Using deep sequencing with a primer ID to resolve the structure of viral populations and reveal pre-existing drug resistance mutations in the HIV and HCV protease genes

Jabara, Cassandra Baheeya

Using deep sequencing with a primer ID to resolve the structure of viral populations and reveal pre-existing drug resistance mutations in the HIV and HCV protease genes Public Deposited

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March 22, 2019

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Jabara, Cassandra Baheeya
- Affiliation: College of Arts and Sciences, Department of Biology

Abstract

Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) are among the most deadly chronic viral diseases affecting the human population. The rich genetic diversity produced within a host includes adaptive resistance alleles that may enable viral escape from drug selective pressures. An in-depth characterization of the intrahost population and the genetic path it takes to escape drug selection may reveal how to prevent the evolution of resistance. Resolving the fine-scale genetic structure of a viral population requires deep sampling of the genetic variation within a viral population. I developed a novel technique, Primer ID, which reproducibly (Chapter 4) captures viral diversity while correcting for PCR biases and error inherent in deep sequencing protocols (Chapter 2). Deep sequencing with a Primer ID was applied to the targeted re-sequencing of protease for two different viral genomes, HIV (Chapter 2-3) and HCV (Chapter 4). The allelic distribution of genetic variation of HIV and HCV was skewed towards low-frequency polymorphisms, some of which were resistance-associated variants (Chapters 2-4). I observed that pre-existing resistance mutations could be directly selected during a drug treatment (Chapter 2). However, the path to resistance was often complex and confounded by variance in the steady-state frequency of resistance alleles, sampling depth, and the effective population size (Chapter 3). Once a population of HIV escaped a drug, it was observed that resistance-associated variants were added de novo in a step-wise manner, not brought together by recombination of pre-existing haplotypes. HCV-HIV co-infection decreased overall population diversity (Chapter 4). This difference did not correlate with a change in the overall frequency of pre-existing resistance mutations, but specific resistance alleles were enriched in either mono- or co-infected populations. Further application of deep sequencing with a Primer ID will result in a greater understanding of the population dynamics of both HIV and HCV and determine if the standing genetic variation can be used to predict if a patient will fail therapy and how a viral population responds to selective pressures. Together, improvements in predictive power will result in an enhancement of therapeutic success rate and sustained virologic response.

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