https://orcid.org/0000-0003-0916-360X
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Abstract
The arthropod-borne chikungunya virus poses a re-emerging global health threat, causing millions of cases worldwide. Neurological complications induced by chikungunya virus are being increasingly reported in vulnerable populations, including infants and young children. However, the mechanisms by which chikungunya virus invades the central nervous system and drives prolonged neurological dysfunction remain poorly defined. Studying neurological chikungunya virus infection has been limited by the lack of an immunocompetent, neurodevelopmentally appropriate small-animal model that reliably develops central nervous system infection and neurological disease by infection routes analogous to natural transmission. Following a screen of ten Collaborative Cross mouse strains, we identified four-to-six-week-old CC041 mice as an immunocompetent, neurodevelopmentally appropriate model that consistently exhibits chikungunya virus neuroinvasion and clinical signs consistent with neurological disease following peripheral inoculation. Central nervous system infection in CC041 mice was dependent on the chikungunya viral strain used, despite comparable viral replication at the inoculation site, and occurred at physiologically relevant inoculation doses without enhancement at higher doses. Comparative analyses with neuroinvasion-resistant C57BL/6J mice demonstrated that susceptibility to neuroinvasion in CC041 mice was associated with enhanced dissemination beyond the site of inoculation, prolonged serum viral load, and lower peripheral type I interferon levels early in infection. In vivo imaging using a reporter chikungunya virus confirmed rapid viral spread and early brain localization in CC041 mice, in contrast to the restricted peripheral infection observed in C57BL/6J mice. Examination of viral antigen expression by immunohistochemistry revealed regionally variable viral localization in the brain, with central nervous system infection occurring independently of virus-induced blood-brain barrier disruption or experimental macrophage depletion. Furthermore, CC041 mice developed clinical signs consistent with neurological disease that persisted beyond the period when infectious virus was detected in the brain. Together, these findings establish CC041 mice as a tractable, immunocompetent model for studying chikungunya virus-associated neurological disease following peripheral infection.
Author summary
Chikungunya virus is a mosquito-borne infection that continues to spread worldwide and can cause long-lasting neurological problems in some individuals. We still do not fully understand how the virus invades the brain or why only certain people develop severe neurological symptoms. A significant challenge has been the lack of a small mammalian model with an intact immune system that develops brain infection via a natural route of exposure. In our study, we identified a mouse strain, CC041, that is uniquely vulnerable to chikungunya virus entering the brain via an inoculation route similar to a mosquito bite. These mice show viral spread throughout the body, including detectable virus in the brain, and clinical signs of neurological disease that persist even after infectious virus is no longer detectable in the brain. By comparing CC041 mice to a mouse strain resistant to brain infection, we found that susceptibility was linked to the virus's ability to spread throughout the body and to the duration of virus circulation in the bloodstream, rather than to differences in virus replication at the infection site. This mouse model provides an essential tool for understanding how chikungunya virus affects the central nervous system.
Citation: Lantz AM, Green FM, Cowan AJ, Miller KA, Saldivar RA, Anderson EJ, et al. (2026) Characterization of an immunocompetent, young adult mouse model for studying chikungunya virus neuroinvasion and central nervous system infection. PLoS Pathog 22(7): e1014395. https://doi.org/10.1371/journal.ppat.1014395
Editor: Julie M. Fox, National Institute of Allergy and Infectious Diseases Laboratory of Viral Diseases, UNITED STATES OF AMERICA
Received: January 14, 2026; Accepted: June 22, 2026; Published: July 10, 2026
Copyright: © 2026 Lantz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The deposited raw data have been updated and are available at: https://doi.org/10.5281/zenodo.21086568.
Funding: This research was supported by the National Institutes of Health grant numbers K01OD026529 (V.K.B), R21NS135258 (V.K.B.), R01AI153209 (W.B.K.), and T32GM145432 (F.M.G.). R.A.S. was supported in part by the Intersection Between Cancer & Infectious Diseases Fellowship (Contract #95928) from the American Cancer Society. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Chikungunya virus (CHIKV) represents a re-emerging global public health threat. This Old-World alphavirus, transmitted by Aedes aegypti and Aedes albopictus mosquitoes, continues to expand its geographic range and cause large-scale outbreaks across India, Southeast Asia, and the Americas. CHIKV causes millions of infections annually, accounting for an estimated average loss of more than 106,000 disability-adjusted life years (DALYs) every year [1]. As of December 2025, CHIKV has been declared a high-risk threat by the World Health Organization due to frequent outbreaks, including in Guangdong Province, China, and multiple countries in South America [2,3]. Since January 2025, more than 240,000 cases and 90 CHIKV-related deaths have been reported across 16 countries and territories, including a locally-acquired infection in New York, USA, in October 2025 [2,4].
CHIKV infection typically presents as an acute febrile illness with arthralgia and rash. However, neurological complications are being increasingly recognized, with up to 25% of cases presenting with neurological manifestations, such as encephalitis, altered consciousness, seizures, cognitive dysfunction, and motor impairment [5,6]. Infants under one year of age are especially at risk, with an estimated 25–28% of infected children developing neurological symptoms [5,7]. Despite the abundance of documented CHIKV neurological disease cases, the mechanisms underlying CHIKV neuropathogenesis remain poorly understood, limiting the development of effective countermeasures.
Until recently, prevention strategies were limited to vector control and personal protection, as no licensed CHIKV vaccines were available. This status changed in late 2023, when the United States Food and Drug Administration (FDA) approved IXCHIQ, a single-dose live-attenuated vaccine for adults, followed soon after by approvals in Canada and Europe [8]. However, early post-market surveillance identified safety concerns, including neurological and cardiac adverse events in older adults, prompting updated guidance and a temporary pause in vaccine use for specific populations in 2025 [9]. More recently, the approval of VIMKUNYATM, a single-dose virus-like particle-based CHIKV vaccine for individuals aged 12 years and older, represents a significant advance. Regardless, limited understanding of CHIKV neuropathogenesis continues to constrain evaluation of vaccine safety, efficacy, and durability, particularly in populations at risk for neurological disease that are not eligible for vaccination [10,11]. These developments underscore the urgent need for a deeper understanding of chikungunya virus neuropathogenesis to inform the development of safer, more effective vaccines and therapeutics, particularly given the absence of approved antiviral therapies and the significant risk of severe or chronic disease accompanying the continued geographic expansion of competent mosquito vectors [5,12].
One significant barrier to advancing such interventions is the limited ability to study CHIKV infection within the human central nervous system (CNS), highlighting the need for robust experimental models. Several animal systems, including zebrafish [13,14] and nonhuman primates (NHPs) [15,16], have provided valuable insights into CHIKV pathogenesis, but each model has fundamental limitations. Zebrafish lack a mammalian immune system, limiting their translational relevance, whereas NHPs provide high-fidelity disease modeling but are costly and typically reserved for late-stage therapeutic evaluation. In contrast, mice offer substantial advantages for studying CHIKV neuropathogenesis, including a fully functional mammalian CNS and immune system, extensive availability of species-specific reagents, and tractable genetic tools that facilitate mechanistic inquiry. Young adult (4–6-week-old) mice, which are neurodevelopmentally equivalent to a 1–2-year-old child [17], one of the human age groups most susceptible to CHIKV encephalitis [7], possess ideal immune and neurodevelopmental statuses for modeling CHIKV-induced neurological disease. Developing an immunocompetent young-adult mouse model capable of developing CNS infection after peripheral inoculation is therefore essential to dissect the mechanisms of CHIKV neuroinvasion and neuropathogenesis.
To address this gap, our team leveraged the genetically diverse Collaborative Cross (CC) recombinant inbred panel and identified a novel mouse strain, CC041/TauUncJ (CC041), that is naturally susceptible to CHIKV neuroinvasion after peripheral inoculation as young adults [18,19]. CC041 mice uniquely supported consistent CNS infection following subcutaneous footpad (SQ FP) injection in a CHIKV strain-, but not a CHIKV dose-dependent manner. These mice also demonstrated viral dissemination and signs of neurological disease that persisted even after infectious virus was no longer detectable in the brain, similar to CHIKV neuropathogenic manifestations in humans [7]. These findings establish CC041 mice as a robust, immunocompetent, developmentally appropriate model for studying CHIKV neuroinvasion and neuropathogenesis following a more natural route of infection.
Results
CC041 mice are susceptible to CHIKV neuroinvasion in a CHIKV strain-dependent manner
Standard inbred mouse strains, such as C57BL/6J (B6), are typically not susceptible to CHIKV neuroinvasion following peripheral infection after weaning age [20]. Therefore, we turned to the genetically diverse Collaborative Cross (CC) recombinant inbred mouse panel to identify alternative susceptible mouse strains. Ten different CC mouse lines were inoculated via SQ FP injection with SL15649, a CHIKV strain previously shown to be neurovirulent in young adult B6 mice following direct intracranial (IC) inoculation [20]. At 5 days post-infection (DPI), brain viral titers varied widely across CC lines, with CC007 and CC041 mice demonstrating consistent rates of neuroinvasion (S1A Fig). While neuroinvasion was not observed in a follow-up experiment in CC007 mice (S1B Fig), longitudinal analysis in CC041 mice confirmed detectable virus in the brain as early as 3 DPI, peaking at 5 DPI, and declining below the assay limit of detection (LOD) by 10 DPI (S1C Fig). Based on their reproducible susceptibility to CHIKV neuroinvasion, CC041 mice were selected for further characterization.
