Intracranial infection of wild-type, Rag1
−/−, and Ifnar1
−/− mice with ZIKV is lethal
To determine if ZIKV causes disease in immunocompetent mice after bypassing the peripheral immune system, WT mice were infected i.c. with a moderately low dose of 2 × 104 PFU. We used the mouse-adapted strain of the African lineage, ZIKV MR766, since murine cells are permissible to this strain and low to moderate doses are sufficient to elicit immune responses in vivo and in vitro. Following i.c. infection, all WT mice had to be euthanized between day 6 and 9 post infection, due to a weight loss of 15% and/or a clinical score of 4 or more, whereas none of the sham-injected mice showed a significant loss of weight or developed signs of disease (Fig. 1a–c). Signs of disease exhibited in ZIKV-infected mice included a hunched posture and reduced activity. However, seizures were not observed. This experiment demonstrated that once ZIKV gains entry to the CNS, it is able to induce lethal disease in the presence of a normal immune system.
To elucidate the disease mechanism and to clarify if key components of the antiviral host response contributed to the disease, we infected Rag1−/− mice, which are deficient in mature T and B cells, and Ifnar1−/− mice, which are unresponsive to all IFN-Is. As expected, sham-injected mice showed mild weight loss and no signs of disease (Fig. 1a–c). When infected peripherally, T and B cells do not play a significant role in host resistance to ZIKV, and Rag1−/− mice do not develop significant weight loss or disease [52]. By contrast, i.c. infection of Rag1−/− mice caused a lethal disease that was similar to the disease seen in WT mice (Fig. 1a–c). Weight loss and onset of signs of disease was slightly delayed in Rag1−/− mice compared with WT mice, but all Rag1−/− mice had to be euthanized on day 8 post infection. In contrast to adaptive immunity, IFN-Is are critical to prevent ZIKV disease in mice [29, 31, 33, 53, 54]. In line with this, i.c. infection of Ifnar1−/− mice resulted in a rapid onset of weight loss from day 2 post infection. Critical weight loss necessitated euthanasia of all Ifnar1−/− mice between days 4 and 5 post infection. These mice had to be euthanized only due to weight loss, which made the survival of Ifnar1−/− mice significantly shorter than in WT or Rag1−/− mice. Other signs of disease in Ifnar1−/− mice were mild and limited to hunched posture and reduced activity.
In wild-type and Rag1
−/− mice, ZIKV is limited to the CNS and predominantly infects neurons but spreads in Ifnar1
−/− mice
Next, we determined the spread of ZIKV by RNase protection assay (RPA). In addition to the CNS, we also assessed viral RNA levels in the liver and testis, as both organs had previously been shown to be a target of ZIKV [29, 33, 55]. In WT mice, ZIKV RNA was detectable in the CNS at low levels at day 4 and had increased by day 6 post infection (Fig. 2a). In line with published observations that ZIKV is rapidly eliminated following peripheral infection, no viral RNA was detectable in the liver or testis of infected WT mice at either day 4 or 6 post infection. By contrast, in Ifnar1−/− mice, ZIKV RNA was detectable at high levels in the CNS, liver, and testis at day 4 post infection (Fig. 2a). In Rag1−/− mice, ZIKV RNA was detectable only in the CNS but not in peripheral organs, and relative levels in the CNS were similar to those seen in WT mice. This demonstrates that IFN-Is but not T or B cells are required to restrict ZIKV spread and replication.
We investigated the spatial distribution of ZIKV RNA in the CNS of mice by in situ hybridization (ISH) at peak disease, i.e., day 6 for WT, day 4 for Ifnar1−/−, and day 8 post infection for Rag1−/− mice. In all three genotypes, ZIKV RNA was present in the cortex, hippocampus, and thalamic region and to a lesser extent in the midbrain and brainstem (Fig. 2b). In addition, in Ifnar1−/− and Rag1−/− mice, ZIKV RNA was also observed in other areas of the cerebrum, and in the midbrain and brain stem, and in Ifnar1−/− mice also in the cerebellum. Overall, while virus distribution was more extensive in Rag1−/− mice than WT mice, ZIKV-infected brain regions were well defined in both genotypes, whereas in Ifnar1−/− mice virus spread appeared more diffuse. Thus, in the absence of a functional IFN-I system, there is an increased virus spread within the CNS, as well as virus spread to other organs.
