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Characterization of Unidentified Viruses from Florida

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Characterization of Unidentified Viruses from Florida University of South Florida Digital Commons @ University of South Florida Graduate Theses and Dissertations Graduate School 7-12-2010 Characterization of Unidentified Viruses from Florida Jessie L. Dyer University of South Florida Follow this and additional works at: https://digitalcommons.usf.edu/etd Part of the American Studies Commons Scholar Commons Citation Dyer, Jessie L., "Characterization of Unidentified Viruses from Florida" (2010). Graduate Theses and Dissertations. https://digitalcommons.usf.edu/etd/3556 This Thesis is brought to you for free and open access by the Graduate School at Digital Commons @ University of South Florida. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Digital Commons @ University of South Florida. For more information, please contact [email protected]. Characterization of Unidentified Viruses from Florida by Jessie L. Dyer A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health Department of Global Health College of Public Health University of South Florida Major Professor: Thomas Unnasch, Ph.D. Lillian M. Stark, Ph.D. Azliyati Azizan, Ph.D. Date of Approval: July 12, 2010 Keywords: Arbovirus, SISPA, Flanders virus, mosquito surveillance Copyright © 2010, Jessie L. Dyer Dedication To Mary and Steve Dyer, Who’s endless supply of biscuits and Thai food has made everything that much better. Acknowledgements Thank you to my major advisor, Dr. Unnasch, for his guidance, time and advice. Sincere thanks to Dr. Christy Ottendorfer. This thesis would not have been possible without her guidance. Thank you to Gregory White for his technical guidance and endless patience. Special thanks to Dr. Stark for providing several of the isolates used in this study and for her expertise in Virology and Public Health. I would like to thank Dr. Azizan for her continual support during my time at USF. Thank you to everyone in the TRU lab for their assistance and contributions to this research. Finally, I thank my parents for instilling in me confidence and curiosity. Table of Contents List of Tables ........................................................................................................ iii List of Figures ....................................................................................................... iv Abstract ........................................................................................................ v Introduction ........................................................................................................ 1 Objectives ...................................................................................................... 12 Materials and Methods ....................................................................................... 14 Viruses ..................................................................................................... 14 Mosquito trapping and sorting .................................................................. 14 Mosquito Processing................................................................................ 17 RT-PCR Screening Panel ........................................................................ 22 SISPA Sample Preparation ...................................................................... 22 Viral Nucleic Acid Isolation and Amplification .......................................... 23 Cloning ..................................................................................................... 24 Sequence Analysis .................................................................................. 27 Virus Culture Confirmation ....................................................................... 28 Results ...................................................................................................... 31 SISPA Validation ..................................................................................... 31 SISPA Optimization ................................................................................. 34 Identification of Viral Isolates ................................................................... 35 Flanders Virus ............................................................................... 35 Phlyogenetic analysis of Flanders virus isolates ........................... 39 Infimatus Virus ......................................................................................... 42 Phylogenetic analysis of Infimatus virus isolates .......................... 44 Discussion ...................................................................................................... 47 References ...................................................................................................... 57 i Appendices ...................................................................................................... 66 Appendix A: Media Components ............................................................. 67 Appendix B: BOL-Tampa environmental isolate screening panel for endemic Arboviruses ..................................................... 69 Appendix C: RT-PCR Assays used for identity confirmation .................... 71 About the Author ..................................................................................... End Page ii List of Tables Table 1: Unidentified viral isolates and control strains ....................................... 15 Table 2: Mosquito abundance at surveillance sites and percentage of mosquitoes screened ........................................................................... 17 Table 3: Alignment of SLEV beAN 156204 fragments obtained from SISPA method identified to genomic segments .................................. 33 Table 4: SISPA Validation using control viral strains ......................................... 34 Table 5: Optimization of SISPA using control viral strains .................................. 35 Table 6: Flanders virus positive mosquito pools, BOL-Tampa archive .............. 38 Table 7: Estimates of Evolutionary Divergence between Sequences of Members of Rhabdoviradae and Flanders virus isolates ................. 41 Table 8: Infirmatus virus positive mosquito pools isolates, Field Surveillance (2008) .............................................................................. 43 Table 9: Infirmatus virus prevalence at the Tampa Bay Downs surveillance site (2008) ........................................................................ 44 Table 10: Estimates of Evolutionary Divergence between Sequences of Members of Bunyaviradae and Infirmatus virus isolates. ................. 45 iii List of Figures Figure1: Schematic representation of RT-PCR .................................................... 7 Figure 2: Schematic of SISPA ............................................................................ 10 Figure 3: Number of mosquitoes collected from Tampa Bay Downs (2008) ....... 19 Figure 4: Number of mosquitoes collected from Eureka Springs (2008) ............ 20 Figure 5: Number of mosquitoes collected from Walton County (2009) ............. 21 Figure 6: An overview of SISPA application ........................................................ 26 Figure 7: Evaluation of SISPA Method................................................................ 32 Figure 8: Flanders Virus RT-PCR Assay ............................................................ 37 Figure 9: Phylogenetic tree of Flanders virus isolates, M gene ........................... 40 Figure 10: Phylogenetic tree of Infimatus virus isolates, M segment .................. 46 iv Characterization of Unidentified Viruses from Florida Jessie L. Dyer Abstract Public Health and clinical laboratories occasionally obtain viral isolates that cannot be typed by routine methods. Therefore, the sequence-independent, single primer amplification (SISPA) technique was adapted to rapidly identify and characterize viral isolates of unknown etiology. A panel of known (West Nile virus and St. Louis encephalitis virus) and unknown viral isolates (environmental samples collected in Florida) were used to develop and refine the SISPA technique. Selectivity for viral genomic sequences was obtained through enriching viral particles by centrifugation, removal of cellular debris by filtration and removal of host genomic material by benzonase application. The SISPA method successfully amplified the panel of known viruses and a previously unknown environmental viral isolate. The previously unknown environmental viral isolate was determined to be closely related, if not identical, to Flanders virus, a member of Rhabdoviradae. A Flanders virus specific RT-PCR assay identified a total of five previously unknown environmental viral isolates as Flanders virus. Unidentified viral isolates were obtained during arbovirus surveillance efforts in Florida, either from the Florida Department of Health program (BOL-Tampa) during 2005 – 2009, or collected during an ongoing project at the University of v South Florida studying the ecology of arthropod-borne encephalitis viruses at sites located in Florida. In a concurrent study, SISPA was successfully used to characterize an unidentifiable virus isolate related to members of the Bunyaviradae family which was designated as Infirmatus virus. Natural mosquito population (10,557 mosquitoes) collected in Florida was screened for Flanders virus and members of Bunyaviradae to determine infection prevalence. Although Flanders virus was not detected in this population, Infirmatus
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