Interaction of bat-derived paramyxoviruses with chiropteran and non

Transcription

University of Veterinary Medicine Hannover
Institute of Virology
Interaction of bat-derived paramyxoviruses with chiropteran
and non-chiropteran cells:
Functional characterization of the African henipavirus and bat-derived
mumps virus fusion and attachment glycoproteins
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY
(PhD)
awarded by the University of Veterinary Medicine Hannover
by
Nadine Krüger
(Marl)
Hannover, Germany 2014
Supervisor:
Prof. Dr. Georg Herrler
Supervision Group:
Prof. Dr. Marion Hewicker-Trautwein
Prof. Dr. Stefan Pöhlmann
1st Evaluation:
Institute of Virology, University of Veterinary Medicine
Hannover
Prof. Dr. Marion Hewicker-Trautwein
Institute of Pathology, University of Veterinary Medicine
Hannover
Prof. Dr. Stefan Pöhlmann
Department of Infection Biology, Deutsches Primatenzentrum
GmbH, Leibniz-Institut für Primatenforschung, Göttingen
2nd Evaluation:
Dr. Veronika von Messling
Federal Institute for Vaccines and Biomedicines, Paul-EhrlichInstitut, Langen
Date of final exam:
06.11.2014
This work was funded by DFG (SFB621 TP B7 and HE 1168/14-1), BMBF (FluResearchNet,
project code 01KI1006D) and Hannover Graduate School for Veterinary Pathobiology,
Neuroinfectiology, and Translational Medicine (HGNI).
Parts of this thesis have been communicated or published previously in:
Publications:
Kruger, N., M. Hoffmann, M. Weis, J. F. Drexler, M. A. Muller, C. Winter, V. M.
Corman, T. Gutzkow, C. Drosten, A. Maisner, and G. Herrler (2013). "Surface
glycoproteins of an African henipavirus induce syncytium formation in a cell line derived
from an African fruit bat, Hypsignathus monstrosus." J Virol 87(24): 13889-13891.
Weis, M., L. Behner, M. Hoffmann, N. Krüger, G. Herrler, C. Drosten, J. F. Drexler, E.
Dietzel, and A. Maisner (2014). "Characterization of African bat henipavirus GH-M74a
glycoproteins." J Gen Virol. 95(Pt 3): 539-548.
Kruger, N., M. Hoffmann, J. F. Drexler, M. A. Muller, V. M. Corman, C. Drosten, and
G. Herrler (2014). „Attachment protein G of an African bat henipavirus is differentially
restricted in chiropteran and non-chiropteran cells.” J Virol. 88(20): 11973-11980.
Kruger, N., M. Hoffmann, J. F. Drexler, M. A. Muller, V. M. Corman, C. Sauder, S.
Rubin, B. He, C. Orvell, C. Drosten, and G. Herrler. „Functional properties and genetic
relatedness of the fusion and hemagglutinin-neuraminidase proteins of a mumps virus-like bat
virus. J Virol. submitted
Oral presentations:
09/07/2012
Zentrumstag, University of Veterinary Medicine Hannover, Germany
Empfänglichkeit respiratorischer Fledermausepithelien für Corona- und
Paramyxoviren. N. Krüger.
12/01/2013
Treffen der Fledermausforscher in Deutschland 2013, Ergenzingen, Germany
Interaction of zoonotic paramyxoviruses with chiropteran cells. N. Krüger,
M. Hoffmann, T. Gützkow, M. A. Müller, J. F. Drexler, A. Maisner, K.-H.
Esser, C. Drosten, G. Herrler.
08/03/2013
23rd Annual Meeting of the Society for Virology, Kiel, Germany
Interaction of zoonotic paramyxoviruses with chiropteran cells: A
comparative analysis of Nipah virus and bat derived Henipa-like virus
glycoproteins. N. Krüger, M. Hoffmann, M. A. Müller, J. F. Drexler, M.
Weis, K.-H. Esser, A. Maisner, C. Drosten, G. Herrler.
12/06/2013
Seminars in Virology and Biochemistry, University of Veterinary Medicine,
Hannover, Germany
Interplay between bat-derived zoonotic paramyxoviruses with
chiropteran and non-chiropteran cells. N. Krüger.
26/05/2014
7th European Meeting on Viral Zoonoses, St. Raphael, France
Similarities and dissimilarities between the glycoproteins F and G of an
African henipavirus and Nipah virus. N. Krüger, M. Hoffmann, M. A.
Müller, J. F. Drexler, C. Drosten, G. Herrler.
03/07/2014
French German Summer School 2014; Oniris National College of Veterinary
Medicine, Food Science and Engineering; Nantes, France.
Functional characterization of the glycoproteins F and G of an African
henipavirus. N. Krüger
Poster presentations
16/03/2012
22nd Annual Meeting of the Society for Virology, Essen, Germany
Comparative analysis of the glycoproteins of high containment viruses
(SARS CoV, NiV, MarV, ZEboV) in mediating cell entry by a single-cycle
VSV pseudotype assay focussing on bat cell lines. M. Hoffmann, N. Krüger,
T. Gützkow, C. Losemann, M. A. Müller, J. F. Drexler, A. Maisner, C.
Drosten, G. Herrler.
12/10/2012
National Symposium on Zoonoses Research 2012, Berlin, Germany
Establishment and employment of primary chiropteran organ cultures
and differentiated epithelial cells as a platform for the investigation of the
interplay between viral pathogens and bats. N. Krüger, M. Hoffmann, C.
Losemann, M. A. Müller, J. F. Drexler, K-H. Esser, C. Drosten, G. Herrler.
17/06/2013
15th International Negative Strand Virus Meeting, Granada, Spain
Functional characterization of the surface glycoproteins G and F of an
African bat henipavirus. N. Krüger, M. Hoffmann, J. F. Drexler, M. A.
Müller, M. Weis, A. Maisner, C. Drosten, G. Herrler. Poster presentation by
Georg Herrler
12/09/2013
5th European Congress of Virology, Lyon, France
Interaction of zoonotic paramyxoviruses with chiropteran cells: A
comparative analysis of Nipah virus and bat-derived African henipavirus
glycoproteins. N. Krüger, M. Hoffmann, M. A. Müller, J. F. Drexler, M.
Weis, K.-H. Esser, A. Maisner, C. Drosten, G. Herrler.
19/09/2013
National Symposium on Zoonoses Research 2013, Berlin, Germany
The glycoproteins of an African henipavirus are functionally related to
the glycoproteins of NiV but fusion-active only in chiropteran cells.
N. Krüger, M. Hoffmann, M. A. Müller, J. F. Drexler, M. Weis, A. Maisner,
C. Drosten, G. Herrler.
27/03/2014
24th Annual Meeting of the Society for Virology, Alpbach, Austria
Surface expression of the attachment glycoprotein is a major restriction
factor for the fusion activity of the African Henipavirus M74 surface
glycoproteins. N. Krüger, M. Hoffmann, M. A. Müller, J. F. Drexler, C.
Drosten, G. Herrler.
27/03/2014
24th Annual Meeting of the Society for Virology, Alpbach, Austria
The surface glycoproteins of a bat-derived paramyxovirus show
functional similarities to human mumps virus. N. Krüger, M. Hoffmann,
M. A. Müller, J. F. Drexler, C. Drosten, G. Herrler.
02/06/2014
Junior Scientist Meeting 2014, Hannover, Germany
Chiropteran cells as a tool for the characterization of bat-derived viruses:
The
restricted
functional
activity
of
the
African
Henipavirus
glycoproteins to chiropteran cells. N. Krüger, M. Hoffmann, M. A. Müller,
J. F. Drexler, C. Drosten, G. Herrler.
In Liebe und Dankbarkeit meinen
Eltern gewidmet.
Contents
LIST OF ABBREVIATIONS
I
LIST OF FIGURES
IV
LIST OF TABLES
IV
ABSTRACT
V
ZUSAMMENFASSUNG
VII
1. INTRODUCTION
-1-
1.1. BATS AND ZOONOTIC VIRUSES ............................................................................. - 1 1.1.1. The order Chiroptera ...........................................................................- 1 1.1.2. Zoonoses ..............................................................................................- 2 1.1.3. Bats as reservoir hosts for zoonotic viruses.........................................- 3 1.1.3.1. Detection of novel viruses in bats ......................................- 4 1.2. PARAMYXOVIRUSES ............................................................................................. - 6 1.2.1. Taxonomy ............................................................................................- 6 1.2.2. Morphology and genome organization of paramyxoviruses ...............- 8 1.2.3. Fusion glycoproteins ..........................................................................- 11 1.2.3.1. Cleavage of MuV F .........................................................- 11 1.2.3.2. Cleavage of henipavirus F proteins ................................- 11 1.2.4. Attachment glycoproteins and receptor binding ................................- 13 1.2.4.1. MuV HN and sialic acids ................................................- 14 1.2.4.2. Henipavirus G proteins and Eph receptors .....................- 15 1.2.5. Paramyxovirus-mediated fusion ........................................................- 18 1.2.6. Viral entry and replication of paramyxoviruses ................................- 21 1.3. MUMPS VIRUS INFECTION ................................................................................... - 23 1.3.1. MuV genotypes and strains ...............................................................- 23 1.3.2. Pathogenesis, symptoms and complications of mumps.....................- 24 1.3.3. Medical treatment and prevention of mumps ....................................- 25 1.3.4. Mumps outbreaks...............................................................................- 28 1.4. HENIPAVIRUS INFECTIONS .................................................................................. - 30 1.4.1. NiV genotypes and lineages ..............................................................- 31 1.4.2. Reservoir hosts and geographic distribution of henipaviruses ..........- 31 -
Contents
1.4.3. Transmission of NiV..........................................................................- 37 1.4.4. Symptoms and pathology of NiV infection .......................................- 39 1.4.5. Prevention of NiV infections .............................................................- 40 1.4.6. NiV outbreaks ....................................................................................- 42 1.5. DETECTION OF BAT-DERIVED PARAMYXOVIRUSES IN AFRICAN FLYING FOXES . - 44 1.6. AIM OF THE STUDY ............................................................................................. - 45 -
2. SURFACE GLYCOPROTEINS
OF AN
AFRICAN HENIPAVIRUS
INDUCE
SYNCYTIUM FORMATION IN A CELL LINE DERIVED FROM AN AFRICAN
FRUIT BAT, HYPSIGNATHUS MONSTROSUS
3. ATTACHMENT
PROTEIN
DIFFERENTIALLY
G
OF AN
RESTRICTED
- 47 -
AFRICAN
IN
BAT HENIPAVIRUS IS
CHIROPTERAN
AND
NON-
- 49 -
CHIROPTERAN CELLS
4. CHARACTERIZATION
OF
AFRICAN
BAT HENIPAVIRUS
GH-M74A
GLYCOPROTEINS
5. ANALYSIS
- 51 -
OF THE PROTEOLYTIC CLEAVAGE OF THE FUSION PROTEIN
OF THE AFRICAN HENIPAVIRUS M74
- 53 -
5.1. ABSTRACT .......................................................................................................... - 54 5.2. INTRODUCTION ................................................................................................... - 54 5.3. METHODS ........................................................................................................... - 55 5.4. RESULTS ............................................................................................................. - 58 5.5. DISCUSSION ........................................................................................................ - 61 5.6. ACKNOWLEDGMENTS ......................................................................................... - 64 5.7. REFERENCES ....................................................................................................... - 64 5.8. FIGURES .............................................................................................................. - 68 -
6. FUNCTIONAL PROPERTIES AND GENETIC RELATEDNESS OF THE FUSION
AND HEMAGGLUTININ-NEURAMINIDASE PROTEINS OF A MUMPS VIRUSLIKE BAT VIRUS
- 73 -
Contents
6.1. ABSTRACT .......................................................................................................... - 74 6.2. INTRODUCTION ................................................................................................... - 74 6.3. METHODS ........................................................................................................... - 76 6.4. RESULTS ............................................................................................................. - 78 6.5. DISCUSSION ........................................................................................................ - 80 6.6. ACKNOWLEDGMENTS ......................................................................................... - 83 6.7. REFERENCES ....................................................................................................... - 84 6.8. TABLES AND FIGURES ......................................................................................... - 87 -
7. DISCUSSION
- 93 -
7.1. FUNCTIONAL CHARACTERISTICS OF THE AFRICAN HENIPAVIRUS M74 .............. - 95 7.1.1. M74 G interacts with the henipavirus receptor .................................- 95 7.1.2. Proteolytic cleavage of M74 F ...........................................................- 96 7.1.3. The ability of the M74 surface glycoproteins to mediate
membrane fusion is restricted to chiropteran cells ............................- 99 7.1.4. Inefficient surface expression of M74 G plays a crucial role for
the restricted functional activity ......................................................- 100 7.1.5. Summary ..........................................................................................- 102 7.2. FUNCTIONAL CHARACTERISTICS OF THE BATMUV .......................................... - 103 7.2.1. BatMuV HN shows sialic acid dependent hemadsorption, but no
neuraminidase activity.....................................................................- 103 7.2.2. The batMuV surface glycoproteins mediate syncytium
formation in various mammalian cells ............................................- 104 7.2.3. The differences in the size of syncytia following F and HN
expression
correlate with the origin of the SP-related aa
residues of the F protein ..................................................................- 106 7.2.4. Summary ..........................................................................................- 107 -
8. BIBLIOGRAPHY
- 109 -
9. APPENDIX
- 139 -
9.1. SEQUENCES ....................................................................................................... - 139 -
Contents
9.1.1. M74 F ...............................................................................................- 139 9.1.2. M74 G ..............................................................................................- 139 9.1.3. NiV F ...............................................................................................- 139 9.1.4. NiV G ...............................................................................................- 139 9.1.5. M74 F-HA........................................................................................- 140 9.1.6. M74 G-FLAG ..................................................................................- 141 9.1.7. NiV F-HA ........................................................................................- 142 9.1.8. NiV G-FLAG ...................................................................................- 143 9.1.9. M74 G-FLAG (ED) .........................................................................- 144 9.1.10. NiV G-FLAG (ED) ........................................................................- 145 9.1.11. M74 F-HA (NiVstart) ....................................................................- 146 9.1.12. NiV F-HA (M74start) ....................................................................- 147 9.1.13. batMuV F .......................................................................................- 148 9.1.14. batMuV HN ...................................................................................- 148 9.1.15. hMuV F ..........................................................................................- 148 9.1.16. hMuV HN ......................................................................................- 148 9.1.17. batMuV F1/hMuV F2 ....................................................................- 149 9.1.18. hMuV F1/ batMuV F2 ...................................................................- 150 9.1.19. batMuV F (SP) ...............................................................................- 151 9.1.20. hMuV F (SP)..................................................................................- 152 9.2. SUPPLEMENTARY DATA .................................................................................... - 153 9.3. AFFIDAVIT ........................................................................................................ - 154 -
-I-
List of abbreviations
LIST OF ABBREVIATIONS
aa
batMuV
BHK
BSA
Cat
CDC
CFP
CedPV
CHO
CNS
C-terminus
CT
DAPI
DMEM
DMSO
DNA
DTT
ECDC
ED
EDTA
e.g.
EidNi
Endo H
Ephrin
ER
ERGIC
E-64d
F
FCS
Fig.
g
G
GFP
h
H/ HA
HBE
HeLa
HeV
HN
HRP
hum
hMuV
HypLu
HypNi
IF(A)
Ig
kDa
Amino acid residue
BatPV/Epo_spe/AR1/DRC/2009
Baby hamster kidney cells
Bovine serum albumin
Cathepsin
Centers for Disease Control and Prevention
Cyan fluorescent protein
Cedar paramyxovirus
Chinese hamster ovary cells
Central nervous system
COOH terminus of proteins
Cytoplasmic tail
4’,6’-Diamidino-2-phenylindol-dihydrochloride
Dulbecco´s minimum essential medium
Dimethyl sulfoxide
Desoxy ribonucleic acid
1,4-Dithio-DL-threit(ol)
European Centre for Disease Prevention and Control
Ectodomain
Ethylenediaminetetraacetic acid
Exempli gratia (for example)
Eidolon helvum kidney cells
Endoglycosidase H
Eph family receptor interacting protein
Endoplasmic reticulum
ER-Golgi intermediate compartment
(2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester
Fusion glycoprotein
Fetal calf serum
Figure
Gramm or gravity
Glycoprotein
Green fluorescent protein
Hour
Hemagglutinin
Human bronchial epithelial cells
Human cervix adenocarcinoma cells
Hendra virus
Hemagglutinin-neuraminidase
Horseradish peroxidase
Human
Human mumps virus
Hypsignathus monstrosus lung cells
Hypsignathus monstrosus kidney cells
Immunofluorescence (analysis)
Immunglobulin
Kilodalton
L
M
M74
min
ml
mM
mRNA
4-MUNANA
MuV
N
NA
NH4Cl
NiV
nm
nM
nt
N-terminus
ORF
P
PAGE
PBS
PCR
PFA
pH
PNGase F
p.t.
PV
RNA
SARS-CoV
SDS
SH
SP
Tab.
TD
vRNA
VSV
WHO
x
°C
µg
µl
µm
µM
- II -
Polymerase
Molarity, -molar, matrix protein
BatPV/Eid_hel/GH-M74a/GHA/2009
Minute
Milliliter
Millimolar
Messenger RNA
2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid
Mumps virus
Nucleoprotein
Neuraminidase
Ammonium chloride
Nipah virus
Nanometer
Nanomolar
Nucleotide
NH2-terminal end of proteins
Open reading frame
Phosphoprotein
Polyacrylamid gel electrophoresis
Phosphate buffered saline
Polymerase chain reaction
Paraformaldehyde
Negative logarithm of the hydrogen ion concentration
Peptide N-glycosidase F
Post transfection
Paramyxovirus
Ribonucleic acid
Severe acute respiratory syndrome (SARS)-associated coronavirus
Sodium dodecyl sulphate
Small hydrophobic protein
Signalpeptide
Table
Transmembrane domain
Viral RNA
Vesicular stomatitis virus
World Health Organization
-fold
Degree Celsius
Microgramm
Microliter
Micrometer
Micromolar
- III -
ABBREVIATIONS FOR NUCLEOTIDES
A
C
G
T
Adenine
Cytosine
Guanine
Thymine
ABBREVIATIONS FOR AMINO ACIDS
Amino acids
1-letter symbol
3-letter symbol
Alanine
A
Ala
Arginine
R
Arg
Asparagine
N
Asn
Aspartic acid
D
Asp
Cysteine
C
Cys
Glutamic acid
E
Glu
Glutamine
Q
Gln
Glycine
G
Gly
Histidine
H
His
Isoleucine
I
Ile
Leucine
L
Leu
Lysine
K
Lys
Methionine
M
Met
Phenylalanine
F
Phe
Proline
P
Pro
Serine
S
Ser
Threonine
T
Thr
Tryptophan
W
Trp
Tyrosine
Y
Tyr
Valine
V
Val
List of figures and tables
- IV -
LIST OF FIGURES
Fig. 1: Worldwide distribution of chiroptera ........................................................................ - 1 Fig. 2: Classification within the Paramyxoviridae family .................................................... - 7 Fig. 3: Schematic structure of paramyxoviruses ................................................................... - 8 Fig. 4: Schematic genome organization of paramyxoviruses ............................................... - 9 Fig. 5: Triggering of the paramyxovirus fusion proteins .................................................... - 20 Fig. 6: Replication of paramyxoviruses .............................................................................. - 22 Fig. 7: Replication of henipaviruses ................................................................................... - 22 Fig. 8: Mumps vaccination in national immunization programs worldwide, 2012 ............ - 27 Fig. 9: Global distribution of henipaviruses ....................................................................... - 32 Fig. 10: Transmission routes of NiV ................................................................................... - 37 Fig. 11: NiV outbreaks in humans in Southeast Asia, 1998 - 2013 .................................... - 43 Fig. 12: Phylogenetic relatedness of batMuV and the MuV genotypes based on the SH
sequence. ............................................................................................................... - 153 -
LIST OF TABLES
Tab. 1: Worldwide detection of henipaviruses in mammals ............................................... - 34 -
-V-
Abstract
ABSTRACT
“Interaction of bat-derived paramyxoviruses with chiropteran and nonchiropteran cells: Functional characterization of the African henipavirus and batderived mumps virus fusion and attachment glycoproteins”
by Nadine Krüger
This thesis deals with the functional characterization of the surface glycoproteins of two batderived paramyxoviruses, the African henipavirus strain M74 (M74) and a bat-derived
mumps virus (batMuV). RNA sequences of these two viruses were detected in African flying
foxes. Aim of this study is to get information about the zoonotic potential, as well as the
functional relatedness of the bat-derived paramyxoviruses and the Nipah virus (NiV), a
zoonotic member of the henipaviruses, or the human mumps virus (hMuV), two
paramyxoviruses which are able to cause infection in humans and - in the case of NiV - have
been transmitted from bats to humans.
For the African henipavirus the attachment glycoprotein (G) has been shown to interact with
the cellular henipavirus receptor. Furthermore, the proteolytic cleavage of the M74 fusion
glycoprotein (F) was found share some similarities to that of the corresponding NiV protein.
The formation of multinucleated giant cells, which is induced by the co-expression of the F
and G protein and constitutes a common feature for paramyxoviruses, was restricted to
chiropteran cell lines. Further studies revealed that especially an inefficient cellular transport
of the M74 G protein to the cell surface is the major reason for the restricted fusion activity of
the African henipavirus surface glycoproteins.
The functional activities of the fusion (F) and hemagglutinin-neuraminidase (HN)
glycoproteins of the bat-derived MuV were demonstrated by the detection of multinucleated
giant cells in different mammalian, including human, cell lines. In addition, the batMuV
glycoproteins were able to interact with the human MuV proteins and induce the formation of
syncytia. The expression of chimeric F proteins revealed that the signal peptide affects the
size of syncytia, indicated by the number of nuclei within one cell. Furthermore, it was shown
that the batMuV HN binds to sialic acids and possesses hemadsorption activity, whereas no
neuraminidase activity above the background level was measured.
Abstract
- VI -
It could be shown that both bat-derived paramyxoviruses show functional similarities to NiV
or hMuV and that in general a zoonotic potential has to be considered. However, as long as
only viral sequences but no virus isolate are available, it is not possible to evaluate the risk of
a virus transmission from bats to humans or even a following human-to-human transmission.
Nevertheless, the in vitro studies performed in this thesis provided important insights about
the interactions of the bat-derived paramyxoviruses and host cells, as well as fundamental
principles for the evaluation of their zoonotic potential.
- VII -
Abstract
ZUSAMMENFASSUNG
“Interaktion von fledertier-assoziierten Paramyxoviren mit Fledertier- und
anderen Säugetierzellen: Die funktionelle Charakterisierung der Fusions- und
rezeptorbindenden Glykoproteine eines afrikanischen Henipavirus und eines
fledertier-assoziierten Mumpsvirus”
von Nadine Krüger
Ziel dieser Arbeit, die sich mit der funktionellen Charakterisierung der Oberflächenglykoproteine von zwei, in afrikanischen Fledertieren nachgewiesenen Paramyxoviren einem afrikanischen Henipavirus (M74) und einem fledertier-assoziierten Mumpsvirus
(batMuV), deren RNS in Flughunden gefunden wurden - befasst, ist es, eine Aussage über das
zoonotische Potential dieser Paramyxoviren sowie über ihre Verwandtschaft zu dem NipahVirus (NiV), einem zoonotischen Vertreter der Henipaviren, bzw. dem humanen Mumpsvirus
(hMuV) zu machen. Diese Fragestellung ist von besonderem Interesse, da für NiV und hMuV
bekannt ist, dass sie zu klinischen Infektionen in Menschen führen können und im Falle von
NiV eine Übertragung von Flughunden auf den Menschen stattfand.
Hinsichtlich des afrikanischen Henipavirus M74 konnte gezeigt werden, dass das
rezeptorbindende Glykoprotein (G) in der Lage ist, mit dem zellulären Henipavirusrezeptor zu
interagieren. Zudem weist die proteolytische Spaltung des Fusionsproteins (F) Ähnlichkeiten
zu dem korrespondierenden Protein von NiV auf. Die Bildung von Riesenzellen, welche
durch die Coexpression von den beiden Glykoproteinen F und G induziert wird und ein
typischer zytopathogener Effekt von Paramyxoviren ist, war im Falle der beiden M74Glykoproteinen auf Zelllinien von Fledertieren beschränkt. Weitere Versuche ergaben, dass
insbesondere ein ineffizienter zellulärer Transport des G Proteins an die Zelloberfläche für die
eingeschränkte funktionelle Aktivität verantwortlich ist.
Die funktionelle Aktivität des Fusions- (F) und des Hämagglutinin-Neuraminidase- (HN)
Glykoproteins des Fledertier-Mumpsvirus, wurde durch den Nachweis von Zell-zu-Zell
Fusion in verschiedenen Säugetierzelllinien, einschließlich humanen Zellen, nachgewiesen.
Zudem waren die batMuV Glykoproteine in der Lage mit humanen MuV-Glycoproteinen zu
interagieren und die Bildung von Riesenzellen hervorzurufen. Die Expression von chimären F
Abstract
- VIII -
Proteinen zeigte, dass das Signalpeptid für die Größe der Riesenzellen, womit in diesem Fall
die Anzahl der Zellkerne innerhalb einer Zytoplasmamembran gemeint ist, verantwortlich ist.
Desweiteren wurde nachgewiesen, dass das batMuV HN Protein an Sialinsäuren bindet und
hämadsorbierende
Eigenschaften
besitzt,
während
keine
Neuraminidaseaktivität
nachgewiesen werden konnte.
In dieser Arbeit wurde gezeigt, dass beide fledertier-assoziierte Paramyxoviren funktionelle
Ähnlichkeiten zu NiV bzw. hMuV aufweisen, sodass ein grundsätzliches zoonotisches
Potential vorhanden ist. Solange allerdings nur Virussequenzen und keine Virusisolate
verfügbar sind, können keine genauen Angaben über das tatsächliche Gefahrenpotential einer
Infektion des Menschen bzw. einer Mensch-zu-Mensch-Übertragung gemacht werden.
Durch die verwendeten in vitro-Techniken konnten jedoch bereits bedeutende Erkenntnisse
über die Virus-Wirts-Interaktion der untersuchten fledertier-assoziierten Paramyxoviren
gewonnen werden und damit bereits elementare Grundlagen für die Evaluierung ihres
zoonotischen Potentials geleistet werden.
-1-
Introduction
1. INTRODUCTION
1.1. Bats and zoonotic viruses
Zoonotic viruses are pathogens which can be transmitted from animals to humans. Small
mammals of the orders Rodentia and Chiroptera are the reservoir hosts of a variety of
emerging zoonotic viruses (Heisch, 1952; Lee et al., 1982; Williams, 2005; Calisher et al.,
2006; Wong et al., 2007; Smith and Wang, 2013).
1.1.1. The order Chiroptera
The name Chiroptera is derived from cheir (greek, hand) and pteron (greek, wing). The order
Chiroptera comprises more than 1200 highly diverse species within two suborders - the
Mega- and Microchiroptera (proposed new terms: Yangochiroptera, Yinpterochiroptera) and comprises about 20% of all recognized species in mammalians (Teeling et al., 2005). The
suborder Megachiroptera comprises only one family, the Pteropodidae. All members of this
family are fruit- and nectar-feeding bats, also called flying foxes. Their geographical
distribution ranges from Africa to Eastern Mediterranean, India, Indonesia, Southeast Asia,
Malaysia, Australia, Southern Japan, the Phillipines, and islands of the Central and South
Pacific (Pierson and Rainey, 1992). The Microchiroptera are divided into 16 families with
about 759 species. Their distribution is similar to that of Megachiroptera; in addition some
members of the Microchiroptera exist in parts of Central and South America, and subpolar
regions (Hill and Smith, 1984). Fig. 1 shows the nearly worldwide - with the exception of the
Antarctic - distribution of the order Chiroptera.
Fig. 1: Worldwide distribution of chiroptera
Adapted from Hill and Smith, 1984
Bats are the most diverse and geographically dispersed mammalian species and the only
mammalians which are capable of active flight. All microchiropteran bats use echolocation
for their orientation, whereas for the subfamily Megachiroptera only bats of the genus
Introduction
-2-
Rousettus are able to produce echolocating signals (Nowak, 1994). Bats roost in colonies of
variable size, depending on the species. The numbers of individuals in roosting colonies
ranges between less than a dozen and about a million of animals (Davis et al., 1962; Barbour
and Davis, 1969; Kingdon, 1974; Lekagul and McNeely, 1977; McCracken and Gustin, 1991;
Pierson and Rainey, 1992). Roosting sites may be caves, mines, tree trunks and branches, or
human buildings (Verschuren, 1957; Kunz, 1982; Stebbings and Walsh, 1988; Kunz et al.,
1994). Most species roost during the day and feed at night (Kunz, 1982). Whereas
megachiropteran bats feed on fruits, flowers, and nectar (Marshall, 1983; Nowak, 1994), the
diet of Microchiroptera is very diverse. The majority of microchiropteran bats are
insectivorous and feeds on insects and arthropods (Zinn and Humphrey, 1976; Fascione et al.,
1991; Whitaker, 1993; Kalko, 1995; McCracken et al., 2008; Clare et al., 2009; Bohmann et
al., 2011; Clare et al., 2011; Siemers et al., 2011). Some species, e.g. Carollia perspicillata
(Cloutier and Thomas, 1992), feed on fruits (frugivorous) and nectar (nectarivorous) of
flowers and trees. A carnivorous or sanguinivorous diet is very rare among bats. Carnivorous
species feed on small birds, fish, frogs, lizards, rodents, and smaller bats (Page and Ryan,
2005; Reid, 2009; Santana et al., 2011; Aizpurua et al., 2013). Some examples for carnivorous
bats are Nycteris thebaica and Vampyrum spectrum (Vehrencamp et al., 1977; Nowak, 1994).
There are only three sanguinivorous species feeding on mammals (Desmodus rotundus) or
small birds (Diaemus youngi and Diphylla ecaudata) (Morton and Janning, 1982; MachadoSantos et al., 2013).
1.1.2. Zoonoses
Zoonoses are diseases which can be transmitted between vertebrate animals and humans
(WHO). Zoonoses can be divided into two groups: Anthropozoonoses and zooanthroponoses.
Whereas
anthropozoonotic
diseases
are
transmitted
from
humans
to
animals,
zooanthroponoses cause disease in humans after being transmitted from animals. Zoonotic
agents may be bacteria, viruses, fungi or prions. Taken together more than 800 zoonotic
pathogens have been defined and about 60 % of human pathogens are zoonotic (Taylor et al.,
2001; Woolhouse and Gowtage-Sequeria, 2005). Zoonotic agents can be transmitted directly
via contact between infected animals and humans, e.g. rabies, or indirectly via vectors like
arthropods (e.g. West Nile virus), food (e.g. Salmonella sp.), and water (diverse parasites)
(Schofield, 1945; WHO, 2006; Ciota and Kramer, 2013).
-3-
Introduction
In general, the animal reservoir host harbouring zoonotic viruses do not show any clinical
symptoms, whereas infection of humans can lead to severe and even lethal diseases as in the
case of henipavirus infections (Rahman et al., 2010; Sohayati et al., 2011).
1.1.3. Bats as reservoir hosts for zoonotic viruses
Bats are the major source of zoonotic pathogens, followed by rodents (Dobson, 2005;
Calisher et al., 2006; Luis et al., 2013). They have some properties making them suitable
hosts for zoonotic pathogens, e.g. their abundance and nearly worldwide distribution (Hill and
Smith, 1984), a long life span, roosting in large colonies, and co-roosting of different species
(Calisher et al., 2006). The active flight results in a higher body temperature and an increased
metabolism. Therefore, it has been suggested that the ability to fly led to changes of the innate
immune system, e.g. to repair DNA damage mediated by the high metabolism rate, which
may affect the virulence of viral pathogens and enable bats to serve as reservoir hosts for
viruses (Baker et al., 2013b; Zhang et al., 2013; O'Shea et al., 2014).
Overall, more than 60 virus species of nine classified genera (Lyssa-, Henipa-, Rubula-,
Flavi-, Alpha-, Bunya-, Phlebo-, Orbi-, Orthoreovirus) and some unclassified viruses of the
Rhabdo-, Bunya-, and Herpesviridae family were detected in bats (Calisher et al., 2006).
One well known examples for a bat-borne zoonotic disease is rabies, a fatal encephalitis
caused by a virus of the genus Lyssavirus (Malaga Alba, 1954). Furthermore, pteropodid bats
(Pteropus ssp.) were found to be the reservoir hosts for the henipaviruses Nipah (NiV) and
Hendra (HeV) virus (Hooper and Williamson, 2000; Field, 2009; Halpin et al., 2011; Chua,
2012; Clayton et al., 2013) and the recently isolated Cedar paramyxovirus (CedPV,
unclassified Henipavirus) (Marsh et al., 2012). So far, the zoonotic potential of CedPV is
unknown. The severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a
pathogen causing severe respiratory disease in humans, has its natural reservoir in Chinese
horseshoe bats of the genus Rhinolophus (Lau et al., 2005; Li et al., 2005a; Poon et al., 2005;
Field, 2009). SARS-CoV related viruses were detected in bats in Europe (Drexler et al.,
2010), Slovenia (Rihtaric et al., 2010), China (Lau et al., 2005), and Nigeria (Quan et al.,
2010). In 2012 a novel coronavirus, the Middle East respiratory syndrome coronavirus
(MERS-CoV) led to cases of severe respiratory disease in humans (Zaki et al., 2012). So far it
is not known whether bats or dromedary camels serve as the major reservoir host for MERSCoV. Recent studies reported the circulation of MERS-CoV related coronaviruses in bats
(Reusken et al., 2010; Annan et al., 2013; Cui et al., 2013; Ithete et al., 2013). On the other
Introduction
-4-
hand, serological studies indicate that dromedary camels in Saudi Arabia and Egypt have
antibodies that neutralize MERS-CoV (Hemida et al., 2013; Perera et al., 2013; Alagaili et al.,
2014; Meyer et al., 2014). Further studies have to be performed to get more information about
the role of bats and dromedary camels for the MERS-CoV infection cycle. Fruit bats of the
species Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata have been
suggested to be the reservoir hosts of Ebola virus (EBOV) (Leroy et al., 2005). Furthermore,
bats are the reservoir hosts for different hepaci-, pegi-, and hantaviruses (Weiss et al., 2012b;
Guo et al., 2013; Quan et al., 2013).
