A Zika Virus Primary Isolate Induces Neuroinflammation, Compromises the Blood-Brain Barrier, and Upregulates CXCL12 in Adult Macaques

Zika virus (ZIKV) is a neurotropic virus that can cause neuropathy in adults and fetal neurologic malformation following 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 neuropathy. We find that the virus causes prevalent acute and chronic neuroinflammation and chronic disruption of the blood-brain barrier (BBB) in adult animals. Infection results in significant, targeted, and sustained upregulation of the chemokine, CXCL12, in the central nervous system (CNS). 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 neuropathy in adults. Author summary Zika virus (ZIKV) is a virus that can cause neurological problems in adults and damage to the fetal brain. Nonhuman primates (NHPs) are usually superior animal models for recapitulating human neurological disease because their brain, nervous system structure and immune response to virus infection are very similar to that of humans. We have studied the effect of ZIKV infection on the adult NHP brain and made several significant observations. Infection resulted in a high incidence of mild to moderate brain inflammation that persisted for a surprisingly long period of time. We also found that the virus disrupted the blood brain barrier, which is important for controlling transport of material from blood to the brain. It appears that the central nervous system expresses a specific substance in response to virus infection called a chemokine. This specific chemokine may be involved in virus-induced inflammation and/or in repair of virus-induced brain damage. Our data are significant since they help in understanding the mechanism of brain damage caused by ZIKV in adults.


Zika virus (ZIKV) is a neurotropic virus that can cause neuropathy in adults and fetal
23 neurologic malformation following infection of pregnant women. We used a nonhuman primate 24 model, the Indian-origin Rhesus macaque (IRM), to gain insight into virus-associated hallmarks 25 of ZIKV-induced adult neuropathy. We find that the virus causes prevalent acute and chronic 26 neuroinflammation and chronic disruption of the blood-brain barrier (BBB) in adult animals. 27 Infection results in significant, targeted, and sustained upregulation of the chemokine, CXCL12, 28 in the central nervous system (CNS). CXCL12 plays a key role both in regulating lymphocyte 29 trafficking through the BBB to the CNS, and in mediating repair of damaged neural tissue including 30 remyelination. Understanding how CXCL12 expression is controlled will likely be of central 31 importance in the definition of ZIKV-associated neuropathy in adults.

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Author summary 33 Zika virus (ZIKV) is a virus that can cause neurological problems in adults and damage to 34 the fetal brain. Nonhuman primates (NHPs) are usually superior animal models for recapitulating 35 human neurological disease because their brain, nervous system structure and immune response 36 to virus infection are very similar to that of humans. We have studied the effect of ZIKV infection 37 on the adult NHP brain and made several significant observations. Infection resulted in a high 38 incidence of mild to moderate brain inflammation that persisted for a surprisingly long period of 39 time. We also found that the virus disrupted the blood brain barrier, which is important for 40 controlling transport of material from blood to the brain. It appears that the central nervous system 41 expresses a specific substance in response to virus infection called a chemokine. This specific 71 lymphocyte migration into the CNS parenchyma, and in multifarious functions during development 72 and immunity, are mediated through interaction of this chemokine with its primary receptor, 73 CXCR4 (13). Stimulation of CXCR4 by CXCL12 results in activation of an interwoven set of 74 downstream effector pathways (14,15). 75 Nonhuman primates (NHPs) are good animal models for recapitulating human 76 neurological disease since they are genetically and physiologically similar to humans and exhibit 77 CNS and PNS elaboration and brain morphology closely resembling that of humans. We have delivered by C-section, or pregnancy was experimentally terminated, or pregnancy ended through 5 97 ZIKV-mediated demise. As outlined in Fig. 1, necropsy and collection of neural tissue was 98 obtained from the dams at various times before or after parturition or fetal termination. Four of the 99 dams (EL21, ID92, JR20, and JI20) were infected and necropsied 16 or 17 days after infection 100 (acute), and the remainder were maintained for significantly longer periods of time after infection 101 (3.5 to 10 months). The three adult males (HP17, HP87, and JP58) were infected and necropsied 102 after 30 days.  Table 1 provides a summary of observations from neural tissue in individual animals. 108 ZIKV infection disrupts the adult blood-brain barrier. Since ZIKV caused perivascular 109 inflammation with accompanying lymphocytic infiltration, this suggested that inflammation is likely 110 to arise through BBB dysregulation. Fibrinogen is a protein normally restricted to serum. Thus, 111 extravasation of fibrinogen into the perivascular space of CNS vessels is indicative of disruption 112 of the BBB. To detect and quantify ZIKV-associated extravasated fibrinogen we performed multi-113 label immunofluorescence staining using anti-fibrinogen Ab. To visualize microvascular 114 endothelial cells we used anti-GLUT-1 Ab. GLUT-1 is a plasma membrane protein found in 115 abundance on vessel endothelial cells at the BBB (18) (Fig. 3a). Using dual overlay histograms 116 of twenty five vessels per animal we quantified the percent of vessels with fibrinogen 117 extravasation in ZIKV-infected and control groups in both cortical brain tissue and spinal cord 118 (Fig. 3b). There was a significant increase in extravasated fibrinogen with infection in both brain 119 and spinal cord (Fig 3c,d). This indicates that the integrity of the microvascular endothelium and 120 the BBB was compromised following ZIKV infection. RT-PCR to attempt detection of virus in the CSF of the males used in the study (HP17, HP87, 128 and JP58). In addition, some CSF samples were available from unrelated studies with ZIKV-129 infected male and female IRMs, and we also attempted to detect CSF-associated ZIKV in these 130 samples. This analysis indicated that virus was present in the CNS/PNS in a majority of the 131 animals during acute infection and was generally, but not always cleared, within two weeks (

