Journal: PLoS ONE

Article Title: Molecular Diversity of HIV-1 among People Who Inject Drugs in Kuala Lumpur, Malaysia: Massive Expansion of Circulating Recombinant Form (CRF) 33_01B and Emergence of Multiple Unique Recombinant Clusters

doi: 10.1371/journal.pone.0062560

Figure Lengend Snippet: A , Schematic representation of the full length recombinant structure of CRF33_01B with the four unique recombination breakpoints labelled as 1–4 (HXB2∶2053–2063 nt, 2375–2416 nt, 2538–2540 nt and 2841–2875 nt, respectively) in the gag-pol region . B , Bootscanning plots of three novel CRF candidates (1 to 3) generated from the 1.6 kb gag-pol gene (HXB2∶1753–3440 nt) using SimPlot version 3.5.1 . HIV-1 reference strains CRF01_CM240 (CRF01_AE) and B′_RL42 (Thai subtype B′) were selected as the putative parental genotypes by similarity plotting and C_95IN21068 (subtype C) as an outgroup, with a window size of 200 nucleotides moving along the alignment in increments of 50 nucleotides to define the recombination structures. The shared recombination breakpoints between the URFs were highlighted and in cases where the breakpoints were also shared with CRF33_01B, these breakpoints were further numbered (1 to 4). Each sub-region of these CRF candidates was also indicated and described further in Figure 3 . C, Bootscanning plots of unique recombinant forms (URFs) –11MY1MR851 (sharing a similar recombination breakpoint with 07MYKLD49 ), 10MYKJ086, 11MY2NF831, 11MY3HA741 and 11MY1IM781 displaying distinct recombinant structures in the 1.6 kb gag-pol sequences amplified among the PWIDs study population. For clarity, bootscanning plots of partial RT and gag- PR sequences (11MY1JJ741 and 10MYKJ084, respectively) were not displayed here but their recombination structures have been discussed in detail in the text.

Article Snippet: In effect, multiple novel unique recombinants were identified in this study among 13% of PWIDs by analysing the 1.6 kb gag-pol and partial PR/RT sequences using an extensive array of recombination analysis tools (RIP, SimPlot, bootscan and informative sites), in which ten unique recombinants characterised from seemingly non-epidemiologically linked individuals shared one or more recombination breakpoints with CRF33_01B (CRF33_01B comprised of two subtype B′ fragments recombined with CRF01_AE in the gag-pol region ).

Techniques: Recombinant, Generated, Amplification

Journal: PLoS ONE

Article Title: Molecular Diversity of HIV-1 among People Who Inject Drugs in Kuala Lumpur, Malaysia: Massive Expansion of Circulating Recombinant Form (CRF) 33_01B and Emergence of Multiple Unique Recombinant Clusters

doi: 10.1371/journal.pone.0062560

Figure Lengend Snippet: The gag-pol genes were sequenced, codon-aligned and manually adjusted with the HIV-1 reference subtypes and circulating recombinant forms (CRFs) retrieved from the Los Alamos HIV database ( http://www.hiv.lanl.gov/ ). The 1.6kb gag-pol sequences of the unique recombinant clusters were subdivided into regions (denoted by Roman letters) in relation to the unique recombination breakpoints estimated by informative site analysis. Sub-region neighbour-joining trees were constructed in MEGA 5.05 using Kimura 2-parameter method for nucleotide substitutions to estimate pair-wise evolutionary distance. The reliability of the branch nodes were assessed by bootstrap analysis of 1000 replicates. Bootscan plots and sub-region neighbour-joining trees for each CRF candidates were shown: A , novel CRF candidate 1 (10MYKJ036, 11MY1RJ704, 11MY1ZK731 and 11MY1EP794), B , novel CRF candidate 2 (10MYKJ067, 10MYKJ079 and 04MYKL016) and C , novel CRF candidate 3 (10MYKJ016, 10MYKJ052, 11MYIYC672 and 11MY1JJ741). HIV-1 reference strains CRF01_CM240 (CRF01_AE) and B′_RL42 (Thai subtype B′) were selected as the putative parental genotypes by similarity plotting and included in the bootscan plots (from Figure 2 ). The novel CRF candidate 1 or CRF33_01B were highlighted where the sequences formed a clade in the respective sub-region trees. In cases where the sub-region of CRF33_01B clustered with the reference strains of either putative parental genotypes - CRF01_AE or B′ showing the parental origin, the parental genotype was highlighted. The HXB2 nucleotide positions and sequence length of individual sub-regions were indicated in the illustration. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar for respective sub-region trees was also indicated (in substitutions per site).

Article Snippet: In effect, multiple novel unique recombinants were identified in this study among 13% of PWIDs by analysing the 1.6 kb gag-pol and partial PR/RT sequences using an extensive array of recombination analysis tools (RIP, SimPlot, bootscan and informative sites), in which ten unique recombinants characterised from seemingly non-epidemiologically linked individuals shared one or more recombination breakpoints with CRF33_01B (CRF33_01B comprised of two subtype B′ fragments recombined with CRF01_AE in the gag-pol region ).

Techniques: Recombinant, Construct, Sequencing