Fig 1.
Weight change, viremia and serum IFN-α in JUNV and GTOV infected guinea pigs.
Guinea pigs (n = 16 per virus) were infected with the indicated viruses at 2,000 PFU by the IP route. Three uninfected animals were included as controls. A. Weight loss was monitored over the course of the study and individual weights were calculated based on Day 0 starting weight. B. Viremia (n = 4 guinea pigs per virus per time point) was determined by plaque assay. Log10 titer (pfu/ml) were calculated and graphed with standard deviation. C. Levels of serum IFN-α were determined by guinea pig-specific ELISA. Uninfected guinea pigs (N = 3) were used as a baseline control. Dashed line indicates mean titer of uninfected animals plus three standard deviations.
Fig 2.
Temporal severity of histology lesions in lymph nodes and spleen for both JUNV and GTOV groups.
In graphs A–I select histology lesions are graphed for the lymph nodes and spleen to display changes in lesion severity over time between JUNV and GTOV. A single dot or square represents a single animal and the blue or red shaded region represents the median.
Table 1.
Summary of major histopathology and ISH findings by number of animals affected, for both viruses, at each time point.
Fig 3.
Comparison of the mandibular lymph node (LN) of day 12 JUNV and GTOV infected animals.
Blue boxes in rows 1 and 2 represent enlarged areas in rows 3 and 4. Row 1 represents H&E at 4X magnification; note the decreased density of lymphocytes in the cortex (and medullary cords), including follicles and parafollicular areas. The control LN is mildly hyperplastic with follicles containing prominent germinal centers. Row 2 represents ISH at 4X magnification and shows marked labeling of viral RNA (in same area shown in row 1) in the depleted lymph nodes including cortex, medullary cords, and prominent outlining of vessels in the GTOV group. Row 3 represents H&E at 20X magnification; there is depletion of lymphocytes with apoptosis/necrosis, which is more prominent in the GTOV image, increased tingible body macrophages (blue ovals) and vessels with hypertrophied endothelium (blue arrows). Row 4 represents ISH at 20X magnification; there is marked viral RNA labeling in same area shown in row 3, with labeling throughout the cortex including in areas of apoptosis/necrosis and in vessels.
Fig 4.
Temporal severity of ISH/viral RNA labeling in select organs for both JUNV and GTOV groups.
In graphs A–I severity of viral RNA labeling is graphed for select organs to display changes in labeling severity over time between JUNV and GTOV. A single dot or square represents a single animal and the blue or red shaded region represents the median.
Fig 5.
Comparison of the spleen of day 12 JUNV and GTOV infected animals.
Blue boxes in rows 1 and 2 represent enlarged areas in rows 3 and 4. Row 1 represents H&E at 4X magnification; note decreased density of lymphocytes in white pulp, including follicles, PALS and marginal zone (most obvious in GTOV animal). Control spleen shows normal red pulp and white pulp architecture and lymphocyte density. Row 2 represents ISH at 4X magnification; there is marked labeling of viral RNA (in same area as shown in row 1) in both the white and red pulp, with prominently outlining red pulp vascular spaces. Row 3 represents H&E at 20X magnification; there is decreased density of lymphocytes in white pulp (nearly absent in GTOV animal), abundant deposition of fibrin in the marginal zone of the white pulp (black asterisk) and in the red pulp, scattered apoptotic/necrotic debris and hemorrhage in the white pulp. Row 4 represents ISH of spleen at 20X magnification; marked viral RNA labeling is present (same area shown in row 3), with labeling in white and red pulp and prominent outlining of red pulp vascular spaces (between blue arrows).
Fig 6.
Temporal severity of histology lesions in select organs and liver ISH/viral RNA labeling (by ISH) for both JUNV and GTOV groups.
In graphs A–H select histology lesions are graphed for a subset organs to display changes in lesion severity over time between JUNV and GTOV; viral RNA labeling by ISH is also graphed for the liver (graph H) to show changes in labeling severity for both viruses over time. A single dot or square represents a single animal and the blue or red shaded region represents the median.
Fig 7.
Images from the brain of a JUNV infected day 12 animal.
