人类免疫缺陷病毒储存库评估测定技术研究进展

Research advances in evaluation and measurement techniques of latent human immunodeficiency virus reservoirs

HIV整合至CD4＋T细胞、脾脏、淋巴结、胃肠相关淋巴组织中，成为HIV潜伏感染的储存库。学界提出“shock and kill”的治疗策略，即先激活释放再杀灭病毒。而实施该策略的前提是评估和测定HIV储存库的大小。本文介绍目前常用的测定HIV储存库方法如Alu-gagPCR、定量病毒产物分析及tat／rev诱导有限稀释法。Alu．gagPCR可区别整合与未整合病毒基因，但不能区分缺陷与有功能的前病毒，因此易高估储存库。定量病毒产物分析在纯化静止CD4＋T细胞中进行，被誉为HIV储存库检测的“金标准”，但实验成本高，技术要求高，所需标本量大，仅由一轮激活并不能完全诱导产物释放，因此会低估储存库。Tat／rev诱导有限稀释法主要测定经由刺激物诱导表达多拼接RNA等生物学标志的潜伏感染细胞频数，具有所需血标本少，不用抽提病毒RNA，实验时间短，对于不同大小储存库都适用等优点，但可能高估潜伏储存库。

Latent reservoir （LR） of HIV is the cells （such as CD4＋T cell） where HIV is able to hide. These cellular reservoirs are located throughout the body,including the spleen, lymph nodes, gastrointestinal lymphoid tissues, and become the major obstacle to cure HIV infection. To truly cure patients, a new strategy ＂shock and kill＂ was put forward by scientists, which is to shock HIV-infected ceils out of hidden reservoirs in the body and then kill them. Quantitatively evaluating the size of long-lived LR is essential to this strategy. This paper reviews assays that measure the magnitude of the latent HIV reservoir, including Alu-gag PCR, quantitative viral outgrowth assay （ Q- VOA） and tat/rev induced limiting dilution assay （TILDA）. Alu-gag PCR can differentiate the integrated and un-integrated HIV DNA, however, it cannot distinguish defective virus from competent virus, leading to overestimate the real size of LR. Q- VOA is based on cell culture, and is the golden standard for measuring the LR since it provides a definitive minimal estimate of reservoir size. Its disadvantages are being more costly, large amount of blood sample required, and underestimating the true size, which was resulted from particles being not released after one round of stimulation. TILDA measures ceils with inducible msRNA as these transcripts are absent in latently infected cells but induced upon viral reactivation. It requires small blood sample size, does not need extraction of viral nucleic acids, can be completed in 2 d and covers a wide dynamic range of reservoir sizes, but has the disadvantage of overestimating the true size of LR.