Table Of ContentEstablishment and characterization of three new
embryonic Spodoptera littoralis cell lines and testing their
susceptibility to SpliMNPV
Der Technischen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Grades
DOKTOR – INGENIEUR
vorgelegt von
Ibrahim Abdulla Ahmed, M.Sc.
aus Diyala, Irak
II
Etablierung und Charakterisierung von drei Zelllinien aus
Spodoptera littoralis Embryonen und Überprüfung deren
Infizierbarkeit mit dem Baculovirus SpliMNPV
Der Technischen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Grades
DOKTOR – INGENIEUR
vorgelegt von
Ibrahim Abdulla Ahmed, M.Sc.
aus Diyala, Irak
III
Als Dissertation genehmigt
von der Technischen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der müdlichen Prüfung: 25.02.2015
Vorsitzende des Promotionsorgans: Prof. Dr.-Ing. habil. Marion Merklein
Gutachter: Prof. Dr. Rainer Buchholz
Prof. Dr. Thomas Brück
Acknowledgements
I would like to express my sincere gratitude to my supervisor Prof. Dr. Rainer Buchholz for
giving me the opportunity to do this work at his chair and also for his unlimited support and
guidance. I also would like to express my sincere gratitude to my second supervisor Dr. Ing.
Holger Huebner for his encouragement and guidance.
Also, I express my gratitude to Dr. Andreas Perlick for his help. I especially would like to
thank: Anette Amtmann, Dr. Anna Becker, Petra Klemenz and Elke Heidenreich for their
scientifically coopration.
I also would like to thank all my collegues and the staff members of institute of bioprocess
engineering especially: Tobias Weidner, Adam Mletzko, Kenny Zambrano, Mathias Stach,
Björn Sommerfeldt, Josef Taucher, Ricarda Friebe, Martin Heining, Konstantin Präbst,
Juliane Richter, Dr. Stephanie Stute, Dr. Roman Breiter, Philipp Schwerna, Jae-Young, Najah
al-Mhanna, Hannes Engelhardt, Ludwig Körber, Stefan Ringgeler, Bertram Geinitz and
Stefan Popov.
I would like also to thank Ingrid Callies, Katja Steinbach and Maria Rehberger.
I thank Dr. Roland Reist and Mr. Oliver Kindler (Syngenta Crop Protection, Switzerland)
for providing S. littoralis eggs and Dr. David Grzywacz (University of Greenwich, UK)
for providing S. littoralis NPV isolates.
I would also like to thank the German Academic Exchange Service (DAAD) and the
Ministry of Higher Education in Iraq for providing personal financial stipend that helped
make this project possible. Special thanks are attributed to Prof. Dr. Cynthia Goodman
(USDA/ARS/BCIRL, USA) and Prof. Elizabeth Didier (Tulane University/USA) and Dr.
Daniel Gilbert (Medical Biotechnology Institute/ Erlangen University).
I thank also Prof. Dr. Bernhard Fleckenstein and Dr. Klaus Korn (Molecular virology
Institute/ Erlangen) for the assistance in qPCR experiments.
I would like to express my respect and sincere gratitude to my wife Nisreen and my family
and my friends in Iraq for their encouragement and support.