To identify the cellular target of ZIKV in the CNS, we performed immunofluorescence with an antibody targeting flavivirus NS1. Independent of the genotype of mice, ZIKV infected neurons primarily (Fig. 2c). By contrast, astrocytes and microglia were only rarely NS1-positive in WT, Rag1−/−, and Ifnar1−/− mice. This was also seen by dual-label ISH, where ZIKV RNA co-localized to NeuN-positive neurons but rarely to GFAP-positive astrocytes or tomato lectin-positive microglia (Additional file 2).
ZIKV infection causes prominent perivascular encephalitis in wild-type and Rag1
−/− mice and diffuse infiltration by polymorphonucleated cells in Ifnar1
−/− mice
To assess whether i.c. infection with ZIKV results in tissue pathology, we analyzed tissue sections from the CNS of infected mice at peak disease by routine histology and immunohistochemistry. Compared with sham-injected controls (Fig. 3a–e), ZIKV infection at peak disease (day 6) was associated with extensive infiltration of leukocytes that had accumulated around blood vessels in the CNS parenchyma of WT mice (Fig. 3f). This was accompanied by increased staining of microglia with Iba1 (Fig. 3g) compared with sham-injected controls (Fig. 3b). Also, microglia from infected WT mice had thick and short cellular processes characteristic of activated microglia (i.e., microgliosis). Astrocyte activation (i.e., astrogliosis) was only moderate in the CNS of infected WT mice (Fig. 3h). Further, Iba1 and CD3 stains revealed numerous monocytes/macrophages (Fig. 3i) and T cells (Fig. 3j), respectively, surrounding the intracerebral blood vessels. In contrast to infected and diseased WT mice, the CNS of infected Ifnar1−/− mice at peak disease (day 4) showed only minor inflammatory infiltrates (Fig. 3k). However, unlike WT mice, the brain parenchyma of infected Ifnar1−/− mice contained large numbers of diffusely distributed polymorphonucleated cells (PMNs) (Fig. 3k, arrowheads in the inset). Also, microgliosis (Fig. 3l) and astrogliosis (Fig. 3m) was almost completely absent in the CNS of infected Ifnar1−/− mice. In line with this, only few monocytes/macrophages (Fig. 3n) and some T cells (Fig. 3o) were seen surrounding blood vessels. The brains from infected Rag1−/− mice at peak disease (day 8) showed a phenotype intermediate to that observed in diseased WT and Ifnar1−/− mice (Fig. 3p–t) with moderate inflammatory changes and few PMNs (Fig. 3p, arrowhead in the inset). Expectedly, T cells were absent from the brain of infected Rag1−/− mice (Fig. 3t).
The liver and testis were also examined for histopathological changes at peak disease in all genotypes. No observable features of pathology were present in the liver or the testis of WT mice following i.c. infection with ZIKV (not shown). In two out of seven Ifnar1−/− mice, infiltrating leukocytes were present around the blood vessels of the portal triad. Infection of the liver was characterized by hepatocytes with dark or enlarged nuclei, commonly observed in inflammatory responses (not shown). Furthermore, in Ifnar1−/− mice, the epithelial layer of the testis was disordered and had a noticeable lack of spermatids compared to sham-injected controls (not shown). PMNs were observed in some mice. The liver or testis of Rag1−/− mice showed no overt morphological changes, except for occasional cells that resembled PMNs.
Expression of pro-inflammatory cytokine genes correlates with virus spread in wild-type and Rag1
−/− mice but not in Ifnar1
−/− mice
We determined the expression of pro-inflammatory cytokine genes in the CNS, liver, and testis of mice. In the CNS of WT mice, increased IFN-α1, IFN-β, TNF, IL-1α, IL-1β, IL-6, and IFN-γ mRNA levels were seen at day 4 post infection, and with the exception of IFN-α1, which decreased slightly, cytokine mRNA levels increased further by day 6 post infection (Fig. 4). In the livers of infected WT mice, only TNF mRNA was significantly increased at day 6 post infection, whereas mRNA levels for the other cytokine genes were comparable to the sham-injected mice and IFN-γ mRNA was undetectable. In the testis, no differences were seen between sham-injected and ZIKV-infected mice. Finally, mRNA for IL-2, IL-3, IL-4, and IL-5 was not detectable in any organs or time points in WT mice (data not shown).
