that in case of IBV the cleavage site of
the spike protein does not define the serotype or the pathogenicity of a certain strain
(JACKWOOD et al. 2001). It is therefore plausible that mainly the S1-subunit is
responsible for the stimulation of immune responses.
Introduction
14
____________________________________________________________________
1.2.4 The receptor-binding domains on the spike protein
As mentioned before (see chapter 1.2) the receptor binding activity of the coronavirus
spike protein resides in the S1-subunit. In general, this subunit may feature two
different receptor binding domains (RBD). However, many coronaviruses only use
one of these domains. Usually, though not without exception, the more N-terminally
located RBD is used for mediating binding to carbohydrate structures whereas the
more C-terminally located RBD is commonly responsible for binding to protein
receptors (LI 2012). Each coronavirus RBD characteristically comprises several
hundred amino acids and folds independently from the rest of the spike molecule
(GODET et al. 1994; GRAHAM a. BARIC 2010; PROMKUNTOD et al. 2014).
1.3 IBV
Infectious bronchitis virus belongs to the genus Gammacoronavirus and presents the
prototype avian coronavirus species. It is a pathogen that mainly infects domestic
fowl, predominantly chickens (gallus gallus), and other galliforme birds. The disease
caused by IBV is one of the leading causes of economic losses for the poultry
industry worldwide. It was first described by SCHALK and HARRIS (1931). They had
noticed a new respiratory disease of chickens in North Dakota with mortality rates of
40-90%. It was also noticed that the disease could be easily transmitted by contact
exposure or application of bronchial exudate. Later, the responsible virus could be
propagated in embryonated eggs (BEAUDETTE 1937; FABRICANT 1998). Over the
years different strains were discovered and the fact that not only the respiratory tract
but also the oviduct and the kidney can be affected was uncovered (COOK et al.
2012).
1.3.1 Classification
IBV is characterized by a huge variety of different strains, but IBV isolates can not
only be characterized by their affiliation to certain strains but also to different
genotypes, virtually dozens of serotypes (reviewed by CAVANAGH (2007)) and also
by pathotypes. Genotyping relies on sequencing and pathotypes are defined by a
common clinical picture caused by different strains. Serotyping is based on the
induction of specific antibody responses, though in some cases this does not
15
Introduction
correspond to crossprotection. Therefore, additionally the term “protectotype” was
introduced by Cook and Lohr in the 1990s, which describes not only the antibody
response but the complete immune response of a chicken against an IBV strain.
Thus, “strains that induce protection against each other in chickens belong to the
same protectotype” (DE WIT et al. 2011). De Wit et. al. characterize protectotyping
and serotyping as functional classification and genotyping as non-functional tests.
Although genotyping might appear to be a very accurate way of distinguishing
different isolates, several problems must be pointed out. First, since for genotyping
mostly the highly variable S1-subunit of the spike protein is used, even small
changes in genotype might result in a change of the serotype, so that there often is
no correlation between homology of the genetic sequences and serotype
(CAVANAGH et al. 1992). Furthermore different diagnostic laboratories sequence
sections of different sizes and locations within the spike protein for genotyping,
ranging from only parts of the S1-subunit to the full-length subunit up to the fulllength spike (SJAAK DE WIT et al. 2011). Depending on whether the hypervariable
regions 1 and 2 are included or not, quite different levels of homology might be
found. Especially due to the occurrence of recombination the sequencing of different
regions might result in assignment of the same isolate to variable geno- and
serotypes by different laboratories (WANG et al. 1993; JIA et al. 1995; DOLZ et al.
2008).
1.3.2 Epidemiology
Today, the infectious bronchitis of chickens is globally distributed (DE WIT et al.
2011; JACKWOOD 2012). Over the time, hundreds of strains have been identified
(CAVANAGH 2007). However, only few strains have been established as a cause of
disease in poultry populations for longer periods of time, whereas many strains have
appeared and shortly afterwards disappeared again (COOK et al. 2012). Another
puzzling phenomenon is the fact that some IBV strains have spread rapidly over
great distances whereas others remain confined to restricted areas or even single
countries (COOK et al. 2012). The way how different variants of IBV spread from
country to country is still unknown. Several AvCoV strains have been found in wild
Introduction
16
____________________________________________________________________
birds (CAVANAGH 2005; CHU et al. 2011), but these strains were not identical with
IBV strains with importance for commercial chicken husbandry.
1.3.3 Pathogenesis and clinical signs
Infection with IBV occurs via the oronasal route. First target cells for the virus are
cells of the respiratory epithelium of the upper respiratory tract. From there, IBV
spreads through viraemia to other organs. Replication of IBV has been detected in
many tissues, including kidneys, the alimentary tract, the oviduct and testes as has
been reviewed before (CAVANAGH 2007). The severities of clinical signs as well as
the clinical manifestations differ between different isolates, ranging from mild
respiratory symptoms to severe kidney and oviduct disease (CAVANAGH 2007).
Dyspnea, rales, coughing, serous nasal discharge have been described. In
pathological investigations, tracheitis, inflammation of the lungs and frequently cloudy
airsacs can be noticed. Histopathological findings include loss of ciliated epithelial
cells, infiltration of subepithelial layers with lymphocytes and heterophils (COOK et al.
2012). Especially manifestations in the oviduct, like generation of cysts have a great
economic impact, since drastic drops in egg production and poor egg quality may
occur. The so-called false layer syndrome describes the phenomenon that birds
infected early in live grow up apparently healthy but are unable to produce eggs due
to damage at the oviduct. Pathological findings in this case include intact ovaries, in
combination with non-patent and cystic oviducts. As a consequence, eggs cannot be
laid externally. Some strains, e.g. the strain B1648, show a predilection for the
kidneys and cause minor or major nephritis and mortality (LAMBRECHTS et al. 1993;
COOK et al. 2001; LIU a. KONG 2004). Particularly the infection of young animals
with involvement of the kidneys can be the cause of high mortality rates. Replication
in the gastrointestinal tract has been observed but was not connected to
development of disease.
1.3.4 Control strategies
IBV spreads readily within poultry populations so that it is hard to control with
biosecurity measures and one-age-systems. Therefore the control of IBV ultimately
relies on the application of different vaccines, especially live vaccines.
17
Introduction
The introduction of a protective immunity by vaccination against IBV infections has
been hampered by some characteristic features of this virus mentioned before: the
existence of a great variety of different strains with partially low crossprotection and
the rapid appearance of new strains and serotypes, respectively. The consequence
is that commonly used vaccines do not confer protection against all strains
(CAVANAGH 2007) and might suddenly become ineffective when new strains are
introduced into poultry husbandry. Even for homologous challenge, the protection
conferred by vaccination is only short-lived (GOUGH a. ALEXANDER 1979;
DARBYSHIRE a. PETERS 1984), so that frequent revaccination is necessary.
Commonly, layers are vaccinated several times during their life with live vaccines and
shortly before onset of lay with an inactivated vaccine.
The most frequently used vaccine strains, H120 and H52, derive from the
Massachusetts strain. Classically attenuated strains have been obtained by repeated
passaging in embryonated eggs but today also inactivated vaccines are used.
Introduction
18
____________________________________________________________________
19
Material
2 Material
2.1 4.1 Cell lines, primary cell cultures and tissue cultures
Table 1: Immortalised cell lines
Identifier
Species
Tissue
Growth Medium
Vero
Vero 7n6
BHK-21
ST
Chlorocebus sp.
Chlorocebus sp.
Mesocricetus auratus
Sus scrofa domestica
Kidney
Kidney
Kidney
Testis
DMEM + 5% FCS
DMEM + 5% FCS
EMEM + 5 % FCS
DMEM + 5 % FCS
Table 2: Primary cell and tissue cultures
Identifier
Species
Tissue
Growth Medium
CKC
Gallus gallus domesticus
Kidney
Tracheal cells
Traquea
TOCs
Traquea
Medium 199
Earle’s + 5% FCS
+ Pen/Strep
Medium 199
Hanks’ + 5% FCS
+ Pen/Strep
Medium 199
Hanks’ +
Pen/Strep
2.2 SPF eggs
Specific pathogen free eggs (SPF) were purchased from VALO SPF (Cuxhaven).
2.3 Chicken trachea and kidney
The tracheas for preparation of primary chicken trachea cells originated from 16-19
weeks old SPF chickens. The tracheas and kidneys for preparation of cryosections
derived from 14 weeks old commercial chickens of the laying type.
2.4 Chicken erythrocytes
Chicken erythrocytes were provided by the clinic for poultry (TiHo) in form of a
solution containing 1% erythrocytes in PBS.
Material
20
____________________________________________________________________
2.5 Virus
The IBV strain B1648 was kindly provided by Dave Cavanagh (Institute for Animal
Health, Compton, UK).
2.6 Bacteria
Escherichia coli (E.coli) MRF´ XL-1 blue
Stratagen, La Jolla USA
2.7 Cell and tissue culture media
2.7.1 DMEM (Dulbecco´s Minimal Essential Medium), pH 6.9
DMEM powder
13.53 g/l
GIBCO/Invitrogen,
Karlsruhe
NaHCO3
2.20 g/l
Merck, Darmstadt
2.7.2 EMEM (Eagle´s Minimal Essential Medium), pH 7.0
EMEM powder
9.60 g/l
GIBCO/Invitrogen,
Karlsruhe
NaHCO3
2.20 g/l
Merck, Darmstadt
Fetal calf serum
10%
Biochrom AG, Berlin
Glycerol (sterile)
10%
AppliChem, Darmstadt
2.7.3 Medium 199 Earle’s
Biochrom AG, Berlin
2.7.4 Medium 199 Hanks’
Biochrom AG; Berlin
2.7.5 Fetale calve serum (FCS)
Biochrom AG, Berlin
2.7.6 Freezing Medium
DMEM / EMEM
21
Material
2.7.7 Trypsin/EDTA
NaCl
8.00 g/l
KCl
0.20 g/l
Na2HPO4 x 12 H2O
2.31 g/l
KH2HPO4 x 2 H2O
0.20 g/l
CaCl2
0.13 g/l
MgSO4 x 7 H2O
1.10 g/l
Trypsin
(3 U/mg) 1.25 g/l
EDTA
1.25 g/l
Streptomycin
0.05 g/l
Penicillin
0.06 g/l
2.8 Bacteria media
2.8.1 Luria-Bertani (LB) media
Tryptone
10 g/l
NaCl
10 g/l
Yeast extract
5 g/l
Roth, Karlsruhe
Tryptone
10 g/l
NaCl
10 g/l
Yeast extract
5 g/l
Roth, Karlsruhe
Agar Agar
20 g/l
Roth, Karlsruhe
Tris 1 M
300 ml/l
Roth, Karlsruhe
Ethanol
200 ml/l
2.8.2 LB agar
2.9 Buffers and solutions
2.9.1 Anode buffer I, pH 9.0
Material
22
____________________________________________________________________
adjust pH with KCl
2.9.2 Anode buffer II, pH 7.4
Tris
1 M 25 ml/l
Roth, Karlsruhe
Ethanol
200 ml/l
adjust pH with HCl
2.9.3 Cathode buffer, pH 9.0
Tris 1 M
25 ml/l
Roth, Karlsruhe
Aminocaproic acid
5.25 ml/l
Sigma-Aldrich, München
Ethanol
200 ml/l
adjust pH with HCl
2.9.4 DAPI staining solution
Ethanol 100%
4′,6-Diamidin-2-phenylindol (DAPI) 1 mg/l
2.9.5 Ethidium bromide staining solution
TAE buffer
Ethidium bromide
10 g/l
2.9.6 Mowiol
Mowiol 4-88
120 g/l
Calbiochem, Heidelberg
Glycerol
300 g/l
Roth, Karlsruhe
DABCO
25 g/l
Tris/HCl
120 mM
2.9.7 Paraformaldehyde (PFA), pH 7.4
PBSM
Paraformaldehyde
30 g/l
23
Material
2.9.8 Phosphate buffered saline, supplemented with Calcium and magnesium
(PBSM), pH 7.5
NaCl
8.00 g/l
KCl
0.20 g/l
Na2HPO4
1.15 g/l
Merck, Darmstadt
KH2PO4
0.20 g/l
Merck, Darmstadt
MgCl2 x 6 H2O
0.10 g/l
Merck, Darmstadt
CaCl2 x 2 H2O
0.13 g/l
Merck, Darmstadt
2.9.9 Phosphate buffered saline (PBS), pH 7.5
NaCl
8.00 g/l
KCl
0.20 g/l
Na2HPO4
1.15 g/l
Merck, Darmstadt
KH2PO4
0.20 g/l
Merck, Darmstadt
2.9.10 FACS-buffer
PBS
EDTA
1,12 g/L
BSA
10 g/L
2.10 Plasmids
2.10.1 Expression plasmids
2.10.1.1
pCG1
This plasmid was originally provided by R. Cattaneo (Mayo Clinic College of
Medicine, Rochester, Minnesota, USA). It contains a promoter region deriving from
human cytomegalovirus, an intron from the rabbit β–tubulin gene which acts as bait
for cellular spliceosomes, and an ampicillin resistance gene for selection in bacterial
cultures (CATHOMEN et al. 1995).
