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. 2020 Aug 26;10:454. doi: 10.3389/fcimb.2020.00454

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Copyright © 2020 Becker, Fink, Podlech, Giese, Schmiedeke, Bukur, Reddehase and Lemmermann.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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2C-ISH analysis of liver infection. Reanalysis of stored liver specimens from a previously performed experiment [lung virus titers shown in Holtappels et al. (2006), Figure 8B]. BALB/c mice were immunocompromised by γ-irradiation (6.5 Gy) and infected at one footpad. Liver tissue sections were taken on day 12 after infection (A,B) Virus spread in liver tissue. Infection was performed with 10⁵ PFU of mCMV-Δm06^W, now identified to represent a mixture of correct virus mCMV-Δm06 and a “large deletion” mutant mCMV-Δm06m145-158 that includes deletion of the m152 gene. (A) 2C-ISH performed with probe m152-P (red staining) specific for mCMV-Δm06 and probe BAC-P (black staining) specific for mCMV-Δm06m145-158. (Center panel) overview image showing foci of infection for both viruses in the mixture representing mCMV-Δm06^W. (Left panel) red-stained mCMV-Δm06-infected cell resolved to greater detail. (Right panel) black-stained mCMV-Δm06m145-158-infected cell resolved to greater detail. (B) 2C-ISH performed with probe M55/gB (red staining) specific for both viruses and probe BAC-P (black staining) specific for mCMV-Δm06m145-158. (Center panel) overview image showing foci of infection for both viruses in the mixture representing mCMV-Δm06^W. (Left panel) red-stained mCMV-Δm06-infected cell resolved to greater detail. (Right panel) red-black speckled cells infected with mCMV-Δm06m145-158, resolved to greater detail. Frames in the overview images indicate tissue section areas resolved to greater detail in the left and right images. Note the stained CMV-typical inclusion bodies in the nuclei of infected hepatocytes. Counterstaining was performed with hematoxylin. Bar markers: 50 μm. (C) vRAP expression-dependent control of liver tissue infection with the indicated viruses on day 12 after adoptive transfer of 10⁴ antiviral CD8 T cells specific for the viral epitope M45-D^d. 2C-ISH was performed with probes M55/gB-P (red symbols) and BAC-P (black symbols). (no AT) no adoptive transfer. (AT) adoptive transfer. vRAPs actually expressed by the viruses as well as their proposed impact on antigen presentation (AP, arrows up or down) are indicated. Data represent counts of infected liver cells in representative 10 mm² tissue section areas. The dashed line indicates the detection limit of the assay, which is one infected cell per selected counting area. Symbols represent mice tested individually. Median values are marked and connected for the groups “no AT” and “AT” to highlight the strength of antiviral control. For statistical analysis, data were log-transformed and P-values were calculated by using the two-sided unpaired t-test with Welch's correction of unequal variances. P < 0.05 indicates statistical significance of the difference between “no AT” and “AT” groups. Linked data are connected by dotted lines, which reveals a correlation between the numbers of cells infected with the correct mutant mCMV-Δm06 and the “large deletion” mutant mCMV-Δm06m145-158 after infection with mCMV-Δm06^W.