Genetically-barcoded SIV facilitates enumeration of rebound variants and estimation of reactivation rates in nonhuman primates following interruption of suppressive antiretroviral therapy

HIV and SIV infection dynamics are commonly investigated by measuring plasma viral loads. However, this total viral load value represents the sum of many individual infection events, which are difficult to independently track using conventional sequencing approaches. To overcome this challenge, we generated a genetically tagged virus stock (SIVmac239M) with a 34-base genetic barcode inserted between the vpx and vpr accessory genes of the infectious molecular clone SIVmac239. Next-generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. In vitro infection of rhesus CD4+ T cells and in vivo infection of rhesus macaques revealed levels of viral replication of SIVmac239M comparable to parental SIVmac239. After intravenous inoculation of 2.2x105 infectious units of SIVmac239M, an average of 1,247 barcodes were identified during acute infection in 26 infected rhesus macaques. Of the barcodes identified in the stock, at least 85.6% actively replicated in at least one animal, and on average each barcode was found in 5 monkeys. Four infected animals were treated with combination antiretroviral therapy (cART) for 82 days starting on day 6 post-infection (study 1). Plasma viremia was reduced from >106 to

HIV与SIV感染动态通常通过检测血浆病毒载量（plasma viral loads）开展研究。然而，该总病毒载量数值代表了诸多独立感染事件的总和，而传统测序方法难以独立追踪此类事件。为破解这一难题，我们构建了带有遗传标签的病毒株SIVmac239M，其在传染性分子克隆SIVmac239的vpx与vpr辅助基因之间插入了一段34碱基的遗传条形码（genetic barcode）。对该病毒株开展下一代测序（next-generation sequencing）后，共鉴定出至少9336个独立条形码或克隆型（clonotypes），任意两个条形码之间的平均遗传距离为7个碱基。对恒河猴CD4+ T细胞的体外感染实验以及对恒河猴的体内感染实验均证实，SIVmac239M的病毒复制水平与亲本株SIVmac239相当。在静脉接种2.2×10^5个感染性单位的SIVmac239M后，26只感染恒河猴在急性感染阶段平均可检测到1247个条形码。在病毒株中鉴定出的条形码里，至少85.6%可在至少一只动物体内实现主动复制，且平均每个条形码可在5只猴子体内被检出。其中4只感染动物于感染后第6天开始接受联合抗逆转录病毒治疗（combination antiretroviral therapy，cART），持续时长为82天（研究1）。血浆病毒血症（plasma viremia）水平从>10^6降至