We next evaluated whether neuroinvasion in CC041 mice varied by CHIKV strain. Young adult (4–6-week-old) male and female CC041 mice were infected via SQ FP with a physiologically relevant dose of 103 plaque-forming units (PFU) [21] with one of three CHIKV strains: two East/Central/South African (ECSA) lineage strains, La Réunion (LR) and SL15649, and one Asian lineage strain, SM2013. CC041 mice infected with ECSA lineage CHIKV strains exhibited consistent viral titers in the brain at 5 DPI, with CHIKV LR generating the highest titers (Fig 1A). In contrast, CC041 mice infected with the Asian lineage strain SM2013 rarely showed detectable CNS infection. Ipsilateral foot and serum viral titers did not differ significantly across CHIKV strains (Fig 1B and 1C), indicating that the CHIKV strain-specific neuroinvasive capacity is not attributable to differences in peripheral replication or systemic viral load. Because the ECSA-lineage CHIKV strains, LR and SL15649, consistently resulted in neuroinvasion, these two strains were used for subsequent studies. For clarity and consistency, CHIKV LR data are denoted with red or maroon symbols (CC041 mice) and black and gray symbols (B6 mice), whereas CHIKV SL15649 data are denoted by bright blue (CC041 mice) and navy (B6 mice) symbols throughout the figures.
CC041 mice were inoculated SQ via FP with 103 PFU of either CHIKV LR (red symbols), SL15649 (blue symbols), or SM2013 (orange symbols). At 5 DPI, brain (A), ipsilateral foot (B), and serum (C) were collected, and viral titers were determined by plaque assay. Assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the plaque assay LOD were assigned a value of half of the LOD. * p < 0.05, *** p < 0.001, Kruskal-Wallis test.
We next assessed whether the dose of CHIKV inoculated in the foot influenced subsequent viral load in the brain. CC041 mice were infected with CHIKV LR or SL15649 at either the physiologically relevant 103 PFU dose as before, or at 105 PFU, a 100-fold higher dose. For CHIKV LR, brain titers at 5 DPI were comparable across doses (S2A Fig), and no dose-dependent differences were observed in foot or serum titers (S2B and S2C Fig). Similar patterns were observed for CHIKV SL15649, although brain titers trended lower in mice infected with the 103 PFU dose (S2D–S2F Fig). Because of these findings, for subsequent studies, infections were performed using the physiologically relevant 103 PFU inoculum dose.
CHIKV dissemination is widespread in CC041 mice, but not in B6 mice, following SQ FP inoculation
We next compared the dynamics of CHIKV dissemination and neuroinvasion in susceptible CC041 mice and resistant B6 mice. CC041 mice infected with CHIKV LR exhibited consistent CNS infection at 5 DPI, whereas B6 mice showed no detectable virus in the brain (Fig 2A), despite comparable viral replication in the inoculated foot (Fig 2B). This finding is consistent with previous studies in B6 mice using other CHIKV strains [20]. Although not statistically significant, CHIKV remained detectable in the serum of most CC041 mice at 5 DPI; in contrast, virus in the serum had largely cleared in B6 mice by that time (Fig 2C), suggesting prolonged systemic viral presence in CC041 mice.
CC041 and C57BL/6J mice were infected SQ via FP with 103 PFU of CHIKV nLuc-expressing CHIKV LR strain (CHIKV-nLuc) or the wild-type (WT) CHIKV LR strain. A-C) Brain (A), ipsilateral foot (B), and serum (C) viral titers in CC041 (red squares) and B6 mice (black circles) at 5 DPI. D) Viral brain titers of WT CHIKV LR- (red squares) and CHIKV-nLuc (maroon squares)-infected CC041 mice at 3, 5, and 7 DPI. E,F) Representative images of CC041 (E) and B6 (F) mice imaged via the IVIS spectral imaging system. G) Quantification of nLuc signal from 1 to 7 DPI in CC041 (maroon squares) and B6 (gray circles) mouse heads from IVIS spectral images. Assay limit of detection (LOD) is depicted by the horizontal dotted line (A-D); all samples that fell below the plaque assay LOD were assigned a value of half of the LOD. *p < 0.05, **p < 0.01, Multiple Mann-Whitney (A) and Šidák’s multiple comparisons (G) tests.
To visualize viral dissemination in vivo, we employed a recombinant nanoluciferase-expressing CHIKV LR (CHIKV-nLuc) [22]. Brain titers were slightly lower in the CHIKV-nLuc-infected mice than those infected with WT CHIKV LR at 3 DPI but were otherwise comparable through 7 DPI (Fig 2D), validating the reporter virus for imaging. In CC041 mice, in vivo imaging system (IVIS) measurements revealed that at 1 DPI, luminescent signal was restricted to the inoculated foot; by 2 DPI, signal appeared at distal anatomical sites consistent with draining inguinal lymph nodes and was observed in the head for the first time (Fig 2E). By 3 and 5 DPI, the CHIKV-nLuc signal was widespread throughout the body before beginning to decline by 7 DPI. In stark contrast, B6 mice rarely demonstrated detectable CHIKV-nLuc signal outside the inoculated foot at any time point (Fig 2F). While whole body imaging precluded precise anatomical localization of signal, particularly in distinguishing signal originating from the brain versus adjacent structures, quantification of cranial radiance revealed significantly higher viral signal in the heads of CC041 mice compared to B6 mice at 5 and 7 DPI (Fig 2G), further supporting the enhanced neuroinvasive susceptibility of CC041 mice.
Systemic and peripheral viral replication and type I interferon levels differ between CHIKV neuroinvasion-susceptible and neuroinvasion-resistant mouse strains during early infection
After determining that neuroinvasion begins as early as 2 DPI in CC041 mice, we next evaluated early peripheral events that may influence susceptibility to CNS infection. CHIKV SL15649 (Fig 3A) and LR (S3A Fig) replication in the inoculated foot was comparable between CC041 and B6 mouse strains at 1 and 3 DPI, indicating that differences in neuroinvasion are not attributable to local replication efficiency. In contrast, CHIKV SL15649 induced significantly higher serum viral loads at 3 DPI in CC041 mice (Fig 3B). Serum viral loads similarly trended higher in CHIKV LR-infected mice at 1 and 3 DPI (S3B Fig), consistent with the prolonged serum viral load observed with CHIKV LR infection at 5 DPI (Fig 2C).
CHIKV titers and IFN-α levels early after CHIKV infection in the periphery of CC041 and B6 mice. CC041 (bright blue squares) and C57BL/6J (navy circles) mice were infected SQ via FP with 103 PFU of CHIKV SL15649. Infectious virus was quantified by plaque assay (A, B), and IFN-α was quantified by Luminex (C, D) in clarified left hind foot homogenates and sera. Assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the assay LOD were assigned a value of half of the LOD. *p < 0.05, **p < 0.01, Šidák’s multiple comparisons tests.
Type I interferons (IFNs), IFN-alpha (IFN-α) and IFN-beta (IFN-β), are essential early antiviral cytokines that limit alphavirus replication and systemic spread by inducing IFN-stimulating genes (ISGs) that establish an antiviral state in infected and neighboring cells [23]. As type I IFN responses help control alphavirus dissemination [24], we quantified IFN-α and IFN-β levels in non-neurologic compartments early after infection. CHIKV-infected B6 mice exhibited significantly higher IFN-α levels in both the ipsilateral foot (Fig 3C, 17-fold increase from baseline to peak levels) and serum (Fig 3D, 26-fold increase from baseline to peak levels) at 1 DPI. In contrast, CC041 mice showed only a sevenfold increase in IFN-α levels in the ipsilateral foot at peak viral load. CC041 mice also had a delay in peak serum IFN-α concentrations, although the magnitude was similar to that in B6 mice (26-fold increase from baseline to peak levels). Interferon beta (IFN-β) concentrations in the ipsilateral foot followed similar kinetics to IFN-α in both CC041 and B6 mice (S3C Fig); however, IFN-β levels in both strains were largely undetectable in serum (S3D Fig). These measurements demonstrate that peripheral type I IFN response dynamics, particularly IFN-α, differ in magnitude and timing between CC041 and B6 mice, with the neuroinvasion-resistant B6 mice exhibiting higher IFN levels at the site of initial CHIKV replication.
