Hunting Viruses in Crocodiles Viral and endogenous retrovira detection and characterisation in farmed crocodiles JULY 2012 RIRDC Publication No. 12/011 Hunting Viruses in Crocodiles Viral and endogenous retroviral detection and characterisation in farmed crocodiles Lorna Melville, Steven Davis, Cathy Shilton, Sally Isberg, Amanda Chong, Jaime Gongora July 2012 RIRDC Publication No. 12/011 RIRDC Project No. PRJ-002461 © 2012 Rural Industries Research and Development Corporation. All rights reserved. ISBN 978-1-74254-366-6 ISSN 1440-6845 Hunting Viruses in Crocodiles: Viral and endogenous retroviral detection and characterisation in farmed crocodiles Publication No. 12/011 Project No. PRJ-002461 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165. Researcher Contact Details Lorna Melville Department of Resources GPO Box 3000 Darwin NT 0801 Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au Electronically published by RIRDC in July 2012 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313 ii Foreword Since the introduction of management changes to control parasitic diseases, the farmed crocodile industry has remained relatively free of diseases capable of causing high levels of losses in well managed operations. Despite these improvements, a condition of ‘runtism’, where a proportion of hatchling crocodiles fail to grow, remains a source of loss to the industry. Also, in June 2006, a syndrome of eye and throat infection (‘conjunctivitis/pharyngitis syndrome’) simultaneously emerged on two farms in the Northern Territory, causing death in up to 96% of hatchling/juvenile crocodiles. Both these issues were addressed through previous RIRDC projects NAP05-16 and PRJ-000301. Both projects found evidence that viral infections could be contributing to these conditions. This could not be confirmed due to the lack of a suitable system for isolating viruses from crocodiles. The objective of this project was to develop cell lines suitable for the isolation of viruses from crocodiles, and methods to maintain and cryopreserve these cell lines. The cell lines would then be used to study the viral fauna of farmed and wild C. porosus in the Northern Territory. A second component of the project, conducted at the University of Sydney, was to characterise endogenous retroviruses (ERVs) in C. porosus and assess their level of expression, particularly in runts. Development of the crocodile cell lines has enabled the first major investigation of viral diseases in crocodiles in Australia, and has improved diagnostic and research capability in crocodile diseases. Twenty different crocodile cell lines have been maintained and used in virus isolation trials. Isolation of viruses from diseased crocodiles has been very successful, with herpesviruses isolated from three different disease syndromes, and adenoviruses from a fourth syndrome. Serology has demonstrated the presence of at least one herpesvirus in wild populations, and that exposure on farms is common. Definitive experimental work to confirm the role of these viruses in the disease syndromes has not been attempted, however, experience in other species suggests they do pose a risk to farmed crocodiles. PCR based screening of crocodiles from across the Northern Territory for ERVs has identified 18 DNA sequences as being potentially functional. This suggests that functional and replication competent ERVs are present in the saltwater crocodile. Further work is required to determine the significance of these viruses in farmed crocodiles and to develop specific control measures. In the absence of this information, good farm management is essential for prevention and control. Measures should be implemented to prevent exposure of young crocodiles to older animals and farm biosecurity plans enforced. The development of disease following exposure to these viruses should be minimized by reducing stress on the animals. This project was funded from RIRDC Core Funds which are provided by the Australian Government. This report is an addition to RIRDC’s diverse range of over 2000 research publications and it forms part of our New Animal Products R&D program, which aims to accelerate the development of viable new animals industries. Most of RIRDC’s publications are available for viewing, free downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313. Craig Burns Managing Director Rural Industries Research and Development Corporation iii Acknowledgments The management and staff of Porosus Pty Ltd provided the hatchlings for the cell line development, collected samples from the sentinel crocodiles, submitted diseased crocodiles for necropsy and virology, and collected blood samples from slaughtered crocodiles. Without their help, this project could not have proceeded. Grahame Webb and Charlie Manolis from Wildlife Management International and Craig Moore from Lagoon Crocodile Farm also submitted samples for investigation. Berrimah Veterinary Laboratory staff, especially Susan Walsh and Richard Weir, contributed to development and implementation of tests especially in relation to virus identification. Tim Hyndman and Mark Bennet at Murdoch University did the original PCR work to confirm the herpesvirus and adenovirus and sequenced the herpesvirus to establish the relationship with published herpesviruses. Alex Hyatt and the staff of the AAHL electron microscope unit performed the EM work to indentify that the viruses belonged to Herpesviridae and Adenoviridae families. Tom Nichols and staff of the crocodile management group of the Department of Natural Resources, Environment, the Arts and Sport collected blood samples from adult crocodiles captured for relocation. Honours student Sarah Atkinson assisted Amanda Chong at the University of Sydney with the molecular characterisation of the ERVs. iv Abbreviations BVL Berrimah Veterinary Laboratory BAC Bacterial artificial chromosome AAHL Australian Animal Health Laboratory ERVs Endogenous retroviruses PCR Polymerase chain reaction ORF Open reading frame CPE Cytopathic effect VNT Virus neutralisation test KUNV Kunjin virus EM Electron microscopy FBS Foetal bovine serum LTR Long tandem repeat Env envelope Gag group specific antigen pro-pol protease-reverse transcriptase region bp base pairs v Contents Foreword ............................................................................................................................................... iii Acknowledgments ................................................................................................................................. iv Abbreviations ......................................................................................................................................... v Executive Summary.............................................................................................................................. ix Introduction ........................................................................................................................................... 1 Objectives ............................................................................................................................................... 3 Methodology ........................................................................................................................................... 4 Cell line development ....................................................................................................................... 4 Virus isolation ................................................................................................................................... 5 Virus identification ..........................................................................................................................