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A real‐time PCR‐based method to independently sample single simian immunodeficiency virus genomes from macaques with a range of viral loads
Author(s) -
Williams Dawnnica,
Overbaugh Julie
Publication year - 2004
Publication title -
journal of medical primatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.31
H-Index - 42
eISSN - 1600-0684
pISSN - 0047-2565
DOI - 10.1111/j.1600-0684.2004.00073.x
Subject(s) - biology , simian immunodeficiency virus , viral load , virology , population , genome , viral quasispecies , virus , lentivirus , macaque , genetics , viral disease , gene , medicine , paleontology , hepatitis c virus , environmental health
The generation of a diverse population of viral variants is a hallmark of simian immunodeficiency virus (SIV) infection. In order to address what role this diversity plays in disease progression, accurate sampling of the viral population is necessary. However, traditional PCR‐based methods often rely on amplification of multiple genomes in one reaction, leading to resampling of viral genomes and potential errors in the estimations of viral diversity, especially when sequences from only one or a small number of PCRs are examined and/or viral copy number is low. Here we describe a method to amplify one viral envelope gene per PCR, thereby avoiding resampling. For this purpose we developed a highly accurate real‐time PCR method to quantify SIV copy number, then used a single SIV template in a sensitive, high‐fidelity full‐length envelope PCR. Using this method, we have estimated the intra‐animal viral diversity for a cohort of five pig‐tailed macaques ( Macaca nemestrina ) infected with SIVMne variants, which displayed a broad range of viral loads at setpoint.