Fig 1.
Design of HSV-1 viruses that express eGFP/Cre.
(A) Schematic representation of the HSV-1 genome (to scale). (B) Schematic representation of the UL3/UL4 intergenic region, with the position at which the eGFP/Cre cassette is inserted indicated in green, and the base pair position indicated using coordinates from the HSV-1 KOS genome (JQ673480). Schematic representation of the eGFP/Cre gene under the control of the (C) ICP6 promoter, (D) gB promoter, (E) ICP47 promoter or (F) ICP0 promoter inserted into the HSV-1 genome, with the sequences of the promoter indicated based on HSV-1 KOS genome coordinates. (G) Schematic representation of the ROSA26 locus of ROSA26 mice. A splice acceptor sequence (SA) is located upstream of a neomycin gene (neo) followed by a triple polyadenylation site, all of which is flanked by loxP sites. The ROSA26 promoter is located upstream of this transgene, while the lacZ gene is located downstream. Following Cre-mediated recombination, the neo transgene is removed and lacZ is constitutively expressed from the ROSA26 promoter.
Fig 2.
HSV continues to spread beyond the acute infection before latency is stably established.
Groups of ROSA26 mice were infected with 1 × 108 PFU/mL HSV-1 pC_eGC by tattoo on the flank and were culled at indicated times p.i. The innervating DRG were removed (from spinal levels L1 to T5) and the total number of β-gal+ cells per mouse was determined. (A) Representative photomicrographs of DRG at spinal levels T13, T10 and T7 of a single mouse for each day p.i. taken at a 40× magnification (scale bar = 300 μm, as indicated on top left image). (B) Total number of β-gal+ cells per mouse (data from some mice have been published previously [24]) and (C) spread of virus as indicated by the number of DRG containing β-gal+ cells. Each point represents a single mouse and the bar represents the mean cell count (n = 9–18 per day p.i.). The results of four independent experiments were pooled. (D) Total number of β-gal+ cells per mouse and (E) spread of virus as indicated by the number of DRG containing β-gal+ cells, as measured at two day intervals between days 3 and 15 p.i. Data from two independent experiments were pooled. (*p < 0.05, **p < 0.01, ***p < 0.001; red denotes differences of particular note).
Fig 3.
Conventional reporter genes show only minor lytic gene expression after the peak of acute infection and none in latency.
Groups of C57Bl/6 mice were infected with KOS6.β by tattoo on the flank and were culled at the indicated times p.i. for the determination of the number of β-gal+ cells per DRG. (A) Representative photomicrographs of DRG at spinal levels T12, T10 and T8 of a single mouse for either 4 or 10 days p.i. taken at 40× magnification (scale bar = 300 μm, as indicated on the top left image). (B) The total number of β-gal+ cells per mouse was estimated. (C) The spread of virus as determined by the number of DRG which contain at least one β-gal+ cell. The results of two independent experiments are pooled (n = 8), with each point representing a single mouse and the bar representing the mean cell count. (**p < 0.01, ***p < 0.001).
Fig 4.
The promoter for ICP0 is active through the establishment phase but not true latency.
Groups of ROSA26 mice were infected with HSV-1 pICP0_eGC by tattoo on the flank and were culled at the indicated times p.i. and the number of β-gal+ cells in each DRG was determined. (A) Representative photomicrographs of DRG at spinal levels T12, T10 and T8 of a single mouse for each day p.i. taken at 40× magnification (scale bar = 300 μm, as indicated on top left image). (B) The total number of β-gal+ cells per mouse and (C) the spread of virus as indicated by the number of DRG containing β-gal+ cells was determined. The data were pooled from three independent experiments (n = 10–11 for each time point). (*p < 0.05, **p < 0.01)
Fig 5.
The promoter for gB drives protein expression during latency.
Groups of ROSA26 mice were infected with HSV-1 pgB_eGC by tattoo on the flank and were culled at either (B, C) 5, 10, 20 and 40, or (D, E) 10, 21 and 100 days p.i. for determination of the number of β-gal+ cells per DRG. (A) Representative photomicrographs of DRG at spinal levels T12, T10, and T8 of a single mouse for each day p.i. taken at a 40× magnification (scale bar = 300 μm, as indicated on top left image). To examine the accumulation of β-gal+ cells throughout the acute infection and establishment of latency, (B) the total number of β-gal+ cells per mouse and (C) the spread of virus as indicated by the number of DRG containing β-gal+ cells was determined. The data are pooled from 5 independent experiments, with each point represents a single mouse and the bar represents the mean cell count (n = 16–18 per day p.i.). To examine the accumulation of β-gal marked cells in ROSA26 mice infected with HSV-1 pgB_eGC over the long term the (D) total number of β-gal+ cells per mouse and (E) the spread of virus as indicated by the number of DRG containing at least one β-gal+ cell is shown. The data are pooled from 3 independent experiments (n = 14–15). (**p < 0.01, ***p < 0.001; red denotes differences of particular note).
Fig 6.
The promoter for ICP6 drives protein expression during latency.
Groups of ROSA26 mice were infected with HSV-1 pICP6_eGC by tattoo on the flank and were culled at 5, 10, 20 and 40, or 10, 20, and 100 days p.i. for determination of the number of β-gal+ cells per DRG. (A) Representative photomicrographs of DRG at spinal levels T11, T10, and T9 of a single mouse for each day p.i. taken at 40× magnification (scale bar = 300 μm, as indicated on top left image). (B) The total number of β-gal+ cells per mouse and (C) the spread of virus as indicated by the number of DRG containing β-gal+ cells was determined. The data were pooled from six independent experiments, with each point representing a single mouse and the bar representing the mean cell count (n = 11–24 per day p.i.). Differences between groups were assessed using Kruskal Wallis test with Dunn’s posttest for pairwise comparison (**p < 0.01, ***p < 0.001; red denotes differences of particular note).
Fig 7.
The promoter for ICP47 drives protein expression during the establishment of latency.
Groups of ROSA26 mice were infected with HSV-1 pICP47_eGC by tattoo on the flank and were culled 5, 10, 20 and 40, or 10, 20 and 100 days p.i. for determination of the number of β-gal+ cells per DRG. (A) Representative photomicrographs of DRG at spinal levels T12, T10 and T8 of a single mouse for each day p.i. taken at 40× magnification (scale bar = 300 μm, as indicated on top left image). (B) The total number of β-gal+ cells per mouse and (C) the spread of virus as indicated by the number of DRG containing β-gal+ cells was determined. The data were pooled from six independent experiments, with each point representing a single mouse and the bar representing the mean cell count (n = 14–22 per day p.i.). (D) The total number of β-gal+ cells per mouse and (E) the spread of virus as indicated by the number of DRG containing β-gal+ cells confirmed the gradual accumulation of β-gal marked cells during the establishment of latency. The data were pooled from two independent experiments, with each point representing a single mouse and the bar representing the mean cell count (n = 6–8 per day p.i.). (*p < 0.05, **p < 0.01, ***p < 0.001; red denotes differences of particular note).
Fig 8.
Data from all experiments where β-gal-marked neurons were counted in DRG after infection of mice with recombinant viruses. The viruses are identified by the gene that was the source of promoter used to drive Cre, or by name (in the case of KOS6β). (A) Total numbers of marked neurons and (B) numbers expressed as a percentage of the maximum counted at the peak time point.