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Retrovirus Life Cycles01:10

Retrovirus Life Cycles

Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host’s genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host’s genome is transcribed, allowing the retrovirus to...

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An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
19:57

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

Published on: March 30, 2014

HIV drug resistance surveillance using pooled pyrosequencing.

Hezhao Ji1, Nathalie Massé, Shaun Tyler

  • 1National HIV and Retrovirology Laboratories, National Microbiology Laboratory, Public Health Agency of Canada, Ottawa, Canada.

Plos One
|February 23, 2010
PubMed
Summary

Pooling surveillance specimens with pyrosequencing offers a cost-effective method for detecting HIV transmitted drug resistance (TDR) mutations in protease. This approach accurately identifies TDR rates in large populations, improving HIV drug resistance surveillance.

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Area of Science:

  • Virology
  • Genetics
  • Public Health

Background:

  • HIV transmitted drug resistance (TDR) surveillance typically relies on individual specimen genotyping.
  • Pyrosequencing enables massive parallel sequencing, allowing simultaneous analysis of numerous specimens.

Purpose of the Study:

  • To demonstrate the feasibility of determining HIV protease TDR rates by interrogating a single pooled sample of surveillance specimens using pyrosequencing.
  • To compare the efficacy of pooled pyrosequencing with conventional Sanger sequencing for TDR detection.

Main Methods:

  • Genotyping of the protease region from 96 treatment-naïve HIV+ serum specimens using Sanger sequencing.
  • Pooling equimolar concentrations of protease amplicons and re-sequencing using the GS FLX Titanium pyrosequencing system.
  • Comparing nucleotide and amino acid differences, and TDR mutations detected by both methods.

Main Results:

  • Pyrosequencing identified more nucleotide (345 vs. 212) and amino acid (168 vs. 81) differences compared to Sanger sequencing.
  • Polymorphisms at frequencies >/=5% were detected by both methods with highly correlated variation rates.
  • Two TDR mutations (M46L, I84V) detected by Sanger sequencing at 1.04% frequency were also identified by pyrosequencing (0.29% and 0.34%).

Conclusions:

  • Pooled pyrosequencing is a cost-competitive method for detecting protease TDR mutations compared to conventional techniques.
  • The method can be adapted to determine population rates of TDR in both protease and reverse transcriptase.
  • This pooled pyrosequencing technique shows potential for broader application in surveying drug resistance rates for other infectious agents.