In the CNS of Ifnar1−/− mice, IFN-α1, IFN-β, TNF, IL-1α, IL-1β, and IL-6 mRNA levels were increased at day 4 post infection when compared with sham-injected controls (Fig. 4). IFN-γ mRNA was only marginally detectable. In addition, in the livers, IFN-α1, IFN-β, TNF, and IL-1β mRNA and, in the testes, IFN-α1, IFN-β, TNF, IL-1β, and IL-6 mRNA levels increased following infection of Ifnar1−/− mice. Notably, at peak disease, IFN-β, TNF, and IL-1α mRNA levels were significantly lower in the CNS of Ifnar1−/− mice than in WT mice, whereas in the liver, TNF and IL-1β mRNA and, in the testis, TNF, IL-1β, and IL-6 mRNA were significantly higher, concordant with the presence of virus RNA (Fig. 2a). Also, IFN-γ mRNA was not detectable in the livers or testes of sham-injected or ZIKV-infected Ifnar1−/− mice. Comparable to WT mice, IL-2, IL-3, IL-4, and IL-5 mRNA was undetectable in the CNS, liver, or testis of Ifnar1−/− mice (data not shown).
Similar to WT mice, in the CNS of Rag1−/− mice IFN-α1, IFN-β, TNF, IL-1α, IL-1β, IL-6, and IFN-γ mRNA levels increased following i.c. infection with ZIKV (Fig. 4), and IL-2, IL-3, IL-4, and IL-5 mRNA was not detectable (data not shown). Interestingly, TNF, IL-1α, IL-6, and IFN-γ mRNA levels were significantly lower in the CNS of Rag1−/− mice compared with WT mice at peak disease, correlating with the milder histological changes in these mice, while IFN-α1 and IFN-β mRNA levels were comparable. None of the cytokine genes investigated showed significantly increased mRNA levels in the liver and testis following infection.
Inflammatory infiltrates in the CNS are dominated by inflammatory macrophages, NK cells, and resident microglia in wild-type mice and by neutrophils in Ifnar1
−/− mice
Histological analysis had shown prominent perivascular cuffs in the CNS of WT mice and mostly diffuse infiltrating PMNs in Ifnar1−/− mice, following infection with ZIKV (Fig. 3d, g). To further characterize the infiltrating leukocytes, we isolated leukocytes from the CNS of sham-injected and ZIKV-infected mice at day 3 post infection. This early time point was necessary as Ifnar1−/− mice became sick at that day and had to be euthanized. Therefore, all mice were euthanized on day 3 post infection for accurate comparison. Following i.c. infection of WT mice, there was only a small increase in the total number of leukocytes (including microglia) in the CNS (Fig. 5a). However, the proportion of inflammatory macrophages and natural killer (NK) cells was significantly increased in the CNS of infected WT mice, compared with sham-injected mice (Fig. 5a, b). In agreement with the immunohistochemistry (Fig. 3j), a significant increase in the numbers of CD4+ T cells and CD8+ T cells was seen, while the number of microglia and B cells did not significantly change (Fig. 5b). Only very few neutrophils were detected in the CNS of sham-injected or ZIKV-infected WT mice.
In contrast to WT mice, in Ifnar1−/− mice, the total number of leukocytes had almost doubled by day 3 post infection (Fig. 5a). Both by proportion and absolute numbers, neutrophils were by far the most abundant leukocyte cell type in the CNS of ZIKV-infected Ifnar1−/− mice (Fig. 5a, b). Further, neutrophils and CD4+ T cells were the only cell type that increased significantly in the brains of Ifnar1−/− mice following infection. When comparing infected WT and Ifnar1−/− mice, the number of inflammatory macrophages was significantly higher in the CNS of WT mice, whereas the number of neutrophils was significantly higher in Ifnar1−/− mice. Of note, the increase in CD4+ T cells and the low number of inflammatory macrophages identified by flow cytometry fits well to the immunohistochemical findings that showed an increased number of CD3+ T cells (Fig. 3o) but only few monocytes/macrophages surrounding blood vessels (Fig. 3n).
To confirm our results, we performed computational analysis of the dataset through clustering (using FlowSOM) and dimensionality reduction (using tSNE), using the CAPX script in R [47]. Using this approach, we were able to identify the same subsets of cells as in the manual gating analysis (Fig. 5c, d, and Additional file 3), including microglia, infiltrating macrophages, neutrophils, CD4+ T cells, CD8+ T cells, NK cells, NKT cells, and B cells, in addition to other phenotypes. When applying the gating strategy on the clustered cells, we found overall agreement between the cell identities determined by manual gating and those determined by exploration of the clustering results (Additional file 1: Figure S1B). The overall changes in these populations were consistent with those identified by manual gating (Additional file 4a, b) with the most striking change being the appearance of a large number of neutrophils in the ZIKV-infected Ifnar1−/− brains (Fig. 5e). When investigating these cells more closely, we found that a large number of these cells exhibited a less mature phenotype of neutrophils, with lower Ly6G and CD11b expression (Additional file 3 A and B, and C, cluster #12).