Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


Dissertation_fnl.pdf (12.12 MB)

ETD Abstract Container

Abstract Header

Rapid Enumeration, Sorting and Maturation Analysis of Single Viral Particles in HIV-1 Swarms by High-Resolution Flow Virometry

Bonar, Michal Mateusz
http://orcid.org/0000-0003-3624-1183
http://rave.ohiolink.edu/etdc/view?acc_num=case149944467787067

Abstract Details

2017, Doctor of Philosophy, Case Western Reserve University, Molecular Virology.
HIV is the most studied human retrovirus whose life cycle presents continuing challenges to virologists that aim to eradicate it. Although viruses are small obligatory parasites that encode a minimum number of proteins, they hijack host proteins and employ highly ordered structural rearrangement termed maturation to ensure successful infection. We do not currently fully understand the role either of these processes has on viral particle diversity and infectivity. Cytometry has increasingly illuminated the complexity of our immune systems, though it remains an underutilized tool amongst microbiologists, mainly due to the physical limitations of small particle detection and analysis by light scatter-dependent methods. However, recently established standards for extracellular vesicle detection that leveraged fluorescence triggering to circumvent these obstacles have reinvigorated interest in high-throughput, quantitative evaluation of viral diversity. An introduction to HIV biology and flow cytometry is presented in Chapter 1. We hypothesized that we could use fluorescence triggering to observe brightly colored HIV-like particles. In this work, we present evidence of quantitative detection of single fluorescent HIV-1 virions rivaling the sensitivity of typical PCR-based methods (Chapter 2). The live viruses can further be sorted into component populations, and retain infectivity. The quality of the samples can be assessed for aggregation rates. We therefore believe that high-resolution flow cytometry, or flow virometry, is an attractive platform for rapid quantification of viral titers and measuring viral heterogeneity. Finally, in Chapter 3, we introduce a novel, versatile reporter of intraviral proteolytic cleavage, MermaidHIV, for the assessment of maturation in HIV-1 particles based on protease-dependent changes in fluorescence. This reporter is specifically incorporated into nascent viruses and can be used with any number of HIV genotypes to study the impact mutations may have on the maturation phenotype. Separation of live viruses based on those factors may reveal viral vulnerabilities and inform therapeutic or vaccine directions, as well as help answer basic virology questions regarding the dynamics of the viral life cycle.
John Tilton, MD (Advisor)
Scott Sieg, PhD (Committee Chair)
Jacobberger James, PhD (Committee Member)
David McDonald, PhD (Committee Member)
193 p.
Biology; Immunology; Virology
hiv; virometry; virometric; maturation; detection; quantification; virology; fluorescent reporter; cytometry; aggregation

Recommended Citations

Citations

  • Bonar, M. M. (2017). Rapid Enumeration, Sorting and Maturation Analysis of Single Viral Particles in HIV-1 Swarms by High-Resolution Flow Virometry [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case149944467787067

    APA Style (7th edition)

  • Bonar, Michal. Rapid Enumeration, Sorting and Maturation Analysis of Single Viral Particles in HIV-1 Swarms by High-Resolution Flow Virometry. 2017. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case149944467787067.

    MLA Style (8th edition)

  • Bonar, Michal. "Rapid Enumeration, Sorting and Maturation Analysis of Single Viral Particles in HIV-1 Swarms by High-Resolution Flow Virometry." Doctoral dissertation, Case Western Reserve University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case149944467787067

    Chicago Manual of Style (17th edition)

case149944467787067
257
© 2017, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.