Fig 1.
Schematic illustration of viral spread of PrV in mice after intranasal infection.
After initial replication in the respiratory epithelium (RE) PrV primarily invades sensible/sensoric fibers of the trigeminal nerve (red) and is transported via 1st order neurons to trigeminal nuclei (Sp5, Pr5, Me5). From there, 2nd order neurons project to the thalamus (TH). Eventually, thalamic fibers end up as third order neurons in the somatosensory cortex (SSC) located in the parietal lobe (PL). PrV does also infect the nasal glands (NG), oral mucosa (OM), nasopharynx (NP), and salivary glands (SG) which are all innervated by the trigeminal nerve. Viral spread further occurs via sympathetic (green) or parasympathetic fibers (blue) and the corresponding autonomic ganglia such as the superior cervical ganglion (SCG) and pterygopalatine ganglion (PtG), otic ganglion (GO), geniculate ganglion (GG), and distal ganglion (GD), respectively. Although not yet reported in mice, herpesviral infection may be facilitated through the olfactory epithelium (OE) and the olfactory nerve (yellow) to finally infect the olfactory bulb (OB) (insert).
Fig 2.
In vitro growth properties and cell-to-cell spread of mutant viruses.
(A) RK13 cells were infected with PrV-Ka and the respective virus mutants at a MOI of 3, and harvested 24 h p.i. Plaque assays performed on RK13 cells were used to determine the titer of virus progeny. (B) Plaque diameters of the different virus mutants were assessed on RK13 cells. 20 plaques each per virus mutant were measured and compared to plaque diameter of PrV-Ka set as 100%. Average values and standard deviations of three independent experiments are shown. Significant differences of virus mutants compared to PrV-Ka are indicated by an asterisk (*).
Fig 3.
Kaplan-Meier curve showing the relative survival rates of mice after inoculation with PrV-Ka and mutants.
Mice (PrV-Ka, n = 4; PrV-US3Δkin, n = 4; PrV-ΔUL21, n = 4; PrV-ΔUL21/US3Δkin, n = 6) were inoculated intranasally with a total of 10μl virus suspension containing 1x104 PFU. The animals were daily monitored and sacrificed in the moribund stage. In contrast to mice infected with PrV-Ka, PrV-US3Δkin and PrV-ΔUL21, PrV-ΔUL21/US3Δkin-infected animals survived the infection except for one animal that had to be euthanized at 239 h p.i. The survival rates differ significantly (log-rank test, p < 0,0001) between the groups.
Fig 4.
Prevalence of diseased animals after infection with PrV-Ka, PrV-US3Δkin, PrV-ΔUL21 and PrV-ΔUL21/US3Δkin in the kinetic trial.
Starting 24 h p.i. at least 3 mice per indicated time point were sacrificed and submitted for pathohistological investigation. The frequency of sick animals infected with PrV-Ka (A), PrV-US3Δkin (B), PrV-ΔUL21 (C) or PrV-ΔUL21/US3Δkin (D) per time is indicated by bars. Based on a scoring system (S1 Table) animals were categorized into either mildly (green), moderately (yellow) or severely affected (red). Prevalence is given by the black line on the right Y-axis.
Fig 5.
Viral antigen score in mice infected with mutant viruses.
Histological specimen of mice infected with PrV-Ka (A), PrV-US3Δkin (B), PrV-ΔUL21 (C) and PrV-ΔUL21/US3Δkin (D) were scored for viral antigen distribution with negative (0), focal to oligofocal (1), multifocal (2) and diffuse (3). The final average value resulted from points given for each tissue section analyzed. Significant differences of mutant viruses compared to the mock-infected control group are indicated with an asterisk (*).
Fig 6.
Colored boxes indicate viral antigen in the respective areas after infection with PrV-Ka (red), PrV-US3Δkin (green), PrV-ΔUL21 (yellow) and PrV-ΔUL21/US3Δkin (blue). TG = trigeminal ganglion, PtG = pterygopalatine ganglion, SCG = superior cervical ganglion, Sp5 = spinal trigeminal nucleus, Pr5 = principal sensory trigeminal nucleus, Sol = solitary tract, IO = inferior olive, Cu = cuneate nucleus, FR = reticular formation, MET = metencephalic regions others than Pr5, MES = mesencephalic regions others than Me5, DI = diencephalic regions, HYPO = hypothalamus, TH = thalamus, TEL = telencephalic regions, FL = frontal lobe, PL = parietal lobe, MTL = mesiotemporal lobe, PIR = piriform lobe, HIP = hippocampus.
Fig 7.
Detection of viral antigen in mice infected with PrV-ΔUL21/US3Δkin.
Immunohistochemistry (anti PrV-gB) of the trigeminal ganglion (TG), brainstem (BS) and mesiotemporal lobe (MTL) are shown in A) 132–168 h p.i., B) 192–240 h p.i., C) 264 h p.i. and D) 288–360 h p.i. Positive staining is highlighted by arrows. A higher magnification of single infected neurons is shown in the inset in B) MTL.
Fig 8.
Inflammation score in infected mice.
Histological specimen of mice infected with PrV-Ka (A), PrV-US3Δkin (B), PrV-ΔUL21 (C) and PrV-ΔUL21/US3Δkin (D) were scored for inflammation with 0 = no inflammation, 1 = mild inflammation, 2 = moderate inflammation, 3 = severe inflammation. The final average value resulted from points given for each tissue section analyzed. Significant differences of mutant viruses compared to the mock-infected control group are indicated with an asterisk (*).
Fig 9.
Sites of inflammatory response.
Colored boxes indicate inflammation in the respective areas after infection with PrV-Ka (red), PrV-US3Δkin (green), PrV-ΔUL21 (yellow) and PrV-ΔUL21/US3Δkin (blue). TG = trigeminal ganglion, PtG = pterygopalatine ganglion, SCG = superior cervical ganglion, Sp5 = spinal trigeminal nucleus, Pr5 = principal sensory trigeminal nucleus, Sol = solitary tract, IO = inferior olive, CU = cuneate nucleus, FR = reticular formation, SC = spinal cord, MET = metencephalic regions others than Pr5, MES = mesencephalic regions others than Me5, DI = diencephalic regions, HYPO = hypothalamus, TH = thalamus, TEL = telencephalic regions, FL = frontal lobe, PL = parietal lobe, MTL = mesiotemporal lobe, PIR = piriform lobe, HIP = hippocampus, AMY = amygdala.
Fig 10.
Detection of inflammation in mice infected with PrV-ΔUL21/US3Δkin.
Hematoxylin and eosin stains of the trigeminal ganglion (TG), brainstem (BS) and mesiotemporal lobe (MTL) are shown in A) 132–168 h p.i., B) 192–240 h p.i., C) 264 h p.i. and D) 288–360 h p.i. Perivascular and parenchymal inflammatory infiltrates consisting of lymphocytes and histiocytes are highlighted by arrows.
Fig 11.
Inflammatory response in the mesiotemporal lobe.
(A) Necrotic neurons (arrow) are surrounded by inflammatory cells and activated microglia. (B and C) Necrotic neurons in the cerebral cortical layer (arrow) with satellitosis and gliosis, as well as parenchymal infiltration of the adjacent neuropil (C).