Material
24
____________________________________________________________________
2.10.1.2
pCG1-Fc and pCG1-Fc-ATG
These plasmids are derivates of the pCG1 plasmid that have the open reading frame
of the Fc fragment of human immunoglobulin G inserted into the multiple cloning site
(MCS). When a coding sequence of a protein is inserted into the MCS of the former
plasmid in frame with the Fc fragment sequence and without STOP-codon
sequences, a chimeric protein is expressed consisting of the Fc fragment and the
protein of choice. The latter possesses an in frame ATG-codon in front of the Fc
sequence, which allows to express the Fc tag alone. Fc tagged proteins or Fc
fragments alone can be easily detected by anti-human IgG antibodies. Both plasmids
have been prepared and provided by Dr. Jörg Glende.
2.10.2 cDNA Constructs
2.10.2.1
pCG1-B1648S1Fc
This plasmid contains the sequence of the S1 subunit (amino acid 1-543) of the spike
protein of the avian coronavirus strain B1648, which is a field-isolate from Belgium
(genbank accession number: X87238). By the beginning of the project it had already
been prepared by Christine Winter.
2.10.2.2
pCG1-TGEVS1Fc
The cDNA coding for the S1 subunit (amino acid 1-858) of the TGEV-strain Madrid
(genbank accession number: M94101) has been inserted into the MCS of the pCG1Fc plasmid. This plasmid was provided by Dr. Jörg Glende.
2.10.2.3
pCG1-Fra1-S1-Fc
This is a pCG1-Fc plasmid with the S1 domain (amino acid 1-667) of the Frankfurt-1
SARS-CoV spike protein (THIEL et al. 2003) inserted into the MCS. The sequence is
found in GenBank under the accession number AAP33697.1. It was provided by Dr.
Jörg Glende.
2.10.2.4
pCG1-H7Fc
The sequence of H7, an influenza hemagglutinin, was inserted into the pCG1-Fcplasmid by Dr. Maren Bohm. The virus strain, which served as template,
A/chicken/Netherlands/621557/2003 (H7N7) HPAI, was supplied by Ben Peeters,
Wageningen University and Research Center, Leylstatd, The Netherlands.
25
Material
2.10.3 Oligonucleotides
Name
IBV1648BamH1se
nse
IBV1648S1BamH1
AS
B1648DelDAS
B1648DelDS
B1648S1Del21-27S
Sequence
TTTGGATCCTGTACTATTAGTGAGTTTAACATAAAACTG
ACC TGC AGT GCA TGT CAA AAT AGC ACT ACA TAG TGC
TGT AGT GCT ATT ACA TGC ACT GCA GGT GTT ATT
TGT AGT GCT ATT TTG TAC TAC CAA AGT GCC TCC AGA
CCA CCC TCA
GGC ACT TTG GTA GTA CAA AAT AGC ACT ACA TAG TGC
B1648S1Del2127AS
B1648S1Del28-34S CCA CCC TGA GGG TGG ATA ATT ATA ATT ATC ATT GAA
CAA AAT AGC
B1648S1Del28GAT AAT TAT AAT TAT CCA CCC TCA GGG TGG CAT TTA
34AS
B1648S1Del35-41S CAA AGT GCC TTC AGA CAT GGG GGT GCT TAT CAA GTG
B1648S1Del35ATA AGC ACC CCC ATG TCT GAA GGC ACT TTG GTA GTA
41AS
ATA ATT ATA
B1648S1Del42-48S TCA GGG TGG CAT TTA GTC AAT GTT ACT AAT GAA AAT
AAT AAT GCA
B1648S1Del42ATT AGT AAC ATT GAC TAA ATG CCA CCC TGA GGG TGG
48AS
TCT GAA GGC
B1648S1Del49-55S GGT GCT TAT CAA GTG AAT AAT GCA GGT TCA TCA CCA
B1648S1Del49TGA ACC TGC ATT ATT CAC TTG ATA AGC ACC CCC ATG
55AS
TAA ATG CCA
B1648S1Del56-72S GTT ACT AAT GAA AAT ACA TGC ACT GCA GGT GTT ATT
B1648S1Del56ACC TGC AGT GCA TGT ATT TTC ATT AGT AAC ATT GAC
62AS
B1648S1Del63-69S GCA GGT TCA TCA CCA TAT TAT AGT AAA AAT TTT ACT
(Del 73-80)
B1648S1Del70ATT TTT ACT ATA ATA TGG TGA TGA ACC TGC ATT ATT
76AS
B1648S1Del77-83S ACT GCA GGT GTT ATT GCT TCT TCT GTA GCC ATG CAT
(Del 81-86)
B1648S1Del84GGC TAC AHA AGA AGC AAT AAC ACC TGC AGT GCA TGT
90AS
B1648S1Del91-97S AGT AAA AAT TTT ACT GCA CCA CCA CCA GGA ATG TCA
(Del 87-92)
B1648S1Del98TCC TGG TGG TGG TGA AGT AAA ATT TTT ACT ATA ATA
104AS
Mutant 5 V1ASe
CTT ATC AAG TGG CCA ATG TTA CTA A
Mutant 5 V2A AS
TTA GTA ACA TTG GCC ACT TGA TAA G
Mutant 5 N1A SE
A TCA AGT GGT CGC TGT TAC TAA TGA
Mutant 5 N1 A AS
TCA TTA GTA ACA GCG ACC ACT TGA T
Mutant 5 V2 A Se
A AGT GGT CAA TGC TAC TAA TGA AAA
Material
26
____________________________________________________________________
Mutant 5V2 A AS
Mutant 5 TA Se
Mutant 5 TA AS
Mutant 5 N2A Se
Mutant 5 N2A A
Mutant 5 EA Sen
Mutant 5 EA AS
Mutant 5 N3A SE
Mutant 5 N3A AS
TTT TCA TTA GTA GCA TTG ACC ACT T
T GGT CAA TGT TGC TAA TGA AAA TAA
TTA TTT TCA TTA GCA ACA TTG ACC A
T CAA TGT TAC TGC TGA AAA TAA TAA
TTA TTA TTT TCA GCA GTA ACA TTG A
A TGT TAC TAA TGC AAA TAA TAA TGC
GCA TTA TTA TTT GCA TTA GTA ACA T
T TAC TAA TGA AGC TAA TAA TGC AGG
CCT GCA TTA TTA GCT TCA TTA GTA A
2.11 Enzymes
2.11.1 Restriction enzymes
BamHI
Fermentas, St. Leon-Rot
XbaI
EcoRV
2.11.2 Other enzymes
Phusion High Fidelity polymerase
Taq polymerase
FastAP
T4 DNA ligase
Neuraminidase, Vibrio cholerae
Roche, Mannheim
Protease from Streptomyces grisesus Type XIV
Sigma-Aldrich, Steinheim
Trypsin
Biochrome AG, Berlin
2.12 Antibodies
The following table 3 lists the secondary antibodies used in immunofluorescence (IF),
Western Blot (WB) or FACS analysis.
Table 3: Antibodies
Name
Goat anti-human Cy3
Dilution
1:500
Usage
IF
Company
Sigma-Aldrich,
Munich
27
Material
Goat anti-human IgG,
Horseraddish peroxidase
Goat F(ab’)2 anti-human IgG
PE
Ch/IBV 48.4
1:20000
WB
1:200
FACS
1:100
IF
Rabbit anti-mouse FITC
1:500
Sigma-Aldrich,
Munich
Beckmann-Coulter;
Marseille, France
Prionics, Lelystad,
the Netherlands
Sigma-Aldrich,
Munich
2.13 Kits
QIAquick PCR Purification Kit
Qiagen, Hilden
QIAquick Gel Extraction Kit
Qiagen, Hilden
NucleoBond Xtra Midi Kit
Macherey-Nagel, Düren
RNeasy Mini Kit
Qiagen, Hilden
BCA Protein Assay Kit
Thermo-Scientific,
Dreieich
2.14 Chemicals
1,4-Dithiotreitol (DTT)
Roth, Karlsruhe
Acrylamide solution 30% (Rotiphorese Gel 30)
Roth, Karlsruhe
Agar Agar
Roth, Karlsruhe
Agarose
Biozym, Hess. Oldendorf
Ammonium persulfate (APS)
Bio-Rad, Munich
Boric acid
Roth, Karlsruhe
Bovine Serum albumin
Roth, Karlsruhe
DEPC treated water
Roth, Karlsruhe
Disodium hydrogen phosphate
Roth, Karlsruhe
dATP
Fermentas, St. Leon.Rot
dCTP
dGTP
dTTP
Material
28
____________________________________________________________________
DTSSP
Thermo
Dreieich
Scientific,
Ethylenediaminetetraacetic acid (EDTA)
Roth, Karlsruhe
Acetic acid
Roth, Karlsruhe
Ethanol
Merck, Darmstadt
Ethidiumbromide
Sigma-Aldrich, Munich
Pencillin/Streptomycin (100x)
PAA, Pasching (Austria)
Glucose
Roth, Karlsruhe
Glycerine
Roth, Karlsruhe
Glycerol
Glycin
Roth, Karlsruhe
Hydrochloric acid
Roth, Karlsruhe
HEPES
Roth, Karlsruhe
Isopropanol
Roth, Karlsruhe
Magnesium chloride
Roth, Karlsruhe
Magnesium sulfate
Roth, Karlsruhe
Methanol
Roth, Karlsruhe
Mowiol
Calbiochem, Heidelberg
N,N,N’,N’-Tetramethylene diamine (TEMED)
Roth, Karlsruhe
NuSerum
Fisher Scientific; Schwerte
Opti-MEM
Life
Technologies,
Darmstadt
Paraformaldehyde
FLUKA, Basel
Polyethylenimine
Polysciences, Eppelheim
Potassium chloride
Roth, Karlsruhe
Potassium dihydrogen phosphate
Roth, Karlsruhe
29
Material
Sodium acetate
Merck, Darmstadt
Sodium chloride
Roth, Karlsruhe
Sodium desoxycholate
Roth, Karlsruhe
Sodium phoshate
Roth, Karlsruhe
Sodium dihydrogen phosphate
Roth, Karlsruhe
Sodiumdodecylsulfat (SDS)
Roth, Karlsruhe
Sodium hydrogen phosphate
Roth, Karlsruhe
Sodium hydroxide
Roth, Karlsruhe
Tissue freezing medium
Jung, Heidelberg
Tris-Hydroxymethylaminomethan (TRIS)
Roth, Karlsruhe
Trypton
Roth, Karlsruhe
Tween 20
Roth, Karlsruhe
Yeast extract
Roth, Karlsruhe
Ampicillin
AppliChem, Darmstad
2.15 Other substances
Blocking reagence
Roche, Mannheim
Super Signal West Dura Extended Duration
Pierce, Rockford (USA)
Substrate
Super Signal West Femto Extended Sensitivity
Pierce, Rockford (USA)
Substrate
Spectra multicolour high range protein ladder
(300 kDa)
Gene Ruler 1 kb DNA Ladder plus
Material
30
____________________________________________________________________
2.16 Equipment
2.16.1 Agarose gel electrophoresis
Electrophoresis Box, Gel Mold, Gel Comb
Keutz, Reiskirchen
Microwave MWS 2820
Bauknecht, Schorndorf
UV-Transluminator
UVP, Upland, (USA)
Swiveling table
Keutz, Reiskirchen
Power Supply
Bio-rad, München
2.16.2 Photometer
Eppendorf BioPhotometer Plus
Eppendorf, Hamburg
2.16.3 Bacteria culture
Petri dishes, 100mm
Greiner, Nürtingen
Erlenmeyer flask, 100ml, 300ml, 500ml
Jürgens, Hannover
Shaking incubator Type 3033
GFL, Burgwedel
Incubator Type B16
Heraeus, Osterode
2.16.4 Cell culture
Tissue culture flasks 75 cm2
Greiner, Nürtingen
24-well plates
Greiner, Nürtingen
6-well plates
Greiner, Nürtingen
145 mm cell culture dishes
Greiner, Nürtingen
CO2 incubator
Heraeus, Hanau
Swiveling table
Keutz, Reiskirchen
Coverslips
Roth, Karlsruhe
Microscope slide
Roth, Karlsruhe
2.16.5 Centrifuges
Megafuge 1,0R
Heraeus, Hanau
31
Material
Centrifuge 5417C/R
Eppendorf, Hamburg
OptimaTM LE-80K Ultracentrifuge
Beckmann
Coulter,
Marseille (France)
2.16.6 Magnetic stirrer
Magnetic stirrer, RCT basic I
KA Labortechnik, Staufen
2.16.7 Microscope and image processing software
Eclipse Ti
Nikon, Düsseldorf
NIS Elements Imaging Software
Nikon, Düsseldorf
(64bit, 3.22.11; Build 728)
GNU Image Manipulation Program
Open Source
ImageJ
Open Source
Inkscape
Open Source
2.16.8 PCR
Prismus 25/96 Thermocycler
MWG Biotech, Ebersberg
0.2 ml PCR reaction tube
4.12.9 pH-Meter
Jürgens, Hess. Oldendorf
2.16.9 Pipettes and pipette helpers
10 µl, 100 µl, 1000 µl
Eppendorf, Hamburg
10 µl, 100 µl SafeSeal-Tips
1 ml, 2 ml, 5 ml, 10 ml, 20 ml glas pipettes
Jürgens, Hannover
AccuJet Pipette Helper
Brand, Wertheim/Main
2.16.10
Reaction tubes and sterile filters
FP 30/0.2 CA-S sterile filter
Schleicher
Dassel
&
15 mL and 50 mL reaction tubes
Greiner, Nürtingen
Schuell,
Material
32
____________________________________________________________________
1.5 mL reaction tubes
Eppendorf, Hamburg
2.0 mL safe seal reaction tubes
Eppendorf, Hamburg
2.16.11
Centrifugal Filters
Amicon® - Ultra-15 Centrifuge filter
2.16.12
Merk Millipore Ltd., Cork
(Ireland)
Flow cytometry
Beckmann Coulter Epics XL flow cytometer
Beckmann-Coulter,
Marseille (France)
EXPO32 analysis software
Beckmann-Coulter,
Marseille (France)
5 mL tubes
Sarstedt, Nümbrecht
2.16.13
Safety cabinettes
NuAire Class II
Nuaire, Plymouth (USA)
Hera Safe
Heraeus, Hanau
NuAire Class II Type A/B3
Nuaire, Plymouth (USA)
KOJAIR KR-130 BW MSC CL II EN12469
KOJAIR, Vilppula, Finland
2.16.14
SDS-PAGE and Semi-dry Western-Blot
Slab Gel chamber
Keutz, Reiskirchen
Filter paper
Schleicher
Dassel
&
Schuell,
Nitrocellulose transfer membrane
Schleicher
Dassel
&
Schuell,
Transfer chamber
Biometra, Analytic Jena,
Ober-Ramstadt
ChemiDoc EQ
Bio-rad, München
Quantity One V 4.4.0 (Software)
Bio-rad, München
33
2.16.15
Material
Vortex
Reax top
Heidolph, Kehlheim
Reax 2000-05-20
Heidolph, Kehlheim
2.16.16
Scales
Electronic analysis scale, Type 1712 MP 8
Sartorius, Göttingen
Sartorius Portable scale Lauda A100
Sartorius, Göttingen
2.16.17
Homogenisator
Dounce-Homogenisator
2.16.18
Water bath
Water bath
2.16.19
GFL, Burgwedel
Cryostat
Reichert-Jung
Nußloch
Material
34
____________________________________________________________________
35
Methods
3 Methods
3.1 Cell and organ culture technique
Continuous cell cultures were grown in 75 cm² cell culture flasks and incubated at
37°C and 5% CO2. Depending on the growth behavior, the cells were passaged 1-3
times a week. For this purpose cells were washed with 2 mL Trypsin/EDTA.
