Paramyxoviruses in Australian Snakes

PARAMYXOVIRUSES IN AUSTRALIAN SNAKES Timothy Hall Hyndman BSc, BVMS This thesis is presented for the degree of Doctor of Philosophy of Murdoch University 2012 I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. .................................................................................... Tim Hyndman, 2012 Table of Contents TABLE OF CONTENTS ............................................................................................................................................. I ACKNOWLEDGEMENTS ....................................................................................................................................... III ABSTRACT ................................................................................................................................................................. V CONFERENCE PROCEEDINGS AND PUBLICATIONS ARISING FROM THIS WORK ..........................VII LIST OF ABBREVIATIONS ................................................................................................................................... IX LIST OF FIGURES .................................................................................................................................................. XV LIST OF TABLES ............................................................................................................................................... XXIII ACKNOWLEDGEMENT OF WORK NOT PERFORMED BY THE AUTHOR ...................................... XXVII CHAPTER 1 – PURPOSE OF PROJECT AND OUTLINE OF THESIS ............................................................ 1 1.1 Purpose of Project ........................................................................................................................... 1 1.2 Outline of Thesis .............................................................................................................................. 2 CHAPTER 2 – LITERATURE REVIEW ................................................................................................................ 5 2.1 Introduction..................................................................................................................................... 5 2.1.1 General Information on Paramyxoviruses .................................................................................................. 5 2.1.2 Ophidian Paramyxovirus or Ferlavirus ........................................................................................................ 7 2.1.3 Specific Information about Ferlaviruses ...................................................................................................... 8 2.2 The Disease ................................................................................................................................... 12 2.2.1 Clinical Signs and Differential Diagnoses .................................................................................................. 12 2.2.2 Gross Pathology ........................................................................................................................................ 18 2.2.3 Histopathology ......................................................................................................................................... 20 2.2.4 Non-Ferlaviral Microbiological Findings ................................................................................................... 23 2.2.5 Transmission ............................................................................................................................................. 24 2.2.6 Treatment and Prognosis .......................................................................................................................... 26 2.2.7 Prevention ................................................................................................................................................. 27 2.2.8 Ferlavirus in Non-Captive Free-Ranging Snakes ........................................................................................ 32 2.2.9 Ferlavirus in Non-Snake Reptilian Hosts ................................................................................................... 33 2.2.10 Zoonotic Potential ..................................................................................................................................... 36 2.3 Diagnostic Tests ............................................................................................................................. 37 2.3.1 Virus Isolation ........................................................................................................................................... 37 2.3.2 Electron Microscopy .................................................................................................................................. 44 2.3.3 Haemagglutination and Haemadsorption Assays ..................................................................................... 48 2.3.4 Neuraminidase Assay ................................................................................................................................ 49 2.3.5 Non-haemagglutination Inhibition Antibody Assays ................................................................................ 49 2.3.6 Haemagglutination Inhibition ................................................................................................................... 50 2.3.7 Immunohistochemistry ............................................................................................................................. 54 2.3.8 In Situ Hybridisation .................................................................................................................................. 55 2.3.9 Polymerase Chain Reaction....................................................................................................................... 55 2.3.10 Commercially Available Diagnostic Tests .................................................................................................. 58 CHAPTER 3 – PARAMYXOVIRUSES IN AUSTRALIAN SNAKES ............................................................... 61 3.1 Australian Snake Virology (excluding paramyxoviruses) ............................................................... 62 3.2 Findings Consistent with Paramyxovirus Infection in Australian Snakes ...................................... 64 3.3 Findings Suggestive of Paramyxovirus Infection in Australian Snakes .......................................... 72 3.4 The Status of Paramyxovirus in Australian Snakes Based on the Results of Others ..................... 75 CHAPTER 4 – DEVELOPMENT AND VALIDATION OF METHODS ......................................................... 77 4.1 Sample Collection .......................................................................................................................... 77 4.2 Virus Isolation and Propagation .................................................................................................... 79 4.2.1 Cell culture ................................................................................................................................................ 79 4.2.2 Embryonated chicken eggs ....................................................................................................................... 92 4.3 Haemagglutination Activity ........................................................................................................... 97 4.4 Haemadsorption .......................................................................................................................... 100 4.5 Haemagglutination Inhibition ..................................................................................................... 100 4.5.1 Eluting Anti-ferlaviral Antibodies from Filter Paper ................................................................................ 103 i | P a g e 4.5.2 Concentrating Anti-ferlaviral Antibodies by Ultrafiltration ..................................................................... 106 4.5.3 Unusual Clumping of Erythrocytes .......................................................................................................... 107 4.6 Neuraminidase Activity ............................................................................................................... 112 4.7 Electron Microscopy .................................................................................................................... 113 4.7.1 Transmission Electron Microscopy .......................................................................................................... 113 4.7.2 Scanning Electron Microscopy ................................................................................................................ 118 4.8 Polymerase Chain Reaction ......................................................................................................... 120 4.9 In situ Hybridisation .................................................................................................................... 130 CHAPTER 5 – SAMPLE EXAMINATION LEADING TO THE ISOLATION OF A VIRUS NAMED BHP1-LUNG .......................................................................................................................................................... 133 5.1 Sample Collection .......................................................................................................................

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