1. Volume 20, Issue 1, Pages 251-263 (July 2017)
Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method 
Dan Dou, Iván Hernández-Neuta, Hao Wang, Henrik Östbye, Xiaoyan Qian, Swantje Thiele, Patricia Resa-Infante, Nancy Mounogou Kouassi, Vicky Sender, Karina Hentrich, Peter Mellroth, Birgitta Henriques-Normark, Gülsah Gabriel, Mats Nilsson, Robert Daniels 
Cell Reports 
Volume 20, Issue 1, Pages (July 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 251-263DOI: (10.1016/j.celrep.2017.06.021)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1 Single-Cell IAV Gene Segment vRNA Labeling Approach
(A) Diagram of an IAV and the eight single-stranded vRNA gene segments.
(B) PLP design for detecting the IAV gene segments. Each PLP contains ∼20-nucleotide 5′ and 3′ arms that hybridize to adjacent sequences in a gene segment and barcodes for identifying the gene segment (colored) and strain (brown).
(C) In situ labeling of IAV gene segments. cDNAs are synthesized from each vRNA in fixed cells. Templates are digested using RNase H. PLPs are hybridized and ligated to produce a template for synthesizing a rolling-circle product (RCP), which creates a concatemer of complementary barcode copies that are detected using complementary fluorescently labeled barcode probes.
(D) Single-cell RCP visualization and quantification. RCPs are detected by cycles of barcode probe hybridization (up to five probes at a time), fluorescence microscopy imaging, and stripping. RCP images from different cycles are compiled and aligned, and the RCPs are assigned to the nuclei and cell segments defined by nuclei staining and cell autofluorescence.
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4. Figure 2 PLPs Detect IAV Gene Segments during Entry and Replication
(A) The IAV genome (vRNA) replication cycle. After endocytosis, IAVs fuse with the endosomal membrane, releasing the vRNAs into the cytosol. The vRNAs are replicated in the nucleus by the vRNA-dependent RNA polymerase (vRdRP), assembled, and exported for packaging at the plasma membrane. BFLA1 inhibits endosomal acidification and fusion. LMB inhibits the nuclear export.
(B) qPCR analysis of cellular and extracellular (media) NP and NA vRNA levels. vRNA levels were normalized to 0 hr p.i. (cellular) or 4 hr p.i. (extracellular). Mean values with the SD from two independent experiments are displayed.
(C) The in situ labeling of IAV gene segments in untreated and BFLA1-treated (100 nM) MDCK cells 2 hr p.i. The percentage of gene segment RCPs that co-localize with the nucleus and the SD were determined from two independent experiments analyzing ∼1,000 cells. The box-and-whisker plot (5%–95% CI) shows the nucleus image occupancy from the corresponding image fields.
(D) In situ labeling of IAV gene segments in untreated or LMB-treated (10 nM) MDCK cells at 8 hr p.i. LMB was added 5 hr p.i. The RCP nuclear co-localization and SD were determined from three independent experiments.
(E) Representative image of the eight IAV gene segment RCPs generated in MDCK cells 8 hr p.i. with an MOI of ∼0.3. Cell nuclei were stained with Hoechst. Inset shows the higher magnification of a cell.
(F) Graph showing the percentage of ∼1,000 cells that contained the indicated number of distinct gene segment RCPs in the nucleus at 8 hr p.i.
(G) Correlation plot showing the relative increases in the NP vRNA levels versus the number of NP gene segment RCPs in ∼500 infected cells at the indicated times post-infection. Values normalized to 4 hr p.i. and are shown as ± SD.
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5. Figure 3
Detection of IAV Gene Segments with Single-Nucleotide Variations
(A) Diagram depicting the single-nucleotide differences in each WSN and WSNIso gene segment. Amino acid codons for the indicated IAV gene are given as a position reference.
(B) Viral titers obtained during the rescue of WSN and WSNIso by reverse genetics in co-cultured 293T and MDCK cells.
(C) Representative immunoblots showing the relative NA, HA, NP, and M1 protein levels in isolated WSN and WSNIso particles.
(D) PLP pair design to differentiate IAV gene segments that differ by one nucleotide. In each gene segment PLP pair, the terminal 3′ nucleotide (strain-specific nucleotide) is designed to hybridize with the respective vRNA nucleotide substitution in the WSNIso or WSN sequence.
(E) The recognition for each gene segment PLP pair was determined in solution (Figure S3) by quantifying the number of RCPs generated from the same amount of vRNA.