Subsequently 4 mL Trypsin/EDTA were added and 3 mL removed again. One mL
was left in the flasks for incubation until all cells had detached from the surface. Then
0.5 mL of FCS was added. At last the cells were resuspended in medium in ratios of
1:10 to 1:20.
3.1.1 Mycoplasma detection test
Permanent cell lines were tested for mycoplasma contamination using a mycoplasma
specific PCR by our lab technician every two months.
3.1.2 Cryoconservation
If not in culture, cells were stored at -80°. Therefore suspended cells were
centrifuged for 5 minutes at 4° and 1000 rpm (173 rcf). The medium was discarded
and the pellet was resuspended in freezing medium at a density of 1x105 cells/mL.
The suspension was portioned at portions of 1 mL and frozen slowly.
To take the cells back in culture aliquots were thawed at room temperature and
suspended in culture medium.
3.1.3 Preparation of primary embryonic chicken kidney cells
Primary chicken kidney cells were prepared as has been described by (WINTER et
al. 2006). The embryonic tissues for this preparation originated from SPF eggs that
had been incubated for 20 days at 37.5-38°C. After this incubation period the eggs
were disinfected with alcohol, opened at the blunt end and the embryos were
removed. Then the animals were decapitated. Afterwards the abdomen of each
embryo was opened. The esophagus was caught with forceps, then severed and
used to pull out the organ convolute. Now the kidneys were exposed and could be
Methods
36
____________________________________________________________________
removed. After the explanted kidney tissue had been washed two times with ice-cold
PBSM, Trypsin/EDTA was added. The mixture was stirred on a magnetic stirrer at
37°C, until the kidney cells had been released. One part medium, containing 5% FCS
was added to two parts of the kidney-Trypsin-mixture. Following a centrifugation step
of 10 minutes at 1000 rpm (173 rcf) the cells were resuspended with culture medium
and subsequently pipetted through a cell sieve. The cells were ultimately seeded in
6-well-plates or on coverslips in 24-well plates so that a confluent cell layer was
produced. The cell cultures were incubated for 24 hours before they were used for
further experiments.
3.1.4 Preparation of primary chicken trachea cell cultures
Organ material for preparation of primary trachea cell cultures was provided by the
Clinic for Poultry (TiHo). The SPF animals were specifically reared for preparation of
organ material for experiments carried out in the clinic. After the animals had been
stupefied and killed by exsanguination the trachea was uncovered by opening the
skin and tissues starting at the beak. The tracheas and larynx were removed in full
length and transported to the Institute of Virology in ice-cold Medium Hanks’ 199. The
organs were cleaned from muscles and connective tissues and cut open
longitudinally. After washing the tracheas with medium they were cut into pieces and
incubated in a freshly prepared solution of 0.1g pronase derived from Streptomyces
griseus in 100mL Medium Hanks’ 199 for four hours at room temperature. Afterwards
the trachea pieces and the pronase mixture were put into a glass petri dish and the
tracheal epithelial cells were scratched from the surfaces with a rubber policeman.
After centrifugation at 1000 rpm (173 rcf) and 4°C for 10 minutes, the medium was
discarded and the pellet was resuspended. The centrifugation step was repeated and
after the cells had been resuspended again they were pressed through a cell sieve.
Finally they were diluted in medium containing 5 % FCS and seeded into six-well
plates. After incubation overnight at 37°C ciliated cells were floating in the medium
while all other cells had settled to the bottom of the wells. Therefore ciliated cells
could be removed from the plates selectively, transferred to reaction tubes and
centrifuged for further usage.
37
Methods
3.1.5 Preparation of tracheal organ culture
Embryonated SPF Eggs were incubated and the embryos were killed as described
above. The trachea was caught with forceps at the anterior end and severed from the
neck tissues and the lung near the bifurcation. The organ was then cleaned from
connective tissue and muscles and cut into rings of 2-3mm length. Every ring was
incubated in a tube for 24 h on a rotator, then mounted with tissue freezing medium
on filterpapers, frozen in liquid nitrogen and finally stored at -80°C.
3.2 Preparation of virus stocks
To obtain virus stocks, B1648 was propagated in SPF eggs. Therefore a virus stock
was supplemented with 1% Penicillin and Streptomycin was applied into the allantoic
cavity by use of sterile syringe. The hole was sealed with glue. After three days of
incubation, the eggs were cooled at -20° for 15 minutes to kill the embryos.
Afterwards, the eggs were opened at the blunt end. Then the allantoic fluid was
harvested, clarified by low-speed centrifugation and stored in aliquots at -80°. The
viral titer was determined by titration in chicken embryonic kidney cells (CEKC).
3.3 Preparation of Cryosections
Tracheas and kidneys of 14 weeks old chickens for the preparation of cryosections
were provided by the Clinic for Poultry, Hannover. The material originated from
chickens that were autopsied for routine diagnostics and that were free of IBV
infections, respiratory or renal findings and of pathological findings of the
reproductive tract. The animals were stupefied and killed by severing of the carotids.
Directly after slaughtering pieces of trachea and kidney were removed, mounted on
filter papers using cryomatrix and frozen in liquid nitrogen. Embryonic TOCs were
frozen after 24 hours of incubation in the same way. Until cutting with the microtome
the organ pieces were stored at -80°C.
The organ compounds were cut into slices of 10µm thickness and mounted on
microscope slides. The slides were air-dried overnight at room temperature and then
stored at -20°C until immunofluorescence staining was performed.
Methods
38
____________________________________________________________________
3.4 Molecular Biology
3.4.1 Polymerase chain reaction
Polymerase chain reaction (PCR) is a biochemical method to amplify DNA. Specific
primers, small strands of nucleic acid, serve as starting points for the amplification
and therefore ensure the selective amplification of certain sequence segments.
Depending on the purpose of a certain PCR, two different polymerases were used.
The phusion high fidelity polymerase possesses a highly efficient proof reading
function that reduces inadvertent nucleotide exchanges. It was therefore applied for
cloning purposes. TheTaq polymerase lacks such a proof reading function and for
this reason it was only used for analytic PCRs, mainly colony-PCR. In the following
table the composition of reaction mixes and temperature protocols are listed for both
enzymes, respectively. These two polymerases also require specific adaptations of
PCR protocols. Furthermore PCR protocols varied in the annealing temperature
needed by different primers and the elongation time depending on the length of the
DNA sequence that should be amplified.
Phusion PCR 50 µl reaction mix:
5x Phusion reaction buffer
10 µL
dNTP (10 mM)
1 µl
Sense primer
2.5 µl
Antisense primer
2.5 µl
Template DNA
0.1 µg
Phusion polymerase
0.5 µl
DEPC treated water
Add to 50 µl
39
Methods
Taq PCR reaction mix for 1 sample:
10x Taq reaction buffer
1.5 µl
MgCl2
1.2µl
dNTP (10 mM)
0.3 µl
Sense primer
0.45 µl
Antisense primer
0.45 µl
Taq polymerase
0.1 µl
DEPC treated water
Add to 15 µl
PHUSION PCR temperature profile:
10 cycles*
18 cycles*
Temperature (°C)
Time (sec)
98
120
98
30
58 (-0.5 per cycle)
30
72
60 / 1 kb of amplificate
98
30
56
30
72
72
(+0.05 per cycle)
7
4
pause
* The number of cycles was increased if the amount of the desired PCR product was
not sufficient after running this program the first time.
Methods
40
____________________________________________________________________
Taq PCR temperature profile:
35 cycles
Temperature (°C)
Time (sec)
95
60
95
30
54
30
72
72
5
4
pause
3.4.2 Agarose gel electrophoresis
After completing the PCR, a mixture of DNA segments is present. To separate PCR
products according to size, agarose gel electrophoresis was used. If DNA was to be
used for further cloning steps, it was separated in TAE-gels, containing 1% agarose.
If the DNA separation was just for analytic purposes, 1% TBE-Gels were used. TAEgels were run at 90 V, TBE-gels at 120 V. The gels were stained in an ethidium
bromide bath for 10 minutes and rinsed in water for 5 minutes. Then the gel was
observed over UV-light for the presence of a specific band.
3.4.3 DNA gel extraction
If specific bands of DNA segments were detected under UV-light, they could be cut
out of the gel with a scalpel and transferred into reaction tubes. Subsequently the
DNA was extracted from the gel pieces using the gel extraction kit. In the last step
the DNA was eluted in DEPC-treated water and stored at -20°C.
3.4.4 Hybridization of DNA fragments
In some cases DNA fragments produced by Phusion-PCR needed to be hybridized.
Therefore, 100 ng of the smaller fragment and an equimolar amount of the bigger
fragment was applied. For the hybridization step no primers were added. Apart from
that the composition of the reaction mix was consistent with that of the Phusion-PCR.
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Methods
This step was followed by an amplification of the hybridization product by PhusionPCR. The following table gives the temperature protocol of the hybridization step.
Temperature (°C)
Time (sec)
95
95
2 cycles
54
72
72
5
4
pause
3.4.5 Restriction Enzyme Digest
Before desired DNA sequences could be inserted into the MCS of plasmids both had
to be digested with the same restriction enzymes. Restriction enzyme digest creates
overhangs of 1-3 nucleotides by cutting DNA at specific sequence sites. These
overhangs can be used for re-ligation. For the digestion of 5 µg plasmid 10 U of the
restriction enzyme, for digesting PCR amplified DNA fragments 20 U of the enzyme
per sample were applied. The total volume of the reaction mix amounted to 50 µl and
contained enzyme buffer (5x) and DEPC treated water. The mixture was incubated
overnight at 37°C and the DNA was afterwards purified by DNA gel extraction or
PCR purification.
3.4.6 PCR Purification
The buffers used in PCR reactions or restriction enzyme digests may interfere with
the further steps of the cloning process. Therefore the remaining buffer and enzymes
were removed using the QIAquick PCR purification Kit (Qiagen). In this study PCRpurification was mainly performed after restriction enzyme digestion of empty
plasmids. The DNA was eluted in DEPC treated water and, if not used immediately,
stored at -20°C.
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3.4.7 Dephosphorylation
If plasmids are opened with a restriction enzyme cutting a singular cleavage site,
religation of the vector is possible. To prevent that, it is necessary to enzymatically
dephosphorylate the plasmid. The digested and purified plasmid was therefore mixed
with 2 U alkaline Phosphatase (FastAP) and 2 µL corresponding buffer. DEPC
treated water was added to obtain a total volume of 20 µL. The mixture was
incubated at 37° for 20 minutes.
3.4.8 Ligation
The respective 5'-phosphate and 3'-hydroxyl termini of desired double strand DNA
sequences and plasmids that have been digested with the same restriction enzymes
can be ligated by usage of T4-DNA-Ligase to form a circular plasmid. We added 4µL
(4 WeissU) T4-DNA-Ligase and 4 µL buffer to 100 ng plasmid and 3-5 times more
moles of the insert. DEPC treated water was added so that the components added
up to a total volume of 40 µL. The reaction can take place at 14°C forabout 16 hours
or at room temperature fortwo hours. We applied both protocols and could not see
differences in the results.