1.1.3.1. Detection of novel viruses in bats
In the last years, many publications reported about the detection of novel viruses in bats,
when faecal, urine or organ samples from different chiropteran species were screened for the
presence of viral RNA via PCR techniques. In most cases, only RNA sequences of ostensible
viruses were detected, but no infectious virus could be isolated from chiropteran samples. One
of the few successful virus isolations from bats was that of CedPV from urine samples of
Australian bats of the species Pteropus alecto and Pteropus poliocephalus (Marsh et al.,
2012). Recent studies of Ge et al. described the isolation of a SARS-like CoV from faecal
samples of Chinese horseshoe bats (Ge et al., 2013). Baker et al. isolated two novel
rubulaviruses, which are phylogenetically related to Menangle virus and Tioman virus, from
the straw-coloured fruit bat Eidolon helvum (Baker et al., 2013a). Furthermore, two novel
morbillivirus-related paramyxoviruses were isolated from bats of the genus Miniopterus in
Comoros and Madagascar (Wilkinson et al., 2012).
In the following cases, virus isolation was not successful or has not been performed: Recent
studies described the detection of novel Influenza A viruses in bats. Tong et al. identified
genetic material of two novel Influenza A viruses: H17N10 from little yellow-shouldered bats
(Sturnira lilium) in Guatemala (Tong et al., 2012) and H18N11 from fruit bats of the species
Artibeus planirostris in Peru (Tong et al., 2013). Different studies reported about the detection
of various coronaviruses in European, German, and neotropical bats (Gloza-Rausch et al.,
2008; Reusken et al., 2010; Falcon et al., 2011; Corman et al., 2013). Furthermore, viral RNA
of paramyxoviruses was detected in chiropteran samples. Drexler et al. and Baker et al.
described the detection of a variety of novel paramyxoviruses in bats, some of which showed
a closely relation to already known viruses causing disease in humans and/ or domestic
animals (Drexler et al., 2009; Baker et al., 2012; Drexler et al., 2012; Baker et al., 2013a).
-5-
Introduction
Three novel paramyxoviruses, related to the genus Rubulavirus or the proposed new genus
Jeilongvirus, were detected in European bats (Kurth et al., 2012). Morbillivirus-related
viruses were detected in samples from bats in Comoros, Madagascar, and Mauritius
(Wilkinson et al., 2012). Lau et al. reported the detection of three rubulaviruses, Tuhoko virus
1 - 3, in fruit bats in China (Lau et al., 2010).
Introduction
-6-
1.2. Paramyxoviruses
Paramyxoviruses (PV) are RNA-viruses that mainly infect mammals - including humans - and
birds. Some members of the Paramyxoviridae family lead to infection in fish (Mitchell and
Rodger, 2011) or reptiles (Clark et al., 1979; Hyndman et al., 2013). In general,
paramyxovirus infections result in respiratory diseases. Some viruses, e.g. Rinderpest virus
and Newcastle disease virus, lead to severe systemic infections in animals (Rolle, 2007). HeV
and NiV are known for their zoonotic potential and can lead to severe and often fatal
infections in humans.
1.2.1. Taxonomy
Paramyxoviruses belong to the order Mononegavirales together with the families Borna-,
Filo-, and Rhabdoviridae. All members of this order are enveloped viruses with a singlestranded (mono), unsegmented ribonucleoid acid (RNA) genome with negative (nega)
orientation.
The family Paramyxoviridae is devided into two subfamilies mainly based on phylogenetic
relatedness and morphological characteristics. The subfamily Pneumovirinae comprises the
two genera Pneumovirus und Metapneumovirus, whereas the Paramyxovirinae subfamily is
divided into seven genera: Rubulavirus, Avulavirus, Respirovirus, Henipavirus, Morbillivirus,
Ferlavirus, and Aquaparamyxovirus (Fig. 2).
Further, there are some so far unclassified members of the family Paramyxoviridae like
different bat and rodent paramyxoviruses, lizard paramyxovirus, snake paramyxovirus,
sunshine virus, Mossman virus, and tortoise paramyxovirus (King et al., 2012).
-7-
Fig. 2: Classification within the Paramyxoviridae family
(*: unclassified henipavirus)
Introduction
Introduction
-8-
1.2.2. Morphology and genome organization of paramyxoviruses
Paramyxoviruses are enveloped viruses with a non-segmented single-stranded RNA genome
with negative orientation and a genomic size between 13.000 - 19.000 nucleotides. The
genome of NiV has a length of 18.246 nucleotides (nt), whereas the MuV genome comprises
15.384 nt. All paramyxoviruses with the exception of the Pneumovirinae subfamily follow the
rule of six (Kolakofsky et al., 1998). The genome length is a multiple of six nt, due to the
association of one nucleocapsid protein with six nucleotides.
Paramyxovirus particles are mainly spherical, occasionally filamentous or pleomorphic in
shaped with a size of 150 - 250 nm in diameter (Modrow et al., 2009). Goldsmith et al.
reported the detection of NiV virions with an average of 500 nm in diameter and size
variations between 180 - 1900 nm (Goldsmith et al., 2003).
The viral envelope contains at least two glycoproteins which are expressed on the viral
surface: the fusion and the attachment glycoprotein (Fig. 3). Some members of the
Paramyxoviridae express a third glycoprotein on their surface: the small hydrophobic (SH)
protein.
Fig. 3: Schematic structure of paramyxoviruses
Modified from Chang and Dutch, 2012
-9-
Introduction
The viral genome codes for at least six structural proteins from 3´ to 5´ (Fig. 4): nucleoprotein
(N), phosphoprotein (P), matrix protein (M), fusion protein (F), attachment protein
(glycoprotein (G), hemagglutinin (H), or hemagglutinin-neuraminidase (HN)), and the RNAdependent RNA polymerase (L). Further, some members of the genera Rubula-, Avula-,
Pneumo-, and Metapneumovirus code for an additional structural protein, the small
hydrophobic protein (SH).
Fig. 4: Schematic genome organization of paramyxoviruses
NDV: Newcastle disease virus, NiV: Nipah virus, MeV: Measles virus, SeV: Sendai virus,
MuV: Mumps virus, HMPV: Human metapneumovirus, HRSV: Human respiratory syncytial
virus. N: nucleoprotein, P: phosphoprotein, M: matrix protein, F: fusion protein, HN:
hemagglutinin-neuraminidase, H: hemagglutinin, G: attachment glycoprotein, L: RNA
polymerase, SH: small hydrophobic protein, NS-1, NS-2: non-structural protein
The N protein encapsidates the viral RNA and forms the ribonucleoprotein (RNP). The
phosphoprotein binds to the N and the L protein to form the RNA transcriptase complex,
which is important for RNA transcription and replication. The RNA-dependent RNA
polymerase uses a viral RNA template to catalyze the synthesis of complementary RNA.
The M protein, which is present at the inner coat of the virus envelope, is responsible for the
rigidity and structure of virions. Therefore, the matrix protein interacts with the cytoplasmic
tails of the surface glycoproteins and the RNP complex (Lamb and Parks, 2007). The M
protein is further required for virus assembly and budding. (Tanabayashi et al., 1990; Schmitt
et al., 2002; Wang et al., 2010; Battisti et al., 2012).
Introduction
- 10 -
The fusion and attachment glycoproteins are surface proteins, which are responsible for virus
entry. They are described in detail in the following chapters 1.2.3 and 1.2.4.
For parainfluenza virus 5 (PIV 5) and the bovine respiratory syncytial virus (BRSV) it was
shown that the SH protein inhibits the signalling of tumour necrosis factor alpha (Goldsmith
et al., 2003; Fuentes et al., 2007). The function of MuV SH protein is so far not known, but
the protein is not necessary for virus growth in cell cultures (Afzal et al., 1990; Takeuchi et
al., 1996). Wilson et al. suggested that the function of the MuV SH protein is similar to that of
PIV 5 (Wilson et al., 2006).
Taken together, the genomes of all members of the Paramyxoviridae family code for six
different non-structural proteins (Samal, 2011; King et al., 2012). It has been suggested that
the non-structural proteins play an important role for the virulence of paramyxoviruses
(Yoneda et al., 2010; Mathieu et al., 2012). The following non-structural proteins are
alternative products of the P protein and are expressed from an alternative ORF within the P
gene or by transcriptional RNA editing (Vidal et al., 1990; Eaton et al., 2006). The most
frequent non-structural protein is the V protein, which can be found in all genera of the
Paramyxovirinae subfamily. Paramyxovirus V proteins are able to inhibit the interferon (IFN)
production and signalling to circumvent the innate immune response (Andrejeva et al., 2004;
Childs et al., 2012; Xu et al., 2012b; Motz et al., 2013). The C protein is present in some
members of the Respiro-, Morbilli-, Henipa-, and Aquaparamyxovirus genus. Mathieu et al.
showed that NiV C proteins are able to inhibit the IFN production as already described for the
V proteins (Mathieu et al., 2012). A third non-structural protein, the W protein, is expressed
by the Atlantic salmon paramyxovirus (Nylund et al., 2008) and NiV (Harcourt et al., 2000)
and has an influence on the signalling pathway during innate immune response (Shaw et al.,
2005) .
An unknown open-reading frame was detected in the genome of the Fer-de-Lance
paramyxovirus (U protein) (Kurath et al., 2004). Members of the genus Metapneumovirus
harbor the non-structural M2 protein; Pneumoviruses expresses three different non-structural
proteins, namely the M2, NS-1, and NS-2 protein (Fig. 4).
- 11 -
Introduction
1.2.3. Fusion glycoproteins
The fusion proteins (F) of enveloped viruses are responsible for the fusion of viral and host
cell membrane or the fusion between the membranes of neighbouring infected cells (Bossart
et al., 2001; Wang et al., 2001; Tamin et al., 2002). This event is necessary to enable the entry
of virus particles into host cells and for virus spread starting from infected cells. An
interaction between the two surface proteins, the fusion and the attachment protein, has to
occur to induce the fusion reaction.
Paramyxovirus F proteins are type I integral membrane proteins which assemble into trimers
on the cell surface. Fusion proteins are synthesized as inactive, non-fusiogenic F0 precursor
proteins, which have to be cleaved into the active and fusiogenic disulfide-linked F1 and F2
subunits (Lamb et al., 2006; Lamb and Parks, 2007). The larger F1 subunit comprises a
hydrophobic fusion peptide at the N-terminal end, two heptad repeat regions, a
transmembrane domain, and a cytoplasmic tail at the C-terminal end.
Cleavage and activation of the F proteins differ within the Paramyxoviridae. The following
section deals with the cleavage of the MuV and NiV F protein.
1.2.3.1. Cleavage of MuV F
The MuV F has a molecular weight of 65-74 kDa and a size of 538 amino acids (Flynn and
Mahon, 2003). The cleavage of MuV F has not been studied in detail, but Waxham et al.
suggested that the multibasic amino acid sequence R-R-H-K-R (aa residue 98 - 102) directly
upstream of the fusion peptide is the cleavage site of MuV F (Waxham et al., 1987). This
finding leads to the conclusion that a cleavage by furin is very likely (Rubin, 2011). The
predicted cleavage site of furin is R-X-K/R-R (Molloy et al., 1992; Walker et al., 1994). Furin
belongs to the subtilisin-like proprotein convertases and is a cellular endoprotease. It is
ubiquitously expressed in all vertebrates and some invertebrates (Seidah et al., 1998) where it
is localized in the Golgi apparatus. Furin is responsible for the cleavage of proteins like
β-nerve growth factor in the secretory pathway (Molloy et al., 1999).
1.2.3.2. Cleavage of henipavirus F proteins
NiV F has a molecular weight of 60 kDa and a length of 546 amino acids (Harcourt et al.,
2000). After synthesis NiV F is transported to the cell surface as an inactive F0 form. This
uncleaved precursor has to undergo clathrin-mediated endocytosis to be cleaved by cellular
proteases in the endosomes (Diederich et al., 2005). After this recycling step, the fusionactive protein consisting of the F1 and F2 subunits is expressed on the cell surface (Fig. 7).
Introduction
- 12 -
Sequence analysis of the F1 subunits predicted that the cleavage of NiV and HeV occurs at a
monobasic cleavage site at position 109 (Michalski et al., 2000; Moll et al., 2004). In the case
of NiV the predicted cleavage site consists of an arginine (R), whereas HeV expresses a single
lysine (K) at this position. Further studies of Moll et al. showed that the R at position 109 is
not necessary for cleavage of NiV F: The replacement of R by an alanine had no effect on the
cleavage or the functional activity of NiV F, suggesting that NiV cleavage does not require a
monobasic cleavage site (Moll et al., 2004). It has been shown that cleavage of henipavirus F
proteins is furin-independent and requires an intracellular low pH (Pager et al., 2004;
Diederich et al., 2005). Pager et al. reported that NiV F can be cleaved by cathepsin (Cat) B
or L, but only cleavage mediated by Cat L resulted in the correct cleavage products (Pager et
al., 2006). Similar results were described for the cleavage of HeV F, which is mediated by Cat
L (Meulendyke et al., 2005; Pager and Dutch, 2005). However, Diederich et al. showed that
the cleavage of NiV F in Madin-Darby canine kidney (MDCK) cells is mediated by Cat B
(Diederich et al., 2012).
Cathepsins are cysteine proteases which are present in lysosomes or the endosomal
compartment (Piovan et al., 2011; Turk et al., 2012). They need an acidic environment to be
fully active; a neutral pH can lead to irreversible deactivations of many cathepsins. Cat B and
L are expressed in different human tissues. Cysteine proteases play an important role for the
adaptive immunity (Cresswell, 1996; Villadangos and Ploegh, 2000; Turk et al., 2002; Conus
and Simon, 2008), bone remodelling, prohormone activation (Hook et al., 2008), tumor
progression and invasion (Turk et al., 2002), and angiogenesis (Lecaille et al., 2002; Turk et
al., 2012).
- 13 -
Introduction
1.2.4. Attachment glycoproteins and receptor binding
The attachment glycoproteins of paramyxoviruses are expressed on the viral surface - together
with the fusion proteins. They mediate the binding of virus particles to cellular receptors and in a concerted action with the fusion proteins - the membrane fusion. Furthermore, it has been
shown that the attachment protein of human parainfluenza virus 3 stabilizes the F protein in
the pretriggered state to prevent an inadvertent activation (Porotto et al., 2012b). Some
members of the Paramyxoviridae family use sialic acid-containing receptors, whereas others
recognize specific cellular receptors.
All paramyxovirus attachment glycoproteins have in common that they are type II integral
membrane proteins (Fields et al., 2007). They consist of an N-terminal cytoplasmic tail, the
hydrophobic transmembrane domain, a stalk region, which is responsible for the
tetramerization (Crennell et al., 2000; Lawrence et al., 2004; Zaitsev et al., 2004) and the
interaction with the F protein (Deng et al., 1995; Tanabayashi and Compans, 1996; Deng et
al., 1999; Melanson and Iorio, 2006), and a six-bladed β-propeller domain (Crennell et al.,
2000; Lawrence et al., 2004; Yuan et al., 2005; Bowden et al., 2008; Xu et al., 2008; Bowden
et al., 2010), which is involved in receptor binding. Attachment proteins are expressed as
disulfide-linked dimers, which are further linked to another dimer resulting in the formation
of tetramers (Crennell et al., 2000; Yuan et al., 2005; Bishop et al., 2008; Xu et al., 2008;
Bowden et al., 2010; Hashiguchi et al., 2011; Maar et al., 2012).
The attachment glycoproteins of paramyxoviruses are divided into three classes. Members of
the genera Avulavirus, Respirovirus and Rubulavirus have hemagglutinin-neuraminidase
(HN) proteins that bind to sialic acids. They further agglutinate red blood cells and cleave
sialic acid linkages to release virions from the cell surface and to prevent self-association
(Villar and Barroso, 2006). The hemagglutinin (H) protein which is expressed by members of
the genus Morbillivirus shares similarities with the HN proteins, but lacks neuraminidase
activity. It binds to specific cellular receptors like CD46 (Dorig et al., 1993; Naniche et al.,
1993), CD150 (Tatsuo et al., 2000; Hsu et al., 2001) or nectin-4 (Muhlebach et al., 2011;
Noyce et al., 2011). The third kind of attachment proteins is the glycoprotein (G) which lacks
hemagglutionation and neuraminidase activity. G proteins are expressed by henipaviruses and
members of the Pneumovirinae and bind to specific cellular receptors such as Eph B2/ B3
(Negrete et al., 2005) and heparin sulphate proteoglycans (Krusat and Streckert, 1997;
Thammawat et al., 2008).
Introduction
- 14 -
1.2.4.1. MuV HN and sialic acids
The HN protein of mumps virus has a molecular weight of 74-80 kDa and a length of 582
amino acids (Flynn and Mahon, 2003). MuV HN is the main target of neutralizing antibodies
directed against three amino acid regions on the extracellular part of the HN protein (Server et
al., 1982; Kovamees et al., 1990; Orvell et al., 1997a). The MuV attachment protein shows
hemagglutination and neuraminidase activity. Hemagglutination is a common feature of many
ortho- and paramyxoviruses like influenza viruses (Burnet and McEwin, 1945; Shubladze and
Soloviev, 1945; Soloviev et al., 1945), Newcastle disease virus (Florman, 1947; Granoff and
Henle, 1954), measles virus (Demeio and Gower, 1961; Rosen, 1961), and MuV (Burnet and
McEwin, 1945; Beveridge and Lind, 1946; Challut et al., 1956). Hemagglutination is the
ability of virus particles (or infected cells, hemadsorption) to bind red blood cells via
interaction between sialic acids on the erythrocytes and the surface proteins on the virus
surface (Cook et al., 1961; Belyavin, 1963). The ability of MuV HN to bind erythrocytes of
different species was analyzed and confirmed for chicken, sheep, monkey, human, guinea pig,
mouse, horse, cow, and pig erythrocytes (Morgan et al., 1948; Chu and Morgan, 1950).
The MuV attachment protein utilizes cellular sialic acids as a receptor determinant (Samal,
2011). The receptor binding of MuV HN has not been studied in detail, but it was shown that
MuV (Urabe strain) binds to sialyl-galactose linkage receptors of group B II Streptococcus
(Hosaka et al., 1998). The capsular polysaccharides of these bacteria contain sialic acid side
chains, namely 5-N-acetyl neuraminic acid α2,3 galactose (Neu5Acα2,3Gal). Furthermore,
Brostrom et al. reported that MuV neuraminidase preferred fetuin with α2-3-linked sialic
acids as a substrate to α1 (orosomucoid) with sialic acid in the same linkage type and bovine
submaxillary mucin containing α2-6 linked sialic acids (Brostrom et al., 1971). Similar results
were reported by Leprat et al.: Four substrates were tested in a neuraminidase activity assay.
Fetuin and N-acetyl neuraminlactose - two substrates with α2-3-linked sialic acids - were
hydrolysed by MuV neuraminidase. In contrast to this, the α2-6 linkages present in bovine
submaxillary glands mucin I and porcine stomach mucine II were not hydrolysed (Leprat and
Aymard, 1979). It was further shown that different variants of the Urabe AM9 strains
interacted with α2-3, α2-6, or α2-8 linked sialic acids (Santos-Lopez et al., 2009). From these
findings it was suggested that α2-3, α2-6, or α2-8 linked sialic acids serve as cellular receptors
for human mumps virus with different affinities depending on the different strains and
variants.
- 15 -
Introduction
Sialic acids are derivates of the monosaccharide neuraminic acid. They are present in the
terminal position of N-glycans, O-glycans, and glycosphingolipids on the surface of cells.
Overall, there are more than 50 different derivates known which occur from substitutions at
the carbons or phosphorylation, acetylation, sulfation, or methylation of the hydroxyl groups.
N-acetylneuraminic acids are the most common sialic acids in mammalian cells (Varki and
Schauer, 2009).
Sialic acids play an important role concerning the development of the central nervous system
(Wang, 2012; Scholtz et al., 2013), the development of cancer (Fuster and Esko, 2005; Cui et
al., 2011; Miyagi et al., 2012), and control of the cell cycle by regulating proliferation and
apoptosis (Mandal et al., 2012). Further, sialic acids are known to be the receptor determinant
for influenza viruses (Carroll et al., 1981; Rogers and Paulson, 1983; Rogers et al., 1986;
Wilks et al., 2012; Xiong et al., 2013).
1.2.4.2. Henipavirus G proteins and Eph receptors
Henipaviruses express an attachment glycoprotein (G) lacking both, hemagglutination and
neuraminidase activity. The molecular weight of NiV G amounts to 72-75 kDa (Bossart et al.,
2002).
NiV and HeV interact with cellular receptors Eph B2 and B3 to mediate viral entry
(Bonaparte et al., 2005; Negrete et al., 2005; Negrete et al., 2006; Bishop et al., 2007; Lee,
2007; Xu et al., 2012a). Eph B2 is the main receptor for NiV G, whereas Eph B3 is used as
an alternative receptor in regions where Eph B2 is not expressed, e.g. the corpus callosum
(Liebl et al., 2003) and the spinal cord (Kullander et al., 2001; Yokoyama et al., 2001). The
binding affinity of NiV G to Eph B3 is weaker compared to Eph B2 (Negrete et al., 2006).
It has been shown that the aa W504, E505, Q530, T531, A532, E533, and N557 in the NiV G
protein are involved in the binding of Eph B2 (Guillaume et al., 2006). These seven aa are
conserved among NiV and HeV. Furthermore, Negrete et al. reported that the amino acid
residues at position 507 (V for NiV G, T for HeV G) and 533 (E) play an important role for
the usage of Eph B2 or B3 as a cellular receptor (Negrete et al., 2007).
Eph B2 and B3 are type I transmembrane proteins with a length of ~330 amino acids (Bennett
et al., 1995). Eph B2 is encoded on human chromosome 13, whereas Eph B3 is encoded on
human chromosome 17 (Bennett et al., 1995; Bergemann et al., 1995). Eph receptors belong
to the receptor tyrosine kinase (RTKs) class VIII (Eph receptor family). They are highly
Introduction
- 16 -
conserved (96-98%) among different mammalian species, as well as fish and amphibians.
Their high level of conservation is also reflected by the fact that the G proteins of
henipaviruses are able to interact with Eph B2 molecules of different species with comparable
efficiencies (Bossart et al., 2008). The Eph family comprises 14 members, which are divided
into the subclasses A and B based on the relatedness of extracellular domain sequences and
binding (Eph Nomenclature Committee, 1997; Boyd and Lackmann, 2001; Kullander et al.,
2001) affinities. The Eph receptor family interacting proteins (ephrins), ligands which bind to
the Ephs, are also divided into two subclasses: GPI-linked class A ephrins and transmembrane
class B ephrins. Ephrins bind to the Ephs of the same subclass, with the exception of Eph A4,
which can interact with both ephrin subclasses.
Eph B2 plays an important role for the embryonic development and regulates the axon
guidance (Orioli and Klein, 1997) and neuronal development (Pasquale et al., 1992; Flanagan
and Vanderhaeghen, 1998; Egea and Klein, 2007), chemotaxis, cell migration and adhesion
(Davy and Soriano, 2005; Meyer et al., 2005), the cardiovascular development (Adams et al.,
1999), and angiogenesis (Wang et al., 1998; Kuijper et al., 2007; Korff et al., 2008). The
important role of Eph B2 for the embryonic development was confirmed by the fact that Eph
B2 knock-out mice died with underdeveloped heart, vessels, and vasculature (Gerety and
Anderson, 2002). Further, Eph receptors have an important role in stem cell differentiation
and the development of cancer (Genander and Frisen, 2010), as well as for the proliferation of
osteoclasts (Zhao et al., 2006; Edwards and Mundy, 2008).
Eph B3 is involved in the regulation of the axon guidance (Orioli et al., 1996; Bergemann et
al., 1998; Kullander et al., 2001; Benson et al., 2005; Kadison et al., 2006).
Eph B2 is mainly expressed in endothelial and smooth muscle cells in arterial vessels,
especially in the brain (Gale et al., 2001). The highest expression levels of Eph B2 were
detected in the prefrontal areas of the cortex. Furthermore, Eph B2 is expressed in lungs,
placenta, and prostate, as well as in bronchial epithelial cells and cardiomyocytes (Liebl et al.,
2003; Su et al., 2004). Eph B3 expression is restricted to the central nervous system,
especially to the spinal cord and the brain (Liebl et al., 2003; Su et al., 2004; Benson et al.,
2005). The highest expression of Eph B3 was detected in the occipital lobe, the amygdala, the
prefrontal cortex and in lower amounts in the pons, the temporal lobe, the hypothalamus, the
subthalamic nucleus, the globus pallidus, the hippocampus, and the corpus callosum (Liebl et
al., 2003; Su et al., 2004; Benson et al., 2005).
- 17 -
Introduction
The usage of a specific cellular receptor, which is highly conserved within different - not only
mammalian - species, may be a reason for the broad host range of henipaviruses. The
susceptibility of immortalized cell lines depends on the expression of Eph B2 or B3. For
example, Chinese hamster ovary cells (CHO-K1) and HeLa-USU cells are not permissive to
NiV infection due to their endogenous lack of any ephrin expression on the surface
(Bonaparte et al., 2005; Negrete et al., 2005). Most cell lines, with a detectable expression of
Eph B2, are permissive to NiV infection with the exception of mouse mast cells (P815) and
rat embryonic fibroblasts (208f) (Yoneda et al., 2006).
The distribution of Eph B2 and B3 receptors in the central nervous system and the lungs is
consistent with the symptoms caused by NiV infection, namely neurological and respiratory
symptoms (Aguilar and Lee, 2011).
Introduction
- 18 -
1.2.5. Paramyxovirus-mediated fusion
Paramyxoviruses induce fusion of virus and cell membrane to enable the release of the viral
genome into the cytoplasm of host cells. A common feature of paramyxoviruses is the fusion
of infected cells with neighbouring cells, which results in the formation of syncytia,
multinucleated giant cells. This cell-to-cell-fusion supports virus spread in infected tissues.
Two surface glycoproteins, the fusion and the attachment protein, are responsible for the
membrane fusion. Paramyxovirus-mediated fusion occurs at neutral pH (Lamb, 1993; Lamb
et al., 2006) with the exception of some strains of the human Metapneumovirus (HMPV),
which mediate fusion at low pH (Schowalter et al., 2006; Herfst et al., 2008). In general, the
inactive F0 proteins have to be proteolytically cleaved into the disulfide-linked F1 and F2
subunits. In the F1-subunit, the cleavage generates a (new) hydrophobic N-terminal fusion
peptide which is buried intra-molecularly (White et al., 2008). To be able to mediate fusion
with the host cell membrane, the F proteins have to undergo conformational changes which
are triggered by the interaction of fusion and attachment proteins which results in the
refolding of the cleaved, prefusion F protein to the postfusion conformation (Smith et al.,
2009).
The fusion process differs within the paramyxoviruses due to differences in the viral
attachment and the activation and triggering of the F proteins. For many paramyxoviruses it
has been shown that the specific interaction of fusion and attachement protein is requiered for
triggering of the fusion activity (Stone-Hulslander and Morrison, 1997; Yao et al., 1997;
Takimoto et al., 2002; Bishop et al., 2007; Navaratnarajah et al., 2011; Porotto et al., 2011;
Porotto et al., 2012a). This interaction mainly takes place at the head region of the F proteins
(Lee et al., 2008) and leads to irreversible conformational changes which result in the
repositioning of the two heptad repeat regions (HRA, HRB) to a stable six-helix bundle
(Smith et al., 2009). In the case of measles virus (MeV) the interaction between fusion and
attachment glycoprotein takes place intracellularly (Plemper et al., 2001), whereas the other
members of the paramyxoviruses interact on the viral surface (Li et al., 2004; Whitman et al.,
2009). Triggering of the fusion proteins without interaction with the attachment protein has
been reported for Sendai virus (SeV) and parainfluenza virus 5 (PIV-5) (Dutch et al., 1998;
Leyrer et al., 1998).
- 19 -
Introduction
So far, there are five different models of paramyxovirus F triggering (Chang and Dutch,
2012). MeV surface glycoproteins interact intracellular in the endoplasmic reticulum (Fig. 5
A). Both proteins are dissociated until the attachment protein interacts with the cellular
receptor and releases the fusion protein for triggering (Plemper et al., 2001; Plemper et al.,
2002). HeV and NiV F and G proteins are independently transported to the surface where the
interaction occurs (Fig. 5 B). The F protein undergoes conformational changes after
disruption of the interaction by binding of the attachment protein to the cellular receptor. The
strength of F-G interaction and fusion activity of HeV and NiV are inversely correlated
(Aguilar et al., 2006; Bishop et al., 2007). Studies of Newcastle disease virus (NDV) F
triggering showed that the F and HN proteins interact with each other only after the
attachment protein has bound to the receptor (Corey et al., 2003; Melanson and Iorio, 2004)
(Fig. 5 C). Some members of the subfamily Pneumovirinae such as RSV and HMPV do not
require an interaction of fusion and attachment protein. Two different models for F triggering
are described for those viruses: The attachment protein binds to a cellular receptor to enable
the binding of the fusion protein to a second receptor (Fig. 5 D) or the F protein binds to a
cellular receptor and mediates fusion without any receptor binding of the attachment protein
(Fig. 5 E) (Techaarpornkul et al., 2002; Thammawat et al., 2008; Kwilas et al., 2009; Chang
et al., 2012).
The fusion mechanism of MuV has not been studied in details, but it has been shown that the
surface expression of both glycoproteins is required for cell fusion, suggesting that the F
triggering is forced by interaction of the F and HN glycoprotein (Tanabayashi et al., 1992).
The triggering of the F results in the formation of a six-helix bundle. Especially the heptad
repeat regions HRA and HRB are affected by the conformational changes and form coiled
coils (Yin et al., 2005; Swanson et al., 2010; McLellan et al., 2011). In the prefusion state
HRA forms long, extended coiled coils which are projected towards the host cell membrane
(Kim et al., 2011). Further conformational changes of HRA and HRB in an anti-parallel
fashion enable the formation of a fusion pore which allows the release of viral RNA into the
host cell cytosol.
Introduction
- 20 -
Fig. 5: Triggering of the paramyxovirus fusion proteins
The triggering is exemplarily shown for MeV (A), NiV and HeV (B), NDV (C), RSV and
HMPV (D, E). Modified from Chang and Dutch, 2012
- 21 -
Introduction
1.2.6. Viral entry and replication of paramyxoviruses
Viruses use different mechanisms to enter their host cells. Viruses with pH-independet fusion
proteins can enter the cells by fusion of viral and plasma membrane (Bissonnette et al., 2006;
Lamb and Jardetzky, 2007). For SeV, NiV, NDV and RSV it has been shown that fusion of
viral particels and cell membranes can occur at the plasma membrane or at intracellular
membranes (Rasmusson et al., 1998). Before these viruses are able to fuse with intracellular
membranes, they have to enter the cells via endocytosis after the receptor binding of the
attachment protein has taken place (Cantin et al., 2007; Kolokoltsov et al., 2007; Diederich et
al., 2008; Schowalter et al., 2009). Further, Pernet et al. showed that NiV can enter cells via
macropinocytosis follwed by intracellular membrane fusion (Pernet et al., 2009).
After receptor binding and fusion of viral and host cell membrane occured, the replication of
paramyxoviruses takes place in the cytoplasm (Fig. 6). The viral nucleocapsid, which is
associated with the matrix protein, is released into the cytoplasm. The RNA genome serves as
a template for the RNA-dependent RNA polymerase which transcribes the viral genome into
messenger RNA (mRNA). The mRNAs are translated into viral proteins. With the exception
of NiV and HeV F, paramyxovirus glycoproteins are synthesized in the ER and mature or - in
the case of F proteins - are cleaved during transport through the Golgi to the host cell
membrane (Garten et al., 1994; Ortmann et al., 1994; Watanabe et al., 1995; Gonzalez-Reyes
et al., 2001; Begona Ruiz-Arguello et al., 2002). Furthermore, the RNA polymerase replicates
the viral RNA (vRNA) and produces complemantary copies of the viral genome. The
synthesized surface proteins are transported to the surface of the infected host cell, whereas
the other newly synthesized proteins assembly together with the RNA replicates at the plasma
membrane. The M protein plays an important role during virus assembly. It lines the inner
surface of the plasma membrane and interacts with the cytoplasmic tails of the envelope
proteins to form the virus envelope during budding (Cathomen et al., 1998; Garoff et al.,
1998). The budding, the release of enveloped viral particles from the host cell membrane
completes the replication cycle (Takimoto and Portner, 2004; Harrison et al., 2010; Aguilar
and Lee, 2011).
Introduction
- 22 -
Fig. 6: Replication of paramyxoviruses
Adapted from Takimoto and Portner, 2004
The clathrin-mediated endocytosis of the F is a unique feature of henipaviruses (Fig. 7). The
inactive precursor F0 is expressed on the surface, followed by endocytosis and proteolytic
cleavage in the endosomal compartment, before the cleaved and active F1 and F2 subunits are
expressed on the cell surface (Diederich et al., 2005; Pager et al., 2006; Diederich et al., 2008)
Fig. 7: Replication of henipaviruses
Modified from Aguilar and Lee, 2011
- 23 -
Introduction
1.3. Mumps virus infection
The designation mumps is derived from mumble and the swollen cheek lumps. The medical
term for MuV infection is parotitis epidemica and describes the most common pathological
effect of mumps. So far, humans are the only known hosts for MuV, but it was shown that
rhesus macaques develop mumps symptoms after experimental infection and therefore serve
as an animal model to study mumps virus infection (Flanagan et al., 1971; Genco et al., 1973;
Rozina et al., 1984). Mice and ferrets can be experimentally infected with MuV, but they do
not show any clinical signs (Xu et al., 2013). Wolinsky and Stroop showed that newborn
hamsters develop severe disease of the central nervous system when infected with
neuroadapted MuV strains (Wolinsky and Stroop, 1978).
