Characterization of LGTV infection
To characterize the pathogenicity of LGTV infection in mice, wildtype and Irf-7−/− mice were infected intraperitoneally. Both wildtype and Irf-7−/− mice survived the infection. Wildtype mice showed no signs of infection, and the body weight was constant over time. However, Irf-7−/− mice showed a mild reduction in body weight at day 8 of infection (Fig. 2a). Analysis of viral replication in the CNS by qRT-PCR revealed very low level of viral replication in the olfactory bulb of wildtype mice on day 7 of infection (Fig. 2b). There was only very low viral replication detectable in the hippocampus of wildtype mice (Fig. 2c). An elevated amount of viral RNA was detected in the olfactory bulb and the hippocampus of Irf-7−/− mice as compared to wildtype mice (Fig. 2b, c). We further analyzed the expression of the proinflammatory cytokines IL-6 and TNF-α by qRT-PCR. Cytokine expression was induced upon infection in WT and Irf-7−/− in the olfactory bulb. In the hippocampus, cytokine expression was only detectable in Irf-7−/− mice (Fig. 2c). To evaluate the effect of infection on cell activation and infiltration of immune cells, we performed flow cytometry. Infection with LGTV leads to a decrease of microglia cells in WT mice, whereas no obvious change was determined in the percentage of infiltrating immune cells (Fig. 2d, e, f). In contrast, Irf-7−/− mice showed massive infiltration of lymphocytes and monocytes to the brain upon LGTV infection (Fig. 2d. e). Histological analyses of virus-infected cells in the olfactory bulb revealed that the virus was cleared in wildtype mice, whereas infected cells were still detectable in Irf-7−/− mice 16 days post-infection (Fig. 2f). Thus, these data indicate that infection with LGTV leads to virus replication in the brain without signs of disease in WT mice, whereas Irf-7−/− mice showed high viral replication and mild disease signs. Surprisingly, low viral replication in the hippocampus was not able to induce cytokine expression.
Open field
To evaluate a potential effect of the LGTV infection on basal locomotor behavior (total distance traveled) and anxiety-related behavior (immobility, time spent in the center), the open field test was performed (Fig. 3). Total distance traveled and average speed were comparable between infected and non-infected in both wildtype and Irf-7−/− mice (Fig. 3a, b) (total distance: WT d0 1.96 ± 0.20 m, WT d14 2.29 ± 0.11 m, p = 0.20; Irf-7−/− d0 2.70 ± 0.24 m, Irf-7−/− d14 2.58 ± 0.17 m, p = 0.66; average speed WT d0 0.006 ± 0.0007 m/s, WT d14 0.007 ± 0.0004 m/s, p = 0.19; Irf-7−/− d0 0.009 ± 0.0008 m/s, Irf-7−/− d14 0.008 ± 0.0006 m/s, p = 0.45). However, non-infected Irf-7−/− mice in general showed an increased total distance traveled (p = 0.01) and average speed (p = 0.007) as compared to non-infected wildtype mice (Fig. 3a, b). The natural tendency of mice is to spend more time in the border zone of the open field arena. However, increased time spent in the border zone indicates anxiety-related behaviors. Therefore, to initially screen for anxiety-related behavior, the time spent in the border and the center zone of the arena was analyzed [31]. Although the time spent in the border zone of the open field arena did not change after infection in both wildtype and Irf-7−/− mice (WT d0 58.33 ± 2.51 %, WT d14 52.19 ± 2.78 %, p = 0.09; Irf-7−/− d0 75.11 ± 2.78 %, Irf-7−/− d14 76.39 ± 2.26%, p = 0.74), it was higher in non-infected and infected Irf-7−/− mice as compared to the respective wildtype mice (p < 0.001, Fig. 3c). However, infected wildtype mice showed significantly more core entries than uninfected wildtype mice, which was not the case for Irf-7−/− mice (Fig. 3d) (WT d0 15.10 ± 1.87, WT d14 24.10 ± 1.32, p < 0.001); Irf-7−/− d0 15.38 ± 1.44, Irf-7−/− d14 13.67 ± 1.87, p = 0.49). Thus, the infection did not influence locomotor activity of mice, regardless of the genotype. In general, Irf-7−/− mice showed increased anxiety-related behavior as compared to non-infected WT mice regardless of virus infection. Infected WT mice showed signs of reduced anxiety-related behavior as the number of core entries were increased as compared to uninfected mice (Fig. 3e).