3.4.9 Measurement of DNA-concentration
Measurement of DNA concentration was carried out in a photometer at 280 nm. For
this purpose DNA was diluted in a ratio of 1:100.
3.4.10 Heat shock transformation of bacteria
Heat shock competent E. coli were incubated with 10µL of the ligation product
containing the mutated gene inserted in a PCG1-Fc-plasmid. A negative control was
incubated with 10 µL of the PCG1-Fc without the inserted gene. After 30 minutes the
reaction tubes with the bacteria were heated in a waterbath at a temperature of 42°C
for two minutes and were subsequently incubated back on ice for another 5 minutes.
Then, 1 mL of LB medium was added and the bacteria were incubated at 37°C for
one hour on a shaking incubator. Afterwards the bacteria were plated on LB agar
plates supplemented with Ampicillin, which were then placed in an incubator at 37°
overnight. The next day, the plates were examined for colony growth and
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Methods
contamination. If colony-PCR was planned, the number of colony forming units of
bacteria transformed with the ligation product was compared with those transformed
with the negative control. A high number of cfu in the negative control is indicative for
an ineffective restriction enzyme digest of the plasmid but does not necessarily
exclude the existence of few clones carrying the plasmid with the desired DNA
sequence inserted.
3.4.11 Colony-PCR
To check for the presence of the desired plasmid in colonies of E. coli, a PCR was
performed. Therefore the reaction mixture was prepared as described above (see
3.8.1) and portioned in PCR tubes. Reaction tubes were filled with 250 µL LB
medium supplemented with ampicillin. Then, a pipette was dipped into single
colonies, subsequently into the PCR tubes and at last used to inoculate the tubes
filled with LB medium. While the PCR was carried out in a thermocycler and
afterwards analyzed by agar gel electrophoresis, the inoculated LB-medium was
cultivated at 37° on a shaker. If a specific DNA band was found for any of the
samples, the corresponding LB medium culture was used to inoculate a larger
amount of medium for an overnight cultivation.
3.4.12 DNA preparation
For DNA preparation, overnight cultures of a clone tested positive for the desired
plasmid by PCR, were prepared. Therefore 5 mL or 50 mL of LB medium,
respectively, supplemented with ampicillin was used. After overnight incubation at
37°C the samples were centrifuged at 4500 rpm at 4°C for 15 minutes. The depleted
medium was discarded and the resulting bacteria pellet was used for DNA extraction
with the Mini prep kit or the Midi prep kit according to manufacturer’s instructions.
The concentration of the plasmid DNA was measured in a photometer. The solution
was diluted to achieve a concentration of 1000 µg/mL and then stored at -20°C. If
necessary the DNA was sent in for sequence analysis.
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3.4.13 Sequence Analysis
For sequence analysis of cloning products samples were sent to Eurofins Genomics
GmbH (Ebersberg). The samples contained 15µl purified DNA at a concentration of
50-100 ng/µl and 2 µL of the sequencing primer with a concentration of 10pmol/µl (10
µM).
3.5 Transient transfection of mammalian cells
BHK-21 cells were seeded in 145 cm² cell culture dishes at a density of 2x105 per
milliliter. The next day the depleted medium was replaced with 14 mL of medium
containing 3% NuSerum. Then 6 mL of Optimem per dish was mixed with 50 µg of
Plasmid-DNA. The mixture was vortexed and incubated for 5 minutes before 40 µl of
polyethylenimine (PEI) per dish were added. After 15 minutes of incubation the
complete Optimem-plasmid-PEI-mix was added in a drop-wise fashion to the seeded
cells while the dishes were carefully rocked back and forth.
Sixteen hours post tranfection medium was harvested for the first time and replaced
with serum free medium. The collected medium was centrifuged at 4500 rpm for 15
minutes and stored at 4°. The following day, medium was harvested for the second
time from the transfected cells. It was centrifuged and pooled with the medium
collected the day before. The success of the transient transfection was checked by
detection of the desired proteins in concentrated supernatants in western blot (see
chapter 2.9.7 and 2.9.8).
3.6 Neuraminidase treatment
Neuraminidase treatment of cells was carried out at 37°C for 1 h. Coverslips
containing cells and cryosections of chicken organs were treated with vibrio cholerae
neuraminidase after quenching with glycine and prior to immunostaining.
Neuraminidase was used at a concentration of 10 mU per 100 µL. Of this solution,
100 µL were applied per coverslip. For cryosections, as much neuraminidase solution
was used as necessary to cover the organ sections completely, usually 250-350 µL.
For FACS analysis cells were treated with neuraminidase after detachment from the
45
Methods
six-well-plates and prior to protein incubation. Each sample was treated with 35 mU
of the enzyme.
3.7 Protease-treatment
To cleave off cellular surface proteins, cells were treated with proteases. This method
has been used before (FINKELSHTEIN et al. 2013). Therefore, CEKC were either
treated with trypsin or with pronase derived from Streptomyces griseus. The day
before the experiment, CEKC or ST cells or Vero 76 cells as controls were seeded in
six-well-plates. To separate the cells from the cell culture dish they were treated with
400 µL accutase at 37° on a shaker, until they had completely detached. Accutase
was then removed using centrifugation at 2000 and 4° for 5 minutes.
3.7.1 Trypsin-treatment
For trypsin-treatment a solution of 0.125% Trypsin and 0.01%EDTA in PBS and
0.01% EDTA in PBS as a control was prepared and 400 µl were added to each
sample. After an incubation period of 1h in case of CEKC and 45 minutes in case of
ST-cells or Vero 76 cells at 37°, 600 µL of cell culture medium containing 5% FCS
was added. The cells were then washed and stained with soluble spike proteins (see
chapter 3.10). The binding of the soluble spike proteins was quantified in FACS
analysis.
3.7.2 Pronase-treatment
1% pronase was added to cell culture medium. Medium only was used for the control
samples. Each sample of cells was incubated with 400 µL for 1h (CEKC) or 45
minutes (ST cells, Vero 76 cells), respectively. After washing the cells were stained
with soluble spike proteins and submitted to FACS analysis (see chapter 3.10).
3.8 Infection of continuous cell lines with IBV
BHK21-cells were seeded at a density of 1.5x105/mL, Vero 76 cells at a density of
2.0x105/mL and ST cells at a density of 3.0x105/mL in 24-well-plates. The next day
the cells were washed with cell culture medium, before virus diluted in medium was
added at a multiplicity of infection (MOI) of 0.1. The cells were incubated with the
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virus (strain B1648) on a swiveling table for 2 hours at 37°. Afterwards, the medium
was changed and the cells incubated for further 24 hours. Then the cells were fixed
with PFA. After quenching with glycine diluted in PBSM, the cells were stained with
Ch/IBV 48.4, an anti-N-IBV-antibody, followed by anti-mouse-FITC before the
coverslips with the cells were on mounted microscope slides with the help of Mowiol.
3.9 Immunofluorescence
3.9.1 Cell culture
Cells were seeded on coverslips placed in 24 well plates. CEKC were prepared and
seeded as described before (see 3.1.3). Vero cells, Vero 76 cells and BHK-21 cells
were seeded at a concentration of 2x105/mL whereas for ST cells a concentration of
3x105/mL was used. FCS was supplied at a concentration of 10 % in case of the
former and 20% in case of the latter. When the cells had formed a confluent
monolayer, usually the day after seeding, they were washed three times with PBSM,
fixed with PFA and stored at 4°C until the immunofluorescence staining was
performed. Before staining t the PFA was removed and the cells were quenched with
glycine in PBSM two times.
Drops of 30 µL of soluble protein or diluted antibody respectively, were loaded on a
strip of parafilm. The coverslips with the cells were placed on top of the droplets with
the cellular surface facing the fluid. After one hour of incubation at 4°C (soluble
proteins) or at room temperature (antibody) the coverslips were put back in the plate
and washed three times with PBSM. After that, the cells were incubated with the
secondary antibody for 1 h at room temperature. Subsequently they were washed
two times with PBSM and one time with ultrapure water. The cells were then
incubated with 200µL DAPI per well for 10 minutes at 37°C before they were washed
two times with PBS and two times with ultrapure water. At last, drops of Mowiol were
put on microscope slides and the coverslips were placed on top.
3.9.2 Cryosections
Before the immunofluorescence staining, microscope slides with organ sections were
thawed and dried at room temperature. The organ slices were then fixed with PFA for
47
Methods
15 minutes at room temperature, quenched two times with glycine in PBSM, washed
and finally air-dried again. After the slices were incubated with the soluble protein at
4°C for 1 h, they were washed and incubated with the secondary antibody for 1 h at
room temperature. After the organ sections had been washed with PBSM and
ultrapure water they were covered with DAPI and incubated at 37° for 10 minutes.
Two washing steps with PBS and 2 steps with ultrapure water followed. The slices
were left to air-dry before Mowiol was added and covered with coverslips.
3.9.3 Image analysis
Cryosections and cell cultures were analyzed with the Nikon Eclipse Ti microscope.
Pictures were taken with the NIS Elements Imaging Software. For picture editing
GIMP and Image J software were used.
3.10 FACS
Cells were seeded in 6-well-plates. When the cell layer had reached confluence or, in
case of CKC the day after seeding, the cells were used for FACS analysis. To this
end, the cells were washed with PBSM and detached with 400 µL Trypsin/EDTA per
well. The Trypsin/EDTA was neutralized with 200 µL FCS. The cells were
resuspended several times and transferred into reaction tubes. After the tubes had
been centrifuged at 2000 rpm at 4°C for 5 minutes the cells were washed with FACS
buffer and then incubated with 300 µL of soluble protein for 1 h at 4°C on a rotator.
After the next incubation step with the secondary antibody under the same
conditions, the cells were washed two times, centrifuged and resuspended in 500 µL
FACS buffer containing only 0.5% BSA. For measurements the cells were transferred
into special tubes.
3.11 Protein biochemistry
3.11.1 Centrifugation of Cell culture supernatants for concentration
The cell culture supernatant of cells transfected with plasmids coding for soluble
proteins were collected 16 and 40 h post transfection and pooled (see 2.4). After
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48
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centrifugation at 4500 rpm for 15 minutes the liquid was stored at 4°C. For
concentration of the soluble proteins, spinning through a 100 kilo Dalton filter device
(Millipore) was used. Therefore 15 mL were transferred into the filter unit and the
devices were centrifuged at 4000 rpm for 15 minutes. Afterwards the flow through
was discarded and the concentrated medium collected. The process was repeated
until all of the supernatant was concentrated 35-40 fold.
3.11.2 Overlay binding assay
Viral glyco-proteins may not only bind to receptor proteins on intact cells but also to
proteins in linearized form, for instance after SDS PAGE and western blot.
For this experimental setting, lysates of cells with biotinylated surface proteins were
precipitated with streptavidin-agarose overnight. After washing the samples with
PBSM the proteins were eluted from the agarose in SDS-buffer at 96°C. Now they
could be separated according to size in SDS-PAGE and transferred to a nitro
cellulose membrane by western blot. Isolated membrane proteins of cells sensitive to
virus infection could be directly loaded on SDS-gels and transferred to western blot.
The blots were then incubated with soluble proteins corresponding to the respective
virus at 4°C overnight, before PO-labelled secondary antibodies were used to detect
binding.
3.11.3 SDS-PAGE
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a method
to separate proteins according to their molecular weight. For separation of soluble
proteins from cell culture supernatants gels were prepared consisting of a separating
gel with an acrylamide concentration of 8% and a stack gel. Samples were mixed
with 2xSDS- or 4xSDS-sample buffer in a ratio 1:1 or 1:3, respectively, and then
loaded on a gel. The electrophoresis was performed at 80 volts until the proteins
accumulated at the boundary of both gels and then continued at 135 volts.
For separation of isolated membrane proteins, cellular lysates or precipitates of
cellular lysates we used commercially available gels, because they allow for a better
separation of proteins.
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Methods
3.11.4 Western Blot
Semi-dry western blot is a method to transfer proteins from a gel to a membrane,
where they can be detected by antibodies. Two filter membranes drenched in anodebuffer I, one filter membrane drenched in anode-buffer II and a nitrocellulose
membrane activated by water application were layered on the anode of the slab
chamber. The gel was placed on top of this stack and covered with three filter
membranes drenched in kathode-buffer before the lid was shut. The blotting was
carried out at 350 mA for 1 hour. Afterwards blocking reagent was used to saturate
unspecific binding sites. It was applied overnight at 4°C on a swiveling table. The
next day the blot was washed with PBS containing 0.1% Tween for 8 minutes and
two times with PBS only for 8 minutes each. After incubation with an antibody, these
washing steps were repeated. If necessary, the application of a secondary antibody
and a repetition of the washing steps followed. One mL of Super Signal West Dura
Extended Duration Substrate was prepared according to manufacturer’s instructions
and applied to the blot membrane which was then covered with foil. After 5 minutes
of
incubation
the
staining
was
visualized
in
the
chemi-imager.
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Results
4 Results
4.1 Binding properties of the IBV spike protein
4.1.1 Structure and expression of chimeric soluble proteins
For investigation of the binding activities of the IBV spike protein, a soluble form of
this molecule was expressed in mammalian cells. The S2-subunit of the molecule,
the membrane anchor, and cytoplasmic tail were deleted and to facilitate the
detection of these proteins they were connected to a human IgG FC-tag (see Fig. 4).
