Study population characteristics
A total of 60 participants, 19 (31.7%) men and 41 (68.3%) women, were selected. In addition, 13 (21.7%) samples had <200 CD4+ T cell counts and fell under the World Health Organization (WHO) clinical stages 1-4; 14 (23.3%) samples had 200-349 CD4+ T cell count and WHO clinical stages 1-4; 12 (20.0%) samples had 350-500 CD4+ T cell counts and WHO clinical stages 1-2; and 21 (35.0%) samples had >500 CD4+ T cell count and WHO clinical stages 1-2. The median age of the study participants was 34.5 (IQR: 30.0-40). Seven (11.7%) of the study participants were at WHO clinical stage ¾ and 13 (21.7%) were at AIDS stage with CD4+ T cell count <200 cells/uL (Supplement 1). WHO clinical stages were not associated with HIV-1 tropism (p > 0.05). However, the four groups of CD4+ T cell counts (<200-, 200-349-, 350-500- and >500 cells/uL) and HIV RNA load (<10,000- and ≥10,000 copies/mL) were associated with viral tropism (p < 0.05). The median viral load in all 60 study participants was 8704.0 copies/mL (IQR: 3280.75-106955.50 copies/mL). And, the median CD4+ T cell count was 368.5 cells/uL (IQR: 251.25 - 571.75 cells/uL). The median values of CD4+ T cell count and HIV RNA load between phenotypes determined by Geno2Pheno were compared using Mann-Whitney test. It is found that [CXCR4 median 209.00 cells/uL (IQR: 126.00-288.50 cells/uL); R5 median 364.00 cells/uL (IQR: 261.50-525.00 cells/uL); p = 0.003] for CD4+ T cell count, and [CXCR4 median 107267.00 copies/mL (IQR: 74976.50-475570.00 copies/mL); R5 median 8734.00 copies/mL (IQR: 5092.50-106646.00 copies/mL); p = 0.003] for HIV RNA load (Table 1).
Table 1. Summary of demographic, hematological and virological characteristics of study participants.
Variables
|
Number (%)
|
Gender
Male
Female
|
19 (31.7)
41 (68.3)
|
Age (years)
18-29
30-39
40-79
|
9 (15.0)
34 (56.7)
17 (28.3)
|
WHO clinical stage
Stage 1
Stage 2
Stage 3/4
|
27 (45.0)
26 (43.3)
7 (11.7)
|
CD4+ cell count (cells/mm3)
<200
≥200
|
13 (21.7)
47 (78.3)
|
HIV RNA load (copies/mL)
<10000
≥10000
|
33 (55.0)
27 (45.0)
|
Subtyping and circulating recombinant forms
Among the total 60 HIV whole genomes sequenced, 49 were subtype C (81.7%), one was subtype A1 (1.7%) six were recombinant C/A1 (10%) and three were recombinant C/A1/D (5.0%) were identified. One of the sequences could not be assigned to a subtype by REGA (Figure 1 & 2; Table 2). The phylogenetic tree generated by the N-J method indicated that all viruses of 58 sequences belonged to subtype C, except for the virus from subject AL-062 who was infected with HIV-1 subtype A. The branch length in the phylogenetic tree between sequences of plasma was not the same in all subjects as indicated in the tree (Figure 2).
Figure 1. Percentage of subtypes, circulating recombinant forms, and R5 and CXCR4 coreceptor uses.
Tropism and glycosylation sites in V3 loop
Among the total 60 HIV genomes sequenced, 10 (16.7%) of them were not with good sequence in V3 loop to determine the coreceptor usage (tropism). From 50 of the sequences where coreceptor usage was determined by PhenoSeq, 44 (88.0%) and 6 (12.0%) were R5 and X4, respectively. In addition, phenotype by Geno2Pheno showed that 41 (82.0%) and 9 (18.0%) were R5 and X4, respectively. There was no any positively charged amino acid arginine (R) or lysine (K) at position 11. At position 24, lysine (K) was recorded for ZM-028 with net positive charge of +5. At position 25, lysine was recorded for AL-108 with the net positive charge of +5 and SP-065 with net positive charge of +6. Therefore, all three of the samples (AL-108, SP-065 and ZM-028) carried X4 tropic viruses. The other possible X4 phenotype virus with net positive charge of +5 was identified in SP-064 (Figure 1, Table 2). The NNNT 35 (70%) motif at the beginning of the loop was identified as the most dominant potential N-linked glycosylation site. Furthermore, it was found that GNNT 8 (16%), SNNT 4 (8%), GNNI 1 (2%), NNNR 1 (2%) and QNNT 1 (2%) were also identified as additional glycosylation sites (Table 2).