To further compare the peripheral and systemic immune response to CHIKV infection between CC041 and B6 mice, we evaluated cytokine and chemokine levels in the ipsilateral foot and serum by qPCR and Luminex. Baseline expression of Infa4, Infb1, Ccl2, Il10, Tnf, Ifng, and Il6 in the foot did not signifcantly differ between mock-infected CC041 and B6 mice (S1 Table). Following infection with CHIKV LR, expression of all genes increased in both mouse strains (S3E Fig), with B6 mice demonstrating signficantly upregulated Tnf expression at 1DPI and CC041 mice demonstrating significantly upregulated Il6 experession at 3 DPI. While expression of Ifna4, Ifnb1, and Il6 returned to baseline levels by 7–14 DPI, Ccl2, Il10, Tnf and Ifng expression remained upregulated at 14 DPI.
Quantification of cytokines and chemokines in the foot and serum up to 5 days following CHIKV LR infection by Luminex (S4 Fig) revealed increased levels of several of the 23 analytes measured. Increased analytes included IL-4, IL-5, IL-10, IL-12p40, CCL4/MIP-1β, CXCL1/KC, and TNF-α in the foot alone and CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, and G-CSF in both the foot and serum. Although a few analytes were significantly higher in mock-infected B6 mice compared to CC041 mice, following CHIKV infection, only limited strain-specific differences were observed. Specifically, B6 mice exhibited higher levels of IL-1β and CCL5/RANTES in the foot at 1 DPI and CCL11/Eotaxin in the foot at all time points, whereas CC041 mice demonstrated significantly higher G-CSF levels in the foot at 5 DPI. Collectively, these findings indicate that CC041 and B6 mice mount broadly comparable local and systemic proinflammatory cytokine and chemokine responses following CHIKV infection.
Evaluation of CHIKV localization and entry into the CNS
We next returned our attention to the CNS and evaluated the location(s) where CHIKV infects the CC041 mouse brain by labeling formalin-fixed, paraffin-embedded brain sections for CHIKV antigen by immunohistochemistry (IHC). Even at 5 DPI, when brain viral titers peak in CC041 mice (S1C Fig), CHIKV antigen (brown chromogen) was detected only sporadically in brain sections. Additionally, when present, CHIKV antigen labeling varied across brain regions. Regions where CHIKV antigen was detected included the cerebellar nuclei and the cerebral cortex, where antigen-positive cells extended from the meninges into the brain parenchyma (Fig 4A). This nonspecific pattern of CHIKV localization is consistent with hematogenous entry of virus into the CNS, as observed in fatal human CHIKV cases and zebrafish models [14,25].
A) CC041 mouse brain sections of cerebellum (left image) and cerebral cortex (right image) at 5 DPI following 103 PFU CHIKV LR infection SQ via FP showing positive CHIKV antigen labeling (brown chromogen, insets) by IHC. B) Cerebral brain section of a CHIKV-LR-infected CC041 mouse at 5 DPI labelled with 3plex-chromogenic IHC showing CHIKV antigen-positive cells (teal chromogen) colocalizing (dark blue color denoted by red arrows) with astrocyte marker (GFAP) identified by purple chromogen. Oligodendrocytes (OLIG2) are denoted with yellow chromogen. C) Cerebral brain section of a CHIKV-LR-infected CC041 mouse at 5 DPI labelled with 4plex-chromogenic IHC showing CHIKV antigen-positive cells (teal chromogen) colocalizing (green color denoted by black arrows) with oligodendrocyte marker (SOX-10) identified by yellow chromogen. Neurons (NeuN) are identified by brown chromogen, and microglia (IBA1) are identified by purple chromogen. D) CC041 (red symbols) and B6 (black symbols) mice were infected with 103 PFU of CHIKV LR (closed symbols) or mock-infected with PBS (open symbols) and euthanized at 5 DPI following an intraperitoneal injection of 100 mg/mL of sodium fluorescein (NaF). NaF and total protein were quantified in 20% w/v brain homogenates immediately following necropsy. E) CHIKV titers at 5 DPI in brains of CC041 mice who received clodronate liposomes to deplete macrophages (“Clodronate”) or control liposomes (“Control”) 2 days prior to infection with 103 PFU CHIKV-nLuc (maroon squares) via SQ FP inoculation. Plaque assay limit of detection (LOD) is depicted by the horizontal dotted line. ***p < 0.001, Mann-Whitney test (D).
We next performed multiplex-chromogenic IHC to determine which resident CNS cells (e.g., neurons, astrocytes, oligodendrocytes, and microglia) were targeted by CHIKV (Fig 4B and 4C). CHIKV antigen, labelled with teal chromogen, frequently colocalized with GFAP+ astrocytes labeled with purple chromogen (colocalized areas seen as dark blue and denoted with red arrows, Fig 4B). CHIKV was also found to occasionally colocalize with the oligodendrocyte marker labeled with a yellow chromogen (colocalized areas shown in green and denoted with black arrows, Fig 4C) but was not detected in NeuN+ neurons (Fig 5C, brown chromogen). CHIKV was also not detected in IBA1+ microglia (Fig 5C, purple chromogen), in line with previous reports that microglia are not permissive or only poorly permissive to CHIKV replication [26]. After quantifying comparable numbers of astrocytes and oligodendrocytes in brain sections, we found 0.66% of astrocytes were infected with CHIKV compared to 0.093% of oligodendrocytes, indicating that astrocytes were infected approximately 7X more frequently than oligodendrocytes. These findings suggest astrocytes are the primary resident CNS cellular target of CHIKV in CC041 mice, consistent with previous reports [27].
CC041 mice were infected with 103 PFU CHIKV LR or mock-infected with PBS SQ via FP. A) Mock-infected (open symbols, n = 25) and CHIKV-infected (closed symbols, n = 49) mice were weighed daily. Clinical scores in mock-infected (B, n = 9) and CHIKV-infected (C, n = 13) CC041 mice were assigned to each mouse daily as follows: 0 = clinically normal; 1 = mild abnormal gait and tail posture; 2 = hunched posture, abnormal gait, unkempt hair coat; 3 = marked hunched posture, and decreased ambulation (humane endpoint); 4 = moribund/dead/euthanized. During daily clinical observations, qualitative descriptors were recorded, including facial grimace, hyperactivity, abnormal behaviors (e.g., digging, excessive grooming), altered gait or posture (e.g., wide-based gait, wide rear-limb posture), and abnormal tail carriage (e.g., stiff-tail, tail-flagging). D) Spider plot illustrating results of a Key Terms analysis of the qualitative descriptors observed in CHIKV LR- (red shading) and mock-infected (black shading) CC041 mice. E) Foot measurements were measured daily in sedated mock-infected (open symbols, n = 5) or CHIKV LR-infected (closed symbols, n = 6) CC041 mice. F) Viral RNA was quantified by qPCR in mock-infected (open symbols, n = 1-6 mice/timepoint) or CHIKV LR-infected (closed symbols, n = 2-6 mice/timepoint) CC041 left brain hemispheres. Data are presented as the mean SEM; in (A) and (E), the horizontal dotted line represents the 0 DPI value to which values at all subsequent timepoints were normalized; in (F), the assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the plaque assay LOD were assigned a value of half of the LOD; * p < 0.05, ** p < 0.01, **** p < 0.0001, Šidák’s multiple comparisons test.
Because several viruses (e.g., human immunodeficiency virus, Japanese encephalitis virus, and Zika virus [28–30]) increase blood-brain barrier (BBB) permeability to facilitate CNS entry, we assessed BBB integrity by measuring sodium fluorescein (NaF) accumulation in brain homogenates of neuroinvasion-susceptible CC041 mice and neuroinvasion-resistant B6 mice. NaF levels did not differ significantly between CHIKV-infected and mock-infected mice in either CC041 or B6 strains at 2 DPI (S5A Fig) or 5 DPI (Fig 4D). However, CC041 mice exhibited significantly higher baseline NaF brain accumulation than B6 mice, regardless of infection status, suggesting that CC041 mice possess an inherently more permeable BBB.
C-C motif chemokine ligand 2 (CCL2) signaling has also been found to promote the transmigration of CHIKV-infected monocytes across the BBB in fatal human CHIKV cases and in vitro [25], suggesting that CHIKV may disseminate throughout the body via infected circulating monocytes and enter the CNS via a “Trojan horse” mechanism. Therefore, we next assessed macrophage involvement in CHIKV neuroinvasion in CC041 mice by depleting monocytes and macrophages with clodronate liposomes or mock-depleting with control liposomes two days before infection with CHIKV LR (S2 and S3 Tables). Monocyte and macrophage depletion did not significantly alter brain titers at 5 DPI (Fig 4E), nor did it affect clinical scores compared to mice administered control liposomes (S5B and S5C Fig), indicating that macrophages are not required for CHIKV entry into the CNS in CC041 mice.
