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Szent István University Postgraduate School of Veterinary Science Detection and Characterisation of Adeno-, Irido- and Paramyxo

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Szent István University Postgraduate School of Veterinary Science Detection and Characterisation of Adeno-, Irido- and Paramyxo Szent István University Postgraduate School of Veterinary Science Detection and characterisation of adeno-, irido- and paramyxoviruses in reptiles Ph.D. dissertation Tibor Papp 2012 Supervisors and consultants: ..................................... Prof. Dr. Balázs Harrach, D.Sc. Institute for Veterinary Medical Research Centre for Agricultural Research Hungarian Academy of Sciences supervisor ..................................... Dr. Rachel E. Marschang P.D., FTÄ Mikrobiologie, ZB Reptilien Institut für Umwelt- und Tierhygiene University of Hohenheim, Stuttgart, Germany supervisor Prof. Dr. Arthur Pfitzner, Ph.D. Prof. Dr. Mária Benkő, D.Sc. Fg. Allgemeine Virologie, Institute for Veterinary Medical Institut für Genetik Research University of Hohenheim, Centre for Agricultural Research Stuttgart, Germany Hungarian Academy of Sciences consultant consultant Copy …. of eight. …………………………………. Tibor Papp 2 Table of contents Abbreviations ............................................................................................................. 5 1. Summary ................................................................................................................. 7 2. Introduction ............................................................................................................ 9 3. Review of literature ...............................................................................................11 3.1. Reptile virology .................................................................................................11 3.1.1. Viruses occurring in reptiles........................................................................ 11 3.1.2. Detection of viruses in reptiles .................................................................... 13 3.2. Adenoviruses ....................................................................................................15 3.2.1. General introduction to the family Adenoviridae .......................................... 15 3.2.2. Characteristics of Atadenovirus genus members ........................................ 18 3.2.3. Adenovirus infections in reptiles ................................................................. 18 3.3. Iridoviruses .......................................................................................................20 3.3.1. Iridovirids, general introduction to the family Iridoviridae ............................. 20 3.3.2. Invertebrate iridescent viruses, characteristics of the genus Iridovirus ........ 22 3.3.3. Iridovirid infections in reptiles ...................................................................... 24 3.4. Paramyxoviruses ..............................................................................................26 3.4.1. General introduction to the family Paramyxoviridae .................................... 26 3.4.2. Paramyxovirus infections in reptiles ............................................................ 28 3.4.3. Characteristics of the members of genus Ferlavirus ................................... 29 4. Aims of the studies ...............................................................................................31 5. Materials and methods ..........................................................................................32 5.1. Samples ............................................................................................................32 5.1.1. Samples for adenovirus screening.............................................................. 32 5.1.2. Samples for iridovirus studies ..................................................................... 33 5.1.3. Samples for paramyxovirus studies ............................................................ 35 5.2. Cell culture-based methods ..............................................................................37 5.2.1. Virus isolation ............................................................................................. 37 5.2.2. Virus propagation, titration and purification ................................................. 37 5.3. Animal infection studies ....................................................................................38 5.3.1. Cricket bioassay ......................................................................................... 38 5.3.2. Bearded dragon transmission study ........................................................... 39 5.4. Microscopic techniques .....................................................................................41 5.4.1. Light microscopy based techniques ............................................................ 41 5.4.2. Electron microscopy ................................................................................... 42 5.5. Molecular biological techniques ........................................................................43 5.5.1. DNA/RNA extraction ................................................................................... 43 5.5.2. Molecular cloning ....................................................................................... 43 5.5.3. Conventional PCRs (nPCR) ....................................................................... 44 5.5.4. Sequencing ................................................................................................ 45 5.5.5. Analysis of sequences ................................................................................ 46 5.5.6. Real-time PCR (qPCR) ............................................................................... 46 6. Results ...................................................................................................................48 6.1. Adenovirus survey ............................................................................................48 6.1.1. Screening with consensus nested PCR ...................................................... 48 6.1.2. Isolation of reptilian adenoviruses .............................................................. 49 3 6.1.3. Sequence analysis ..................................................................................... 50 6.2. Iridovirus studies ...............................................................................................56 6.2.1. Real-time PCR sensitivity and specificity .................................................... 56 6.2.2. Sequence comparison of isolates, similarities to CIV .................................. 57 6.2.3. Comparing isolates in cricket infection study .............................................. 62 6.2.4. Transmission study with bearded dragons .................................................. 64 6.3. Paramyxovirus studies ......................................................................................65 6.3.1. Analyses of three genes of rPMV from snakes, lizards and a tortoise ......... 65 6.3.2. PMV survey in snakes and a tortoise .......................................................... 69 7. Discussion .............................................................................................................73 7.1. Adenovirus studies............................................................................................73 7.1.1. Novel reptilian adenoviruses ....................................................................... 73 7.1.2. Partial genome analysis of isolated lizard AdVs.......................................... 76 7.2. Iridovirus studies ...............................................................................................79 7.2.1. The qPCR as a diagnostic tool ................................................................... 79 7.2.2. Comparison of genome fragments of different IIV isolates .......................... 80 7.2.3. Comparison of three IIV isolates in a cricket bioassay ................................ 84 7.2.4. Bearded dragon transmission study ........................................................... 85 7.3. Paramyxovirus studies ......................................................................................86 7.3.1. Studies on three genes of rPMV isolates .................................................... 86 7.3.2. Chelonid and squamatid ferlaviruses; novel grouping ................................. 88 7.3.3. PMV survey in snakes and a tortoise; multiple infections ............................ 90 8. New scientific results ............................................................................................94 9. References .............................................................................................................94 10. Publications ....................................................................................................... 112 10.1. Related articles ............................................................................................. 112 10.2. Unrelated articles .......................................................................................... 113 11. Acknowledgements ........................................................................................... 114 12. Appendix ............................................................................................................ 115 12.1. Primers used in the study .............................................................................. 115 Suppl. Table 1. Primers used for adenovirus studies. ......................................... 115 Suppl. Table 2. (continue next page) Primers used for iridovirus studies. ........... 116 Suppl. Table 3. Primers used for paramyxovirus studies. ..................................
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