Table 2. HIV-1 V3 loop consensus sequences, co-receptor use, and subtypes and circulating recombinant forms.
Env name
|
V3 HIV aligned sequences
|
charge
|
Coreceptor usage
|
Subtype/recombinant
|
PhenoSeq
|
Geno2Pheno
|
|
* 11 24 25 *
|
|
|
|
|
AL-008
|
CTRPSNNTRK S IRIGPGQAFYAT G D VTGDIRQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
AL-017
|
CTRPNNNTRE S IRIGPGQTFYAT G A IIGDIRQAHC
|
+2
|
R5
|
R5
|
Subtype C
|
AL-045
|
CTRPGNNTRE S IRIGPGQAFYAT G D IIGDIRQAYC
|
+1
|
R5
|
R5
|
Subtype C
|
AL-062
|
CTRPSNNTRT S IRIGPGQAFFAT G D IIGDIRQAHC
|
+2
|
R5
|
R5
|
Subtype A (A1)
|
AL-075
|
CIRPNNNTRK S MRIGPGQTFYAT G G IIGDIRAAYC
|
+4
|
R5
|
R5
|
Subtype C
|
AL-077
|
CTRPNNNTRK S VRIGPGQTFYAT G D IIGDIRQAYC
|
+3
|
R5
|
R5
|
Subtype C
|
AL-105
|
CTRPNNNTRK S IRIGPGQTFYAT G E IIGDIREAHC
|
+2
|
R5
|
R5
|
Subtype C
|
AL-108
|
CTRPNNNTIK S MRIGPGQTFYAT G K IVGNIRQAHC
|
+5
|
CXCR4
|
CXCR4
|
Subtype C
|
AL-115
|
CTRPNNNTRR S IRIGPGQAFYAT E D VIGDIRQAYC
|
+2
|
R5
|
CXCR4
|
Subtype C
|
AL-124
|
CIRPGNNTRK S IRIGPGQTFYAT G D IIGNPRKAYC
|
+4
|
R5
|
R5
|
Subtype C
|
AL-127
|
CTRPNNNTRQ S IRIGPGQTFYAT G E IIGDIRQAHC
|
+2
|
R5
|
R5
|
Subtype C
|
AL-128
|
CTRPNNNTRK S MRIGPGQTFYAT - D IIGDIRQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
AL-134
|
CVRPNNNTRK S IRIGPGQAFYAT G A IIGDIRQAYC
|
+4
|
R5
|
R5
|
Subtype C
|
Al-136
|
CTRPNNNTRK S VRIGPGQAFFAT G D IIGDIRQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
AL-137
|
CMRPGNNTRT S VRIGPGQTFYAT G D IVGDIKQAHC
|
+2
|
R5
|
R5
|
Subtype C
|
AL-149
|
CARPNNNTRK S VSVGPGQAIYAT G D IIGDIRQAHC
|
+2
|
CXCR4
|
R5
|
Subtype C
|
AL-161
|
CTRPNNNTRK S VRIGPGQTFYAT G A IIGEIRQAHC
|
+4
|
R5
|
R5
|
Subtype C
|
AL-182
|
CTRPNNNTRK S IRIGPGQTFYAT - D IIGDIRQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
AL-185
|
CTRPNNNTRQ S IRIGPGQTFYAT G E IIGDIRQAHC
|
+2
|
R5
|
R5
|
Subtype C
|
AL-205
|
CTRPNNNTRE S IRIGPGQTFYAT G D IIGDIRQAHC
|
+1
|
R5
|
R5
|
Subtype C
|
SP-008
|
CIRPNNNRRK S VRIGPGQTFYAT G D IIGDIRAAFC
|
+4
|
R5
|
R5
|
Subtype C
|
SP-013
|
CTRPGNNTRK S VRIGPGQTFYAT G D IIGDIKQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
SP-022
|
CTRPSNNTRK S VRIGPGQTFYAT G D IIGNIRQAYC
|
+4
|
R5
|
R5
|
Subtype C
|
SP-052
|
CTRPNNNTRE S IRIGPGQTFYAT G D IIGDIRQAYC
|
+1
|
R5
|
R5
|
Subtype C
|
SP-064
|
CTRPNNNTRK Y VRIGRGQVFHAT G E IIGDIRKAYC
|
+5
|
CXCR4
|
CXCR4
|
Subtype C
|
SP-065
|
CTRPNNNTRK S VRIGPGQTFYTT - K IIGNIRLAHC
|
+6
|
CXCR4
|
R5
|
Recombinant of C, A1
|
SP-067
|
CTRPGNNTRE S VRIGPGQAFYAT G E IIGDIRKAHC
