Development and characterization of a mouse model of HSV-2 infection during pregnancy

iii ACKNOWLEDGMENTS v LIST OF FIGURES AND TABLES viii LIST OF ABBREVIATIONS ix DECLARATION OF ACADEMIC ACHIEVEMENT xi CHAPTER 1: INTRODUCTION 2 1.1 INTRODUCTION 2 1.2 PREGNANCY COMPLICATIONS 2 1.2. 1 PRETERM BIRTHS 4 1.2. 2 FETAL INFLAMMATORY RESPONSE SYNDROME 5 1.3 IMMUNOLOGY OF THE FEMALE REPRODUCTIVE TRACT 6 1.4 IMMUNOLOGY OF PREGNANCY 9 1.5 MOUSE PLACENTAL DEVELOPMENT 12 1.6 INFECTIONS IN PREGNANCY 16 1.6. 1 TORCH INFECTIONS 17 1.6. 2 HSV-2 IN PREGNANCY 18 1.7 HERPES SIMPLEX VIRUS 2 (HSV-2) 19 1.7. 1 EPIDEMIOLOGY AND PATHOGENESIS 19 1.7. 2 INNATE IMMUNE RESPONSES 22 1.7. 3 ADAPTIVE IMMUNE RESPONSES 25 1.8 MOUSE MODELS 26 1.8. 1 MOUSE MODEL OF HSV-2 INFECTION 26 1.8. 2 HSV-2 MOUSE MODELS IN PREGNANCY AND GAPS IN THE LITERATURE 28 CHAPTER 2: RATIONALE 32 CHAPTER 3: MATERIALS AND METHODS 34 3.1 MICE 34 3.2 HSV-2 INOCULATION 34 3.3 VIRAL TITERING AND PATHOLOGY MONITORING 35 3.4 VIRAL DNA EXTRACTION 37 3.5 HSV-2 REAL-TIME POLYMERASE CHAIN REACTION (QPCR) 38 3.6 MATERNAL SERUM AND AMNIOTIC FLUID COLLECTION 39 3.7 FETAL HOMOGENATE SUPERNATANT COLLECTION 39 3.8 MULTIPLEX CYTOKINES/CHEMOKINES ANALYSIS 40 3.9 TISSUE STAINING 41 3.9. 1 TISSUE FIXING AND EMBEDDING 41 M.Sc. Thesis – Philip V. Nguyen McMaster University – Medical Sciences vii 3.9. 2 HEMATOXYLIN AND EOSIN STAINING 41 3.9. 2 PERIODIC-ACID SCHIFF STAINING 42 3.9. 3 HSV-2 STAINING 43 3.10 STATISTICAL ANALYSIS 45 3.11 EXPERIMENTAL DESIGN 46 CHAPTER 4: RESULTS 38 4.1 INCREASED SUSCEPTIBILITY OF HSV-2 INFECTION IN MICE POSITIVE FOR VAGINAL PLUGS 38 4.2 INCREASED PATHOLOGY SCORE IN MATED MICE INFECTED WITH HSV-2 43 4.3 PREGNANCY OUTCOMES 45 4.4 ASSESSING TISSUE HISTOPATHOLOGY IN VAGINAL PLUG POSITIVE, HSV-2 INFECTED MICE 47 4.5 REAL-TIME PCR ASSESSMENT OF HSV-2 VIRAL DNA IN THE VAGINAL TRACT, SPLEEN, PLACENTA AND/OR WHOLE IMPLANTATIONS 52 4.5 CHARACTERIZING MATERNAL SERUM IN HSV-2 INFECTED MICE 57 4.6 CHARACTERIZING AMNIOTIC FLUID IN HSV-2 INFECTED MICE 60 CHAPTER 5: DISCUSSION 63 5.1 SUMMARY 63 5.2 ADVANTAGES AND DISADVANTAGES COMPARED TO RELATED STUDIES 71 5.3 LIMITATIONS 76 5.4 IMPLICATIONS 80 5.5 FUTURE DIRECTIONS 81 CHAPTER 6: CONCLUSION 83 APPENDICES 106 APPENDIX A – SUMMARY OF MATED MICE (GESTATIONAL DAY 8) 107 APPENDIX B – SUMMARY OF MATED MICE (GESTATIONAL DAY 13) 108 APPENDIX C – COPYRIGHTS NATURE REVIEW GENETICS 109 APPENDIX D – COPYRIGHTS NATURE REVIEW IMMUNOLOGY 110 APPENDIX E – COPYRIGHTS JOURNAL OF CLINICAL INVESTIGATION 111 M.Sc. Thesis – Philip V. Nguyen McMaster University – Medical Sciences viii List of Figures and Tables Figure 1 – Anatomical and immunological components of the female reproductive tract. Figure 2 – Structure of the placenta. Figure 3 – Hemachorial