| ObjectiveAIDS is one of the major cause of adult death nowadays. By far, no treakthrough progression on the treatment of AIDS has been made, the high mutation rate of the genome of HIV— 1 maybe one of the major cause. The extensive variability of HIV—1 has a huge impact on the epidemiology, diagnosis , disease pathogenesis and on the development of effective vaccine , long lasting antiretroviral therapy. To make clear the relationship of the gene polymorphism of HIV—1 and HIV—1 infection, nearly whole HIV— 1 genome has been studied. Most such studies were focus on subtype B strains epidemic in Europe and America. There are little data on other subtype strains by far. Because many HIV — lsybtypes of M group (A to G) and HIV—2 are prevalant in China, and the continue mutation and recombination during the transmition, which make the HIV—1epidemic pattern differ from any other countries,so we cannot copy the result of abroad studies. So, we are in great need to make clear the HIV— 1 mutation and recombinant pattern.Antiretroviral therapy can induce the emergence of drug resistance mutation. In the Unites States, nearly 21%— 40% new infections are with HIV—1 strains harboring resistance to at least one of three classes of antiretroviral drugs. Now, 5 domestically manufacture drugs, comprised of 3 NRTIs , 1 NNRTI and 1 PI are available. More and more HIV/AIDS patients are and will receive HAART treatment. We can predict the same situation is inevitable after the HAART treatment on largescale. Bacause different subtype HIV—lstrains may have different mutation pattern. And many HIV—1 sybtypes of M groupCA to G) and HIV— 2 are prevalant in China, which may predict the HIV—1 drug resistance mutation may more complex than abroad. We must survey the epidemic of drug resistance HIV—1 as soon as possible, get the data of HIV— ldrug redistance in China, in order to give a scientific guideline to physicians to select antiretroviral drugs, and prevent the epidemic of rug resistance HIV—1 strains.Materials and methodsStudy populationHIV antibody testing was performed by commercial enzyme—linked immunosorbent assay (Vironstike HIV uniform II plus O, biomerieux bv), with confirmation by Western blotting (HIV BLOT 2. 2, GENELABS) method in AIDS Research Center, 1st Affiliated Hospital, China Medical University and CDC of Jilin province and Henan province. The patients of Liaoning province and Jilin province began to reveive HAART at June 2002 and Sep. 2003 respectively. The enrollment standard: CD4 T cell lower than 350 cells per microliter. We collected blood samples before and after treatment for 6 months. Some patients of Henan province had receive HAART before our first sampling, so in the trand-sect sampling, 17 patients were grouped as treatment experience group, 12 patients were grouped as treatment naive group. After 3 months, we collected blood samples again. The 11 patients of Liaoning province reveived EFV+ IDV regimen, the 24 patients of Jilin province reveived d4T + ddI-f-EFV regimen and the 29 patients of Henan province reveived d4T +ddI+NVP regimen. According to the priciple of know and consent , all HIV/AIDS patients filled the uniform questionary and collected EDTA— 3K anticoagulated whole blood, CD4 T cell were detected within 24 hours. Serum and plasma samples were aliquot within 4 hours of collection and stored at — 80°C.2. Absolute CD4+ T cell countsAbsolute CD4+ T cell counts were measured by flow cytometry. 50ul EDTA3K anticoagulant whole blood was incubated with 20ul BD TriT-EST CD4/CD8/CD3 reagent. Incubation was for 15 minutes in the dark at room temperature, after lysing red blood cells, then incubated for another 15 minutes in the dark at room temperature and acquired and analyzed. Absolute CD4+ T cell counts and CD4/CD8 were automatically reported by BD MultiSET? software.3. Plasma HIV viral loadHIV—1 RNA in plasma was measured by RT —PCR using the CO BAS Amplicor. HIV—1 Monitor 1. 