Journal: Journal of Neuroinflammation

Article Title: Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4 + T-cells and IFN-γ

doi: 10.1186/s12974-025-03422-6

Figure Lengend Snippet: Tropism of mouse cytomegalovirus in the brain. A-C Newborn C57BL/6 mice were infected with MCMV. A Viral titers in the cortex, hippocampus, and cerebellum were determined by plaque assay at the indicated days post-infection (dpi) ( n = 10–13 mice/time point). Results for individual mice are shown (circles, squares, and triangles). Black horizontal lines indicate the median values. D.L., detection limit. Kruskal–Wallis test was used. B GFAP (astrocyte marker), IBA1 (microglia marker) or MAP2 (neuron marker, all cells stain brown) and MCMV IE1 protein (red) co-staining of paraffin-embedded brain sections on 11 dpi. Arrows point to MCMV-infected cells. Representative images are shown (40x magnification, 100x magnification insert). C MCMV genome-equivalent copy numbers per gram of tissue in indicated brain regions determined by qPCR at 180 dpi ( n = 10 mice/time point). Mock, mock-infected mice. Results for individual mice are shown (circles, squares, and triangles). Black horizontal lines indicate the median values. D.L., detection limit. Kruskal–Wallis test was used. p values indicate statistically significant differences

Article Snippet: Viral antigen IE1 was detected using biotinylated (Thermofisher Scientific, #A39256) anti-IE1 antibody (clone IE1.01; Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, #HR-MCMV-12).

Techniques: Infection, Plaque Assay, Marker, Staining

Journal: Journal of Neuroinflammation

Article Title: Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4 + T-cells and IFN-γ

doi: 10.1186/s12974-025-03422-6

Figure Lengend Snippet: Neurons are the major source of infectious virus during the late phase of acute infection in the brain. A Schematic representation of the reporter system for quantitative tracking of virus progeny in vivo. Following infection of a Cre-expressing cell with MCMV-flox, the loxP-flanked (floxed) STOP cassette is removed from the viral genome, allowing constitutive EGFP expression under the control of the major immediate early promoter (MIEP) of HCMV. B Newborn GFAP-Cre +/- , Sall1CreERT2 +/- and BAF53b-Cre +/- transgenic mice were infected i.p with MCMV-flox. Tamoxifen (TAM) was administered for 3 consecutive days intragastrically to Sall1-CreERT2 +/- mice. Mice were euthanized at 7, 10, 14, and 17 dpi, and organs were collected. Titers in brain ( B ), spleen (Sp), liver (Li), salivary glands (Sg) and lungs (Lu) of individual mice are shown (circles). Mean percentage of EGFP + plaques per organ are shown (bars). D.L., detection limit. C Representative MAP2 (brown) and MCMV IE1 (red) co-staining of paraffin-embedded brain sections on 17 dpi. Arrows point to MCMV-infected cells. Representative MCMV-infected neuron in the hippocampus is shown (40x magnification, 100x magnification insert). D Quantification of IE1 protein-positive MAP2 neurons. Columns indicate median values ( n = 3 mice, 10 sections/mouse)

Article Snippet: Viral antigen IE1 was detected using biotinylated (Thermofisher Scientific, #A39256) anti-IE1 antibody (clone IE1.01; Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, #HR-MCMV-12).

Techniques: Virus, Infection, In Vivo, Expressing, Control, Transgenic Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4 + T-cells and IFN-γ

doi: 10.1186/s12974-025-03422-6

Figure Lengend Snippet: CD4 + T-cells are essential for the resolution of productive MCMV infection in neurons. A-B Newborn wild-type C57BL/6, CD4- (CD4 -/- ) and CD8-deficient (CD8 -/- ) mice were infected i.p. with MCMV. Viral titers in the brain ( n = 10 mice) were determined on A 14, 30, 70, and B 120 dpi. Titers in brains of individual mice are shown (circles). Black horizontal lines indicate the median values. D.L., detection limit. A Kruskal–Wallis test and B Mann–Whitney two-tailed test were used. C-H Immunohistochemical analysis of MCMV infection in neurons, astrocytes and microglia of CD4 -/- mice at 37 dpi ( n = 6 mice, 10 sections/mouse). C A simplified 2D sagittal view of the location of IE1 + cells in the brain regions. Each colored dot represents individual IE1 + cells in six individual mice. D Representative MAP2 (neuron marker, brown) and MCMV IE1 (virus infection, red) co-staining of paraffin-embedded brain sections. Arrows point to the MCMV-infected neurons (40x magnification, 100x magnification insert). E Quantification of IE1 + MAP2 + neurons in the brain. F Quantification of IE1 + GFAP + astrocytes in the brain. G Representative GFAP (astrocyte marker, brown) and MCMV IE1 (virus infection, red) co-staining of paraffin-embedded brain sections. Arrow points to the MCMV-infected astrocyte (40x magnification, 100x magnification insert). H Quantification of IE1 + IBA1 + microglia in the brain. E-F , H Columns indicate median values. Mann–Whitney two-tailed test was used. p values indicate statistically significant differences

Article Snippet: Viral antigen IE1 was detected using biotinylated (Thermofisher Scientific, #A39256) anti-IE1 antibody (clone IE1.01; Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, #HR-MCMV-12).