Elevated plus maze
For a more detailed analysis on the impact of LGTV infection on anxiety behavior, the elevated plus maze test was performed (Fig. 4). In this test as well, the natural tendency of mice is to spend more time in the closed arms of the maze, however, an increase in closed arm activity (duration and/or entries) indicates anxiety-like behavior in rodents [32].
Although all tested animals spent more time in the closed arms than in the open arms of the elevated plus maze, LGTV infection in wildtype mice led to a significant reduction in the time spent in the closed arm (Fig. 4a) and increased numbers of entries to the open arms (Fig. 4b) compared to uninfected wildtype mice (time in closed arms: uninfected WT 87.06 ± 2.84 %, infected WT 74.02 ± 3.55%, p = 0.007; entries in open arms: uninfected WT 6.30 ± 1.11%, infected WT 11.70 ± 1.28%, p = 0.008). In contrast, LGTV infection of Irf-7−/− mice had no impact on time spent in the closed arms (and number of entries in the open arms) compared to uninfected Irf-7−/− mice (Fig. 4a–c) (time in closed arms uninfected Irf-7−/− 79.24 ± 3.75, infected Irf-7−/− 77.83 ± 3.40, p = 0.77; entries in open arms: uninfected Irf-7−/− 9.25 ± 1.57, infected Irf-7−/− 12.33 ± 1.66, p = 0.14). Overall, LGTV infection of wildtype mice diminished anxiety-like behavior.
Morris water maze
To investigate the effects of LGTV infection on cognitive function, training in the Morris water maze task was performed. During 3 days of pre-training, swimming ability and visual acuity were intact in all animals as the swimming speed and escape latency were comparable in all tested groups (data are not shown).
In the Morris water maze test, swimming time (escape latency) and path length (swim distance) to reach the platform can be used as measures for memory formation over consecutive days [26, 27]. During 8 days of acquisition training, the escape latency and swim distance to reach the platform declined and thereby indicated hippocampus-dependent spatial learning and memory formation in all tested groups, (Fig. 5) (escape latency: repeated measure one-way ANOVA: FUninfected WT (7, 56) = 14.24, p < 0.001; FInfected WT (7, 63) = 8.38, p < 0.001; FUninfected Irf-7−/− (7, 42) = 6.87, p < 0.001; FInfected Irf-7−/− (7, 56) = 6.18, p < 0.001; swim distance: repeated measure one-way ANOVA: FUninfected WT (7, 56) = 15.06, p < 0.001; FInfected WT (7, 63) = 8.56, p < 0.001; FUninfected IrfF-7−/− (7, 42) = 8.32, p < 0.001; FInfected Irf-7−/− (7, 56) = 6.58, p < 0.001). Yet, the escape latency (Fig. 5a) and swim distance (Fig. 5c) in LGTV-infected wildtype mice were increased on day 3, day 4, and day 5 of acquisition training in LGTV-infected WT mice as compared to uninfected wildtype mice (escape latency: day 3 p = 0.024; day 4 p = 0.0002, day 5 p = 0.001; swim distance day 3 p = 0.038; day 4 p = 0.0002, day 5 p = 0.0007). These results suggest an impairment in spatial learning and memory formation following LGTV infection in wildtype animals (escape latency—two-way RM ANOVA: FTreatment (1, 74) = 29.40, p < 0.001; swim distance—two-way RM ANOVA: FTreatment (1, 74) = 26.23, p < 0.001). In contrast, analysis of the escape latency (Fig. 5b) and swim distance (Fig. 5d) in Irf-7−/− mice did not reveal any significant differences (escape latency: two-way RM ANOVA: FTreatment (1, 62) = 0.18, p = 0.66; swim distance: two-way RM ANOVA: FTreatment (1, 62) = 1.19, p = 0.27). Furthermore comparison of the swim distance to reach the platform between wildtype and Irf-7−/− uninfected control mice did not show any significant changes (Fig. 5e) (swim distance—two-way RM ANOVA: FTreatment (1, 62) = 1.81, p = 0.18). However, LGTV infection in mice led to an increased swim distance in wildtype mice as compared to Irf-7−/− mice (Fig. 5f) (swim distance—two-way RM ANOVA: FTreatment (1, 74) = 7.26, p = 0.008).