|
+2
|
R5
|
R5
|
Subtype C
|
SP-073
|
CTRPNNNTRK S VRIGPGQTFFAT G E IIGNIRKAYC
|
+5
|
R5
|
R5
|
Subtype C
|
SP-078
|
CTRPGNNTRK S VRIGPGQTFYAT G D IIGDIRQAHC
|
+3
|
R5
|
R5
|
Not identified
|
SP-079
|
CTRPNNNTRK S VRIGPGQTFYAT G A IIGEIRQAHC
|
+5
|
R5
|
R5
|
Subtype C
|
SP-090
|
CTRPNNNTRK S VRIGPGQVFYAT G D IIGDIRQAHC
|
+3
|
R5
|
R5
|
Recombinant of C, A1, D
|
SP-095
|
CTRPNNNTRR S VRIGPGQTFYAT G E IIGDIKQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
SP-109
|
CTRPGNNIRK S MRIGPGQAFYAT G D IIGDLRQAHC
|
+3
|
R5
|
CXCR4
|
Subtype C
|
SP-146
|
CTRPNNNTRQ S MRIGPGQAFYAM G D IIGDIRQAHC
|
+2
|
R5
|
R5
|
Subtype C
|
SP-151
|
CTRFNNNTRK S IRIGPGQAFYTA G E IIGEIRQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
YK-003
|
CTRPGNNTRK S VRIGPGQTFYAT G A ----------
|
-
|
R5
|
CXCR4
|
Subtype C
|
YK-019
|
CTRPGNNTRR S VRIGPGQTFYAT G D IIGDIRQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
YK-052
|
CTRPQNNTRR S VRIGPGQAFYTT G D IIGDIRQAHC
|
+3
|
R5
|
R5
|
Subtype C
|
ZM-005
|
CTRPNNNTRK S IRIGPGQAFYAR G D IIGDIRQAHC
|
+4
|
R5
|
CXCR4
|
Subtype C
|
ZM-019
|
CTRPNNNTRK S MRIGPGQVFYAT E D IIGDIRQAHC
|
+2
|
R5
|
CXCR4
|
Subtype C
|
ZM-028
|
CMRPNNNTRK S IRIGPGQTFYAT K D IIGNIRQAHC
|
+5
|
CXCR4
|
CXCR4
|
Subtype C
|
ZM-058
|
CTRPNNNTRE S VRIGPGQTFFAT G D IIGDIRQAHC
|
0
|
R5
|
R5
|
Subtype C
|
ZM-075
|
CTRPGNNTRR S VRIGPGQTFFAT G E IIGDIRQAYC
|
+3
|
R5
|
R5
|
Subtype C
|
ZM-086
|
CTRPNNNTRK S VRIGPGQTFYAT G D IIGNIRQAHC
|
+4
|
R5
|
R5
|
Subtype C
|
ZM-102
|
CTRPNNNTRT S IRIGPGQSFHAT G A ITGRIRQAHC
|
+4
|
CXCR4
|
R5
|
Subtype C
|
ZM-121
|
CTRPNNNTRK S VRIGPGQAFYAT G D IIGNIRQAYC
|
+4
|
R5
|
R5
|
Subtype C
|
ZM-134
|
CTRPNNNTRK S VRIGPGQTFYAT G D IIGNIRQAHC
|
+4
|
R5
|
R5
|
Subtype C
|
ZM-136
|
CERPNNNTRE S IRIGPGKTFYAT G E IIGDIRQAYC
|
+1
|
R5
|
R5
|
Subtype C
|
ZM-151
|
CTRHSNNTRK S IRIGPGQAFFAT G E VIGDIRLAHC
|
+3
|
R5
|
R5
|
Subtype C
|
ZM-156
|
CMRPNNNTRK S IRIGPGQAFFAT G A VTGDIRQAHC
|
+4
|
R5
|
CXCR4
|
Subtype C
|
Detection of drug resistance-associated mutations
No drug resistance-associated mutations were detected by the Stanford HIV Drug Resistance Database.
Figure 2. Neighbor-joining tree demonstrating the evolutionary relationship and the distance of the HIV-1 genome consensus sequences. Sixty sequences from plasma samples, subtype C, Subtupe A1, Subtype D and AC, AD, CD, ACD circulating recombinant forms as reference sequences from the Los Alamos database were used. The scale bar represents a genetic distance of 2%.