Postnatal Zika virus infection is associated with persistent abnormalities in brain structure, function, and behavior in infant macaques

Abstract

The Zika virus (ZIKV) epidemic is associated with fetal brain lesions and other serious birth defects classified as congenital ZIKV syndrome. Postnatal ZIKV infection in infants and children has been reported; however, data on brain anatomy, function, and behavioral outcomes following infection are absent. We show that postnatal ZIKV infection of infant rhesus macaques (RMs) results in persistent structural and functional alterations of the central nervous system compared to age-matched controls. We demonstrate ZIKV lymphoid tropism and neurotropism in infant RMs and histopathologic abnormalities in the peripheral and central nervous systems including inflammatory infiltrates, astrogliosis, and Wallerian degeneration. Structural and resting-state functional magnetic resonance imaging (MRI/rs-fMRI) show persistent enlargement of lateral ventricles, maturational changes in specific brain regions, and altered functional connectivity (FC) between brain areas involved in emotional behavior and arousal functions, including weakened amygdala-hippocampal connectivity in two of two ZIKV-infected infant RMs several months after clearance of ZIKV RNA from peripheral blood. ZIKV infection also results in distinct alterations in the species-typical emotional reactivity to acute stress, which were predicted by the weak amygdala-hippocampal FC. We demonstrate that postnatal ZIKV infection of infants in this model affects neurodevelopment, suggesting that long-term clinical monitoring of pediatric cases is warranted.

Figure 1.. ZIKV RNA in blood and tissues.

(A) Plasma ZIKV RNA concentration as measured by RT-PCR. (B) Assessment of ZIKV RNA concentration by RT-PCR in lymphoid (top) and non-lymphoid tissues (bottom) at day 3 or 14-15 post-infection. Low RNA indicates < 20 ng/μl was run in the PCR. (C) Representative detection of ZIKV RNA by RNAscope in situ hybridization in nervous system tissues. Arrowheads indicate some of the most intense RNAscope signals (red). No arrowheads were placed on the bottom left panel as the RNAscope signal was diffusely positive in the cerebellum at this magnification. The scale bars represent 100μm and 50μm in the left and right panels, respectively. 4-5 images per brain area were examined.

Figure 2.. Humoral immune response to ZIKV-infection.

(A) ZIKV E-specific IgM plasma concentrations measured by ELISA. (B) ZIKV E-specific IgG plasma concentrations measured by ELISA. (C) ZIKV neutralization activity of RM plasma measured by focus reduction neutralization test (FRNT). The dashed line indicates the limit of detection for the respective assays (i.e., the lowest dilution tested). Undetectable values were set at half the lower limit of detection for visualization purposes.

Figure 3.. Neurohistopathology of postnatal ZIKV infection in infant RMs.

Hematoxylin and eosin (H&E) staining of nervous tissue sections from ZIKV-infected infant RM (left) and age-matched controls (right). (A) Occipital cortex with multifocal meningeal infiltrates (arrowheads) in ZIKV-infected RM. (B) Anterior thoracic spinal cord with circumscribed inflammatory focus (arrowhead 1), dilated axons (arrowhead 2) and spheroid formation (arrowhead 3) in ZIKV-infected RM. (C) Basal ganglia showing hypercellularity with glial cells in the underlying stroma of the ependymal epithelial cell lining (arrowhead) in ZIKV-infected RM. (D) cauda equina with multifocal perivascular inflammatory cells (arrowheads) in ZIKV-infected RM. Scale bars, 200μm (10X), 100μm (20X). 4-5 images per brain area were examined.

Figure 4.. Astrogliosis and apoptosis of brain regions in postnatally-infected infant RMs.

Staining of central nervous system tissue sections from ZIKV-infected infant RM (left) and age-matched controls (right). (A) H&E staining with moderate astrogliosis in the white matter in the periventricular region of the occipital cortex and glial nodule in the grey matter of the basal ganglia in ZIKV-infected RM. (B) GFAP (Glial Fibrillary Acidic Protein) staining of the occipital cortex and basal ganglia demonstrating reactive astrocytes in ZIKV-infected RM. (C) Caspase-3 staining of the occipital cortex and basal ganglia demonstrating increased apoptosis in ZIKV-infected RM. Scale bars, 200μm (10X), 100μm (20X). 4-5 images per brain area were examined.

Figure 5.. Structural neuroimaging at three and six months of age after postnatal ZIKV infection of infant RMs.

(A) Sagittal T1-weighted structural MRI images through the corpus callosum (top) and hippocampus (bottom) at six months of age; yellow arrows illustrate the increased lateral ventricle volume in ZIKV-infected RMs. (B) Structural MRI measurements in cubic millimeters for total brain volume and lateral ventricle volume as well as specific grey and white matter areas corrected for total brain volume (TBV) at three and six months of age in ZIKV-infected RMs and controls. The correction used was specific brain region/TBV x 100.

Figure 6.. Functional neuroimaging at three and six months of age after postnatal ZIKV infection of infant RMs.

(A) Depiction of Amygdala-Hippocampus resting state functional connectivity (FC) measured by rs-fMRI at six months of age displayed in the UNC-Emory RM infant atlas (35). Images show the same two coronal slice series in each subject with the seed (green) placed in the left amygdala and the FC r-values in the hippocampus colored according to the scale provided. Results for the right amygdala seed not shown for clarity, but are provided in Table S4. L=left hemisphere; R=right hemisphere; r-values=raw FC correlations. (B) rs-fMRI longitudinal measurements of FC between specific regions of interest at three and six months of age in ZIKV-infected RM and controls. V3 = visual area 3; OFC = orbital frontal cortex.

Figure 7.. Assessment of the behavioral response to acute stress using the Human Intruder Paradigm at six and twelve months of age after postnatal ZIKV infection of infant RMs.

(A) Description of the Human Intruder task measuring the ability to modulate emotional behavior based on the salience of the threat presented. ALONE: the animal is placed alone in a novel room; PROFILE: a human intruder wearing a mask presents his profile to the animal; STARE: the intruder makes direct eye contact with the animal. (B) Comparison of behaviors using a standard ethogram (described in Table S5) in response to the three stress levels in ZIKV-infected infant RMs and age-matched uninfected controls at six months of age. Bottom right graph shows and pre- and post-stress plasma cortisol concentrations. (C) Correlations between the observed behavioral responses to stress with functional connectivity (r-values) between specific brain regions of interest in ZIKV-infected infant RMs and age-matched uninfected controls at six months of age. (D) Comparison of behaviors using a standard ethogram (described in Table S5) in response to the three stress levels in ZIKV-infected infant RMs and age-matched uninfected controls at twelve months of age. Bottom right graph shows pre- and post-stress plasma cortisol concentration.