Protocol for nearly full-length sequencing of HIV-1 RNA from plasma

PloS One
Yuka NadaiJean K Carr

Abstract

Nearly full-length genome sequencing of HIV-1 using peripheral blood mononuclear cells (PBMC) DNA as a template for PCR is now a relatively routine laboratory procedure. However, this has not been the case when using virion RNA as the template and this has made full genome analysis of circulating viruses difficult. Therefore, a well-developed procedure for sequencing of full-length HIV-1 RNA directly from plasma was needed. Plasma from U.S. donors representing a range of viral loads (VL) was used to develop the assay. RNA was extracted from plasma and reverse-transcribed. Two or three overlapping regions were PCR amplified to cover the entire viral genome and sequenced for verification. The success of the procedure was sensitive to VL but was routinely successful for VL greater than 10(5) and the rate declined in proportion to the VL. While the two-amplicon strategy had an advantage of increasing the possibility of amplifying a single species of HIV-1, the three-amplicon strategy was more successful in amplifying samples with low viral loads. This protocol provides a useful tool for molecular analysis to understand the HIV epidemic and pathogenesis, as well as diagnosis, therapy and future vaccine strategies.

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Citations

Mar 8, 2014·AIDS Research and Human Retroviruses·Hongxiong GuoPing Ding
Mar 13, 2009·PloS One·Yuka NadaiJean K Carr
Sep 1, 2015·AIDS Research and Human Retroviruses·Santosh KaradeRaman R Gangakhedkar
Aug 18, 2012·Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology·Roni SarkarSekhar Chakrabarti
May 15, 2015·AIDS Research and Human Retroviruses·Yan-Heng ZhouYong-Tang Zheng
Nov 8, 2014·PloS One·Christelle Mbondji-WonjeIndira K Hewlett
Mar 18, 2017·Scientific Reports·John R CounsellOlivier Danos
Nov 22, 2016·Journal of Medical Virology·Adhikarimayum Lakhikumar SharmaLisam Shanjukumar Singh
Mar 3, 2017·Scientific Reports·John R CounsellOlivier Danos
Jun 8, 2013·AIDS Research and Human Retroviruses·Sudhanshu PandeyRamesh Paranjape
Dec 20, 2011·AIDS Research and Human Retroviruses·Fernando Lucas MeloPaolo Marinho de Andrade Zanotto
Sep 29, 2019·Scientific Reports·Shuhei UedaMasanori Kameoka
Apr 8, 2015·AIDS·Thibault MesplèdeMark A Wainberg
Aug 18, 2020·AIDS Research and Human Retroviruses·Ying-Na XieXin Chen
Nov 23, 2019·Scientific Reports·Maja OrozSnježana Židovec Lepej
Jun 14, 2019·Frontiers in Microbiology·Beatriz BeamudFernando González-Candelas
Jun 4, 2019·AIDS Research and Human Retroviruses·Tatsuya OkaMasanori Kameoka
Aug 5, 2020·AIDS Research and Human Retroviruses·Si-Wei ChengYan-Heng Zhou
Dec 9, 2020·AIDS Research and Human Retroviruses·Ying-Na XieHong Li
Sep 29, 2019·Journal of Virological Methods·Laura HebberechtChris Verhofstede

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Datasets Mentioned

BETA
K03455

Methods Mentioned

BETA
PCR
Assay

Software Mentioned

Primalign HIV

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