Characterisation of Cherax Quadricarinatus Densovirus: the First Virus Characterised from Australian Freshwater Crayfish
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ResearchOnline@JCU This file is part of the following reference: Bochow, Shaun (2016) Characterisation of Cherax quadricarinatus densovirus: the first virus characterised from Australian freshwater crayfish. PhD thesis, James Cook University. Access to this file is available from: http://researchonline.jcu.edu.au/48889/ The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] and quote http://researchonline.jcu.edu.au/48889/ Characterisation of Cherax quadricarinatus densovirus; the first virus characterised from Australian freshwater crayfish Thesis submitted by Shaun Bochow Bsc Science (Aquaculture) Hons Class 1 (Microbiology & Immunology) August 2016 For the degree of Doctor of Philosophy in Microbiology & Immunology College of Public Health, Medical and Veterinary Science James Cook University STATEMENT OF ACCESS DECLARATION I, the undersigned, author of this work, understand that James Cook University will make this thesis available for use within the University Library and, via the Australian Digital Theses Network, for use elsewhere. I understand that, as an unpublished work, a thesis has significant protection under Copyright Act and, I do not wish to place any further restriction on access to this work. Signature Date: i STATEMENT OF SOURCES DECLARATION I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of references is given Signature Date: ii STATEMENT OF SOURCES ELECTRONIC COPY DECLARATION I, the undersigned, the author of this work, declare that the electronic copy of this thesis provided to the James Cook University Library is an accurate copy of the print thesis submitted, within limits of the technology available. Signature Date: iii DECLARATION OF ETHICS The research presented and reported in this thesis was conducted within the guidelines for research ethics outlined in the National Statement on Ethics Conduct in Research Involving Human (1999), The Joint NHMCR/AVCC Statement and Guidelines on Research Practice (1997), the James Cook University Policy on Experimentation Ethics, Standard Practices and Guidelines (2001), and the James Cook University Statement and Guidelines on Research Practice (2001). The proposed research methodology received clearance from the James Cook University Experimentation Ethics Review (Approval numbers A1922 and A2053). Signature Date: iv STATEMENT OF Co-AUTHORS Bochow, S., Condon, K., Elliman, J. and Owens, L. (2015) First complete genome of an Ambidensovirus; Cherax quadricarinatus densovirus, from freshwater crayfish Cherax quadricarinatus. Marine Genomics 24, Part 3: 305-312 Author Contribution Shaun Bochow Infected, isolated and purified the virus from Cherax quadricarinatus at James Cook University Extracted viral DNA Sequenced genome using PCR with random and specific primers Performed the bioinformatics analysis of the genome Prepared manuscript drafts Kelly Condon Provided the original virus inoculum Assisted with DNA extraction and PCR Assisted with manuscript preparation Jennifer Elliman Consulted on PCR trouble shooting and optimising PCR Manuscript revision Leigh Owens Provided funding to complete work Manuscript direction and revision Coordinated the Aquatic Fish Pathology Laboratory where the work was undertaken v STATEMENT OF THE CONTRIBUTION OF OTHERS The staff at the Queensland Government’s Tropical and Aquatic Animal Health Laboratory provided the original tissue infected with the virus that was the focus of this project. I was the recipient of an Australian Postgraduate Award. This study was partially funded by Indo-Australia Biotechnology Grant BF50090. Funding was provided in the form of competitive grants from Graduate Research Scheme Grants-in aid Conference Funding Dr Ian Hewson of Cornell University provided unpublished echinoderm sequences. Rusaini contributed to the histology of CqDV used in Chapter 4. Associate Professor Leigh Owens, Dr Jennifer Elliman and Kelly Condon provided editorial support. The northern Queensland farms provided the C. quadricarinatus for this project at no cost. Dr Kathryn Green and Mr Richard Webb from the Centre for Microscopy and Microanalysis at the University of Queensland processed samples from caesium chloride bands and gills and took transmission electron microscopy pictures used in this project. vi ACKNOWLEDGEMENTS The staff of the Queensland Government’s former Tropical and Aquatic Animal Health Laboratory are gratefully acknowledged for providing CqDV infected tissue for this project. I would like