CHIKV-infected CC041 mice develop persistent clinical signs consistent with neurological dysfunction
To evaluate the clinical impact of peripheral CHIKV infection in CC041 mice, animals were assessed for weight loss and clinical disease through 28 DPI using a clinical scoring system adapted from that for IC CHIKV infection in B6 mice [20]. No weight loss was observed in mock-infected or CHIKV-infected mice at any time during the period of observation (Fig 5A). Mock-infected CC041 mice remained clinically normal throughout the study, rarely scoring above a ‘0’ (i.e., no clinical signs of disease) (Fig 5B), whereas CHIKV-infected CC041 mice exhibited progressively elevated clinical scores. Although no mortality was observed, approximately half of the infected CC041 mice reached a clinical score of “2” by 10 DPI, and all CHIKV-infected CC041 mice reached this score between 25 and 28 DPI (Fig 5C). In contrast, B6 mice, whether mock-infected or CHIKV infected, showed comparable weight gain (S6A Fig) and remained largely clinically normal through 14 DPI, with only occasional sporadic clinical scores of “1” observed (S6B and S6C Fig).
Several clinical phenotypes displayed by CHIKV-infected CC041 mice (S1 Video) but not observed in mock-infected mice (S2 Video) were consistent with neurological dysfunction, including postural abnormalities, gait abnormalities, hyperactivity, facial grimace, and hair coat condition alterations (Fig 5D). Abnormal tail posture (S7 Fig), characterized by rigidity or a persistent upward curvature, was an especially notable feature frequently observed in CHIKV-infected CC041 mice. Although the specific neurological phenotypes varied among mice, CHIKV-infected animals consistently exhibited these signs until the study concluded at 28 DPI. In contrast, neurological phenotypes in B6 mice, whether CHIKV-infected or mock-infected, were limited to rare, sporadic observations (S6D Fig). These results demonstrate that young-adult CC041 mice develop prolonged signs consistent with neurological disease following peripheral CHIKV exposure.
To assess whether development of the clinical signs observed in CHIKV-infected CC041 mice could be attributed to local inflammation at the site of inoculation, footpad swelling was measured. We focused on CC041 mice, as swelling kinetics following footpad injections in B6 mice following CHIKV infection are already well characterized [31,32]. Foot size increased significantly in CHIKV-infected mice relative to mock-infected mice by 2 DPI, several days before increased clinical scores were observed, and returned to baseline by 9 DPI (Fig 5E). Furthermore, clinical signs consistent with neurological dysfunction persisted well beyond the resolution of measurable swelling, suggesting that local swelling at the site of inoculation alone does not account for the observed clinical signs in CC041 mice.
To determine if persistent or reactivated virus is associated with the development of clinical signs, we quantified both infectious virus by plaque assay and viral RNA by qPCR. Infectious virus was undetectable at 10, 14, and 28 DPI (S8 Fig); however, detectable viral RNA persisted in the brain through 28 DPI (Fig 5F). These data suggest that persistent viral RNA could be contributing to the development of clinical signs consistent with neurological dysfunction.
Discussion
In this study, we identified young adult CC041 mice as a unique immunocompetent small mammalian model that supports CHIKV neuroinvasion following physiologically relevant peripheral infection. The CC041 mouse model addresses a longstanding limitation in the field, as CNS involvement in CHIKV infection has been challenging to study in systems that preserve immune competence, neurodevelopmental maturity, and natural routes of exposure. Although prior work has established that mice are susceptible to CHIKV infection in the CNS [27,33,34], susceptibility is dependent on host age and viral strain [20], and most observations derive from experimental systems that do not reflect typical routes of human exposure.
Historically, studies of CHIKV neuropathogenesis have relied on neonatal mice, severely immunocompromised mice, or direct IC inoculation to achieve neurological CHIKV infection, because post-weanling-age, immunocompetent, standard inbred mouse strains do not develop CNS infection following peripheral (e.g., footpad or subcutaneous) inoculation. However, each of these approaches has significant limitations. Neonatal mice, while permissive to CNS infection by CHIKV, are neurodevelopmentally equivalent to a third-trimester fetus or a newborn human [17]. While helpful for studying susceptibility to neurological infection, their lack of neurodevelopmental translatability makes them poorly suited for modeling neuroinvasion, age-appropriate immune responses, or neurological disease progression. Immunocompromised mouse strains tend to develop more robust CNS infection but distort key aspects of host-virus interactions that shape neuropathogenesis in immunocompetent hosts. Direct IC inoculation allows controlled CNS infection but artificially bypasses peripheral viral replication and consequent local and systemic host inflammatory responses, hematogenous dissemination, and natural barriers to neuroinvasion. In vitro systems, such as human microglial cell lines and neurospheres, provide valuable insights into cellular responses and viral replication dynamics but lack the complex interactions of multiple cell types, immune responses, and the BBB, all of which are critical for understanding CHIKV neuroinvasion and disease progression in humans [35–37]. While these models have provided valuable insights into CHIKV pathogenesis, they cannot fully recapitulate the systemic viral dissemination, immune-mediated pathology, or the natural route of infection, limiting their ability to predict clinical outcomes or evaluate therapeutics effectively. Given the frequency of neurological complications in infants and young children infected with CHIKV, evaluating novel models that preserve intact antiviral immunity, age-appropriate neurodevelopment, and peripheral routes of inoculation are critical for understanding how viral and host factors contribute to neuroinvasion and disease.
The Collaborative Cross (CC) is a multi-parent recombinant inbred mouse reference population designed to capture genetic diversity comparable to that of the human population, providing a reproducible platform for both novel phenotype discovery and high-resolution genetic mapping [38,39]. As several genetic factors have been associated with susceptibility to CHIKV-induced disease in humans, including genetic polymorphisms in human leukocyte antigens (HLAs), pathogen-recognition receptors (PRRs), and cytokines [40–45], the ability to evaluate how host genetic variation influences disease manifestations is critical for understanding pathogenesis and identifying mechanisms underlying differential disease outcomes. For our CHIKV neuropathogenesis studies, the CC framework enabled identification of a permissive yet immunocompetent host background that was not represented by standard inbred mouse strains, such as B6. This finding highlights the ability of CC strains to reveal extreme responder phenotypes and supports expanded screening of the remaining available CC strains to facilitate downstream genetic dissection.
CC041 mice have been previously used to study complex genetic traits, with their resistance to cocaine-induced behavioral sensitization being particularly well documented [39,46–48]. However, to our knowledge, CC041 mice have not been reported to be uniquely sensitive to viral infection. While defining the specific genetic determinants that distinguish CC041 mice from neuroinvasion-resistant B6 mice will require dedicated genetic mapping studies, the strain divergence observed here underscores the central value of the CC framework for uncovering host factors that modulate viral neuroinvasion. The CC041 mouse model, therefore, provides a tractable platform for dissecting mechanisms that have remained largely inaccessible in human populations and other model systems.
Our findings support our hypothesis that permissive host genetics, in combination with viral strain-specific properties, govern susceptibility to CHIKV neuroinvasion following peripheral infection, with CC041 mice demonstrating CNS infection following SQ FP infection with CHIKV strains belonging to the ECSA, but not Asian, phylogenetic clade. Viral lineage has also been implicated in human neurological CHIKV disease, with encephalitis reported in association with both Asian and ECSA lineage strains, but not West African lineage strains, supporting the inclusion of multiple CHIKV strains in experimental models of neuroinvasion [49]. In contrast to the CHIKV strain-dependent susceptibility observed in CC041 mice, the widely used B6 mouse strain is resistant to CNS infection across multiple CHIKV strains following physiologically relevant doses (i.e., 103 PFU) and routes of infection [20,50,51]. Notably, increasing the inoculum dose by 100-fold did not significantly increase brain viral titers for CHIKV LR in CC041 mice, suggesting that once systemic infection occurs, CNS seeding may be constrained by host barriers or innate immune control that cannot be overcome by additional virus input. This observation further supports the concept that neuroinvasion in CC041 mice reflects intrinsic host susceptibility rather than simple dose-dependent spillover.
A defining difference observed between infected CC041 and B6 mice was the extent and duration of systemic viral burden following peripheral infection. Although viral replication at the inoculation site was comparable between mouse strains, CC041 mice maintained significantly higher serum viral loads and exhibited widespread viral dissemination beginning as early as 2 DPI. In contrast, B6 mice effectively restricted infection to the inoculation site, showing minimal viral dissemination. Sustained serum viral load and broad dissemination are well-recognized risk factors for neuroinvasion in alphavirus infections [33,24], and the elevated CNS susceptibility observed in CC041 mice is consistent with this infection profile.
Type I IFN is critical for early alphavirus control, inducing antiviral effectors such as IFIT1, which inhibits translation of alphavirus genomic RNA [52], IFITM3, which restricts viral fusion and entry [53], and PKR, which suppresses viral protein synthesis. During the 2005–2006 CHIKV outbreak in La Réunion, infected patients exhibited serum IFN-α concentrations that were, on average, greater than twofold higher than those of uninfected controls [54,55]. In this context, the sevenfold and twenty-sixfold increase in IFN-α levels relative to baseline observed in CC041 and B6 mice, respectively, suggest that IFN-α responses measured here fall within a physiologically meaningful range.
