Whole-Genome Characterization of Human and Simian Immunodeficiency Virus Intrahost Diversity by Ultradeep Pyrosequencing

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103

h Simian immunodeficiency virus (SIV) stocks for in vivo nonhuman primate models of AIDS are typically generated by transfection of 293T cells with molecularly cloned viral genomes or by expansion in productively infected T cells. Although titers of stocks are determined for infectivity in vitro prior to in vivo inoculation, virus production methods may differentially affect stock features that are not routinely analyzed but may impact in vivo infectivity, mucosal transmissibility, and early infection events. We performed a detailed analysis of nine SIV stocks, comprising five infection-derived SIVmac251 viral swarm stocks and paired infection-and transfected-293T-cell-derived stocks of both SIVmac239 and SIVmac766. Representative stocks were evaluated for (i) virus content, (ii) infectious titer, (iii) sequence diversity and polymorphism frequency by single-genome amplification and 454 pyrosequencing, (iv) virion-associated Env content, and (v) cytokine and chemokine content by 36-plex Luminex analysis. Regardless of production method, all stocks had comparable particle/infectivity ratios, with the transfected-293T stocks possessing the highest overall virus content and infectivity titers despite containing markedly lower levels of virion-associated Env than infection-derived viruses. Transfected-293T stocks also contained fewer and lower levels of cytokines and chemokines than infection-derived stocks, which had elevated levels of multiple analytes, with substantial variability among stocks. Sequencing of the infection-derived SIVmac251 stocks revealed variable levels of viral diversity between stocks, with evidence of stock-specific selection and expansion of unique viral lineages. These analyses suggest that there may be underappreciated features of SIV in vivo challenge stocks with the potential to impact early infection events, which may merit consideration when selecting virus stocks for in vivo studies.

“…Pyrosequencing of viral stocks was done, essentially, as described previously (28). Viral RNA was isolated using the QIAamp MinElute virus spin kit (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

Comparative Characterization of Transfection- and Infection-Derived Simian Immunodeficiency Virus Challenge Stocks for In Vivo Nonhuman Primate Studies

Prete¹,

Scarlotta²,

Newman³

et al. 2013

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103

“…High-throughput (deep) sequencing has been widely used to characterize the diversity of virus variants in natural infections as well as their sequence level adaptations to antiviral drugs (27)(28)(29)(30)(31)(32)(33)(34)(35).…”

mentioning

confidence: 99%

Quantitative Characterization of Defective Virus Emergence by Deep Sequencing

Timm

Akpinar

Yin

2014

Populations of RNA viruses can spontaneously produce variants that differ in genome size, sequence, and biological activity. Defective variants that lack essential genes can nevertheless reproduce by coinfecting cells with viable virus, a process that interferes with virus growth. How such defective interfering particles (DIPs) change in abundance and biological activity within a virus population is not known. Here, a prototype RNA virus, vesicular stomatitis virus (VSV), was cultured for three passages on BHK host cells, and passages were subjected to Illumina sequencing. Reads from the initial population, when aligned to the fulllength viral sequence (11,161 nucleotides [nt]), distributed uniformly across the genome. However, during passages two plateaus in read counts appeared toward the 5= end of the negative-sense viral genome. Analysis by normalization and a simple slidingwindow approach revealed plateau boundaries that suggested the emergence and enrichment of at least two truncated species having medium (ϳ5,900 nt) and short (ϳ4,000 nt) genomes. Relative measures of full-length and truncated species based on read counts were validated by quantitative reverse transcription-PCR (qRT-PCR). Limit-of-detection analysis suggests that deep sequencing can be more sensitive than complementary measures for detecting and quantifying defective particles in a population. Further, particle counts from transmission electron microscopy, coupled with infectivity assays, linked the rise in smaller genomes with an increase in truncated particles and interference activity. In summary, variation in deep sequencing coverage simultaneously shows the size, location, and relative level of truncated-genome variants, revealing a level of population heterogeneity that is masked by other measures of viral genomes and particles. IMPORTANCEWe show how deep sequencing can be used to characterize the emergence, diversity, and relative abundance of truncated virus variants in virus populations. Adaptation of this approach to natural isolates may elucidate factors that influence the stability and persistence of virus populations in nature.

“…An alternate hypothesis for explaining the differential timing of immune escape is that the HIV/SIV genome can tolerate only a limited amount of simultaneous variation, such that variants accumulate in epitopes sequentially. Pyrosequencing has enabled investigators to quantify genome-wide variation within CD8 ϩ T cell epitopes and explore the progression of immune escape with unprecedented resolution (2,3,11,26). Using this approach, we tested the hypothesis that there is an upper limit to the number of SIV genomic sites in which simultaneous variation can be tolerated, such that escape within a given epitope may be delayed by the selection of variants in another epitope.…”

mentioning

confidence: 99%

Conditional CD8⁺T Cell Escape during Acute Simian Immunodeficiency Virus Infection

O’Connor

Becker

Weinfurter

et al. 2012

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CD8 ؉ T cell responses rapidly select viral variants during acute human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. We used pyrosequencing to examine variation within three SIV-derived epitopes (Gag 386-394 GW9, Nef 103-111 RM9, and Rev 59-68 SP10) targeted by immunodominant CD8 ؉ T cell responses in acutely infected Mauritian cynomolgus macaques. In animals recognizing all three epitopes, variation within Rev 59-68 SP10 was associated with delayed accumulation of variants in Gag 386-394 GW9 but had no effect on variation within Nef 103-111 RM9. This demonstrates that the entire T cell repertoire, rather than a single T cell population, influences the timing of immune escape, thereby providing the first example of conditional CD8 ؉ T cell escape in HIV/SIV infection.