1.3.1. MuV genotypes and strains
Mumps viruses are divided into 12 genotypes (A - N, excluding E and M) based on genetic
variations of the SH proteins (Yeo et al., 1993; Afzal et al., 1997; Orvell et al., 1997b; Tecle
et al., 1998; Wu et al., 1998; Inou et al., 2004; WHO, 2012). The different genotypes show a
global distribution: Genotypes C, D, G, H, J, and K are present in the Western Hemisphere,
whereas B, F, G, I, and L were detected in Asia (WHO, 2005).
Each genotype comprises different MuV strains, which show sequence differences, especially
for the F and HN proteins (McCarthy and Johnson, 1980). A consequence of the variations
within the two surface glycoproteins is a different ability to mediate cell-to-cell fusion (Merz
and Wolinsky, 1981; Merz et al., 1983; Elango et al., 1989): Avirulent strains are not able to
mediate cell-to-cell fusion, whereas virulent strains show fusion activity. The neuraminidase
activity appears to correlate with the ability to fuse cells. Fusion-active strains showed less
neuraminidase activity than strains which do not support cell-to-cell fusion (Merz and
Wolinsky, 1981). Furthermore, the MuV strains differ in their virulence and tropism:
McCarthy and Wolinsky reported that fusion-active mumps strains are neurovirulent in
newborn hamsters, whereas non-fusing strains are not (Wolinsky and Stroop, 1978; McCarthy
and Johnson, 1980).
The WHO suggested to designate mumps viruses according to the same procedure used for
measles and rubella viruses (WHO, 2012). Sequences from clinical samples should be
designated MuV/City.Country_ISO3code/week.year/replicate in week [genotype], whereas
samples isolated in cell culture are designated “MuVi” instead of “MuV”. According to this
Introduction
- 24 -
nomenclature the Enders strain should be named MuVi/Boston.USA/0.45 [A]. If the isolates
contain sequences of a vaccine MuV strain, the term “(VAC)” should be added, e.g.
MuVi/Pennsylvannia.USA/13.63 [A] (VAC).
1.3.2. Pathogenesis, symptoms and complications of mumps
In general, MuV infection is a highly contagious childhood disease with mild symptoms
which occurs worldwide through the whole year with peaks in winter and spring. MuV is
transmitted via respiratory droplets, mucus or saliva of the mouth, nose or throat of infected
humans. An indirect transmission via contact to contaminated surfaces is also possible.
Infected humans can transmit viruses from three days before to four days after the onset of
parotitis. Asymptomatically infected individuals are able to transmit MuV as well. The
replication of MuV occurs in the nasopharynx and the regional lymphnodes. The incubation
time ranges from 14 to 25 days. During this time a viremia occurs and MuV can be detected
in various tissues and organs like salivary glands, meninges, pancreas, testes, and ovaria
resulting in inflammations of the infected tissues (CDC, 2012).
The uni- or bilateral parotitis is the hallmark of mumps and occurs in 90 % of all clinical
cases (CDC, 2012). Further unspecific symptoms are fever, headache, tiredness, anorexia or
myalgia. About 40-50 % of MuV infections result in respiratory symptoms. Generally, the
symptoms resolve after 1 - 2 weeks without any complications. About 20 % of MuV
infections are asymptomatic (CDC, 2012; WHO, 2012). Sometimes mumps results in
complications like aseptic meningitis (15%) (Russell and Donald, 1958), encephalitis (0.1 %),
orchitis (up to 50 %) (Beard et al., 1977), oophoritis (0.5 - 7 %), pancreatitis (2 - 5 %) (Falk et
al., 1989; Taii et al., 2008), deafness and high frequency hearings loss (4 %) (Vuori et al.,
1962; Stratton et al., 1994), or myocarditis (3 - 15 %) (Ozkutlu et al., 1989). MuV can
increase the risk of abortion during the first trimester (Siegel et al., 1966), but there is no
association between mumps in pregnant women and congenital malformation in newborns
(Siegel, 1973; Ornoy and Tenenbaum, 2006). The fatility rate of mumps is very low and
deaths are mainly caused by encephalitis. The USA reported 2 deaths per 10000 mumps cases
during 1966 - 1971 (Fiebelkorn et al., 2012); 93 deaths were registered in the United
Kingdom from 1962 - 1981 (Galbraith et al., 1984).
- 25 -
Introduction
1.3.3. Medical treatment and prevention of mumps
Diseased patients receive symptomatic treatment like analgesic or antipyretic drugs. So far,
there is no specific antiviral treatment against MuV available. Therefore, the prevention of
MuV infection plays an important role. Some easy ways to prevent MuV transmission via
droplets are washing hands, cleaning of often touched surfaces like door handles, and
covering nose and mouth while coughing or sneezing.
Vaccination plays the most important role in the prevention of mumps. Mumps vaccines are
available as monovalent vaccines or combined with measles, measles and rubella (MMR) or
measles, rubella, and varicella (MMRV) vaccines. The Centers for Disease Control and
Prevention (CDC) proposes to vaccinate children using MMR or MMRV vaccines at the age
of 12 - 15 month with the first dose, followed by a second dose at the age of 4 - 6 years (CDC,
2012). Children between 13 and 18 years should be vaccinated by one dose of MMR vaccine.
The MMRV vaccine is not licensed for children older than 12 years or adults. Adults, who
were born during or after 1957, should receive on dose of MMR vaccine. In general, adults
born before 1957 do not need to be vaccinated. The mumps vaccines were routinely
introduced in 1967 and MuV infections mainly occured during the age of 5-9 years,
suggesting that most of the persons born before 1957 were infected with MuV and are
protected by antibodies. Generally, natural MuV infection confers a longlife protection
against mumps. Nevertheless, healthcare workers, who can get in contact with infected
persons, should receive 2 doses of MMR vaccine within 4 weeks to receive immunity against
MuV infection. Mumps vaccines can not prevent mumps if applied after exposure to virus.
Immunosuppressed people, pregnant women, and people showing allergy against components
of mumps vaccines such as neomycin or gelatine should be excluded from vaccination
(Recommendations of the Immunization Practises Advisory Committee, 1998; Galazka et al.,
1999).
Since 2001, the STIKO (ständige Impfkomission) adviced to vaccinate German children by
two doses of mumps vaccines: the first dose should be administered at an age of 11 - 14
months, the second dose at an age of 15 - 23 months. All licensed mumps vaccines used in
Germany are derived from the Jeryl Lynn strain (Koch, 2013). Furthermore, since March
2013 mumps is a notifiable disease in Germany in accordance to §6, Abs. 1
Infektionsschutzgesetz (IFSG). Confirmed MuV infections and deaths, as well as the
suspicion of disease have to be reported to the public health office.
Introduction
- 26 -
The first MuV vaccine, which was developed in 1948, was used from 1950 to 1978 in the
United States. This inactivated vaccine induced only short-term immunity and is therefore not
used anymore. At the moment, all licensed mumps vaccines are live attenuated vaccines
which are derived from different MuV strains. MuV is serologically monotypic: Mumps
vaccines are able to neutralize other mumps strains, but their efficacy is lower compared to
the strain used for developing the vaccine (Rubin and Beeler, 2013).
A live attenuated Jeryl Lynn strain MuV vaccine was developed by passaging the virus in
embryonated hen´s eggs followed by passaging in chicken embryo cell culture (Buynak and
Hilleman, 1966). This vaccine was licensed in 1967 and used in the United States for more
than 30 years. The seroconversion rates of a single dose of Jeryl Lynn vaccine ranged
between 80 - 100 % (Fahlgren, 1988); 63 - 96 % of vaccinated people were protected against
clinical mumps. More than 135 million people worldwide received the Jeryl Lynn mumps
vaccine (WHO, 1994).
Another vaccine, derived from the Leningrad-3 strain, was developed in the former Soviet
Union in guinea pig kidney cells, followed by passaging in Japanese quail embryo cultures
(Smorodintsev et al., 1970). Leningrad-3 vaccine was used in the former Soviet Union since
1974. The seroconversation in children aged 1 - 7 years ranged between 89 - 98 % and the
protective efficacy against clinical mumps ranged between 92 - 99 % (Smorodintsev et al.,
1970). The Leningrad-Zagreb vaccine was developed by further attenuation of Leningrad-3
strain by adaptation and passaging in chicken embryo fibroblast cells (Beck et al., 1989). It
was used in Croatia, the former Yugoslavia, and India. The seroconversation and preotection
against mumps was similar to the Leningrad-3 vaccine (Beck et al., 1989; Bhargava et al.,
1995).
The Urabe Am9 vaccine, which was produced in embryonated hen´s eggs or chicken embryo
cells, was first licensed in Japan and later in Belgium, France, and Italy (WHO, 1994).
Overall more than 60 million people worldwide received this vaccine. Seroconversation and
clinical protection was similar to Jeryl Lynn vaccine or even a little bit higher (Vesikari et al.,
1983; Boulianne et al., 1995; Miller et al., 1995). Canada, the United Kingdom, and Japan
reported cases of aseptic meningitis associated with the administration of Urabe Am9 vaccine
and therefore withdrew MMR vaccines containing the Urabe Am9 strain from the market
(Furesz and Contreras, 1990; Sugiura and Yamada, 1991; Miller et al., 1993; Ueda et al.,
1995; Brown et al., 1996).
- 27 -
Introduction
The Rubini strain vaccine was licensed in Switzerland in 1985 and used for the vaccination of
more than 4 million people worldwide (WHO, 1994). This strain was first passaged in human
diploid cells and embryonated hen´s eggs before it was adapted to the MRC-5 human diploid
cell line (Gluck et al., 1986). Several studies in Switzerland and Italy showed that the
seroconversion and efficacy of Rubini vaccine was lower compared to Jeryl Lynn and Urabe
Am9 mumps vaccines and suggested that this vaccine should not be used for immunization
programs any more (Paccaud et al., 1995; Chamot et al., 1998; Unknown, 1998).
In December 2005 the WHO reported that 110 (57 %) of the 193 WHO member states are
using mumps vaccines in national immunization programs (WHO, 2007). In contrast to this,
in most regions of Africa and regions of Southeast Asia MuV vaccines are not part of national
immunization programs (Galazka et al., 1999). Fig. 8 gives an overview of countries using
mumps vaccines in national immunization programs in 2012.
Fig. 8: Mumps vaccination in national immunization programs worldwide, 2012
Adapted from WHO, 2013 (Retrieved 2013-12-05, from http://www.who.int/immunization/
monitoring_surveillance/burden/vpd/surveillance_type/passive/Mumps_map_schedule.JPG?u
a=1)
Introduction
- 28 -
1.3.4. Mumps outbreaks
The first reported epidemic mumps outbreaks occurred in the eighteenth century in crowded
areas such as prisons, schools, orphanages, and military barracks (Hirsch, 1886). Before
mumps vaccines were included to national immunization programs, mumps has been a
common disease with more than 100 cases per 100.000 individuals (Galazka et al., 1999); the
USA reported 2000 cases per 100.000 individuals (Levitt et al., 1970). The highest incidence
has been reported in military populations with about 6000 cases per 100.000 (Gordon and
Kilham, 1949). The introduction of mumps vaccines diminished the incidence of mumps.
Countries that did not vaccinate against MuV report a high mumps incidence with epidemic
peaks every 2 - 5 years. Especially children aged 5 - 9 years are infected. Nevertheless,
several mumps outbreaks have been reported in countries using mumps vaccines as part of
immunization programs such as the United States and the United Kingdom. So far, the reason
for these outbreaks is not clearly known (Dayan and Rubin, 2008; Vandermeulen et al., 2009;
Rubin et al., 2012).
The European Centre for Disease Prevention and Control (ECDC) reported a total of 11741
cases including 7103 confirmed mumps cases for 27 European countries in 2010 (ECDC,
2012). The United Kingdom, the Netherlands and the Czech Republic reported the highest
rates. The age of the most infected individuals ranged between 15 and 24 years. 35 % of the
cases were not vaccinated, 34 % received one dose of mumps vaccine, and 26 % received two
doses. A total of 928.949 mumps cases was reported from 2000 to 2007 (ECDC, 2009). The
main age-group infected by MuV ranged between five and nine years.
Germany reported two mumps outbreaks in the last years. In summer 2010 genotype G MuV
caused an outbreak with more than 300 cases in Bavaria (Otto et al., 2010; Robert Koch
Institut, 2012). The infected individuals aged between 16 and 22 years and visited school,
universities or sport clubs. About 43 % of them did not receive any vaccine against mumps,
whereas 35 % received at least one or two doses of mumps vaccine (Otto et al., 2010; Takla et
al., 2013). A recent outbreak occurred in a school in Nuremberg in 2011. The RKI reported a
total of 23 cases which included pupils, teachers, and parents. The age of infected individuals
ranged between 6 and 47 years with a median of eight years (Robert Koch Institut, 2012).
Some 12 individuals were not vaccinated, eight individuals had received two, and three
individuals had received one dose of mumps vaccine.
- 29 -
Introduction
The United States reported two large mumps outbreaks in the last 10 years: A multi-state
outbreak in 2006 and a second outbreak mainly restricted to New York, in 2009 - 2010. In
2006 a total of 6584 mumps cases were reported from 45 states (CDC, 2006b; CDC, 2006a).
5 % of infected individuals developed complications, but no deaths were reported (Dayan et
al., 2008). Infection mainly occurred among college students and persons aged 18 to 24 years.
63 % of all mumps cases had received one or two doses of mumps vaccine (CDC, 2006b).
The second mumps outbreak started in June 2009 in a summer camp for Jewish boys. Source
of infection was a boy who returned from a trip to the United Kingdoms where an outbreak
with more than 7000 mumps cases was ongoing (CDC, 2009; CDC, 2010). From June 2009 to
June 2010 a total of 3502 cases of mumps were reported from New York and New Jersey
(Barskey et al., 2012). 65 infected individuals developed complications; no deaths have
occurred. Most cases occurred in male individuals aged 7 - 18 years and 89 % of individuals
with a known vaccination status had received two doses of mumps vaccination (CDC, 2009;
CDC, 2010).
Introduction
- 30 -
1.4. Henipavirus infections
The genus Henipavirus comprises the zoonotic Nipah virus (NiV) and Hendra virus (HeV).
The recently discovered Cedar paramyxovirus (CedPV) is supposed to be a member of the
genus Henipavirus, but has not been classified. So far, there are no recognized outbreaks
caused by CedPV.
HeV emerged in Australia in 1994 and led to severe respiratory and/ or neurological disease
in horses and humans (O'Sullivan et al., 1997; Paterson et al., 1998). The first outbreak
occurred in Mackay in the state of Queensland among a racehorse trainer and his animals
(Murray et al., 1995; Selvey and Sheridan, 1995; Selvey et al., 1995). Initially the new agent
was named equine morbillivirus, before it was renamed as HeV. In the following years,
further outbreaks were reported from Queensland and New South Wales (Playford et al.,
2010). Overall, 86 horses, seven humans, and one dog became infected; 59 horses and 4
humans died from 1994 - 2013 (Australian Veterinary Association, 2011; Australian
Veterinary Association, 2012; Australian Veterinary Association, 2013). All human cases
resulted from the transmission of HeV from infected horses to humans (O'Sullivan et al.,
1997; Hanna et al., 2006; Perkins, 2009; Playford et al., 2010), no person-to-person
transmissions have been observed. Fruit bats of the genus Pteropus (P. alecto, P. scapulatus,
P. poliocephalus, P. conspicillatus) were identified as the reservoir hosts for HeV (Field et
al., 2001; Field et al., 2007). So far, there is no medical treatment or effective vaccine for
humans available to prevent HeV infections.
NiV was isolated in March 1999 from the cerebrospinal fluid of a hospitalized patient from
the village Sungai Nipah in Malaysia Penninsula (Chua et al., 1999). The natural reservoirs of
NiV are fruit bats of the genus Pteropus (Yob et al., 2001); pigs were identified to serve as
amplifying hosts (Chua et al., 2000; Epstein et al., 2006). This virus can infect a wide range
of mammalian species such as horses, pigs, dogs, cats, goats, guinea pigs, and humans
(Hooper et al., 2001; Middleton et al., 2002; Wong et al., 2002). Natural NiV infections were
reported for pigs and rarely for horses, cats, dogs, cows, and goats (Hooper and Williamson,
2000; Field et al., 2001; Mills et al., 2009). NiV infections lead to respiratory illness or
encephalitis with a high fatality rate. Due to the fact that NiV and HeV can lead to fatal
infections and there is no medical treatment or licensed vaccine available, it is classified as a
biosafety level (BSL) 4 pathogen and only selected laboratories are allowed to work with live
- 31 -
Introduction
henipaviruses. NiV has been well studied and serves as a model for henipaviruses. The
following sections describe this virus in more details.
1.4.1. NiV genotypes and lineages
Sequence analysis of NiV isolates from outbreaks in Malaysia, India, and Bangladesh
revealed a molecular diversity of Nipah viruses. Taken together, NiV is divided into two
genotypes: Isolates from Malaysia and Cambodia were designated genotype M, whereas
isolates from India and Bangladesh were designates genotype B (Lee and Rota, 2012). The
nucleotide variations between the complete genome of genotype M and B is about 8 %. The
variation within each genotype ranges between 0.2 % and 3.6 %.
1.4.2. Reservoir hosts and geographic distribution of henipaviruses
Flying foxes of the genus Pteropus were identified to be the natural reservoir for NiV (Young
et al., 1996; Olson et al., 2002; Epstein et al., 2006) and HeV (Field et al., 2001; Field et al.,
2007). Flying foxes comprise 58 species and are distributed in South and Southeast Asia,
Australia, Africa, east of Philippines, and Pacific islands (Koppman, 1992).
So far, all reported HeV outbreaks are restricted to Queensland and New South Wales,
Australia (Murray et al., 1995; Selvey and Sheridan, 1995; Selvey et al., 1995; Playford et al.,
2010). HeV has been isolated from Pteropus alecto, Pteropus poliocephalus, and Pteropus
conspicillatus, the reservoir hosts for HeV, in Australia (Halpin et al., 2000; Breed et al.,
2011; Field et al., 2011; Smith et al., 2011).
NiV infections in humans were reported from Malaysia, Singapore, India, and Bangladesh
(Fig. 9). In these countries, NiV or neutralising antibodies directed against NiV were detected
especially in bats of the species Pteropus giganteus (Hsu et al., 2004; Epstein et al., 2008;
Yadav et al., 2012), Pteropus hypomelanus, and Pteropus vampyrus (Yob et al., 2001; Chua
et al., 2002; Rahman et al., 2010; Sendow et al., 2010; Sohayati et al., 2011; Sendow et al.,
2013). The prevalence of antibodies to NiV in Pteropus hypomelanus and Pteropus vampyrus
is approximately 25 % (Yob et al., 2001). Virus has been detected in saliva, urine, and nasopharyngeal secretions of bats, as well as in uterine and placenta discharges.
However, although NiV has been detected in Pteropus lylei in Cambodia there were no
reported NiV outbreaks in this country (Olson et al., 2002; Reynes et al., 2005). Furthermore,
RNA or neutralising antibodies were detected in Pteropus lylei, Pteropus hypomelanus,
Pteropus vampyrus, and Hipposideros larvatus in Thailand (Reynes et al., 2005;
Introduction
Wacharapluesadee
- 32 et
al.,
2005;
Wacharapluesadee
and
Hemachudha,
2007;
Wacharapluesadee et al., 2010). No NiV outbreaks have been observed in Thailand so far.
Sendow et al. detected NiV RNA in Pteropus vampyrus in Sumatera (Sendow et al., 2013);
seroepidemiologic studies of Yob et al. confirmed the presence of neutralising antibodies
against NiV in two fruit bat species, Cynopterus brachyotis and Eonycteris spelaea, and an
insectivorous bat, Scotophilus kuhli, in Malaysia (Yob et al., 2001). Antibodies directed
against NiV or NiV-like viruses were detected in various bat species in China (Li et al.,
2008).
Fig. 9: Global distribution of henipaviruses
Countries with reported henipavirus outbreaks in humans are shown in dark red; countries at
risk (detection of antibodies and/ or viral RNA in chiroptera) are shown in light red.
The detection of antibodies directed against henipaviruses in fruit bats of the species Pteropus
rufus and Eidolon dupreanum in Madagascar (Iehle et al., 2007), in Pteropus lylei in
Cambodia (Olson et al., 2002), as well as in flying foxes of the species Eidolon helvum in
Ghana (Hayman et al., 2008; Drexler et al., 2009; Drexler et al., 2012) and Eidolon
annobonensis in Equatorial Guinea (Peel et al., 2012) indicates a broader distribution of
henipaviruses, even if those countries did not report any outbreaks of henipaviruses so far
(Tab. 1). Furthermore, henipavirus-related sequences were detected in Eidolon helvum in
Zambia (Muleya et al., 2013), in various African flying foxes in Ghana, Gabon, Democratic
Republic of Kongo, and Central African Republic and in Carollia perspicillata and
- 33 -
Introduction
Pteronotus parnellii in Brazil, Costa Rica, and Panama (Drexler et al., 2012). Further,
antibodies against henipaviruses were found in Ghanaian domestic animals (Hayman et al.,
2011).
A recently published paper described the detection of the Mojiang paramyxovirus, a
henipavirus-related virus, which was detected in rats (Rattus flavipectus) in Mojiang, China
(Wu et al., 2014b). The recently discovered CedPV was isolated from pteropodid bats in
Australia (Marsh et al., 2012).
An overview of the global distribution of henipaviruses and the detection of henipaviruses in
mammalian species is shown in Fig. 9 and Tab. 1.
Introduction
- 34 -
Tab. 1: Worldwide detection of henipaviruses in mammals
- 35 -
Introduction
Introduction
- 36 -
- 37 -
Introduction
1.4.3. Transmission of NiV
Epidemiologic studies illustrated different routes for the transmission of NiV: Transmission
from bats to humans, transmission from domestic animals or pigs as amplifying hosts,
transmission through raw date palm sap, and person-to-person transmissions (Fig. 10).
Fig. 10: Transmission routes of NiV
Bat-to-human transmission (white arrow), transmission via amplifying hosts (light grey
arrow), transmission via contaminated food (black arrow), human-to-human transmission
(dark grey arrow).
Pigs were identified to serve as amplifying hosts during the Malaysian outbreak in 1998/ 99
(Goh et al., 2000). They get infected by eating dropped, partially eaten fruits, contaminated
with saliva of infected fruit bats (Field et al., 2001; Chua et al., 2002) or by being fed with
date palm juice, which is contaminated with bat faces and therefore did not serve as food for
humans (Luby et al., 2009a; Nahar et al., 2010). NiV is extremely contagious between pigs
where it is transmitted via coughing. Humans, especially pig farmers, get infected via direct
contact to pigs.
Further, epidemiologic studies in Bangladesh reported that the risk of NiV infection was
increased eight times for people having contact to sick cows (Hsu et al., 2004). In 2004, a
child got infected after having contact to goats that developed a severe, febrile illness with
neurological symptoms (Luby et al., 2009a).
The outbreaks in Bangladesh and India did not involve amplifying hosts. Instead, the virus
was directly or indirectly transmitted from bats to humans (Epstein et al., 2006; Lo and Rota,
2008). The shedding of virus by infected bats can lead to the contamination of food, which is
Introduction
- 38 -
an important source of infection for humans and animals. Epidemiologic studies identified the
contamination of raw date palm sap via urine as the main pathways of NiV transmission from
bats to humans (Luby et al., 2006). Infrared camera photography confirmed that Pteropus bats
lick the sap during palm juice collection and contaminate the collection pots with urine (Khan
et al., 2010; Rahman et al., 2012). Infected bats shed NiV via saliva and urine, so a
contamination of the raw palm sap is very likely (Reynes et al., 2005; Bossart et al., 2007).
The consumption of contaminated raw palm juice was identified as an important source of
infection during the outbreaks in Bangladesh and India (Luby et al., 2006; Rahman et al.,
2012). Fogarty et al. reported that NiV survives in mango, pawpaw, and lychee juice for up to
three days (Fogarty et al., 2008). The survival of NiV in palm juice has not been tested, but a
similar result can be expected (Aidoo et al., 2006).
In some areas of Asia, the Pacific islands, and West Indian Ocean islands bats of the genus
Pteropus serve as food; Eidolon helvum is hunted as source of food in Africa (Mickleburgh et
al., 2009). This so-called bushmeat is another potential source of infection for humans,
especially if infected bats are consumed as raw meat or after insufficient heat-inactivation.
Weiss et al. detected henipavirus-related RNA in bats of the species Eidolon helvum, which
were offered as meat at markets in the Republic of Congo (Weiss et al., 2012a). In some areas
of Asia fresh bat blood is drunk as an aphrodisiac (Wacharapluesadee et al., 2006). Given that
NiV RNA was detected in serum samples of Pteropus sp., the consumption of bat blood
displays
another
possibility
for
the
transmission
of
henipaviruses
to
humans
(Wacharapluesadee et al., 2005).
Further, humans can get infected after having contact to excretions of bats. During the
outbreak in Bangladesh in 2004, NiV infection mainly occured amoung persons who climbed
on trees to harbour palm sap and thereby got into contact with infectious bat excretions
(Montgomery et al., 2008).
Human-to-human transmission was reported in Bangladesh (Gurley et al., 2007; Luby et al.,
2009a; Luby et al., 2009b) and Siliguri, India (Chadha et al., 2006). Diseased humans,
especially those showing respiratory symptoms, shed NiV mainly by tracheal and
nasopharyngeal secretions (Chua et al., 2001; Homaira et al., 2010). NiV RNA was frequently
detected in saliva of infected individuals (Chua et al., 2001; Harcourt et al., 2005).
Transmission mainly occured amoung individuals, such as family members or health workers,
caring for diseased persons.
- 39 -
Introduction
1.4.4. Symptoms and pathology of NiV infection
Natural infected fruit bats do not show any clinical signs (Rahman et al., 2010; Sohayati et al.,
2011). Experimentally infected Australian Flying foxes of the species Pteropus poliocephalus
did not develop illness or gross pathology although NiV could be isolated from the urine of an
infected bat (Bossart et al., 2007).
In contrast to this, naturally infected pigs developed febrile illness with respiratory or
neurological symptoms, or abortion after an incubation period of 7 - 14 days (Mohd Nor et
al., 2000). In general, only mild illness occurs. Neurological signs such as agitation, spasm,
paralysis, salivation, and dropping of the tongue mainly occurred among young piglets,
whereas young pigs mainly developed respiratory symptoms (Middleton and Weingartl,
2012). The case fatality rate ranged between 1 - 5 % during the Malaysian outbreak but
nevertheless, sudden deaths, mainly amoung piglets, were reported (Mohd Nor et al., 2000).
Pathological examinations demonstrated giant cell pneumonia and multi-nucleated giant cells
in the lungs (Chua et al., 2000). Experimental infected pigs developed respiratory symptoms
with a low mortality rate of 1 - 5 % (Middleton et al., 2002).
Dead-end-hosts such as humans, dogs, or cats develop severe illness after NiV infection.
After an incubation time of 4 - 18 days (Selvey et al., 1995; Goh et al., 2000), NiV mainly
infects two organ systems in humans: the central nervous system (CNS) or the respiratory
tract. After infection of the respiratory tract, NiV can be detected in bronchiolar epithelial
cells. Pathological changes include alveolitis, necrosis, pulmonary edema, and aspiration
pneumonia (Wong et al., 2002). During the late stage of infection NiV can spread from the
epithelium to the endothelium of the lungs where it can infect small blood vessels and
capillaries and induce the specific vasculitis followed by viremia and infection of target
organs such as the brain, spleen, or kidney (Wong et al., 2002; Rockx et al., 2011). A multiorgan failure can be the consequence of this viremia (Selvey et al., 1995). Two pathways to
enter the CNS were described: anterogradely via the olfactory nerve or hematogenous through
cerebral blood vessels (Weingartl et al., 2005; Munster et al., 2012). Infection of the CNS
leads to necrosis, vasculitis, and thrombosis (Wong et al., 2002). The major target cells of
henipaviruses are microvascular endothelial cells (Hooper et al., 1997a). Pathological changes
like vasculitis, perivascular necrosis of small blood vessels, thrombosis, and syncytium
formation of endothelial cells are characteristics for henipavirus infections (Wong et al., 2002;
Maisner et al., 2009). The syncytium formation results in cellular dysfunction and apoptosis.
Introduction
- 40 -
The vasculitis concorded with the expression levels of Eph B2: Vasculitis was mainly seen in
the CNS (80 %) and the lungs (62 %), followed by the heart (31 %) and the kidney (24 %)
(Wong et al., 2002; Hafner et al., 2006).
The clinical symptoms depend on the stage of infection. Generally, NiV infection starts with
unspecific symptoms like fever, headache, influenza-like illness, nausea, vomiting and
diarrhoea, or convulsions (Hsu et al., 2004; Chadha et al., 2006; Hossain et al., 2008).
Infection of the respiratory tract leads to respiratory symptoms such as coughing and
difficulty in breathing, whereas the infection of the CNS leads to encephalitis and
neurological signs. During the Malaysian outbreaks acute neurological symptoms such as
reduced or abnormal reflexes, nystagmus, tremor, reduced consciousness or coma, and tonicclonic seizures were reported (Goh et al., 2000). In 3 - 7 % of infected individuals, NiV
infection caused relapsed or late-onset encephalitis several months to years after infection
(Chua et al., 1999; Tan et al., 2002; Luby et al., 2006; Sejvar et al., 2007; Tyler, 2009). The
case fatality rate of NiV infections ranged between 0 - 100 % with an average of 74.5 %
(WHO, 2013).
1.4.5. Prevention of NiV infections
So far, there is no medical treatment or vaccination against NiV infection available.
Therefore, it is important to prevent virus transmission from bats to humans, domestic
animals, and food. Fresh date palm juice is an important national source of food. That is why
it is easier to prevent contamination of palm juice via bats than to limit the consumption. Pilot
studies used bamboo skirts, jute sticks, polyethen skirts, or lime (calcium carbonat) to inhibit
the access of bats to the juice collection pots (Khan et al., 2012; Nahar et al., 2013; Nahar et
al., 2014).
To reduce the risk of human-to-human transmissions health care workers should avoid getting
in contact with saliva of patients showing respiratory or neurological symptoms. Persons
caring for infected individuals should implement precautions: Persons who washed their
hands with soap after having contact to NiV infected individuals had a lower risk to become
infected (Gurley et al., 2007).
Dropped, partially eaten fruits can be contaminated by the saliva of NiV infected bats. In
Bangladesh, where food is rare and many children suffer from chronic malnutrition, fruits are
consumed irrespective of the risk of NiV transmission (Luby et al., 2009a).
- 41 -
Introduction
Several animal models for henipavirus infections have been established to test potential
therapeutica or vaccines. Henipaviruses do not cause clinical disease in common small animal
models such as mice, rats or rabbits (Westbury et al., 1995; Bossart et al., 2007).
Experimental henipavirus infections of guinea pigs resulted in mild disease which
significantly differs from human infection (Hooper et al., 1997b; Williamson et al., 2001).
NiV infection in pigs is often asymptomatic or causes only mild symptoms (Mohd Nor et al.,
2000; Weingartl et al., 2006; Berhane et al., 2008). In contrast to this, experimentally infected
cats develope severe and lethal henipavirus infection (Hooper et al., 1997b). A subunit
vaccine for NiV has been successfully tested in challenge and protection experiments in cats
(McEachern et al., 2008). Similar results were obtained from henipavirus infection studies of
ferrets. Infected ferrets developed severe respiratory and neurological illness and have
therefore been used for studies to establish vaccines or to test medical treatments of
henipavirus infections (Bossart et al., 2009; Pallister et al., 2009). Experimental henipavirus
infections of African green monkeys resulted in respiratory and neurological symptoms,
generalized vasculitis, and death (Geisbert et al., 2010). The African green monkey models is
supposed to be the best reflection of henipavirus infections in humans (Lee and Rota, 2012).
This model has already been used for challenge and protection studies of HeV (Rockx et al.,
2010; Bossart et al., 2011).
Infection studies of animal models showed that a cross-reactive human monoclonal antibody
(m102.4) protected ferrets and African green monkeys from lethal henipavirus infections
(Bossart et al., 2009; Geisbert et al., 2010; Rockx et al., 2010). Further, a recombinant subunit
vaccine consisting of the ectodomain of HeV G protected ferrets, monkeys, cats, and horses
against HeV and NiV infections (Mungall et al., 2006; McEachern et al., 2008; Balzer, 2011;
Bossart et al., 2011; Pallister et al., 2011).
Introduction
- 42 -
1.4.6. NiV outbreaks
The first recognized NiV outbreaks occurred in Selangor, Negeri Sembilan, and Perak in
peninsular Malaysia during September 1998 to May 1999 (CDC, 1999a; Paton et al., 1999;
Chua et al., 2000; Chua, 2003). A total of 283 cases of febrile encephalitis and 109 deaths (39
%) were reported, most of the patients were in contact to infected pigs (CDC, 1999b; Chua et
al., 2000). During this outbreak no human-to-human transmission has been reported (CDC,
1999b; Parashar et al., 2000). Furthermore, eleven cases of febrile illness were reported from
abattoir workers in Singapore who were in contact with imported pigs from Malaysia in
March 1999 (Ling, 1999; Paton et al., 1999). Only one death was reported in Singapore. The
nucleotide sequence of NiV isolated from the abattoir worker who died was identical to the
NiV isolates from infected humans and pigs from Malaysia (Paton et al., 1999). Overall more
than 1.1 million pigs were compulsory culled to control the outbreak (Chua et al., 2000). The
outbreak ended in May 1999 and so far no further outbreaks have been reported from
Malaysia or Singapore.