The IBV spike protein of the B1648 strain, a Belgian field isolate that does not
possess additional binding activities as it has been reported for the Beaudette strain
(MADU et al. 2007) was chosen for the experiments.
As a negative control for all binding assays with chicken cells and tissues, a chimeric
spike protein from the SARS-CoV strain Frankfurt 1, which consisted also of the S1subunit connected to a human IgG-Fc-tag was used.
For binding assays on non-avian cells, supernatants from cells transfected with
empty vector were used as negative controls.
IBVS
IBBS1Fc
Fig. 4: Schematic drawing of the IBV spike protein. The size of the different
sections does not correspond to molecular weight. NTD indicates a putative Nterminal binding domain, CTD indicates a putative C-terminal binding domain.
The soluble IBV S1 protein has been provided by Christine Winter, the SARSS1Fc
has been kindly given by Dr. Jörg Glende. The expression of the proteins in
transiently transfected BHK-21 cells was verified through analysis of the harvested
supernatants in SDS-PAGE and western blot (see Fig. 5).
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Fig. 5: Detection of soluble chimeric proteins derived from coronaviruses IBVB1648 and SARS-CoV by goat anti-human IgG antibody marked with horseraddish
peroxidase in Western blot.
4.1.2 Detection of IBVS1Fc binding to different cells of chicken origin
The binding ability of the soluble IBV spike was tested on cryo-slices of chicken
kidney and embryonic and juvenile trachea, organs which are in vivo susceptible to
IBV infection. Additionally, fixed CEKC cultures were used for binding tests. As a
negative control the soluble spike protein SARSS1Fc was applied. Binding of the
spike proteins was detected by goat anti-human Cy3 antibody and visualized by
immunofluorescence microscopy. The IBV spike protein was able to bind to epithelial
cells of trachea as well as to those of kidneys in contrast to the SARS spike, as
depicted in Fig. 6. The latter result was confirmed in immunofluorescence binding
assays with primary chicken kidney cells. Here, the binding of the IBV spike could be
observed, although a strong background staining was present (see Fig.7).
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Fig. 6: Binding of IBVS1Fc to chicken tissues. Spike protein binding was detected
by the goat anti-human Cy3 antibody and appears red, cellular nuclei are stained
with DAPI and therefore appear blue.
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Fig. 7: Binding of IBVS1Fc to primary CEKC. Spike protein binding is depicted in
red, cellular nuclei are stained in blue.
4.1.3 Analysis of IBVS1Fc binding to chicken primary cells in FACS analysis
To quantify the binding of the IBV spike protein to chicken cells, a FACS analysis
was carried out. Binding of the IBVS1Fc spike protein was detected by flow
cytometry on CEKC (Fig. 8) and tracheal cells (Fig. 9), but not on chicken
erythrocytes (Fig. 10). The percentage of the cells, which were positively tested for
binding, was subject to variations. In FACS experiments with CEKC and chicken
erythrocytes an influenza hemagglutinin fused to a human Fc-tag was used as a
positive control, since this protein is also a type I viral fusion protein like the
coronavirus spike and host cell entry of influenza viruses is also sialic aciddependent (PAULSON 1985). In these experiments the H7Fc protein showed clearly
stronger binding to the target cells compared to the IBV spike.
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Results
Fig 8: Quantification of the binding capacity of IBVS1Fc to CEKC in FACS
analysis. SARSS1Fc served as negative control, H7 FC as positive control.
Fig. 9: Quantification of the binding capacity of IBVS1Fc to tracheal cells in
FACS-Analysis. SARSS1Fc served as negative control.
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Fig 10: Quantification of the binding capacity of IBVS1Fc to chicken
erythrocytes in FACS Analysis. SARSS1Fc served as negative control, H7Fc as
positive control.
4.1.4 Binding of the IBV spike protein is sialic acid-dependent
The
binding
specificity
of
the
soluble
proteins
was
also
assessed
by
immunofluorescence microscopy and FACS analysis. To this end cryosections (Fig.
11+20) and CEKC cultures (Fig. 19) were treated with neuraminidase to cleave off
the sialic acids from the cellular surface. Afterwards the neuraminidase-treated and
mock-treated cells were incubated with the spike proteins. The binding of IBVS1Fc
was clearly reduced on enzyme-treated organ sections and cell cultures in
comparison to the mock-treated controls, but never completely abolished. The same
was observed in FACS analysis. Here, neuraminidase-treated samples showed a
reduction compared to the untreated controls of up to 32% (see Fig. 12).
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Results
Fig. 11: Reduction of IBVS1Fc binding to cryosections of chicken kidneys after
neuraminidase treatment. Spike protein binding is depicted in red, cellular nuclei
are stained in blue.
Fig. 12: Reduction of IBVS1Fc binding to neuraminidase-treated CEKC in FACSanalysis. SARSS1Fc served as negative control.
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4.1.5 The IBV spike is able to bind to permanent cell cultures
The IBVS1Fc protein was also examined for its ability to bind to three permanent cell
lines. Since SARSS1Fc is known to bind to Vero cells this construct was used here
as a positive control. Likewise, the TGEVS1Fc protein served as a positive control for
binding to ST cells. For BHK 21 cells we had no positive control available.
Concentrated supernatants of cells transfected with empty vector were used as a
negative control. As shown in Fig. 13, all cell cultures incubated with the IBV spike
showed a signal in immunofluorescence microscopy in contrast to the cells that were
incubated with medium of mock-transfected cells only. However, the signal of the IBV
spike protein on all cell lines was weaker in comparison to the positive controls.
SARSS1Fc
TGEVS1Fc
Fig. 13: Binding of IBVS1Fc to permanent cell lines. Spike protein binding is
depicted in red.
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4.1.6 The IBV strain B1648 does hardly infect permanent cell lines
The B1648 virus was not able to infect BHK 21 cells and Vero cells efficiently.
Despite a high MOI, only very few infected cells were identified. For ST cells no
infected cells could be found (Fig. 14).
Fig. 14: Infection of permanent cell line with IBV B1648. Infection was detected by
staining with the Ch/IBV 48.4 monoclonal antibody and made visible with the rabbit
anti-mouse FITC antibody in fluorescence microscopy. It is depicted in green.
Cellular nuclei are stained with DAPI and appear blue.
4.2 Analysis of the sialic acid binding domain within the IBV S1
4.2.1 Deletion of 42 AA near the N-terminus of the spike protein results in a
reduced binding of the IBV spike protein
To localize the sialic acid binding site within the S1 domain of the IBV spike, the
amino acids 21 to 62 were deleted. It is known that a corresponding sequence in the
D274 strain contains the epitope for hemagglutination inhibiting antibodies (KOCH et
al. 1990).
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Del DS1Fc
Fig. 15: Schematic drawing the B1648 deletion mutant Del D-S1Fc. Close to the
N-terminus of the molecule 42 amino acids are deleted. The white band depicts the
deleted putative NTD, here named domain D.
Fig. 16: Detection of soluble chimeric proteins derived from IBV-B1648 deletion
mutant DelD by Western blot.
The construct Del D-S1Fc (see Fig. 15), which lacks the amino acids 21-62, was
expressed efficiently in BHK21 cells (see Fig. 16.). It showed reduced binding to
chicken cells and tissues in immunofluorescence binding tests compared to the
parental protein. The deletion did not completely abolish the ability to bind to CEKC
cultures and cryosections of chicken trachea, as can be seen in figures 19 and 20.
FACS analysis revealed that fewer cells were positive for the binding of the deletion
mutant compared to the parental protein (Fig. 17). Although the percentage of
positive cells for both constructs varied strongly between experiments, the ratio
between cells positive for IBVS1Fc and Del D-S1Fc binding remained more or less
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Results
constant. Usually about twice as many cells were positive for the parental protein
compared to the mutant.
Fig. 17: Quantification of the binding capacity to CEKC of the deletion mutant
Del D-S1Fc compared to the parental protein in FACS analysis. SARS S1 served
as negative control.
4.2.2 The residual binding of the Del D mutant is not sialic acid dependent
To determine the cause of the residual binding capacity of the Del D mutant,
immunofluorescence tests were carried out. Cryosections of chicken organs or CEKC
cultures were treated with neuraminidase before incubation with the different soluble
spike proteins to examine if the binding of Del D-S1Fc was still sialic-aciddependent. The enzymatic removal of sialic acids did not result in an apparent
reduction of the binding of Del DS1Fc. Moreover it appears as if the binding might
even be increased on neuraminidase-treated cells and cryosections (Fig. 19 and 20).
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This result is confirmed by the results of the FACS analysis. Here, the binding of Del
DS1Fc on neuraminidase-treated cells was also not reduced compared to the binding
to untreated cells but enhanced (Fig. 18).
Fig. 18: Quantification of the reduction of Del D-S1Fc binding to CEKC after
neuraminidase-treatment in FACS-analysis. SARS S1 served as negative control.
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Fig. 19: Binding of IBVS1Fc and Del D-S1Fc to neuraminidase-treated and
untreated CEKC cell cultures. Spike protein binding is depicted in red, cellular
nuclei are stained in blue.
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Fig. 20: Binding of IBVS1Fc and Del D-S1Fc to neuraminidase-treated and
untreated cryosections of chicken trachea. Spike protein binding is depicted in
red, cellular nuclei are stained in blue.
4.2.3 Analysis of shorter amino acid sequences responsible for the sialic acid
binding capacity
To narrow down the amino acids responsible for the sialic acid-binding, mutants were
generated which lacked only seven amino acids within the peptide sequence that had
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Results
been deleted in the mutant Del DS1Fc (see Fig. 21). These chimeric proteins were
efficiently expressed in BHK21 cells (see Fig. 22).
IBVS1Del21-27
IBVS1Del28-34
IBVS1Del35-41
IBVS1Del42-48
IBVS1Del49-55
IBVS1Del56-62
Fig 21: Schematic drawing of IBVS1Fc deletion mutants.
seven amino acids.
marks deletions of
The deletion mutants were tested for their binding characteristics in FACS analysis.
All mutants showed a reduced binding capacity in comparison to the parental protein.
The greatest reduction was seen with mutant Del 49-55. Here, the reduction
amounted to about 50%, comparable with the reduction seen with the Del D-S1Fc
mutant. The other mutants showed less reduction of the binding capacity (see Fig.
23). To determine whether amino acids outside the scope of domain D were involved
in host cell attachment and could thus be responsible for the residual binding
capacity, which we observed for Del D-S1Fc, three further mutants were produced. In
each of these mutants the subsequent seven amino acids were deleted. However,
these deletion mutants did not show a reduced binding to CEKC in FACS analysis
(see Fig. 24).
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Fig. 22: Detection of different IBVS1Fc deletion mutants by Western blot.
Fig. 23: Quantification of the reduction of different IBVS1Fc deletion mutants’
binding to CEKC in FACS-analysis. SARSS1Fc served as negative control.
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Results
Fig. 24: Quantification of the reduction of three further IBVS1Fc deletion
mutants’ binding to CEKC in FACS-analysis. SARSS1Fc served as negative
control.
4.2.4 Binding abilities of alanine mutants of the IBVS1Fc construct
Fig. 25: Alanine exchange mutants of the IBVS1Fc chimeric protein. The
respective amino acids along with their specific positions in the sequence of the spike
protein are given. (V= Valine, N= Asparagine, T= Threonine, E= Glutamic acid)
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The mutant IBVS1Del 49-55 showed the lowest binding affinity to CEKC ( see Fig.
23). Therefore it should be examined if any of the seven amino acids is especially
important for the binding of sialic acids. To test this, seven mutants were produced.
In each of these mutants one respective amino acid was exchanged for alanine (see
Fig. 25). All of the mutants were expressed after transient transfection in BHK21 cells
(see Fig. 26)
Fig. 26: Detection of seven alanine exchange mutants in supernatants of
transfected BHK21 cells by Western blot.
Binding abilities of these alanine exchange mutants were examined in FACS analysis
with CEKC. Here, all mutants showed reduced binding compared to the parental
protein. However, this reduction was only slight and none of the mutants stood out
from the others (see Fig. 27).
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Results
Fig. 27: Quantification of the reduction of different alanine exchange mutants’
binding capacities to CEKC in FACS-analysis. SARSS1Fc served as negative
control.
4.3 No evidence for a protein binding property of the IBV spike
protein
4.3.1 Overlay
To search for a cellular surface protein interacting with the IBV spike protein, an
overlay-assay was carried out. To this end isolated membrane proteins had been
separated by SDS-PAGE and were transferred to a nitrocellulose membrane by
Western blot. The blot membrane was then incubated with concentrated soluble Del
D-S1Fc overnight (see Fig. 28). However, binding of the spike protein to the
membrane proteins could not be detected. The control viral spike protein TGEV S1Fc
was detected on blotted membrane proteins of ST-cells in a similar experimental
setting. The mobility of the isolated membrane proteins detected here corresponded
roughly to the mobility of pAPN, the protein receptor of TGEV, of approximately 150
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kDa (see Fig. 28). Additionally a high molecular band was detected that has also
been detected in a similar experiment by Shahwan (2012).
Fig. 28: Search for cellular receptors with soluble proteins. Blots were incubated
with TGEVS1Fc or Del D-S1Fc, respectively.
