Fig. 4.

Prevalence of VLT and ORF63 transcript in human TG. a PCR amplification of cDNA (n = 5 TGs, Supplementary Table 1), synthesized in the presence (+) or absence (−) of reverse transcriptase. Sanger sequencing of all five purified PCR products yielded identical sequences corresponding to VLT (Fig. 3b). b Integrative Genomics Viewer (IGV) screenshot showing representative RNA-Seq data from a single TG. Paired-end reads are shown as fragments mapped across the VLT locus. Grey boxes (exons) connected by black lines (introns) indicate individual read pairs with distinct read-pairs separated by white space. Each fragment spans between 2 and 5 exons and there was no evidence of additional upstream exons. c In lytically VZV-infected MeWo cells, Sanger sequencing of amplicons generated through rapid amplification of cDNA ends revealed multiple VLT_ly isoforms, visualized using IGV. Sequencing of 29 clones identified the three most abundant VLT_ly isoform groups (red boxes) A (35%), B (19%) and C (15%). Note that the specific VLT isoform observed in latently infected TG was not observed among VLT_ly isoforms. d Quantification of ORF63 transcript and VLT isoforms in lytically VZV-infected ARPE-19 cells (red; n = 3) and latently VZV-infected TG (blue; n = 19 TG), using primers/probes spanning VLT splice junctions between exons 3→4, A→1, B→1 and C→1 (Fig. 4c and Supplementary Table 5). Data represent mean (±SEM) relative transcript levels normalized to β-actin RNA. nd not detected. e Levels of paired ORF63 transcript and VLT (primers/probe spanning exon 2→3) in the same VZV^POS TG (VZV^POS; n = 15) determined by RT-qPCR. ***p < 0.001; Wilcoxon signed rank test. f Correlations (Spearman) between relative ORF63 transcript and VLT levels in VZV^POS TG (n = 15), as determined by RT-qPCR. g–j In situ hybridization (ISH; red signal) analysis of ORF63 RNA and VLT in latently VZV-infected TG (n = 12). g Frequency of neurons positive for ORF63 RNA and VLT in consecutive TG sections of the same donor. *p < 0.05; paired Student’s t-test. h Nuclear and cytoplasmic ORF63 RNA and VLT expression in consecutive TG sections from individual donors. ns not significant; paired Student's t-test. i, j Representative ISH images of VZV^POS TG sections (i) and two VZV naive human fetal dorsal root ganglia (j). Nuclei were stained with haematoxylin. Magnification: ×400 (with 3× digital zoom for insets). Bars = 50 µm