to thank Associate Professor Leigh Owens and Dr Jennifer Elliman who have been my supervisors during Honours and now PhD. They have been a valuable source of information and have helped keep me on track to complete this project. I am grateful for the opportunity that they have given me. To Kelly Condon, my friend and mentor, you have been a rock throughout this project. To have someone of your calibre in my corner, in this, a very difficult environment to operate in, has been immeasurable. I have enjoyed all the talks, laughs and rough times that we have shared over the last few years. Because of your training and tutelage at the bench, I now have skills applicable to the real world. I wish you all the very best of luck in your PhD. Stay calm, breath, and don’t let the voices in your head lead you down the path that leads to a lab on fire. A special thankyou to Alyssa Budd from the MEEL lab. Working with you has been very insightful and I learned a lot in a very short time. I am glad my ‘slaughtered’ RNA made you laugh. To the wonderful technicians at the vet school that I have worked with over the years: you guys are awesome! You deserve a medal for keeping the labs and the equipment in a ‘working state’ despite having no time or money to do so. I hope things improve for you in the future. Thankyou to James Cook University for the Australian Postgraduate Award and the conditions attached. Finally I would like to thank my family, my parents Kathy and Kevin, my brothers Matthew and Simon. My family have provided financial support throughout my PhD, vii particularly when I was funding the research myself. I have appreciated being able to just turn up on your front door step with a bag of washing, looking for a hot meal. Having somewhere to retreat to, to clear my head and refocus on my goals has been invaluable in helping me through the hard times. viii ABSTRACT Early disease investigations in Cherax spp. were observational in nature. Investigators found a variety of pathogens including fungi, ciliates, platyhelminthes, nematodes and viruses. To date, the viral flora identified have been studied using histopathology and transmission electron microscopy. There have been two transmission trials carried out using Cherax spp. infecting viruses, a Reo-like virus and a parvovirus-like virus. Despite multiple detections of viruses in Cherax spp., there has been no molecular characterisation of any of these pathogens. Of the various viral flora identified in Cherax spp., four were classified as parvovirus-like. These include Cherax destructor systemic parvo-like virus, Cherax quadricarinatus gill parvo-like virus, spawner-isolated mortality like virus and Cherax quadricarinatus parvo-like virus (now Cherax quadricarinatus densovirus, CqDV). The focus of this project was to characterise, for the first time, a Cherax spp. infecting virus, CqDV. The isolate was obtained from the staff at the Queensland Government’s former Tropical and Aquatic Animal Health Laboratory. Infected tissue was used to recreate the disease at James Cook University. As well as confirming the results of Bowater et al. (2002), we extended these results, characterising the tissue tropism of CqDV using quantitative real-time PCR (qPCR). We revealed that CqDV preferentially targets ectodermal tissues, consistent with histopathology observations, and that infection is systemic. CqDV was detected in the heart and muscle, most likely from infected haemocytes in the haemocoel. The genome of CqDV was sequenced using primer walking. The CqDV genome is 6,334 nucleotides in length (GenBank: KP410261) and has four open reading frames (ORFs), three (non structural proteins, NS3, NS1 and NS2) on the sense strand and one (viral protein, VP) on the anti-sense strand, indicating an ambisense organisation. Bioinformatics analysis of the ORFs identified highly conserved motifs characteristic of the family Parvoviridae, including endonuclease and helicase motifs in the NS1 protein and a phospholipase A2 motif in the VP. Phylogenetic analysis firmly placed CqDV in the subfamily Densovirinae, genus Ambidensovirus, species Decapod ambidensovirus, virus variant Cherax quadricarinatus densovirus. The CqDV isolate grouped with the blattodean-infecting densoviruses but its genome was architecturally similar to the Lipidopteran ambidensovirus 1 group. The CqDV genome shared 75 % amino acid homology across the entire genome with sea star associated densovirus (SSaDV). Although CqDV and SSaDV are phylogenetically similar, they are geographically and ix environmentally distinct from each other, infecting two different orders, a unique feature not observed