Since 2001 there are nearly annual NiV outbreaks in Bangladesh with case fatality rates up to
100 %. Overall, the WHO reported 209 cases and 161 deaths during January 2001 to February
2012 (WHO, 2013). Contact to infected individuals and the consumption of raw date palm sap
were identified as the main risk factors for NiV infection in Bangladesh (Hsu et al., 2004;
Rahman and Chakraborty, 2012). A recent outbreak during January and May 2013 with 24
cases and 21 deaths was reported by the Institute of Epidemiology, Disease Control and
Research, Bangladesh (IEDCR, 2013).
India reported two outbreaks of NiV encephalitis from the West Bengal region. During
January and February 2001, 66 cases and 45 deaths were reported from Siliguri (Chadha et
al., 2006; Harit et al., 2006). In April 2007, five cases and five deaths were reported from
Nadia (Mandal and Banerjee, 2007).
So far, all recognized NiV outbreaks showed a geographically restriction to regions of
Southeast Asia, especially Malaysia and Bangladesh (Fig. 11).
- 43 -
Fig. 11: NiV outbreaks in humans in Southeast Asia, 1998 - 2013
Introduction
Introduction
- 44 -
1.5. Detection of bat-derived paramyxoviruses in African flying foxes
RNA of an African henipavirus named BatPV/Eid_hel/GH-M74a/GHA/2009 (M74, GenBank
accession number: HQ660129) was detected in spleen samples of the straw-coloured fruit bat,
Eidolon helvum in 2009 in Ghana (Drexler et al., 2012).
The straw-coloured fruit bat is the second largest chiropteran species in Africa with a
wingspan of 75 - 95 cm and a weight of 250 - 350 g (DeFrees, 1988). This African flying fox
feeds on flowers, and fruits including palm fruits. The distribution of Eidolon helvum
comprises the savanna and lowland rainforest areas of Africa from Senegal to Ethiopia and
South Africa, as well as the southwest of Saudi Arabia and Jemen (Mickleburgh et al.,
2008d). In West and Central Africa, E. helvum is hunted and consumed as bushmeat
(Mickleburgh et al., 2008d).
The RNA of a bat-derived mumps virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV,
GenBank accession number: HQ660095) was sampled from the spleen of an African flying
fox of the species Epomophorus (Epauletted fruit bat) in 2009 in the Democratic Republic of
Congo (Drexler et al., 2012).
The Epauletted fruit bats feed on fruits and figs (Acharya, 1992). They have a wingspan of 51
- 60 cm (males)/ 45 - 54 cm (females) and a body mass of 60 - 124 g (E. wahlbergi). Three
different species of Epomophorus occur in Africa and especially the Democratic Republic of
Congo: E. crypturus, E. labiatus, and E. wahlbergi. E. crypturus is distributed from the
Democratic Republic of Congo to Tanzania and the eastern coast of South Africa
(Mickleburgh et al., 2008a); E. labiatus is distributed in Central and South Africa and parts of
Ethiopia (Mickleburgh et al., 2008b); E. wahlbergi mainly occurs in South and East Africa
(Acharya, 1992; Mickleburgh et al., 2008c).
- 45 -
Introduction
1.6. Aim of the study
Aim of this study is the functional characterization of a bat-derived African henipavirus
(M74) and a bat-derived mumps virus (batMuV), two viruses which show sequence
similarities to the zoonotic henipaviruses Nipah virus (NiV) and Hendra virus (HeV) or the
human mumps virus (hMuV). Only RNA sequences comprising the complete genome of M74
and batMuV were detected in bats, but no infectious virus has been isolated.
Henipaviruses are known to be zoonotic agents leading to severe and fatal infections in
humans. Flying foxes of the species Pteropus were identified to be the reservoir host for NiV
and HeV. So far, NiV outbreaks were restricted to Southeast Asia, whereas all recognized
HeV outbreaks occured in Australia. The recent detection of the African henipavirus strain
M74 in Ghana raises the question if there is any relation between M74 and already known
henipaviruses and if such a virus is able to cross the species barrier and cause infection in
humans or other animals.
Mumps is a human disease and until now humans are the only known hosts of MuV. The
amino acid sequences of the glycoproteins of the bat-derived mumps virus, which was
detected in the Democratic Republic of Congo, show high similarities to the corresponding
proteins of different human MuV strains. The relatedness between human and bat-derived
MuV and the possibility of a transmission from bats to humans is so far not known.
To gain information about the African henipavirus and the bat-derived mumps virus, the open
reading frames (ORFs) of the M74 fusion (F) and attachment glycoprotein (G), as well as the
ORFs of the batMuV fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein were
cloned into expression plasmids and expressed in different immortalized chiropteran and nonchiropteran cell lines. At the beginning of this study, the glycoproteins were analyzed
concerning their functional activity in immortalized cell lines from different species and
organs. In this context, the ability to mediate cell-to-cell fusion - a feature which is common
to all paramyxoviruses - was analyzed in homo- and heterologous fusion assays.
The proteolytic cleavage of the M74 F protein was investigated by the treatment of
glycoprotein expressing cells with different protease inhibitors and the expression of chimeric
glycoproteins and mutants. To gain information whether the M74 G is able to utilize Eph B2,
the cellular receptor for henipaviruses, the interaction of M74 G and a recombinant receptor
Eph B2 or the ligand ephrin B2 was analyzed.
Introduction
- 46 -
Furthermore, it was analyzed whether the batMuV HN protein interacts with sialic acids and
shows hemadsorption and neuraminidase activity like some human MuV strains.
The results obtained for the M74 and batMuV glycoproteins were compared to the already
well studied and characterized glycoproteins of NiV or a human MuV isolate from a
hospitalized patient to gain information about the functional relatedness of these viruses and
their bat-derived counterparts. This information will be helpful for a risk assessment of a
potential spill-over from bats to humans.
- 47 -
Manuscript 1
2. SURFACE GLYCOPROTEINS OF AN AFRICAN HENIPAVIRUS
INDUCE
SYNCYTIUM FORMATION IN A CELL LINE DERIVED
FROM AN AFRICAN FRUIT BAT, HYPSIGNATHUS MONSTROSUS
Nadine Krüger1, Markus Hoffmann1, Michael Weis2, Jan Felix Drexler3, Marcel Alexander
Müller3, Christine Winter1, Victor Max Corman3, Tim Gützkow1, Christian Drosten3, Andrea
Maisner2, Georg Herrler1#
1
Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany
2
Institute of Virology, Philipps University Marburg, Marburg, Germany
3
Institute of Virology, University of Bonn Medical Centre, Bonn, Germany
#Correspondence to:
Georg Herrler
Institute of Virology, University of Veterinary Medicine Hannover
Bünteweg 17
D-30559 Hannover
fon: +49 511 953 8857
fax: +49 511 953 8898
email: [email protected]
State of publication: published
J. Virol. 2013, 87(24):13889-13891. doi: 10.1128/JVI.02458-13.
Available at: http://jvi.asm.org/content/87/24/13889.full.pdf+html
Abstract
Serological screening and detection of genomic RNA indicates that members of the genus
Henipavirus are present not only in Southeast Asia but also in African fruit bats. We
demonstrate that the surface glycoproteins F and G of an African henipavirus (M74) induce
syncytium formation in a kidney cell line derived from an African fruit bat, Hypsignathus
monstrosus. Despite a less broad cell tropism, the M74 glycoproteins show functional
similarities to the glycoproteins of Nipah virus.
- 48 -
- 49 -
Manuscript 2
3. ATTACHMENT PROTEIN G OF AN AFRICAN BAT HENIPAVIRUS IS
DIFFERENTIALLY RESTRICTED IN CHIROPTERAN AND NONCHIROPTERAN CELLS
Nadine Krüger1, Markus Hoffmann1, Jan Felix Drexler2, Marcel Alexander Müller2, Victor
Max Corman2, Christian Drosten2, Georg Herrler1#
1
2
#Correspondence to:
Georg Herrler
Bünteweg 17
D-30559 Hannover
fon: +49 511 953 8857
fax: +49 511 953 8898
J. Virol. 2014, 88(20): 11973-11980. doi: 10.1128/JVI.01561-14
Available at: http://jvi.asm.org/content/88/20/11973.full.pdf+html
Abstract
Henipaviruses are associated with pteropodid reservoir hosts. The glycoproteins G and F of an
African henipavirus (strain M74) have been reported to induce syncytium formation in kidney
cells derived from a Hypsignathus monstrosus bat (HypNi/1.1), but not in the non-chiropteran
cells BHK-21 and Vero76. Here we show that syncytia are also induced in two other
pteropodid cell lines from Hypsignathus monstrosus and Eidolon helvum bats upon coexpression of the M74 glycoproteins. The G protein was transported to the surface of
transfected chiropteran cells, whereas surface expression in the non-chiropteran cells was
detectable only in a fraction of cells. By contrast, the G protein of Nipah virus is transported
efficiently to the surface of both chiropteran and non-chiropteran cells. Even in chiropteran
Manuscript 2
- 50 -
cells, M74-G was predominantly expressed in the ER as indicated by colocalization with
marker proteins. This result is consistent with the finding that all N-glycans of the M74-G
proteins are of the mannose-rich type as indicated by sensitivity to endo H treatment. These
data indicate that the surface transport of M74-G is impaired in available cell culture systems
with larger amounts of viral glycoprotein present on chiropteran cells. The restricted surface
expression of M74-G explains the reduced fusion activity of the glycoproteins of the African
henipavirus. Our results suggest strategies for the isolation of infectious viruses which is
necessary to assess the risk of zoonotic virus transmission.
Importance
Henipaviruses are highly pathogenic zoonotic viruses associated with pteropodid bat hosts.
Whether the recently described African bat henipaviruses have a similarly high zoonotic
potential as their Asian and Australian relatives is unknown. We show that surface expression
of the attachment protein G of an African henipavirus, M74, is restricted in comparison to the
G protein of the highly pathogenic Nipah virus.
Transport to the cell surface is more restricted in non-chiropteran cells than it is in chiropteran
cells explaining the differential fusion activity of the M74 surface proteins in these cells. Our
results imply that surface expression of viral glycoproteins may serve as a major marker to
assess the zoonotic risk of emerging henipaviruses.
- 51 -
Manuscript 3
4. CHARACTERIZATION OF AFRICAN BAT HENIPAVIRUS GH-M74A
GLYCOPROTEINS
Michael Weis1, Laura Behner1, Markus Hoffmann2, Nadine Krüger2, Georg Herrler2,
Christian Drosten3, Jan Felix Drexler3, Erik Dietzel1, and Andrea Maisner1#
1
Institute of Virology, Philipps University Marburg, Marburg, Germany
2
3
# Correspondence to:
Andrea Maisner
Philipps University Marburg
Hans-Meerwein-Straße 2
D-35043 Marburg
Phone: +49 6421 28-65360
Fax: +49 6421 28-68962
J Gen Virol. 2014 Mar;95(Pt 3):539-548. doi: 10.1099/vir.0.060632-0
Available at: http://vir.sgmjournals.org/content/95/Pt_3/539.long
Abstract
In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To
evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we
compared the biological functions of the surface glycoproteins G and F of the prototype
African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a wellcharacterized pathogenic member of the henipavirus genus. Glycoproteins are central
determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular
ephrin receptors and functional expression of fusion-competent F proteins are indispensable
prerequisites for virus entry and cell-to-cell spread. In this study, we analysed the ability of
the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily
Manuscript 3
- 52 -
permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat
cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any
fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst
GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and
induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did
not cause evident fusion in the presence of heterotypic NiV G protein. Pulse-chase and
surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression
of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of
GH-M74a showed a functional defect that is most likely caused by impaired trafficking
leading to less efficient proteolytic activation and surface expression.
- 53 -
Manuscript 4
5. ANALYSIS OF THE PROTEOLYTIC CLEAVAGE OF THE FUSION
PROTEIN OF THE AFRICAN HENIPAVIRUS M74
Max Corman2, Christian Drosten2, Georg Herrler1#
1
Institute of Virology, University of Veterinary Medicine Hannver, Hannover, Germany
2
#Correspondence to:
Georg Herrler
Institute of Virology, Stiftung Tierärztliche Hochschule Hannover
Bünteweg 17
D-30559 Hannover
fon: +49 511 953 8857
fax: +49 511 953 8898
State of publication: in preparation
Manuscript 4
- 54 -
5.1. Abstract
The inefficient transport of the attachment glycoprotein (G) of the African henipavirus strain
M74 (M74) to the cell surface has been reported to play an important role concerning the
restricted functional activity of the M74 fusion (F) and attachment glycoprotein, which are
able to mediate cell-to-cell fusion only in chiropteran cells.
No differences between chiropteran and non-chiropteran cells were obtained when the
proteolytic cleavage and expression of the M74 F was analyzed: Cleavage, as well as surface
expression was similar in all tested cell lines, regardless if they showed cell-to-cell fusion or
not. When the proteolytic cleavage of M74 F was compared to that of NiV F, it could be
indicated that also the M74 F has to undergo endocytosis before the cleavage occurs in an
acidic compartment. Whereas the cleavage of NiV F is initiated by cathepsin (Cat) L, the
inhibition of cathepsins had no effect on the proteolytic cleavage of M74 F. Further, the
cleavage of M74 F does not require an R at the predicted monobasic cleavage site at the aa
residue 205. From the expression of truncated mutants of M74 F we suggest, that an
additional cleavage of the M74 F occurs during an early stage of the secretory pathway.
5.2. Introduction
The genus Henipavirus within the Paramyxoviridae family comprises two highly pathogenic
viruses, Nipah virus (NiV) and Hendra virus (HeV) that can cause severe encephalitis in
humans with case fatality rates of 40 to 100 %.
HeV emerged in 1994 in Queensland, Australia, and causing severe respiratory and/ or
neurological disease in horses and sporadically humans who had direct contact to infected
animals [1-3]. NiV was isolated in March 1999 from the cerebrospinal fluid of a hospitalized
patient from Malaysia, where NiV has been transmitted from pigs to farmers and abattoir
workers [4, 5]. Since 2001, nearly annual NiV outbreaks in humans were reported from
Bangladesh; two NiV outbreaks occurred in India [6]. Epidemiological studies revealed that
NiV was transmitted from flying foxes to humans via the consumption of contaminated palm
sap [7-9]. Also human-to-human transmission was reported [10, 11]. Asian fruit bats of the
genus Pteropus have been identified as the natural reservoir of NiV and HeV [12-16].
The detection of genomic RNA in bats as well as of cross-neutralizing antibodies in bats and
domestic animals (goat, sheep, pig) indicated that henipaviruses are also present in Africa
[17-23]. So far, all efforts to isolate an African henipavirus have failed which makes it
difficult to assess the zoonotic potential of these viruses.
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Manuscript 4
The infection of cells by NiV and HeV is initiated by the binding of the viral attachment
glycoprotein (G) to the ubiquitously expressed cellular receptors Eph B2 or Eph B3 [24-27].
Following the binding of the G protein to the cellular receptor, the fusion protein (F) induces
the fusion of the viral envelope with cellular membranes to release the viral genome into the
cytoplasm. Co-expression of F and G on the surface of infected or transfected cells results in
cell-to-cell fusion of neighbouring cells and thus in the formation of syncytia, i.e.
multinucleated giant cells. The fusion activity of NiV and HeV F depends on prior proteolytic
activation that occurs after internalization from the cell surface during an endosomal recycling
process. Within the acidic environment of vesicular structures, the inactive precursor F0 is
converted into the mature, fusion-active form by cathepsins (Cat) B or L that cleave the F
protein into the subunits F1 and F2 [28-32].
It has been shown recently that the attachment glycoprotein (G) of the African henipavirus
strain M74 (M74, BatPV/Eid_hel/GH-M74a/GHA/2009, GenBank accession number
HQ660129) is able to interact with the henipavirus receptor Eph B2 and B3 or its ligand
ephrin B2 [33-36]. Furthermore, it has been demonstrated that the M74 fusion protein (F) is
proteolytically cleaved into the F1 and F2 subunits in an acidic compartment [33, 34]. The
ability of M74 F and G to mediate cell-to-cell fusion was found to be restricted to chiropteran
cells as a consequence of an inefficient transport of the M74 G to the cell surface [44].
In our further studies we wanted to address the question whether the proteolytic cleavage of
M74 F occurs similar to that of NiV F. We report that the M74 F protein has to undergo
endocytosis before the cleavage occurs in an intracellular, acidic compartment. In contrast to
NiV F, the cleavage of M74 F was not abolished by inhibition of cathepsins. The expression
of mutants of M74 F revealed that the cleavage of this protein does not require an R at the aa
residue 205, the putative cleavage site of henipavirus F proteins.
5.3. Methods
Cells
BHK-21, Vero76, and HypNi/1.1 cells, a kidney cell line derived from Hypsignathus
monstrosus [33], were maintained in Dulbecco´s minimum essential medium (DMEM;
Gibco) supplemented with 5 % (BHK-21, Vero76) or 10 % (HypNi/1.1) fetal calf serum
(FCS; Biochrom). Cells were cultivated in 75 cm² tissue culture flasks (Greiner Bio-One) at
37°C and 5% CO2.
Manuscript 4
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Plasmids
The open reading frame (ORF) of M74 F (GenBank accession number AFH96010.1) was
cloned into the expression vector pCG1, which was kindly provided by R. Cattaneo. The HA
epitope labelled constructs of M74 F (M74 F-HA) and NiV F (NiV F-HA, GenBank
accession number AF212302, kindly provided by A. Maisner), as well as the FLAG epitope
labelled construct of NiV G (GenBank accession number AF212302) and M74 G (GenBank
accession number AFH96011.1) have been described previously [33, 37].
Mutants of NiV F in which (i) the R109 was replaced by A (NiV F-HA (R109A)), (ii) the aa
residues 100 - 108 were replaced by A (NiV F-HA (9A)), or (iii) the N of an motif for
N-linked glycosylation at the aa residue 99 was replaced by Q (NiV F-HA (N99Q)).
Corresponding mutants of M74 F were generated in which (i) the predicted monobasic
cleavage site at aa residue 205 was replaced by an A (M74 F-HA (R205A)), (ii) the aa
residues 196 - 204 or 196 - 205 were replaced by alanines (M74 F-HA (9A), M74 F-HA
(10A), respectively), or (iii) the predicted N-linked glycosylation motif upstream of the
cleavage site was destroyed by the replacement of N at position 195 by Q (M74 F-HA
(N195Q)). A predicted multibasic cleavage site of M74 F was replaced by alanines (M74 F
(92-97A)) or deleted (M74 F (∆92-97)). Additionally, the aa residues 92 -97 and 205 of M74
F were substituted by alanines (M74 F (92-97A+R205A)). Furthermore, chimeric proteins of
NiV and M74 F were generated. Therefore, the aa residues 1 - 12 of NiV F or 1 - 108 of M74
F were replaced by the aa residues 1 - 108 of M74 F or 1 - 12 of NiV F, respectively (NiV FHA (M74start), M74 F-HA (NiVstart)).
Immunofluorescence analysis (IFA)
Cells were grown on coverslips and transfected with the epitope labelled F and G proteins
using the ICAFectin ® 441 transfection reagent (InCellArt). At 24 h post-transfection (p.t.),
cells were fixed and permeabilized with 0.2 % Triton-X-100. To detect the HA-labelled
constructs, cells were incubated with an antibody directed against the HA epitope (anti-HA,
rabbit, Sigma) and an FITC-conjugated anti-rabbit secondary antibody (Sigma), for 1 h or 30
min, respectively. The FLAG-labelled NiV and M74 G proteins were detected by incubation
with an antibody directed against the FLAG epitope (anti-FLAG, mouse, Sigma), followed by
an incubation with anti-mouse IgG F(ab´) 2-fragment Cy3 (Sigma). Finally, cells were treated
with DAPI (Roth) and mounted in Mowiol. Immunofluorescence analysis (IFA) was
performed using a Nikon Eclipse Ti fluorescence microscope and the NIS Elements AR
software (Nikon).
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Manuscript 4
Preparation of cell lysates, SDS PAGE, and Western Blot analysis
Cells were grown in 6-well plates and transfected for expression of the epitope-labelled F
constructs using Lipofectamine ® 2000 transfection reagent (Life Technologies). At 24 h p.t.,
cells were detached from the bottom of the plates using a cell scraper, collected in reaction
tubes, and centrifuged for 15 min and 600 x g at 4 °C. After the removal of the supernatants,
the cell pellet was resuspended in NP-40 lysis buffer with protease inhibitors and incubated
on ice for 1 h. Afterwards, the cell lysates were centrifuged for 30 min and 10.000 x g at 4 °C
and the supernatant was transferred to a new reaction tube. Subsequently, 4x LDS sample
buffer (final concentration 1x, Life Technologies) was added to the samples, which were then
incubated for 10 min at 96 °C in the presence of 0.1 M (final concentration) 1,4-dithio-DLthreitol (DTT) prior to loading onto an SDS gel (10 %) for SDS PAGE and Western blotting.
Proteins were detected by incubation with an anti-HA antibody (rabbit, Sigma), followed by
anti-rabbit horseradish peroxidase (HRP, Dako). Incubation with primary antibodies was
performed at 4 °C overnight, while secondary antibodies were applied for 1 h at 4 °C. For the
visualization of protein bands, membranes containing the immobilized proteins were
incubated with Super Signal West Dura Extended Duration Substrate (Thermo Scientific),
placed in a Chemi doc imager (Biorad), and analyzed with the Quanti One software (Biorad).
Surface biotinylation
At 24 h p.t., transfected cells were washed with ice-cold PBS, followed by incubation with 0.5
mg/ ml LC biotin (Thermo Scientific) dissolved in PBS for 20 min at 4°C on a rocking
platform shaker. Afterwards, the biotin solution was removed, cells were washed with PBS
0.1 M glycine buffer and further incubated with PBS glycine buffer for 15 min at 4°C. Next,
the cells were scraped from the bottom of the plates, collected in reaction tubes, and
centrifuged for 15 min and 600 x g at 4 °C. To determine the relative amounts of the cleaved
and uncleaved forms of M74 F at different time points post surface-biotinylation, cells were
further incubated in cell culture medium at 37 °C for 0 - 24 h post biotinylation, before the
cells were scraped from the plates, collected in reaction tubes, and centrifuged for 15 min and
600 x g at 4 °C. The supernatants were removed by aspiration and the pellets were
resuspended in 500 µl NP-40 lysis buffer with protease inhibitors. Cell lysates were mixed
with streptavidin-agarose (Pierce). After overnight incubation at 4 °C on an overhead shaker,
samples were centrifuged for 5 min and 10.000 x g at 4 °C. Next, the supernatants were
removed by aspiration, the agarose-bound proteins were washed with 250 µl NP40 cell lysis
buffer without protease inhibitor and centrifuged again for 5 min and 10.000 x g at 4°C. This
Manuscript 4
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step was repeated 3 times, before 50 µl of 2x SDS sample buffer were added to the pellet, and
the samples were incubated for 10 min at 96 °C to release bound protein from the agarose
beads. After a final centrifugation for 5 min at 10.000 x g at room temperature, the resulting
supernatants were collected, and subjected to SDS PAGE and Western blotting. Protein
detection and visualization of the protein bands was performed as described above.
Protease inhibitors
Cells were transfected for expression of the epitope-labelled NiV or M74 F proteins. At 4 h
p.t., the medium was removed and cells were washed with PBS for three times. Cells were
further incubated in cell culture medium containing DMSO, chloroquine (80 µM, Promega),
E-64d (20 µM, Sigma), Cat B inhibitor II (30 nM, Calbiochem), or Cat L inhibitor III (20 µM,
Calbiochem). At 24 h p.t., cells were subjected to immunofluorescence microscopy or
Western blotting as described before.
Generation of VSV pseudotype (VSVpp)
The preparation of VSV*ΔG-Luc viruses, as well as the generation of VSV pseudotypes
(VSVpp) have been described elsewhere [38, 39]. We generated VSVpp harbouring VSV G,
NiV F + NiV G, NiV F (M74start) + NiV G, M74 F + M74 G, and M74 F (NiVstart) + M74 G.
To quantify the infection, the luciferase activity of lysates of infected cells was determined
using the Luciferase assay system E1500 Kit (Promega) and the GENiosPro
chemiluminometer (Tecan) in accordance to the manufacturer´s protocol.
5.4. Results
The cleavage efficiency of M74 F is similar in chiropteran and non-chiropteran cell lines.
Western Blot analysis of lysates of transfected cells expressing M74 F revealed that the F
protein is expressed in different mammalian cell lines in similar amounts (Fig. 1).
Furthermore, cleaved F1 subunits of M74 F were detected in cell lysates of chiropteran and
non-chiropteran cells, indicating that the efficiency of the proteolytic cleavage of M74 F did
not differ between these cell lines.
Proteolytical cleavage occurs after M74 F was expressed on the cell surface.
After having shown, that cleaved forms of M74 F are expressed in transfected cells, we
focused our interest on the question whether the cleavage requires a recycling step similar to
that of NiV F. When the relation of the uncleaved F0 and the cleaved F1 subunit on the cell
surface of BHK-21 cells was analyzed by Western blots, most of the M74 F protein was
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Manuscript 4
detected in the uncleaved F0 form, whereas after 3 h post surface biotinylation an increase of
the cleaved F1 subunit and a decrease of the uncleaved F0 were detected (Fig. 2). The same
result was obtained for Vero76 and HypNi/1.1 cells (data not shown). This result indicates
that the M74 F was expressed on the cell surface prior to the proteolytic cleavage event.
Cleavage occurs at a low pH, but is not abolished by inhibitors of cathepsins.
Sequence analysis showed that the amino acid residues at the C-terminus of the fusion peptide
are not highly conserved amoung henipavirus F proteins. The predicted monobasic cleavage
site of NiV F consists of an R at position 109, whereas HeV F expresses a K at the
corresponding position and CedPV an M. M74 F expresses an R at the aa position 206, which
precedes a putative fusion peptide (aa 206 - 225 FAGVIIAGVALGVAAAAQIT). While the
region, starting at R206 and followed by the putative fusion peptide of M74 F has a high
similarity to the corresponding aa residues of all known henipaviruses, the aa residues directly
upstream of the putative cleavage site show a lower conservation (Fig. 3).
It has already been shown that the cleavage of M74 F requires a low pH and thus resembles
NiV F [33]. To gain information whether the proteolytic cleavage is mediated by cathepsins,
cells expressing the M74 F were treated with inhibitors of cysteine protease, Cat B, or Cat L,
as well as chloroquine that prevents the endosomal acidification. As expected from past
studies [28, 29], the cleavage of NiV F was inhibited by E-64d, Cat L inhibitors, and
chloroquine, whereas the inhibition of Cat B had no influence on the proteolytic cleavage
(data not shown). Differences were observed for the M74 F. Only the treatment with
chloroquine abolished the cleavage, whereas neither E-64d nor the Cat inhibitors were active
in this respect (Fig. 4).
The proteolytic cleavage of M74 F does not require an R directly upstream of the fusion
peptide.
Mutants of M74 F in which the predicted monobasic cleavage site R205 or the aa residues
196 - 204 or 196 - 205 were replaced by A (Fig. 5) were analyzed with respect to their
functional activities and proteolytic cleavage. Western blot analysis showed that all three
mutants are cleaved as efficiently as the parental M74 F (Fig. 6A). Furthermore, no
differences in the number or the size of syncytia were obtained for any of the mutants when
compared to those derived from the co-expression of the parental M74 F and M74 G in
HypNi/1.1 cells (data not shown). In contrast to this, differences were obtained for mutants of
NiV F: Whereas NiV F (R109A) was still cleaved and mediated cell-to-cell fusion as already
Manuscript 4
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reported by Moll et al. [40], the replacement of the aa residues 100 - 108 by A abolished the
proteolytic cleavage, as well as the ability to mediate fusion (Fig. 6B).
In consistence with past studies the destruction of the N-linked glycosylation site at the aa
residues 99 of NiV F resulted in an increase of the fusogenicity [37, 41]. For the
corresponding M74 F mutant we did not observe a higher fusogenicity in comparison to the
parental M74 F, even if the total amount of expressed M74 F (N195Q) was increased as
indicated by a stronger band intensity of the detected mutant in Western Blots (Fig. 6A).
Furthermore, the M74 F contains a predicted multibasic cleavage motif (K-R-G-K-R-R) at the
aa residues 92 - 97. This multibasic motif is located far upstream of the putative fusion
peptide (starting at aa residue 207) and might serve as an additional cleavage site. We found
that a deletion or a replacement of these aa residues had no effect on the proteolytic cleavage
(Fig. 6A) or the ability to mediate syncytium formation (data not shown).
The N-terminal region of M74 F is not responsible for the restricted functional activity
Sequence analysis showed that the N-terminal sequence of the M74 F protein is about 106 aa
longer than that of NiV F and contains a potential multibasic cleavage site (K-R-G-K-R-R).
By analyzing the cleavage of the chimeric F proteins NiV F (M74start) and M74 F (NiVstart) via
Western blotting, we observed that both mutants were cleaved with a similar efficiency when
compared to their corresponding parental F proteins (Fig. 7A). Interestingly, the molecular
weight of the detected F0 form did not differ between NiV F (M74start) and NiV F, although
the protein length and the calculated molecular weight of NiV F (M74start) (71.6 kDa) are
higher compared to the parental NiV F (60 kDa). The chimeric M74 F (NiVstart), which is 94
aa shorter in comparison to M74 F and has a calculated molecular weight of 64 kDa, was
detected at the same molecular weight as the parental M74 F protein (70 kDa).
The ability to mediate cell-to-cell fusion was analyzed in chiropteran and non-chiropteran cell
lines. As shown in Fig. 7B, co-expression of NiV F (M74start) and NiV G led to the
formation of syncytia, the size and number of which were similar to syncytia induced by the
co-expression of NiV F and G, in Vero76 and HypNi/1.1 cells. In contrast to this, M74 F
(NiVstart) was not able to mediate syncytium formation in Vero76, but in HypNi/1.1 cells.
These syncytia were phenotypically similar to those obtained after the co-expression of M74
F and G. Furthermore, VSVpp containing NiV F (M74start) and NiV G were able to mediate
infection, however the luciferase activity was lower in comparison the cells infected by
VSVpp containing NiV F and G (Fig. 7C). No luciferase activity above the background level
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Manuscript 4
was measured with cells infected by VSVpp harbouring M74 F and G or M74 F (NiVstart) and
M74 G.
5.5. Discussion
Having shown previously, that the proteolytic cleavage of the fusion protein (F) of the
African henipavirus M74 occurs at a low pH [33], similar to that of NiV F, the results from
this study provide novel insights on the proteolytic cleavage of M74 F.
The chiropteran cell line HypNi/1.1, has been shown to be susceptible to cell-to-cell fusion
induced by co-expression of M74 F and G [33]; by contrast, the non-chiropteran cells
BHK-21, and Vero76 cells do not support cell-to-cell fusion. When we analyzed the cleavage
efficiency of M74 F in cell lysates, we found no difference in these cells. A similar result was
reported by Weis et al., 2014: No differences in the surface expression level and cleavage
efficiency of M74 F were detected between HypNi/1.1 cells and MDCK cells, the latter cell
line did not show syncytium formation following co-expression of M74 F and G [34].
For NiV and HeV F it has been demonstrated that, the uncleaved precursor protein F0 which
is expressed at the cell surface of an infected cell undergoes a clathrin-mediated endocytosis
leading to its inclusion into endosomes [31, 42]. There, the low pH enables cathepsin (Cat) L
to further process the F protein by cleaving the polyeptide into the F1/ F2 subunits, which are
connected by disulphide bonds [29, 43]. This cleavage is necessary for the fusogenicity of the
F protein, since only cleaved F protein is able to promote membrane fusion. Following, Cat
L-cleavage, the activated F protein is relocated to the plasma membrane and can either be
incorporated into new viral particles or, in cooperation with the G protein, induce syncytium
formation.
To get information whether the M74 F undergoes a recycling process similar to that of NiV
and HeV F, the shift of the uncleaved precursor F0 to the proteolytic cleavage product F1 on
the surface of M74 F transfected cells was analyzed by surface biotinylation and Western
blotting. In this way, it was demonstrated that - over the time - the amount of the cleaved F1
subunit increased, while, at the same time, the amount of the uncleaved F0-form decreased.
However, the cleavage kinetics of M74 F was slower in comparison to that of NiV F [34].
Our data indicate that the cleavage of M74 F occurs after the protein has been expressed on
the cell surface and therefore, an endocytosis process of M74 F - similar to NiV F - is very
likely.
Manuscript 4
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After having shown that the M74 F is cleaved in a pH dependent manner [33] and that
cleavage occurs after the F0 form has been transported to the plasma membrane, we wanted to
gain information about which kind of cellular protease that is responsible for the cleavage.
Sequence analysis of NiV and M74 F indicated that the M74 F contains an R (R205) as a
potentially monobasic cleavage site at the corresponding aa position of the NiV F cleavage
site R109. We found that an inhibition of the endosomal acidification resulted in an inhibition
of the proteolytic cleavage, as well as the fusogenic activity of M74 F indicating that the
cleavage is pH-dependent and occurs in an acidic environment - similar to that of NiV F.
Differences between NiV and M74 F were observed when transfected cells were treated with
inhibitors of cysteine proteases (E-64d), Cat B, or Cat L: Whereas the cleavage of NiV F was
inhibited by E-64d as well as inhibitors of Cat L, neither E-64d nor the Cat B or L inhibitors
reduced the proteolytic cleavage of M74 F. A possible explanation for this observation may
be that the cleavage is mediated by an uncommon Cat, which is not covered by the spectrum
of the Cat inhibitors. Alternatively, another, non-cathepsine protease, the activity of which
also relies on a low pH, may be responsible for the cleavage of M74 F.
