Competitive ELISAs confirm that equine arteritis virus-infected horses develop antibodies to the M viral envelope protein [Elektronische Ressource] / Heike M. Wagner

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2004
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Competitive ELISAs confirm that equine arteritis virus-infected horses
develop antibodies to the M viral envelope protein





INAUGURAL-DISSERTATION
zur Erlangung des Doktorgrades beim
Fachbereich der Veterinärmedizin
der Justus-Liebig-Universität Gie βen




Heike M. Wagner




Aus dem Department of Pathology, Microbiology and Immunology
der School of Veterinary Medicine,
University of California, Davis
Betreuer: Prof. N.James MacLachlan, BVsc, PhD. DipACVP



Eingereicht über das Institut für Virologie
der Justus-Liebig-Universität Gießen
im Fachbereich vertreten durch: Prof. Dr. Heinz-Jürgen Thiel




Competitive ELISAs confirm that equine arteritis virus-infected horses
develop antibodies to the M viral envelope protein



INAUGURAL-DISSERTATION
zur Erlangung des Doktorgrades beim
Fachbereich der Veterinärmedizin
der Justus-Liebig-Universität Gießen



Eingereicht von
Heike M. Wagner
Tierärztin aus Brühl (NRW)

Gießen 2004











Mit Genehmigung des Fachbereiches der Veterinärmedizin
der Justus-Liebig-Universität Gießen





Dekan: Prof. Dr. Manfred Reinacher




1. Berichterstatter: Prof. Dr. James MacLachlan
2. Berichterstatter: Prof. Dr. Heinz-Jürgen Thiel

Tag der mündlichen Prüfung: 20.10.2004



Ein Teil dieser Arbeit ist in folgende Veröffentlichung mit eingegangen:

Wagner, H.M., Balasuriya, U.B.R. and N.J. MacLachlan. 2003.
The serologic response of horses to equine arteritis virus as determined by competitive
enzyme-linked immunosorbent assays (c-ELISA) to structural and non-structural viral
proteins; CIMID. 26(4)251-260





In memory of
Susan Ellenor Haines
(23.03.1968-18.01.2004)

“You better stand tall
when they are calling you out,
don’t bend, don’t break;
Baby don’t back down.”
(John Bon Jovi)
Table of contents


Table of contents

1. Abbreviations 1
2. Introduction 3
3. Literature review 5
3.1. Equine arteritis virus 5
3.1.1. Classification 5
3.1.2. Morphology and physicochemical properties 6
3.1.3. Growth in cell culture 7
3.1.4. Molecular biology 8
3.1.5. Structural proteins 13
3.1.6. Neutralization determinants of the virus 15
3.1.7. Genetic and phenotypic variation 16
18 3.2. Clinical signs of an EAV infection
3.3. Pathogenesis and pathology of EAV infection of horses 21
3.4. Equine immune response to an infection with EAV 23
3.5. Epidemiology 24
3.6. Diagnostics 29
3.7. Prevention and control 33
4. Materials 35
4.1. Cell lines 35
4.2. Media and media supplement for cell culture 35
4.3. Buffers and solutions 36
4.4. Enzymes other proteins 40
4.5. Antibodies and antisera 40
4.6. Equine sera 41
4.7. Chemicals 41
4.8. Equipment 42
4.9. Other materials 42
5. Methods 43
5.1. Cell culture maintenance 43
Table of contents


