Date of Award
Doctor of Philosophy
Comparative and Experimental Medicine
John C. New, Jr.
Stephen Kania, Robert Donnell, Robert Moore, Dorcas O'Rourke
Hantaviruses are the etiologic agents of hemorrhagic fever with 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 in Peromyscus maniculatus (deer mouse) although other strains have been found in association with a district rodent host and geographical region. Reservoir hosts are asymptomatically, persistently infected and shed virus particles intermittently in urine, saliva and feces. Thus, the primary transmission route for hantavirus from infected small mammals to humans is inhalation of aerosolized excreta.
Between September 13, 2000 and December 1, 2000; November 28 and 29, 2001; May 27, 2002 through July 24, 2002; and May 1, 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 of 345 animals) were captured. Blood samples were obtained from 305 animals and subsequently tested for anti-hantavirus antibodies with enzyme linked immunosorbent assays (ELISAs) and immunofluorescent assay (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 for the detection of anti-hantavirus antibody in rodent and insectivore sera. The sensitivity and specificity of the ELISA were comparable to those of an IFA using virus infected cells. The only seropositive animals detected were deer mice and white-footed mice (Peromyscus leucopus). The overall estimated seroprevalance in GSMNP was 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/nested PCR amplification products.
We dectected our target genome sequence in one Sorex fumeus (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 was identical to that previously taken from a deer mouse. This is the first reported case of a New World hantavirus being detected in a shrew and the first evidence of a hantavirus detected in a Clethrionomys spp.
Lastly, we sequenced G1 and G2 segments of the M gene and performed phylogenetic analysis with these segments and the N segment. We have designated this hantavirus strain Newfound Gap virus (NGV). The G1 and G2 segments demonstrated homology to New York virus, a pathogenic strain maintained in white-footed mice 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 have occurred. NGV possesses unique characteristics and is closely related to pathogenic strains that have resulted in HCPS case fatalities in this region.
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.