Viral Titers in Sera and Brains of ZIKV-Infected Mice
Prior to PET scanning at days 3, 6, and 10 post-infection, mice designated for imaging from each treatment group were bled and viral titers in sera were determined by qRT-PCR (Fig. 1a). For the PBS + 5A3 and ZIKV + PBS treatment groups, no viremia was detected in any mouse at any time point tested. In the group susceptible to ZIKV infection (ZIKV + 5A3), average viremia was highest (~ 5.5 log10 PFUe/ml; n = 5) at day 3 PI and decreased at both days 6 (~ 4.2 log10 PFUe/ml; n = 5) and 10 PI (~ 3.0 log10 PFUe/ml; n = 4). These data demonstrate a productive infection in the subjects challenged with ZIKV and treated with 5A3 with the exception of one mouse at day 10 PI.
At the conclusion of each PET scan, mice were euthanized and brains were harvested for viral titer determination (Fig. 1b). As expected, the mice in the PBS + 5A3 treatment group showed no detectable viral titers in brain at days 3, 6, or 10 post-infection. In the ZIKV + PBS treatment group, four out of the five mice tested at each time point also showed no detectable viral titers in brain, however, a single mouse at each time point tested (days 3, 6, and 10 post-infection) had viral brain titers of ~ 3.9, ~ 6.8, and ~ 4.9 log10 PFUe/g, respectively, suggesting perhaps some level of viral CNS penetration and subsequent clearance with no detectable viremia. In contrast with the decreasing viremia that was measured in the susceptible ZIKV + 5A3 animals, brain titers in this group were shown to increase over time as determined by qRT-PCR from brain homogenates. From day 3 PI to day 10 PI, brain viral titers increased from an average of ~ 5.3 log10 PFUe/g at day 3 PI to an average of ~ 6.95 log10 PFUe/g in the remaining living mice (n = 4/5) by day 10 PI. It should be noted, however, that at days 3 and 10 post-infection, a single mouse at each time point, respectively, had brain viral titers below the limit of detection for our assay. To confirm the presence of infectious virus in the brain, qRT-PCR titers were verified by conventional plaque assay. Altogether, these data are consistent with our previous findings [24] and those of others [20,21,22,23] and demonstrate a substantial increase in ZIKA neuroinvasion when IFN-I signaling is blocked.
[18F]DPA-714 Uptake in Brains of Control and ZIKV-Infected Mice
To evaluate the ability of in vivo PET imaging to detect ZIKV-induced neuroinflammation in mouse brain, we used [18F]DPA-714, a PET radiotracer that is specific for TSPO [11], a biochemical marker of neuroinflammation that is highly upregulated in activated microglia, CNS macrophages, and reactive astrocytes [12,13,14,15,16,17,18,19]. By as early as day 3 PI, ZIKV-infected mice treated with MAb-5A3 showed a modest but significant (approximately twofold) increase in mean whole-brain [18F]DPA-714 binding (2.21 ± 0.14 %ID/g; n = 5) compared with historic PBS controls (Fig. 2a, b). By days 6 and 10 post-infection, the increase in mean whole-brain [18F]DPA-714 binding in ZIKV + 5A3 mice was approximately four- (4.48 ± 1.35 %ID/g; n = 5) and sixfold (5.70 ± 3.19 %ID/g; n = 3), respectively, compared with historic PBS controls. The changes in whole-brain [18F]DPA-714 binding was highly correlated with brain viral titer, i.e., r = 0.716. Based on brain subregion analysis, all brain regions evaluated (olfactory bulb, striatum, hippocampus, thalamus, hypothalamus, cortex, and cerebellum) were increased similar to the whole brain (details are provided in Table 1 of the ESM). Of note, one of the three mice from the ZIKV + 5A3 group at day 10 PI had [18F]DPA-714 binding (2.25 %ID/g) similar to the ZIKV + PBS (2.53 ± 0.41 %ID/g, n = 5) and PBS + 5A3 (1.97 ± 0.37 %ID/g, n = 5) control groups at day 10 PI, which reduced the mean whole-brain [18F]DPA-714 binding for this group. Moreover, viral RNA was not detected in the brain of this mouse at day 10 PI (Fig. 1b). This subject was not excluded from our analyses because viremia was not assessed in this subject at day 3 and/or day 6 PI allowing for the possibility that this subject was infected and cleared the virus prior to CNS penetration, i.e., it recovered from the neuroinflammatory disease prior to day 10 or it never became infected.