Prior studies in Sindbis virus (SINV) [56] and Venezuelan equine encephalitis virus (VEEV) [57] mouse models have demonstrated that failure to activate IFN signaling early in infection permits uncontrolled systemic viral replication and markedly increases CNS invasion [33]. Consistent with this framework, differences in systemic viral burden between CC041 and B6 mice found in our study appear to be driven by the timing and magnitude of the type I IFN response. B6 mice mounted a rapid IFN-α and IFN-β response early after CHIKV infection (1–3 DPI), whereas CC041 mice exhibited delayed induction and lower peak IFN-α and IFN-β levels at the site of initial viral replication. Taken together, these findings support a mechanistic model in which delayed type I IFN induction in CC041 mice leads to less regulation of early viral replication, resulting in prolonged serum viral titers and extensive dissemination, thereby increasing the likelihood of CNS seeding [33]. Conversely, the rapid IFN-α and IFN-β response in B6 mice may restrict viral amplification early enough to prevent systemic spread and subsequent neuroinvasion [24]. Although correlative, these results emphasize that the timing of innate immune activation, rather than its magnitude alone, may be a critical determinant of susceptibility to CHIKV neuroinvasion.
The adaptive immune system plays a critical role in viral clearance and disease pathogenesis in both CHIKV and other encephalitic alphaviruses [20,58]. Previous studies characterizing immune cell populations across available CC strains at baseline have demonstrated that CC041 mice possess major adaptive immune cell populations, including CD4+ T cells, CD8+ T cells, and B cells, and antibodies [59,60]. Although an in-depth evaluation of the adaptive immune response was beyond the scope of this study, we observed a >94-fold increase in Ifng expression in CC041 mouse feet by 7 DPI, suggesting activation of antiviral immune responses involving NK and/or T cells. Additionally, the increased expression and protein production of multiple proinflammatory cytokines and chemokines in the inoculated feet of CC041 mice, at levels comparable to those observed in B6 mice, indicate that CC041 mice are capable of mounting a multifaceted immune response to CHIKV infection similar to that of standard immunocompetent inbred mouse strains. Nevertheless, future studies characterizing the adaptive immune response to CHIKV infection in CC041 mice and determining whether and how that response differs from that in neuroinvasive-resistant strains, such as B6 mice, are warranted.
Viruses can access the CNS through multiple routes, including hematogenous spread across the BBB (e.g., Epstein-Barr virus, hepatitis C virus, West Nile virus) [61–63], axonal transport along peripheral nerves (e.g., poliovirus, rabies virus, alphaherpesviruses) [64–66], and leukocyte-mediated “Trojan horse” mechanisms (e.g., human immunodeficiency virus, human polyomavirus 2) [67,68]. Recent studies in humans have demonstrated CHIKV-associated perivascular and meningeal inflammation, with additional in vitro human BBB models supporting CCL-2-dependent transmigration of CHIKV-infected monocytes across the endothelium [25]. To define how CHIKV gains access to the CNS in CC041 mice, we evaluated virus localization, BBB integrity, and myeloid cell involvement during CHIKV infection. While not conclusive, several lines of evidence from our studies support hematogenous dissemination as the predominant route of CNS entry in the CC041 model. First, CHIKV antigen was detected sporadically across multiple brain regions, including the cerebral cortex and cerebellar nuclei, with antigen-positive cells extending from the meninges into the parenchyma. This distribution argues against entry via discrete neural pathways and instead supports vascular or meningeal access, consistent with observations from fatal human CHIKV cases and zebrafish models of CHIKV [14,25]. Second, despite consistent CNS infection in CC041 mice, CHIKV inoculation via SQ FP did not result in detectable increases in bulk BBB permeability, as measured by NaF accumulation. However, CC041 mice exhibited modestly higher baseline BBB permeability than B6 mice regardless of infection status. These inherent mouse strain-specific differences may lower the threshold for viral entry during sustained viremia, even in the absence of overt BBB disruption. Third, depletion of circulating monocytes and macrophages did not reduce brain CHIKV titers. Studies in zebrafish infected with CHIKV also showed that macrophages are not productively infected and depletion does not delay CNS invasion [14]. Together, these findings argue against a leukocyte-mediated “Trojan horse” mechanism of CNS infection in CC041 mice.
Although CHIKV did not increase BBB permeability in CC041 mice, the virus may still access the CNS through alternative mechanisms, such as endothelial cell infection or transcytosis across the BBB. Other alphaviruses, including VEEV, can traverse the BBB via caveolin-1-dependent transcytosis before detectable barrier disruption [69], and CHIKV LR can infect human brain microvascular endothelial cells in vitro [70]. Definitive resolution of these potential mechanisms will require endothelial-targeted in vivo analyses and ultrastructural imaging. Future studies that experimentally modulate BBB stability (e.g., pharmacologically enhancing tight junction integrity) may be particularly informative for determining whether baseline barrier properties influence susceptibility to CHIKV neuroinvasion.
CHIKV-infected CC041 mice developed clinical signs consistent with neurological disease, including gait abnormalities, ataxia, hunching, abnormal tail posture, and pronounced hyperactivity, that persisted well after infectious virus was cleared from the CNS. These phenotypes mirror neurological sequelae reported in human pediatric CHIKV survivors [71] and in other alphavirus mouse models [72], including the persistence of behavioral and motor abnormalities beyond the CNS viral replication period [73]. Importantly, our data suggest that these clinical signs are not solely attributable to local inflammation at the site of inoculation. Significant footpad swelling in CC041 mice was first noted at 2 DPI, when all mice were clinically normal, and resolved by 9 DPI, whereas clinical signs persisted through 28 DPI. In addition, infection of B6 mice, which do not develop CNS infection following peripheral inoculation, resulted in only infrequent clinical findings that were not clearly associated with infection. Together, these findings support a neurological basis for the observed phenotype in CC041 mice. However, a partial contribution from peripheral inflammation cannot be excluded, as expression of several proinflammatory genes in the ipsilateral foot remained elevated at 14 DPI. Therefore, mechanical (e.g., von Frey) and thermal (e.g., hot plate) nociception assays [74] in the ipsilateral and contralateral feet of CHIKV-infected mice will be evaluated in future studies to determine the extent to which foot pain and inflammation contribute to the observed clinical signs.
The persistence of neurological signs after viral clearance suggests that post-acute mechanisms, such as sustained neuroinflammation or neural circuit dysfunction, may contribute to long-term disease. The continued presence of viral RNA, even in the absence of active infectious virus production, requires continuous immune surveillance and control in the brain [75–78]. The effects of this ongoing local immune presence, such as low-level inflammation or changes to neuronal function and signaling, could be contributing to the persistent clinical signs observed in CC041 mice [72, 79,80]. Neuroimmune interactions during and after viral clearance can induce alterations in CNS physiology, potentially driving these sequelae [15,81,26]. Comprehensive behavioral and neuropathological analyses will be necessary to define the nature, mechanisms, and chronicity of these deficits.
Our study has several limitations that guide future work. CNS viral titers in CC041 mice were lower than those achieved with mouse-adapted encephalitic alphaviruses, such as SINV, likely limiting sensitivity for antigen detection and constraining definitive cell-type tropism analyses [20,82–84]. In addition, BBB assessment with NaF at peak infection did not reveal changes in overall permeability; however, it may not have detected transient or regional barrier alterations or distinguished endothelial infection from transcytosis-based entry mechanisms. Although macrophage depletion did not reduce brain titers, further characterization of affected myeloid subsets and trafficking will strengthen conclusions about leukocyte-mediated contributions to CHIKV infection in the CNS. The spinal cord was not evaluated in this study, as the primary objective was to characterize neuroinvasion and neuropathology in the brain. However, CHIKV and other encephalitic alphaviruses can cause widespread CNS infection, including involvement of the spinal cord. For example, SINV infects motor neurons within the anterior horn of the spinal cord [83,85], while VEEV infection in mice results in spinal cord inflammation and white matter demyelination [86]. Therefore, determining whether spinal cord pathology contributes to the motor coordination-related phenotypes observed during CHIKV infection represents an important direction for future studies. Finally, while CC041 mice displayed persistent neurological signs after clearance of infectious virus, comprehensive behavioral testing and longitudinal evaluation of neuropathological changes will be required to define the mechanisms and chronicity of post-acute deficits. Future studies combining endothelial-targeted viral localization, ultrastructural imaging, and experimental modulation of BBB stability, together with standardized behavioral and neuropathological assessments, will clarify how host susceptibility permits CNS entry and drives long-term neurological outcomes in the CC041 mouse model.
