Graduation Year

2010

Document Type

Thesis

Degree

M.S.P.H.

Degree Granting Department

Global Health

Major Professor

Thomas Unnasch, Ph.D.

Co-Major Professor

Lillian M. Stark, Ph.D.

Committee Member

Azliyati Azizan, Ph.D.

Keywords

Arbovirus, SISPA, Flanders virus, mosquito surveillance

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 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 virus was identified in 14 mosquito pools with the highest infection prevalence in Cx. quinquefasciatus mosquitoes. The SISPA technique was successful for the genetic identification of unknown viral isolates and application of this method to samples with suspected or unidentified viral etiologies may be used to enhance public health surveillance of emerging or re-emerging viruses in Florida.

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