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Genetic Diversity, Molecular Evolution and Classification of the Viruses In Genetic diversity, molecular evolution and classification of the viruses in the genus Amdoparvovirus circulating in free-ranging mink in Nova Scotia and detection of novel species By Faezeh Kharazyan Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia December 2016 © Copyright by Faezeh Kharazyan, 2016 TABLE OF CONTENT LIST OF TABLES .................................................................................................vi LIST OF FIGURES ...............................................................................................ix ABSTRACT ......................................................................................................... xii LIST OF ABBREVIATIONS USED ..................................................................... xiii ACKNOWLEDGMENTS ..................................................................................... xiv CHAPTER 1. INTRODUCTION ............................................................................ 1 CHAPTER 2. LITERATURE REVIEW .................................................................. 3 2.1 Permissive and restricted infections of AMDV ..................................... 3 2.2 Pathogenicity of AMDV strains ............................................................ 5 2.3 Transmission, host range and epidemiology of AMDV ........................ 6 2.4 Genome structure and viral proteins .................................................... 8 2.4.1 Caspase recognition sites in the NS1 and VP2 proteins .......... 10 2.4.2 Determinants of in vitro and in vivo replication in VP2 protein.. 11 2.4.3 Hypervariable regions in the NS1 and VP2 proteins ................ 12 2.5 Multiple infection and genetic recombination ..................................... 14 2.6 Methods of molecular evolutionary analysis ...................................... 16 2.6.1 Identifying selection pressures ................................................. 17 2.6.2 Phylogenetic analysis ............................................................... 19 2.6.3 Genetic recombination analysis ............................................... 25 2.6.4 Impacts of genetic recombination on phylogenetic estimation . 27 2.7 Virus classification ............................................................................. 28 2.7.1 Taxonomic classification of the family Parvoviridae ................. 28 2.7.2 Classification of the viruses in the genus Amdoparvovirus....... 30 CHAPTER 3. MATERIALS AND METHODS ...................................................... 31 ii 3.1 Animal specimens .............................................................................. 31 3.2 Viral nucleic acid extraction ............................................................... 31 3.3 PCR amplification of viral DNA .......................................................... 33 3.3.1 PCR primer design and optimization ........................................ 33 3.3.2 Primer preparation and optimization of PCR conditions ........... 34 3.3.3 PCR conditions ........................................................................ 37 3.3.4 DNA preparation for PCR amplification .................................... 38 3.3.5 PCR amplification for sequencing ............................................ 39 3.4 PCR product purification and sequencing .......................................... 39 3.5 Molecular cloning ............................................................................... 40 3.6 Nucleotide sequence editing and assembly ....................................... 41 3.7 Amino acid sequence editing ............................................................. 42 3.8 Sequence analysis ............................................................................. 43 3.8.1 Dataset construction and multiple sequence alignment ........... 43 3.8.2 Inferring nucleotide and amino acid compositions .................... 46 3.8.3 Selection pressure analysis...................................................... 47 3.8.4 Entropy measurement .............................................................. 47 3.8.5 Pairwise sequence identity and genetic distance analyses ...... 48 3.9 Recombination analysis ..................................................................... 50 3.9.1 Recombination breakpoint analysis .......................................... 50 3.9.2 Recombination hotspot test ...................................................... 51 3.9.3 Proof of recombination using phylogenetic analysis ................. 52 3.10 Phylogenetic analyses of the genus Amdoparvovirus ...................... 53 3.10.1 Finding the best substitution models ...................................... 53 3.10.2 Phylogenetic analyses of the original sequences ................... 53 iii 3.10.3 Phylogenetic analyses of the modified sequences ................. 54 3.10.4 Identifying a phylogenetic marker ........................................... 55 CHAPTER 4. RESULTS ..................................................................................... 56 4.1 Genome characterization ................................................................... 56 4.1.1 Ambiguous codes and mixed amino acids ............................... 56 4.1.2 Nucleotide and amino acid indels ............................................. 58 4.1.3 Nucleotide and amino acid variable sites ................................. 61 4.1.4 Rates of synonymous and non-synonymous substitutions ....... 63 4.1.5 Number of total and unique amino acid substitutions ............... 63 4.1.6 Sequence motifs ...................................................................... 65 4.1.7 Hypervariable regions .............................................................. 67 4.2 Molecular evolution ............................................................................ 68 4.2.1 Recombination analysis ........................................................... 68 4.2.2 Pairwise sequence identity and genetic distance analyses ...... 74 4.2.3 Phylogenetic analyses of the genus Amdoparvovirus .............. 80 4.2.3.1 Phylogenetic analyses of the original sequences .............. 80 4.2.3.2 Phylogenetic analyses of the modified sequences ............ 86 4.2.3.3 Phylogenetic analyses of the partial fragments ................. 96 4.2.3.4 Identification of phylogenetic marker ............................... 101 CHAPTER 5. DISCUSSION ............................................................................. 103 5.1 Heterogeneity and taxonomy of the species Amdoparvovirus ......... 104 5.2 Epidemiology of the Amdoparvoviruses in free-ranging mink .......... 107 5.3 Multiple infection and genetic recombination ................................... 111 5.4 Predominance of purifying selection on evolution of the AMDV ....... 114 5.5 Genetic characterization of the AMDV genes and proteins .............. 115 iv 5.6 Amdoparvovirus classification accuracy .......................................... 118 5.6.1 Effect of the rooting method on topology of phylogenies ........ 119 5.6.2 Effect of ambiguous codes on phylogenetic analysis ............. 120 5.6.3 Effects of recombination and outlier isolates on phylogenies . 121 5.6.4 Effect of target genome on phylogenetic analysis .................. 123 5.6.5 Identifying a phylogenetic marker for Amdoparvoviruses ....... 124 CHAPTER 6. CONCLUSIONS ......................................................................... 126 APPENDICES .................................................................................................. 127 BIBLIOGRAPHY ............................................................................................... 197 v LIST OF TABLES Table 2.1. Determinants of in vitro and in vivo replication in VP2 protein ........... 12 Table 3.1. Information on 34 mink samples used for AMDV sequencing ........... 32 Table 3.2. Primer pairs for PCR amplification of AMDV and sequencing ........... 35 Table 3.3. Primers designed based on the AMDV sequences in free-ranging mink .................................................................................................................... 36 Table 3.4. Sequencing primers .......................................................................... 40 Table 3.5. Samples and primers used for cloning of the local AMDV isolates ... 44 Table 3.6. Published Amdoparvovirus sequences used for analysis .................. 45 Table 4.1. Positions and lengths of the sequences of the 25 local isolates ........ 57 Table 4.2. Nucleotide indels in local isolates and GenBank sequences ............. 59 Table 4.3. Sequence alignment of the Glycine-rich region ................................. 60 Table 4.4. Position and types of amino acid indels in the 25 local isolates ........ 61 Table 4.5. Variant nucleotide positions in different genome regions .................. 62 Table 4.6. Variant amino acid positions in all proteins ....................................... 62 Table 4.7. Averages of pairwise comparisons of non-synonymous and synonymous substitutions in all genes of the local isolates and GenBank sequences .......................................................................................................... 63 Table 4.8. NS1 amino acid residues shared by the most distinct isolates .......... 66 Table 4.9. Hypervariable regions of the NS1 and VP2 proteins ......................... 71 Table 4.10. Information on the 18 significant recombination events in the entire coding region of the genus Amdoparvovirus ............................................
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