Similar to a previous study reporting that an exchange of the R109 of NiV F did not affect the
proteolytic cleavage [40], the exchange of the predicted cleavage site of M74 F (R205) to A
did not reduce or abolish the proteolytic cleavage of the F protein or the ability to mediate
cell-to-cell fusion following co-expression with M74 G. In contrast to NiV F, even the
substitution of nine aa residues directly upstream of the fusion peptide, had no effect on the
proteolytic cleavage of M74 F. From these findings, it is concluded that either the cellular
protease which is responsible for the proteolytic cleavage of the M74 F does not recognize a
specific monobasic cleavage motif or the motif is not located in the nine aa residues directly
upstream of the predicted fusion peptide.
The M74 F protein is about 106 aa longer than NiV F and contains a potential multibasic
cleavage site (K-R-G-K-R-R) within the first 100 aa residues (aa 92 - 97). To analyze whether
these N-terminal aa residues affect the processing of the M74 F protein, two different sets of
mutants were generated: (i) A truncated M74 F in which the aa residues 1 - 108 were replaced
by the aa residues 1 - 12 of NiV F, M74 F (NiVStart), together with a chimeric mutant of NiV
F in which the aa residues 1 - 12 of NiV F were replaced by the aa residues 1 - 108 of M74 F,
NiV F (M74Start), and (ii) mutant M74 F proteins where the aa residues of the multibasic motif
were either replaced by A, M74 F (92 - 97A), or completely deleted, M74 F (∆92 - 97).
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Manuscript 4
Western Blot analysis and cell-to-cell fusion assays of these mutants showed, (i) that no
differences in the molecular weight were detected between the parental M74 F and the M74 F
(NiVStart), as well as between the parental NiV F and the NiV F (M74Start). Co-expression with
the corresponding G proteins revealed that the chimeric NiV F (M74start) mediates cell-to-cell
fusion in HypNi/1.1 and Vero76 cells as efficiently as the parental NiV F protein. In contrast
to this, M74 F (NiVstart) mediated syncytium formation only in HypNi/1.1, but not in Vero76
cells. Furthermore, the phenotype of the syncytia was similar to that obtained from the coexpression of M74 F and G. Whereas VSVpp containing NiV F (M74start) and NiV G were
able to mediate infection of different cell lines, only a low luciferase activity was measured
for VSVpp harbouring M74 F (NiVstart) and M74 G. In accordance with the just described
observation, (ii) a replacement of the aa residues of the multibasic motif by A or their deletion
did not result in differences in F protein cleavage or fusogenicity compared to parental M74
F.
A recent report claimed that the additional N-terminal aa of M74 F, which are not present in
the henipaviruses NiV or HeV, are the result of a sequencing error at an adenine-thymine-rich
region, and supported this statement by the lack of a typical signal peptide sequence [35]. In
this study, the introduction of an alternative start codon by the insertion of a nucleotide at
position 303 created an in frame ATG, resulting in a truncated M74 F protein that has an Nterminus more similar to NiV F [35]. In that study it was also shown by Western blotting that
the molecular weight of the truncated M74 F did not differ from the parental M74 F, but the
authors did not comment on this.
From these data we suggest that in addition to the proteolytic cleavage required for M74 F
fusogenicity, an additional cleavage occurs within the first 108 aa during an early stage of the
secretory pathway. The N-terminal aa residues of the M74 F may be removed by proteolytic
cleavage before the protein is expressed on the cell surface to interact with the M74 G protein.
The N-terminal aa residues do not have an effect on the phenotype of syncytia which are
restricted to chiropteran cells.
In summary, we have shown, that the M74 F protein is proteolytically cleaved in chiropteran
and non-chiropteran cell lines with a similar efficiency - regardless whether they showed
cell-co-cell fusion after co-expression of M74 F and G or not. These data support the idea that
the inefficient surface expression of the M74 G rather than the cleavage or expression of the
M74 F protein is mainly responsible for the phenotype of syncytia induced by the
glycoproteins of the African henipavirus M74.
Manuscript 4
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Furthermore, evidence was obtained which supported the notion that the M74 F undergoes a
recycling process to be cleaved enabling proteolytic cleavage within endosomes. However,
the cellular protease which mediates the proteolytic cleavage of M74 F, as well as the
cleavage motif are still unknown. Further studies e.g. the incubation of (soluble) M74 F
protein with different intra- or extracellular proteases will help to elucidate the identity of the
M74 F-cleaving protease.
Our data provide important information concerning the risk of a zoonotic transmission event.
Given that the cleavage efficiency is similar in chiropteran and non-chiropteran cell lines, the
proteolytic activation of the F protein does not represent a restriction factor for a spillover to
new, non-chiropteran hosts.
5.6. Acknowledgments
Part of this work was performed by N.K. in partial fulfillment of the requirements for the
doctoral degree from Stiftung Tierärztliche Hochschule Hannover. N.K. was funded by a
fellowship of the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology,
and Translational Medicine (HGNI). We are grateful to Roberto Cattaneo for providing
expression plasmids. This work was supported by grants to GH from DFG (HE 1168/14-1 and
SFB621 TP B7) and from Bundesministerium für Bildung und Forschung (Ecology and
Pathogenesis of SARS, project code 01Kl1005B and FluResearchNet, project code
01KI1006D).
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20. Hayman DT, Suu-Ire R, Breed AC, McEachern JA, Wang L, Wood JL, and Cunningham AA:
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V: Surface glycoproteins of the recently identified African Henipavirus promote viral entry and cell
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Herrler G, Feldmann H, Drosten C, and Pohlmann S: Comparative analysis of Ebola virus
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attachment glycoprotein of an African henipavirus is differentially restricted in chiropteran and nonchiropteran cells. J Virol 2014, 88:11973-11980
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5.8. Figures
Fig 1: Cleavage of M74 F in chiropteran and non-chiropteran cells. BHK-21, Vero76, and
HypNi/1.1 cells were transfected for expression of M74 F-HA. At 24 h p.t, cells were lysed
and subjected to SDS PAGE under reducing conditions and Western blotting. Proteins were
detected by incubation with an anti-HA antibody, followed by incubation with anti-rabbit
HRP. Molecular mass markers are indicated on the left (kDa).
Fig. 2: Endocytosis of M74 F. BHK-21 cells were transfected with M74 F-HA. At 24 h p.t.,
surface biotinylation was performed. Cells were further incubated with cell culture medium
before the lysates were loaded on streptavidin-agarose. Cell lysates were subjected to SDS
PAGE and Western blotting. M74 F was detected by incubation an anti-HA antibody,
followed by incubation with anti-rabbit HRP. Molecular mass markers are indicated on the
left (kDa).
Fig. 3: Cleavage site and fusion peptide of henipavirus F proteins. The aa residues 100 129 of NiV F were aligned to the corresponding aa residues of HeV F (aa 100 - 129), CedPV
F (99 - 128) and M74 F (196 – 225). The monobasic cleavage site is indicated by bold letters.
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Fig 4: Effect of cathepsin inhibitors on the cleavage of M74 F. BHK-21 cells were
transfected for expression of M74 F-HA. At 4 h p.t., cells were incubated with cell culture
medium containing DMSO, 20 µM E-64d, 30 nM Cat B inhibitor II, 20 µM Cat L inhibitor III,
or 80 µM chloroquine. At 24 h p.t, cells were lysed and subjected to SDS PAGE under
reducing conditions and Western blotting. Proteins were detected by incubation with an antiHA antibody, followed by incubation with anti-rabbit HRP. Molecular mass markers are
indicated on the left (kDa).
Fig 5: Schema of NiV and M74 F cleavage mutants. The predicted cleavage sites are
indicated by bold letters. Substitutions are shown in red letters.
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Fig 6: Cleavage and fusion activity of NiV and M74 F mutants. (A) BHK-21 cells were
transfected with NiV or M74 F-HA and mutants of NiV and M74 F. At 24 h p.t, cells were
lysed and subjected to SDS PAGE under reducing conditions and Western blotting. Proteins
were detected by incubation with an anti-HA antibody, followed by incubation with antirabbit HRP. Molecular mass markers are indicated on the left (kDa). (B) Cells were cotransfected with NiV F-HA or mutants of F and NiV G-FLAG. At 24 h p.t., cells were fixed,
permeabilized and incubated with an antibody directed against the HA-tag and another one
against the FLAG epitope, followed by incubation with FITC- and Cy3-conjugated secondary
antibodies
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Fig. 7: Chimeric F proteins. (A) Cells were transfected with NiV or M74 F-HA (wt), or the
chimeric F proteins (M74start/ NiVstart). At 24 h p.t, cells were lysed and subjected to SDS
PAGE under reducing conditions and Western blotting. Proteins were detected by incubation
with an anti-HA antibody, followed by incubation with anti-rabbit HRP. Molecular mass
markers are indicated on the left (kDa). (B) Cells were co-transfected with the parental or
chimeric NiV or M74 F-HA proteins and NiV or M74 G-FLAG. At 24 h p.t., cells were fixed,
permeabilized and incubated with primary antibodies directed against the HA- or the FLAGtag, followed by incubation with FITC- and Cy3-conjugated secondary antibodies. (C) VSVpp
harbouring pCG1, VSV G, NiV F+G, NiV F (M74 start)+G, M74 F+G, or M74 F (NiV
start)+G were generated in BHK-21 cells. Undiluted supernatants were used for the infection
of BHK-21, Vero76, and HypNi/1.1 cells. VSVpp infection was analyzed via measurement of
the luciferase activity. Luciferase activity is given in x-fold changes compared to
VSVpp(pCG1).
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6. FUNCTIONAL PROPERTIES AND GENETIC RELATEDNESS OF THE
FUSION AND HEMAGGLUTININ-NEURAMINIDASE PROTEINS OF A
MUMPS VIRUS-LIKE BAT VIRUS
Max Corman2, Christian Sauder3, Steven Rubin3, Biao He4, Claes Örvell5, Christian Drosten2,
Georg Herrler1#
1
2
3
Division of Viral Products, Center for Biologics Evaluation and Research, United States Food and
Drug Administration, Bethesda, Maryland, USA
4
Department of Infectious Diseases, University of Georgia, Athens, Georgia, USA
5
Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden
#Correspondence to:
Georg Herrler
Bünteweg 17
D-30559 Hannover
fon: +49 511 953 8857
fax: +49 511 953 8898
State of publication: submitted, J Virol, 2014
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6.1. Abstract
Recently, genomic sequences of a paramyxovirus have been detected in samples derived from
an African bat. As sequence analysis had revealed a high phylogenetic relatedness to human
mumps virus (MuV), we analyzed the functional activities of the hemagglutininneuraminidase (HN) and the fusion protein (F) of the bat virus (batMuV) and compared them
to those of the human counterpart. Transfected cells expressing the F protein of batMuV were
recognized by monoclonal antibodies directed against the fusion protein of MuV indicating
that both viruses are serologically related. The HN protein of batMuV was found to have
sialic acid binding activity as indicated by a hemadsorption assay; however, it was devoid of
neuraminidase activity above detection level. The F and the HN glycoproteins of batMuV
were shown to induce syncytium formation when co-expressed in mammalian cell lines,
including chiropteran, human, simian, and rodent cells. The size of the syncytia was smaller
and they were present in lower numbers when compared to those observed after co-expression
of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The batMuV
glycoproteins were able to functionally interact with the glycoproteins of hMuV as indicated
by the formation of syncytia when the respective partner proteins were expressed in the same
cells. Co-expression of HN with chimeric F proteins indicated that the phenotypic differences
in syncytia formation between the bat and the human MuV glycoproteins are determined by
the signal peptide (SP) of the F proteins. These results indicate that the surface glycoproteins
of batMuV are serologically and functionally related to those of hMuV but show some
striking differences. The relevance for the biology of batMuV and for the risk of zoonotic
infections are discussed.
6.2. Introduction
Mumps virus (MuV) belongs to the genus Rubulavirus within the Paramyxoviridae family.
Mumps viruses are divided into 12 genotypes comprising different MuV strains based on
genetic variations of the small hydrophobic (SH) proteins [1-5].
In general, mumps is a highly contagious childhood disease with mild symptoms, such as
fever, headache, and uni- or bilateral parotitis, which is the hallmark of mumps and occurs in
90% of all clinical cases [6]. Sometimes, mumps results in complications like meningitis or
orchitis [7, 8], but the fatalitiy rate of mumps is very low. So far, humans are the only known
host of MuV.
Recently,
the
detection
of
genomic
RNA
of
a
MuV-related
paramyxovirus
(BatPV/Epo_spe/AR1/DCR/2009, batMuV, GenBank accession number: HQ660095), in an
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Manuscript 5
African flying fox of the genus Epomophorus (Epauletted fruit bat) in 2009 in the Democratic
Republic of Congo has been reported [9]. So far, all efforts to isolate a bat-derived MuV
(batMuV) have failed which makes it difficult to evaluate the zoonotic potential of these
viruses.
The infection of cells by MuV is initiated by the binding of the hemagglutinin-neuraminidase
glycoprotein (HN), a type II membrane protein, to sialic acids of cell surface macromolecules
[10]. MuV has been shown to bind to erythrocytes from different mammalian and avian
species [11-15], but the binding activity of MuV HN has not been studied in detail. Sialic
acids present in α2-3, α2-6, and/ or α2-8-linkages may serve as receptor determinants for
MuV. The affinity of the HN interaction with sialic acid varies depending on the strain used
[16-19].
The release of the viral genome into the cytoplasm is mediated by the action of the MuV
fusion protein (F), which induces the fusion of the viral envelope with the membrane of the
target cell. Co-expression of F and HN on the surface of infected cells results in the fusion of
neighboring cells and thus in the formation of syncytia, i.e. multinucleated giant cells [20,
21]. The ability to fuse cells correlates with the neuraminidase activity of the MuV HN
[20,21], suggesting that a high neuraminidase activity promotes a rapid release of virions
without the occurrence of cell-to-cell fusion, whereas a low neuraminidase activity supports
the extracellular association of virions with infected cells, thereby increasing the likelihood of
cell-to-cell fusion [22]. Sequence analysis of MuV F suggests that the multibasic amino acid
sequence R-R-H-K-R directly upstream of the fusion peptide represents the cleavage site of
the F protein [23] and that cleavage may be induced by furin-like enzymes [24].
The amino acid sequences of the batMuV F and HN proteins are closely related to the
glycoproteins from human MuV strains. To address the question whether these surface
glycoproteins are functionally related to their human counterparts, the open reading frames
(ORF) were cloned into expression plasmids to be further analyzed with respect to their
ability to mediate cell-to-cell fusion, hemadsorption, and neuraminidase activity.
We report that the surface glycoproteins F and HN of batMuV are able to mediate syncytium
formation following co-expression, even after heterologous co-expression with either of the
glycoproteins of a human isolate of MuV. The phenotype of the syncytia was determined by
the signal peptides of the respective F proteins as indicated by expression studies with
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chimeric F proteins. Furthermore, batMuV HN was found to have a sialic acid-binding
activity but no neuraminidase activity above detection level.
6.3. Methods
Cells
BHK-21, Vero76, and HeLa cells (kindly provided by A. Maisner) were maintained in
Dulbecco´s minimum essential medium (DMEM; Gibco) supplemented with 5% fetal calf
serum (FCS; Biochrom). Chiropteran cells derived from different species and organs were
used in this study: HypNi/1.1 (Hypsignathus monstrosus kidney cells), HypLu/2
(Hypsignathus monstrosus lung cells), and EidNi/41 cells (Eidolon helvum kidney cells) were
maintained in DMEM containing 10% FCS. All cells were cultivated in 75 cm² tissue culture
flasks (Greiner Bio-One) at 37°C and 5% CO2.
Expression plasmids
The ORFs of batMuV F and HN, as well as the ORFs of the F and HN proteins of a human
MuV isolate from a hospitalized patient (hMuV) were cloned into the expression vector pCG1
kindly provided by R. Cattaneo. In addition, chimeric MuV fusion proteins with an exchange
of the F1 subunit (hF1/batF2: batMuV F aa 1 - 102, hMuV F aa 103 - 538; batF1/hF2: hMuV
F aa 1 - 102, batMuV F aa 103 - 538) or the predicted signal peptide (SP, amino acids 1 - 20)
were generated (hSP: hMuV F aa 1 - 20, batMuV F aa 21-538; batSP: batMuV F aa 1 - 20,
hMuV F aa 21 - 538).
Transfection and immunofluorescence analysis (IFA)
Cells were grown on coverslips and transfected for expression of MuV glycoproteins using
Lipofectamine ® 2000 transfection reagent (Life Technologies). At 24 h post-transfection
(p.t.), cells were fixed with PBS/ 3% PFA, permeabilized with 0.2 % Triton-X-100, incubated
with either of the following primary antibodies: (i) mouse monoclonal antibodies directed
against
the F protein of the Enders strain (antibodies F-2117 and F-5418) or the
Iowa.US/2006[G] strain (antibody IA-F) of MuV or (ii) rabbit antibodies directed against the
cytoplasmic tails of the F protein or the HN protein (designated anti-mumps F(CT) or antimumps HN(CT), respectively). Cy3-conjugated sheep or goat antibodies directed against
rabbit or mouse IgG F(ab´) 2-fragment (both purchased from Sigma) served as secondary
antibodies. Incubation with primary and secondary antibodies was performed in humidity
chambers for 1 h or 30 min, respectively. Finally, the cells were incubated with DAPI (Roth)
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and mounted in Mowiol (DABCO). Immunofluorescence analysis (IFA) was performed using
the Nikon Eclipse Ti microscope and the NIS Elements AR software (Nikon).
Hemadsorption assay
HypNi/1.1 cells were grown in 24-well plates and transfected with h/batMuV HN or empty
vector pCG1. At 18 h p.t., the medium was removed and cells were washed carefully with
PBS three times, followed by incubation with 250 µl 2% chicken erythrocytes (Clinic for
Poultry, University of Veterinary Medicine Hannover) per well for 10 min at 4°C. With some
samples, erythrocytes had been pre-incubated with Clostridium perfringens neuraminidase
(NA, Sigma-Aldrich; 0.1 µunits/ µl) at 37°C for 30 min and then added. Afterwards, cells
were washed with PBS several times to remove unbound erythrocytes. Finally, cells were
fixed with PFA and analysed via light microscopy. The assays were performed at least five
times.
Neuraminidase activity assay
2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA, Sigma-Aldrich) was
used to measure the neuraminidase activity of the HN proteins. HypNi/1.1 cells were grown
in black 96-well plates and transfected with MuV glycoproteins or the empty expression
plasmid pCG1. At 24 h p.t., the medium was removed and cells were washed with cold PBS
three times. Transfected cells were incubated with 0.3 mM 4-MUNANA at 37°C for 1 h. As a
control, Cl. perfringens neuraminidase (Sigma-Aldrich) was added to non-transfected cells at
different concentrations (0.5 µU, 2.5 µU, 5.0 µU, 10.0 µU) and incubated as described for the
transfected cells. The reaction was stopped by addition of a stop solution containing 0.5 M
NaOH and 25% ethanol. Neuraminidase activity was measured by a chemiluminometer
(GENios pro, Tecan) using an excitation wavelength of 355 nm and an emission wavelength
of 460 nm. The assays were performed at least five times, with each value determined in
quadruplicates.
Fusion assays
Cells were grown in 24-well plates and cotransfected for expression of human- or bat-derived
MuV F and HN proteins, as well as chimeric proteins. At 12 h p.t., the medium was removed,
cells were washed with PBS three times, and fixed by incubation with ice-cold methanol/
acetone (1:1) for 30 sec. Subsequently, the cells were washed with PBS twice and incubated
with May Grünwald solution (Sigma) for 5 min, followed by washing with PBS and
incubation with Giemsa staining solution (1:10 diluted with Aqua bidest., Sigma) for 20 min.
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Finally, cells were washed with Aqua bidest., air-dried and analyzed via light microscopy for
the formation of syncytia. The assays were performed at least five times.
6.4. Results
Expression of bat-derived MuV F and HN glycoproteins in mammalian cells
To analyze the F and HN proteins of batMuV, HypNi/1.1 cells and Vero76 cells were
transfected with the respective expression plasmids. The former cell line has been shown
recently to support the functional activity of glycoproteins derived from an African
henipavirus [25]. To visualize the glycoproteins, different available antibodies were used for
immunostaining. As shown in Fig. 1 for HypNi/1.1 cells, three monoclonal antibodies
directed against the F protein of the Enders strain [26] or the Iowa.US/2006 strain recognized
the F proteins of both the hMuV isolate used in our study and of batMuV. A rabbit antiserum
directed against the cytoplasmic tail of MuV F also recognized both F proteins. There were no
antibodies against the ectodomain of the HN protein available, that recognized either of the
two HN proteins. On the other hand, the rabbit antiserum directed against the cytoplasmic tail
of HN could be used to visualize HN from both hMuV and batMuV. A similar result was
obtained with Vero76 cells (data not shown). This result indicates that the F proteins of hMuV
and batMuV are serologically related.
Hemadsorption of batMuV HN proteins
To get information about the functional activities of the glycoproteins of batMuV, the HN
protein was analyzed for sialic acid binding activity. A convenient way to determine the
interaction of viruses with sialic acids is a hemagglutination assay. As no virus particles were
available for batMuV, we performed a hemadsorption assay with cells expressing the HN
protein on their surface. Transfected HypNi/1.1 cells expressing either of the HN proteins
were incubated with 2% chicken erythrocytes. No hemadsorption was observed with control
cells transfected with an empty vector. By contrast, cells expressing the HN proteins of either
hMuV or batMuV bound erythrocytes that could not be washed away (Fig. 2). The binding of
the erythrocytes to the HN-expressing cells was sialic acid-dependent as no hemadsorption
activity was detectable with erythrocytes that had been pretreated with neuraminidase. This
result indicates that the HN protein of batMuV - like the counterpart from hMuV - has sialic
acid binding activity.
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Neuraminidase activity
Apart from binding to sialylated sialoglycoconjugates, the HN protein of paramyxoviruses is
also known for its neuraminidase activity. To analyze the HN protein of batMuV for its ability
to release sialic acid, transfected HypNi/1.1 cells expressing the HN protein of either hMuV
or batMuV were incubated with 4-MUNANA. This substrate is converted into a fluorescent
compound after release of sialic acid. Incubation of transfected HypNi/1.1 cells expressing the
HN protein of hMuV resulted in fluorescent signals similar to those induced by incubation of
the substrate with 10 µU of neuraminidase from Cl. perfringens (Fig. 3). No fluoresecence
above background level was measurable with cells expressing either no foreign protein
(empty vector) or the F protein of hMuV and batMuV. Interestingly, the HN protein of
batMuV was also devoid of detectable neuraminidase activity in the assay used.
Co-expression of batMuV F and HN results in syncytium formation in different
mammalian cell lines
Co-expression of F and HN glycoproteins is required for cell-to-cell fusion. To analyze the
glycoproteins of batMuV for fusion activity, chiropteran (HypNi/1.1, HypLu/2, EidNi/41),
human (HeLa), simian (Vero76), and rodent (BHK-21) cells were co-transfected for
expression of both HN and F proteins and then screened at 12 h p.t. for the presence of
syncytia with a minimum of five nuclei within one cell. Co-expression of F and HN proteins
from hMuV resulted in the formation of huge syncytia in all cell lines analyzed (Fig. 4). The
batMuV glycoproteins were also able to mediate fusion in all chiropteran and non-chiropteran
cell lines. However, the size of the syncytia was smaller compared to those observed after the
co-expression of the hMuV glycoproteins (Fig. 4). The size of the syncytia induced by the
batMuV glycoproteins did not increase significantly at later times p.t.; cells expressing the
glycoproteins of hMuV could not be analyzed at later time points because of the cytopathic
effect resulting in cell detachment.
Heterologous expression of human- and bat-derived MuV glycoproteins mediates
syncytium formation
After having shown that the glycoproteins of batMuV are able to induce the formation of
syncytia, a heterologous fusion assay was performed to find out whether human and batderived MuV glycoproteins are able to interact with each other. Co expression of hMuV F and
batMuV HN as well as the co-expression of batMuV F and hMuV HN, induced syncytium
formation (Fig. 5). Syncytia obtained from the heterologous expression were smaller
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compared to those observed after homologous expression of hMuV F and HN proteins. In the
heterologous glycoprotein combinations, those comprising the F protein of hMuV induced
larger syncytia than those comprising the counterpart from batMuV. This result indicates that
the size difference between the syncytia induced by the glycoproteins of hMuV and batMuV
has to be attributed to the F protein.
The signal peptide of the MuV fusion proteins determines the size of syncytia
The amino acid sequence of batMuV F was compared to that of a consensus sequence derived
from 26 full length sequences of human MuV F (Tab. 1). The conservation of the F sequence
of our clinical isolate was within the range of variation observed among the published
sequences. Overall, the amino acid similarity of the fusion protein of batMuV to that of the
MuV consensus sequence was very high with homology values of 94.6 for the full-length
protein, 96.5 for F1 and 86.5 for the F2 subunit (Tab. 1). A striking difference was noticed in
the first 20 amino acids of the F2 subunit comprising the putative signal peptide. There, at the
aminoterminus of F2, the amino acid homology between the MuV consensus sequence and
batMuV was only 69 % (Tab. 1). In the case of our clinical isolate, this predicted signal
peptide differs only at position 5 (S-L) from the consensus sequence. Therefore, chimeric F
proteins with an exchange of the first 20 (predicted signal peptide) or 102 (F2 subunit) amino
acids were generated and further analyzed in a fusion assay. F proteins containing the hMuV
F2 subunit or only the hMuV F signal peptide induced the formation of huge syncytia,
whereas syncytia mediated by the F proteins containing the bat-derived F2 subunit or signal
peptide were much smaller (Fig. 6). This result indicates that the signal peptide is responsible
for the differences between F proteins of hMuV and batMuV in the fusion activity.
6.5. Discussion
The identification of genomic sequences of a bat virus related to MuV was a unique finding
because it had revealed that a virus that has established a human lineage many years ago still
has substantial similarity to a counterpart in bats (Drexler et al., 2012). Sequence analysis and
serologic assays suggested that hMuV and batMuV are conspecific and belong to one
serogroup. The antigenic relatedness was confirmed in this study. Monoclonal antibodies
directed against the F proteins of different MuV strains efficiently interacted with the
corresponding protein of batMuV as indicated by immunofluorescence microscopy of
transfected cells. As the F proteins of paramyxoviruses have been shown to be targets of
neutralizing antibodies, it is conceivable that the protective immunity elicited by MuV
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Manuscript 5
infection or vaccination might apply to batMuV. As there was no antibody against the
ectodomain of MuV HN available we could not analyze the cross-reactivity of antibodies
directed against HN. The sequence of HN of batMuV is very similar to that of the human
counterpart with a homology value of 91% with respect to a consensus sequence of human
strains of MuV; for comparison, the homology among the HN proteins of MuV was 99%.
Though an infectious batMuV is not yet available, one may predict that this virus will not
succeed in infecting humans that carry neutralizing antibodies against hMuV.
In addition to the serological similarity, the results presented in this study also demonstrate a
functional relatedness between the glycoproteins of hMuV and batMuV. Like the human
counterpart, the HN protein of batMuV has a sialic acid binding activity that is evident in a
hemadsorption activity. In the case of MuV, adaptation to different hosts (humans and bats)
did not involve a major change in the receptor binding activity. This is different from
coronaviruses. Though the receptor for SARS-like bat viruses is not known, the S proteins of
these viruses appear to interact with a receptor different from human ACE2 (Becker et al.,
2008; Ren et al., 2008; Hou et al., 2010). The same may apply to influenza viruses. Genomic
sequences detected in bats from Central America were used to generate a recombinant bat
influenza virus from cDNA (Juozapaitis et al., 2014). These attempts were only successful,
when the glycoproteins of the bat virus were replaced by those of an avian influenza virus.
The receptor for bat influenza viruses is not known; however, available evidence indicates
that the hemagglutinins of these viruses are not able to use sialic acid as a receptor
determinant (Tong et al., 2012; Sun et al., 2013; Tong et al., 2013; Zhu et al., 2013; Wu et al.,
2014a). Whereas the HN protein of batMuV resembled the corresponding protein of hMuV in
its sialic acid binding activity, it differed from most MuV strains as far the second activity of
paramyxovirus HN proteins is concerned. It was devoid of neuraminidase activity above
detection level. This finding does not exclude that the HN protein has low enzyme activity;
but it demonstrates that the neuraminidase activity is much lower than that of the hMuV
counterpart. Variations in the neuraminidase activity of different strains of mumps virus are
well documented (Merz and Wolinsky, 1981). Usually, they have a high or a low enzyme
activity. Variants of MuV devoid of neuraminidase activity have been obtained when
selection protocols were applied involving neuraminidase inhibitors (Waxham and
Aronowski, 1988). The two mutations, Ile181Thr and Gln261Lys, identified in those studies
do not explain the phenotype of batMuV HN because the respective amino acids in this
glycoprotein are Ile181 and Thr261. It will be interesting in the future, when more bat MuV
Manuscript 5
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sequences are available, to find out whether lack of neuraminidase or low enzyme activity is a
general feature of the HN proteins of batMuV.
Similar to most paramyxoviruses, the fusion activity of batMuV requires the cooperation of
the F and the HN protein. The functional relatedness between hMuV and batMuV is
convincingly demonstrated by the finding that each of the two glycoproteins of batMuV can
cooperate with the respective partner protein of hMuV to induce syncytium formation.
Among paramyxoviruses, the fusion protein usually requires the HN protein of viruses from
the same species to induce membrane fusion. An exception is SV5 which can cooperate with
HN proteins of other paramyxoviruses (Bagai and Lamb, 1995). However, the F protein of
SV5 is unusual because it has fusion activity even in the absence of HN protein. Though there
are substantial differences in the SH protein between batMuV and MuV with a homology
value 61% with respect to a consensus sequence for MuV strains, the similarity of the other
viral proteins is rather high with homology values of about 90%. This similarity is sufficient
to allow cooperation of the glycoproteins in the induction of syncytia formation. However,
there are also differences between hMuV and batMuV in their fusion activity. Syncytia
induced by the bat virus glycoproteins were substantially smaller than those observed after
co-expression of the glycoproteins of hMuV. A similar difference has recently been
demonstrated when the syncytium formation by a putative African henipavirus was compared
to that induced by the Nipah virus glycoproteins. Cooperation of the F and G proteins of the
latter virus results in large syncytia in all mammalian cells. By contrast, syncytium formation
induced by the glycoproteins of the African henipavirus is restricted to chiropteran cells and
not observed in cells of other mammalian species (Kruger et al., 2013; Lawrence et al., 2014;
Weis et al., 2014). This differential fusion activity has been traced back to the surface
transport of the G protein, which was inefficient in all cells but the inefficiency was less
pronounced in chiropteran cells (Kruger et al., 2014). The fusion activity of the batMuV
glycoproteins differs from that of the African henipavirus as it is not restricted to chiropteran
cells. The available reagents did not allow to compare the surface expression of the different
glycoproteins of hMuV and batMuV. However, different expression levels do not explain the
differences in the induction of syncytia observed between the hMuV and batMuV
glycoproteins. HN of batMuV was highly fusion-active when co-expressed with F protein of
hMuV. The same is true for the chimeric F proteins containing the F2 or the signal peptide of
the fusion protein of the human virus. For hMuV it is known that the neuraminidase activity
of the HN protein affects the size of the syncytia. Strains with high neuraminidase activity in
general induce no or only small syncytia, whereas variants or mutants with low enzyme
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Manuscript 5
activity are associated with the formation of large syncytia (Merz and Wolinsky, 1981). In
this respect, one might expect large syncytia from the batMuV glycoproteins because of the
lack of neuraminidase activity or low enzyme activity, respectively, and small syncytia from
the hMuV glycoproteins as the respective HN has a much higher enzyme activity. However,
the neuraminidase is not the only factor that affects the fusion activity. This is evident from
the syncytia induced by the heterologous MuV glycoproteins. Large syncytia were observed
with the HN glycoprotein of bat MuV when co-expressed with the F protein of hMuV. Thus a
negative factor associated with the fusion protein of batMuV overrides the positive effect
exerted by the lack of neuraminidase activity in the HN protein. As shown by analysis of the
chimeric proteins, the signal peptide is the determinant which down-regulates the fusion
activity of the F protein of batMuV. How the signal peptide of the fusion protein affects the
formation of syncytia is not known. Possible reasons may involve the efficiency of protein
processing, the transport along the secretory pathway, the halftime of the protein or slight
conformational changes. However, at present we do not have experimental data to support
any of these explanations. We also do not know the biological importance of the signal
peptide-dependent down-regulation of the fusion activity. In this context, it is interesting to
note, that several bat viruses show restrictions in the entry strategies. As mentioned above,
SARS-like bat coronaviruses and bat influenza viruses are restricted at the level of interaction
with cellular receptors, the African henipavirus and batMuV are restricted in their fusion
activity. Therefore, replication of the respective bat viruses may not be characterized by
production of a large number of progeny virions but rather by a persistent type of infection
characterized by cell-to-cell spread. The difficulty in isolating these viruses in an infectious
form is consistent with this idea. To get more knowledge about the replication of these bat
viruses, it is however, indispensable to isolate infectious viruses from the respective bats in
the future.
6.6. Acknowledgments
Part of this work was performed by N.K. in partial fulfillment of the requirements for the
doctoral degree from Stiftung Tierärztliche Hochschule Hannover. N.K. was funded by a
fellowship of the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology,
and Translational Medicine (HGNI). We are grateful to Roberto Cattaneo for providing
expression plasmids. This work was supported by grants to GH from DFG (HE 1168/14-1 and
SFB621 TP B7) and from Bundesministerium für Bildung und Forschung (Ecology and
Manuscript 5
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Pathogenesis of SARS, project code 01Kl1005B and FluResearchNet, project code
01KI1006D).
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Manuscript 5
6.8. Tables and figures
Tab. 1: Sequence homology between the F proteins of MuV and batMuV.
