Immunological and physiological effects of Piscine orthoreovirus infection in Atlantic salmon (Salmo salar) Philosophiae Doctor (PhD) Thesis Morten Lund Department of Food Safety and Infection Biology Faculty of Veterinary Medicine Norwegian University of Life Sciences Adamstuen 2017 Thesis number 2017:33 ISSN 1894-6402 ISBN 978-82-575-1994-0 1 Table of contents Acknowledgements --------------------------------------------------------------------------------------------------------------------------------- 4 Abbreviations ------------------------------------------------------------------------------------------------------------------------------------------ 6 List of papers ------------------------------------------------------------------------------------------------------------------------------------------- 8 Summary ------------------------------------------------------------------------------------------------------------------------------------------------- 9 Sammendrag (Summary in Norwegian) -------------------------------------------------------------------------------------------------- 11 1 Introduction ------------------------------------------------------------------------------------------------------------------------------------ 13 1.1 General background --------------------------------------------------------------------------------------------------------------- 13 1.2 The life cycle of farmed Atlantic salmon ------------------------------------------------------------------------------- 14 1.3 Common infections in Norwegian aquaculture ---------------------------------------------------------------------- 16 1.4 Piscine orthoreovirus and heart and skeletal muscle inflammation --------------------------------------- 18 Piscine orthoreovirus (PRV) ------------------------------------------------------------------------------------------ 18 Heart and skeletal muscle inflammation ------------------------------------------------------------------------ 19 1.5 Responses to hypoxia ------------------------------------------------------------------------------------------------------------ 24 Introduction to cardiorespiratory physiology in teleost fish ------------------------------------------ 24 Responses triggered by hypoxia in teleost fish ------------------------------------------------------------- 29 Hypoxia tolerance in salmonids ------------------------------------------------------------------------------------- 32 1.6 Antiviral immune responses -------------------------------------------------------------------------------------------------- 34 Components of the immune system ------------------------------------------------------------------------------ 34 Organization of the immune system in teleost fish ------------------------------------------------------- 40 The innate immune response to viral infection in teleosts --------------------------------------------- 41 The adaptive antiviral immune response in salmonids -------------------------------------------------- 47 Environmental effects on the immune response ------------------------------------------------------------ 47 1.7 Effects of PRV infection in Atlantic salmon ------------------------------------------------------------------------- 49 Hypoxic stress during PRV infection ---------------------------------------------------------------------------- 49 The host response to PRV infection ------------------------------------------------------------------------------ 50 2 Aims of the study -------------------------------------------------------------------------------------------------------------------------- 53 3 Summary of papers ------------------------------------------------------------------------------------------------------------------------ 54 4 Methodological considerations -------------------------------------------------------------------------------------------------------57 4.1 Experimental virus infections in Atlantic salmon ------------------------------------------------------------------57 4.2 Quantitative PCR ------------------------------------------------------------------------------------------------------------------- 63 4.3 Histopathological examinations -------------------------------------------------------------------------------------------- 66 4.4 Physiological measurements and tests performed in the hypoxia challenge trial ----------------- 68 4.5 Microarray analysis ----------------------------------------------------------------------------------------------------------------75 2 4.6 Immunohistochemistry ----------------------------------------------------------------------------------------------------------- 77 4.7 Virus neutralization test --------------------------------------------------------------------------------------------------------- 78 4.8 Field data ------------------------------------------------------------------------------------------------------------------------------- 80 4.9 Statistical methods ------------------------------------------------------------------------------------------------------------------ 81 5 Results and general discussion ------------------------------------------------------------------------------------------------------ 84 5.1 PRV infection compromise hypoxia tolerance in Atlantic salmon --------------------------------------- 84 5.2 Transient hypoxic episodes improve the hypoxia tolerance in PRV infected Atlantic salmon but did not affect HSMI development ----------------------------------------------------------------------------------------------- 87 5.3 Hemoglobin levels are reduced at peak PRV RNA levels in the heart --------------------------------- 90 5.4 PRV infection reduces cardiac performance -------------------------------------------------------------------------- 91 5.5 Hemoglobin oxygen affinity is reduced in PRV-infected fish exposed to hypoxic episodes 92 5.6 Experimental PRV infection protects against pancreas disease -------------------------------------------- 94 5.7 Interaction between co-infecting viruses ------------------------------------------------------------------------------- 97 5.8 Immunological mechanisms of the PRV-SAV protection ---------------------------------------------------- 99 5.9 PRV and SAV RNA levels in field samples from co-infected farmed Atlantic salmon ------ 102 5.10 Implications for the aquaculture industry ----------------------------------------------------------------------------- 104 6 Main conclusions -------------------------------------------------------------------------------------------------------------------------- 106 7 Future perspectives ----------------------------------------------------------------------------------------------------------------------- 107 8 References ------------------------------------------------------------------------------------------------------------------------------------ 110 9 Scientific papers I - III ------------------------------------------------------------------------------------------------------------------ 144 3 Acknowledgements The study was performed during 2014 – 2017 at the Norwegian Veterinary Institute (NVI), section of Immunology and at the Department of Food Safety and Infection Biology at the Norwegian University of Life Sciences (NMBU), Campus Adamstuen. The work was funded by the Research Council of Norway (RCN #235788/E40: PRV-PROTect) and the Norwegian Seafood Research Fund (FHF #901001: HSMImore). I am sincerely grateful to my supervisors Maria Krudtaa Dahle, Espen Rimstad and Vidar Aspehaug for your guidance, interesting discussions, good humor and massive support. By being included in the PRV group at Lindern, I have had the opportunity to attend interesting discussions, get an overview of the molecular research on PRV and gather a lot of inspiration in my work. This thesis is a result of the collaboration between NMBU, the Norwegian Veterinary Institute, PatoGen Analyse AS and NOFIMA, which have been a colorful and interesting group to work with. Working with Sven Martin Jørgensen, Gerrit Timmerhaus and Aleksei Krasnov at NOFIMA have been truly inspiring and fruitful. I am also very grateful that professor Mark Powell at the University of Bergen/Norwegian Institute for Water Research joined the project and added expertise on respiratory physiology. This collaboration has inspired and encouraged me in the planning and performance of the challenge trials, the analyses and finally, at the writing of the papers and this thesis. Thanks to all co-authors for your contributions and interesting discussions. A special thanks to Magnus Vikan Røsæg for a great collaboration on the co-infection trial, discussing the results and writing the manuscripts from the co-infection trial. It has been very nice working with you and your analytical abilities have provided valuable input in my work. This work would not have been successfully accomplished without the help from Trine Kolstad and Linda Ramsevik Teigene at PatoGen AS. Furthermore, thanks to the team at VESO Vikan, led by Christian Wallace, for your expertise and patience in performing the challenge trials and for sharing your knowledge and experience. Thanks to all the colleagues at the Immunology department at the NVI, Marta Alarcon, Torunn Taksdal, Ingvild Berg Nyman Turhan Markussen, Øystein Wessel and Anja B. Kristoffersen for your support and guidance during my laboratory work and when learning R. 4 Finally, I would express my gratitude to my family and friends for your support and patience