In vivo validation of the viral barcoding of SIVmac239 and the development of new barcoded SIV and subtype B and C SHIVs.

Genetically barcoded viral populations are powerful tools for evaluating overall viral population structure as well as assessing the dynamics and evolution of individual lineages 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 SIVmac239, resulting in a synthetic swarm (SIVmac239M) containing approximately 10,000 distinct viral clonotypes for which all genetic differences where within a 34-base barcode that can 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 infectious inoculum dose, and the primary viral growth rate was similar in all infected animals regardless of 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 (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 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. 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 (SIVmac239M2), with over 16-times the number of barcoded variants of the original stock, and a barcoded stock of SIVmac239Opt5M that has 5 changes from wild type SIVmac239. We also generated 4 barcoded stocks from subtype B and C SHIV clones each containing an HIV-1 envelope. These virus models are functional and can be useful for studying viral transmission and HIV cure/reservoir research.