5.1.1. Propagation of continuous cell lines (RK-13, BHK-21) 43
5.1.2. Counting cells 43
5.1.3. Freezing 44
5.1.4. Recovery of frozen cells 44
5.2. Cell culture and virology techniques 44
5.2.1. Infection of RK-13 and BHK-21 cells with EAV
5.2.2. Production of working virus stock 45
5.2.3. Plaque purification of EAV 45
5.2.4. Serum neutralization 46
5.3. Production and characterization of monoclonal antibodies 46
5.3.1. Collection of blood and separation of serum from blood 46
5.3.2. Immunization of mice 47
5.3.3. Thawing of P3x63Ag.8.653 cells 47
47 5.3.4. Propagation of
5.3.5. Counting of P3x63Ag.8.653cells 48
5.3.6. Freezing P3x63Ag.8.653 cells 48
5.3.7. Harvesting of mouse spleen cells and fusion protocol 48
5.3.8. Propagation of hybrid cell line 49
5.3.9. Screening of hybrids for positive cells 49
5.3.10. Expansion of hybridoma cells 50
5.3.11. Cloning of hybridoma cell lines by limiting dilution 50
5.3.12. Production of ascitic fluid 51
5.3.13. Purification MAb 52
5.3.14. Mouse immunoglobulin isotyping ELISA 53
5.4. Molecular biology 54
5.4.1. Characterization of EAV expressed proteins 54
5.4.2. Immunofluorescense assay 55
5.4.3. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis of proteins 55
5.4.4. Western immunoblotting assay 57
5.4.5. d-ELISA 59
5.4.6. c-ELISA 60
Table of contents


6. Results 61
6.1. Prodcution of antibodies to nsp1, 2, 4 an the M protein of EAV 61
6.1.1. Production of MAbs to nsp1 of EAV 61
6.1.2. Production of antibodies to nsp2 of EAV 65
6.1.3. Production of antibodies to nsp4 of EAV 65
6.1.4. Production of MAbs to the M protein of EAV 66
6.2. Development of a d-ELISA using the nsp1 MAb 67
6.3. Screening of other protein specific MAbs with the d-ELISA 71
6.4. Development of a c-ELISA 75
7. Discussion 82
7.1. Production of MAbs to nsp1 and antibodies to nsp2 and 4 82
7.2. Production of MAbs to the M protein 83
7.3. Development of a d-ELISA using the nsp1 MAb 86
87 7.4. Screening of other EAV protein-specific MAbs with the d-ELISA
7.5. Evaluation of the c-ELISA 88
7.6 Conclusions 90
8. Summary 92
9. Zusamenfassung 93
10. References 94
11. Acknowledgments 122
Abbreviations


1. Abbreviations

aa amino acids
Ala alanine
Arg arginine
Asn asparagine
ATTC American Type Culture Collection
BALB/c “Bagg Albino” inbred mice strain
˚C degree Celsius
cDNA complementary deoxyribonucleic acid
c-ELISA competitive enzyme-linked immunosorbent assay
Cys cysteine
d-ELISA direct enzyme-linked immunosorbent assay
O distilled water dH2
EAV equine arteritis virus
EDTA ethylene-diamine tetra-acetic acid
ELISA enzyme linked immunobsorbent assay
EVA viral arteritis
g relative centrifugal force
Gln glutamine
Gly glycine
Gp glycoprotein
h hour
His histidine
IFA immunofluorescense assay
Ig immunoglobulin
Ile isoleucine
kDa kilodalton
l liter
Leu leucine
Lys lysine
- 1 - Abbreviations


M Mol
MAb monoclonal antibody
MEM minimal essential medium
Met ethionine
min minute(s)
ml milliliter
MLV modified live virus
M.O.I. multiplicity of infection
mRNA messenger ribonucleic acid
nsp non structural protein
OD optical density
OPD o-phenyldiamine
ORF open reading frame
PCR polymerase chain reaction
PEG polyethylene glycol
PFU plaque forming units
pH pondus hydrogenii (measurement of hydrogen ion concentration)
Phe phenylalanine
Pro proline
qs quantitate to
RNA ribonucleic acid
rpm rounds per minute
RT-PCR reverse transcription-polymerase chain reaction
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
Ser serine
sg subgenomic
T temperature
Thr threonine
TRS transcription regulating sequence
Tyr tyrosine
µl microliter
- 2 -