Domain
Variations
within MuV
strains [%]
Homology to the consensus
sequence a [%]
hMuV F b
batMuV F
Full length F
97.7
98.9
94.6
F1 subunit
98.3
99.2
96.5
F2 subunit
95.3
97.4
86.5
Signal peptide c
86.5
96.0
69.0
Cleavage site
96.7
100
77.8
Fusion peptide
100
100
100
Ectodomain
Transmembrane
domain
Cytoplasmic
domain
97.7
98.8
94.3
96.0
99.2
96.8
99.3
100
97.1
a
The homology of hMuV F and batMuV F was compared to a MuV F consensus sequence
which was derived from an alignment of the aa sequence of the F proteins of 26 MuV strains
(GenBank accession numbers: EU884413.1; FJ556896.1; AF467767.2; GU980052.1;
AF338106; AY681495.1; AY508995; AB600843.1; JX287386.1; EU370206.3; JX287388.1;
AY309060.1; AB600942.1; AF314561.1; AF314558.1; AB823535.1; JN012242.1;
KF481689.1; AF280799.1; EU370207.1; JN635498.1; JX287385.1; JX287391.1;
JX287390.1; JX287387.1; JX287389.1), using the PRofile ALIgNEment (PRALINE)
software.
b
hMuV F designates the fusion protein of the clinical isolate used in this study.
c
The sequence of the signal peptide:
consensus:
hMuV:
batMuV:
M K A F L V T C L G F A V F S S S I C V
. . . . S . . . . . . . . . . . . . . .
M R K T . A I G . . . M I . . L . V . I
Manuscript 5
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Fig. 1: Expression of human and bat-derived MuV F and HN. HypNi/1.1 cells were
transfected for expression of human or bat-derived MuV F (a-d) or HN (e) proteins. Proteins
were detected in permeabilized cells via MuV specific antibodies ( a) anti-mumps F 2117, b)
anti-mumps F 5418, c) anti-mumps IA F, d) anti-mumps F (CT), e) anti-mumps HN (CT))
and indocarbocyanine labelled anti-rabbit or anti-mouse IgG. Scale bar indicates 25 µm.
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Manuscript 5
Fig. 2: Hemadsorption of chicken erythrocytes in transfected HypNi/1.1 cells. HypNi/1.1
cells were transfected with the empty vector, hMuV HN or batMuV HN. At 18 h p.t., cells
were incubated with untreated (-NA) or Cl. perfringens NA pre-incubated (+NA) chicken
erythrocytes and analyzed via light microscopy.
Fig. 3: Neuraminidase activity of hMuV and batMuV HN proteins in HypNi/1.1 cells.
Transfected HypNi/1.1 cells and Cl. perfringens NA were incubated with 4-MUNANA. The
emission was measured using an excitation wavelength of 355 nm and an emission
wavelength of 460 nm. Emission is given in relative fluorescence units (RFU).
Manuscript 5
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Fig. 4: Homologous co-expression of human or bat-derived MuV F and HN proteins. Cells
were transfected with the empty vector pCG1, hMuV F + HN, or batMuV F + HN. At 12 h
p.t., cells were fixed with methanol/ acetone (1:1) and incubated in May Grünwald and
Giemsa staining solution. Scale bar indicates 25 µm.
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Manuscript 5
Fig. 5: Heterologous co-expression of human and bat-derived MuV F and HN. BHK-21
cells were grown on coverslips and transfected for the co-expression of hMuV F and hMuV
HN/ batMuV HN or batMuV F and hMuV HN/ batMuV HN. At 12 h p.t., cells were fixed with
methanol/ acetone (1:1) and incubated in May Grünwald and Giemsa staining solution. Scale
bar indicates 25 µm.
Manuscript 5
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Fig. 6: Co-expression of signal peptide chimera and MuV HN proteins cells. BHK-21 cells
were grown on coverslips and transfected for the co-expression of hMuVF2, batMuVF2, hMuV
SP, or batMuV SP and hMuV HN/ batMuV HN. At 12 h p.t., cells were fixed with methanol/
acetone (1:1) and incubated in May Grünwald and Giemsa staining solution. Black arrows
indicate syncytia. Scale bar indicates 50 µm.
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Discussion
7. DISCUSSION
Zoonotic viruses are pathogens which can be transmitted from an animal reservoir host to
humans. The transmission to humans can occur either via direct contact to the reservoir host
(e.g. by biting) or via indirect contact - for example via an intermediate/ amplifying host as it
has been described for the transmission of NiV to humans. In all cases, the virus has to cross
the species barrier to infect a new host. The spread of infection from a reservoir host to a new
host is called spillover and can result in the successful production of infectious progeny
viruses leading to subsequent infections in the new host species. Not every spillover event is
followed by the establishment of a virus lineage in the newly infected species. Successful
virus transmission depends on several pathogen- and host-specific factors, which together
form the species barrier.
Depending on the factors involved in the species barrier (e.g. taxonomic relationship between
the original reservoir and the new host species, receptor expression, etc.) it may be easy or
difficult to establish a productive infection in the new host: (i) Ecological factors such as the
excretion of pathogens or physical proximity determine the likelihood whether a new host
gets in contact with the reservoir host and its pathogens. (ii) Also, different pathogen-related
factors play an important role for the adaptation to a new host. Pathogens with a broad host
range (e.g. receptor molecules that are highly conserved among different species) and/ or a
high genetic variability can adapt to new hosts much easier in comparison to pathogens with
specializations on a certain species, organ or cell type (Woolhouse et al., 2001; Parrish et al.,
2008). (iii) In addition, host-specific factors, such as the expression of cellular receptor
molecules or the immune response can affect the likelihood of a spillover event.
Aim of this study was to analyze the interaction of bat-derived paramyxoviruses related to the
henipaviruses Nipah virus (NiV) and Hendra virus (HeV), or mumps virus (MuV), with
chiropteran and non-chiropteran cells. The main focus was put on the initial steps of the viral
life cycle, the entry process (receptor binding and membrane fusion). In this context, the viral
entry process of enveloped RNA viruses and its particular share of the species barrier will be
explained in more detail.
Viral infection starts with the attachment to a susceptible host cell and the overcoming of the
plasma membrane barrier (penetration). Following binding to an appropriate receptor
molecule, the penetration of enveloped RNA viruses - like NiV or MuV - is achieved by the
fusion of the viral membrane (envelope) with cellular membranes of the host. This step is
Discussion
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indispensable to proceed to viral replication which takes place inside the cell - the cytoplasm
(NiV, MuV) or the nucleus (influenza A viruses, FLUAV). Some viruses, e.g. NiV and HeV,
use highly conserved and ubiquitously expressed cellular receptor molecules for the
attachment step (Bossart et al., 2008), so that binding to cells of new species is achieved
without difficulties. In contrast to this, other viruses such as coronaviruses, which mostly have
a very narrow host range, interact with specific receptor molecules: For the transmissible
gastroenteritis virus (TGEV) and the human coronavirus 229E (HCoV-229E) it is known that
the susceptibility of cells to viral infection is restricted by the presence of porcine or human
aminopeptidase N (APN), respectively (Delmas et al., 1992; Yeager et al., 1992). Similar
results were reported for the spike (S) protein of severe acute respiratory syndrome-associated
coronavirus (SARS-CoV): Only cells expressing the angiotensin-converting enzyme 2
(ACE2) of human, (some) chiropteran, or certain other mammalian species were susceptible
to infection (Li et al., 2003; Moore et al., 2004; Li et al., 2005b; Qu et al., 2005; Hoffmann et
al., 2013). In general, the adaptation of highly specified viruses to new hosts most commonly
requires genetic changes of the viral genome/ viral components. These can be obtained via
spontaneous mutations in the reservoir host or via (stepwise) adaptation in an intermediate/
amplifying host.
Once the virus has bound to the target cell, the fusion of the viral envelope and host cell
membranes is necessary to release the viral genome into the cytoplasm. The fusion process of
paramyxoviruses is mediated by the fusion glycoprotein (F), which has to be proteolytically
activated by cellular proteases. This activation depends on a specific cleavage motif that is
recognized by a cellular protease, leading to the proteolytic cleavage of the F protein. The
distribution and activity of F protein-cleaving proteases within a new host defines in which
organs or tissues a virus is able to replicate. The proteolytic cleavage is a prerequisite for the
F protein to adopt - upon interaction with the attachment glycoprotein - a fusion-active
conformation.
These virus entry factors, the presence of an appropriate cellular receptor, which enables the
attachment of virions, as well as the proteolytic cleavage of the F protein by cellular
proteases, play crucial roles for a spillover event of paramyxoviruses from their reservoir
hosts to a new host species. Nevertheless, the ability of a virus to successfully enter the cell of
a new host species does not necessarily result in a productive infection and the generation of
infectious progeny virus.
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Discussion
7.1. Functional characteristics of the African henipavirus M74
The RNA of the African henipavirus strain M74 (M74) was detected in an African flying fox
of the species Eidolon helvum in Ghana (Drexler et al., 2012).
The amino acid (aa) identity of the M74 polymerase, fusion protein, nucleoprotein, and matrix
protein in comparison to NiV and HeV ranged between 53.0 % and 62.82 %, whereas only
25.8 % and 26.0 % identity was observed for the phosphoprotein and the attachment
glycoprotein, respectively (Drexler et al., 2012).
7.1.1. M74 G interacts with the henipavirus receptor
Henipavirus infections are initiated by the interaction of the attachment glycoprotein (G) with
its cellular receptor Eph B2 or Eph B3 (Negrete et al., 2005; Negrete et al., 2006; Xu et al.,
2012a). Further, the ephrin B2 ligand can serve as a functional receptor for NiV and HeV
(Bonaparte et al., 2005). Seven amino acid residues (aa) in the NiV G protein, which are
involved in the binding of Eph B2, were identified - W504, E505, Q530, T531, A532, E533,
and N557 (Guillaume et al., 2006) - and M74 G shares five of them, W514, E515, Q540,
A542, and E543. In addition, also the aa V507 (NiV G) / T507 (HeV G) and E533 have been
shown to play an important role for the interaction with Eph B2 or B3 (Negrete et al., 2007).
In the case of M74 G, the aa at positions 517 (T) and 543 (E) are in consistence with the
corresponding aa of HeV G.
In summary, those aa of NiV and HeV G, which are responsible for the binding to Eph B2/
B3, are highly conserved among the so far known henipaviruses, and are also present in M74
G. Concluding from this, it is very likely that M74 G can use the henipavirus receptor Eph B2
as a cellular receptor.
To analyze whether the African henipavirus attachment glycoprotein is able to interact with
the henipavirus receptors in-vitro, a coimmunoprecipitation assay was performed, in which it
was demonstrated for the first time, that M74 G indeed can interact with the ephrin B2 ligand
(Kruger et al., 2013). This result was confirmed by another study, in which the interaction of
M74 G with Eph B2 – also in a coimmunoprecipitation assay - as well as the binding of
soluble Eph B2 to M74 G-expressing, Eph-negative HeLa cells was shown (Weis et al.,
2014). Consistent with our observations, Pernet et al. revealed the binding of soluble ephrin
B2 to M74 G via fluorescence-activated cell sorting (FACS) (Pernet et al., 2014).
Furthermore, the interaction of M74 G with Eph B2 and Eph B3 was confirmed by Lawrence
et al. (2014): When CHO cells lacking endogenous Eph expression, or expressing Eph B1,
Discussion
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Eph B2, or Eph B3 were co-transfected with M74 G and NiV F, fusion was only obtained in
those cells expressing either Eph B2 or Eph B3 (Lawrence et al., 2014).
Conclusively, these data show that the African henipavirus utilizes the cellular receptors Eph
B2 and Eph B3 that are employed also by the highly pathogenic henipaviruses NiV and HeV
for attachment of viral particles. The usage of the ubiquitously expressed Eph receptors,
which enables a broad host range for the henipaviruses, may increase the chances of a
spillover event of the African henipavirus M74 from Eidolon helvum to other mammalian
species.
7.1.2. Proteolytic cleavage of M74 F
After having shown that the M74 G interacts with the known henipavirus receptor, next, the
question was addressed whether also the cleavage of M74 F shares similarities with that of
NiV F. For NiV F it has been demonstrated that cleavage occurs - following a recycling
process - within endosomes at low pH and is mediated by the cysteine protease cathepsin
(Cat) L. In this process, the uncleaved F protein (F0) which is expressed at the cell surface of
an infected cell undergoes a clathrin-mediated endocytosis leading to its uptake into
endosomes. There, the low pH enables Cat L to further process the F protein by cleaving the
polypeptide into the F1/ F2 subunits, which are connected by disulphide bondages. This
cleavage is necessary for fusogenicity of the F protein, since only cleaved F protein is able to
promote membrane fusion due to a highly hydrophobic fusion peptide which can insert into
the host cell membrane. Following cleavage by Cat L, the activated F protein is relocated to
the plasma membrane and can either be incorporated into new viral particles or, in cooperation with the G protein, induce syncytium formation.
When cell lysates of HypNi/1.1 cells expressing M74 F were analyzed by Western blotting
and antibody staining, a cleaved form, which is suggested to represent the F1 subunit, was
detected in addition to the uncleaved F0-form (Kruger et al., 2013). Furthermore, it has been
shown that the M74 F protein is cleaved in cells derived from different mammalian species
with a similar efficiency (Kruger et al., 2013; Weis et al., 2014; Kruger et al., in preparation).
To find out whether the M74 F undergoes a recycling process similar to NiV or HeV F, the
relation of the uncleaved F0-form and cleaved F1 subunit on the cell surface was analyzed at
different time points following labelling with biotin. In this way it was demonstrated that over the time - the amount of the cleaved F1 subunit increases, while, at the same time, the
amount of the uncleaved F0-form decreases (Kruger et al., in preparation). However, the
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Discussion
cleavage kinetics of M74 F was slower in comparison to that of NiV F (Weis et al., 2014). In
summary, the data obtained from the surface biotinylation experiment indicates, that the
cleavage of M74 F occurs after the protein has been expressed on the cell surface and
therefore, an endocytosis process of the M74 F (similar to NiV F) is very likely.
After having shown that the M74 F is cleaved in transfected cell lines and cleavage occurs
after the F0-form has been transported to the plasma membrane, we wanted to gain
information about which kind of cellular protease is responsible for the cleavage. First, an
alignment of the aa sequences of NiV and M74 F indicated that the M74 F contains an R
(R205) as a potential monobasic cleavage site at the corresponding aa position of the NiV F
cleavage site R109. Therefore, it was analyzed whether the cleavage of M74 F takes place in
the endosomes and is mediated by cathepsins. An inhibition of the endosomal acidification
resulted in a clear reduction of the cleavage and fusogenic activity of the M74 F, indicating
that the cleavage is pH-dependent and occurs in an acidic environment (Kruger et al., 2013;
Kruger et al., in preparation). Differences between NiV and M74 F were obtained when
transfected cells were treated with inhibitors for cysteine proteases (E-64d), Cat B, or Cat L.
Whereas the cleavage of NiV F was inhibited by E-64d as well as inhibitors of Cat L, the
inhibition of Cat B had no influence on the proteolytic cleavage. For M74 F however, neither
E-64d nor the different Cat inhibitors reduced the proteolytic cleavage (Kruger et al., in
preparation). A possible explanation for this observation is that the cleavage is mediated by an
uncommon Cat, which is not covered by the spectrum of the Cat inhibitors. Alternatively,
another, non-cathepsine protease, the activity of which also relies on a low pH, may be
responsible for the cleavage of M74 F.
To narrow down the cleavage motif within the sequence of the M74 F, mutants were
generated where the aa residues immediately upstream of the predicted fusion peptide were
replaced by alanines (A). Similar to the results published for the NiV F with an R109Asubstitution, the R205A mutation did not reduce or abolish the proteolytic cleavage of the
M74 F protein or the ability to mediate cell-to-cell fusion following co-expressing with M74
G (Kruger et al., in preparation). Even the substitution of nine (aa 196 - 204) or ten aa (aa 196
- 205) located between a predicted N-glycosylation site (N195) and the fusion peptide,
including or excluding the R205 residue, had no effect on the proteolytic cleavage. In contrast
to this, the replacement of nine aa (aa 96-104) directly upstream of the fusion peptide of NiV
F completely abolished F cleavage and its ability to mediate syncytium formation when co-
Discussion
- 98 -
expressed with NiV G. From these findings, it is concluded that either the cellular protease
which is responsible for the cleavage does not recognize a specific monobasic cleavage motif
or the motif is not located in the aa residues directly upstream of the predicted fusion peptide.
Sequence analysis showed that the M74 F protein is about 106 aa longer than NiV F and
contains a potential multibasic cleavage site within the first 100 aa residues (aa 92 - 97, K-RG-K-R-R). Furthermore, a recently published study argued that the additional N-terminal aa
of the M74 F (in comparison to NiV F) might be a result of a sequencing error at an adeninethymine-rich region, and supported this statement by the lack of a typical signal peptide
sequence - that are classically characterized by the presence of aliphatic and hydrophobic aa
such as L and I, within the first 30 aa (Pernet et al., 2014). In that study, the introduction of an
alternative start codon by the insertion of a nucleotide at position 303 created an in frame
ATG, resulting in an M74 F protein that has an N-terminus more similar to NiV F (both, in
length and aa composition) (Pernet et al., 2014). However, although this modified M74 F now
possesses a classical signal peptide sequence, the underlying data do not provide proof for the
incorrectness of the published M74 F sequence and more importantly, do not explain the
different phenotypes, when M74 F and G are co-expressed in chiropteran or non-chiropteran
cells. Actually, sequencing of more samples confirmed the correctness of the published
sequences (Jan Felix Drexler, personal communication).
Interestingly, when M74 F was analyzed by Western blotting, the uncleaved F0-form was
detected at a molecular weight of approximately 70 kDa, which is lower than the expected
calculated (unmodified; e.g. glycosylations) molecular weight of 74 kDa (Kruger et al., 2013).
Furthermore, no differences in the molecular weight were detected between the native M74 F
and a truncated mutant of M74 F where the aa residues 1 - 108 were replaced by the aa
residues 1 - 12 of NiV F (Kruger et al., in preparation). From these data it is suggested that
besides the proteolytic cleavage necessary for M74 F fusogenicity, an additional cleavage
occurs within the first 108 aa during a very early stage of the secretory pathway and that it
was not possible to detect the “complete” F0-form of the M74 F protein with the methods
used. Such a cleavage might result in an F protein that looks similar to the F protein generated
by Pernet et al.. To confirm our hypothesis, we are going to performed mass spectrometry
analysis of the uncleaved F0–form of the M74 F to unravel the aa composition of its Nterminus.
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Discussion
In summary, it has been shown, that the M74 F protein is proteolytically cleaved in a pHdependent (low pH) manner. Furthermore, evidence was obtained which supports the notion
that the M74 F undergoes a recycling process similar to that of NiV F, to be cleaved within
endosomes. So far, the cellular protease which mediates M74 F-cleavage, as well as the
cleavage motif is unknown. Further studies e.g. the incubation of (soluble) M74 F protein
with different intra- or extracellular proteases will help to elucidate the identity of the M74 Fcleaving protease. These data provide important information concerning the risk of a zoonotic
transmission event: Given that the cleavage efficiency is similar in chiropteran and nonchiropteran cell lines, the proteolytic activation of the F protein does not represent a
restriction factor for a spillover to new, non-chiropteran hosts.
7.1.3. The ability of the M74 surface glycoproteins to mediate membrane fusion is
restricted to chiropteran cells
Paramyxoviruses enter their target cells via a fusion process involving the viral envelope and
the host cell plasma membrane. The fusion reaction is mediated by a cooperation of the
attachment glycoprotein and the fusion protein. The subsequent spread of infection in infected
tissues is achieved not only by progeny virions but also by the fusion of infected cells
expressing the viral glycoproteins on the cell surface with neighbouring cells, leading to the
formation of multinucleated giant cells - syncytia. The fusion process and especially the
conformational changes in the F protein are irreversible; therefore it is important that the
activation of the F protein takes place at the right time and at the right place. The binding of
the attachment glycoprotein to the cellular receptor, as well as the interaction between fusion
and attachment glycoproteins, which leads to conformational changes in the F protein, are a
prerequisite to activate the F protein. Furthermore, it is required that the F protein undergoes a
proteolytic cleavage, mediated by host cell proteases, to promote fusion. All three events have
to occur to induce the fusion process.
After having shown that M74 G interacts with the henipavirus receptors and that the F protein
is cleaved by cellular proteases, it was analyzed whether the African henipavirus surface
glycoproteins are able to mediate cell-to-cell fusion. For this purpose, chiropteran and nonchiropteran cells were co-transfected to express the NiV or M74 F and G and screened for the
presence of syncytia at 24 h p.t. Whereas the co-expression of the NiV glycoproteins resulted
in the formation of syncytia in all tested cell lines, the ability of the M74 F and G to mediate
cell-to-cell fusion was restricted to chiropteran cells: Fusion was observed only in HypNi/1.1,
Discussion
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HypLu/2, and EidNi/41 cells - chiropteran cell lines derived from the lung (HypLu/2) or the
kidney of Hypsignathus monstrosus (HypNi/1.1) or Eidolon helvum (EidNi/41)- but not in
NiV-permissive BHK-21 (hamster), HBE (human) and Vero76 (African green monkey) cells
(Kruger et al., 2013; Kruger et al., 2014). Similar results were published in collaboration with
Weis et al.: Following co-expression of M74 F and G, no cell-to-cell fusion was obtained in
henipavirus-permissive cell lines (Vero76, A549, BHK-21, MDCK, and PAEC-EB2) and a
chiropteran cell line derived from Eidolon helvum (EidNi/41.3), whereas HypNi/1.1 cells
supported syncytium formation (Weis et al., 2014). The result obtained for the EidNi/41.3
cells is not a contradiction to the findings obtained in the course of this thesis: The Eidolon
cell line used in the Weis et al. study (EidNi/41.3) is a selected subclone of the EidNi/41 cell
line that was used during this thesis and which was shown to fuse when M74 F and G were
co-expressed (Kruger et al., 2014). Therefore, a plausible explanation for the result of Weis et
al. (2014) is that the subcloning process has resulted in the selection of a cell clone that has
lost the ability of syncytium formation following M74 F and G co-expression. Additionally, a
recent study conducted by another group confirmed that the functional activity of M74 F and
G is restricted to chiropteran cells: Cell-to-cell fusion was obtained in chiropteran (EidNi)
cells co-expressing M74 F and G, but not in human (HeLa, HEK293T) or simian (VeroE6)
cells (Lawrence et al., 2014).
Taken together, these findings indicate that in general the surface glycoproteins of M74 are
able to mediate cell-to-cell fusion, although the fusion event is restricted to chiropteran cells.
Given that syncytium formation was obtained not only in Eidolon helvum cells, the host
species from which the RNA of M74 was isolated, but also in cells derived from an African
flying fox of the species Hypsignathus monstrosus, the restricted functional activity seems to
be rather an effect related to chiropteran cells than a restricted tropism for a certain reservoir
host species. The inability to mediate fusion in non-chiropteran cells might represent a
potential barrier for a spillover event from bats to humans or other mammalian species.
7.1.4. Inefficient surface expression of M74 G plays a crucial role for the
restricted functional activity
It has been reported, that the ability of the M74 surface glycoproteins to mediate cell-to-cell
fusion is restricted to chiropteran cells, but no explanation has been provided (Kruger et al.,
2013; Lawrence et al., 2014; Weis et al., 2014). Even the assumption, that the sequence of the
M74 F protein might be wrong - due to an error during sequencing (Pernet et al., 2014) - does
- 101 -
Discussion
not explain the restricted functional activity. Given that neither the cleavage nor the surface
expression of the M74 F protein differed between chiropteran and non-chiropteran cell lines
(Weis et al., 2014; Kruger et al., in preparation), the focus of further studies was laid on the
question, whether differences in the expression of the M74 G protein may explain the
restricted fusion activity?
Recently, it has been demonstrated, that the majority of the M74 G protein is localized in the
ER of both, transfected chiropteran cell lines, which show syncytium formation following coexpression of M74 F and G and non-chiropteran cell lines which do not (Kruger et al., 2014).
Consistent with this finding it was shown that M74 G does not contain detectable amounts of
complex N-glycans, that are modified from mannose-rich precursors during the transport of
the glycoproteins along the secretory pathway at a pre-Golgi and the Golgi compartment
(Kruger et al., 2014). In this context, the expression of chimeric proteins containing portions
of the NiV G and M74 G revealed, that the ectodomain of the M74 G is responsible for the
phenotypic expression pattern (Kruger et al., 2014). Whereas the results concerning the
intracellular localization of the M74 G were similar in chiropteran and non-chiropteran cells,
differences were obtained when the amount of M74 G on the cell surface was analyzed: It has
been demonstrated by immunofluorescence microscopy, surface biotinylation and Western
blotting, as well as by flowcytometric analysis, that the amount of surface expressed G protein
was higher in chiropteran cells (HypNi/1.1, HypLu/2, EidNi/41) than in non-chiropteran cells
(BHK-21, Vero76, HBE) (Kruger et al., 2014). However, even in the chiropteran cells the
amount of surface expressed M74 G was considerably lower compared to the total expression
level of the M74 G protein.
Taken together, these results demonstrate that the general transport of the M74 G protein from
the ER to the cell surface is inefficient in all cell lines tested. Nevertheless, more G protein
reached the cell surface of chiropteran cells in comparison to non-chiropteran cells. These
findings explain why the ability to mediate syncytium formation is restricted to chiropteran
cells. As described for NiV, fusion takes places after an interaction of the F and G protein at
the cell surface, leading to a conformational change of the F protein into a fusion-active form.
Without an efficient surface expression of the G protein, the triggering of the F protein is not
possible and therefore the fusion event cannot be initiated. It has been described that the
(heterologous) co-expression of M74 G and NiV F resulted in cell-to-cell fusion of nonchiropteran cells (Lawrence et al., 2014; Pernet et al., 2014; Weis et al., 2014), suggesting that
Discussion
- 102 -
the NiV F protein has a higher fusion activity than the M74 F. This suggestion is supported by
the finding that the size and number of syncytia obtained after the co-expression of NiV F and
G is higher in comparison to the syncytia obtained from the co-expression of M74 F and G
(Kruger et al., 2013).
7.1.5. Summary
In summary, it has been demonstrated that the surface glycoproteins of the African
henipavirus strain M74 share some similarities with the corresponding glycoproteins of NiV
and/ or HeV, two zoonotic paramyxoviruses which have succeeded in crossing the species
barrier. Especially the usage of the cellular receptor Eph B2 or Eph B3 may facilitate the
infection of other mammalian species. The proteolytic cleavage of the M74 F was
demonstrated in different chiropteran, human, simian, and rodent cell lines, suggesting that
the M74 F protein has the ability to mediate fusion. In general, there is evidence for a
zoonotic potential of M74. However, the ability to induce syncytium formation is restricted to
chiropteran cells and an inefficient surface expression of the M74 G appear to be a major
factor for the restricted functional activity and furthermore, may represent a potential barrier
for the transmission of M74 from bats to other mammalian species. On the other hand, the
inefficient surface expression of the M74 G, as well as the enhanced fusogenicity may be
explained by a peculiar replication strategy of the African henipavirus: Viral infections in bats
often do not result in a productive infection with a release of high amounts of virions, but
rather in a persistent infection, in which the virus spreads from cell to cell.
Further studies are needed to evaluate the zoonotic potential of this bat-derived henipavirus.
Especially, information about the replication strategy of M74 is missing, but without the
isolation of infectious viruses it is not possible to study the virus in more detail. Based on the
results obtained during this study, chiropteran cells, e.g. HypNi/1.1, are the cells of choice for
future attempts to isolate chiropteran (henipa-) viruses. Furthermore, cell-associated virus
transmission may be more likely than the infection by free virions released from the infected
cell.
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Discussion
7.2. Functional characteristics of the batMuV
During a survey, aiming to estimate the paramyxoviral flora of bats, the RNA of a bat-derived
mumps virus (batMuV) has been detected in an African flying fox of the genus Epomophorus
(Epomophorus spec.) in the Democratic Republic of Congo (Drexler et al., 2012). The viral
genome organization, as well as the length of each of its protein coding open reading frames
was identical to the reference strain MuVi/Boston.USA/0.45 [A] (previous name: Enders
strain); the general amino acid (aa) identity ranged between 72.6 % (phosphoprotein) to 94.2
% (RNA-dependent RNA polymerase), with the exception of the small hydrophobic (SH)
protein, which only showed 38.6 % aa identity (Drexler et al., 2012). In general, the SH
proteins show the lowest conservation among the MuV strains and are therefore used to
separate the strains into different genotypes. Based on this background information, a
phylogenetic analysis of the aa sequences of the SH proteins of batMuV as well as two
representative MuV strains of each genotype was performed, which revealed, that batMuV
did not cluster within any of the established MuV genotypes (Fig. 12).
7.2.1. BatMuV HN shows sialic acid dependent hemadsorption, but no
neuraminidase activity
The attachment glycoproteins of all known human MuV strains have in common, that they
promote virus binding to sialic acids which are present on the surface of target cells and/ or
erythrocytes of different mammalian and avian species, whereas the presence or extent of a
neuraminidase activity differs remarkably between different MuV strains. However, the
number of previous publications on the functional characteristics of MuV HN is very low and
only a few MuV strains have been evaluated in this respect.
It could be demonstrated that both, hMuV and batMuV HN are able to adsorb to chicken
erythrocytes in a sialic acid dependent manner to HN-expressing cells: This hemadsorption
was abolished when the erythrocytes were pre-incubated with a commercially available
neuraminidase (isolated from Clostridium perfringens) to remove surface bound sialic acids
(Kruger et al., submitted). This result clearly indicates that the HN of batMuV is functional
active with respect to binding to sialic acids.
Whereas the batMuV HN protein resembled the corresponding hMuV protein in its sialic acid
binding activity, it differed from the hMuV HN as far the second activity of paramyxovirus
HN proteins, the neuraminidase activity, is concerned. Whereas a high enzymatic activity was
Discussion
- 104 -
obtained for hMuV HN, no neuraminidase activity was detected with batMuV HN (Kruger et
al., submitted). It will be interesting to test in further studies, when more bat MuV sequences
are available, whether the lack of neuraminidase activity is a general feature of the HN
proteins of bat-derived MuV.
7.2.2. The batMuV surface glycoproteins mediate syncytium formation in various
mammalian cells
The ability to mediate membrane fusion differs within the various MuV strains: Whereas
some strains cause extensive cell-to-cell fusion in vitro, others do not cause fusion or other
cytopathic effects (Wolinsky et al., 1974; Wolinsky and Stroop, 1978; McCarthy and
Johnson, 1980). Past studies have shown, that the processing of the MuV F0 protein did not
differ between both kinds of strains and that the MuV F of both, fusion-active and fusioninactive strains, are readily cleaved in vivo; moreover, even the proteolytic cleavage sites are
identical (Merz et al., 1983). It has further been shown, that some MuV strains, which did not
mediate cell-to-cell fusion, have a higher neuraminidase activity in comparison to fusionactive strains, suggesting that a high neuraminidase activity promotes a rapid release of
virions without the occurrence of cell-to-cell fusion, whereas a low neuraminidase activity
supports the extracellular association of virions with infected cells, thereby increasing the
likelihood of cell-to-cell fusion (Merz and Wolinsky, 1981). The inhibition of the viral
neuraminidase activity, as well as the proteolysis of the HN has been shown to create a
fusion-mediating phenotype from a naturally non-fusion-mediating MuV strain (Merz and
Wolinsky, 1983; Waxham and Wolinsky, 1986). Taken together, these studies indicate that
the MuV HN, in particular its neuraminidase activity, modulates the fusogenicity of MuV.
The F proteins of all MuV strains contain a multibasic cleavage motif (aa 98 - 102, R-R-H-KR) directly upstream of the fusion peptide, that is suggested to be a substrate for proteolytic
cleavage mediated by the subtilisin-like proprotein convertase furin (Waxham et al., 1987;
Rubin, 2011). During this study it has been revealed by sequence analysis that also the
batMuV F contains a multibasic cleavage motif (aa 98 - 102, R-R-R-K-R) and that its aa
residues comprising the fusion peptide (aa 103 - 128) are completely conserved to those of
different MuV strains on aa level (Kruger et al., submitted). From this finding it is concluded,
that the proteolytic cleavage of the batMuV F follows a similar pattern to that of human MuV
F proteins.
- 105 -
Discussion
By in vitro co-expression studies of the batMuV F and HN it was analyzed, whether the
batMuV glycoproteins have the ability to mediate cell-to-cell fusion, leading to the formation
of syncytia. For comparison, the corresponding glycoproteins of a virulent human MuV strain
(hMuV), which was isolated from a hospitalized patient showing symptoms of mumps, were
included in this study. The glycoproteins F and HN of the hMuV isolate showed a homology
of 98.9 % or 99.2 % to a MuV consensus sequence, respectively.
The surface glycoproteins of hMuV were able to mediate the formation of huge syncytia in a
variety of mammalian cell lines. Also, the co-expression of the batMuV F and HN proteins
resulted in cell-to-cell fusion in chiropteran (HypNi/1.1, HypLu/2, EidNi/41), human (HeLa),
simian (Vero76), and rodent (BHK-21) cell lines (Kruger et al., submitted). As a general
finding, the size of syncytia induced by the batMuV glycoproteins was smaller compared to
those observed after co-expression of hMuV F and HN.
For some genera of the family Paramyxoviridae it has been reported, that closely related
viruses - e.g. the henipaviruses NiV and HeV - are still able to induce membrane fusion when
one of the two surface glycoproteins was replaced by the corresponding glycoprotein of the
related virus (heterologous expression) (Bossart et al., 2002). After having shown that the
homologous co-expression of the human or bat-derived MuV glycoproteins resulted in cellto-cell fusion, it was analyzed whether hMuV and batMuV F and HN proteins are also able to
interact with each other in a heterologous fashion and still are capable of mediating syncytium
formation. The co-expression of hMuV F and batMuV HN, as well as batMuV F and hMuV
HN resulted in cell-to-cell fusion, indicating that the batMuV glycoproteins are able to
interact with those of human MuV and that especially the batMuV HN triggers the hMuV F
(Kruger et al., submitted). However, based on the size of syncytia, the heterologous
co-expression was less efficient as the homologous expression of the hMuV F and HN.
