The Study On Relationship Between HIV-1 Tropism And The Disease Progression Of Chinese HIV-infected Individuals

The Study on Relationship Between HIV-1 Tropism and the Disease Progression of Chinese HIV-infected IndividualsObjectiveTo isolate HIV-1 virus stains from HIV/AIDS with microseparation of the whole blood specimens, and to identify the association between HIV-1 cell tropism and HIV/AIDS disease progression. We detected and Statistical Analysis the Virus tropism in GHOST-CCR5 and GHOST-CXCR4 cell line according to viral transmission, disease progression and subtype. The study provide important information for prodiction of HIV-1 cellular tropism and disease progression using bioinformatics approaches.Methods1. Subjects404 HIV-infected individuals confirmed by western blot were recruited from LiaoningHenan, Yunnan and Jilin province. HIV-infected subjects were classified into 3 clinial stages. The first stage was long term non-progressors (LTNPs) and included subjects with persistent CD4+T-cell counts more than 500 cells/μl, no antiretroviral therapy, and no clinical sign of disease for at least 10 years. The second stage was symptomless HIV-infected subjects and included patients who had CD4⁺ T-cell counts less than 500 cells/μl, and no AIDS-defining condition without antiretroviral therapy. The third stage was AIDS and consisted of patients with an AIDS-defining condition according to the World Health Organization classification, including CD4⁺ T-cells less than 200 cells/μl, present or previous opportunistic infections or HIV-related neoplasms. We choose 63 LTNPs, 265 HIV, and 76 AIDS. Blood was collected in EDTA tubes (Becton Dickinson) and processed within 12 hours.2. Sample collection50ml sterile whole blood were collected with EDTA-K3 anticoagulant.3. The origin of HIV-1 stains and HIV-1 virus isolatedOne was lab-virus strain SF33 (donated by CDC in China ). 134 were isolated in this work. HIV-1 virus was cultured by the method of mini-whole blood. PBMC(Peripheral blood mononuclear cell) of healthy donators were isolated by standard Ficoll-hypaque density centrifugation. The cells were stimulated with PHA for 72h in RPMI-1640. Moderating PBMC concentration to 1 million per well. Adding 25, 50, 100μl whole blood respectively to per well and cultured in 37℃. Collected supernatant per 3 days until 30 days and detected p24 and store in -70℃.4. Virus tropism assayCocultured GHOST-CCR5, GHOST-CXCR4 and MT2 cells with 100TCID₅₀ HIV-1 virus in 37℃for 3 hours. Washed with PBS twice at 300g for 10 minutes and cultured with RPMI-1640 and observed cytopathogenic cells and detected p24-antigen per 3 days until 15 days.5. HIV viral load assayHIV-1 viral load(VL) was measured in-thawed plasma samples using a quantitative RT-PCR assay (COBAS AMPLICOR, Roche Company, Switzerland) according to the instructions provided with the HIV-1 RNA monitor Test Kit version 1.5. The detectable range was 400～7.5×10⁵ copies/ml.6. p24 detectionp24 antigen was detected by Biomerieux kit according to instruction. We culture and read the p24 plates in the Triturus automatic Enzyme Immunoassay Analyzer made in Spain. 7. Statistical AnalysisThe data were analyzed with SPSS 11.5 software. Pearson-Chi-Square was used for statistical analysis.Results1. The characters of HIV-seropositive and HIV strains: We had successfully isolated 134 strains of infected patients in our experiments. 41 virus strains were M-tropisc. 17 virus strains were Double-tropisc and 28 were T-tropisc.2. Route of infection, disease progression, viral load and subtype influence the viral isolation. Route of infection may influence the rate of viral isolation. Route of blood and drug infection have the similar rate of viral isolation. Patients with route of drug infection are easier to solute virus. The viral isolation was also obviously increased by the disease progression. The higher viral load, the higher the rate of the viral isolation. The rate of viral isolation was higher in subtype B′/C than in B′.3. M tropism virus is predominate in the route of blood infection. M tropism and Double tropism virus are the main strains in the route of sexual infections. The rate of T tropism is higher in the route of drug infection. The rate of Double tropism is higher in the route of sexual infections and blood infections than the drug infections. M tropism virus is the most, the second is Double tropism virus, T tropism virus is less. With the disease progression, the rate of M tropism virus and Double tropism virus decreased in AIDS than in HIV and LTNP. The rate of T tropism virus is higher in AIDS than in HIV and LTNP (P＜0.05) The rate of T tropism virus is higher in subtype B′/C than in subtype B′.ConclusionsThe rate of viral isolation was possible related to the way of infection. The rate of viral isolation was higher following the disease progression. The viral isolation was higher with the higher viral load. The rate of viral isolation was higher in subtype B′/C than in B′. It is possible that there is some relationship between HIV disease progression and viral tropism. T tropic viral strains is higher in drug infection than in sexual and blood ways. The rate of M tropic is higher in the LTNP and HIV group than the AIDS group. T tropic viral strains is higher in AIDS. The rate of viral tropic may have some relationship with subtype. The rate of T tropism virus is higher in subtype B′/C than in subtype B′. It is possible that virology factor is one of the causes that affecting the disease progression.