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To investigate the mechanism of ZIKV-induced neural damage and repair we carried out 146 Luminex-based quantification of cytokine levels in the CSF in four female IRMs (EL21, JI20, JR20, 7 147 and ID92) and three male IRMs (HP17, HP87, and JP58) using a macaque-specific panel 148 designed to detect cytokines found during infection and inflammation (a list of the cytokine panel 149 is provided in Table S1). Surprisingly, of the 37 cytokines screened and quantified in this initial 150 analysis only two, CXCL12 and IL1RN, were significantly affected by virus infection (Fig. S3).

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CXCL12 is a chemokine important in multiple processes including neural repair and maintenance  To determine the location of CXCL12 expression in neural tissue following ZIKV infection 163 we carried out immunohistochemistry using an anti-CXCL12 antibody. As expected, the majority 164 of CXCL12 was detected in association with microvascular endothelia, stained with GLUT-1.

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Expression by vascular endothelial cells is consistent with the role of CXCL12 in maintaining BBB 166 integrity and restriction of lymphocytes into the CNS parenchyma during homeostasis. Parallel 167 evalutation of control and ZIKV-infected animals by semi-quantitative immunohistochemistry 168 using an anti-CXCL12 antibody was undertaken to investigate whether the increase in CXCL12 169 seen in the CSF was mirrored in neural tissue. Suprisingly, we did not see a significant difference 170 in cortical tissue between control and ZIKV-infected animals. However, expanding the analysis to 171 spinal cord, there was a significant increase in CXCL12 staining in ZIKV infected tissue consistent 172 with the increase in CXCL12 seen in the CSF (Fig. 4d). These results show that CXCL12 is   smaller plaques on Vero cell monolayers than other strains. In addition, Rio-U1 is more 236 pathogenic than other commonly used American strains in AG129 mice (32). It will be interesting 237 to see whether other ZIKV strains elicit identical neuropathology to that described in this study.

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While the mechanism of ZIKV-induced neuropathology in the IRM model remains to be 239 elucidated, the neural damage and repair we observe is likely to overlap with pre-clinical or clinical   Animal care staff conduct routine husbandry procedures (e.g., cleaning, feeding and watering), histograms. The histograms were then analyzed to determine whether the fibrinogen was above 307 background levels outside of the two primary GLUT-1 peaks; vessels that displayed this phenotype 308 were considered to be extravasated. A total of 25 vessels were examined from each animal via 309 random imaging, but were required to meet the following criteria; vessels must be less than 10µm  Triple-label immunofluorescence was performed with anti-CXCL12, GLUT-1, and CD206.

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As described above, sections were de-paraffinized and rehydrated, followed by antigen retrieval.

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Following another wash, sections were incubated with 5% normal goat serum in PBS for 30 min 339 at room temperature before incubation for 1 h at room temperature with primary antibodies diluted The data sets generated during and/or analysed during the current study are available from 370 the corresponding author on reasonable request.      Table S1. Initial analysis of several animals was carried out using an NHP-specific panel designed 537 to detect key cytokines (Cytokine/Chemokine/Growth Factor 37 Plex NHP ProcartaPlex Panel).

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The specific cytokines detected in the panel are listed above.