Fig 1.
Neuroinvasion of the CSG viruses.
A, C, E, G, I) Brain tissue was evaluated for viral RNA via RT-qPCR with virus-specific primers. Dotted lines indicate mock sample average from n = 4. B, D, F, H, J) Brain tissues were evaluated for infectious virus via plaque assays of brain homogenates in Vero cells. Dotted lines represent the limit of detection. No plaques were detected in any mock controls, n = 4. Individual dots represent individual mice. For all viruses and time points, n = 6, except for: LACV n = 4 for 6 dpi, no mice survived to 7 dpi; SSHV n = 5 for 5 dpi, two of the designated 7 dpi mice developed neurological disease at 6 dpi and had to be euthanized, so n = 2 for 6 dpi, n = 4 for 7 dpi; for TAHV, one mouse in the 5 dpi group developed neurological disease at 4 dpi and had to be euthanized, so n = 1 for 4 dpi and n = 5 for 5 dpi. (G) Dunnett’s multiple comparisons tests was done for JCV viral RNA compared to mock, using Log2(%gapdh) values. Nonclinical = mice with no signs of disease; Clinical = mice displaying neurological signs or other endpoint criteria as described in the methods.
Fig 2.
A) Viremias were assessed via plaque assays of plasma samples in Vero cells. Dotted line represents the limit of detection. B-C) Viral RNA levels were analyzed via RT-qPCR with virus-specific primers in lymph nodes (B) and spleens (C). Dotted lines represent mock average from n≥3. Sample numbers are the same as described in Fig 1. Individual points represent individual mice. Dunnett’s multiple comparisons tests were done for each virus, tissue, and time point compared to mock, using Log2(%gapdh) values. Asterisks indicate sample days that had significantly higher viral RNA expression compared to mock. For mice that developed clinical signs prior to their designated time point, those values were grouped in with their designated time point (6dpi with 7dpi for SSHV, 4 dpi with 5 dpi for TAHV). Nonclinical = mice with no signs of disease; Clinical = mice displaying neurological signs or other endpoint criteria. *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009.
Fig 3.
IFN response to CSG viruses in 1 dpi lymph nodes.
IFN mRNA expression was evaluated via RT-qPCR for a panel of 10 Type I IFNs in lymph nodes taken at 1 dpi from mice inoculated with A) LACV; B) SSHV; C) TAHV; D) JCV; E) INKV. Expression is plotted for each samples as the fold change in %gapdh from the mock average. N = 6 for all viruses, n = 7 for mock. Each individual point represents an individual mouse. Dotted lines indicate fold change = 1. Fold change to mock is plotted on the left axis, viral RNA plotted on the right axis. One-way ANOVA analyses were performed on Log2(%gapdh) with Dunnett’s multiple comparison test performed between mock and each virus. Asterisks denote Ifn expression that was significantly different from mock for that IFN and virus (gray = significantly higher, black = significantly lower than mock): *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009.
Fig 4.
IFN response to LACV and INKV in the brains.
A) Viral RNA values are the same as Fig 1A and 1I, but are represented here for a direct comparison of virus in LACV-infected and INKV-infected mice at 1, 3, and 5 dpi. IFN mRNA expression of 10 IFNs in the brains of LACV- and INKV-infected mice at (B) 1 dpi, (C) 3 dpi, and (D) 5 dpi. ISG mRNA expression in the brain was compared between LACV- and INKV-infected mice at (E) 1 dpi, (F) 3 dpi, and (G) 5 dpi. B-G) Dotted lines indicate fold change = 1. One-way ANOVA analyses were performed on Log2(%gapdh) values comparing the expression of each individual IFN and ISGs for mock (n≥4), LACV (n = 6), and INKV (n = 6), with Dunnett’s multiple comparison test performed between mock and each virus, and follow-up analyses with Sidak’s multiple comparsions test between LACV and INKV at any day with any IFN or ISG mRNA expression levels significantly higher than mock. Asterisks below the line represent expression significantly different from mock for that virus and IFN or ISG, and asterisks above the line represent a significant difference between LACV and INKV for that IFN or ISG mRNA at that dpi. If no asterisks are present above the line, this indicates there was no significant difference. The † represents significantly higher expression of Ifna2 between INKV at 5 dpi and LACV at 3 dpi (p<0.0001). *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009. Individual points represent individual mice. Nonclinical = mice with no signs of disease; Clinical = mice displaying neurological signs or other endpoint criteria.
Fig 5.
Immune responses to LACV and INKV infection in the brain.
