In Vivo Validation of the Viral Barcoding of Simian Immunodeficiency Virus SIVmac239 and the Development o New Barcoded SIV and Subtype B and C Simian-Human Immunodeficiency Viruses
In Vivo Validation of the Viral Barcoding of Simian Immunodeficiency Virus SIVmac239 and the Development o New Barcoded SIV and Subtype B and C Simian-Human Immunodeficiency Viruses
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DOI：
10.1128/jvi.01420-19
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发表时间：
2020-01-01
影响因子：
5.4
通讯作者：
Keele, Brandon F.
中科院分区：
文献类型：
--
作者：
Khanal, Sirish;Fennessey, Christine M.;Keele, Brandon F.
基因条形码化的病毒群体是用于评估总体病毒群体结构以及评估体内个体谱系随时间推移的动态和进化的有力工具。条形码化病毒通过将小的、遗传上独特的标签插入病毒基因组中而产生，所述标签保留在子代病毒中。我们最近报告了对表征良好的分子克隆猿猴免疫缺陷病毒（SIV）SIVmac 239进行条形码化，从而产生了一个合成群（SIVmac 239 M），其中包含约10，000种不同的病毒克隆型，所有遗传差异都在34个碱基的条形码内，可以使用下一代深度测序进行跟踪。在这里，我们评估了人口规模，分布和真实性的个别病毒克隆型在这个合成群使用样本从120恒河猴静脉感染。血浆中复制条形码的数量与感染性接种物剂量相关，并且无论接种物大小如何，所有感染动物的原代病毒生长速率相似。总体而言，在至少一只感染动物的血浆中鉴定了病毒储备液中97%的可检测克隆型。此外，我们制备了第二代条形码化SIVmac 239储备液（SIVmac 239 M2），其条形码化变体的数量是原始储备液的16倍以上，以及另外的条形码化储备液，其具有校正的次优核苷酸（SIVmac 239 Opt 5 M）。我们还从亚型B和C猿猴-人免疫缺陷病毒（SHIV）克隆产生了四种条形码化的储备物。这些新的SHIV克隆可能是评估Env靶向方法以研究病毒传播或病毒库清除的特别有价值的模型。总的来说，这项工作进一步建立了可靠的条形码病毒的方法，并强调适应这种技术的可行性，以其他viral clones.IMPORTANCE我们最近开发并发表了一个描述的条形码猿免疫缺陷病毒，有一个短的随机序列直接插入到病毒基因组。这使得能够以高保真度和超深灵敏度追踪单个病毒谱系。这种病毒被用来感染120恒河猴，我们在这里报告这些动物在初次感染的条形码分析。我们发现绝大多数条形码在体内是有功能的。然后，我们在第二代SIVmac 239原种（SIVmac 239 M2）中扩展了条形码化方法，其条形码化变体的数量是原始原种的16倍以上，并且SIVmac 239 Opt 5 M的条形码化原种的序列与野生型SIVmac 239序列相比具有5个变化。我们还从亚型B和C SHIV克隆中产生了4种条形码化的原液，每种均含有1型人免疫缺陷病毒（HIV）包膜。这些病毒模型是功能性的，可用于研究病毒传播和HIV治疗/储库研究。
Genetically barcoded viral populations are powerful tools for evaluating the overall viral population structure as well as assessing the dynamics and evolution of individual lineages in vivo over time. Barcoded viruses are generated by inserting a small, genetically unique tag into the viral genome, which is retained in progeny virus. We recently reported barcoding the well-characterized molecular clone simian immunodeficiency virus (SIV) SIVmac239, resulting in a synthetic swarm (SIVmac239M) containing approximately 10,000 distinct viral clonotypes for which all genetic differences were within a 34-base barcode that could be tracked using next-generation deep sequencing. Here, we assessed the population size, distribution, and authenticity of individual viral clonotypes within this synthetic swarm using samples from 120 rhesus macaques infected intravenously. The number of replicating barcodes in plasma correlated with the infectious inoculum dose, and the primary viral growth rate was similar in all infected animals regardless of the inoculum size. Overall, 97% of detectable clonotypes in the viral stock were identified in the plasma of at least one infected animal. Additionally, we prepared a second-generation barcoded SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and an additional barcoded stock with suboptimal nucleotides corrected (SIVmac239Opt5M). We also generated four barcoded stocks from subtype B and C simian-human immunodeficiency virus (SHIV) clones. These new SHIV clones may be particularly valuable models to evaluate Env-targeting approaches to study viral transmission or viral reservoir clearance. Overall, this work further establishes the reliability of the barcoded virus approach and highlights the feasibility of adapting this technique to other viral clones.IMPORTANCE We recently developed and published a description of a barcoded simian immunodeficiency virus that has a short random sequence inserted directly into the viral genome. This allows for the tracking of individual viral lineages with high fidelity and ultradeep sensitivity. This virus was used to infect 120 rhesus macaques, and we report here the analysis of the barcodes of these animals during primary infection. We found that the vast majority of barcodes were functional in vivo. We then expanded the barcoding approach in a second-generation SIVmac239 stock (SIVmac239M2) with over 16 times the number of barcoded variants of the original stock and a barcoded stock of SIVmac239Opt5M whose sequence had 5 changes from the wild-type SIVmac239 sequence. We also generated 4 barcoded stocks from subtype B and C SHIV clones each containing a human immunodeficiency virus (HIV) type 1 envelope. These virus models are functional and can be useful for studying viral transmission and HIV cure/reservoir research.