4.3.2 Binding of different coronavirus spike proteins to protease-treated cells
To address the question if a protein receptor is involved in attachment of IBV to its
host cells, which could explain the residual binding after neuraminidase treatment,
cellular surface proteins of CEKC were cleaved off with either of the serine proteases
trypsin, derived from swine pancreas, or pronase, derived from Streptomyces
grisesus. Afterwards the cells were incubated with IBVS1Fc proteins and submitted
to FACS analysis. However, no reduction of the spike protein binding to trypsin- or
pronase-treated cells compared to the mock-treated control was observed. On the
contrary the binding seemed to be increased after the protease-treatment (see Fig.
71
Results
29 and 30). The same experiment was carried out with trypsin for the deletion mutant
Del D-S1Fc. Here, the increase of the binding after treatment was even stronger (see
Fig. 29).
To verify the effectiveness of the protease-treatment, it was also tested for
SARSS1Fc binding to Vero 76 cells and TGEVS1Fc binding to ST-cells. For both
viruses, SARS-CoV and TGEV, it is known that they attach to their host cells via
binding of the cellular proteins ACE-2 and pAPN, respectively (DELMAS et al. 1992;
LI et al. 2003). The binding of SARSS1Fc was indeed abolished by trypsin- and
pronase-treatment. In contrast, the binding of the TGEV spike to its host cell could
only be reduced by pronase-treatment but not by trypsin-treatment (see Fig. 30).
Fig. 29: Quantification of the reduction of different coronavirus spike proteins’
binding capacity to target cells after trypsin-treatment in FACS-analysis.
Results
72
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Fig. 30: Quantification of the reduction of different coronavirus spike proteins’
binding capacity to target cells after pronase-treatment in FACS-analysis.
73
Discussion
5 Discussion
Host cell attachment is in most cases the precondition for virus infections. It is the
first important step in the viral life cycle and in case of coronaviruses it is mediated by
the spike protein, more precisely the S1-subunit of the spike protein. For several
coronaviruses cellular proteins have been identified as receptors while for others,
including IBV, sialic acids serve as receptor determinants, as has been reviewed
before (SCHWEGMANN-WESSELS a. HERRLER 2006; LI 2014). Sialic acids have
been described previously (VARKI a. VARKI 2007). They constitute a group of ninecarbon monosaccharides, which carry a carboxyl group at the 1-carbon. Sialic acids
typically form the most distal monosaccharide units on the glycan chains of
glycolipids and glycoproteins, which are part of the glycocalyx covering all eukaryotic
cells. The linkage type to underlying sugar chains contributes to the diversity of sialic
acids as recognition sites (SCHAUER 2000; ANGATA a. VARKI 2002). Several
physiological functions of sialic acids have been revealed, most prominently perhaps
their involvement in the immune response (VARKI a. VARKI 2007) and in the
neurological development (RUTISHAUSER 2008).
IBV differs from other sialic acid binding viruses in lacking a receptor-destroying
enzyme and in possessing a lower affinity to these sugars than other viruses
(WINTER et al. 2006; WICKRAMASINGHE et al. 2011). The aim of the present study
was to characterize the binding properties of IBV spike proteins in more detail. To
this end soluble Fc-tagged forms of the protein were used.
5.1 Assessment of the binding characteristics of the construct
IBVS1Fc
Recombinant viral attachment proteins connected to a human IgG Fc tag have been
used before to study the binding behavior of coronavirus spike proteins (SHAHWAN
et al. 2013; MORK et al. 2014) and influenza virus hemagglutinins (SAUER et al.
2014). In the present study, it could be shown that the construct IBVS1Fc is able to
bind to different cells like CEKC, and epithelial cells of trachea and kidney in
immunofluorescence assays and in FACS analysis. Winter and colleagues have
Discussion
74
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demonstrated that the corresponding B1648 strain infects TOCs and CEKC cultures
(WINTER et al. 2008) which correlates with the results of our binding assays. Since
binding is the first step of infection, this finding supports the supposition that the
binding behavior of the soluble spike protein reflects the binding behavior of the viral
particles.
However, our results concerning the binding properties of IBVS1Fc are in contrast to
the observations of WICKRAMASINGHE et al. (2011). This group also constructed a
soluble IBV B1648 S1 protein and analyzed its binding capacity to trachea, lung,
intestine and kidney. In these cases, no binding was observed at all. One might
speculate that this difference in the binding function of the two soluble proteins might
be due to the usage of a different construction. Wickramasinghe et al. used a
trimerisation domain to induce a multimerization and a Strep-tag to detect the protein.
The different constructions might have influence on the folding of the proteins or the
accessibility of the binding domains. It has been argued that for the spike proteins of
some IBV-strains the S1-subunit alone is not sufficient to mediate binding (DE HAAN
et al. 2006; PROMKUNTOD et al. 2013). Promkuntod et al. examined the spike
protein of the Beaudette strain. Whereas the S1-subunit alone did not show binding
to susceptible embryonic chorio-allantoic membrane, a construct of the whole spike
protein ectodomain, containing both subunits could bind to this tissue. It seems
possible that the S2-subunit is not required to enable spike protein binding, but the
presence of a larger molecule might be necessary for correct folding. The Fc-tag
comprises roughly 230 amino acids and is therefore bigger than the Strep-tag used
by Wickramasinghe et al. Indeed, a dimeric, Fc-tagged IBVB1648S1 protein showed
slightly increased binding compared to the trimeric form in the same lab
(WICKRAMASINGHE et al. 2014). It should further be pointed out, that the ability to
bind CEKC and epithelial cells of trachea of the IBVS1Fc-construct, which was used
in the present study, corresponds with the sensitivity of these cells to infection with
the parental virus (WINTER et al. 2008). Binding to host cells is for most
coronaviruses a prerequisite for infection. A lack of binding to chicken kidney or
trachea, as asserted by Wickramasinghe et al. seems unlikely in this context.
Moreover, it could be demonstrated for the IBVS1Fc construct used in this study, that
75
Discussion
its binding property to epithelial cells of the immature chicken oviduct corresponds to
the binding patterns of whole virus particles (MORK et al. 2014).
Compared to the hemagglutinin (HA) of the Influenza subtype virus H7N7 the binding
of the IBV spike protein to tracheal cells was weaker, as was evidenced by FACS
analysis. The same was observed by Wickramasinghe et al. They analyzed the
binding of the IBV S1 protein derived from the M41 strain to different avian tissues
and compared it with an Influenza hemagglutinin (H5N1-HA). The amount of soluble
HA proteins which was required to detect binding in histochemistry was lower than
the amount of soluble IBVS1 needed. These results can be explained by a weaker
binding affinity of the IBV spike to the receptor determinant sialic acid compared to
the influenza virus protein (WINTER et al. 2006).
Remarkable is, that IBVS1Fc was not able to bind to chicken erythrocytes, although
some IBV strains have been shown to agglutinate chicken erythrocytes (BINGHAM et
al. 1975). Due to the fact, that IBV virions need neuraminidase-pretreatment to be
able to hemagglutinate, the hemagglutinating activity of IBV is considered to be quite
weak (BINGHAM et al. 1975; SCHWEGMANN-WESSELS a. HERRLER 2006)
compared to that of other viruses. Perhaps, binding to erythrocytes was below the
detection limit of FACS analysis.
Interestingly, the quantity of bound proteins, as measured in FACS analysis, differed
strongly between different experiments of our study, especially in case of the primary
CEKC. Several explanations seem possible. First, these cells are primary cells. They
are prepared freshly for each experiment. Although glycosylation patterns have so far
only been reported to be altered under certain conditions including embryogenesis,
cancer, injury and inflammation (BOHM 2010) it cannot be excluded that also
different cell preparations feature different glycan patterns. Second, primary CEKC
cannot be cultured purely, since it is not possible to remove all erythrocytes and
fibroblasts from the cultures. In FACS analysis we could demonstrate that there is no
IBVS1Fc attachment to erythrocytes. A varying portion of erythrocytes in the cultures
might partially explain differences in the percentage of cells which are positive for
spike protein binding. In immunofluorescence analysis of binding tests on CEKC, the
Discussion
76
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spike construct binds to the epithelial cells rather than to fibroblasts. Our results
indicate that the spike proteins attach only to the epithelial cells. The portion of these
cells in the culture is variable, which might as well explain the variations between
different experiments.
5.2 Sialic acid dependence of IBVS1Fc binding
The sialic acid dependence of an IBV infection and in particular of infection by the
strain B1648 has been demonstrated before (WINTER et al. 2006; WINTER et al.
2008; ABD EL RAHMAN et al. 2009). In this study we could show that the binding of
the chimeric soluble spike protein IBVS1Fc to CEKC and to epithelial cells of chicken
kidney and trachea is also influenced by the presence of sialic acids on host cell
surfaces. Sialic acid dependent binding of different soluble IBV spike proteins,
including the B1648 spike has been shown in our lab previously (SHAHWAN et al.
2013; MORK et al. 2014) and by others (WICKRAMASINGHE et al. 2011). In the
study of Wickramasinghe et al. binding of the S1-subunit of the IBV M41 strain to
chicken trachea and lung was completely abolished after neuraminidase-treatment of
these tissues. In the present work it was observed that the binding of the IBVS1Fcconstruct was clearly reduced after such a treatment. However it was not abolished
completely. This could be shown in immunofluorescence analysis as well as in
quantitative assessment of the binding in FACS analysis. The residual binding might
be explained by incomplete removal of sialic acids by the neuraminidase used. An
alternative explanation would be that IBVS1Fc possesses an additional binding
activity that is not sialic acid dependent, as has been observed for other
coronaviruses (DELMAS et al. 1992; KREMPL et al. 1997). Such additional binding
might for instance involve cellular proteins or lipids.
5.3 Analysis of the sialic acid binding domain of IBVS1Fc
5.3.1 Assessment of mutant Del D-S1Fc
The receptor binding capacity of the spike protein of different coronaviruses has been
mapped to the S1-subunit. Principally two receptor binding domains exist within this
subunit, one more N-terminally (NTD) and one more C-terminally located (CTD).
77
Discussion
Sugar binding affinities have been assigned to the N-terminus of the spike protein
before (TSAI et al. 2003; PENG et al. 2012). At the beginning of this study the
localization of the receptor binding domain of IBV was unknown. Koch and
colleagues mapped a hemagglutination-inhibiting monoclonal antibody against the
spike protein, which attaches to a region comprising 42 amino acids close to the Nterminus of the spike protein (KOCH et al. 1990). Hemagglutination is based on
binding of the spike protein to sialic acids on the surface of erythrocytes. A deletion of
the corresponding 42 amino acids in the soluble spike protein led to a reduced affinity
to epithelial cells of chicken tissues and CEKC in immunofluorescence and CEKC in
FACS analysis. This substantiated the theory that the deleted region plays a role in
sialic acid dependent attachment to host cells. The reduction was always not
complete. It reached at most a reduction of about 50%. Several possible
explanations for that residual binding need to be considered. First, more than the 42
amino acids are involved in sialic acid binding and therefore the deleted domain is
too small to abolish attachment completely. Usually, RBDs of coronaviruses
comprise several hundred amino acids, but within these RBDs several residues that
are indeed critical for binding can be identified. These critical residues may be
clustered in one or more different regions, thus forming distinct epitopes, which have
been named receptor binding motifs (LIN et al. 2008; LI 2012). Three mutants with
deletions of 7 amino acids each were produced. The deletions were located Cterminally of the domain D. The mutants however did not reveal a reduced affinity to
CEKC in FACS analysis (see chapter 4.2.3) in contrast to mutants with deletions
within the range of epitope D. More importantly, the residual binding of Del D was not
sialic acid dependent which argues against the involvement of more epitopes in sialic
acid binding. A second possible explanation for the partial reduction of binding in the
mutant Del D-S1Fc would be, that IBV does not only make use of its N-terminal RBD
but also of a second RBD with a different binding affinity. At this point it should also
be mentioned that after neuraminidase- treatment of CEKC or TOCs prior to
infection, the infection by IBV was only reduced by ~50% (WINTER et al. 2006; ABD
EL RAHMAN et al. 2009). Taken together these results would suggest that sialic acid
binding is only partially responsible for host cell attachment of IBV.
Discussion
78
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Remarkable is also the binding behavior of Del D-S1Fc to neuraminidase-treated
chicken cells and tissues, which resembled the binding behavior of the soluble
TGEVS1Fc on TGEV-susceptible cells and tissues. Both soluble spike proteins show
an increased affinity to their target cells and tissues if these had been treated with
neuraminidase (SHAHWAN et al. 2013). In contrast to the spike protein incorporated
into whole virions, TGEVS1Fc does not possess sialic acid binding affinity
(SHAHWAN et al. 2013). A lack of sialic acids on target cells might render the protein
receptor, pAPN, more accessible for the TGEV spike, which would explain the
increased binding. One could assume, that increased binding of the IBV spike
without sialic acid binding domain to neuraminidase-treated cells and tissues would analog to TGEVS1Fc- also be due to an increased accessibility of a so far unknown
protein receptor of IBV.
5.3.2 Identification of important sequences and single amino acids for
attachment within domain D
To further narrow down the range of amino acids which are important for sialic acid
binding, mutants with seven deleted amino acids out of the 42 amino acids of the D
epitope have been analyzed for their binding capacity to CEKC in FACS analysis. All
six mutants showed a reduced affinity to CEKC, most prominently mutant
IBVS1Del49-55. For IBVS1Del49-55, the reduction amounted to about 50%
compared to the parental protein IBVS1Fc and thus equaled the reduction observed
for Del D-S1Fc. Therefore, single amino acids out of the seven amino acids deleted
in mutant IBVS1Del49-55 were exchanged for alanine. These exchange mutants
showed only a minor reduction in their affinity to CEKC in FACS analysis compared
to IBVS1Fc. It seems, that the exchange of only one amino acid within this sequence
area is not enough to influence the binding capacity of the spike protein to sialic acid
molecules.
