Yukl, S. A. | Search bioRxiv

bioRxiv2021

Novel RT-ddPCR assays for determining the transcriptional profile of SARS-CoV-2

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics. HighlightsO_LIWe developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2 C_LIO_LIOur panel targets nongenic and genic regions in genomic and subgenomic RNAs C_LIO_LIAll assays detect 1-10 copies and are linear over 3-4 orders of magnitude C_LIO_LIAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load Assay C_LIO_LIClinical samples showed higher copy numbers for targets at the 3 end of the genome C_LI

microbiology↗

bioRxiv2020

The Atlas of the In Vivo HIV CD4 T Cell Reservoir

ABSTRACTThe latent reservoir is a main barrier for curing HIV. But because latently-infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently-infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. Most importantly, by selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells, thereby validating the PP-SLIDE approach for reservoir characterization. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies of HIV persistence.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