Techniques: Infection, MANN-WHITNEY, Two Tailed Test, Immunohistochemical staining, Marker, Virus, Staining

Journal: Journal of Neuroinflammation

Article Title: Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4 + T-cells and IFN-γ

doi: 10.1186/s12974-025-03422-6

Figure Lengend Snippet: IFN-γ and IL-12 promote the resolution of productive MCMV infection in neurons. Newborn C57BL/6, A Prf1 −/− , B Gzma −/− Gzmb −/− , C TNFRp55 −/− , D-G IFN-γ −/− , and H-K Il12rb2 −/− mice were infected with MCMV on PND1. A-D , H Viral titers in the brain were determined by plaque assay at 14, 30 and, 70 dpi ( n = 5–14). Titers in organs of individual mice are shown (circles). Black horizontal lines indicate the median values. D.L., detection limit. Mann–Whitney two-tailed test was used. E-G , I-K Immunohistochemical staining of MCMV infection in neurons at day 37 p.i. in E-G IFN-γ −/− , and I-K Il12rb2 −/− mice. E , I Localization of IE1 protein-positive cells in the murine brain. Median values are shown ( n = 5, 10 sections/mouse). Other refers to IE1 + cells outside the cortex and hippocampus in the brain. Kruskal–Wallis test was used. F , J Representative images of MCMV infected MAP2 + neuron. Arrows point to the MCMV-infected neurons (40x magnification, 100x magnification insert). G , K Quantification MCMV infected MAP2 + neurons ( n = 5 mice, G = 10 sections/mouse, K = 20 sections/mouse). Columns indicate median values. Mann–Whitney two-tailed test was used. p values indicate statistically significant differences

Article Snippet: Viral antigen IE1 was detected using biotinylated (Thermofisher Scientific, #A39256) anti-IE1 antibody (clone IE1.01; Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, #HR-MCMV-12).

Techniques: Infection, Plaque Assay, MANN-WHITNEY, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Journal of Neuroinflammation

Article Title: Neuron-restricted cytomegalovirus latency in the central nervous system regulated by CD4 + T-cells and IFN-γ

doi: 10.1186/s12974-025-03422-6

Figure Lengend Snippet: Neurons are the sites of latent and reactivating MCMV in the CNS. A-C , F Newborn R26 tdTomato mice were infected with MCMV-GFP-Cre i.p. A Brains were collected and analyzed 45 dpi. A representative brain section (10x magnification) containing tdTomato-positive cells (left) and representative magnified (63x magnification) tdTomato-positive cell (right) are shown. B Representative image of tdTomato-positive NeuN + neuron (40x magnification). C Quantification of cell types expressing tdTomato reporter protein 45 dpi ( n = 5 mice, 5 sections/mouse). ND - Not detected. D Newborn C57BL/6 mice were infected with MCMV. Brain regions were isolated and cultured ex vivo for 6 weeks. The reactivation frequency in indicated brain regions was determined ( n = 6 mice). ND - not detected. E Newborn C57BL/6 mice were infected with MCMV. The number of CD4 + T-cells per gram of tissue was analyzed using flow cytometry at 180 dpi. Columns indicate median values ( n = 6–10 mice). Kruskal–Wallis test was used was used. F Mice were depleted of CD4 + T-cells starting on 90 dpi for 1 month. Mice were perfused, sacrificed and brains were collected and frozen. Quantification of co-expression of NeuN and tdTomato is shown ( n = 3–5 mice, 5 sections/mouse). Columns indicate median values. Mann–Whitney two-tailed test was used. G Newborn C57BL/6 mice were infected with MCMV and mice were depleted of CD4 + T-cells starting at 90 dpi for 1 month ( n = 10 mice). Brain regions were separated, homogenized and layered on MEF cells in multiwall plates. The frequency of MCMV-positive wells was determined. Results for individual mice are shown (circles, squares and rectangles). The number of mice from which the virus was recovered is indicated above the graph. H-I Newborn C57BL/6 mice were infected with MCMV and mice were depleted of CD4 + T-cells starting on 90 dpi for 1 month. H Representative image of MCMV-infected MAP2 + neuron. The arrow points to the MCMV-infected neuron (40 magnification, 100x magnification insert). I Quantification of IE1 protein-positive MAP2 neurons ( n = 4 mice, 20 sections/mouse). p values statistically indicate significant differences

Article Snippet: Viral antigen IE1 was detected using biotinylated (Thermofisher Scientific, #A39256) anti-IE1 antibody (clone IE1.01; Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia, #HR-MCMV-12).

Techniques: Infection, Expressing, Isolation, Cell Culture, Ex Vivo, Flow Cytometry, MANN-WHITNEY, Two Tailed Test, Virus