(F) RNA was extracted from MDCK cells (16 hr p.i.) or mouse lung tissue harvested 12 hr p.i. with the indicated viruses and RCPs were generated with the HA PLP pair. The graph shows the percentage of WSN (green) and WSNIso (red) RCPs from each sample.
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6. Figure 4
In Situ IAV Genome Labeling Can Identify Infections with Single-Nucleotide Precision
(A) MDCK cells infected with the indicated virus were labeled 6 hr p.i. using the individual gene segment PLP pairs (Figure S4), and the resulting number of RCPs for each PLP are displayed as a box-and-whisker plot.
(B) MDCK cells infected with WSN or WSNIso were labeled 6 hr p.i. with the eight PLP pairs. Strain-specific barcodes were used for visualization, and the RCP per cell numbers for each PLP set are shown for ∼25,000 cells.
(C) MDCK cells infected with WSN or WSNIso were trypsinized 2 hr p.i., the cells were combined, reseeded on the same slide, and labeled 4 hr later with the eight WSN and WSNIso PLP pairs. A representative image is shown with arrows indicating WSN-infected (green) or WSNIso-infected (red) cells. The an inset shows the nuclei staining.
(D) Representative images of lung cryosections from mice 2 days post-inoculation with WSN or WSNIso, or co-inoculation with WSN and WSNIso, which were labeled with the eight WSN and WSNIso PLP pairs. The insets display the mosaics of the lung sections with the respective image region. The number of RCPs and the SD from the indicated PLP set was obtained from analyzing two entire lung cryosection mosaics. RCP signals are displayed as enlarged circles for better visualization.
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7. Figure 5
Single-Cell Analysis of IAV Entry by In Situ Gene Segment Labeling
(A) Infection procedure for IAV gene segment labeling during viral entry. WSN at either a low (∼0.3) or a high (∼3) MOI was bound to MDCK cells at 4°C, unbound virus was removed, and entry was initiated by shifting the cells to 37°C. Labeling was performed at the indicated times post-binding.
(B) Percentage of cells possessing one or more RCPs plotted with respect to the time post-binding for the low and high MOI infections. For each time point, ∼1,000 cells were examined.
(C) The number of gene segment RCPs per infected cell is shown as a box-and-whisker plot for ∼1,000 cells at each time point following infection with the low (left panel) or high (right panel) MOI.
(D and E) Representative images of the IAV gene segment labeling at the indicated times post-binding with the low (D) and high (E) MOI. The RCPs were visualized with the strain-specific barcode (low and high MOI) and the gene segment barcodes (high MOI).
(F) Cell diagrams of the parameters for classifying the five infection stages based on the number of labeled gene segments and the percentage of nuclear co-localization.
(G) Distribution of the infection stages in the cell population at the indicated times following infection with the low (left panel) or high (right panel) MOI. More than 12,000 cells were analyzed.
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8. Figure 6
Co-labeling of IAV Genomes Reveals the Time Window for Productive Cell Co-infections
(A) Diagram of the co-infection experiment. Cells were bound with the primary IAV (WSN) at 4°C using an MOI of ∼3, unbound virus was removed, and the secondary IAV (WSNIso) was added at an equal MOI at the indicated times (red arrows) after the 37°C temperature shift. Cells were labeled 8 hr p.i. with WSN, resulting in increasingly shorter durations of the WSNIso infections.
(B) Representative control images of the WSNIso gene segment labeling to account for the shorter durations of the infection. Cells were only infected with WSNIso.
(C) Representative images of the WSN (green) and WSNIso (red) gene segment labeling in co-infected cells. The secondary WSNIso infections were initiated at the indicated times after WSN binding (listed on the left). The durations of the WSNIso infections are listed on the right.
(D) Distribution of the WSNIso infection stages in the control samples (no WSN primary infection) as the duration of the infection becomes shorter.
(E) Left: distribution of the WSNIso infection stages as the virus was added at increasing time intervals (listed in parentheses) after the primary WSN infection. For each time point, ∼2,000 cells were examined. Right: combined distribution of the WSN infection stages in the cell populations from all samples.
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9. Figure 7
The Primary IAV Replication Stage Defines the Cell Susceptibility to Secondary Infections
The model displays the distribution of the five IAV replication stages that were observed by gene segment PLP labeling over the course of an infection. Each stage is diagramed with respect to the cell population and the corresponding infection time that was observed using a single (primary) IAV infection. The time window and the stages of a primary IAV infection in which the cell is susceptible to co-infection by a secondary IAV are shown above the graph.
Cell Reports  , DOI: ( /j.celrep )
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