1 INDEX OF CONTENTS LIST OF ABBREVIATIONS...3 ABSTRACT...5 CHAPTER I: INTRODUCTION………7 ANELLOVIRUS………...8
1. Torque teno virus (TTV)………..12
1.1. TTV detection and characterization………….………13
1.2. Variability………...18
1.3. Replication………..19
1.4. Pathogenesis……….………...21
1.5. Association with disease ………....23
1.6. Laboratory diagnosis………...27
1.7. Epidemiology………...……….…..28
2. TTV related viruses…..………...30
2.1. Torque teno mini virus (TTMV)………31
2.2. Torque teno midi virus (TTMDV)………...33
3. Animal TTV ………..………...34
3.1. Torque teno sus virus (TTSuV)………...38
CHAPTER II: AIMS AND OBJECTIVES...40
CHAPTER III: MATERIALS AND METHODS………43
1. Sera samples………...….44
2. Samples purification and DNA extraction...44
2
4. Human TTV DNA Detection and Quantification………..………..48
5. Anello specific RCA/PCR...48
6. Cloning...52
7. Sequencing………...52
8. TTSuVs quantification………...53
CHAPTER IV: RESULTS………...56
1. Sample purification from endogenous DNA contamination………...57
2. RCA-SISPA in swine ……….58
3. Anello-RCA/PCR in swine and human ………61
4. Anello-RCA/PCR in other animal species ………...66
5. Evaluation of Anello-RCA and RCA efficiencies by quantitative PCR...70
CHAPTER V: DISCUSSION AND CONCLUSIONS………71
CHAPTER VI: REFERENCES………77
3 LIST OF ABBREVIATIONS
aa amino acid
AIDS acquired immunodeficiency syndrome ALT alanine amino transferase
βLG βlactoglobulin gene CAV chicken anemia virus CMV cytomegalovirus COX cyclo-oxygenase dNTP deossinucleotide GLC glucagon gene HBV hepatitis B virus HCC hepatocellular carcinoma HCV hepatitis C virus
HIV human immunodeficiency virus HSV herpes simplex virus
HVR hypervariable regions
ICTV International Committee on Taxonomy of Viruses IL interleukin
Kb kilobases
NF-Kb nuclear factor-Kb NK natural killer cells ORF open reading frame
4 PCR polymerase chain reaction
PCV porcine circovirus pLC phospholipase C PI post inoculation
RCA rolling circle amplification
RDA representational difference analysis SARS severe acute respiratory disease SAV small anellovirus
SISPA sequence -independent single -primer amplification ss single stranded
TAIP TTV-derived apoptosis-inducing protein hepatocellular carcinoma TLMV Torque teno like mini virus
TTMDV Torque teno midi virus TTMV Torque teno mini virus TTSuV Torque teno sus virus TTV Torque teno virus UTR untranslated region