Antiviral Substances from Microalgae Applied in Aquacultures

174 Pages·2016·11.42 MB·English

by  Matthias Reichert

Antiviral Substances from Microalgae
Applied in Aquacultures
Anwendung von Antiviralen Substanzen aus
Mikroalgen in Aquakulturen
Der Technischen Fakultät
der Friedrich-Alexander-Universität
Erlangen-Nürnberg
zur
Erlangung des Doktorgrades
Doktor-Ingenieur (Dr.-Ing.)
vorgelegt von
Dipl.-Ing.
Matthias Reichert
aus Forchheim
Als Dissertation genehmigt
von der Technischen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 16.03.2016
Vorsitzender des Promotionsorgans Prof. Dr. Peter Greil
Gutachter: Prof. Dr. rer. nat. Rainer Buchholz
Prof. Dr. rer. nat. Dieter Steinhagen
Personal Foreword
Four years ago I listened to my heart and made a decision which changed my life. And honestly,
I could have never imagined how much this thesis did! In the very beginning, it was just a small
whisper inside of me and the curiosity to be part of something exciting - raising the first German
university in South Korea and doing extraordinary research abroad. But now it is clear, it became
somuchmore.Afterahardtimebackin2012,averyspecialpersonbelievedinmeandofferedme
the possibility to do to my doctoral degree in a research and development project in South Korea.
I remember, putting this news on a tiny letter under the Christmas tree with excitement inside me.
Mydadwasspeechlessandhadsometearsinhiseyes,mybrotherlookedupatmewithpride,and
my mum said:
"Matthias, why are you always setting your goals that high, we are also proud of you when you
are not living always on the edge?!"
I answered: "See mum, you know me better than I know myself, how else should I figure out how
far I can go?"
I guess this drive and ambition somehow – led me to survive the hard times abroad – sepa-
rated from the people I love, and persist through my thesis – even in the difficult situations, these
past years. Reflecting, this work was the most challenging – most difficult, but also the best I have
done in my life so far. Well, the latter you can scientifically judge by your own when reading it.
However, it is certain that I put in all my effort – knowledge – ideas – hard work and entire
heart into it. Despite the fact that I will never write a second doctoral thesis again, I still think
the sky is the limit and I hope I can encourage people not to take the easy way, to endure in the
hard times, to try again or different, to stay curious and go for their goals. This way of thinking to
solveproblems,taughtmesomuchaboutmyself,gavemesomanyopportunities,newexperiences,
different perspectives and all the tools needed for my personal and professional development.
It’s not science without your personal and emotional experiences, because this brings it to life on
the paper.
This thesis gives a resolution for a fundamental research problem of the treatment of viral
diseases in aquacultures, starting from the laboratory and then bringing it round to the effective
application of a finished product in animals.
Baiersdorf, January 2016 Matthias Reichert
Acknowledgments
To whom I am thankful for supporting and assisting me during almost four years of research at
the Institute of Bioprocess Engineering in Busan (South Korea) and about one year in Erlangen
(Germany):
This work was funded by KIAT, South Korea, which I greatly appreciate.
I would like to thank my "Doktorvater" Prof. Dr. Rainer Bucholz for his trust and confidence.
He offered me the opportunity to work on the present research project in Korea.
A very special acknowledgment goes to all stuff of the FAU Busan Campus, including the Insti-
tuteAdministrationinErlangen.Especially,Iwouldliketomention:Mr.Sung-HoBeak,Mr.Heejae
Yang,Mr.HuichihWang,Ms.KatharinaSommer,Mrs.IngridCalliesandMrs.KatjaSteinbach.With-
out their extraordinary measure of sacrifice covering administrative and scientific coordination,
controlling, human resources and finances on two different continents, the ship would have been
sunk.