5 (Roche Diagnostic Systems). The limit of detection of the assay is from 400 to 750,000 copies/ml. HIV—1 RNA copy number was calculated on the basis of the manufacturers reference standards.4. HIV—1 RNA and DNA extractionHIV— 1 RNAs and provirus DNA were extracted from plasma and whole blood with QIAamp Viral RNA Mini Kit and QIAamp whole blood Mini Kitaccording to the manufacturer's recommendations. HIV—1 RNA were diluted in DEPC free H2O and stored at —80 °C.5 . Drug resistance mutation detectionReverse transcription PCR anf nested PCR. Outer primer pairs : MAW-26(TTg gAA ATg Tgg AAA ggA Agg Ac 2028-2050) ,RT-21 ( cTg TAT TTc Tgc TAT TAA gTc TTT TgA Tgg g 3509 - 3539 ) (50pmol/^l) ,inner primer pairs:PRO—1( cAg Age cAA cAg ccc cAc cA 2147-2166),RT-20(cTg ccA gTT cTA gcT cTg cTT c 3441-3462) were used to amplify HIV — 1 protease 1 — 99 amino acid and Reverse transcriptase 1 — 220 amino acid coding region.6 gag—pol 2. 6kb amplificationRT—PCR and nested — PCR, gag—pol 2. 6kb were amplified with three fragments, Outer primer pairs 1:172A:5— ATC TCT AgC AgT ggC gCC CgA ACA g-3',505B:5- ACT CTT gCT TTA Tgg CTg ggT CC-3', inner primer pairs 1:174A:5- CTC TCg ACg CAg gAC TCggCT TgC T-3',506B:5- CCT gAC ATg CTg TCA TCA TTT CTT CTA— 3', to amplify gag P17 —P24 lOOObp fragment. Outer primer pairs 2:507A:5-'AAggAACCC TTT AgA gAC TAT gTAgA-3',503B:5 -'TAT ggA TTT TCA ggC CCA ATT TTT g-3',inner primer pairs 2: 508A: 5- 'gTA AAA AAT Tgg ATg ACA gAA ACC TTg-3\ 504B: 5-'ACT TTT ggg CCA TCC ATT CC-3' to amplify gag P24-PR #J 900bp fragment, Outer primer pairs 3: 325A: 5— 'ggA AAC CAA AAA TgA TAg ggg gAA TTg gAg-3?,326B:5-'CTg TAC TTC TgC TAC TAA gTC TTT TgA Tgg g-3', inner primer pairs 3:327A: 5-'gTg gAA AAA Agg CTA TAg gTA CAg-3',328B: 5-'CTg CCA ACT CTA ATT CTg CTT C-3' ,to amplify PR-PT 900bp fragment.7. SequencingProducts were purified with QIAquick Gel Extraction Kit and se-quenced directly with primers PRO—1 ^ RT— 1 (TTA AAg CCA ggA ATggAT gg 2567-2583) and 174A, 506B, 508A, 504B, 327A, 328B by dideoxy chain termination method with ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and AmpliTaq DNA polymerase (FS;Perkin—Elmer, Roissy, France) on genetic analyzer according to the manufacture.8. Drug resistance mutation analysisSequence framents were linked by Vector NTI software package Con-tig Express program. The sequences were aligned with previously reported HIV—1 strains of various subtypes from the Los Alamos database. Multiple alignments were performed automatically by BIOEDIT software with minor manual adjustments. The amino acid sequences deduced from the protease and RT nucleic acid sequences were compared to the published data on Los Alamos database and Stanford HIV drug resistance database and analyzed for mutations associated with reduced sensitivity to antiretroviral drugs.9. Genetic subtypeSequence framents were linked by Vector NTI software package Con-tig Express program. The sequences were aligned by BIOEDIT software? 11 ?with minor manual adjustments. The interpatient genetic distances were calculated using the Kimura two —parameter distance measurement in the DNA DIST part of the Mega 2. 1 and are expressed as the average genetic distance for all pairwise combinations of nucleotide sequences from patients within each group. Neighbor —joining phelogenitic tree was build with Mega 2. 1.10. Recombinant analysisSequence framents were linked by Vector NTI software package Con-tig Express program. The sequences were aligned by BIOEDIT software with minor manual adjustments and upload to the online recombinant a-nalysis program RIP2. 0 on HIV Sequence Database , then we do recombinant analysis with Simplot software. Simplot, Boot scan and Findsites program were used to determine the breakpoint of recombinant, after chi square test, we confirm the correctness of breakpoint by draw Neighbor —joining phelogenitic tree on either side of breakpoint with Mega 2. 1.11. Statistic AnalysisThe data of HIV viral load were normal school after logarithm transforming. Data were expressed as mean SE. Statistical significance was determined by Wilcoxon test using SPSS software (version 11. 0). P |
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