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A Newly Identified Hantavirus: the Development of Immunologic Diagnostic Assays and Phylogenetic Analysis for Detection and Characterization

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University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2005 A Newly Identified Hantavirus: The Development of Immunologic Diagnostic Assays and Phylogenetic Analysis for Detection and Characterization Shawn Lee Lewis University of Tennessee, Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Medicine and Health Sciences Commons Recommended Citation Lewis, Shawn Lee, "A Newly Identified Hantavirus: The Development of Immunologic Diagnostic Assays and Phylogenetic Analysis for Detection and Characterization. " PhD diss., University of Tennessee, 2005. https://trace.tennessee.edu/utk_graddiss/4368 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Shawn Lee Lewis entitled "A Newly Identified Hantavirus: The Development of Immunologic Diagnostic Assays and Phylogenetic Analysis for Detection and Characterization." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Comparative and Experimental Medicine. John C. New, Jr., Major Professor We have read this dissertation and recommend its acceptance: Stephen Kania, Robert Donnell, Robert Moore, Dorcas O'Rourke Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I amsubmitting herewith a dissertation written by ShawnLee Lewis entitled "A Newly IdentifiedHantavirus: The Development of Immunologic Diagnostic Assays and Phylogenetic Analysis for Detection and Characterization." I have examinedthe final paper copy of thisdissertation forform and content and recommend thatit be accepted in partialfulfillment of the requirements forthe degreeof Doctor of Philosophy, with a major in Comparative and Experimental Medicine. We have read this dissertation and recommend its acceptance: � KoW-URobert Moore M� Dokasa'Rourke1---.--.___----------- Acceptancefor the Council: A NEWLY IDENTIFIED HANT A VIRUS: THE DEVELOPMENT OF IMMUNOLOGIC DIAGNOSTIC ASSAYS AND PHYLOGENETIC ANALYSIS FOR DETECTION AND CHARACTERIZATION A Dissertation Presented forthe Doctor of Philosophy Degree The University of Tennessee, Knoxville Shawn Lee Lewis December 2005 Abstract Hantaviruses are the etiologic agents of hemorrhagic feverwith renal syndrome (HFRS) in Europe and Asia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. As of July 2005, 396 HCPS cases in 30 U.S. states with a 36% mortality rate have been confirmed since reporting began in 1993. The primary rodent host of numerous U.S. hantaviruses is Peromyscus maniculatus( deer mouse) although other strains have been foundin association with a distinct rodent host and geographical region. Reservoir hosts are asymptomatically, persistently infectedand shed virus particles intermittently in urine, saliva and feces. Thus, the primary transmission route forhantaviruses from infectedsmall mammals to humans is inhalation of aerosolized excreta. Between September 13, 2000 and December l, 2000; November 28 and 29, 2001; May 27, 2002 through July 24, 2002; and May l, 2004 through May 8, 2004 trapping was conducted in the Great Smoky Mountains National Park (GSMNP). A total of 310 rodents and 35 insectivores (a total of345 animals) were captured. Blood samples were obtained from 305 animals and subsequently tested for anti-hantavirus antibodies with enzyme linked immunosorbent assays (ELISAs) and immunofluorescentassay (IFA). We performed RT-PCR, PCR and sequencing analysis encoding the immunodominant region of the nucleocapsid (N) protein contained within the S gene. A 59 amino acid peptide was synthesized based on the deduced amino acid sequence of the 59 residue epitope region of the N protein. This 59-mer was applied as a serodiagnostic antigen in an ELISA forthe detection of anti-hantavirus antibody in rodent and insectivore sera. The sensitivity and specificityof this ELISA were comparable to those 11 of an IFA using virus infected cells. The only seropositive animals detected were deer mice and white-footedmice (Peromyscus leucopus). The overall estimated seroprevalence in GSMNPwas 8. 9% (27 /305). In our study, we also developed a one-step real-time detection-PCR (RTD-PCR) assay based on Superscript III reverse-transcriptase-Platinum Taq polymerase enzyme mixture. PCR amplicons were detected in real time with the use of a 5'hybridization probe. Results were compared to RT-PCR/nestedPCR amplification products. We detected our target genome sequence in one Sorexfumeus (smoky shrew), one Clethrionomys gapperi (Southern red-backed vole) and 16 mice of Peromyscus spp. by RTD-PCR. The overall estimated viral prevalence in GSMNP was 5.2% (18/345). Sequence analysis of the amplicon detected in the smoky shrew �as identical to that previously taken froma deer mouse. This is the firstreported case of a New World hantavirus being detected in a shrew and the firstevidence of a hantavirus detected in a Clethrionomys spp. Lastly, we sequenced Gt and G2 segments of the M gene and performed phylogenetic analysis with these segments and the N segment. We have designated this hantavirus strain NewfoundGap virus (NGV). The Gt and G2 segments demonstrated ho�ology to New York virus, a pathogenic strain maintained in white-footedmice while the N segment demonstrated greater homology to Monongahela and Sin Nombre viruses, also pathogenic strains maintained in deer mice. The incongruous homologies of the NGV S and M segments to other closely related hantaviruses suggest that genetic reassortment resulting in a hybrid virus may lll have occurred. NGV possesses unique characteristics and is closely related to pathogenic strains that have resulted in HCPS case fatalitiesin this region. IV Table of Contents Part l ................................................................................................................................... l INTRODUCTION........................................... .............................................................. l 1. LITERATURE REVIEW .................................................................................... 2 1.1 History of Hemorrhagic Fever with Renal Syndrome in Eurasia .............. 2 1.2 Hantaan Agents ............................................................................................... 6 1.3 Pathogenesis of Human Disease................................................................... l 0 HFRS Clinical Features .................................................................................... 10 HFRS PathologJJ...................................... ......................................................... 12 HPS Clinical Features and PathologJJ.............................................................. 14 1.4 Rodent-Human Hanta virus Transmission ................................................. 18 1.5 Hantavirus Epidemiology ............................................................................. 21 1.6 Viral Maintenance in Rodent Reservoirs ................................................... 29 Deer Mouse BiologJJ.............................. ·........................................................... 29 Rodent-Rodent Hantavirus Transmission ....................� .................................... 34 Rodent Hantavirus PathologJJ................... ........................................................ 38 l.7 Hantavirusand Tennessee............................................................................... 44 1.8 Molecular Methods forthe Detection of Hanta viruses ............................. 45 Serolo g}'.................................................... ........................................................ 45 Real-Time Detection and Quantitative PCR ..................................................... 49 Phylogenetic Analysis ....................................................................................... 5 l LITERATURE CITED .................................................................................................. 54 Part2 ................................................................................................................................. 68 THE DEVELOPMENT OF A SYNTHETIC-PEPTIDE BASED ELISA FOR SCREENING SERA FOR ANTIBODIES AGAINST NEWFOUND GAP VIRUS- A NEWLY IDENTIFIED HANTA VIRUS ............................................................... 68 1. INTRODUCTION...................................... ......................................................... 69 1.1 Hantaviruses in North America ................................................................... 71 1.2 CDC Survey of the Great Smoky Mountains National Park (GSMNP) .. 71 1.3 Nucleocapsid and Glycoprotein Epitopes ................................................... 72 1.4 Current Serologic Tests ................................................................................ 73 1.5 Synthetic Peptide ELISAs ............................................................................ 74 1.6 UTCVM Synthetic Peptide ELISA.............................................................
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