PROMKUNTOD et al. (2014) made a different approach to map the sialic acid
binding activity of the IBV spike protein. These authors detected a difference in the
binding behavior of a soluble spike protein derived from the M41 strain and the one
derived from the Beaudette strain. Whereas the former could bind to chicken trachea
79
Discussion
and lung, the latter could not. Since the Beaudette strain has been originated from
the M41 strain by passaging in egg culture, they are closely related and differ in their
S1-sequence only by 26 out of the total of 532 amino acids (PROMKUNTOD et al.
2014). Prokumtod and colleagues in this study examined whether any of the
mentioned amino acids would be responsible for the difference in binding behavior.
When they introduced the 15 most N-terminal amino acids unique to the M41
sequence into the Beaudette spike protein, the binding characteristics of the M41
spike were also transferred. The same was observed, when only the first seven
amino acids (A19V, N38S, H43Q, S56F; P63S, I66T T69I), which are unique to M41
were introduced into the Beaudette spike protein, but not, when the following eight
amino acids unique to M41 were replaced.
The introduction of a single M41 specific amino acid did not alter the binding
characteristics of the Beaudette spike. However, the binding ability of the M41 spike
was abolished, when some single Beaudette specific amino acids were introduced
into the M41 background N38S, H43Q, P63S, T69I. The other way around,
introducing the respective M41 specific amino acids into the Beaudette spike had no
effect. These results are in contrast with our findings that domain D, comprising aa
21-62, and especially the aa 49-55 within this range, contribute decisively to the
binding capacity of the spike protein. Moreover in contrast to the trimeric soluble
Beaudette spike, which was used by Prokumtod et al., the dimeric Fc-tagged
Beaudette S1 protein did bind to chicken trachea and to CEKC (HESSE 2012). The
different results regarding the binding capacity of the Beaudette spike obtained by
these two groups could be due to different conformations induced by the trimerisation
domain or the tags used. However, the binding behavior of the dimeric protein
corresponds with the ability of the Beaudette virus to infect TOCs and CEKC in vitro
(WINTER et al. 2006; ABD EL RAHMAN et al. 2009) and therefore reflects the
infectivity of the virus better than the results of Prokumtod et al.
Discussion
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5.4 Possibility of involvement of further main- or co-receptors in
IBV host cell attachment
So far no protein receptors for any of the gammacoronaviruses have been identified
(BELOUZARD et al. 2012). However, the notion, that i) the binding of IBVS1Fc to its
target tissues could not be completely abolished by neuraminidase-treatment of the
cells, that ii) the deletion mutant Del D-S1Fc showed only a reduction of the binding
capacity and not an complete abolishment, and that iii) neuraminidase-treatment of
target tissues increased the affinity of Del D-S1Fc, led to the question, whether an
additional binding activity next to the sialic acid binding property is present in the IBVspike protein. This supposition is supported by the fact, that the tissue tropism and
host range of coronaviruses is commonly attributed to the binding abilities of their
spike proteins. Sialic acids, which have been shown to be the receptor determinants
of IBV, are present in many species and tissues, which are not infected by the virus.
On the other hand, it has to be considered, that IBV spike proteins have been
demonstrated to bind only to a narrow range of different sugars in glycan arrays
(WICKRAMASINGHE et al. 2011), which might be not so ubiquitously present.
Another argument for our supposition has been made by CASAIS et al. (2003). They
pointed out, that an abolishment of the infectibility of vero cells after switching the
Beaudette spike for the M41 spike in a recombinant IBV, is also an indication for the
involvement of a secondary receptor that could be recognized by the Beaudette but
not by the M41 spike. We therefore tried to identify cell surface proteins interacting
with the spike protein using different biochemical methods. However, no specific
binding partners could be detected in pulldown and overlay assays. For this reason
we tried a more direct approach to address the question whether or not a protein
receptor is involved in binding of IBV to host cells. FINKELSHTEIN et al. (2013) have
answered this question for VSV by showing, that protease treatment of normally
susceptible cells abolished the ability of VSV to infect these cells. According to that,
we treated detached CEKC with two different proteases. Subsequently, instead of
trying to infect the cells with IBV, we incubated them with soluble spike proteins. In
FACS analysis we could not detect a diminishment of IBVS1Fc-binding to CEKC
after treatment with trypsin or pronase compared to the control. However, this does
81
Discussion
not exclude the involvement of a protein receptor in IBV host cell binding, which is
resistant to trypsin- or pronase-treatment. The cellular receptor of TGEV, pAPN, for
instance could be destroyed by pronase but was resistant to trypsin-treatment. At
least it seems unlikely, that a protein is involved. The residual binding capacity of
IBVS1Fc to neuraminidase-treated cells and of the deletion mutant Del D-S1Fc might
actually also be explained by use of a glycolipid rather than a protein as a secondary
receptor. For Sendai virus for instance it has been shown that specific gangliosides,
which indeed are glycolipids function as host cell receptors (MARKWELL et al. 1981).
5.5 Binding of the IBV spike to continuous cell lines
The IBV spike can bind to BHK 21 cells, ST cells and Vero cells, though only weakly
compared to positive controls (TGEVS1Fc for ST cells, SARSS1Fc for Vero76 cells).
Most coronaviruses infect only cell cultures derived from their host species and most
IBV strains can only be cultivated in primary cell or tissue cultures of chicken origin.
The IBV Beaudette strain is generally thought to be an exception, because it is a cell
culture adapted strain which infects BHK and Vero cells (OTSUKI et al. 1979; MADU
et al. 2007). Since receptor binding is an important factor determining host range and
tissue tropism, the attachment of the soluble IBV spike protein of the B1648 strain to
the three permanent cell lines was analyzed and also infection of these cells by the
virus itself. Only very few BHK 21 cells and Vero cells and no ST cells could be
infected although a high MOI was applied, but binding was observed to all three cell
lines. On the one hand, B1648 is a field strain, which has not undergone cell culture
adaptation (MEULEMANS et al. 1987). Thus its poor ability to infect permanent cell
lines is not surprising. On the other hand, a difference between the ability of a virus to
infect cells and the ability of the corresponding spike protein to bind to these cells
contrasts with reports that the RBD is the principal factor determining the host range
of coronaviruses (ENJUANES et al. 2006; PERLMAN a. NETLAND 2009; GRAHAM
a. BARIC 2010). This finding might also suggest that a further main or co-receptor
participates in host cell entry of IBV, which is not as ubiquitously distributed as sialic
acids. Cavanagh and colleagues have noted before that neuraminic acids are
present on cells, which are resistant to infection with IBV and also suggested the
Discussion
82
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involvement of a secondary receptor as a possible explanation (CAVANAGH 2007).
Whereas sugar binding might enable a first attachment of IBVS1Fc to permanent cell
lines, a secondary receptor might be required for further steps, which is not present
on the examined permanent cell lines. Also the residual binding to neuraminidasetreated cells and the residual binding of mutant Del-DS1Fc argue for the participation
of further main or co-receptors in host cell attachment.
However, for the infection process also other restricting factors are conceivable.
Although binding has been identified as an important restraining factor for host range
(ENJUANES et al. 2006; PERLMAN a. NETLAND 2009; GRAHAM a. BARIC 2010),
other steps of the viral life cycle have been proposed to be restricting factors for host
range and tissue tropism as well. These factors shall be discussed in the following
paragraphs.
5.6 Further factors possibly determining host range of IBV
Spike proteins are often cleaved by host cell proteases and host range might
therefore be limited by the distribution of host cell proteases. KLENK and GARTEN
(1994) asserted that for some viruses, which are not cleaved by ubiquitous
intracellular proteases but by secreted proteases, the host range is influenced by the
occurrence of those host cell proteases. Most IBV spike proteins are cleaved by furin,
a serine protease. Despite the widespread occurrence of furin, it might well be a
restraining factor since the expression in many cells is low (SHAPIRO et al. 1997;
MILLET a. WHITTAKER 2014), possibly too low for effective cleavage of the spike.
It has been observed, that the in vitro cell tropism of IBV can also be affected by the
S2 subunit of the spike protein. When the S1 subunit of the H120 strain, a vaccine
strain without extended tropism for continuous cell lines, was introduced to form a
recombinant virus with Beaudette backbone and the S2-subunit of the Beaudette
strain, it retained the ability to grow in Vero cells (WEI et al. 2014). In vivo however,
this recombinant was also able to infect and replicate in the chicken respiratory tract,
an ability that is derived from the S1 donor strain H120 rather than from the
Beaudette backbone. A possible explanation for this is, that not the sialic acid binding
which resides in the S1-subunit, is responsible for the extended host range of the
83
Discussion
Beaudette strain. IBV Beaudette possesses also an additional binding capacity for
heparan sulfate (MADU et al. 2007) and additional furin cleavage site (MILLET a.
WHITTAKER 2014), both located in the S2-subunit, which might both also play a role
in defining host range. Also mutations that drive cell-cell fusion have been made
responsible for determining the tropism of IBV (YAMADA et al. 2009). In vivo, the
introduction of the M41 spike protein into Beaudette backbone resulted in an
apathogenic recombinant virus (HODGSON et al. 2004)
Also proteins of the replicase complex might present a determining factor of host
range in vivo, as has been summarized elsewhere (WICKRAMASINGHE et al. 2014).
AMMAYAPPAN et al. (2009) showed that the main sequence differences between a
virulent and an avirulent variant of the IBV-strain ArkDPI IBV occurred not only in the
spike protein but also in ORF1a, a region that is coding for replicase proteins. It was
also observed that the introduction of the replicase gene of Beaudette into a M41
backbone yielded a chimeric, apathogenic virus that did not replicate in the trachea
(ARMESTO et al. 2009) and thus reflects the behavior of the Beaudette strain. In
contrast, other studies found identical sequences for replicase genes in vaccine and
virulent strains and thus no correlation of this gene with the pathotype (MONDAL a.
CARDONA 2004).
5.7 Conclusion
It could be demonstrated that IBVS1Fc soluble proteins are valuable tools for the
characterization of the IBV spike protein binding. Their binding to target tissues is
partially sialic acid dependent and the binding to respiratory tract epithelial cells and
chicken kidney cells reflects the ability of the virus to infect these cells and tissues.
Deletion of certain amino acids could reveal a region important for the sialic acid
binding ability of the IBV spike protein. However, some open questions remain. It
remains to be elucidated, if further main or co-receptors are involved in IBV-spike
protein binding to host cells. In the present study some indications in favor of this
perception have been presented. First, the binding of the deletion mutants to target
tissues was merely reduced, not abolished. The fact that the residual binding
capacity was shown not to depend on sialic acids argues for the existence of
Discussion
84
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additional RBDs for yet unknown main- or co-receptors. Moreover, the difference
between binding to and infection of continuous cell lines might as well indicate the
participation of further receptors. This view is also supported by the fact that
neuraminidase-treatment of target cells and tissues seems to increase the affinity of
mutant Del D. The same phenomenon has been observed for the TGEVS1Fc
protein, which has lost the sialic acid binding capacity but retained the ability to bind
to protein receptors (SHAHWAN et al. 2013). However in case of IBV, it is unlikely
that a protein constitutes an additional receptor. Binding of neither IBVS1Fc nor Del
D-S1Fc to protease-treated cells was reduced. This might be explained by the
existence of protease-resistant protein receptors or alternatively by the use of a
glycolipid as receptor. For a better understanding of IBV attachment to host cells it
would be desirable to clarify the crystal structure of possibly the whole spike protein
or at least the S1 subunit. Then, it would be possible to compare the spike protein
with other type I transmembrane proteins, like influenza hemagglutinin. The
knowledge of crystal structures would also be helpful to identify the accessibility of
RBDs directly. However, as MILLET a. WHITTAKER (2014) have stated, so far there
has been no complete crystal structure determinations identified for any coronavirus
spike protein. Only individual structures of key domains as the RBDs of several
coronavirus spike proteins have been clarified (XU et al. 2004b; LI et al. 2005a; WU
et al. 2009; PENG et al. 2011; PENG et al. 2012). For the determination of the
significance of the IBV spike protein for the in vivo tropism of the virus further clinical
studies are needed.
