Background
La Crosse virus (LACV), family
Peribunyaviridae (genus
Orthobunyavirus), is a leading cause of pediatric arboviral encephalitis in the USA [
1]. The primary vector of LACV is the eastern tree-hole mosquito (
Ochlerotatus triseriatus). LACV was responsible for 665 confirmed cases of encephalitis from 2003 to 2012, although the true incidence of disease is thought to be underestimated [
2]. Endemic areas of infection include the Midwest and Appalachian regions, with county-level incidence of 0.2–228 cases per 100,000 children under the age of 15, but LACV is also becoming an important emerging pathogen of the Southern and Western United States [
3]. Despite the threats posed, there are currently no approved therapeutics or vaccines available against LACV.
LACV encephalitis is almost exclusively found in children under 15 years of age [
4]. Like other arboviruses, the majority of cases present as mild febrile illness, but in a minority of cases, LACV causes severe neuroinvasive disease including encephalitis, meningitis, and meningoencephalitis [
5]. Neuroinvasive LACV typically presents with fever, headache, lethargy, and vomiting, and nearly half of patients experience seizures [
4,
5]. While the disease is rarely (< 1%) fatal, neurological deficits such as epilepsy (in 10–28% of cases), reduced IQ, and attention-deficit-hyperactivity disorder (ADHD) are not uncommon [
4‐
6].
LACV replicates peripherally and likely invades the central nervous system (CNS) via the olfactory bulb in the mouse model of LACV encephalitis after the compromise of the blood-brain barrier (BBB) [
7]. In human infection, cortical and basal ganglia neurons appear to be the primary target of infection in the CNS leading to foci of neuronal necrosis [
8]. Additionally, inflammatory lesions with largely monocytic infiltration and lymphocytic perivascular cuffing are noted [
8]. The understanding of LACV neuropathogenesis has been advanced by studies using the suckling mouse model which closely resembles human disease including age-related susceptibility [
9,
10]. Infection of adult mice and rhesus macaques result in asymptomatic infections and antibody responses [
9,
10]. Most studies agree that neurons comprise the main target cell in the CNS [
9,
11]. Infected neurons appear to undergo apoptosis via mitochondrial antiviral-signaling protein (MAVS)-induced oxidative stress [
12]. However, some groups report low levels of astrocyte infection in vitro and in vivo [
1,
11]. Especially interesting is the finding that when NSs, a LACV encoded interferon (IFN) antagonist, is deleted, astrocytes significantly increase production of IFN, suggesting that IFN production in astrocytes is antagonized by LACV [
11]. Regarding the inflammatory component of the disease, a recent study showed that lymphocytes play a protective role during LACV infection of adult mice and do not contribute to the pathogenesis of weanling mice [
13]. The majority of inflammatory cells noted in human and mouse brains during LACV infection are monocytes and macrophages. Recent work has demonstrated that in the mouse model, CCL2 is important for inflammatory monocytic migration within the brain and that astrocytes are a source of CCL2 in the brain [
8,
14]. Importantly, it is becoming increasingly clear that CNS parenchymal cells play a major role in the development of innate immune responses during LACV infection [
15‐
17]. Additionally, cytokine responses can also negatively impact BBB integrity and lead to worsened neuroinvasion [
18,
19]. While our knowledge on the pathogenesis and molecular mechanisms of LACV-induced disease using animal models is increasing, there is still a need to verify many of these results with a human-based system.
Primary human neurons are terminally differentiated, post-mitotic, and difficult to obtain. Most studies of encephalitic viruses rely on primary rat or mouse neuronal cells or human neuroblastoma cell lines. While these models are strong tools for understanding pathogenesis, species differences and the genetic and signaling abnormalities found in these models require validation using human cells without genetic modification. Furthermore, most studies rely on the use of a single cell type, although it has been shown that neuronal cells behave differently in co-culture compared to monoculture [
20,
21]. In recent years, human neural stem cells (hNSC), embryonic stem cells (hESCs), and induced pluripotent cells (iPCs) have become important tools in studying neurologic diseases, including encephalitic viruses. Varicella zoster virus (VZV) has been extensively studied using such systems, which has provided accurate models for VZV productive infection, latency, and reactivation. [
22‐
26].
In this study, we use a well-validated hNSC-derived neuron/astrocyte co-culture system which has previously been used in the study of neurodegenerative diseases [
27‐
29]. Importantly, this primary human neural cell system was recently used to assess Zika virus-induced changes in hNSC differentiation, although this study mainly focused on the direct infection of hNSCs rather than differentiated neuronal cells [
30]. We have reported susceptibility of neuron/astrocyte co-cultures to infection with henipaviruses, but an in-depth characterization of the cellular responses to infection has not been reported yet [
31]. In the present study, we infected hNSC-derived neuron/astrocyte co-cultures with LACV. Our results indicate that both neurons and astrocytes are highly susceptible to LACV, and that LACV infection induces strong proinflammatory responses, which likely play a major role in the observed neuroinflammation and breakdown in the BBB.