placentation Figure 4 – Comparative anatomy of human and mouse placentas Figure 5 – Viral titers of diestrus and mated mice infected with HSV-2 (333) Figure 6 – Survival curves Figure 7 – Genital pathology scores Figure 8 – Percentage of mice with plugs that had fetuses at time of sacrifice Figure 9 – Percentage of resorption Figure 10 – Histopathology and localization of leukocytic infiltration in the vaginal tract of mated mice Figure 11 – Placental Histopathology of mated mice Figure 12 – Localization of NK cells in whole implantation of pregnant mice Figure 13 – Localization of HSV-2 in vaginal tract of mated mice Figure 14 – Absolute HSV-2 real-time PCR quantification (GD 8) Figure 15 – Absolute HSV-2 real-time PCR quantification (GD 13) Figure 16 – Detection of cytokines and chemokines in maternal sera Figure 17 – Detection of cytokines and chemokines in amniotic fluid (AF) M.Sc. Thesis – Philip V. Nguyen McMaster University – Medical Sciences ix List of Abbreviations AF amniotic fluid ANOVA analysis of variance AREB animal research ethics board Ct crossing threshold d.p.i days post infection ELISA enzyme linked immunosorbent assay GAPDH glyceraldehyde 3-phosphate dehydrogenase GD gestational day H2O water H&E hematoxylin and eosin HRP horseradish-peroxidase HSV-1 herpes simplex virus 1 HSV-2 herpes simplex virus 2 IFN-γ interferon gamma IHC immunohistochemistry IL interleukin IVAG intravaginal KC keratinocyte-derived chemokine LIX LPS induced CXC chemokines LPS lipopolysaccharide MDG millennium development goal mRNA messenger ribonucleic acid NaCl sodium chloride NK natural killer NS not significant PAS periodic acid-Schiff stain PBS phosphate buffered saline M.Sc. Thesis – Philip V. Nguyen McMaster University – Medical Sciences x PFU plaque forming unit p.i post infection PMN polymorphonuclear qPCR quantitative real-time polymerase chain reaction rpm revolutions per minute SEM standard error of the mean STI sexually transmitted infection TGF-β transforming growth factor beta Th T helper lymphocyte TNF-α tumour necrosis factor-alpha UN United Nations M.Sc. Thesis – Philip V. Nguyen McMaster University – Medical Sciences xi Declaration of Academic Achievement All experiments were conceived and designed by Philip Nguyen and Charu Kaushic. Kathy Luinstra and Marek Smieja helped with the design of real-time PCR (qPCR) primers and protocols. John Mayberry histology facility staff optimized and performed all histology staining. Philip Nguyen performed all mouse and qPCR experiments. Philip Nguyen and Charu Kaushic performed data analysis. Philip Nguyen wrote this dissertation with contributions from Charu Kaushic. M.Sc. Thesis – Philip V. Nguyen McMaster University – Medical Sciences 2 CHAPTER 1: INTRODUCTION 1.