In summary, CC041 mice represent the first immunocompetent, developmentally appropriate small mammalian model in which CHIKV consistently invades the CNS following a physiologically relevant peripheral infection route and dose. This model recapitulates key features of human pediatric CHIKV disease, including systemic dissemination, CNS infection, and persistent neurological abnormalities, and provides a tractable platform for elucidating the mechanisms of CHIKV neuroinvasion. As CHIKV continues to expand into previously unaffected geographic regions, disproportionately impacting young children, the CC041 model offers a powerful and urgently needed tool to accelerate the development of vaccines, therapeutics, and biomarkers that prevent or mitigate CHIKV-associated neurological disease.
Materials and methods
Ethics statement
All procedures were conducted under an animal use protocol (1865MU) approved by the Institutional Animal Care and Use Committee at Texas Biomedical Research Institute, an AAALAC International-accredited institution. All procedures were performed in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals and complied with institutional and federal regulations. All work conducted with live CHIKV was performed under Biosafety Level 3 (BSL3) containment at Texas Biomedical Research Institute in accordance with the guidelines outlined in the 6th edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL). Euthanasia was performed in accordance with the 2020 edition of the AVMA Guidelines for the Euthanasia of Animals.
Cell and virus stocks
CHIKV stocks were made from infectious clones SM2013 (GenBank: MT228631.1) and SL15649 (GenBank: GU189061.1) provided by the Heise laboratory (University of North Carolina at Chapel Hill). The infectious clone of La Réunion (LR) strain (GenBank: DQ443544.2) was generated by Scott Weaver’s laboratory at the University of Texas Medical Branch. The nanoluciferase (nLuc)-expressing CHIKV LR strain reporter virus (CHIKV-nLuc) was generated by William Klimstra’s Laboratory (University of Pittsburgh) [22]. Virus stocks were grown as previously described [20]. Briefly, RNA was electroporated into BHK-21 cells (ATCC stock CCL-10) and supernatant was clarified and collected at 36 hours post-electroporation. Viral stock titers were established by plaque assay on Vero-81 cells (ATCC stock CCL-81).
Mice
C57BL/6J (B6) mice were originally obtained from Jackson Laboratories, and CC041/TauUncJ (CC041) mice were obtained from the University of North Carolina System Genetics Core Facility. Experimental mice were bred in-house at Texas Biomedical Research Institute. Mice were housed in an ABSL2 facility under a 12:12-hour light-dark cycle in autoclaved cages containing autoclaved Teklad 7097A bedding, sterile Teklad 2920X chow, autoclaved water, and standard environmental enrichment. At least 24 hours prior to infection, mice were transferred to the Animal Biosafety Level 3 (ABSL3) facility to allow for acclimation. Both male and female mice were included in all experiments, with each sex represented at every collection time point. Mice were randomly assigned to experimental groups.
mouse infections and clinical monitoring
Mice were infected at 4–6 weeks of age under light isoflurane anesthesia. Infections were performed by subcutaneous injection into the left hind footpad using a 29-gauge insulin syringe, delivering 10 μL PBS containing 103 plaque-forming units (PFU) of CHIKV. Mock-infected controls received 10 μL sterile PBS.
Mice were weighed and clinically evaluated daily by two independent observers blinded to infection status. Clinical scoring was performed using a system adapted from Anderson et al. [20] and expanded to capture a broader range of neurological and behavioral abnormalities. Scores were assigned to each mouse daily as follows: 0 = clinically normal; 1 = mild abnormal gait and tail posture; 2 = hunched posture, abnormal gait, unkempt hair coat; 3 = marked hunched posture, and decreased ambulation (humane endpoint); 4 = moribund/dead/euthanized. Observers additionally recorded qualitative descriptors including facial grimace, hyperactivity, abnormal behaviors (e.g., digging, excessive grooming), altered gait or posture (e.g., wide-based gait, wide rear-limb posture), and abnormal tail carriage (e.g., stiff-tail, tail-flagging). A clinical score of 3 or ≥30% weight loss constituted a humane endpoint, though no animals reached a humane endpoint in any experiment.
Rear footpad swelling was assessed longitudinally using digital calipers. Mice were anesthetized with continuous-flow isoflurane, and rear paw measurements were taken daily while the animals remained under anesthesia via a nose-cone. For each paw, footpad height was measured from the dorsal surface to the plantar surface, and footpad width was measured from the medial to lateral edge at the widest point just above the phalanges. Measurements were collected by a single investigator for both the ipsilateral and contralateral rear paws, with care taken to maintain consistent paw positioning, caliper placement, and pressure across timepoints. These measurements were used to quantify changes in footpad swelling following subcutaneous footpad inoculation.
tissue collection and processing
Mice were euthanized by isoflurane overdose. Blood was collected by cardiocentesis into serum separator tubes and centrifuged to remove red blood cells and isolate serum. Mice were transcardially perfused with 15 mL ice-cold PBS to remove circulating blood. Brains were harvested and hemisected sagitally. The right hemisphere was fixed in 4% paraformaldehyde (PFA) for 24 hours at 4°C, washed, and stored in 1X PBS at 4°C. The left brain hemisphere and left hind footpad were flash-frozen on dry ice and stored at -80 °C until processing.
infectious virus quantification
Infectious virus in brain, left hind foot, and serum was quantified by plaque assay as previously described [20]. Briefly, brains and feet were homogenized for 2 min to 20% w/v in ice-cold PBS and clarified by centrifugation at 10,000 RPM for 15 min. Homogenates and serum were diluted 10-1 to 10-6 for plaque assays. Two hundred microliters of each dilution were applied in duplicate to confluent Vero-81 (ATCC CCL-81) monolayers in 12-well plates. Following 1 hour incubation at 37°C with 5% CO2, cells were overlaid with 1 × αMEM (Gibco) containing 0.2 mM L-glutamine (Gibco), 5% FBS (Gibco), 1 mM HEPES (Gibco), 1% Penicillin-Streptomycin (Gibco), and 1.25% carboxymethylcellulose sodium (Sigma), and incubated at 37°C with 5% CO2 for 48 hours. Plates were fixed with 4% PFA for at least 24 hours, rinsed with tap water, and stained with 1% crystal violet. The assay limit of detection (LOD) was 125 PFU/ g tissue for brain and foot and 50 PFU/mL for serum. Samples below the LOD were assigned a value half the LOD (brain and foot: 62.5 PFU/ g tissue; serum: 25 PFU/mL).
In vivo bioluminescence imaging
Mice infected with CHIKV-nLuc as described above were imaged at 6 hours post-infection and on 1, 2, 3, 4, 5, and 7 DPI using the IVIS Spectrum multispectral in vivo imaging system (IVIS) as described previously [87]. Mice were anesthetized with 50 µL of ketamine (86 mg/kg) /xylazine (5 mg/kg) cocktail via intraperitoneal (IP) injection and injected retro-orbitally with 100 μL Nano-Glo substrate (Promega) diluted 1:10 in sterile PBS prior to imaging. Images were acquired using standardized exposure time, binning, and field-of-view settings. Both dorsal and ventral views were initially assessed, and consistent with previous findings by others [22], the ventral view produced equal-to-more intense signal (S9 Fig) and so was used for all subsequent experiments. IVIS was performed across four independent experiments. Images were analyzed using the manufacturer-recommended Aura Analysis Software (Spectral Instruments Imaging). Identical square regions of interest (ROIs) were applied to each mouse head using a standardized gating strategy that included the entire cranial area while carefully excluding the forepaws. nLuc intensity was quantified for both CC041 and B6 mice infected with either the nLuc or wild-type (WT) CHIKV LR strain, which served as the baseline control. For each experimental animal, raw nLuc values (two measurements per mouse per time point) were exported from Aura Analysis. Corresponding WT CHIKV LR controls, matched by mouse strain and sex where possible, were used to account for background nLuc. The nLuc intensity of each nLuc-infected mouse was corrected by subtracting the value of its matched WT CHIKV LR control. The two measurements per mouse were then averaged to obtain a single mean nLuc value per time point. To facilitate visualization of relative changes in signal intensity across mouse strains, background-corrected nLuc values were log10-transformed before analysis.
IFN-α and IFN-β measurements
IFN‑α and IFN-β levels in left foot homogenates and serum were quantified using the ProcartaPlex Mouse IFN‑alpha/IFN‑beta Panel (Invitrogen, EPX02A-22187-901) according to the manufacturer’s instructions in a BSL‑3 facility with an established decontamination protocol. Following the addition of Streptavidin‑PE and subsequent plate washes, assay plates were decontaminated with freshly prepared 4% PFA for 30 min at room temperature (RT). The plates were then transferred to a BSL‑2 facility, washed three additional times, and the remaining steps of the manufacturer’s protocol were completed. Cytokines were analyzed on a Luminex 200 Analyzer instrument and quantified by comparison to a standard curve. All reported analyte values correspond to Luminex-derived concentrations calculated from individual calibration curves for each analyte. The assay LOD for IFN-α was 14.65 pg / g tissue for foot homogenates and 2.93 pg / mL for sera, and for IFN-β was 36.25 pg / g tissue for foot homogenates and 7.25 pg / mL for sera. Samples that fell below the assay LOD were assigned a value of half of the LOD (foot homogenates: 7.33 pg / g for IFN-α, 18.13 pg / g for IFN-β, sera: 1.47 pg / mL for IFN-α, 3.63 pg / mL for IFN-β).