Representative whole-brain sagittal sections from (left column, top to bottom), 3 dpi LACV, 5 dpi LACV, 3 dpi INKV, 5 dpi INKV and mock-inoculated mice were immunohistochemically labeled for the myeloid/microglia-specific marker ionized calcium binding adaptor molecule 1 (IBA1, magenta), virus (white) and cell nuclei (blue). The white scale bar in the mock-inoculated panel represents 1mm and corresponds to all other images. The yellow boxes in the 3 dpi LACV (A), 5 dpi INKV (B), and mock-inoculated (C) sections correspond to high magnification insets shown in the right column highlighting the myeloid cellular morphology in the olfactory bulb of each respective animal.
Table 1.
Scoring of Virus, Microglial Activation, and Perivascular Cuffing from Immunohistochemistry.
Fig 6.
Disease curves and virus loads for C57BL/6 mice and Ifnar1-/- mice.
Mice were inoculated IP with 105 PFU of A) INKV, n = 6 for C57BL/6, n = 9 for Ifnar1-/- or B) JCV, n = 5 for C57BL/6, n = 7 for Ifnar1-/-, and followed for neurological signs, other endpoint criteria, or death. Timepoint analysis of infectious virus via plaque assay of tissue homogenates in Vero cells of tissues from INKV-inoculated C) WT B6 mice and D) Ifnar1-/- mice.
Fig 7.
Disease curves in immune deficient mouse strains.
Mice were inoculated IP with 105 PFU of A) INKV or B) JCV, and followed for neurological signs, other endpoint criteria, or death. For Irf3-/-xIrf7-/- n = 8 INKV and JCV; for Myd88-/- n = 5 INKV, n = 8 JCV; for Unc93b1.3D n = 11 INKV, n = 10 JCV; for Mavs-/- n = 14 INKV, n = 10 JCV; for Mavs-/-xUnc93b1.3D n = 9 INKV, n = 10 JCV.
Fig 8.
Comparison of the IFN mRNA response in LN at 1 dpi of wild type C57BL/6 mice and immune deficient DKO mice.
Viral RNA was compared in the lymph nodes (LN) at 1 dpi of mice inoculated with JCV (A) or INKV (C). B,D) IFN mRNA expression was evaluated via RT-qPCR in LN at 1 dpi of JCV-inoculated mice (B) or INKV-inoculated mice (D) in B6 and DKO mice. A,C) One-way ANOVA was performed for each tissue on Log2(%gapdh) values and Dunnett’s multiple comparisons test done between B6 and DKO mice. Asterisks represent significant ANOVA and multiple comparisons p-values. B, D) There were significantly different levels in some basal Ifn expression between mock-inoculated B6 and DKO mouse strains, therefore Log2(fold change in %gapdh from mock) values were used to normalize expression to mock for analyses. One-sample t tests were done to analyze if the expression for each mouse strain and Ifn was significantly different from 0 (equivalent to fold change = 1 = mock). One-way ANOVA with Dunnett’s multiple comparison test between mock and the DKO strains was then run. Asterisks below the lines represent expression significantly different from mock (gray = significantly higher, black = significantly lower), and asterisks above the line represent a significant difference between B6 and one (bracketed) or both (flat line) DKO strains. Asterisks denote *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009, and values for the multiple comparisons are reported as the higher value from the two DKO strains. Dotted lines indicate fold change to mock = 1.
Fig 9.
Comparison of the IFN and ISG response in brains of INKV-inoculated C57BL/6 mice versus immune deficient DKO mice.
A) Viral RNA in the brains of WT C57BL/6 mice and the DKO strains, data for B6 from Fig 1I. mRNA expression levels of IFNs (B) and Ifits (C), data for B6 the same as Fig 4C and 4D. A) One-way ANOVA was performed on Log2(%gapdh) values and Tukey’s multiple comparisons test done between B6 and DKO mice. Asterisks represent significant ANOVA and multiple comparisons p-values. B & C) There were significantly different levels in basal Ifn and Ifit expression between mock-inoculated B6 and DKO mouse strains, therefore Log2(fold change in %gapdh from mock) values were used to normalize expression to mock for analyses. One-sample t tests were done to analyze if the expression for each Ifn and Ifit was significantly different from 0 (equivalent to fold change = 1 = mock average). One-way ANOVA with Dunnett’s multiple comparison test between mock and the DKO strains was then run. Asterisks below the lines represent expression significantly different from mock, and asterisks above the line represent a significant difference between B6 and one (bracketed) or both (flat line) DKO strains. Asterisks denote *p = 0.05–0.01, **p = 0.009–0.001, ***p≤0.0009, and for the multiple comparisons are reported as the higher value from the two DKO strains. Dotted lines indicate fold change to mock = 1. Nonclinical = mice with no signs of disease; Clinical = mice displaying neurological signs or other endpoint criteria.