Computational protocol: Viral Diversity and Diversification of Major Non-Structural Genes vif, vpr, vpu, tat exon 1 and rev exon 1 during Primary HIV-1 Subtype C Infection

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Protocol publication

[…] The single genome amplification was based on the method of limiting dilutions , and was used with minor modifications. A median (IQR) of 2.5 (2 to 4) timepoints were sequenced for each patient, and a median of 11.5 (10.4 to 13.5) sequences were generated per patient. The median (IQR) time range of sampling post-seroconversion was 174 (55–349) days. Briefly, the cDNA produced was diluted in 96 well plates with the aim to yield <30% positive reactions of PCR-amplified product. The targeted region spanned from HXB2 nt position 5,041 to 6,310, and included HIV-1 sequence encoding overlapping non-structural viral genes vif (nt positions 5,041 to 5,619; HXB2 numbering), vpr (nt positions 5,559 to 5,850), tat exon1 (nt positions 5,831 to 6,045), rev exon 1 (nt positions 5,970 to 6,045) and vpu (nt positions 6,062 to 6,310). The diluted cDNA corresponding to about 30% of positive PCR was used as a template for two rounds of nested PCR. The first round PCR was conducted using primers Vif1bw- 5′ – GGG TTT ATT ACA GAG ACA GCA GAG- 3′ (HXB2 coordinates 4900 to 4923) and OFM19, and second-round PCR primers Fvif- 5′ - AGA CCC TAT TTG GAA AGG ACC AGC - 3′ (HXB2 coordinates 4922 to 4945) and Rvpu- 5′-CTT CTT TCC ACA CAG GTA CCC CAT- 3′ (HXB2 coordinates 6366 to 6343). The amplicons were sequenced on both strands. Sequencing primers included Rvpu, Fvif, 5198L- 5′ - TCC AGG GCT CTA GGT TAG - 3′, 4679L- 5′ - GCC CAG GGT CTA CTT GTG - 3′; an additional primer 1466L- 5′ - TCA TTG CCA CTG TCT TCT GCT CTT - 3′ was used to increase coverage in cases primer 5198L failed. Amplicons were Exo-SAP purified , and sequenced directly using BigDye technology on the ABI 3730 DNA Analyzer. The sequence fragments were assembled and edited using SeqScape v2.6 (Applied Biosystems). The sequences generated were tested using HYPERMUT v 2.0 , and all hypermutated sequences were excluded from the analysis. A total of 6 out of 667 (0.88%) sequences were found to be hypermutated.During the preliminary analysis of viral quasispecies a link was identified between sequences of subjects OI and OK. We ruled out a potential contamination by (1) similar clustering patterns observed in structural genes, env and gag, of the same subjects (env/gag unpublished data from same cohort); (2) similar branching topology of viral sequences obtained at multiple timepoints of sampling; (3) nucleic acid isolation, PCR amplification and sequencing were separated by place and time; and (4) proper quality controls were used in each experiment. [...] Nucleotide sequences were codon aligned using the MUSCLE algorithm implemented in Mega 5 followed by manual adjustment in Bioedit . For HIV-1 subtyping, three sequences per patient were randomly selected from the pool of generated viral quasispecies. The selection criteria included earliest timepoint available and sequence length with more than 90% coverage of the targeted region (5,041 to 6,310 nt position in HXB2). To determine phylogenetic relationships and clustering patterns of generated viral sequences, the phylogenetic tree reconstruction was performed using Neighbor joining, Maximum likelihood, and Bayesian methods. A standard set of HIV-1 subtype references from LANL was included in the analysis. The sequence CPZ.CM.98.CAM3.AF115393 was used as an outgroup. The online Rega-2 subtyping tool was used in parallel for confirmation . […]

Pipeline specifications

Software tools SeqScape, MUSCLE, MEGA, BioEdit
Applications Phylogenetics, Sanger sequencing, Nucleotide sequence alignment
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases Infection, HIV Infections