RNA isolation and quantitative polymerase chain reaction
One mL of TRIzol (Invitrogen) was added to homogenized foot pellets (after removing clarified homogenate for infectious virus quantification as stated above) and homogenized twice at 60Hz for one minute each using a MagNA Lyser (Roche). Samples were then centrifuged at 15,000 × g for 5 min at 4ºC, and then the clarified supernatant was transferred to another tube. RNA was isolated using the RNeasy Tissue Mini Kit (Qiagen) with a chloroform separation according to the manufacturer’s protocol as described previously [20]. A 15 min DNase I (Qiagen) digest was used to purify samples. cDNA was generated using 1 µg of RNA and the high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s protocol. For the qPCR reaction, the TaqMan Fast Advanced Master Mix (ThermoFisher) was used according to the manufacturer’s protocol. An amount of 4 μL of cDNA and 1 μL of primer probe (S4 Table) were added to each reaction. The reaction was run for 40 cycles using the QuantStudio 5 (Applied Biosystems). All samples that fell below the limit of detection were assigned a Ct value of 40 for all primer probe sets. All Ct values were normalized to Gapdh, and data were reported as fold change (2−∆∆Ct) compared to mock-infected samples from each mouse strain (CC041 and B6).
CHIKV viral RNA was quantified by qPCR in the cDNA prepared above using primers and a probe specific to the CHIKV E1 gene (S5 Table). Standard curves were made by making 10-fold serial dilutions of Synthetic CHIKV RNA (VR-3246SD, ATCC) and Rodent Gapdh (4308313, Applied Biosystems), and CHIKV E1 and Gapdh copies in each sample were calculated using the standard curves. CHIKV RNA copies were normalized to Gapdh copies and reported as Log(CHIKV E1 copies / 106 Gapdh copies).
Cytokine and chemokine quantification by luminex
Cytokines and chemokines were quantified in ipsilateral left foot and serum samples using the BioPlex Pro Mouse Cytokines 23-Plex assay (Bio-Rad) according to the manufacturer’s protocol. Briefly, 50µL of clarified left foot samples or 1:4 diluted serum samples were used and incubated with magnetic capture beads, detection antibodies, and SA-PE with washes in between. The Bio-Plex System (Bio-Rad) was used to acquire cytokine levels by standard curve. IL-6, IL-9, and IL-13 were not detectable in samples, so they were excluded from analyses.
Immunohistochemistry
Mouse tissue sections for IHC staining were cut to 4µm thickness, mounted onto positively charged slides, and allowed to air-dry overnight. For single chromogen CHIKV IHC, slides were loaded onto the Discovery Ultra IHC/ISH automatic stainer, and deparaffinization occurred using Discovery Wash (Roche cat. # 950-510). Cell conditioning occurred using Discovery CC1 (Roche cat. # 950-500) at 95°C for 64 min. The blocking of endogenous peroxidase was performed using Discovery Inhibitor (Roche cat. # 760-4840) for 8 min. Slides were then incubated with CHIKV rabbit polyclonal antibody (1:500 dilution, Novus Biologicals cat. # NBP3-13436) for 1 hour at RT. Detection was performed using Anti-rabbit HQ (Roche cat. # 760-4815) for 8 min followed by Anti-HQ HRP (Roche cat. # 760-4820) for 8 min at 36°C. CHIKV was then visualized using ChromoMAP DAB (Roche cat. # 760-159). The slides were counterstained using Hematoxylin (Roche cat. # 760-2021) for 8 min followed by Bluing Reagent (Roche cat. # 760-2037) for 4 min.
For the (3-plex) multiplex IHC involving OLIG2, CHIKV, and GFAP markers, slides were loaded onto the Roche Ventana Discovery Ultra IHC/ISH automated stainer. Deparaffinization occurred using Discovery Wash (Roche cat. # 950-510). Cell conditioning was performed using Discovery CC1 (Roche cat. # 950-500) at 95°C for 64 min. The blocking of endogenous peroxidase occurred using Discovery Inhibitor (Roche cat. # 760-4840) for 8 min. Slides were then incubated with OLIG2 (EP112) predilute rabbit monoclonal antibody (Roche cat. # 760-5050) for 16 min at 36°C. Detection was performed using Anti-rabbit HQ (Roche cat. # 760-4815) for 8 min at 36°C followed by anti-HQ HRP (Roche cat. # 760-4820) for 8 min at 36°C. OLIG2 (EP112) was visualized by applying Discovery Yellow HRP chromogen kit (Roche cat. # 760-250) for 64 min. Slides were then denatured using ULTRA Cell Conditioning Solution (ULTRA CC2, Roche cat. # 950-223) at 95°C for 8 min. Next, slides were incubated with CHIKV rabbit polyclonal antibody (1:500 dilution, Novus Biologicals cat. # NBP3-13436) for 1 hour at RT. Detection was performed using anti-rabbit HQ for 8 min at 36°C followed by anti-HQ HRP for 8 min at 36°C. Visualization of CHIKV was achieved by applying Discovery Teal HRP chromogen kit (Roche cat. # 760-247) for 32 min. Slides were denatured a second time using ULTRA CC2 at 95°C for 8 min and then incubated with anti-GFAP rabbit polyclonal antibody (1:1000 dilution, Sigma-Aldrich cat. # AB5804) for 1 hour at RT. Detection was performed using anti-rabbit HQ for 8 min at 36°C followed by anti-HQ HRP for 8 min at 36°C. Visualization was achieved by applying Discovery Purple chromogen kit (Roche cat. # 760-229) to the slides for 32 min. The slides were counterstained using Hematoxylin (Roche cat. # 760-2021) for 4 min followed by Bluing Reagent (Roche cat. # 760-2037) for 4 min. Infected oligodendrocytes and astrocytes were quantified using the HALO system, and the percentage of cells infected was calculated as the number of CHIKV+ cells divided by the total number of cells for each cell type, multiplied by 100.
For the (4-plex) multiplex IHC involving SOX-10, CHIKV, IBA1, and NeuN markers, slides were loaded onto the Roche Ventana Discovery Ultra IHC/ISH automated stainer. Deparaffinization occurred using Discovery Wash (Roche cat. # 950-510). Cell conditioning was performed using Discovery CC1 (Roche cat. # 950-500) at 95°C for 64 min. The blocking of endogenous peroxidase occurred using Discovery Inhibitor (Roche cat. # 760-4840) for 8 min. Slides were then incubated with SOX-10 (SP267) rabbit monoclonal antibody (Roche cat. # 760-4968) for 32 min at 36°C. Detection was performed using anti-rabbit HQ (Roche cat. # 760-4815) for 8 min at 36°C followed by anti-HQ HRP (Roche cat. # 760-4820) for 8 min at 36°C. SOX-10 (SP267) was visualized by applying Discovery Yellow HRP chromogen kit (Roche cat. # 760-250) for 72 min. Slides were then denatured using ULTRA Cell Conditioning Solution (ULTRA CC2, Roche cat. # 950-223) at 95°C for 8 min. Next, slides were incubated with CHIKV rabbit polyclonal antibody (1:500 dilution, Novus Biologicals cat. # NBP3-13436) for 1 hour at RT. Detection was performed using anti-rabbit HQ for 8 min at 36°C followed by anti-HQ HRP for 8 min at 36°C. Visualization of CHIKV was achieved by applying Discovery Teal HRP chromogen kit (Roche cat. # 760-247) for 32 min. Slides were denatured a second time using ULTRA CC2 at 95°C for 8 min and then incubated with IBA1 rabbit polyclonal antibody (1:800 dilution, Proteintech cat. # 10904-1-AP) for 1 hour at RT. Detection was performed using anti-rabbit HQ for 8 min at 36°C followed by anti-HQ HRP for 8 min at 36°C. IBA1 visualization was achieved by applying Discovery Purple chromogen kit (Roche cat. # 760-229) to the slides for 32 min. Slides were denatured a third time using ULTRA CC2 at 95°C for 8 min and then incubated with anti-NeuN rabbit polyclonal antibody (1:500 dilution, Abcam cat. # ab104225) for 1 hour at RT. Detection was performed using anti-rabbit HQ for 8 min at 36°C followed by anti-HQ HRP for 8 min at 36°C. Visualization of NeuN was achieved by applying Discovery ChromoMap DAB chromogen (Roche cat. # 760-159). The slides were counterstained using Hematoxylin (Roche cat. # 760-2021) for 4 min followed by Bluing Reagent (Roche cat. # 760-2037) for 4 min.
