A Zika virus primary isolate induces neuroinflammation, compromises the blood-brain barrier and upregulates CXCL12 in adult macaques

Abstract

Zika virus (ZIKV) is a flavivirus that can cause neuropathogenesis in adults and fetal neurologic malformation following the infection of pregnant women. We used a nonhuman primate model, the Indian-origin Rhesus macaque (IRM), to gain insight into virus-associated hallmarks of ZIKV-induced adult neuropathology. We find that the virus causes prevalent acute and chronic neuroinflammation and chronic disruption of the blood-brain barrier (BBB) in adult animals. ZIKV infection resulted in specific short- and long-term augmented expression of the chemokine CXCL12 in the central nervous system (CNS)of adult IRMs. Moreover, CXCL12 expression persists long after the initial viral infection is apparently cleared. CXCL12 plays a key role both in regulating lymphocyte trafficking through the BBB to the CNS and in mediating repair of damaged neural tissue including remyelination. Understanding how CXCL12 expression is controlled will likely be of central importance in the definition of ZIKV-associated neuropathology in adults.

Keywords: CXCL12; Zika virus; blood-brain barrier; flavivirus; nonhuman primate.

Figure 1

NHP study design. Fifteen female IRMs (26) and three male IRMs were infected with 10⁴ PFU of ZIKV strain Rio‐1. Pregnancy in the Rhesus macaque is divided into three 55‐day trimesters, which are equivalent in developmental landmarks to the trimesters in human pregnancy (6). These trimesters are depicted in the timeline at the top, as are relative times of infection, sample collection and necropsy for the dams. Acute viremia was detected in all animals (Figure S1).

Figure 2

Neuroinflammation in the spinal cord of ID92 (A and B) and the spinal cord and brain of HE27 (C‐F). ID92 had multifocal glial nodules (arrows) within both the white (A) and grey (B) matter of the spinal cord. HE27 had widespread perivascular inflammation (arrows) at multiple levels of the cerebrum (C and D), cerebellum (E) and spinal cord (F). H&E, Bar = 100um.

Figure 3

ZIKV disrupts the BBB in adult IRMs as evidenced by an increase in extravasated vessel fibrinogen during infection. Vessel endothelial cells were detected using Alexa Fluor 488‐labeled anti‐GLUT‐1 Ab (green) and fibrinogen detected using Alexa Fluor 594‐labeled anti‐fibrinogen Ab (red). Nuclei were detected with DAPI. A. Cross‐section and immunofluorescence analysis of a stained vessel without extravasation and associated dual overlay histogram. B. Immunofluorescence stained vessel with extravasation and associated dual overlay histogram. Percent of extravasated vessels in cortical brain tissue (C) and spinal cord (D).

Figure 4

Upregulation of CXCL12 in the CSF of adult IRMs. A. Total protein concentrations in the CSF of acutely and chronically infected IRMs. For acutely infected animals (HP87, EL21, JI20 and JR20) a two‐tailed paired T‐test was used to compare protein concentrations with match pre‐infection samples. For samples derived from later times following infection, comparison with the pre‐infection samples using a two‐tailed unpaired T‐test indicated that the means of the two samples were below the threshold of significance (P = 0.12). B and C. Concentrations of CXCL12 and IL1RN in CSF samples following ZIKV infection, respectively. For purposes of statistical analysis samples were placed in four groups based on time of collection relative to virus infection. P values were determined by comparison to preinfection controls using two‐tailed unpaired T‐tests. Exact times of CSF sample collection are displayed in Figure S4. D. Immunohistochemistry using anti‐CXCL12. CXCL12 was visualized in cortical brain or spinal cord tissue for several uninfected controls and animals acutely or chronically infected with ZIKV. ImageJ was used to quantify the area of CXCL12‐positive staining in 20 random images per animal. A.U. – arbitrary units.