I kindly thank Dr. Magdalena Stachnik, Department of Fish Diseases, National Veterinary Re-
search Institute in Pulawy (Poland), for providing the virus isolate KHV-P. Foremost, I thank Dr.
habil. Sven M. Bergmann, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health,
Greifswald,Germany,forconductingtheanimalexperiments.Hisenormousknowledgeandadvice
was certainly fundamental throughout parts of my work. I am truly thankful for his dedication.
I would like to express my gratitude to my students and project partners AmBio and DSU
(South Korea). They are, Ms. Yun-sun Song, Mr. Vilas Pujar, Mr. General Thiyam, Mr. Binod Prasad
and his wife Mrs. Nithya Vadakkedath who all contributed in conducting parts of the experiments.
Without their hard work, many of the present results would probably not have been achieved.
I have to extend my gratitude to my former colleagues at FAU Busan Campus, Mr. Marc
Williams, Dr. Soebiakto "Subi" Loekman and especially Ms. Sunhwa Jung and Mr. Joachim Hahm.
They helped in implementing and running the laboratories in Korea. Without their effort, this
research would not have been possible.
I cannot forget to thank also my former colleagues at the Institute of Bioprocess Engineering
in Erlangen. I thank Mr. Hannes Engelhardt for sharing the office with me in Germany and the
excellent scientific discussions. Further, I thank Mrs. Anette Amtmann for conducting virus
experiments, the shipment of cells to Busan, our fruitful discussions about cell technology, and
all that knowledge she gave me in this field. In particular, I thank Dr. Anna M. Becker for always
providing me highly valuable advice.
I also would like to mention the magnificent country of South Korea, I fell in love with it
and that became my second home. It gave me the opportunity to meet such precious people from
different nationalities around the world in a professional and personal manner. It made me to see
things from different perspectives, which are not limited to the strict German attitude of thinking.
Especially, I want to mention my friends that I met abroad and that brought so much joy into my
life. Here, want to name the Hadan group, and in particular Dr. Giovanni Luzi, Mr. Randy Lander,
Ms. Bridget Blahosky, and Mr. Brendan Shea and Mrs. Naomi Shea. They are just amazing people
and I am sure we would have been childhood friends, if we would have grown up in the same
country. We stole horses and we experienced unbelievable moments together, which I will never
forget. I always felt very connected to them, accepted, pushed, supported and loved like a family
member, especially in the hard times away from home. I have to extend my gratitude to my best
friend, Mr. Kai Rebhan. He always supported me as true best friend, gave me the feeling of home
during his visits in Switzerland and South Korea, while I was climbing the academic ladder. Every-
one contributed so much for my academic and personnel success because they made me stronger
as a person.
A very special acknowledgment goes to my fiancée and future wife, Ms. Mun Juong "MJ" Kim,
forherpatienceandconstantsupportduringthefinal,criticalandtoughmonthsofmydissertation.
She always encouraged me to work hard for my goals. She is, and she will always be my base of
strength and support throughout my life.
Words cannot express, no matter in which language, how deeply grateful I am to my whole
family, especially to my mother Brigitte Reichert, my father Fred Reichert and my brother Michael
"Bruderherz" Reichert. They are my biggest supporters, they always believe in me, they shaped my
life, taught me to listen to my heart and made me who I am today.
I also want to thank my grandfather Mr. Erich Krieg. He showed me the ideals of family and
truesportsmanship,whichmeanstoworkhardformyaims,theneedtostandupandtodealwith
the adversity and to learn from my mistakes. It is painful to think that these words of thanks have
against his promise no room anymore in his memory. This thesis work is dedicated to him.
Finally,Itrytoputmydeepestrespectandgratitudeformymentor,mysupervisor,myboss,my
formerroommate,mysurrogatefatherabroadandmyfriendDr.ChristophLindenbergerintowords.
Without him this thesis undoubtedly would be a non-existing empty piece of paper. His vision, his
trust,hisbelieveinme,hispatienceandhisenormousknowledgewasfundamentalthroughoutmy
doctoral degree and beyond. He contribute not only with excellent scientific discussion, insightful
comments and advice but also shaped me in leadership, as scientist and engineer. Written words
will not do justice for what he did for me but I am truly thankful from the bottom of my heart for
his selfless dedication to both my academic and personal development.