List of contents
Abstract ...................................................................................................................................... 1
Zusammenfassung ...................................................................................................................... 3
1. Introduction .................................................................................................................... 5
2. Literature review ............................................................................................................ 7
2.1 Pesticides ....................................................................................................................... 7
2.1.1 Chemical pesticides ................................................................................................ 7
2.1.2 Harmful effects of chemical pesticides .................................................................. 8
2.1.3 Biopesticides .......................................................................................................... 8
2.2 Baculoviruses ............................................................................................................. 9
2.2.1 Nomenclature and classification ............................................................................ 9
2.2.2 Baculovirus life cycle ........................................................................................... 11
2.2.3 S. littoralis multiple nuclear polyhedrosis virus (SpliMNPV) ............................. 14
2.3 Cotton leaf worm S. littoralis ................................................................................... 15
2.3.1 Nomenclature and taxonomy ............................................................................... 15
2.3.2 Host range ............................................................................................................ 15
2.3.3 Distribution ........................................................................................................... 15
2.3.4 Life history ........................................................................................................... 16
2.3.5 Pest control ........................................................................................................... 16
2.4 Development of insect cell lines .............................................................................. 17
2.4.1 Primary culture ..................................................................................................... 17
2.4.2 Cell lines ............................................................................................................... 18
2.4.3 Cell line characterization ...................................................................................... 20
2.4.3.1 Cell morphology ........................................................................................... 20
2.4.3.2 Karyotyping .................................................................................................. 21
2.4.3.3 Isoenzyme profiles ....................................................................................... 21
2.4.3.4 DNA analysis ............................................................................................... 22
2.5 Insect cell culture ..................................................................................................... 23
2.5.1 Insect cell culture media ....................................................................................... 23
2.5.2 Nutritional requirements of insect cells ............................................................... 26
2.5.3 Insect cell culture conditions ................................................................................ 27
2.5.3.1 Effects of temperature .................................................................................. 27
II
2.5.3.2 Effects of pH ................................................................................................ 28
2.5.3.3 Effects of oxygen ......................................................................................... 28
2.5.3.4 Effects of carbon dioxide ............................................................................. 29
2.6 Insect cell culture applications ................................................................................. 29
2.6.1 Bio-insecticide production ................................................................................... 29
2.6.1.1 In vivo production ......................................................................................... 31
2.6.1.2 In vitro production ........................................................................................ 32
2.6.2 Recombinant proteins and vaccine production .................................................... 33
2.7 Large-scale production ............................................................................................. 34
2.8 Cell immobilization .................................................................................................. 37
2.8.1 Immobilization techniques ................................................................................... 37
2.8.1.1 Adsorption .................................................................................................... 37
2.8.1.2 Covalent binding .......................................................................................... 37
2.8.1.3 Entrapment ................................................................................................... 38
2.8.1.4 Encapsulation ............................................................................................... 38
2.8.1.5 Cross-linking ................................................................................................ 38
2.8.2 Insect cell immobilization .................................................................................... 40
2.9 Enhancing the insecticidal properties of baculoviruses ........................................... 40
3. Materials and methods ................................................................................................. 42
3.1 Equipment and Materials ......................................................................................... 42
3.1.1 Laboratory equipment used in this study ............................................................. 42
3.1.2 Chemicals and reagents ........................................................................................ 43
3.1.3 Insect cell lines ..................................................................................................... 44
3.1.4 Viral strains .......................................................................................................... 44
3.1.5 Kits ....................................................................................................................... 44
3.1.6 Protein markers .................................................................................................... 44
3.1.7 DNA markers ....................................................................................................... 45
3.1.8 List of solutions and buffers ................................................................................. 45
3.1.9 Insect cell media ................................................................................................... 49
3.1.10 Oligonucleotides ................................................................................................... 49
3.1.11 Software ............................................................................................................... 51