ZIKV alone (ZIKV + PBS) showed a similar modest (2.25 ± 0.11 %ID/g; n = 5) but significant increase in mean whole-brain [18F]DPA-714 binding at day 3 PI relative to historic PBS controls. Mean whole-brain [18F]DPA-714 binding in ZIKV + PBS remained at modest levels at day 6 PI (2.51 ± 0.30 %ID/g, n = 5) and returned to the level of historic PBS controls by day 10 PI suggesting that ZIKV infection in the presence of IFN signaling is sufficient to produce a mild yet transient neuroinflammatory effect measureable by [18F]DPA-714 PET imaging. Importantly, 5A3 treatment in the absence of ZIKV had no detectable effect on mean whole-brain [18F]DPA-714 uptake and shared a similar tracer uptake profile as the historic PBS control mice at all three time points tested. These results demonstrate for the first time the ability of [18F]DPA-714 PET imaging to detect and quantify ZIKV-related neuroinflammation disseminated throughout the brains of infected mice.
Histopathology Findings
To confirm the in vivo findings of ZIKV-induced neuroinflammation as determined by [18F]DPA-714 PET imaging, brain sections from the PET imaged mice were assessed for pathology, in situ hybridization (ISH) to detect viral RNA, and IR and immunofluorescent detection of Iba-1, a microglia/macrophage-specific calcium-binding protein that is highly upregulated during neuroinflammation [31]. Consistent with our imaging results, brains from the susceptible ZIKV + 5A3 treatment group exhibited histopathological lesions consistent with encephalitis with minimal microgliosis beginning as early as day 3 PI (Fig. 3a). By days 6 and 10 PI, microgliosis was more pronounced, with the presence of mononuclear cell perivascular cuffs peaking at day 6 PI and waning by day 10 PI (Fig. 3b, c). Other histological findings consistent with encephalitis include minimal necrotic cellular debris scattered throughout the parenchyma (days 6 and 10 PI) and neuronal degeneration and necrosis observed in the cerebrum as early as day 6 PI and becoming more pronounced and widespread on day 10 PI (cerebrum, hippocampus, and thalamus). Additionally, variable minimal perivascular edema and hemorrhage was present on both days 6 and 10 PI. Moreover, ZIKV RNA was detected by ISH in the cerebral cortex on days 6 and 10 PI and in the hippocampus on day 10 PI in mice administered ZIKV + 5A3 (Fig. 3e, f). No ZIKV RNA was detected on day 3 PI in mice administered ZIKV + 5A3 (Fig. 3d). Mice administered ZIKV + PBS exhibited no signs suggestive of ZIKV infection on day 3 or 6 PI; however, 1/5 animals exhibited minimal lesions consistent with encephalitis (perivascular cuffing, microgliosis) on day 10 PI that may be attributable to ZIKV infection, though no viral RNA was detected in this animal by ISH. As expected, animals administered 5A3 + PBS exhibited no CNS lesions suggestive of ZIKV infection and no viral RNA was detected by ISH at any of the time points tested.
In terms of Iba-1 expression, no statistical difference was observed by IR imaging at days 3 and 6 PI for any of the treatment groups tested, PBS + 5A3, ZIKV + PBS, or ZIKV + 5A3 (Fig. 4a, b, first two rows). At day 6 PI, however, whole-brain Iba-1 expression in the susceptible ZIKV + 5A3 group is modestly, though not significantly, increased. By day 10 PI, and in agreement with the [18F]DPA-714 findings, Iba-1 expression was significantly increased (approximately fourfold) in the ZIKV + 5A3 group compared with either of the control groups (Fig. 4a, b, bottom row). These findings were confirmed by immunofluorescent labeling of Iba-1 in these brains (Fig. 4c, d). Importantly, the single animal in the ZIKV + 5A3 treatment group at day 10 post-infection that showed no appreciable Iba-1 labeling by IR imaging was the same animal that at day 10 PI showed no detectable virus in the brain (Fig. 1b) and no significant whole-brain [18F]DPA-714 binding over controls (Fig. 2a, b). These data are consistent with and serve to confirm our in vivo PET imaging results. These results also demonstrate the ability of in vivo [18F]DPA-714 PET imaging to provide a more sensitive measure of ZIKV-induced neuroinflammation than Iba-1 IR or immunofluorescent imaging.