In summary, it could be demonstrated that the co-expression of batMuV F and HN resulted in
cell-to-cell fusion in various cell lines of different mammalian species (including humans),
proving, that they are functionally active. Given that the bat-derived paramyxovirus
glycoproteins are able to mediate fusion of neighbouring cells, it is very likely that it would
also be able to mediate fusion of the viral envelope and plasma membrane of target cells. This
finding provides a strong evidence for a zoonotic potential of batMuV.
Discussion
- 106 -
7.2.3. The differences in the size of syncytia following F and HN expression
correlate with the origin of the SP-related aa residues of the F protein
Co-expression of batMuV F and HN resulted in the formation of syncytia, which were
noticeably smaller in comparison to those obtained after the co-expression of hMuV F and
HN. When compared to a MuV consensus sequence, that is generated by sequence analysis of
26 different MuV strains and isolates, the corresponding glycoproteins of both human and
bat-derived MuV show high levels of sequence homology on aa level: The hMuV F and HN
proteins showed 98.9 % or 99.2 % homology to the consensus sequence; batMuV F and HN
showed 94.6 % or 91 % homology, respectively (Kruger et al., submitted). Even in the case
that a different aa residue is present at a certain position, it is mostly an aa residue with
similar characteristics. For the batMuV F protein it was observed, that the total aa homology
of the F1 subunit (96.5 %) is higher than that of the F2 subunit (86.5%). In the latter,
especially the first 20 aa of the N-terminus of batMuV F, which have the characteristics of a
signal peptide (SP; calculated by the SignalIP software), shined out in regard of aa
conservation (69 %) in comparison to the corresponding aa residues of the consensus
sequence. In contrast to this, the hMuV F2 subunit and the SP showed a high homology to the
MuV consensus sequence (97.4 % and 96 %, respectively). To analyze whether the
differences in this part of the polypeptides have an influence on the fusogenicity, chimeric F
proteins with an exchange of the complete F2 subunit or only the predicted SP were generated
und investigated in a fusion assay. In this way it was shown, that F proteins harbouring either
the complete hMuV F2 subunit linked to the batMuV F1, or only the hMuV F SP-related aa
residues linked to the remaining portion of the batMuV F, mediated the formation of huge
syncytia following co-expression with both hMuV or batMuV HN (Kruger et al., submitted).
Those syncytia were larger in size when compared to those obtained after co-expression of
batMuV F an HN, and comparable to those which were formed after hMuV F and HN
co-expression. In contrast, F protein chimeras comprising the batMuV F2 subunit and the
hMuV F1, or the batMuV F SP-related aa residues followed by the aa residues of the hMuV F
(without the SP-related portion) induced the formation of smaller syncytia, resembling the
phenotype achieved by co-expression of batMuV F and HN (Kruger et al., submitted).
From these results it is concluded, that the SP of the hMuV F enables a more efficient
processing and transport of the F protein along the secretory pathway than the batMuV F SP.
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Discussion
7.2.4. Summary
It has been shown that there is a high relatedness between the glycoproteins of batMuV and
human MuV strains, not only based on the highly conserved aa sequences, but also on the
serological similarity and the functional activity. So far, no infectious MuV has been isolated
from bats, but nevertheless the herein before mentioned studies provide some indications for a
zoonotic potential of batMuV: (i) The fusion activity is not restricted to the reservoir host. (ii)
Based on past studies, the ability to mediate cell-to-cell fusion is associated with the virulence
of MuV strains and therefore, the syncytium formation induced after batMuV F and HN coexpression should be considered an important factor for the estimation of the virulence of the
batMuV. (iii) Furthermore, the interaction with sialic acids (and the potential usage of them as
a cellular receptor for virus entry) provides a widely distributed receptor determinant which is
usually present in many animal species.
The in silico analysis of the phylogenetic relatedness of the small hydrophobic protein (SH) of
batMuV and MuV genotypes indicates that the batMuV is not a variant of an already known
human strain (that might have been introduced into the chiropteran species by accident), but
rather a novel MuV strain of an origin different from humans.
In this light, it is noteworthy to mention that a MuV vaccination is not included into national
immunization programs in African countries and therefore, the people are not protected
against MuV infections. Given that monoclonal antibodies directed against the F protein of
different MuV strains were able to detect both, hMuV and batMuV F, it is likely that hMuV
and batMuV are conspecific and belong to one serogroup and that further a vaccination with
the common MuV vaccine could elicit a protective immunity against the batMuV strain
investigated in this study.
Based on the data obtained during this study it is feasible, that a batMuV has already been
able to infect humans in local areas, although there are no data that support this assumption.
But this is a general problem for infectious diseases with non-specific symptoms on the
African continent, as febrile illnesses are often attributed to malaria, yellow fever, dengue
fever, etc., treated with therapeutics against the disease with the most-fitting symptoms.
Further testing for very rare, uncommon, or new infectious diseases is either not performed or
impossible due to the high costs, missing equipment, lack of medical/ or immunological
training, or a lack of tests (especially for so far unknown pathogens).
Discussion
- 108 -
To get more information about the prevalence of (bat)MuV in Africa, it would be necessary to
screen serum samples from humans as well as different mammalian species for the presence
of MuV antibodies or RNA. This would give an overview about the distribution and diversity
of (bat)MuV strains in Africa and would provide valuable informations for possible
epidemiological follow-up studies.
- 109 -
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Appendix
9. APPENDIX
9.1. Sequences
9.1.1. M74 F
Construct: full length fusion protein of BatPV/Eid_hel/ GH-M74a/GHA/2009
GenBank accession number: AFH96010.1
Expression plasmid: pCG1/ pCAGGS
Labelling: Reference: Drexler et al., 2012
9.1.2. M74 G
Construct: full length attachment glycoprotein of BatPV/Eid_hel/ GH-M74a/GHA/2009
Expression plasmid: pCG1
9.1.3. NiV F
Construct: synthetic fusion protein of NiV/MY/HU/1999/CDC
GenBank accession number: AF212302 (complete genom), Protein ID: AAK29087.1
Expression plasmid: pCAGGS
Labelling: Reference: Chua et al., 2000; Lamp et al., 2013
9.1.4. NiV G
Construct: codon optimized attachment glycoprotein protein of NiV/MY/HU/1999/CDC
GenBank accession number: AF212302 (complete genome), Protein ID: AAK29088.1
Labelling: Reference: Chua et al., 2000; Lamp et al., 2013
Appendix
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9.1.5. M74 F-HA
Construct: HA-labelled M74 F
Expression plasmid: pCG1/ pCAGGS
Labelling: C-terminal HA epitope
Reference: Krüger et al., 2013, Weis et al., 2014
Nucleotide sequence:
ATGAAGAAAAAGACGGACAATCCCACAATATCAAAGAGGGGTCACAACCATTCTCGAGGAATCAAATC
TAGAGCGCTACTCAGAGAGACAGATAATTATTCCAATGGGCTAATAGTTGAGAATTTAGTTAGAAACT
GTCATCATCCAAGTAAGAACAATCTAAACTATACTAAGACACAAAAAAGAGATTCTACAATCCCTTAT
CGTGTGGAAGAGAGAAAAGGACATTATCCAAAGATTAAACATCTTATTGATAAATCTTACAAGCATAT
AAAAAGAGGGAAGAGAAGAAATGGTCATAATGGGAACATTATAACTATAATTCTGTTGTTGATTTTAA
TTTTGAAGACACAGATGAGTGAAGGTGCTATCCATTACGAGACTCTAAGTAAGATCGGATTAATAAAG
GGAATCACCAGAGAGTACAAAGTCAAAGGAACTCCGTCAAGTAAAGACATAGTCATCAAATTGATTCC
GAATGTCACCGGTCTTAACAAGTGCACGAACATATCAATGGAAAACTATAAAGAACAACTTGACAAAA
TACTAATTCCTATTAACAACATAATTGAATTGTATGCAAACTCAACTAAATCAGCCCCTGGGAATGCA
CGTTTTGCTGGCGTTATAATTGCAGGAGTGGCATTAGGTGTTGCAGCGGCAGCCCAAATAACTGCCGG
CATTGCACTGCATGAAGCTCGACAGAATGCAGAGAGAATTAATCTCTTAAAGGATAGCATTTCTGCCA
CTAACAACGCAGTAGCAGAACTCCAGGAAGCAACTGGTGGAATAGTAAATGTCATTACAGGAATGCAA
GATTACATCAATACAAATCTAGTCCCGCAGATTGACAAACTGCAATGTAGTCAGATCAAAACGGCATT
AGACATATCTCTCTCCCAATACTATTCAGAAATATTAACAGTGTTCGGTCCAAACCTTCAAAATCCAG
TAACTACTTCCATGTCAATACAAGCCATATCACAATCCTTTGGGGGAAATATAGATTTGCTCTTAAAC
CTACTAGGTTACACTGCAAACGACTTATTGGATTTGCTCGAAAGTAAAAGTATAACAGGCCAAATAAC
ATACATAAATCTTGAACATTACTTCATGGTAATCAGAGTATATTATCCTATAATGACAACAATCAGCA
ATGCTTATGTCCAGGAATTGATCAAAATTAGCTTCAATGTCGATGGCAGTGAATGGGTATCTCTTGTA
CCCTCGTATATATTGATTAGAAACTCATATCTCTCAAACATAGACATATCAGAATGTCTCATAACCAA
AAATTCAGTGATATGTCGTCATGACTTTGCAATGCCAATGAGTTACACCTTAAAGGAATGCCTAACTG
GAGACACTGAAAAGTGTCCGAGAGAGGCTGTTGTAACCTCATATGTCCCAAGATTTGCTATCTCCGGG
GGAGTGATTTATGCTAATTGTCTAAGTACAACATGTCAATGCTATCAAACTGGCAAAGTAATTGCTCA
AGACGGCAGCCAAACATTGATGATGATCGATAATCAAACATGTTCAATAGTAAGAATTGAAGAAATCC
TCATATCAACAGGGAAATATCTGGGAAGTCAGGAGTACAATACGATGCATGTGTCAGTCGGCAATCCT
GTCTTCACTGACAAGCTGGACATAACAAGTCAAATTTCCAACATCAACCAATCCATTGAACAATCCAA
ATTTTATCTAGATAAGTCTAAGGCTATACTTGACAAGATAAATCTCAACTTAATTGGCTCTGTACCGA
TATCAATACTTTTCATAATTGCGATCTTATCATTGATTCTCTCTATTATAACTTTTGTGATTGTGATG
ATAATTGTCAGAAGATATAACAAATACACTCCTCTTATAAACTCTGATCCATCCAGTAGGAGGAGTAC
TATACAGGACGTATATATCATCCCGAACCCCGGAGAACATTCGATTAGATCAGCTGCTCGATCAATTG
ACAGAGATCGAGATTATCCTTATGATGTTCCTGATTATGCTTGA
Amino acid sequence:
MKKKTDNPTISKRGHNHSRGIKSRALLRETDNYSNGLIVENLVRNCHHPSKNNLNYTKTQKRDSTIPY
RVEERKGHYPKIKHLIDKSYKHIKRGKRRNGHNGNIITIILLLILILKTQMSEGAIHYETLSKIGLIK
GITREYKVKGTPSSKDIVIKLIPNVTGLNKCTNISMENYKEQLDKILIPINNIIELYANSTKSAPGNA
RFAGVIIAGVALGVAAAAQITAGIALHEARQNAERINLLKDSISATNNAVAELQEATGGIVNVITGMQ
DYINTNLVPQIDKLQCSQIKTALDISLSQYYSEILTVFGPNLQNPVTTSMSIQAISQSFGGNIDLLLN
LLGYTANDLLDLLESKSITGQITYINLEHYFMVIRVYYPIMTTISNAYVQELIKISFNVDGSEWVSLV
PSYILIRNSYLSNIDISECLITKNSVICRHDFAMPMSYTLKECLTGDTEKCPREAVVTSYVPRFAISG
GVIYANCLSTTCQCYQTGKVIAQDGSQTLMMIDNQTCSIVRIEEILISTGKYLGSQEYNTMHVSVGNP
VFTDKLDITSQISNINQSIEQSKFYLDKSKAILDKINLNLIGSVPISILFIIAILSLILSIITFVIVM
IIVRRYNKYTPLINSDPSSRRSTIQDVYIIPNPGEHSIRSAARSIDRDRDYPYDVPDYA-
- 141 -
Appendix
9.1.6. M74 G-FLAG
Construct: FLAG-labelled M74 G
Labelling: C-terminal FLAG epitope
Reference: Krüger et al., 2013
ATGCCGCAGAAGACTGTGGAATTCATTAACATGAATTCCCCTCTAGAAAGAGGGGTCAGCACTCTTTC
AGATAAGAAGACCCTCAATCAATCTAAAATCACCAAGCAGGGGTATTTTGGGTTAGGATCCCACAGTG
AGAGAAATTGGAAGAAGCAGAAGAATCAAAATGATCATTACATGACTGTTTCAACCATGATTCTTGAG
ATATTAGTTGTCCTGGGCATCATGTTTAATCTCATAGTTTTAACTATGGTGTATTATCAGAATGACAA
CATCAATCAAAGGATGGCAGAACTTACAAGCAATATCACAGTCCTGAATTTAAATCTTAATCAATTGA
CAAACAAAATTCAAAGAGAAATTATTCCTAGGATCACTCTTATTGACACAGCAACCACCATTACAATT
CCTAGTGCCATTACTTACATATTAGCAACTCTGACAACCAGAATCTCGGAATTATTGCCGTCAATCAA
CCAAAAGTGTGAGTTCAAGACACCGACACTTGTCCTGAATGACTGCAGAATAAACTGTACCCCACCAC
TAAACCCGTCTGATGGAGTGAAAATGAGTTCTCTTGCCACTAACTTGGTTGCACATGGGCCCTCTCCC
TGTAGAAACTTTTCATCCGTACCTACAATTTACTATTATCGGATTCCAGGATTATACAATAGAACAGC
ATTGGACGAAAGATGTATACTAAACCCGAGATTGACAATAAGCAGTACAAAATTTGCTTATGTCCACT
CTGAATATGATAAAAATTGCACCAGAGGATTCAAATACTATGAATTGATGACATTTGGAGAAATACTG
GAGGGTCCGGAAAAAGAACCCAGAATGTTTTCTAGGTCATTTTATTCGCCCACAAATGCTGTGAACTA
TCATTCTTGTACGCCGATCGTGACTGTCAATGAAGGATATTTTCTTTGCCTTGAATGCACCTCCTCAG
ATCCCTTGTACAAAGCAAATCTATCTAATAGCACATTCCATTTGGTGATACTGAGGCATAACAAGGAT
GAGAAAATAGTTTCAATGCCTAGCTTTAACCTTTCTACTGATCAAGAGTATGTTCAGATAATCCCTGC
AGAAGGTGGCGGCACAGCAGAGAGTGGCAATCTTTACTTCCCTTGTATTGGAAGGCTCTTACACAAAC
GAGTCACCCATCCTTTATGCAAAAAGTCAAATTGTTCGCGAACTGATGATGAATCTTGCCTGAAAAGT
TATTACAATCAAGGGTCGCCTCAGCACCAAGTAGTCAACTGTCTGATAAGGATCAGAAATGCACAGAG
AGATAATCCAACCTGGGATGTTATCACAGTTGATCTGACTAATACATACCCAGGATCAAGGAGCAGGA
TCTTTGGAAGCTTCTCCAAACCGATGCTTTATCAATCATCAGTATCATGGCATACTCTTCTTCAGGTA
GCAGAGATAACAGACCTAGATAAGTATCAATTGGACTGGTTGGATACACCCTATATATCTCGTCCTGG
AGGATCTGAGTGCCCTTTCGGAAATTATTGTCCAACGGTATGCTGGGAAGGGACATATAATGATGTCT
ATAGCTTAACTCCAAATAACGATCTTTTTGTCACTGTGTATTTGAAGAGTGAACAAGTTGCAGAGAAC
CCTTATTTCGCAATCTTCTCCCGGGATCAAATCTTGAAAGAATTCCCTCTTGATGCATGGATAAGCAG
TGCACGAACTACGACAATATCGTGCTTCATGTTCAACAATGAAATTTGGTGTATAGCTGCATTAGAGA
TCACAAGATTGAATGATGACATCATAAGACCAATTTATTACTCTTTCTGGCTGCCTACTGATTGCCGG
ACACCATATCCCCACACCGGTAAGATGACCAGGGTTCCCTTGCGCTCCACATATAACTACGATTATAA
AGATGATGATGATAAATAA
MPQKTVEFINMNSPLERGVSTLSDKKTLNQSKITKQGYFGLGSHSERNWKKQKNQNDHYMTVSTMILE
ILVVLGIMFNLIVLTMVYYQNDNINQRMAELTSNITVLNLNLNQLTNKIQREIIPRITLIDTATTITI
PSAITYILATLTTRISELLPSINQKCEFKTPTLVLNDCRINCTPPLNPSDGVKMSSLATNLVAHGPSP
CRNFSSVPTIYYYRIPGLYNRTALDERCILNPRLTISSTKFAYVHSEYDKNCTRGFKYYELMTFGEIL
EGPEKEPRMFSRSFYSPTNAVNYHSCTPIVTVNEGYFLCLECTSSDPLYKANLSNSTFHLVILRHNKD
EKIVSMPSFNLSTDQEYVQIIPAEGGGTAESGNLYFPCIGRLLHKRVTHPLCKKSNCSRTDDESCLKS
YYNQGSPQHQVVNCLIRIRNAQRDNPTWDVITVDLTNTYPGSRSRIFGSFSKPMLYQSSVSWHTLLQV
AEITDLDKYQLDWLDTPYISRPGGSECPFGNYCPTVCWEGTYNDVYSLTPNNDLFVTVYLKSEQVAEN
PYFAIFSRDQILKEFPLDAWISSARTTTISCFMFNNEIWCIAALEITRLNDDIIRPIYYSFWLPTDCR
TPYPHTGKMTRVPLRSTYNYDYKDDDDK-
Appendix
- 142 -
9.1.7. NiV F-HA
Construct: aa residues 538 - 546 of NiV F were replaced by an HA-epitope
Expression plasmid: pCAGGS
Reference: Moll et al., 2004
ATGGTAGTTATACTTGACAAGAGATGTTATTGTAATCTTTTAATATTGATTTTGATGATCTCGGAGTG
TAGTGTTGGGATTCTACATTATGAGAAATTGAGTAAAATTGGACTTGTCAAAGGAGTAACAAGAAAAT
ACAAGATTAAAAGCAATCCTCTCACAAAAGACATTGTTATAAAAATGATTCCGAATGTGTCGAACATG
TCTCAGTGCACAGGGAGTGTCATGGAAAATTATAAAACACGATTAAACGGTATCTTAACACCTATAAA
GGGAGCGTTAGAGATCTACAAAAACAACACTCATGACCTTGTCGGTGATGTGAGATTAGCCGGAGTTA
TAATGGCAGGAGTTGCTATTGGGATTGCAACCGCAGCTCAAATCACTGCAGGTGTAGCACTATATGAG
GCAATGAAGAATGCTGACAACATCAACAAACTCAAAAGCAGCATTGAATCAACTAATGAAGCTGTCGT
TAAACTTCAAGAGACTGCAGAAAAGACAGTCTATGTGCTGACTGCTCTACAGGATTACATTAATACTA
ATTTAGTACCGACAATTGACAAGATAAGCTGCAAACAGACAGAACTCTCACTAGATCTGGCATTATCA
AAGTACCTCTCTGATTTGCTTTTTGTATTTGGCCCCAACCTTCAAGACCCAGTTTCTAATTCAATGAC
TATACAGGCTATATCTCAGGCATTCGGTGGAAATTATGAAACACTGCTAAGAACATTGGGTTACGCTA
CAGAAGACTTTGATGATCTTCTAGAAAGTGACAGCATAACAGGTCAAATCATCTATGTTGATCTAAGT
AGCTACTATATAATTGTCAGGGTTTATTTTCCTATTCTGACTGAAATTCAACAGGCCTATATCCAAGA
GTTGTTACCAGTGAGCTTCAACAATGATAATTCAGAATGGATCAGTATTGTCCCAAATTTCATATTGG
TAAGGAATACATTAATATCAAATATAGAGATTGGATTTTGCCTAATTACAAAGAGGAGCGTGATCTGC
AACCAAGATTATGCCACACCTATGACCAACAACATGAGAGAATGTTTAACGGGATCGACTGAGAAGTG
TCCTCGAGAGCTGGTTGTTTCATCACATGTTCCCAGATTTGCACTATCTAACGGGGTTCTGTTTGCCA
ATTGCATAAGTGTTACATGTCAGTGTCAAACAACAGGCAGGGCAATCTCACAATCAGGAGAACAAACT
CTGCTGATGATTGACAACACCACCTGTCCTACAGCCGTACTCGGTAATGTGATTATCAGCTTAGGGAA
ATATCTGGGGTCAGTAAATTATAATTCTGAAGGCATTGCTATCGGTCCTCCAGTCTTTACAGATAAAG
TTGATATATCAAGTCAGATATCCAGCATGAATCAGTCCTTACAACAGTCTAAGGACTATATCAAAGAG
GCTCAACGACTCCTTGATACTGTTAATCCATCATTAATAAGCATGTTGTCTATGATCATACTGTATGT
ATTATCGATCGCATCGTTGTGTATAGGGTTGATTACATTTATCAGTTTTATCATTGTTGAGAAAAAGA
GAAACACCTACAGCAGATTAGAGGATAGGAGAGTCAGACCTACAAGCTATCCGTACGACGTCCCGGAT
TACGCTTAG
MVVILDKRCYCNLLILILMISECSVGILHYEKLSKIGLVKGVTRKYKIKSNPLTKDIVIKMIPNVSNM
SQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYE
AMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALS
KYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLS
SYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVIC
NQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQT
LLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKE
AQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFIIVEKKRNTYSRLEDRRVRPTSYPYDVPD
YA-
- 143 -
Appendix
9.1.8. NiV G-FLAG
Construct: FLAG-labelled NiV G
ATGCCCGCCGAGAACAAGAAAGTGCGGTTCGAGAACACCACCTCCGACAAGGGCAAGATCCCCAGCAA
GGTGATCAAGAGCTACTACGGCACCATGGACATCAAGAAGATCAACGAGGGCCTGCTGGACAGCAAGA
TCCTGAGCGCCTTCAACACCGTGATCGCCCTGCTGGGCAGCATCGTGATCATTGTGATGAACATCATG
ATCATCCAGAACTACACCAGAAGCACCGACAACCAGGCCGTGATCAAGGACGCTCTCCAGGGGATCCA
GCAGCAGATCAAGGGCCTGGCCGACAAGATCGGCACCGAGATCGGCCCCAAGGTGTCCCTGATCGACA
CCAGCAGCACCATCACCATCCCCGCCAACATCGGCCTGCTGGGGTCCAAGATCAGCCAGAGCACCGCC
AGCATCAACGAGAACGTGAACGAGAAGTGCAAGTTCACCCTGCCCCCCCTGAAGATCCACGAGTGCAA
CATCAGCTGCCCCAACCCCCTGCCCTTCCGGGAGTACCGGCCCCAGACCGAGGGCGTGAGCAACCTGG
TCGGCCTGCCCAACAACATCTGCCTGCAGAAAACCAGCAACCAGATCCTGAAGCCCAAGCTCATTTCC
TACACCCTGCCCGTGGTGGGCCAGAGCGGCACCTGCATCACCGACCCCCTGCTGGCCATGGACGAGGG
CTACTTCGCCTACAGCCACCTGGAACGGATCGGCAGCTGCAGCAGGGGCGTGTCCAAGCAGCGGATCA
TCGGCGTGGGCGAGGTGCTGGACCGGGGCGACGAGGTGCCCAGCCTGTTCATGACCAACGTGTGGACC
CCCCCCAACCCCAACACCGTGTACCACTGCAGCGCCGTGTACAACAACGAGTTCTACTACGTGCTGTG
CGCCGTGAGCACCGTGGGCGACCCCATCCTGAACAGCACCTACTGGTCCGGCAGCCTGATGATGACCC
GGCTGGCCGTGAAGCCTAAGAGCAATGGCGGCGGATACAACCAGCACCAGCTGGCCCTGCGGAGCATC
GAGAAGGGCAGATACGACAAAGTGATGCCCTACGGCCCCAGCGGCATCAAGCAGGGCGACACACTGTA
CTTCCCCGCCGTGGGCTTCCTGGTCCGGACCGAGTTCAAGTACAACGACAGCAACTGCCCCATCACCA
AGTGCCAGTACAGCAAGCCCGAGAACTGCAGACTGAGCATGGGCATCCGGCCCAACAGCCACTACATC
CTGCGGAGCGGCCTGCTGAAGTACAACCTGAGCGACGGCGAGAACCCCAAAGTCGTCTTTATTGAGAT
CAGCGACCAGCGGCTGTCCATCGGCAGCCCCAGCAAGATCTACGACAGCCTGGGCCAGCCCGTGTTCT
ACCAGGCCAGCTTCAGCTGGGACACCATGATCAAGTTCGGCGACGTGCTGACCGTGAACCCCCTGGTG
GTGAACTGGCGGAACAATACCGTGATCAGCAGACCCGGCCAGAGCCAGTGCCCCCGGTTCAACACCTG
CCCCGAGATCTGCTGGGAGGGCGTGTACAACGACGCCTTCCTGATCGACCGGATCAACTGGATCTCTG
CCGGCGTGTTCCTGGACTCCAACCAGACCGCCGAGAATCCCGTGTTCACCGTGTTTAAGGACAACGAG
ATCCTGTACCGGGCCCAGCTGGCCAGCGAGGACACCAACGCCCAGAAAACCATCACCAACTGCTTTCT
GCTGAAGAACAAGATCTGGTGCATCAGCCTGGTGGAGATCTACGATACCGGCGACAACGTGATCAGGC
CCAAGCTGTTCGCCGTGAAGATCCCCGAGCAGTGCACCGATTATAAAGATGATGATGATAAATAA
MPAENKKVRFENTTSDKGKIPSKVIKSYYGTMDIKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIM
IIQNYTRSTDNQAVIKDALQGIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTA
SINENVNEKCKFTLPPLKIHECNISCPNPLPFREYRPQTEGVSNLVGLPNNICLQKTSNQILKPKLIS
YTLPVVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWT
PPNPNTVYHCSAVYNNEFYYVLCAVSTVGDPILNSTYWSGSLMMTRLAVKPKSNGGGYNQHQLALRSI
EKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPITKCQYSKPENCRLSMGIRPNSHYI
LRSGLLKYNLSDGENPKVVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVLTVNPLV
VNWRNNTVISRPGQSQCPRFNTCPEICWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNE
ILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCTDYKDDDDK-
Appendix
- 144 -
9.1.9. M74 G-FLAG (ED)
Construct: chimeric G protein, cytoplasmic tail and transmembrane domain of NiV G,
ectodomain of M74 G
ATGCCCGCCGAGAACAAGAAAGTGCGGTTCGAGAACACCACCTCCGACAAGGGCAAGATCCCCAGCAA
GGTGATCAAGAGCTACTACGGCACCATGGACATCAAGAAGATCAACGAGGGCCTGCTGGACAGCAAGA
TCCTGAGCGCCTTCAACACCGTGATCGCCCTGCTGGGCAGCATCGTGATCATTGTGATGAACATCATG
ATCATCCAGAATGACAACATCAATCAAAGGATGGCAGAACTTACAAGCAATATCACAGTCCTGAATTT
AAATCTTAATCAATTGACAAACAAAATTCAAAGAGAAATTATTCCTAGGATCACTCTTATTGACACAG
CAACCACCATTACAATTCCTAGTGCCATTACTTACATATTAGCAACTCTGACAACCAGAATCTCGGAA
TTATTGCCGTCAATCAACCAAAAGTGTGAGTTCAAGACACCGACACTTGTCCTGAATGACTGCAGAAT
AAACTGTACCCCACCACTAAACCCGTCTGATGGAGTGAAAATGAGTTCTCTTGCCACTAACTTGGTTG
CACATGGGCCCTCTCCCTGTAGAAACTTTTCATCCGTACCTACAATTTACTATTATCGGATTCCAGGA
TTATACAATAGAACAGCATTGGACGAAAGATGTATACTAAACCCGAGATTGACAATAAGCAGTACAAA
ATTTGCTTATGTCCACTCTGAATATGATAAAAATTGCACCAGAGGATTCAAATACTATGAATTGATGA
CATTTGGAGAAATACTGGAGGGTCCGGAAAAAGAACCCAGAATGTTTTCTAGGTCATTTTATTCGCCC
ACAAATGCTGTGAACTATCATTCTTGTACGCCGATCGTGACTGTCAATGAAGGATATTTTCTTTGCCT
TGAATGCACCTCCTCAGATCCCTTGTACAAAGCAAATCTATCTAATAGCACATTCCATTTGGTGATAC
TGAGGCATAACAAGGATGAGAAAATAGTTTCAATGCCTAGCTTTAACCTTTCTACTGATCAAGAGTAT
GTTCAGATAATCCCTGCAGAAGGTGGCGGCACAGCAGAGAGTGGCAATCTTTACTTCCCTTGTATTGG
AAGGCTCTTACACAAACGAGTCACCCATCCTTTATGCAAAAAGTCAAATTGTTCGCGAACTGATGATG
AATCTTGCCTGAAAAGTTATTACAATCAAGGGTCGCCTCAGCACCAAGTAGTCAACTGTCTGATAAGG
ATCAGAAATGCACAGAGAGATAATCCAACCTGGGATGTTATCACAGTTGATCTGACTAATACATACCC
AGGATCAAGGAGCAGGATCTTTGGAAGCTTCTCCAAACCGATGCTTTATCAATCATCAGTATCATGGC
ATACTCTTCTTCAGGTAGCAGAGATAACAGACCTAGATAAGTATCAATTGGACTGGTTGGATACACCC
TATATATCTCGTCCTGGAGGATCTGAGTGCCCTTTCGGAAATTATTGTCCAACGGTATGCTGGGAAGG
GACATATAATGATGTCTATAGCTTAACTCCAAATAACGATCTTTTTGTCACTGTGTATTTGAAGAGTG
AACAAGTTGCAGAGAACCCTTATTTCGCAATCTTCTCCCGGGATCAAATCTTGAAAGAATTCCCTCTT
GATGCATGGATAAGCAGTGCACGAACTACGACAATATCGTGCTTCATGTTCAACAATGAAATTTGGTG
TATAGCTGCATTAGAGATCACAAGATTGAATGATGACATCATAAGACCAATTTATTACTCTTTCTGGC
TGCCTACTGATTGCCGGACACCATATCCCCACACCGGTAAGATGACCAGGGTTCCCTTGCGCTCCACA
TATAACTACGATTATAAAGATGATGATGATAAATAA
MPAENKKVRFENTTSDKGKIPSKVIKSYYGTMDIKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIM
IIQNDNINQRMAELTSNITVLNLNLNQLTNKIQREIIPRITLIDTATTITIPSAITYILATLTTRISE
LLPSINQKCEFKTPTLVLNDCRINCTPPLNPSDGVKMSSLATNLVAHGPSPCRNFSSVPTIYYYRIPG
LYNRTALDERCILNPRLTISSTKFAYVHSEYDKNCTRGFKYYELMTFGEILEGPEKEPRMFSRSFYSP
TNAVNYHSCTPIVTVNEGYFLCLECTSSDPLYKANLSNSTFHLVILRHNKDEKIVSMPSFNLSTDQEY
VQIIPAEGGGTAESGNLYFPCIGRLLHKRVTHPLCKKSNCSRTDDESCLKSYYNQGSPQHQVVNCLIR