Image group 1 represents an area of cerebral cortex at level of central hippocampus/thalamus/hypothalamus (images 1c and 1d correspond to the blue box in images 1a and 1b respectively and images 1b/d show the same area as images 1a/c). In image 1a (4x magnification H&E) at center is a lateral ventricle with choroid plexus (corpus callosum is below) and an infiltrate of lymphocytes is present in the choroid plexus (black arrow). Image 1b (4X magnification ISH; same area shown in 1a) shows an area of strong viral RNA labeling in the choroid plexus and in vessels in the adjacent neuroparenchyma (black arrows). Image 1c (20X magnification H&E) shows inflammation/necrosis in/around vessels of the choroid plexus (with endothelial hypertrophy and leakage of fibrin/edema). Image 1d (20X magnification ISH) shows strong viral RNA labeling in the center/vessels of the choroid plexus–note labeling is predominantly isolated to the center and not in the lining epithelium. Image group 2 shows an area of cerebral cortex (images 2c and 2d correspond to the blue box in images 2a and 2b respectively and images 2b/d show the same area as images 2a/c). In image 2a (4X magnification H&E) there are no appreciable histologic lesions. In image 2b (4X magnification ISH; same area shown in 2a) there is strong viral RNA labeling in vessels and in a large focal area of peripheral neuroparenchyma underlying the meninges (black arrow). In image 2c (20X magnification H&E) there are also no appreciable histologic lesions. In image 2d (20X magnification ISH; same area shown in 2c) there are areas of viral RNA labeling in the neuroparenchyma (glia, neuropil and potentially neurons) as well as strong labeling in vessels, including the endothelium.
Fig 8.
Images from the eye of a JUNV infected day 12 animal.
Images a and b are from the same region and images c and d represent blue boxes in images a and b respectively. Image a (4X magnification, H&E) shows a thickened choroid; retina is artificially detached from choroid. In image b (4X magnification, ISH) there is strong viral RNA labeling (red) in choroid extending anterior toward the anterior uvea (black arrow); note also the small section of lacrimal gland with a minimal amount of labeling (blue arrow); part of the sclera is artifactually separated from the choroid. Image c (20X magnification, H&E) shows the choroid (between thick black arrows) thickened with infiltrating mononuclear inflammatory cells; note the retina is artifactually separated with only thin layer of outer retina remaining attached. Image d (20X magnification, ISH) shows strong viral RNA labeling in the inflamed, thickened choroid; note the sclera has partially, artifactually separated from the choroid).
Fig 9.
Images from the lung of a GTOV infected day 12 animal.
Images a and b are from the same region; images c and d represent blue boxes in images a and b respectively. In image a (4X magnification, H&E) there are multifocal areas of minimal alveolar inflammation (blue arrow and blue box) that are often associated with small vessels, and areas of mononuclear infiltrate surrounding larger vessels (black arrow). In image b (4X magnification, ISH) there is strong viral RNA labeling in areas of alveolar inflammation and labeling in mononuclear infiltrates surrounding vessels. In image c (20X magnification, H&E) there is minimal mononuclear alveolar inflammation, at the center of which is a small vessel surrounded by a mononuclear infiltrate (perivascular). Image d (20X magnification, ISH) is the corresponding ISH image and shows strong viral RNA labeling in the area of alveolar inflammation and surrounding the small vessel at center.
Fig 10.
Images from the small intestine (jejunum or ileum) of a GTOV infected day 12 animal.
Images a and b are from the same region; images c and d represent blue boxes in images a and b respectively. In image a (4X magnification, H&E) there is minimal dilation of villar lamina propria with small amounts of apoptotic debris. In image b (4X magnification, ISH), the corresponding ISH images there is strong viral RNA labeling in the lamina propria and submucosa with occasional labeling in the muscular layers; there is prominent labeling in intestinal nerve plexi (submucosal and myenteric) (black arrows). In image c (20X magnification, H&E) there are small amounts of apoptotic debris in the lamina propria (blue arrows); black arrows indicate submucosal and myenteric nerve plexi. In image d (20X magnification, ISH), the corresponding ISH image, there is strong viral RNA labeling in the lamina propria, extending into the submucosa; note rare labeling in the inner muscle layer and labeling in nerve plexi (black arrows).