Open Access Open Badges Research article

Characterizing the emergence and persistence of drug resistant mutations in HIV-1 subtype C infections using 454 ultra deep pyrosequencing

Vijay Bansode1, Grace P McCormack1, Amelia C Crampin23, Bagrey Ngwira23, Ram K Shrestha4, Neil French35, Judith R Glynn3 and Simon A Travers4*

Author Affiliations

1 Molecular Evolution and Systematics laboratory, Zoology, Ryan Institute, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland

2 Karonga Prevention Study, Chilumba, Malawi, South Africa

3 Faculty of Epidemiology and Population Health, London School of Hygiene and Tropical Medicine, London, UK

4 South African National Bioinformatics Institute, University of the Western, Cape, Bellville, South Africa

5 Institute of Infection & Global Health, University of Liverpool, Liverpool, UK

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BMC Infectious Diseases 2013, 13:52  doi:10.1186/1471-2334-13-52

Published: 30 January 2013



The role of HIV-1 RNA in the emergence of resistance to antiretroviral therapies (ARTs) is well documented while less is known about the role of historical viruses stored in the proviral DNA. The primary focus of this work was to characterize the genetic diversity and evolution of HIV drug resistant variants in an individual’s provirus during antiretroviral therapy using next generation sequencing.


Blood samples were collected prior to antiretroviral therapy exposure and during the course of treatment from five patients in whom drug resistance mutations had previously been identified using consensus sequencing. The spectrum of viral variants present in the provirus at each sampling time-point were characterized using 454 pyrosequencing from multiple combined PCR products. The prevalence of viral variants containing drug resistant mutations (DRMs) was characterized at each time-point.


Low abundance drug resistant viruses were identified in 14 of 15 sampling time-points from the five patients. In all individuals DRMs against current therapy were identified at one or more of the sampling time-points. In two of the five individuals studied these DRMs were present prior to treatment exposure and were present at high prevalence within the amplified and sequenced viral population. DRMs to drugs other than those being currently used were identified in four of the five individuals.


The presence of DRMs in the provirus, regardless of their observed prevalence did not appear to have an effect on clinical outcomes in the short term suggesting that the drug resistant viral variants present in the proviral DNA do not appear to play a role in the short term in facilitating the emergence of drug resistance.

HIV-1; Drug resistance; Subtype C; Malawi; Ultradeep sequencing; Proviral DNA