| Objective:Owing to the high replication rate and variation rate of HIV-1, there are highly diverse viral quasispecies throughout infection under both of the pressure of patients'immune system and the pressure of the antiretroviral therapy drug, the component of the virul quasispecis changes over time . The method we used for HIV-1isolation is bulk virus isolations,This method will result in the rapid outgrowth of one or few of the infectious HIV-1variants with the most fit phenotype under these isolation conditions ,some slowly replicating variant will be lost,we can not obtain a more complete picture of the diversity of the HIV-1 quasispecies present in an individual in vivo. Now , a limiting dilution virus isolation protocol was developed by foreign scientists, In order to obtain the diversity of the HIV-1 quasispecies present in a chinese Individual. then study the virus biology characteristic of the diversity of the HIV-1 present in Chinese Individual.So far. There has been 27 antiretroviral therapy drug for HIV-1 approved by the U.S.FDA.Only 5 of them are used in free anti-viral treatment for AIDS patients in China. The emergence of resistant strains and drug toxicity, resulting in failure of antiretroviral therapy in AIDS patients .Therefore, it is opportunities and challengesr for the Scientists developing high efficient, low toxicity anti-HIV drug . there are plenty of plants resources in our country, chinese herbal medicine in the application of anti-HIV therapy has been widely reported. the study the anti-HIV antivity and cytotoxicity of potentilla anserine L. extracts in vitro, it is helpful for the development of anti-HIV drugs that have Our independent intellectual property rights.Method:HIV-1 infect individuals were selected from xinjiang province and beijing ,PBMCs were isolated from the patients'whole blood,the PBMCs were diluted into a series of concentration ,then the diluted PBMCs were co-cultured with PHA-stimulated donor PBMCs.HIV-1 P24 was quantified using a commercial enzyme-linked immunosorbent assay kit .The biological clonal HIV-1 variants were determined by fraction of positive cultures. P24 antigen of the biological clonal HIV-1 variants were measured every week by ELISA. HIV-1 V1-V5 region was amplified from HIV-1 biological clonal HIV-1 variants and further was analyzed the differences between the biological clonal HIV-1 variants.Potentilla anserine L. extracts was extracted in our laboratory . cell counting kit-8 (CCk-8)was used in Cytotoxicity assay of potentilla anserine L extracts. The CASY-TT was used in Cytotoxicity assay of potentilla anserine extracts on TZM-bl and MT-4.The anti-SF33 activity of potentilla anserine L. extracts was tested on TZm-bl,MT-4and PBMCs by detecting luciferase activity and P24 expression .Then,the activity of potentilla anserine L. extracts for anti HIV-1 in clinical strains and pseudovirus was tested on TZm-bl by measure luciferase activity.In the anti-HIV activity experiment .TZM-bl,MT-4 and PBMCs were used for detecting the anti-SF33 activity of potentilla anserine L. extracts.the TZM-bl,Mt-4 and PBMCs were infected by 100TCID50 virus,potentilla anserine L.extracts was diluted into 40,20,10,5,2.5,1.25μg/mL,Respectively at the third day and the seventh day. luciferase activity and P24 expression were used to detecte the condition that virus enter the cell and reproduct.the 50% inhibitory concentration(IC50) was calculate by Reed-Muench method. Then the same tests were taken on HIV-1 clinical strains and pseudovirus.Results:1 Multiple HIV-1 variants had been successfμLly isolated from a single PBMCs sample by the limiting dilution-coculture .HIV-1 can be isolated from small amounts infected PBMCs.2 The P24 was quantified every 7 day, General trend of P24 expression is increase over time,which shows us the HIV-1 variants isolated by this method have the ability to infect other PBMCs;in the same conditions,the biological clonal HIV-1 product different content of P24.this Suggest that the virus isolated from a single blood sample are different in the ability to infect and reproduct.3 The analysis of V1-V5 env sequences shows us that a series of virus isolated from a single blood sample are different in Amino acid sequence, especially in V1 and V2 region. Amino acid mutation, insertion, and deletion has been seen in those region. This proves the quasispecies theory.4The cytotoxicity are first measure by cck-8 kit, 50% Cytotoxicity concentration(CC50) of potentilla anserine L. extracts on PBMCS is 92.5μg/mL ,the CC50 of potentilla anserine L. extracts on TZM-bl is 147.5μg/mL . the CC50 on MT-4 is163.4μg/mL then Cytotoxicity experiment were measured by CASY-TT ,the CC50 on TZM-bl is 220μg/mL5 The IC50 of potentilla anserine L. extracts on SF33 is 6.17μg/mL ,which is detected by P24 Assay;the IC50 on SF33 is 4.67μg/mL which is detected by luciferase activity assay; the average IC50 of potentilla anserine L. extracts on HIV-1 Clinical strains is 1.99μg/mL. the average IC50 of potentilla anserine L. extracts on Pseudovirus is 2.43μg/mLConclusions:1 A series of the HIV-1variants can be isolated from the blood sample in a patient,s sample.2 The HIV-1variants isolated by this virus isolation protocol is different for each other in both structure and properties3 potentilla anserine L. extracts can inhibit HIV-1 with the IC50 between 1.99μg/mL and 6.17μg/mL4 some constituents of potentilla anserine L.we known could inhibit HIV-1 by inhibiting HIV-1 protease,there mey has been other constituents in potentilla anserine L could inhibit HIV-1 by inhabiting HIV-1 from entrying cell or reverse transcrtion...
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