85
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99
Appendix
7 Appendix
7.1 Amino acids
Amino acid
Alanine
Cysteine
Aspartic acid
Glutamic acid
Phenylalanine
Glycine
Histidine
Isoleucine
Lysine
Leucine
Methionine
Asparagine
Proliine
Glutamine
Arginine
Serine
Threonine
Selenocysteine
Valine
Tryptophan
Tyrosine
Three-Letter-Code
Ala
Cys
Asp
Glu
Phe
Gly
His
Ile
Lys
Leu
Met
Asn
Pro
Gln
Arg
Ser
Thr
Sec
Val
Trp
Tyr
One-Letter-Code
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
U
V
W
Y
7.2 Sequences
7.2.1 B1648S1 nucleotide sequence
ATGTTGGTAGTGCAACTTTCAGTAGTGACTCTTTTGTTTGCACTATGTAGTGCTA
TTTTGTTCAATGATAATTATAATTATTACTACCAAAGTGCCTTCAGACCACCCTCA
GGGTGGCATTTACATGGGGGTGCTTATCAAGTGGTCAATGTTACTAATGAAAAT
AATAATGCAGGTTCATCACCAACATGCACTGCAGGTGTTATTTATTATAGTAAAA
ATTTTACTGCTTCTTCTGTAGCCATGACTGCACCACCACCAGGAATGTCATGGTC
CACTTCAGAATTTTGTACGGCCCACTGTAATTTTAGTACATTTACAGTGTTCGTTA
CACATTGTTTTAAAAGCGGTGCAGGCCAATGTCCTTTAACTGGTTTAATACAACA
GGGATATATTCGTGTCTCTGCTATGAAAACGGAAGGTGAATCATACCTATTTTAT
AATTTAACATTGCCAACGACTAAACATCCTAAGTTTAGGTCGCTACAATGTGTTA
ATAATCAAACATCTGTGTATTTAAATGGTCATCTTGTCTTTACTTCTAATGAGACT
TTAGATGTTAGTGCGGCAGGTGTTTATTTTAAATCTGGTGGACCTATAACTTATA
AAGTTATGAGAGAAGTTAAAGCCCTTGCTTATTTTGTTAATGGTACTGCACATGA
Appendix
100
____________________________________________________________________
TGTAATTTTGTGTGACAATTCACCAAGGGGTTTGTTAGCATGCCAGTACAACACT
GGTAATTTTTCAGATGGATTTTATCCTTTTACTAATTCTTCTTTAGTTAAGGAAAA
GTTTATTGTTTATCGTGAAAGTAGTGTGAATACTACATTAGAGTTAACTAACTTCA
CATTTTCAAATCAAAGTAATGCTACACCCAATAGTGGTGGTGTTAATACCTTTTCA
TTATATCAAACACACACAGCTCAGAGTGGTTATTATAATTTTAACTTTTCTTTTCT
GAGTGGGTTTACGTATAAACCATCCGATTTTATGTATGGGTCATATCACCCACGT
TGTAATTTTAGACCAGAGAATATTAATAATGGCTTATGGTTTAATTCATTAACTGT
GTCACTTACTTATGGACCTCTTCAAGGTGGTTGTAAGCAATCGGTTTTTAGTAAT
AGAGCAACTTGTTGTTATGCTTATTCTTATAATGGACCCATATCTTGTAAAGGTGT
TTATTCAGGTGAATTAAACCAATATTTTGAATGTGGTTTGTTGGTTTATGTTACTA
AGAGCGATGGCTCTCGCATACAAACGGCAACAGAACCACCCATTTTTACGGAAA
ATTATTATAACAACATTACTTTAGGTAAGTGTGTTGAGTATAATATATATGGTAGA
TTTGGTCAAGGTTTTATTACTAATGTAACTGATTCAGCTGCTAATTTTAATTATTTA
GCAGATGGTGGCTTAGCTATTTTAGATACGTCTGGAGCCATAGACATCTTTGTTG
TTCAAGGTGACTATGGTCTTAATTATTATAAGGTTAATCCCTGTGAAGATGTAAAT
CAGCAGTTTGTAGTCTCTGGTGGTAATATAGTAGGTGTCCTTACATCAATTAATG
AAACTGGTTCTCAATTTGTTGGGAATCAGTTTTATGTTAAACTCACTAATAGTACA
7.2.2 B1648S amino acid sequence
MLVVQLSVVTLLFALCSAILFNDNYNYYYQSAFRPPSGWHLHGGAYQVVNVTNENN
NAGSSPTCTAGVIYYSKNFTASSVAMTAPPPGMSWSTSEFCTAHCNFSTFTVFVTH
CFKSGAGQCPLTGLIQQGYIRVSAMKTEGESYLFYNLTLPTTKHPKFRSLQCVNNQ
TSVYLNGHLVFTSNETLDVSAAGVYFKSGGPITYKVMREVKALAYFVNGTAHDVILC
DNSPRGLLACQYNTGNFSDGFYPFTNSSLVKEKFIYRESSVNTTLELTNFTFSNQSN
ATPNSGGVNTFSLYQTHTAQSGYYNFNFSFLSGFTYKPSDFMYGSYHPRCNFRPE
NINNGLWFNSLTVSLTYGPLQGGCKQSVFSNRATCCYAYSYNGPISCKGVYSGELN
QYFECGLLVYVTKSDGSRIQTATEPPIFTENYYNNITLGKCVEYNIYGRFGQGFITNV
TDSAANFNYLADGGLAILDTSGAIDIFVVQGDYGLNYYKVNPCEDVNQQFVVSGGNI
VGVLTSINETGSQFVGNQFYVKLTNST
7.2.3 Nucleotide sequence of the linking sequence and of the Fc-tag
GGATCCTTAATTAAGTCTAGAGTCGACTGTTTAAACCTGCAGGCATGCAAGCTT
GATATCATCGAAGGTCGTGGTGGTGGTGGTGGTGATCCCAAATCTTGTGACAAA
CCTCACACATGCCCACTGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA
GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA
GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC
AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCA
GCCTGACCTGCCTAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGG
101
Appendix
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGGCCACGCCTCCCGTGCTGG
ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGT
GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
7.2.4 Amino acid sequence of the linking part and the Fc-tag
GSLIKSRVDCLNLQACKLDIIEGRGGGGGDPKSCDKPHTCPLCPAPELLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
7.2.5
Nucleotide sequences of different B1648S1 deletion mutants
atgttggtagtgcaactttcagtagtgactcttttgtttgcactatgtagtgctattttgttcaatgataattataattattactacc
aaagtgccttcagaccaccctcagggtggcatttacatgggggtgcttatcaagtggtcaatgttactaatgaaaataat
aatgcaggttcatcaccaacatgcactgcaggtgttatttattatagtaaaaattttactgcttcttctgtagccatgactgca
ccaccaccaggaatgtcatggtccacttcagaattttgtacggcccactgtaattttagtacatttacagtgttcgttacaca
ttgttttaaaagcggtgcaggccaatgtcctttaactggtttaatacaacagggatatattcgtgtctctgctatgaaaacg
gaaggtgaatcatacctattttataatttaacattgccaacgactaaacatcctaagtttaggtcgctacaatgtgttaata
atcaaacatctgtgtatttaaatggtcatcttgtctttacttctaatgagactttagatgttagtgcggcaggtgtttattttaaat
ctggtggacctataacttataaagttatgagagaagttaaagcccttgcttattttgttaatggtactgcacatgatgtaatttt
gtgtgacaattcaccaaggggtttgttagcatgccagtacaacactggtaatttttcagatggattttatccttttactaattct
tctttagttaaggaaaagtttattgtttatcgtgaaagtagtgtgaatactacattagagttaactaacttcacattttcaaatc
aaagtaatgctacacccaatagtggtggtgttaataccttttcattatatcaaacacacacagctcagagtggttattata
attttaacttttcttttctgagtgggtttacgtataaaccatccgattttatgtatgggtcatatcacccacgttgtaattttagacc
agagaatattaataatggcttatggtttaattcattaactgtgtcacttacttatggacctcttcaaggtggttgtaagcaatc
ggtttttagtaatagagcaacttgttgttatgcttattcttataatggacccatatcttgtaaaggtgtttattcaggtgaattaa
accaatattttgaatgtggtttgttggtttatgttactaagagcgatggctctcgcatacaaacggcaacagaaccaccca
tttttacggaaaattattataacaacattactttaggtaagtgtgttgagtataatatatatggtagatttggtcaaggttttatt
actaatgtaactgattcagctgctaattttaattatttagcagatggtggcttagctattttagatacgtctggagccatagac
atctttgttgttcaaggtgactatggtcttaattattataaggttaatccctgtgaagatgtaaatcagcagtttgtagtctctgg
tggtaatatagtaggtgtccttacatcaattaatgaaactggttctcaatttgttgggaatcagttttatgttaaactcactaat
agtaca
Underlined = nucleotides deleted in mutant Del D; green = nucleotides deleted in
mutant Del 21-27; yellow = nucleotides deleted in mutant Del 28-34; red =
nucleotides deleted in mutant Del 35-41; light blue = nucleotides deleted in mutant
Del 42-48; blue = nucleotides deleted in mutant Del 49-55; pink = nucleotides deleted
in mutant Del 56-62; grey = nucleotides deleted in mutant Del 63-68; bluegreen =
Appendix
102
____________________________________________________________________
nucleotides deleted in mutant Del 69-76; olive = nucleotides deleted in mutant Del
77-83
7.2.6 Amino acid sequences of different B1648S1 deletion mutants
MLVVQLSVVTLLFALCSAILFNDNYNYYYQSAFRPPSGWHLHGGAYQVVNVTNENN
NAGSSPTCTAGVIYYSKNFTASSVAMTAPPPGMSWSTSEFCTAHCNFSTFTVFVTH
CFKSGAGQCPLTGLIQQGYIRVSAMKTEGESYLFYNLTLPTTKHPKFRSLQCVNNQ
TSVYLNGHLVFTSNETLDVSAAGVYFKSGGPITYKVMREVKALAYFVNGTAHDVILC
DNSPRGLLACQYNTGNFSDGFYPFTNSSLVKEKFIVYRESSVNTTLELTNFTFSNQS
NATPNSGGVNTFSLYQTHTAQSGYYNFNFSFLSGFTYKPSDFMYGSYHPRCNFRP
ENINNGLWFNSLTVSLTYGPLQGGCKQSVFSNRATCCYAYSYNGPISCKGVYSGEL
NQYFECGLLVYVTKSDGSRIQTATEPPIFTENYYNNITLGKCVEYNIYGRFGQGFITN
VTDSAANFNYLADGGLAILDTSGAIDIFVVQGDYGLNYYKVNPCEDVNQQFVVSGG
NIVGVLTSINETGSQFVGNQFYVKLTNST
Underlined = amino acids deleted in mutant Del D; green = amino acids deleted in
mutant Del 21-27; yellow = amino acids deleted in mutant Del 28-34; red = amino
acids deleted in mutant Del 35-41; light blue = amino acids deleted in mutant Del 4248; blue = amino acids deleted in mutant Del 49-55; pink = amino acids deleted in
mutant Del 56-62; grey = amino acids deleted in mutant Del 63-68; bluegreen =
amino acids deleted in mutant Del 69-76; olive = amino acids deleted in mutant Del
77-83
7.2.7 Alignment of the N-terminus of B1648S1, M41S1 and BeaudetteS1
B1648
BdS1
M41
MLVVQLSVVTLLFALCSAILFNDN-YNYYYQSAFRPPSGWHLHGGAYQVVNVTNENNNAG 59
MLVTPLLLVTLLCALCSAVLYDSSSYVYYYQSAFRPPSGWHLQGGAYAVVNISSEFNNAG 60
MLVTPLLLVTLLCVLCSAALYDSSSYVYYYQSAFRPPNGWHLHGGAYAVVNISSESNNAG 60
***. * :**** .**** *::.. * **********.****:**** ***::.* ****
B1648
BdS1
M41
SSPTCTAGVIYYSKNFTASSVAMTAPPPGMSWSTSEFCTAHCNFSTFTVFVTHCFKSGAG 119
SSSGCTVGIIHGGRVVNASSIAMTAPSSGMAWSSSQFCTAHCNFSDTTVFVTHCYKHGG- 119
SSPGCIVGTIHGGRVVNASSIAMTAPSSGMAWSSSQFCTAHCNFSDTTVFVTHCYKYDG- 119
**. * .* *: .: ..***:*****..**:**:*:********* *******:* ..
Green= domain D; lightblue = eight N-terminally amino acids differing between M41
and Beaudette spike protein; red = amino acids that, if introduced into M41S1 as
single mutations, abolish binding of the protein to the virus’ target cells and tissues
103
Acknowledgements
8 Acknowledgements
I am particularly grateful to Prof. Dr. Georg Herrler and Dr. Christine Winter for
providing this very interesting topic. Thank you for your continuous advice,
encouragement and support throughout my research and Ph.D. study.
Prof. Dr. Silke Rautenschlein and Prof. Dr. Wolfgang Garten I would like to thank for
interesting discussions and helpful remarks during our meetings and for evaluation of
this thesis. Also, I thank Prof. Dr. ………. for external review of this work.
For the provision of the IBV-strain B1648 I would like to thank Dave Cavanagh. Dr.
Maren Bohm kindy gave the expression plasmid coding for H7S1Fc, Dr. Jörg Glende
provided the plasmids coding for SARSS1Fc and TGEVS1Fc and Dr. Christine
Winter the plasmid B1648S1Fc and several B1648S1Fc deletion mutants.
Furthermore, I thank the Clinic for Poultry for providing incubated SPF eggs, chicken
erythrocytes and organ material.
I am very grateful to Martina Kaps for her assistance. Markus Hoffmann I would like
to thank for his advice, his help, and for sharing his incredible knowledge.
Also I like to thank all recent and former members of our institute, in particular AnnKathrin, Jana, Anne, Fandan, Wei, Katarina, Karim, Sabine, Tanja, Moni, Nadine,
Nai-Huei, Sandra B., Sandra S., Christel, Anna, Tim, Holger and Günther. Working
with all of you has been a great experience.
At the end I like to thank Til for his patience, support and for motivating me and last
but not least for his adcive in the use of software tools. ;-)

Characterization of the binding properties of the Avian Coronavirus

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