title: A new approach for sequencing virion genome of Chinese HIV-1 strains subtype B and BC from plasma.
abstract: BACKGROUND
  Although it was widely accepted that full-length HIV genome sequences is important in studying virus genetic evolution and variation as well as developing vaccine candidate, to directly sequencing HIV-1 genome of virion RNA remains as a challenge worldwide. Up to date, no published genomic sequences from virion RNA are available for Chinese prevalent HIV-1 strains due to the absence of specialized protocol and appropriate lab equipments. In this study we developed a straightforward approach for amplifying and sequencing HIV virion RNA from plasma by modifying published protocols and further confirmed it is suitable to process Chinese samples.
  
  
  METHODS
  The methods for viral RNA extraction and gene amplification was modified and optimized as could be widely used in most Chinese labs. Gene alignment of Chinese HIV-1 strains was employed for designing specialized primer sets for Thai-B and BC recombinant strains. Based on comprehensively consideration of high variable gene region and recombinant breakpoints in BC recombinant strains, a three-amplicon strategy (including 4.3-kb gag-pol, 2.9-kb pol-env and 2.7-kb env-nef) was developed. In addition, one amplicon (9 kb near full-length genome) was also used in 32 samples with varied viral loads. All amplicons were directly sequenced by DNA automated sequencer.
  
  
  RESULTS
  Twenty-five percent (8/32) amplification efficiency was achieved by the one-amplicon strategy and 65.6% (21/32) by three-amplicon strategy. For one amplicon strategy, none of complete near full-length genome sequences was obtained by DNA sequencing. For three-amplicon strategy, 75% sequences were achieved in DNA sequencing. Amplification efficiency but not sequencing efficiency was closely associated with viral loads.
  
  
  CONCLUSIONS
  Three-amplicon strategy covering all encoding regions of HIV-1 is suitable for Thai-B and BC recombinant strains and could be potentially employed in less-well equipped Chinese labs.
authors.affiliation: Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology of Ministry of Health, Shanghai Medical College, Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Zhe-Feng
authors.initials: ZF
authors.lastname: Meng
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Zhang
authors.forename: Ruo-Lei
authors.initials: RL
authors.lastname: Xin
authors.forename: Hui
authors.initials: H
authors.lastname: Xing
authors.forename: Xiang
authors.initials: X
authors.lastname: He
authors.forename: Jian-Qing
authors.initials: JQ
authors.lastname: Xu
authors.forename: Yi-Ming
authors.initials: YM
authors.lastname: Shao
mesh_terms: D016679:Genome, Viral / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014771:Virion / Q000235:genetics*
chemical_list: D012367:RNA, Viral