Epidemiology and molecular biology of Elephant Endotheliotropic Herpesvirus 1 in the Asian elephant Elephas maximus Laura Bennett, BSc Submitted: August 2016 Resubmitted: January 2018 Abstract Abstract Herpesviruses are ubiquitous and are found worldwide, most animal species can be infected with multiple herpesviruses. Some cause clinical disease and others remain symptomatic throughout life. Herpesviruses are found in both captive and wild animals including Asian elephants (Elephas maximus). Elephant Endothelioltropic Herpesvirus (EEHV) has been reported in both captive and wild Asian elephants, with a number of cases being reported in North America, Europe and Asia. It has been suggested that EEHV is associated with haemorrhagic disease, which has been attributed to a number of Asian elephant deaths, affecting mostly juveniles and calves. Clinical signs can vary from weight loss, lethargy, depression, cyanosis of the tongue and sudden death. Molecular testing using qPCR has enabled the detection of individual variants of EEHV, this thesis investigates the EEHV1 variant. EEHV1 has been highlighted as the variant that is more frequently associated with deaths. This thesis includes five studies investigating different aspects of EEHV. Including, the relationship between pregnancy and EEHV viral shedding, the use of an amended human protocol for culturing endothelial cells, EEHV tissue tropism, a potential genetic or familial link between EEHV associated deaths and the detection of potential co-pathogens. The main findings from this thesis include: 1) the use of a longitudinal study investigating a potential link between the physiological stress of pregnancy and EEHV viral shedding. i Abstract This study suggested there was no link between pregnancy and EEHV viral shedding however other stressors may be involved. 2) Using an amended human umbilical vein endothelial cell protocol, the culture of Asian elephant endothelial cells was successful. The cells from this study may be used in subsequent drug testing and vaccine development. 3) Quantitative PCR was used to determine EEHV1 tropism in tissues from two deaths associated with the virus. Tropism appeared to be for the heart and liver. 4) This thesis provides results from a preliminary study into a potential link between EEHV associated deaths. The data from an Asian elephant genogram shows there is the possibility of a genetic or familial link, which requires further investigation. 5) A number of tissues from deaths associated with EEHV and or death from other causes were investigated for the presence of potential co-pathogens, including the presence of encephalomyocarditis virus (EMCV), using microarray technology. The results indicated there were no co-pathogens present in the tissues. This thesis adds to the current published data, and includes the first known preliminary study investigating a potential genetic link between elephant deaths due to EEHV. ii Acknowledgements Acknowledgements I would like to express my gratitude to all my supervisors, without them this would not have been possible. Thank you to Lisa, Steve and Rachael for all the help and advice that you have given me, and for your patience. Thank you to the BBSRC and The University of Nottingham for funding this PhD. This project would not have been possible without the cooperation of zoos both in the UK and further afield. I would like to thank the staff at Twycross Zoo, Whipsnade Zoo, Woburn Safari Park, Belfast Zoo, Dublin Zoo, Zurich Zoo and Oklahoma Zoo. I would also like to thank Dr Paul Ling at Baylor College of Medicine for his help and advice. A special thank you to the staff and postgraduate students at the Vet School, specifically Rebecca Sumner and Jennifer Edwards for being there when I needed help and a shoulder to cry on. I would also like to thank Katy Brown whose help and advice has been invaluable throughout my PhD. A big thank you to WildTech, specifically Dr Tim Giles and Dr Abu-Bakr, whose help with the Microarray technique was invaluable throughout this project. I would like to thank my family for their support and help throughout another four years of being a student. A huge thank you to my mum who went out of her way to help me collect a sample from a zoo in the UK at 4am. Finally, I would like to thank my husband who has supported me every step of the way even when I doubted myself, and our three guinea pigs Davina, Phyllis and Clementine whose company helped me through the stressful times. iii Declaration Declaration Unless otherwise acknowledged the work presented in this thesis is original. No part has been submitted for another degree at The University of Nottingham or elsewhere. Any views expressed in the dissertation are those of the author. Signed………………………………………………… Date……………………………………………………. iii Table of contents Table of contents Abstract …………………………………………………………………………………….. i Acknowledgements ………………………………………………………………….. ii Declaration ………………………………………………………………………………. iii Table of contents ……………………………………………………………………… iv List of figures ………………………………………………………………………… xiii List of tables …………………………………………………………………………… xvi List of abbreviations …………………………………………………………….. xviii Introduction ..................................................................... 