While the escape latency and swim distance provide a read-out for memory acquisition, the reference memory test (probe trial) provides quantification for the retrieval of the specific memory. The probe trial tests were performed at days 3, 6, and 9 before the actual training began (Fig. 6). The probe trial tests revealed that the percentage of time spent in the target quadrant (T) increased over the training time in all groups (WT uninfected—one-way RM ANOVA: FTreatment (2, 16) = 9.007, p = 0.002; WT infected—one-way RM ANOVA: FTreatment (2, 18) = 13.13, p < 0.001; Irf-7−/−uninfected—one-way RM ANOVA: FTreatment (2,12) = 11.94, p = 0.001; Irf-7−/− infected—one-way RM ANOVA: FTreatment (2, 16) = 10.42, p = 0.001). The quadrant preference was comparable between all tested groups, regardless of the genotype and whether the mice had been infected (day 3—one-way ANOVA: FTreatment (3, 31) = 0.47, p = 0.70, Fig. 6a; day 6—one-way ANOVA: FTreatment (3, 31) = 0.10, p = 0.95, Fig. 6b; day 9—one-way ANOVA: FTreatment (3, 31) = 0.69, p = 0.56, Fig. 6c). However, according to the heat maps of the animals’ center position, it seems that LGTV infection in wildtype mice caused less concentration for searching in the target area on probe trial day 9 (Fig. 6d).
A detailed analysis of the swimming path allows for a qualitative assessment of learning in mice. Over time, healthy animals progressively switch from egocentric (hippocampus-independent: chaining, scanning, and random swimming) to allocentric (hippocampus-dependent: directed search) strategies to navigate to the hidden platform while a spatial map of the maze is formed (Fig. 7a) [29, 30]. All groups of control and LGTV-infected mice showed an augmentation of the hippocampus-dependent searching strategy during the 8 days of training (Fig. 7b). However, this progression seemed to be decreased for LGTV-infected wildtype mice compared to wildtype control mice (Fig. 7c) (two-way RM ANOVA: FWT (1, 17) = 4.49, p = 0.04). No significant differences were detectable in the relative percentage of hippocampus-dependent strategy used between Irf-7−/− LGTV-infected and control mice (Fig. 7d) (two-way RM ANOVA: FIrf-7−/− (1, 14) = 0.50, p = 0.48). Thus, LGTV infection impacts hippocampus-dependent searching strategies only in WT mice.
Once a spatial map including the distal cues is formed, a new platform position only need to be updated in the already existing cognitive map and can therefore be memorized faster. However, depending on the cognitive flexibility of the animal, the two memories can also compete with each other, which can be seen especially during the probe trial test [20]. In the reversed Morris water maze paradigm, the hidden platform was moved to the opposite quadrant (south west). During 3 days of training, the escape latency and swim distance to reach the new platform position decreased in all tested groups (Fig. 8a–d) (escape latency: repeated measure one-way ANOVA: FUninfected WT (2, 16) = 44.95, p < 0.001; FInfected WT (2, 18) = 18.65, p < 0.001; FUninfected Irf-7−/− (2, 12) = 14.36, p = 0.001; FInfected IrfF-7−/− (2, 16) = 19.64, p < 0.001; swim distance: repeated measure one-way ANOVA: FUninfected WT (2, 16) = 48.22, p < 0.001; FInfected WT (2, 18) = 16.44, p < 0.001; FUninfected Irf-7−/− (2, 12) = 12.79, p = 0.001; FInfected Irf-7−/− (2, 16) = 19.11, p < 0.001). However, LGTV-infected wildtype mice showed an increased escape latency (Fig. 8a) and swim distance (Fig. 8b) as compared to uninfected controls (escape latency: two-way RM ANOVA: FTreatment (1, 74) = 12.15, p = 0.0008; swim distance: two-way RM ANOVA: FTreatment (1, 74) = 12.42, p = 0.0007). No significant differences in escape latency (Fig. 8c) and swim distance (Fig. 8d) were detectable between LGTV infected and uninfected Irf-7−/− mice (escape latency: two-way RM ANOVA: FTreatment (1, 62) = 0.14, p = 0.70; swim distance two-way RM ANOVA: FTreatment (1, 62) = 0.86, p = 0.35). Therefore, LGTV infection led to a reduced ability to memorize the new location of the hidden platform only in wildtype mice.