IRNAQRDNPTWDVITVDLTNTYPGSRSRIFGSFSKPMLYQSSVSWHTLLQVAEITDLDKYQLDWLDTP
YISRPGGSECPFGNYCPTVCWEGTYNDVYSLTPNNDLFVTVYLKSEQVAENPYFAIFSRDQILKEFPL
DAWISSARTTTISCFMFNNEIWCIAALEITRLNDDIIRPIYYSFWLPTDCRTPYPHTGKMTRVPLRST
YNYDYKDDDDK-
- 145 -
Appendix
9.1.10. NiV G-FLAG (ED)
Construct: chimeric G protein, cytoplasmic tail and transmembrane domain of M74 G,
ectodomain of NiV G
ATGCCGCAGAAGACTGTGGAATTCATTAACATGAATTCCCCTCTAGAAAGAGGGGTCAGCACTCTTTC
AGATAAGAAGACCCTCAATCAATCTAAAATCACCAAGCAGGGGTATTTTGGGTTAGGATCCCACAGTG
AGAGAAATTGGAAGAAGCAGAAGAATCAAAATGATCATTACATGACTGTTTCAACCATGATTCTTGAG
ATATTAGTTGTCCTGGGCATCATGTTTAATCTCATAGTTTTAACTATGGTGTATTATCAGAACTACAC
CAGAAGCACCGACAACCAGGCCGTGATCAAGGACGCTCTCCAGGGGATCCAGCAGCAGATCAAGGGCC
TGGCCGACAAGATCGGCACCGAGATCGGCCCCAAGGTGTCCCTGATCGACACCAGCAGCACCATCACC
ATCCCCGCCAACATCGGCCTGCTGGGGTCCAAGATCAGCCAGAGCACCGCCAGCATCAACGAGAACGT
GAACGAGAAGTGCAAGTTCACCCTGCCCCCCCTGAAGATCCACGAGTGCAACATCAGCTGCCCCAACC
CCCTGCCCTTCCGGGAGTACCGGCCCCAGACCGAGGGCGTGAGCAACCTGGTCGGCCTGCCCAACAAC
ATCTGCCTGCAGAAAACCAGCAACCAGATCCTGAAGCCCAAGCTCATTTCCTACACCCTGCCCGTGGT
GGGCCAGAGCGGCACCTGCATCACCGACCCCCTGCTGGCCATGGACGAGGGCTACTTCGCCTACAGCC
ACCTGGAACGGATCGGCAGCTGCAGCAGGGGCGTGTCCAAGCAGCGGATCATCGGCGTGGGCGAGGTG
CTGGACCGGGGCGACGAGGTGCCCAGCCTGTTCATGACCAACGTGTGGACCCCCCCCAACCCCAACAC
CGTGTACCACTGCAGCGCCGTGTACAACAACGAGTTCTACTACGTGCTGTGCGCCGTGAGCACCGTGG
GCGACCCCATCCTGAACAGCACCTACTGGTCCGGCAGCCTGATGATGACCCGGCTGGCCGTGAAGCCT
AAGAGCAATGGCGGCGGATACAACCAGCACCAGCTGGCCCTGCGGAGCATCGAGAAGGGCAGATACGA
CAAAGTGATGCCCTACGGCCCCAGCGGCATCAAGCAGGGCGACACACTGTACTTCCCCGCCGTGGGCT
TCCTGGTCCGGACCGAGTTCAAGTACAACGACAGCAACTGCCCCATCACCAAGTGCCAGTACAGCAAG
CCCGAGAACTGCAGACTGAGCATGGGCATCCGGCCCAACAGCCACTACATCCTGCGGAGCGGCCTGCT
GAAGTACAACCTGAGCGACGGCGAGAACCCCAAAGTCGTCTTTATTGAGATCAGCGACCAGCGGCTGT
CCATCGGCAGCCCCAGCAAGATCTACGACAGCCTGGGCCAGCCCGTGTTCTACCAGGCCAGCTTCAGC
TGGGACACCATGATCAAGTTCGGCGACGTGCTGACCGTGAACCCCCTGGTGGTGAACTGGCGGAACAA
TACCGTGATCAGCAGACCCGGCCAGAGCCAGTGCCCCCGGTTCAACACCTGCCCCGAGATCTGCTGGG
AGGGCGTGTACAACGACGCCTTCCTGATCGACCGGATCAACTGGATCTCTGCCGGCGTGTTCCTGGAC
TCCAACCAGACCGCCGAGAATCCCGTGTTCACCGTGTTTAAGGACAACGAGATCCTGTACCGGGCCCA
GCTGGCCAGCGAGGACACCAACGCCCAGAAAACCATCACCAACTGCTTTCTGCTGAAGAACAAGATCT
GGTGCATCAGCCTGGTGGAGATCTACGATACCGGCGACAACGTGATCAGGCCCAAGCTGTTCGCCGTG
AAGATCCCCGAGCAGTGCACCGATTATAAAGATGATGATGATAAATAA
MPQKTVEFINMNSPLERGVSTLSDKKTLNQSKITKQGYFGLGSHSERNWKKQKNQNDHYMTVSTMILE
ILVVLGIMFNLIVLTMVYYQNYTRSTDNQAVIKDALQGIQQQIKGLADKIGTEIGPKVSLIDTSSTIT
IPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYRPQTEGVSNLVGLPNN
ICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEV
LDRGDEVPSLFMTNVWTPPNPNTVYHCSAVYNNEFYYVLCAVSTVGDPILNSTYWSGSLMMTRLAVKP
KSNGGGYNQHQLALRSIEKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPITKCQYSK
PENCRLSMGIRPNSHYILRSGLLKYNLSDGENPKVVFIEISDQRLSIGSPSKIYDSLGQPVFYQASFS
WDTMIKFGDVLTVNPLVVNWRNNTVISRPGQSQCPRFNTCPEICWEGVYNDAFLIDRINWISAGVFLD
SNQTAENPVFTVFKDNEILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAV
KIPEQCTDYKDDDDK-
Appendix
- 146 -
9.1.11. M74 F-HA (NiVstart)
Construct: chimeric F protein, the aa residues 1 - 108 of M74 F were replaced by the aa
residues 1 - 12 of NiV F
ATGGTAGTTATACTTGACAAGAGATGTTATTGTAATCTGTTGTTGATTTTAATTTTGAAGACACAGAT
GAGTGAAGGTGCTATCCATTACGAGACTCTAAGTAAGATCGGATTAATAAAGGGAATCACCAGAGAGT
ACAAAGTCAAAGGAACTCCGTCAAGTAAAGACATAGTCATCAAATTGATTCCGAATGTCACCGGTCTT
AACAAGTGCACGAACATATCAATGGAAAACTATAAAGAACAACTTGACAAAATACTAATTCCTATTAA
CAACATAATTGAATTGTATGCAAACTCAACTAAATCAGCCCCTGGGAATGCACGTTTTGCTGGCGTTA
TAATTGCAGGAGTGGCATTAGGTGTTGCAGCGGCAGCCCAAATAACTGCCGGCATTGCACTGCATGAA
GCTCGACAGAATGCAGAGAGAATTAATCTCTTAAAGGATAGCATTTCTGCCACTAACAACGCAGTAGC
AGAACTCCAGGAAGCAACTGGTGGAATAGTAAATGTCATTACAGGAATGCAAGATTACATCAATACAA
ATCTAGTCCCGCAGATTGACAAACTGCAATGTAGTCAGATCAAAACGGCATTAGACATATCTCTCTCC
CAATACTATTCAGAAATATTAACAGTGTTCGGTCCAAACCTTCAAAATCCAGTAACTACTTCCATGTC
AATACAAGCCATATCACAATCCTTTGGGGGAAATATAGATTTGCTCTTAAACCTACTAGGTTACACTG
CAAACGACTTATTGGATTTGCTCGAAAGTAAAAGTATAACAGGCCAAATAACATACATAAATCTTGAA
CATTACTTCATGGTAATCAGAGTATATTATCCTATAATGACAACAATCAGCAATGCTTATGTCCAGGA
ATTGATCAAAATTAGCTTCAATGTCGATGGCAGTGAATGGGTATCTCTTGTACCCTCGTATATATTGA
TTAGAAACTCATATCTCTCAAACATAGACATATCAGAATGTCTCATAACCAAAAATTCAGTGATATGT
CGTCATGACTTTGCAATGCCAATGAGTTACACCTTAAAGGAATGCCTAACTGGAGACACTGAAAAGTG
TCCGAGAGAGGCTGTTGTAACCTCATATGTCCCAAGATTTGCTATCTCCGGGGGAGTGATTTATGCTA
ATTGTCTAAGTACAACATGTCAATGCTATCAAACTGGCAAAGTAATTGCTCAAGACGGCAGCCAAACA
TTGATGATGATCGATAATCAAACATGTTCAATAGTAAGAATTGAAGAAATCCTCATATCAACAGGGAA
ATATCTGGGAAGTCAGGAGTACAATACGATGCATGTGTCAGTCGGCAATCCTGTCTTCACTGACAAGC
TGGACATAACAAGTCAAATTTCCAACATCAACCAATCCATTGAACAATCCAAATTTTATCTAGATAAG
TCTAAGGCTATACTTGACAAGATAAATCTCAACTTAATTGGCTCTGTACCGATATCAATACTTTTCAT
AATTGCGATCTTATCATTGATTCTCTCTATTATAACTTTTGTGATTGTGATGATAATTGTCAGAAGAT
ATAACAAATACACTCCTCTTATAAACTCTGATCCATCCAGTAGGAGGAGTACTATACAGGACGTATAT
ATCATCCCGAACCCCGGAGAACATTCGATTAGATCAGCTGCTCGATCAATTGACAGAGATCGAGATTA
TCCTTATGATGTTCCTGATTATGCTTGA
MVVILDKRCYCNLLLILILKTQMSEGAIHYETLSKIGLIKGITREYKVKGTPSSKDIVIKLIPNVTGL
NKCTNISMENYKEQLDKILIPINNIIELYANSTKSAPGNARFAGVIIAGVALGVAAAAQITAGIALHE
ARQNAERINLLKDSISATNNAVAELQEATGGIVNVITGMQDYINTNLVPQIDKLQCSQIKTALDISLS
QYYSEILTVFGPNLQNPVTTSMSIQAISQSFGGNIDLLLNLLGYTANDLLDLLESKSITGQITYINLE
HYFMVIRVYYPIMTTISNAYVQELIKISFNVDGSEWVSLVPSYILIRNSYLSNIDISECLITKNSVIC
RHDFAMPMSYTLKECLTGDTEKCPREAVVTSYVPRFAISGGVIYANCLSTTCQCYQTGKVIAQDGSQT
LMMIDNQTCSIVRIEEILISTGKYLGSQEYNTMHVSVGNPVFTDKLDITSQISNINQSIEQSKFYLDK
SKAILDKINLNLIGSVPISILFIIAILSLILSIITFVIVMIIVRRYNKYTPLINSDPSSRRSTIQDVY
IIPNPGEHSIRSAARSIDRDRDYPYDVPDYA-
- 147 -
Appendix
9.1.12. NiV F-HA (M74start)
Construct: chimeric F protein, the aa residues 1 - 12 of NiV F were replaced by the aa
residues 1 - 108 of M74 F
ATGAAGAAAAAGACGGACAATCCCACAATATCAAAGAGGGGTCACAACCATTCTCGAGGAATCAAATC
TAGAGCGCTACTCAGAGAGACAGATAATTATTCCAATGGGCTAATAGTTGAGAATTTAGTTAGAAACT
GTCATCATCCAAGTAAGAACAATCTAAACTATACTAAGACACAAAAAAGAGATTCTACAATCCCTTAT
CGTGTGGAAGAGAGAAAAGGACATTATCCAAAGATTAAACATCTTATTGATAAATCTTACAAGCATAT
AAAAAGAGGGAAGAGAAGAAATGGTCATAATGGGAACATTATAACTATAATTCTTTTAATATTGATTT
TGATGATCTCGGAGTGTAGTGTTGGGATTCTACATTATGAGAAATTGAGTAAAATTGGACTTGTCAAA
GGAGTAACAAGAAAATACAAGATTAAAAGCAATCCTCTCACAAAAGACATTGTTATAAAAATGATTCC
GAATGTGTCGAACATGTCTCAGTGCACAGGGAGTGTCATGGAAAATTATAAAACACGATTAAACGGTA
TCTTAACACCTATAAAGGGAGCGTTAGAGATCTACAAAAACAACACTCATGACCTTGTCGGTGATGTG
AGATTAGCCGGAGTTATAATGGCAGGAGTTGCTATTGGGATTGCAACCGCAGCTCAAATCACTGCAGG
TGTAGCACTATATGAGGCAATGAAGAATGCTGACAACATCAACAAACTCAAAAGCAGCATTGAATCAA
CTAATGAAGCTGTCGTTAAACTTCAAGAGACTGCAGAAAAGACAGTCTATGTGCTGACTGCTCTACAG
GATTACATTAATACTAATTTAGTACCGACAATTGACAAGATAAGCTGCAAACAGACAGAACTCTCACT
AGATCTGGCATTATCAAAGTACCTCTCTGATTTGCTTTTTGTATTTGGCCCCAACCTTCAAGACCCAG
TTTCTAATTCAATGACTATACAGGCTATATCTCAGGCATTCGGTGGAAATTATGAAACACTGCTAAGA
ACATTGGGTTACGCTACAGAAGACTTTGATGATCTTCTAGAAAGTGACAGCATAACAGGTCAAATCAT
CTATGTTGATCTAAGTAGCTACTATATAATTGTCAGGGTTTATTTTCCTATTCTGACTGAAATTCAAC
AGGCCTATATCCAAGAGTTGTTACCAGTGAGCTTCAACAATGATAATTCAGAATGGATCAGTATTGTC
CCAAATTTCATATTGGTAAGGAATACATTAATATCAAATATAGAGATTGGATTTTGCCTAATTACAAA
GAGGAGCGTGATCTGCAACCAAGATTATGCCACACCTATGACCAACAACATGAGAGAATGTTTAACGG
GATCGACTGAGAAGTGTCCTCGAGAGCTGGTTGTTTCATCACATGTTCCCAGATTTGCACTATCTAAC
GGGGTTCTGTTTGCCAATTGCATAAGTGTTACATGTCAGTGTCAAACAACAGGCAGGGCAATCTCACA
ATCAGGAGAACAAACTCTGCTGATGATTGACAACACCACCTGTCCTACAGCCGTACTCGGTAATGTGA
TTATCAGCTTAGGGAAATATCTGGGGTCAGTAAATTATAATTCTGAAGGCATTGCTATCGGTCCTCCA
GTCTTTACAGATAAAGTTGATATATCAAGTCAGATATCCAGCATGAATCAGTCCTTACAACAGTCTAA
GGACTATATCAAAGAGGCTCAACGACTCCTTGATACTGTTAATCCATCATTAATAAGCATGTTGTCTA
TGATCATACTGTATGTATTATCGATCGCATCGTTGTGTATAGGGTTGATTACATTTATCAGTTTTATC
ATTGTTGAGAAAAAGAGAAACACCTACAGCAGATTAGAGGATAGGAGAGTCAGACCTACAAGCTATCC
GTACGACGTCCCGGATTACGCTTAG
MKKKTDNPTISKRGHNHSRGIKSRALLRETDNYSNGLIVENLVRNCHHPSKNNLNYTKTQKRDSTIPY
RVEERKGHYPKIKHLIDKSYKHIKRGKRRNGHNGNIITIILLILILMISECSVGILHYEKLSKIGLVK
GVTRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDV
RLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQ
DYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLR
TLGYATEDFDDLLESDSITGQIIYVDLSSYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIV
PNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSN
GVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPP
VFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFI
IVEKKRNTYSRLEDRRVRPTSYPYDVPDYA-
Appendix
- 148 -
9.1.13. batMuV F
Construct: full length F protein of BatPV/Epo_spe/AR1/DRC/2009
GenBank accession number: HQ660095.1 (full genome)
9.1.14. batMuV HN
Construct: full length HN protein of BatPV/Epo_spe/AR1/DRC/2009
9.1.15. hMuV F
Construct: full length F protein of a human MuV isolate
GenBank accession number: KM519599
Labelling: Reference: Krüger et al., 2014 submitted
9.1.16. hMuV HN
Construct: full length HN protein of a human MuV isolate
GenBank accession number: KM519600
Labelling: Reference: Krüger et al., 2014 submitted
- 149 -
Appendix
9.1.17. batMuV F1/hMuV F2
Construct: chimeric MuV F protein containing the batMuV F1 and the hMuV F2 subunit
Labelling: Nucleotide sequence:
ATGAAGGCTTTTTCAGTTACTTGCTTGGGCTTCGCAGTCTTTTCATCTTCTATATGTGTGAACATCAA
CATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCCCAAAGTTCAA
GCTCCTACATAGTGGTCAAACTTTTACCGAATATCCAACCCCCTGATGACAGCTGTGAATTTAAGAGT
ATAAGTCAATACAATAAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAGAATATAAACAATATTGC
ATCGCCCTCACCTGGGTCAAGACGTCACAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCGCTTG
GTGTTGCAACAGCAGCACAAGTAACTGCCGCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCA
ATAGCAGCGATGAAAAATTCAATACAGGCAACTAATCGGGCAGTATTTGAAGTGAAGGAAGGCACCCA
ACAGTTAGCCATAGCAGTACAAGCTATACAAGACCACATCAACACTATTATGAACACCCAATTGAACA
ATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTYCCTAGGATTATACCTAACAGAATTAACA
ACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTAYACAAGCCTTGAGGTCCTT
GCTTGGGAGTATGACTCCCGCAGTGGTTCAAGCAGCATTATCTACTGCAATTTCTGCTGCTGAAATAC
TTAGCGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAG
ATAAATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTT
TGTTAATAATCAGGAATCCATAATTCAATTGCCAGACAGGATCTTAGAGATCGGGAATGAACAATGGA
GCTATCCAGCTAAGAATTGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGG
CTGAGCCTAGAATCAAAACTATGCCTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCTATAGCAGG
GAGTTATATGAGGCGATTTGTAGCACTGGATGGAACAATTGTTGCAAACTGTCGAAGCCTAACGTGTC
TATGCAAAAGTCCATCTTATCCTATATACCAACCTGACCATCATGCGGTCACGACCATTGATCTAACC
GCGTGTCAAACATTGTCCCTAGACGGATTGGACTTTAGCATTGTCTCTCTAAGCAACATCACTTACGC
TGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGACATATCAACTGAACTGA
GTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAACTCCAATCC
GTGAGTGTAAATTCCAAAATCGGAGCTATAATTGTAGCAGCTTTAGTCTTGAGCATCCTGTCAATTAT
CATTTCACTATTATTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGGATCAACTTCAAAA
CAAATCATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAA
MKAFSVTCLGFAVFSSSICVNINILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPPDDSCEFKS
ISQYNKTLSNLLLPIAENINNIASPSPGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARA
IAAMKNSIQATNRAVFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELT
TVFQPQLINPALSPISTQALRSLLGSMTPAVVQAALSTAISAAEILSAGLMEGQIVSVLLDEMQMIVK
INIPTIVTQSNALVIDFYSISSFVNNQESIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAER
LSLESKLCLAGNISACVFSPIAGSYMRRFVALDGTIVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLT
ACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDISTELSKVNASLQNAVKYIKESNHQLQS
VSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHINTISSSVDDLIRY-
Appendix
- 150 -
9.1.18. hMuV F1/ batMuV F2
Construct: chimeric MuV F protein containing the hMuV F1 and the batMuV F2 subunit
ATGAGAAAAACTCTAGCTATTGGTTTGGGTTTTATGATCTTTTCATTATCAGTATGTATAAATATCAA
TACTCTACAACAAGTTGGTTATATTAAACAGCAGGTTAGACAATTAAGCTACTATTCACAGAGTTCAA
GTGCATATATTGTGGTTAAGCTTCTACCAAATATCAAGCCAAGTAATGGGAATTGTGAATTTAATAGT
GTAGCCCAATACAACAAGACATTGAGTAACCTACTTCTTCCAATCGCAGAGAATATTAACAGCATAGC
ACCGCCCTCATCAAGTAGTCGGCGCAGGAAAAGGTTTGCAGGTATTGCTATCGGAATTGCTGCCCTTG
GCGTCGCAACTGCAGCACAAGTTACAGCTGCAGTTTCATTGGTGCAAGCCCAAACGAATGCACGTGCA
ATTGCAACGCTTAAGAATTCTATTCAGGCCACTAACAAAGCAATTTTTGAAGTGAAAGAAGGAACTCA
ACAGCTAGCCATTGCAGTACAAGCAATTCAAGATCACATCAATACAATCATGAATACACAATTAAACA
ACATGTCTTGTCAAATTCTCGATAATCAGCTTGCAACATCGTTAGGATTGTATTTAACAGAACTAACA
ACAGTTTTCCAACCTCAATTAGTTAATCCGGCACTATCACCTATCAGCATTCAAGCTTTAAGATCTTT
GCTAGGCAGTATGACACCTGCAGTGGTTCAAGCGGCACTATCAACTTCGATATCTGCTGCAGAGATTT
TGAGTGCCGGCCTGATGGAAGGTCAGATAGTTGCAGTTTTTTTAGATCAAATGCAAATGATAGTTAAA
GTTAATGTTCCGACTATCGTTACTCAATCTAATGCACTAGTAATAGATTTCCACTCCATATCAAGCTT
TATCAATAATCAGGAGTCTTTGATCCAGTTACCTAGCAGAATTTTAGAAATCGGAAGTGAGCAATGGA
GTTATCCGGCTGCTAACTGCAAGTTGACAAGAAATCATATATTCTGTCAATATAATGAAGCAGAGATA
ATGAGTCTAGAGTCAAAGCAATGCTTAGCTGGCAATATTAGTGCATGTGTGTTCACACCTATAGCAGG
AAGTTATATGAGGAGGTTTGTTGCATTGGATGGAACTATTGTTGCAAATTGCCGGAGTCTCACATGCC
TTTGCAAAGAACCATCTTATCCTATCTATCAGCCCGACCATCATGCAGTCACAACAATTGATTTAACA
ATATGCAAAACATTGTCTCTGGATGGATTAGACTTCAGTATTGTCTCATTAAGCAACGTTACTTATGC
TGAGAATTTAACAATTTCATTATCTCAAACAATCAATACTCAACCTATAGATATATCAACTGAGCTTA
GTAGGGTCAACGCATCTCTCCAAAATGCTGTCAATTACATAAAGGAGAGCAATAATCAATTGCAATCA
GTCAGTGTTAATTCAAAGTTAGGAGCTATAGTAGTGGCATCATTAGTGCTTAGCATATTATCAATTAT
CATTTCTCTCTTGTTTTGTTGCTGGGCTTATATAGCTACTAAAGAGATTAGACGAATTAATTTCAGAA
CTAATCATATTAACACTGTATCAAGCAGTGTTGATGACCTTGTGAGATACTAA
MRKTLAIGLGFMIFSLSVCININTLQQVGYIKQQVRQLSYYSQSSSAYIVVKLLPNIKPSNGNCEFNS
VAQYNKTLSNLLLPIAENINSIAPPSSSSRRRKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARA
IATLKNSIQATNKAIFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELT
TVFQPQLVNPALSPISIQALRSLLGSMTPAVVQAALSTSISAAEILSAGLMEGQIVAVFLDQMQMIVK
VNVPTIVTQSNALVIDFHSISSFINNQESLIQLPSRILEIGSEQWSYPAANCKLTRNHIFCQYNEAEI
MSLESKQCLAGNISACVFTPIAGSYMRRFVALDGTIVANCRSLTCLCKEPSYPIYQPDHHAVTTIDLT
ICKTLSLDGLDFSIVSLSNVTYAENLTISLSQTINTQPIDISTELSRVNASLQNAVNYIKESNNQLQS
VSVNSKLGAIVVASLVLSILSIIISLLFCCWAYIATKEIRRINFRTNHINTVSSSVDDLVRY-
- 151 -
Appendix
9.1.19. batMuV F (SP)
Construct: chimeric MuV F protein containing aa residues 1 - 20 of batMuV F (predicted
batMuV F signal peptide) and aa residues 21 - 538 of hMuV F
Labelling: -
ATGAGAAAAACTCTAGCTATTGGTTTGGGTTTTATGATCTTTTCATTATCAGTATGTATAAACATCAA
CATCTTGCAGCAAATTGGATATATCAAGCAACAAGTCAGGCAACTAAGCTATTACTCCCAAAGTTCAA
GCTCCTACATAGTGGTCAAACTTTTACCGAATATCCAACCCCCTGATGACAGCTGTGAATTTAAGAGT
ATAAGTCAATACAATAAGACCTTGAGTAATTTGCTTCTTCCAATTGCAGAGAATATAAACAATATTGC
ATCGCCCTCACCTGGGTCAAGACGTCACAAAAGGTTTGCTGGCATTGCCATTGGCATTGCTGCGCTTG
GTGTTGCAACAGCAGCACAAGTAACTGCCGCTGTCTCATTAGTTCAAGCACAGACAAATGCACGTGCA
ATAGCAGCGATGAAAAATTCAATACAGGCAACTAATCGGGCAGTATTTGAAGTGAAGGAAGGCACCCA
ACAGTTAGCCATAGCAGTACAAGCTATACAAGACCACATCAACACTATTATGAACACCCAATTGAACA
ATATGTCTTGTCAGATCCTTGATAACCAGCTTGCAACTTYCCTAGGATTATACCTAACAGAATTAACA
ACAGTGTTTCAGCCACAATTAATTAATCCGGCATTGTCACCGATTAGTAYACAAGCCTTGAGGTCCTT
GCTTGGGAGTATGACTCCCGCAGTGGTTCAAGCAGCATTATCTACTGCAATTTCTGCTGCTGAAATAC
TTAGCGCCGGTCTAATGGAGGGTCAGATTGTTTCTGTTCTGCTAGATGAGATGCAGATGATAGTTAAG
ATAAATATTCCAACCATTGTCACACAATCAAATGCATTGGTGATTGACTTCTACTCAATTTCGAGCTT
TGTTAATAATCAGGAATCCATAATTCAATTGCCAGACAGGATCTTAGAGATCGGGAATGAACAATGGA
GCTATCCAGCTAAGAATTGTAAGTTGACAAGACACCACATATTCTGCCAATACAATGAGGCAGAGAGG
CTGAGCCTAGAATCAAAACTATGCCTTGCAGGCAATATAAGTGCCTGTGTGTTCTCACCTATAGCAGG
GAGTTATATGAGGCGATTTGTAGCACTGGATGGAACAATTGTTGCAAACTGTCGAAGCCTAACGTGTC
TATGCAAAAGTCCATCTTATCCTATATACCAACCTGACCATCATGCGGTCACGACCATTGATCTAACC
GCGTGTCAAACATTGTCCCTAGACGGATTGGACTTTAGCATTGTCTCTCTAAGCAACATCACTTACGC
TGAGAACCTTACCATTTCATTGTCTCAGACAATCAATACTCAACCCATTGACATATCAACTGAACTGA
GTAAGGTTAATGCATCCCTCCAAAATGCCGTTAAGTACATAAAGGAGAGCAACCATCAACTCCAATCC
GTGAGTGTAAATTCCAAAATCGGAGCTATAATTGTAGCAGCTTTAGTCTTGAGCATCCTGTCAATTAT
CATTTCACTATTATTTTGCTGCTGGGCTTACATTGCAACTAAAGAAATCAGAAGGATCAACTTCAAAA
CAAATCATATCAACACAATATCAAGTAGTGTCGATGATCTCATCAGGTACTAA
MRKTLAIGLGFMIFSLSVCININILQQIGYIKQQVRQLSYYSQSSSSYIVVKLLPNIQPPDDSCEFKS
ISQYNKTLSNLLLPIAENINNIASPSPGSRRHKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARA
IAAMKNSIQATNRAVFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATFLGLYLTELT
TVFQPQLINPALSPISTQALRSLLGSMTPAVVQAALSTAISAAEILSAGLMEGQIVSVLLDEMQMIVK
INIPTIVTQSNALVIDFYSISSFVNNQESIIQLPDRILEIGNEQWSYPAKNCKLTRHHIFCQYNEAER
LSLESKLCLAGNISACVFSPIAGSYMRRFVALDGTIVANCRSLTCLCKSPSYPIYQPDHHAVTTIDLT
ACQTLSLDGLDFSIVSLSNITYAENLTISLSQTINTQPIDISTELSKVNASLQNAVKYIKESNHQLQS
VSVNSKIGAIIVAALVLSILSIIISLLFCCWAYIATKEIRRINFKTNHINTISSSVDDLIRY-
Appendix
- 152 -
9.1.20. hMuV F (SP)
Construct: chimeric MuV F protein containing aa residues 1 - 20 of hMuV F (predicted
hMuV F signal peptide) and aa residues 21 - 538 of batMuV F
ATGAAGGCTTTTTCAGTTACTTGCTTGGGCTTCGCAGTCTTTTCATCTTCTATATGTGTGAATATCAA
TACTCTACAACAAGTTGGTTATATTAAACAGCAGGTTAGACAATTAAGCTACTATTCACAGAGTTCAA
GTGCATATATTGTGGTTAAGCTTCTACCAAATATCAAGCCAAGTAATGGGAATTGTGAATTTAATAGT
GTAGCCCAATACAACAAGACATTGAGTAACCTACTTCTTCCAATCGCAGAGAATATTAACAGCATAGC
ACCGCCCTCATCAAGTAGTCGGCGCAGGAAAAGGTTTGCAGGTATTGCTATCGGAATTGCTGCCCTTG
GCGTCGCAACTGCAGCACAAGTTACAGCTGCAGTTTCATTGGTGCAAGCCCAAACGAATGCACGTGCA
ATTGCAACGCTTAAGAATTCTATTCAGGCCACTAACAAAGCAATTTTTGAAGTGAAAGAAGGAACTCA
ACAGCTAGCCATTGCAGTACAAGCAATTCAAGATCACATCAATACAATCATGAATACACAATTAAACA
ACATGTCTTGTCAAATTCTCGATAATCAGCTTGCAACATCGTTAGGATTGTATTTAACAGAACTAACA
ACAGTTTTCCAACCTCAATTAGTTAATCCGGCACTATCACCTATCAGCATTCAAGCTTTAAGATCTTT
GCTAGGCAGTATGACACCTGCAGTGGTTCAAGCGGCACTATCAACTTCGATATCTGCTGCAGAGATTT
TGAGTGCCGGCCTGATGGAAGGTCAGATAGTTGCAGTTTTTTTAGATCAAATGCAAATGATAGTTAAA
GTTAATGTTCCGACTATCGTTACTCAATCTAATGCACTAGTAATAGATTTCCACTCCATATCAAGCTT
TATCAATAATCAGGAGTCTTTGATCCAGTTACCTAGCAGAATTTTAGAAATCGGAAGTGAGCAATGGA
GTTATCCGGCTGCTAACTGCAAGTTGACAAGAAATCATATATTCTGTCAATATAATGAAGCAGAGATA
ATGAGTCTAGAGTCAAAGCAATGCTTAGCTGGCAATATTAGTGCATGTGTGTTCACACCTATAGCAGG
AAGTTATATGAGGAGGTTTGTTGCATTGGATGGAACTATTGTTGCAAATTGCCGGAGTCTCACATGCC
TTTGCAAAGAACCATCTTATCCTATCTATCAGCCCGACCATCATGCAGTCACAACAATTGATTTAACA
ATATGCAAAACATTGTCTCTGGATGGATTAGACTTCAGTATTGTCTCATTAAGCAACGTTACTTATGC
TGAGAATTTAACAATTTCATTATCTCAAACAATCAATACTCAACCTATAGATATATCAACTGAGCTTA
GTAGGGTCAACGCATCTCTCCAAAATGCTGTCAATTACATAAAGGAGAGCAATAATCAATTGCAATCA
GTCAGTGTTAATTCAAAGTTAGGAGCTATAGTAGTGGCATCATTAGTGCTTAGCATATTATCAATTAT
CATTTCTCTCTTGTTTTGTTGCTGGGCTTATATAGCTACTAAAGAGATTAGACGAATTAATTTCAGAA
CTAATCATATTAACACTGTATCAAGCAGTGTTGATGACCTTGTGAGATACTAA
MKAFSVTCLGFAVFSSSICVNINTLQQVGYIKQQVRQLSYYSQSSSAYIVVKLLPNIKPSNGNCEFNS
VAQYNKTLSNLLLPIAENINSIAPPSSSSRRRKRFAGIAIGIAALGVATAAQVTAAVSLVQAQTNARA
IATLKNSIQATNKAIFEVKEGTQQLAIAVQAIQDHINTIMNTQLNNMSCQILDNQLATSLGLYLTELT
TVFQPQLVNPALSPISIQALRSLLGSMTPAVVQAALSTSISAAEILSAGLMEGQIVAVFLDQMQMIVK
VNVPTIVTQSNALVIDFHSISSFINNQESLIQLPSRILEIGSEQWSYPAANCKLTRNHIFCQYNEAEI
MSLESKQCLAGNISACVFTPIAGSYMRRFVALDGTIVANCRSLTCLCKEPSYPIYQPDHHAVTTIDLT
ICKTLSLDGLDFSIVSLSNVTYAENLTISLSQTINTQPIDISTELSRVNASLQNAVNYIKESNNQLQS
VSVNSKLGAIVVASLVLSILSIIISLLFCCWAYIATKEIRRINFRTNHINTVSSSVDDLVRY-
- 153 -
Appendix
9.2. Supplementary data
Fig. 12: Phylogenetic relatedness of batMuV and the MuV genotypes based on the SH
sequence.
Reference strains: A1: MuVi/Boston.USA/0.45 [A]; A2: MuVi/Pennsylvannia.USA/1363 [A] (VAC);
B1: MuVi/Urabe.JPN/0.67[B]; B2: MuVi/Himeji.JPN/24.00[B]; C1: MuVi/Zagreb.HRV/39.98[C];
C2: MuVi/Stockholm.SWE/46.84[C]; D1: MuVi/Ge9.DEU/0.77[D]; D2: MuVi/Nottingham.GBR
/19.04[D]; F1: MuVi/Shandong.CHN/4.05[F]; F2: MuVi/Zhejiang.CHN/11.06/1[F]; G1: MuVi/
Gloucester.GBR/32.96[G]; G2: MuVi/Sheffield.GBR/1.05[G]; H1: MuVi/Bedford.GBR/0.89[H]; H2:
MuVi/Ulaanbaatar.MNG/22.09 [H]; I1: MuVi/Akita.JPN/42.93[I]; I2: MuVi/Dg1062.KOR/46.98[I];
J1: MuVi/Leeds.GBR/ 9.04[J]; J2: MuVi/ Sapporo.JPN/12.00[J]; K1: MuVi/RW154.USA/0.70s[K];
K2: MuVi/Stockholm.SWE/26.83[K]; L1: MuVi/Fukuoka.JPN/41.00 [L]; L2: MuVi/Tokyo.JPN/
6.01[L]; N1: MuVi/Vector.RUS/0.53[N](VAC); N2: MuVi/ LZagreb .HRV/0.71[N](VAC); U1: MuVi/
Taylor.GBR/0.50s; U2: MuVi/Tokyo.JPN/0.93; U3: MuVi/London.GBR/3.02
Appendix
- 154 -
9.3. Affidavit
I herewith declare that I autonomously carried out the PhD-thesis entitled
“Interaction of bat-derived paramyxoviruses with chiropteran and non-chiropteran cells:
Functional characterization of the African henipavirus and bat-derived mumps virus fusion
and attachment glycoproteins”.
No third party assistance has been used.
I did not receive any assistance in return for payment by consulting agencies or any other
person. No one received any kind of payment for direct or indirect assistance in correlation to
the content of the submitted thesis.
I conducted the project at the following institution:
University of Veterinary Medicine Hannover, Foundation
Bünteweg 17
30559 Hannover.
The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation in a
similar context.
I hereby affirm the above statements to be complete and true to the best of my knowledge.
31.08.2014, Nadine Krüger

Interaction of bat-derived paramyxoviruses with chiropteran and non

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