Methods
Cells and viruses
Vero CCL81 cells were acquired from American Type Culture Collection (ATCC, Manassas, VA). Vero cells were propagated using MEM (Corning) supplemented with 10%FBS. K048 hNSCs were originally obtained from the cortex of a 9-week-old male fetus and were propagated as described previously [
27]. Briefly, hNSCs were cultured as nonadherent neurospheres in DMEM/F12 (Corning) media supplemented with epidermal growth factor (EGF) (20 ng/mL) (R&D Systems), fibroblast growth factor (FGF) (20 ng/mL) (R&D Systems), leukocyte inhibitory factor (LIF) (10 ng/mL) (Chemicon), heparin (5 μg/mL) (Sigma-Aldrich), and insulin (25 μg/mL) (Sigma-Aldrich). Cells were passaged every 10 days and maintained at 37 °C and 8.5% CO
2.
hNSCs were plated onto wells coated with 0.01% poly-D-Lysine (Sigma-Aldrich) and 1 μg/cm
2 laminin. Cells were primed for 4 days with a priming media containing EGF (20 ng/mL), LIF (10 ng/mL), and laminin (1 μg/mL) (GIBCO). K048 hNSCs were primed and differentiated into neurons and astrocytes in a roughly 1:1 ratio. The neurons in this system have previously been characterized as being both GABAergic and glutamatergic, and the overall composition is similar to that found in the cerebral cortex [
32]. Cells were then differentiated for 9 days in a differentiation media containing N2 basal media supplemented with glutathione (1 μg/mL) (Sigma-Aldrich), biotin (0.1 μg/mL) (Sigma-Aldrich), superoxide dismutase (2.5 μg/mL) (Sigma-Aldrich), DL-α-tocopherol (1 μg/mL) (Sigma-Aldrich), DL-α-tocopherol acetate (1 μg/mL) (Sigma-Aldrich), and catalase (2.5 μg/mL) (Sigma-Aldrich).
LACV was obtained from the World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch (kindly provided by R. Tesh). The strain used was isolated from a human brain in Wisconsin in 1964. This strain had undergone nine passages in suckling mice and was amplified in our lab in one passage in Vero cells.
Growth curves
Neuron/astrocyte co-cultures were infected with 0.1, 1, or 10 multiplicity of infection (MOI) of LACV for 1 h at 37 °C and 8.5% CO2. Virus inoculum was removed, cells gently washed with PBS, and fresh culture medium re-added. Cells only underwent one PBS wash due to the fragility of the neurons to avoid their detachment. Supernatant aliquots were then collected at various time points after infection. Samples were titrated via standard plaque assay. Cell culture supernatant aliquots were serially diluted in MEM supplemented with 2% FBS and used to infect Vero CCL81 cells for 1 h. Cells were washed with PBS and given a media overlay of MEM with 0.8% tragacanth (Sigma-Aldrich). At 4 days post-infection, the overlay was removed, cells were fixed in 10% formalin (Thermo Fisher) and stained using crystal violet (Thermo Fisher), and plaques were counted.
Immunofluorescence
Neuron/astrocyte co-cultures on glass coverslips were infected with 0.1, 1, or 10 MOI of LACV as described above. Infected cells were fixed in formalin at various times post-infection. Cells were stained with primary antibodies and fluorophore-conjugated secondary antibodies along with DAPI (Sigma Aldrich) as previously described [
20]. Antibodies used were rabbit-anti-microtubule associated protein 2 (MAP2) polyclonal (Millipore, AB5622) used at 1:500, rabbit-anti-glial fibrillary acidic protein (GFAP) polyclonal (Millipore, AB5804) used at 1:2,000, and mouse-anti-LACV Gc monoclonal (ThermoFisher, MA1–10801) used at 1:1,000. Secondary antibodies were Alexa Fluor goat-anti-rabbit 594 and goat-anti-mouse 488 used at 1:500 (ThermoFisher). Images were acquired on an Olympus IX71 fluorescent microscope and cells were quantified visually using six random fields per condition with an average of over 200 cells/field.
Cytotoxicity and apoptosis assays
Neuron/astrocyte co-cultures were grown in 96-well plates and infected with 1 MOI of LACV as in other experiments along with media only controls and 10 μM staurosporine (Abcam) treatment. Cells were then assayed using the ApoTox-Glo triplex assay (Promega) according to the manufacturer’s protocols. Plates were assayed on a BioTek Synergy HT plate reader at 485nmEx/505nmEm for cytotoxicity (extracellular protease activity) and luminescence for apoptosis (caspase 3/7 activity).
