Modeling host responses to tick feeding and tick-borne flavivirus evolution and dispersal. by Dar Heinze, BM Dissertation Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Medical Branch in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Experimental Pathology The University of Texas Medical Branch October, 2012 Dedication To Michelle, my chief supporter, and to Galen and Myla, who enrich my life; and to Jesus, who makes the love we share and the work we do both possible and worthwhile. Acknowledgements The author would like to thank many people that aided the work presented in this dissertation. A special thank-you to Stephen Wikel, Francisco Alarcon-Chaidez, Saravanan Thangamani, Gustavo Valbuena, Venkata Boppana, and Nicole Hausser: without your help none of this would have happened. I would also like to thank Kenneth Escobar, Judith Aronson, Russ Carmical, Natacha Milhano, and Tais Saito for their assistance. For the tick-borne flavivirus work, I am indebted to Naomi Forrester, Ernest Gould, Scott Weaver, Jonathan Auguste, and Nikolaos Vasilakis. I also received funding through the “Emerging and Tropical Infectious Diseases” training grant T32AI007526, Stephen Wikel’s RO1AI062735, and Saravanan Thangamani’s start-up. iv Modeling host responses to tick feeding and tick-borne flavivirus evolution and dispersal. Publication No.____________ Dar Heinze, PhD The University of Texas Medical Branch, 2012 Supervisors: Gustavo Valbuena, Saravanan Thangamani Abstract: Ixodid ticks are hematophagous arthropods that feed on host animals for an extended period of time. To facilitate feeding, ticks secrete a complex mixture of salivary molecules that inhibit host responses such as coagulation, complement, itch responses, and immune responses. Ticks act as vectors for an extremely broad range of human and animal pathogens. Many tick-borne pathogens are significant public health risks throughout the world. In addition, even un-infected ticks cause significant economic losses in the livestock industry. Seminal studies have shown that animals with prior exposure to un-infected ticks can develop an immune response that reduces successful feeding and pathogen transmission by infected ticks in subsequent infestations. Despite the importance of the host response, very few studies have looked at the in vivo response to ticks at the level of the skin. In the following studies, the murine cutaneous response during initial and subsequent infestations with Ixodes scapularis and Dermacentor andersoni nymphs was characterized using PCR-array, microarray, histopathology, and protein-detection methods. These studies show a pro-inflammatory v innate-like immune response characterized by cytokines IL-1b, IL-6, IL-10, chemotaxis of neutrophils and monocytes, wound healing responses, anti-microbial peptides, reactive oxygen species, and C-type lectins. Some down-regulation of transcription during primary infestation was indicated. During secondary infestations, these responses intensified and more cell types infiltrated the bite site. A Th2-type response was suggested for D. andersoni but a mixed Th1/Th2 response was suggested for I. scapularis. The host response to I. scapularis was much more activated during primary infestations than that to D. andersoni, possibly because of differences in the mode of tick attachment. Comparison of these host responses to published literature suggests the cutaneous response to ticks can be characterized as a wound healing response. In a final study, a model of tick-borne flavivirus evolution and dispersal was refined. This study suggests that Powassan encephalitis virus has been in North America for about 12,000 years, and that it will become a more significant public health risk as deer and tick populations increase in the United States. vi Table of Contents Table of Contents List of Tables ........................................................................................................................................ x List of Figures ..................................................................................................................................... xii Chapter 1: Introduction .................................................................................................................... 1 Part 1: An introduction to ticks. .............................................................................................................. 1 What are ticks? .............................................................................................................................................................. 1 Tick classification and evolution. .......................................................................................................................... 1 Tick life cycle and basic behavior. ........................................................................................................................ 2 Tick feeding. ................................................................................................................................................................... 3 Basic biology of the ticks used in my projects................................................................................................. 4 Part 2: Host responses to tick feeding. ................................................................................................. 7 Early studies. .................................................................................................................................................................. 7 Host resistance to tick feeding. .............................................................................................................................. 7 Early host responses to tick feeding. ................................................................................................................... 9 Bite site histopathology........................................................................................................................................... 12 Interaction between innate immune cells and tick saliva. ...................................................................... 15 Interaction between adaptive immune cells and tick saliva. .................................................................. 19 Tick salivary proteins neutralize host cytokines, chemokines, and growth factors. ................... 22 Part 3: Interactions between the tick, pathogen, and host. ....................................................... 25 Ticks aid tick-borne pathogen transmission. ................................................................................................ 25 The timing of tick-borne pathogen transmission. ....................................................................................... 26 How do ticks become infected? ........................................................................................................................... 27 The importance of the host response: prior exposure to ticks can protect from tick-borne disease. ........................................................................................................................................................................... 29 Part 4: The impact of ticks on humans and livestock and tick control strategies. ........... 30 The burden of ticks and tick-borne disease. .................................................................................................. 30 Tick control strategies. ............................................................................................................................................ 31 Part 5: Specific Aims................................................................................................................................... 32 A gap in knowledge. .................................................................................................................................................. 32 Goals................................................................................................................................................................................. 33 Specific aim 1a: Gene expression profiling of the host response to tick feeding. .......................... 34 Specific aim 1b: Histological analysis of tick bite sites.............................................................................. 34 Specific aim 2: Characterize the evolution and dispersal of the tick-borne flaviviruses. .......... 35 Chapter 2: Methods ......................................................................................................................... 40 Ticks. ................................................................................................................................................................. 40 Animals. ........................................................................................................................................................... 40 Time course infestations. ......................................................................................................................... 41 RNA isolation. ............................................................................................................................................... 42 Host gene expression profiling using Affymetrix GeneChips. ................................................... 43 Host gene expression profiling using pathway-specific PCR Array