1 1 Literature review ......................................................... 4 1.1 Taxonomy and distribution .................................... 4 1.1.1 Conservation concerns ....................................... 7 1.1.2 Asian elephant .................................................. 8 1.1.2.1 Conservation concerns ................................ 10 1.2 Genetics ........................................................... 13 1.2.1 Current knowledge .......................................... 13 1.3 General biology ................................................. 18 1.3.1 Behaviour and biology ..................................... 18 1.3.2 Social behaviour ............................................. 19 1.3.3 Reproduction .................................................. 21 1.3.3.1 Captive breeding ........................................ 22 1.3.4 Elephant immune system ................................. 25 1.3.4.1 Immunomodulation and stress ..................... 30 1.4 Diseases of elephants ......................................... 31 1.4.1 Non-infectious diseases.................................... 31 iii Table of contents 1.4.1.1 Obesity ..................................................... 32 1.4.1.2 Foot problems ........................................... 34 1.4.1.3 Degenerative joint disease (DJD) ................. 35 1.4.2 Infectious diseases .......................................... 37 1.4.2.1 Mycobacterium spp .................................... 37 1.4.2.2 Bacillus anthracis ....................................... 41 1.4.2.3 Cowpox virus ............................................. 44 1.4.2.4 Leptospira spp ........................................... 45 1.4.2.5 Yersinia spp............................................... 47 1.4.2.6 Toxoplasma gondii ..................................... 49 1.4.2.7 Encephalomyocarditis virus (EMCV) .............. 50 1.4.2.8 Elephant Endotheliotropic Herpesvirus (EEHV) 53 1.5 Herpesviruses ................................................... 54 1.5.1 Taxonomy ...................................................... 54 1.5.2 Structure ....................................................... 57 1.5.3 Genome organisation ....................................... 59 1.5.4 Replication ..................................................... 62 1.5.4.1 Early and late phases ................................. 62 1.5.4.2 Viral DNA replication .................................. 63 1.5.4.3 Viral protein production .............................. 65 1.5.4.3.1 Viral immunomodulatory proteins .............. 66 1.5.4.4 Virion assembly and egress ......................... 66 1.5.5 Latency and reactivation .................................. 68 1.5.6 Herpes induced disease.................................... 69 1.5.7 Cross species transmission of herpesviruses ....... 73 iv Table of contents 1.5.8 Herpesvirus infections during pregnancy ............ 75 1.5.9 Diagnostic methods ......................................... 75 1.5.10 Treatment ...................................................... 77 1.5.10.1 Nucleoside analogues ................................. 78 1.5.10.2 DNA polymerase inhibitors .......................... 79 1.5.10.3 Synthetic oligonucleotide ............................ 80 1.5.10.4 Vaccines ................................................... 80 1.5.11 Control strategies ............................................ 81 1.6 Herpesviruses of elephants ................................. 82 1.6.1 Bovine herpes ................................................. 83 1.6.2 Elephant gamma herpesviruses ........................ 84 1.6.3 Elephant Endotheliotropic Herpesvirus (EEHV) .... 84 1.6.3.1 EEHV phylogeny......................................... 84 1.6.3.2 Variants .................................................... 85 1.6.3.3 EEHV in African elephants ........................... 86 1.6.3.4 EEHV in Asian elephants ............................. 86 1.6.3.4.1 Clinical disease ........................................ 86 1.6.3.4.2 Reported cases to date ............................. 87 1.6.3.4.3 Current recommendations and treatment .... 90 1.6.3.4.4 Diagnostic methods .................................. 94 1.6.3.4.5 Epidemiology ........................................... 96 1.6.3.4.6 Early detection