低水平病毒载量长期不进展人类免疫缺陷病毒-1感染者的人类免疫缺陷病毒-1的遗传分析

Genetic analysis of human immunodeficiency virus-I in long-term non-progress human immunodeficiency virus-1 infected individuals with low viral load

目的分析低水平病毒载量长期不进展（LTNP）HIV1感染者HIV-1的遗传特征。方法采用有限稀释套式PCR、终点PCR和序列确证分析等技术对5例低病毒载量LTNPHIV1感染者不同随访时间点H1V1前病毒enu基因c2-v3c3区域和gag基因p17区域进行扩增和序列分析，分别计算不同时间点内以及不同时间点与用于分析的最早时间点之间上述两个基因区域的基因多样性和基因离散率，依据基因离散率计算基因的进化率，统计学分析用GraphPadPrism5软件。结果5例患者在21个随访时间点共获得n5条c2-v3-c3序列和173条p17序列。进化树分析表明，不同患者的序列分开，同一患者的序列特异地聚集，序列质量可靠。5例患者env基因c2-v3-c3区域不同时间点基因多样性为0～6．38％，平均为2．1％，病例1、3和5基因多样性随感染时间的增加逐渐升高（r=0．7957、0．4954、0．3288），病例2和4基因多样性随感染时间的增加逐渐下降（r=-0．3759、-0．5028）；基因离散率为0．1％～6．5％，平均为2．9％，除病例1外，基因离散率均随感染时间间隔的增加逐渐升高，进化率分别为每年每位点-0．13％、0．81％、0．09％、0．14％和0．16％，平均为0．21％。gag基因p17区域基因多样性为0～2．5％，平均为1．2％，基因离散率为0．2％～2．7％，平均为1．4％，除病例3基因多样性和基因离散率随感染时间或时间间隔的增加下降外，其余病例均随感染时间或时间间隔的增加而逐渐升高，进化率分别为每年每位点0．087％、0．064％、-0．014％、0．081％和0．087％，平均为0．061％。结论低水平病毒载量LTNPHIV-1感染者H1V1有复制能力，病毒基因维持较低水平的进化；HIV-1-u基因变化的程度大于gag基因。

Objective To analyze the genetic characteristics of human immunodeficiency virus （HIV） 1 in long-term non-progress （LTNP） HIV-1 infected individuals with low viral load. Methods Limiting dilution nested polymerase chain reaction （PCR） and end point PCR were used to amplify the env gene c2 v3-c3 region and gag gene p17 region of HIV-1 provirus DNA extracted from peripheral blood mononuclear cells （PBMC） of 5 LTNP patients （named as patient 1, 2, 3, 4 and 5） at sequential visiting time points. All sequences of c2-v3 c3 and p17 regions were analyzed by sequence confirm analysis technology, gene diversity between different time points and gene divergence from first time point were then calculated separately. Evolution rate of e2 v3-c3/p17 was calculated based on gene divergence. Statistics analysis was performed by GraphPad Prism 5 software. Results Total 115 sequences of c2-v3-c3 region and 173 sequences of p17 region were obtained from 5 LTNP patients at 21 visiting time points. Results of phylogenetic tree analysis showed that the sequences of different patients were separated and the sequences from the same patient gathered together. The diversity of c2-v3 e3 region at different time points varied from 0 to 6. 38% （mean=2. 1%）, which increased gradually along with the prolonged infection duration in patient 1, 3 and 5, but decreased in patient 2 and 4. The divergence of e2-v3-c3 region varied from 0. 1% to 6. 5% （mean=2. 9%）, which also increased with prolonged infection duration in all patients except patient 1. The evolution rate per site per year for c2-v3-c3 region was 0.13%, 0.81%, 0.09%, 0.14% and 0.16% respectively in the 5 LTNP individuals. The average rate was 0.21%. The diversity of p17 region at different time points varied from 0 to 2.5% （mean=1. 2%） and the divergence of p17 region varied from 0.2% to 2.7% （mean= 1. 4%）, which likewise increased gradually with extended infection duration in all LTNP individuals but patient 3. The yearly evolution rate per site for p17 region was 0. 087%, 0. 064%, -0. 014%, 0. 081% and 0. 087% respectively in the 5 LTNP individuals. The average rate was 0.061%. Conclusions HIV-1 in LTNP HIV1 infected individuals with low viral load shows replicative capability and the viral genes keep on evolving slowly. The variation of HIV-1 env gene is more significant than that of gag gene in these 5 LTNP individuals.