Contents
Abbreviations and Formula Symbols with Units iv
Abstract viii
Zusammenfassung x
1 Introduction 1
2 Rewiew of Literature 2
2.1 Worldwide Situation and Problem with Carp . . . . . . . . . . . . . . . . . . . . . . . 2
2.1.1 KHVD and Virus Spread . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1.2 KHV and Virus Replication Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.1.3 Detection and Diagnostic Methods of KHV . . . . . . . . . . . . . . . . . . . . . 10
2.1.4 Virus Containment Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2 Antiviral Standard Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.3 Importance of Microalgae as Antiviral Drug Source . . . . . . . . . . . . . . . . . . . . 13
3 Aims of the Study 15
4 Material and Methods 16
4.1 Microalgae Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.1.1 Identification of Native Microalgae Strains . . . . . . . . . . . . . . . . . . . . . 16
4.1.2 Cultivation of Microalgae and Isolated Fractions . . . . . . . . . . . . . . . . . . 18
4.1.3 Chemical Analysis of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4.2 CCB Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.2.1 CCB Cell Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.2.2 CCB Cell Growth Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4.3 KHV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.3.1 KHV Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.3.2 KHV Infection Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.3.3 KHV Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.3.4 KHV Titer Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.3.5 KHV Degree of Infection Estimation . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.3.6 KHV Infection Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4.3.7 Mathematical Treatment of the Data. . . . . . . . . . . . . . . . . . . . . . . . . 24
4.4 Antiviral Activity Assay of Isolated Fractions . . . . . . . . . . . . . . . . . . . . . . . . 24
4.4.1 Cytoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.4.2 Antiviral Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4.4.3 Infection Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Contents ii
4.4.4 Determination of IC /CC and SI . . . . . . . . . . . . . . . . . . . . . . . . . . 26
50 50
4.4.5 Mechanism and Site of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.5 Effects of BM and EPS on KHV Infection in Aquacultures of Carp . . . . . . . . . . . . 30
4.5.1 Large Scale Cultivation of A. platensis . . . . . . . . . . . . . . . . . . . . . . . . 30
4.5.2 Isolation of BM and EPS from A. platensis . . . . . . . . . . . . . . . . . . . . . . 30
4.5.3 Carp and their Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.5.4 Substances Used in the Experiment and their Application to the Carp . . . . . 31
4.5.5 Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.5.6 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.5.7 Antigen and Antibody Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5 Results 34
5.1 CCB Cell Growth Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
5.2 KHV Infection Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5.2.1 Replication of KHV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5.3 Screening of Native Microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
5.3.1 Antiviral Activity of Native Microalgae Extracts . . . . . . . . . . . . . . . . . . 44
5.3.2 Identification and Chemical Composition of Promising Microalgae Isolates . . 45
5.4 Antiviral Activity of Standard Drugs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
5.4.1 Cytotoxicity and Growth Inhibition of Standard Drugs . . . . . . . . . . . . . . 49
5.4.2 Infection Inhibition of Standard Drugs . . . . . . . . . . . . . . . . . . . . . . . 51
5.5 Cultivation of A. platensis and Isolated Fractions . . . . . . . . . . . . . . . . . . . . . 55
5.5.1 Total Carbohydrate Content of Different Isolated Fractions . . . . . . . . . . . . 56
5.5.2 Antiviral Activity of Different Isolated Fractions . . . . . . . . . . . . . . . . . . 58
5.6 Antiviral Activity of EPS Isolated from A. platensis . . . . . . . . . . . . . . . . . . . . 61
5.6.1 Cyotoxic Effect of EPS on CCB Cells . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.6.2 Infection Inhibition of KHV by EPS . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5.6.3 Mechanism and Site of Action of Infection Inhibition of KHV by EPS . . . . . . 71