3.2 Methods .................................................................................................................... 51
3.2.1 Siliconization ........................................................................................................ 51
3.2.2 Establishment of cell lines .................................................................................... 51
3.2.2.1 Egg disinfection ............................................................................................ 51
3.2.2.2 Initiation of the primary culture ................................................................... 52
3.2.2.3 Cell subculture .............................................................................................. 52
III
3.2.3 Evaluation of cell growth using the MTT assay .................................................. 52
3.2.4 Cryopreservation .................................................................................................. 53
3.2.4.1 Cell freezing ................................................................................................. 53
3.2.4.2 Cells thawing ................................................................................................ 54
3.2.5 Preparation of insect cell culture medium ............................................................ 54
3.2.6 Determination of cell count and viability ............................................................. 55
3.2.7 Heat inactivation of fetal bovine serum ............................................................... 55
3.2.8 Adaptation to serum-free medium ........................................................................ 55
3.2.9 Adaptation to suspension culture ......................................................................... 56
3.2.10 Live/Dead cell viability assay .............................................................................. 56
3.2.11 Cell line characterization ...................................................................................... 56
3.2.11.1 Cell growth curve ......................................................................................... 56
3.2.11.2 Determination of cell growth rate and cell population doubling time ......... 57
3.2.11.3 Cell size measurement .................................................................................. 58
3.2.11.4 DNA fingerprinting ...................................................................................... 59
3.2.11.5 Isoenzyme analysis ....................................................................................... 62
3.2.12 Virus stock preparation ........................................................................................ 66
3.2.12.1 Preparation of virus inoculums .................................................................... 66
3.2.12.2 Large working virus stock ............................................................................ 66
3.2.13 Virus quantification .............................................................................................. 67
3.2.13.1 End point dilution ......................................................................................... 67
3.2.13.2 Plaque assay ................................................................................................. 68
3.2.14 Virus storage ........................................................................................................ 69
3.2.15 SpliMNPV DNA fingerprint ................................................................................ 69
3.2.15.1 DNA extraction from the budded virus ........................................................ 69
3.2.15.2 DNA fingerprint ........................................................................................... 70
3.2.15.3 Polyhedrin gene sequencing ......................................................................... 70
3.2.16 Testing the susceptibility of the S. littoralis cell lines to SpliMNPV and
AcMNPV .......................................................................................................................... 71
3.2.17 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ........ 71
3.2.17.1 Sample preparation ....................................................................................... 71
3.2.17.2 SDS electrophoresis protocol ....................................................................... 71
3.2.17.3 Coomassie blue staining ............................................................................... 73
3.2.17.4 Silver staining ............................................................................................... 73
3.2.18 DNA Fragmentation Assay .................................................................................. 73
3.2.19 Viral polyhedra purification and quantification ................................................... 74
3.2.20 Comparing SpliMNPV OBs production in three S. littoralis cell lines ............... 74
3.2.21 SpliMNPV DNA replication in the three S. littoralis cell lines ........................... 75
3.2.21.1 DNA extraction from budded virus .............................................................. 76
3.2.21.2 Design of primers and probes ....................................................................... 76
3.2.21.3 Real time PCR standard curve ..................................................................... 78
IV
3.2.21.4 QPCR Procedure .......................................................................................... 79
3.2.22 SpliMNPV polyhedrin gene expression ............................................................... 79
3.2.22.1 Total RNA extraction ................................................................................... 79
3.2.22.2 RT-qPCR protocol ........................................................................................ 79
3.2.23 S. littoralis cell encapsulation .............................................................................. 80
3.2.23.1 Encapsulation procedure .............................................................................. 80
3.2.23.2 Determination of cell density in capsules .................................................... 81
4. Results .......................................................................................................................... 82
4.1 Establishment of cell lines ........................................................................................ 82
4.1.1 Disinfection .......................................................................................................... 82
4.1.2 Initiation and maintenance of the primary culture ............................................... 83
4.2 Cell subculture .......................................................................................................... 87
4.3 Cryopreservation and recovery ................................................................................ 89
4.4 Cell growth curve ..................................................................................................... 89
4.5 Cell morphology ....................................................................................................... 92
4.6 Minimum and maximum cell density ....................................................................... 93
4.7 DNA fingerprinting .................................................................................................. 94
4.8 Isoenzyme analysis ................................................................................................... 96
