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. CONCLUSION: 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.
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. CONCLUSION: 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.