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

Multiple genotype <i>P</i>. <i>knowlesi</i> infections and diversity among infections in three host species from Kapit.

<p>All data for the multiple genotype infections and distribution of identical alleles between infections derived from complete genotyping of 10 microsatellite loci. (A) Numbers of different <i>P</i>. <i>knowlesi</i> genotypes per infection (multiplicity of infection, MOI) showing significant difference between human and macaque infections (Fisher’s Exact P < 1 x 10^–15), but not between long-tailed and pig-tailed macaques (Fisher’s Exact P = 0.65). (B) Numbers of identical alleles out of 10 loci in pairwise comparisons of infections, showing a similar diversity among infections in humans and long-tailed macaques, but a higher average identity among infections from pig-tailed macaques. All infections had a different 10-locus genotype, except for 5 of the 167 human infections (there was one pair sharing an identical 10-locus genotype, and a triplet of infections sharing another 10-locus genotype, indicated with asterisks here and shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.s011" target="_blank">S4 Table</a>).</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

Correlation between degree of cluster admixture and multi-locus linkage disequilibrium (standardised index of association).

<p>The degree of <i>P</i>. <i>knowlesi</i> cluster admixture was estimated as the local cluster mixedness (<i>p1</i>*<i>p2</i>) based on the proportions of infections designated as Cluster 1 (<i>p1</i>) and Cluster 2 (<i>p2</i>) at each of 10 sampling sites for human infections across Malaysia (Spearman’s Rho = 0.678, P = 0.015). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.s012" target="_blank">S5 Table</a> gives details of each of these values for each population.</p

Isolation-by-distance model and principal component analysis (PCA) of the human <i>P</i>. <i>knowlesi</i> isolates.

<p>(A) Relationship between transformed genetic differentiation and natural log of geographical distance (Euclidean distances of population pairs ranged from 36 km to 1631 km) for all pairs of sites across Malaysia (Mantel test of matrix correlation P < 0.0001), with a similar relationship when analysing only isolates from the Cluster 1 subpopulation (Mantel test P = 0.0016). Black dots denote pairs of populations within Malaysian Borneo and Peninsular Malaysia, while grey dots denote pairs of populations between Malaysian Borneo and Peninsular Malaysia (all data are given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.s013" target="_blank">S6 Table</a>). Evidence of isolation by distance remained when only sites within Borneo were considered (Mantel test P = 0.0016). (B) PCA of the whole infection haplotype dataset indicated differentiation of Peninsular Malaysia isolates from Malaysian Borneo isolates by the second principal component axis, whereas isolates defined as Cluster 1 and Cluster 2 by STRUCTURE analysis were almost completely differentiated along the first principal component axis.</p

Map of sampling locations of 599 <i>P</i>. <i>knowlesi</i> infections genotyped in this study.

<p>A total of 552 samples were from <i>P</i>. <i>knowlesi</i> malaria patients from 10 geographical locations: Peninsular Malaysia (Kelantan and Pahang), Sarawak (Kapit, Betong, Miri, Kanowit and Sarikei), and Sabah (Kudat, Ranau and Tenom). Additionally, 47 samples were from wild macaques (37 long-tailed and 10 pig-tailed macaques) with <i>P</i>. <i>knowlesi</i> infections in Kapit.</p

Intermediate cluster assignment indices in <i>P</i>. <i>knowlesi</i> infections in humans and macaques.

<p>The index for each infection was based on the proportion of shared ancestry between Cluster 1 and Cluster 2 inferred by the STRUCTURE analysis on all samples genotyped at 10 microsatellite loci. An index below 0.25 signifies the individual parasites were predominantly either Cluster 1 or Cluster 2 while those above 0.25 had more intermediate assignment (up to a maximum index value of 0.5). (A) There was no significant difference between single or multiple genotype human infections in the distribution of the indices (Mann-Whitney U test, P = 0.20). (B) Intermediate cluster assignment indices were lower in infections from macaques in Kapit than in human infections overall (Mann-Whitney U test, P < 0.001), but were not significantly different from human infections in Kapit (P = 0.25). Among geographical sites, the indices for human infections were significantly higher in Kelantan than in Kapit, Betong, Kanowit, Kudat or Ranau (P < 0.05 for each pairwise comparison after Bonferroni correction).</p

Diversity and genetic structure of <i>P</i>. <i>knowlesi</i> in human infections from nine different geographical locations.

<p>All <i>P</i>. <i>knowlesi</i> data for analysing multiple genotype infections, distribution of identical alleles between isolates and STRUCTURE analysis were derived from complete genotyping of 10 microsatellite loci. (A) Proportions of infections containing different numbers of genotypes (multiplicity of infection, MOI). Comparison across all 10 geographical locations including Kapit (data in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.g002" target="_blank">Fig 2</a>) showed no significant differences (Pearson’s <i>X</i>^<i>2</i> with 10000 replicates, P = 0.096). (B) Distribution of numbers of identical alleles out of 10 loci in pairwise comparisons of infections. In six of the populations, a small number of pairs of identical multi-locus genotypes were seen as indicated with a label here (2*) and tabulated in detail in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.s011" target="_blank">S4 Table</a>. (C) Subpopulation clusters inferred by the Bayesian model-based STRUCTURE analysis with Cluster 1 (black) and Cluster 2 (red) corresponding to those identified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.g003" target="_blank">Fig 3</a> (<i>K</i> = 2, <i>ΔK</i> = 174.94). Proportions of isolates assigned as Cluster 2 are highest at the sites in Sarawak (top four panels in the figure; locations of all sites are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004888#ppat.1004888.g001" target="_blank">Fig 1</a>).</p