Blood-brain barrier permeability assay
Following CHIKV infection as described above, at 2 and 5 DPI, mice received a 100 µL IP injection of 100mg/mL NaF diluted in sterile PBS. After 45 min, mice were euthanized and perfused as described above. Left brain hemispheres were homogenized to 20% w/v as described above and 100µL of undiluted homogenate was pipetted in duplicate into a black walled, clear-bottomed plate that contained NaF standards ranging 10-0.3125 µg/mL. All procedures were done with the biosafety cabinet light off. NaF was then quantified using the GloMax 3500 (excitation 475nm, emission 500–550nm). Protein concentration was measured using a bicinchoninic acid (BCA) assay per manufacturer protocol (Pierce). NaF levels were normalized to total protein and presented as µg of NaF in tissue/mg of protein.
Monocyte/macrophage depletion
Macrophages were depleted using clodronate liposome (Liposoma; CP-010-010). Two days prior to infection, mice received 200 μL of clodronate liposomes or control liposomes via IP injection. Monocyte depletion was confirmed on the day of infection by peripheral blood smear and monocyte enumeration (S2 and S3 Tables). A 100 μL booster dose of liposome was administered on 2 DPI. Mice were euthanized at 5 DPI, and the brain, spleen, and left foot were collected. CHIKV brain titers were quantified by plaque assay using the left-brain hemispheres as described above.
Biostatistical analyses
Statistical analyses were performed using GraphPad Prism 9. Biological replicates (individual mice) are indicated in figure legends. Viral titers and nLuc measurements were analyzed by two-way ANOVA with Sidak’s or Tukey’s multiple comparisons tests. All observations were independent and the assumptions of variance were met. Data were log10 transformed where necessary to meet assumptions of normality. A p-value of < 0.05 was considered statistically significant.
Supporting information
S1 Fig. Brain titers in Collaborative Cross (CC) mice following peripheral CHIKV infection.
CHIKV brain titers were measured by plaque assay at 5 DPI in ten CC lines (A) and in CC007 (B) and CC041 (C) mice over time following SQ FP inoculation with 103 PFU CHIKV SL165649. Plaque assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the plaque assay LOD were assigned a value of half of the LOD.
https://doi.org/10.1371/journal.ppat.1014395.s001
(TIF)
S2 Fig. Effect of CHIKV input dose on brain infection in CC041 mice.
Brain (A, D), foot (B, E), and serum (C, F) viral titers were measured by plaque assay at 5 DPI following SQ FP inoculation with 103 PFU (square symbols) or 105 PFU (triangular symbols) of CHIKV LR (A-C, red symbols) or CHIKV SL15649 (D-F, blue symbols) in CC041 mice. Assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the plaque assay LOD were assigned a value of half of the LOD. Note: The 103 PFU titers presented in this figure are the same data as presented in Fig 1.
https://doi.org/10.1371/journal.ppat.1014395.s002
(TIF)
S3 Fig. Viral load, IFN-β, and cytokine/chemokine gene expression CHIKV-infected CC041 and B6 mice.
CC041 and B6 mice were infected with 103 PFU of CHIKV LR (A, B, E) or SL15649 (C, D), and feet and sera were collected following euthanasia. (A,B) Infectious CHIKV LR in CC041 (red squares) and B6 (black circles) mouse feet (A) and sera (B) was quantified by plaque assay. (C,D) IFN-β in CHIKV SL15649-infected CC041 (bright blue squares) and B6 (navy circles) mouse feet (C) and sera (D) was quantified by Luminex. E) RNA was extracted from mock-infected or CHIKV LR-infected CC041 (red squares) and B6 (black circles) mouse feet, and Ifna4, Ifnb1, Ccl2, Il10, Tnf, Ifng, and Il6 expression were quantified by qPCR. Assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the plaque assay LOD were assigned a value of half of the LOD; * p < 0.05; ** p < 0.01, Šidák’s multiple comparisons.
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S4 Fig. Proinflammatory cytokine and chemokine levels in CHIKV-infected CC041 and B6 feet and sera.
CC041 (red squares) and B6 (black circles) mice were infected with 103 PFU of CHIKV LR, and feet and sera were collected following euthanasia. Cytokine and chemokine levels were quantified in feet (A) and sera (B) by Luminex. Assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the plaque assay LOD were assigned a value of half of the LOD; * p < 0.05; ** p < 0.01, ***p < 0.001, ****p < 0.001, Šidák’s multiple comparisons.
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S5 Fig. Evaluating potential mechanisms of neuroinvasion in CHIKV-infected CC041 mice.
A) Mice were mock-infected with PBS (open symbols) or infected with 103 PFU of CHIKV LR (closed symbols) and intraperitoneally injected with sodium fluorescein (NaF) prior to euthanasia at 2 DPI, and NaF and total protein were quantified in 20% w/v brain homogenates. B,C) Clinical scores in CC041 mice injected with clodronate (B) or control (C) liposomes at -2 DPI and infected with 103 PFU of CHIKV-nLuc via SQ FP. Clinical scores are represented as follows: 0 = clinically normal; 1 = mild abnormal gait and tail posture; 2 = hunched posture, abnormal gait, unkempt hair coat; 3 = marked hunched posture, and decreased ambulation (humane endpoint); 4 = moribund/dead/euthanized.
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S6 Fig. Clinical disease evaluation in CHIKV-infected B6 mice.
B6 mice were infected with 103 PFU CHIKV LR (n = 8) or mock-infected (n = 8) with PBS SQ via FP. A) Mock-infected (open symbols) and CHIKV-infected (closed symbols) mice were weighed daily. B,C) Clinical scores in mock-infected (B) and CHIKV-infected (C) B6 mice were assigned to each mouse daily as follows: 0 = clinically normal; 1 = mild abnormal gait and tail posture; 2 = hunched posture, abnormal gait, unkempt hair coat; 3 = marked hunched posture, and decreased ambulation (humane endpoint); 4 = moribund/dead/euthanized. During daily clinical observations, qualitative descriptors were recorded, including facial grimace, hyperactivity, abnormal behaviors (e.g., digging, excessive grooming), altered gait or posture (e.g., wide-based gait, wide rear-limb posture), and abnormal tail carriage (e.g., stiff-tail, tail-flagging). D) Spider plot illustrating results of a Key Terms analysis of the qualitative descriptors observed in CHIKV LR- (red shading) and mock-infected (black shading) B6 mice. Data are presented as the mean SEM; in (A), the horizontal dotted line represents the 0 DPI value to which values at all subsequent timepoints were normalized.
https://doi.org/10.1371/journal.ppat.1014395.s006
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S7 Fig. Representative image of stiff tails seen in CHIKV LR-infected CC041 mice.
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S8 Fig. CHIKV titers in CC041 mouse brains at later timepoints.
Brain viral titers were measured by plaque assay at 10, 14, and 28 DPI following SQ FP inoculation with 103 CHIKV in CC041 mice. Assay limit of detection (LOD) is depicted by the horizontal dotted line; all samples that fell below the plaque assay LOD were assigned a value of half of the LOD.
https://doi.org/10.1371/journal.ppat.1014395.s008
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S9 Fig. Comparison of ventral and dorsal imaging approaches.
Representative IVIS images of ventral (top images) and dorsal (bottom images) views of CC041 mice infected with CHIKV-nLuc- (left images) or CHIKV-WT- (right images) at 5 DPI.
https://doi.org/10.1371/journal.ppat.1014395.s009
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S1 Video. Representative clinical signs in CHIKV LR-infected CC041 mice.
https://doi.org/10.1371/journal.ppat.1014395.s010
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S2 Video. Representative clinical signs in mock-infected CC041 mice.
https://doi.org/10.1371/journal.ppat.1014395.s011
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S1 Table. Gene expression in mock-infected CC041 and B6 mouse feet.
ΔCt values calculated by taking the difference between target gene Ct values and Gapdh Ct values for each mouse. Data presented as mean ± SD. P-values calculated by unpaired t-test.
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S2 Table. Testing monocyte depletion following clodronate liposome dose.
Blood was collected from B6 mice immediately before the first dose of liposomes was administered to establish baseline numbers. Blood smears were performed at baseline, 24, 48 and 96 hours. Mice were re-dosed at 48hours with 100µL (mouse 1) or 200µL (mouse 2) with clodronate liposomes. Total monocytes were counted out of 100 cells across multiple fields. Counts were performed in triplicate.
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S3 Table. Confirmation of monocyte depletion following clodronate liposome administration.
Blood smears were performed from blood collected from CC041 mice immediately before CHIKV infection and at 48 hours after clodronate or control liposome administration to confirm depletion. Total monocytes were counted out of 100 cells across multiple fields. Counts were performed in triplicate.
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Acknowledgments
We thank the University of North Carolina at Chapel Hill Systems Genetics Core Facility for maintenance and distribution of the Collaborative Cross mice. We thank Dr. Mark Heise for helpful scientific discussions and Renee Escalona for technical assistance.
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