PMC9979085_01
The phylogenetic tree is based on NFLG sequences BDD002A and BDD069A. The standard subtype references were downloaded from the Los Alamos National Laboratory HIV Database (www.hiv.lanl.gov). The neighbor-joining phylogenetic tree of BDD002A (8,810 bp, red-filled circle ) and BDD069A (8,944 bp, red-filled square ) was constructed based on the NFLG sequences using Mega6.0. The stability of each node was assessed by bootstrap tests with 1,000 replicates, and only bootstrap values >=90% are shown at the corresponding nodes. The scale bar represents a 5% genetic distance. NFLG, near full-length genome.
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F1
The constructed NFLG N-J tree showed that both BDD002A and BDD069A formed a monophyletic branch with a bootstrap value of 100%, indicating that BDD002A and BDD069A are two novel recombinant forms (Figure 1).
Bootscan results of the novel CRF01_AE/B identified. (A) Bootscan plots of BDD002A using CRF01_AE, subtype B and subtype G as references. The parameters of the bootscan analysis were a window size of 400 bp and a step size of 100 bp. (B) Bootscan plots of BDD069A using CRF01_AE and subtype B as putative parental reference sequences and subtype G as the outgroup. The parameters of the bootscan analysis were a window size of 400 bp and a step size of 100 bp. CRF, circulating recombinant form.
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F2
The recombinant breakpoints analysis revealed that BDD002A and BDD069A were composed of 12 interlaced mosaic gene segments, including six CRF01_AE subregions (I, III, V, VII, IX and XI) and 6 B regions (II, IV, VI, VIII, X and XII), with 11 recombinant breakpoints relative to the HXB2 coordinate (Figure 2; Figure 3; Figure 4).
Recombinant maps of the novel CRF01_AE/B identified. The unique recombinant maps of (A) BDD002A and (B) BDD069A were drawn with the online Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html). Six B segments were inserted into the CRF01_AE backbone, and 11 breakpoints divided the whole genome into 12 unique segments.
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F3
The recombinant breakpoints analysis revealed that BDD002A and BDD069A were composed of 12 interlaced mosaic gene segments, including six CRF01_AE subregions (I, III, V, VII, IX and XI) and 6 B regions (II, IV, VI, VIII, X and XII), with 11 recombinant breakpoints relative to the HXB2 coordinate (Figure 2; Figure 3; Figure 4). Subregion analysis (Figure 3) confirmed that the gene mosaic structure of the two NFLGs are: ICRF01_AE (HXB2, 790-1,172 nt), IIB (HXB2, 1,173-1,818 nt), IIICRF01_AE (HXB2, 1,819-2,098 nt), IVB (HXB2, 2,099-2,755 nt), VCRF01_AE (HXB2, 2,756-4,489 nt), VIB (HXB2, 4,490-4,881 nt), VIICRF01_AE (HXB2, 4,882-5,390 nt), VIIIB (HXB2, 5,391-5,617 nt), IXCRF01_AE (HXB2, 5,618-6,331 nt), XB (HXB2, 6,332-8,285 nt), XICRF01_AE (HXB2, 8,286-8,979 nt) and XIIB (HXB2, 8,980-9,411 nt) for BDD002A (Figure 3A); and ICRF01_AE (HXB2, 790-1,172 nt), IIB (HXB2, 1,173-1,818 nt), IIICRF01_AE (HXB2, 1,819-2,070 nt), IVB (HXB2, 2,071-2,726 nt), VCRF01_AE (HXB2, 2,727-4,489 nt), VIB (HXB2, 4,490-4,881 nt), VIICRF01_AE (HXB2, 4,882-5,390 nt), VIIIB (HXB2, 5,391-5,700 nt), IXCRF01_AE (HXB2, 5,701-6,331 nt), XB (HXB2, 6,332-8,285 nt), XI CRF01_AE (HXB2, 8,286-9,006 nt) and XIIB (HXB2, 9,007-9,411 nt) for BDD069A (Figure 3B). The above data revealed that both NFLGs shared seven almost identical breakpoints except a minor difference within the vif-vpr gene region (Figure 3).
Subregional phylogenetic trees of the novel CRF01_AE/B identified. The trees were constructed by the neighbor-joining method with 1,000 bootstrap replications using Mega6.0. Bootstrap values >=90% are shown at the corresponding nodes. The scale bars indicate a genetic distance of 5%. Each segment of BDD002A and BDD069A is marked by a red-filled circle and a red-filled square, respectively. (A) CRF01_AE regions for both URFs. (B) subtype B regions for both URFs.
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F4
The recombinant breakpoints analysis revealed that BDD002A and BDD069A were composed of 12 interlaced mosaic gene segments, including six CRF01_AE subregions (I, III, V, VII, IX and XI) and 6 B regions (II, IV, VI, VIII, X and XII), with 11 recombinant breakpoints relative to the HXB2 coordinate (Figure 2; Figure 3; Figure 4). The parental origin of all fragments of the two NFLGs were analyzed and the CRF01_AE regions for both URFs were from the CRF01_AE cluster five lineage, which is circulating primarily among MSM in major northern cities of China (Figure 4A). The subtype B regions for both URFs were clustered within the northern China subtype B lineage, which also circulates primarily among MSM in northern China (Figure 4B).