Schematic representation of the NFLGs, partial structure and breakpoint profiles of the BF1 sequences identified in this study from proviral DNA generated by deep sequencing approach and previously published cell free viruses generated by bulk sequencing approach.

<p>Sequences were mapped relative to the HXB2 numbering system. Genetic distances of overlapping regions (marked with orange boxes) between sequences from plasma and PBMCs together with the overall mean coverage depth are demonstrated. Distances were computed using the maximum composite likelihood method in MEGA version 6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112674#pone.0112674-Tamura1" target="_blank">[28]</a>. As depicted in the figure, the intra-individual plasma and proviral sequence variation in four patients (10BR_PE073, 10BR_PE053, 10BR_PE104 and 10BR_PE032) in the partial <i>pol</i> regions (marked with orange boxes) were remarkably high. These results may indicate that the plasma viruses were derived from a population significantly distinct from those of the cellular sources; a result consistent with dual infection with different subtype. In sample 10BR_PE104, MPS data revealed the existence of subtype B NFLGs and a second BF1 recombinant strain (4450 bp) almost identical to the plasma virus in the same region.</p

Split networks for four sequences of CRF_70BF1 (marked with orange circles) and eleven CRF_71BF1 (marked with blue boxes) and representatives of HIV subtypes A–K sequences from the Los Alamos database (http://hiv-web.lanl.gov).

<p>The splits graphs used distances computed under the GTR+I+G model. The scale bar represents 0.01 nucleotide substitution per site. The results demonstrate that sequences within each of CRF did not group directly with each other, but they were present as one group of sequences within a cluster between subtype B and subclade F1.</p

Comparison of the nucleotides of the transcontinental (A) and Japanese (B) sub-subtypes of the cosmopolitan genotype^*.

<p>*The intra- and intergroup comparisons were performed with the new sequences generated in this study.</p

Drug resistance mutations detected with bulk sequencing (plasma) and deep sequencing (PBMCs).

<p>Samples displayed discordant genotypic data between the cell free viruses and the PBMC-viruses are marked by black dots, Major mutations are marked in boldface type, Major drug resistance mutations at prevalence >20% of the sequenced population are underlined, Major drug resistance mutations at prevalence <20% of the sequenced population are marked by star symbol, Mutations detect only in PBMCs are marked in italic face type.</p>1<p>Transmitted drug resistance mutation, Mutations detect only in plasma are marked by pilcrow symbol.</p><p>Drug resistance mutations detected with bulk sequencing (plasma) and deep sequencing (PBMCs).</p

Phylogenetic tree of HTLV-1 sub-subtypes based on Bayesian Inference analysis of the complete coding region sequences (7593 bp, nucleotide 804–8397 according to position in B1033-2009 “GenBank accession no. AB513134”) of 76 participant samples.

<p>Colored (blue, sub-subtype aA; red, sub-subtype aB) and black branches represent patient samples and reference sequences from all verified sub-subtypes, respectively. Sequences displayed simultaneous base substitutions over the complete coding region (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093374#pone-0093374-t002" target="_blank">Table 2</a>) and formed a monophyletic cluster are indicated by yellow box. For clarity, the tree was midpoint rooted. Values at the nodes represent Bayesian probabilities.</p

Comparison of phylogenetic clustering profile of sequences from both plasma (empty circles) and provirus (black circles) and other HIV-1 reference sequences from the Los Alamos HIV-1 database representing 11 genetic subtypes.

<p>Viral sequences from both compartments were aligned with the complete set of reference sequences obtained from the Los Alamos database (<a href="http://hiv-web.lanl.gov" target="_blank">http://hiv-web.lanl.gov</a>). Green, blue and red colored branches represent subtype B, BF1 recombinants, and subclade F1, respectively. Sequence with discordant results between PBMCs and plasma are marked with blue and red oval circles, respectively. For purposes of clarity, the tree was midpoint rooted. The approximate likelihood ratio test (aLRT) values of ≥90% are indicated at nodes. The scale bar represents 0.02 nucleotide substitutions per site. Except subjects 10BR_PE073, 10BR_PE053, 10BR_PE104 and 10BR_PE032, each patient forms a tight cluster and is distinct from other subjects with aLRT SH-like supports >95% for all inter-subject clusters. The results from the ML analysis added further support to the results depicted in figure and were sufficiently robust to confirm the event of dual infection with different subtype. In case 10BR_PE104 (indicated by black arrow), MPS data revealed the existence of subtype B NFLGs and a second BF1 recombinant strain almost identical to the plasma virus in the same region.</p

Phylogenetic tree of HTLV-1 sub-subtypes based on Bayesian Inference analysis from the long terminal repeat (LTR, 664 bp) of 88 participant samples and 279 HTLV-1 LTR sequences from the database representing 5 of the HTLV-1 subtypes.

<p>The representatives of the 5 references are color-coded. Branches with posterior probabilities ≥0.70 are displayed with blank square.</p

The near full-length genomic (NFLG) and partial fragments subtyping of HIV-from plasma and blood samples.

1<p>Variant was erroneously classified as F1 in our previous study.</p><p>The near full-length genomic (NFLG) and partial fragments subtyping of HIV-from plasma and blood samples.</p

Schematic representation of NFLGs structure and breakpoint profiles with confirmatory phylogenetic trees of the twelve sequences identified in this study as CRF70_BF1 (colored circles).

<p>For patient 10BR_PE026, we were unable to amplify fragment designated as B2 and therefore we could not investigate the location of breakpoint at the 5′ of fragment B2. All ML phylogenic trees were constructed using the PHYML v.3.0 package. The region of subclade F1 and subtypes B are indicated at the bottom. Positions of breakpoints are numbered relative to the HXB2 numbering system. For clarity purposes, the trees were midpoint rooted. The approximate likelihood ratio test (aLRT) values of ≥90% are indicated at nodes. The scale bar represents 0.02 nucleotide substitution per site. The results from the ML analysis were sufficiently robust to confirm the structure for the twelve specimens that were suggested by the recombination analysis.</p

ML phylogenetic tree from concatenated regions assigned as subtype B and F1 from four CRF70_BF1 (3A and 3B) and twelve CRF71_BF1 (3C and 3D) isolates as defined by recombination analysis.

<p>Sequences from each region were aligned with reference sequences representing subtypes A–D, F–H, J and K obtained from the Los Alamos database (<a href="http://hiv-web.lanl.gov" target="_blank">http://hiv-web.lanl.gov</a>). For clarity purposes, the trees were midpoint rooted. The approximate likelihood ratio test (aLRT) values of ≥90% are indicated at nodes. The scale bar represents 0.02 nucleotide substitution per site. The results from this analysis revealed that each segment of the CRF70_BF1 (3A and 3B) and twelve CRF71_BF1 (3C and 3D) viruses was found to cluster with corresponding segments of subtype B or F1 viruses in agreement with the subtype assigned by recombination analysis.</p