Table 1.
Genomic regions covered by sequences of CNGZD.
Figure 1.
Subtyping analysis of the genome of CNGZD using Rega HIV-1 subtyping tool (version 2.0).
The recombination pattern was determined by comparing CNGZD sequence with reference sequences of HIV-1 pure subtypes A, B, C, D, F, G, H, J, K. Five DNA segments from subtype B were found to be integrated into the genomic backbone of subtype C by phylogenetic methods of the Rega HIV-1 subtyping tool, which can identify the subtype of a specific sequence. The result was displayed as a virus genomic structure figure with subtype components (A). For subtype recombination analysis, reference sequences of different pure subtypes and CRFs were further applied for the comparison of the virtually full-length sequence of CNGZD by bootscanning methods. Bootscan analysis showed that CNGZD belonged to CRF07_BC (B).
Figure 2.
Analysis of nearly full length genome of CNGZD using Recombinant Identification Program (RIP) (version 3.0).
Mosaic structures of subtype B (in blue) and C (in violet) in both CNGZD and the reference strain of CRF07_BC (97CN54) were figured out by software RIP and the recombinant profile of CNGZD was similar to the reference strain 97CN54. Five segments from subtype B were found in subtype C backbone and the recombination points located at positions in p17/p24, p7/p6, RT, Vpr/Vpu and nef regions of CNGZD and 97CN54, respectively. Window size: 200; threshold for statistical significance: 90%.
Figure 3.
Phylogenetic analysis of clinical isolated virus strain CNGZD.
A neighbor joining phylogenetic tree was built by Mega 4.1 based on full length sequences of clinical isolated virus strain CNGZD and different pure subtypes and CRFs. The Kimura 2-parameter substitution model was used and bootstrap values (1000 replicates) higher than 50 were shown next to the nodes of the tree. J.SE. SE9280 with long distance to CRF07_BC was selected as an outgroup in the phylogentic tree. Subtype references were downloaded from the Los Alamos HIV database (http://hiv-web.lanl.gov). CNGZD located in a clade with the other CRF07_BC isolates. Bar: 0.002 substitutions per site.
Figure 4.
Combination profiles of mutations selected by NVP in CRF07_BC.
Mutations that were bolded are novel mutations. Four different combination profiles initialed with mutations A98G, V108I, Y181C and I135T with I382L were selected by NVP in CRF07_BC.
Table 2.
Mutations developed in RT gene of CRF07_BC strain CNGZD during the in vitro selection with increasing concentrations of NVP.
Figure 5.
Phenotypic analysis of CRF07_BC virus variants selected by NVP.
The NVP EC50 of CRF07_BC virus variants with different mutations were shown. TZM-bl cells were infected with virus variants and treaded with serial diluted NVP. After 48 hrs incubation, luciferase activities representing HIV infection were tested. The EC50 NVP was estimated from the standard curve plotted by the percentage of reduction of luciferase activity and serial concentrations of NVP.
Table 3.
Characterization of NVP resistance mutations in CRF07_BC virus.
Table 4.
Comparison of drug-resistant effects between reported mutations selected in this study and those in HIV drug resistance database of Stanford University.