Genome Sequencing and Phylogenetic Analyses as a Basis for Molecular Subtyping of Male-Specific (FRNA) Coliphages

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  • March 21, 2019
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  • Friedman, Stephanie
    • Affiliation: Gillings School of Global Public Health, Department of Environmental Sciences and Engineering
Abstract
  • Monitoring programs for recreational waters utilize indicator bacteria concentrations as predictors of sewage-exposure related illness risks. However, most illnesses contracted through exposure to recreational waters may be of viral etiology. Identifying the fecal sources (non-human vs human) is also valuable information for risk management and source mitigation. Male-specific (FRNA) coliphages are proposed as sensitive enteric viral indicators for source-tracking fecal pollution in environmental waters. Classified as family Leviviridae of two genera, Levivirus and Allolevivirus, and four genogroups (I, II, III, IV) the genogroups provide information regarding animal or human fecal sources. In order to design an assay for molecular identification of specific genogroups, a genomic sequence database of sufficient size must be generated from several FRNA coliphages collected from diverse sources or locations. The complete genome of 21 FRNA strains was sequenced and compared with 11 strains available in GenBank. Sequences of 30 out of 32 FRNA coliphages demonstrated very similar conserved regions, Open Reading Frame positions, amino acid compositions and gene maps when compared to the FRNA reference strains. The sequence of two strains could be placed in a new subcluster of genogroup I and further analysis suggests that these viruses are natural recombinants. Among viruses within each genogroup, nucleotide sequence similarities ranged from 75-99%, 83-93%, 69-95% and 74- 95% for genogroups I, II, III and IV, respectively. Genogroup II lysis protein tree formed a unique branch that was not observed in the full-length nucleotide tree. Thus, both full-length nucleotide and individual protein sequences need to be evaluated when genotyping or phylogenetically clustering these FRNA coliphages. From conserved regions within each genogroup, four genogroup-specific primer sets were designed for a reverse-transcription polymerase chain reaction (RT-PCR) assay. The assay was then evaluated successfully on a panel of environmental FRNA strains demonstrating their usefulness to assess the sanitary quality of recreational waters and provide data identifying and subsequently eliminating the contamination source.
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  • In Copyright
Advisor
  • Stamm, Lola
  • Vinjé, Jan
  • Pfaender, Frederic K.
  • Ball, Louise
  • Sobsey, Mark
  • Noble, Rachel T.
  • Genthner, Fred
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2008
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Date uploaded
  • April 26, 2017

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