Subsequently, a single probe trial test 24 h after the last day of reversal training was performed (Fig. 8e, f). Uninfected wildtype, uninfected Irf-7−/− and LGTV-infected Irf-7−/− mice spent more time in the new target quadrant (T, south-west) as compared to the average time spent in the non-target quadrants (N, p < 0.05). Whereas in LGTV-infected wildtype animals (p = 0.73), no preference for the new target quadrant could be observed (Fig. 8e, f). Consequently, LGTV infection impaired initial and reversal learning in the Morris water maze; however, the phenotype was compensated in Irf-7−/− mice.
Hippocampal neuron morphology
To investigate the potential cellular basis underlying the observed behavioral changes, hippocampal neuronal morphology was analyzed in uninfected and infected wildtype and Irf-7−/− animals. Spines are tiny, dendritic protrusions that carry the majority of excitatory synapses in the hippocampus, and changes in spine density can provide information about alterations in the connectivity of hippocampal subregions [33]. Using Golgi-cox staining, spines were counted separately on apical and basal dendrites of CA1 and dentate granule cells located in the superior and inferior blade of the granule cell layer in the hippocampus (Fig. 8). In wildtype mice, the spine density in the apical and basal dendrites of CA1 pyramidal neurons decreased upon infection with LGTV (Fig. 9a, b). In the apical region, a significant spine reduction was detectable on day 7 and day 14 post-infection (Fig. 9a) (d7: Δ 23.14%, p < 0.001; d14 Δ 29.34%, F (2, 90) = 18.76, p < 0.001), whereas in the basal region, a significant dendritic spine reduction was detectable only 14 days post LGTV infection (Fig. 9b) (Δ 27.34%, F (2, 85) = 16.34, p < 0.001).
In addition, LGTV infection led to dendritic spine reduction in DG neurons located in the superior blade 14 days post-infection (Fig. 9c) (Δ 13.76%, F (2, 92) = 7.81, p = 0.02). Interestingly, infection with LGTV had no effects on dendritic spine numbers of CA1 and DG hippocampal subregions in Irf-7−/− mice (Fig. 9a–d) (CA1: apical: F (2, 95) = 1.44, p = 0.24, basal: F (2, 84) = 0.67, p = 0.51; DG: superior: F (2, 91) = 0.94, p = 0.22, inferior: F (2, 88) = 0.57, p = 0.56). Taken together, LGTV infection reduced the spine density in the CA1 region in wildtype mice. Irf-7−/− mice have a low spine density, which is not influenced by the LGTV infection.
Microglia and astrocyte density and activity
Since LGTV infection leads to alteration in spine density and behavior, we analyzed hippocampal glia cells for neuroinflammatory processes. The density of astrocytes in the hippocampus was analyzed using GFAP staining (Fig. 10a). Astrocyte density was decreased in the hippocampus of wildtype mice during the course of infection. At 16 days post-infection, when the virus was cleared, the number of activated astrocytes increased (Fig. 9b). In contrast, the infection had no dramatic impact on the activation status of the astrocytes in Irf-7−/− mice (Fig. 10a, b).
For microglia cells, infection with LGTV decreased the overall density of activated IBA1+ cells 9 days post-infection (Fig. 10c, d). In contrast, the number of activated cells increased on day 16 post-infection to higher levels than before infection. These data indicate that LGTV infection in wildtype and Irf-7−/− mice influence the activation of glia cells, although direct viral replication in the hippocampus was hardly detectable.