5.7 Effects of BM and EPS on KHV Infection Used in Aquacultures of Carp . . . . . . . . 77
5.7.1 Large Scale Cultivation of A. platensis . . . . . . . . . . . . . . . . . . . . . . . . 77
5.7.2 BM and EPS and their Application to KHV Infected Carp . . . . . . . . . . . . . 80
6 Significance Analysis 84
7 Discussion 90
7.1 Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
7.2 Antiviral Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
7.3 Antiviral Strategies in Aquacultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
8 Appendix A: Listed Laboratory Procedures 110
8.1 Cultivation of Microalgae A. platensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
8.1.1 Isolation of Different Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
8.1.2 Chemical Analysis of EPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
8.2 CCB Cell Culture and Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
8.2.1 CCB Cell Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Contents iii
8.2.2 CCB Cell Growth Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.3 Koi Herpesvirus (KHV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.3.1 KHV Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.3.2 KHV Titer Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
8.3.3 KHV Infection Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
8.4 Antiviral Activity Assay of Isolated Fractions . . . . . . . . . . . . . . . . . . . . . . . . 119
8.4.1 Cytotoxicity Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
8.4.2 Antiviral Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
8.4.3 Mechanism and Site of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
9 Apendix B: Laboratory Equipment and Material 126
9.1 Cultivation of Microalgae A. platensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
9.2 CCB Cell Culture, KHV and Antiviral Activity Assay . . . . . . . . . . . . . . . . . . . . 128
9.2.1 Mechanism and Site of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
10Appendix C: Supplementary Figures and Tables 133
Bibliography 143
List of publications 158
Abbreviations and Formula Symbols with Units
µ specific growth rate h−1, d−1
µg mikrogramme
µl mikrolitre
A area cm2
A. platensis Arthrospira (Sprulina) platensis
AAHD Aquatic Animal Health Division
Ara-C cytosine β-D-arabinofuranoside
ASS antiviral standard substance
BM biomass
bp base pair
BVT Lehrstuhl für Bioverfahrenstechnik
C conductivity mS
CC cell concentration cellscm-2
CC 50 % cytotoxic concentration µgml−1
50
CCB Common carp brain cells
cDNA complementary DNA
CEFAS The Centre for Environment, Fisheries and Aquaculture
Science
CF concentration factor
CHX cycloheximide
CI continuous illumination
CIDC Centraal Instituut voor Dierziekte Controle
CVI Central Veterinary Institute
CyHV-3 cyprinid herpesvirus-3
d diameter cm, m
d days
Da Dalton
dBM disrupted biomass
DCW dry cell weight gmL−1
DEPC diethylpyrocarbonate
DF dilution factor
DFVF Danish Institute for Food and Veterinary Research
AbbreviationsandFormulaSymbolswithUnits v
DFW dry filter weight
DI demineralised water
DIVA differentiating infected from vaccinated animals
DNA deoxyribonucleic acid
DO dissolved oxygen mg L−1
dNTPs deoxyribonucleotide triphosphates
dsDNA double-stranded DNA
E viral early protein
EDTA ethylenediamine tetraacetic acid disodium salt dihydrate
EICC expected initial cell concentration cellscm-2
EM Electron Microscopy
EPS extracellular polysaccharides
EtBr ethidium bromide
EtOH ethanol
FAU Friedrich-Alexander-Universität Erlangen-Nürnberg
FBS foetal bovine serum
FLI Friedrich-Löffler-Institut
FW filter weight
g gravitation m3kg-1s-2
g gramme
Ganc ganciclovir
g.i. gen of interest
HCMV human cytomegalovirus
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HHV human herpesvirus
HIV human immunodeficiency virus
HPLC High Pressure Liquide Chromatography
HSV herpes simplex virus
IC 50 % inhibitory concentration µgml−1
50
ICC initial cell concentration cellscm-2
IE viral immediate early protein
IPS intracellular polysaccharides
KHV koi herpesvirus
KHVD koi herpesvirus disease
L litre
L viral late protein
L/D cycle light/dark cycle
Lab. Laboratories
LED light-emitting diode
m metre
mg milligramme
min minute