4.9 Adaptation to serum free medium ............................................................................ 97
4.10 SpliMNPV virus stock preparation .......................................................................... 98
4.11 SpliMNPV DNA fingerprint .................................................................................... 99
4.12 Polyhedrin gene sequencing ................................................................................... 100
4.13 Testing the susceptibility of the S. littoralis cell lines to SpliMNPV .................... 100
4.14 SDS-polyacrylamide gel electrophoresis ............................................................... 101
4.15 Infected and uninfected Spli-S cell size distribution .............................................. 102
4.16 Testing the susceptibility of the S. littoralis cell lines to AcMNPV ...................... 103
4.17 AcMNPV and SpliMNPV viral titer ...................................................................... 108
4.18 DNA Fragmentation Assay .................................................................................... 109
4.19 Comparing SpliMNPV OBs production in S. littoralis cell lines .......................... 109
4.20 SDS-PAGE electrophoresis of purified occlusion bodies (OBs) ........................... 112
4.21 SpliMNPV DNA replication in the three S. littoralis cell lines ............................. 113
4.22 SpliMNPV polyhedrin gene expression in the three cell lines ............................... 115
4.23 S. littoralis cell encapsulation ................................................................................ 116
5. Error discussion .......................................................................................................... 120
5.1 Laboratory instruments .......................................................................................... 120
V Abstract
5.2 Determining cell density ........................................................................................ 120
5.3 Viral occlusion bodies (OB) counting .................................................................... 121
5.4 DNA fingerprinting ................................................................................................ 121
5.5 Determining cell density in capsules ...................................................................... 122
5.6 Virus titer ................................................................................................................ 123
5.7 Viral DNA replication ............................................................................................ 123
6. Discussion .................................................................................................................. 124
6.1 Initiation of primary cell culture ............................................................................ 124
6.2 Cell subculture and growth .................................................................................... 126
6.3 Isozyme analysis .................................................................................................... 129
6.4 DNA fingerprinting ................................................................................................ 130
6.5 Testing the susceptibility of the S. littoralis cell lines to AcMNPV ...................... 133
6.6 SpliMNPV DNA fingerprinting and polyhedrin gene sequencing ........................ 136
6.7 Testing the susceptibility of the S. littoralis cell lines to SpliMNPV .................... 137
6.8 SDS-PAGE analysis ............................................................................................... 137
6.9 Comparsion of SpliMNPV OBs production in the S. littoralis cell lines .............. 138
6.10 SpliMNPV DNA replication and polyhedrin gene transcription ........................... 139
6.11 S. littoralis cell encapsulation ................................................................................ 141
6.12 Future aspects ......................................................................................................... 142
7. References .................................................................................................................. 145
8. List of abbreviations ................................................................................................... 168
9. List of Figures ............................................................................................................ 171
10. List of Tables .............................................................................................................. 174
11. Appendix .................................................................................................................... 175
1 Abstract
Abstract
Baculoviruses have a significant potential as biological pesticides. Spodoptera littoralis
multicapsid nucleopolyhedrovirus (SpliMNPV) could thus be applied to protect plants against
the African Cotton Leafworm. For the in vitro production of SpliMNPV a cellular system has
to be established. For this purpose three new continuous cell lines were established from
embryonic tissues of S. littoralis. These three cell lines were designated as Spli-C, Spli-S and
Spli-B. They consist mostly of spherical cells in addition to spindle and giant cells. The
population doubling time for Spli-C, Spli-S and Spli-B were 30.5, 31 and 44.5 h, respectively,
at passage 19, while at passage 120 it was 26, 27 and 32 h, respectively. The DNA
fingerprinting techniques RAPD and DAF confirmed that the cell lines originated from
S. littoralis tissues. Lactate dehydrogenase (LDH) isozyme analysis showed distinguishable
differences between the three new cell lines and the other insect cell lines used in our
laboratory.
Susceptibility tests of the three cell lines showed that all the cell lines were non-permissive to
Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), with the cells dying a
few hours post-infection and forming apoptosis-like bodies. Nuclear DNA fragmentation was
observed in all AcMNPV-infected cell lines by DNA gel electrophoresis analysis.
At same time, the susceptibility of the three cell lines to SpliMNPV showed that all the cell
lines were susceptible, whereby many viral occlusion bodies (OBs) were observed inside the
infected cells. The SpliMNPV OB productivity of the three cell lines showed significant
differences. The cell line Spli-C had the highest susceptibility to SpliMNPV compared to the
other two cell lines. The average of OB production was about 1x107 OBs/ml in the Spli-C cell
line, while in Spli-S and Spli-B it was 3.3x106 and 2x106 OBs/ml, respectively. In addition,
the SpliMNPV polyhedrin gene expression and DNA replication in the three cell lines were
also investigated over time using quantitative PCR (qPCR).
The Spli-C cell line that showed higher OB production than the other two cell lines was
immobilized using sodium cellulose sulfate (NaCS) and poly-diallyldimethylammonium
chloride (PDADMAC) capsules to protect cells from shear stress caused by agitation and gas
sparging during supply of sufficient oxygen. Cell culture in capsules leads to high cell
densities resulting in increased viral OB production per volume. The Spli-C cell densities
were increased from 4-5x106 cells/ml in suspension culture to 1.3x107 cells/ml in capsules.
Description:thank: Anette Amtmann, Dr. Anna Becker, Petra Klemenz and Elke Heidenreich for their scientifically coopration. I also would like to thank all my
