Bootscan and informative sites analyses of the 1.6kb partial <i>gag-pol</i> gene sequences of various HIV-1 unique recombinant forms (URFs) characterised among the blood donors in Kuala Lumpur.

A, Bootscan plot of strain 14MYNBB230 which shares all four recombination breakpoints with a previously reported URF, 05MYKL043 (GenBank accession number: DQ366666) [14]. Both strains also share two identical breakpoints with CRF33_01B, as denoted by square textboxes. B, Bootscan plot of strain 13MYNBB034 which displays a total of six recombinant segments involving CRF01_AE and subtype B' (of Thai origin), whereby three breakpoints were identical to that of CRF33_01B and CRF74_01B (which shares four breakpoints with CRF33_01B). Strain 13MYNBB034 may be another newly-characterised genetic variant of CRF33_01B which differed structurally from CRF74_01B. C, Bootscan plots of four CRF01_AE/B' URFs (13MYNBB048, 13MYNBB128, 13MYNBB059 and 14MYNBB193) displaying distinct mosaic recombination structures and breakpoints, which have yet to be reported in the country. D, Bootscan plots of subtype B'/C recombinants displaying all identical recombination structures and breakpoints amongst each other which potentially represent a novel CRF candidate in Kuala Lumpur. All four strains which are epidemiologically-unlinked (13MYNBB108, 14MYNBB084, 14MYNBB090 and 14MYNBB164) also shared three breakpoints (as denoted by square textboxes) with CRF07_BC which is prevalent in China [32]. These isolates, however were genetically and structurally distinct from CRF08_BC from China. E, Bootscan plots of subtype B'/G recombinants (13MYNBB064 and 13MYNBB065) displaying all identical recombination structures and breakpoints amongst each other. Putative HIV-1 parental reference genotypes were selected by similarity plot, which included 90THCM235 (CRF01_AE), CNRL42 (subtype B' of Thai origin), 95IN21068 (subtype C) and 01NGPL0674 (subtype G). All breakpoints were labelled numerically and identical breakpoints were highlighted with dotted lines. Bootscan was performed in SimPlot version 3.5.1 [24] using a window size of 200 nucleotides moving along the alignment in increments of 20 nucleotides.</p

Phylogenetic reconstruction of 136 partial <i>gag-pol</i> gene sequences of 1.6kb amplified among the blood donors in Kuala Lumpur, Malaysia between 2013 and 2014.

<p>HIV-1 incidence was estimated using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) to identify recent (incident) and long-standing (prevalent) infections as indicated where available. Neighbour-joining tree was constructed in MEGA 5.05 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161853#pone.0161853.ref022" target="_blank">22</a>] using Kimura 2-parameter method of nucleotide substitutions and the reliability of the branching nodes were assessed by bootstrap analysis of 1000 replicates. Eleven partial <i>gag</i>-PR (834bp) and two RT gene sequences (966bp) were genotyped separately using similar methods and their prevalence was reported in this study (figures not shown for clarity). Relevant HIV-1 reference genotypes in Southeast Asia include subtype B, CRF01_AE, CRF33_01B, CRF34_01B, CRF48_01B, CRF52_01B, CRF53_01B, CRF54_01B, CRF58_01B and CRF74_01B. Reference sequences of other genotypes prevalent in China (CRF07_BC, CRF08_BC and other recently-described B'/C CRFs) and Africa (subtype G, CRF02_AG and CRF45_cpx) were also included in the analysis. The reference sequences were labelled in the following order: genotype, country of origin, isolate name and GenBank accession number. A well-supported cluster of Malaysian subtype G strains was also highlighted as G_MY within the subtype G clade of African reference strains. All 12 unique recombinant forms were denoted by closed diamonds and labelled according to incident or prevalent infection status. Clusters of novel B'/C recombinants (strains 13MYNBB108, 14MYNBB084, 14MYNBB090 and 14MYNBB164) and B'/G recombinants (13MYNBB064 and 13MYNBB065) were highlighted in the tree. Simian immunodeficiency virus (SIVcpz) reference strains were included as outgroup. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar represents 1% genetic distance (0.01 substitutions per site).</p

HIV-1 genotype distribution of incident and prevalent infections among 127 blood donors in Kuala Lumpur.

<p>A limiting-antigen avidity enzyme immunoassay (LAg-Avidity EIA) was used to distinguish incident from prevalent HIV-1 infections. Out of 179 samples available for incidence assay testing, 70.9% (n = 127) were successfully genotyped and comprised of 29 (22.8%) incident HIV-1 infections.</p

Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries - Fig 3

<p><b>Sub-region neighbour joining tree analyses of the 1.6kb partial <i>gag-pol</i> genes sequenced in two clusters of (A) subtype B'/C and (B) B'/G recombinants characterised in the population.</b> Based on the informative sites analyses, recombination breakpoints were estimated for each strain and the partial <i>gag-pol</i> sequences (HXB2:1753–3440) were then sub-divided into different regions for phylogenetic reconstruction. Putative HIV-1 parental reference genotypes used in bootscan were 90THCM235 (CRF01_AE), CNRL42 (subtype B' of Thai origin), 95IN21068 (subtype C) and 01NGPL0674 (subtype G). Incident or prevalent HIV-1 infections for each strain, as determined using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA) were identified by orange triangles or red circles, respectively. Bootstrap values of greater than 70% were indicated on the branch nodes. The scale bar represents 1% genetic distance (0.01 substitutions per site).</p