TUNEL assay
Neuron/astrocyte co-cultures were infected with 1 MOI LACV as described previously and fixed at 96 HPI. Additionally, staurosporine-treated cells were fixed at 12 h post-treatment. Formalin-fixed monolayers were prepared for immunofluorescence staining as previously described. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (TACS 2 TdT-Fluor In Situ Apoptosis Detection Kit, R&D systems) was performed as per manufacturer instructions. Briefly, following DAPI stain, cells were incubated in labeling buffer, followed by labeling reaction mix. Strep-FITC was added for 20 min before final washes. Slides were briefly rinsed in ddH2O to remove residual salt and inverted onto Fluoromount G (Southern Biotech) and allowed to cure overnight at 4 °C in the dark. Samples were imaged on an Olympus BX61 microscope.
BioPlex assays
Co-cultures were infected as in previous experiments with 0.1, 1, or 10 MOI of LACV or treated with heat-inactivated LACV (60 °C for 30 min) (1 MOI) or 10 μM polyinosinic:polycytidylic acid (poly I:C) (Sigma Aldrich). Supernatant samples were collected at various time points post-infection and γ-irradiated with a dose of 5 Mrad to inactivate the infectious virus. Cells treated with Poly I:C or heat-inactivated virus were collected at 48 h post-infection (HPI) only. Samples were then used for BioPlex assay analysis (Bio-Plex Pro Human Cytokine, Group 1, 27-Plex, Bio-Plex) according to manufacturer’s protocols. Standard curves were developed using fresh standards provided in each kit. The assays were run on a Bio-Plex 200 system (Bio-Rad), and data was analyzed using Bio-Plex Manager (Bio-Rad).
qRT-PCR
Co-cultures were grown and infected with 0.1, 1, 10 MOI or treated with heat-inactivated virus or poly I:C. Cells were lysed and collected in TRIzol (Thermo Fisher) reagent, and RNA was isolated using Direct-zol RNA Miniprep kits (Zymo Research). cDNA was obtained using High Capacity RNA-to-cDNA kit (ThermoFisher). cDNA was then amplified using SYBR green mix (Bio-Rad) on a Bio-Rad CFX384 instrument. Data was analyzed using CFX Manager (Bio-Rad), and mRNA expression differences were determined via change in threshold cycle (ΔCT) and normalized to 18S RNA (ΔΔCT). PrimeTime qPCR primers (IDT) were used to target MMP7, MMP2, and TIMP2. PrimePCR PCR primers (Bio-Rad) were used to target IL-6, IL-8, CXCL10, CCL2, CCL4, CCL5, and TNF-α. Additional primers (IDT) were used to target 18S (For-GTAACCCGTTGAACCCCATT, Rev-CCATCCAATCGGTAGTAGCG), IFN-α (For-GACTCCATCTTGGCTGTGA, Rev-TGATTTCTGCTCTGACAACCT), IFN-β (For-TCTGGCACAGGTAGTAGGC, Rev-GAGAAGCACAACAGGAGAGCAA) and LACV (For-ATTCTACCCGCTGACCATTG, Rev-GTGAGAGTGCCATAGCGTTG).
MMP activity assay
Supernatants of LACV-infected neuron/astrocyte co-cultures were assayed for the activities of matrix metalloproteinases (MMPs) using MMP Activity Assay Kit (Fluorometric-Green) (Abcam, #ab112146) as per manufacturer’s instructions. Briefly, pro-MMPs in solution are activated by incubating with APMA, immediately prior to enzyme reaction. Samples were read on a Bio-Tek Synergy multimode plate reader at 490/525 nm at 60 min incubation. Substrate and culture media controls were used to reduce background in analyzed values. All conditions were performed in singlicate from biological triplet samples.
Statistical analysis
All experiments were performed in biological triplicate. All statistical analysis and figure preparation was performed with Prism (GraphPad Software). Cytotoxicity and apoptosis assays were subjected to two-way ANOVA with Bonferroni’s multiple comparisons test. BioPlex control experiments were subjected to one-way ANOVA with Tukey’s multiple comparisons test. The qRT-PCR and BioPlex experiments of the stimulated controls were subjected to one-way ANOVA with Dunnett’s multiple comparisons test. The qRT-PCR, BioPlex, MMP activity assays, and immunofluorescence experiments of virus-infected samples were subjected to two-way ANOVA with Tukey’s multiple comparisons test. TUNEL assays were subjected to one-way ANOVA with Tukey’s (percent of cells TUNEL positive) or Sidak’s (percent of TUNEL positive cells expressing GFAP or MAP2) multiple comparisons tests.
Discussion
CNS infections continue to be an important facet of emerging viral disease. Despite this threat, in vitro models of CNS infection remain limited. Clinical samples are rarely obtained outside of autopsy due to the challenging nature of CNS biopsy. Animal models, both in vivo and ex vivo studies with primary cells, have been the primary models and provided many important insights [
43,
44]. However, animal models often do not accurately model all aspects of clinical disease described in patients. Many studies have now demonstrated profound differences between murine and human cellular responses [
45,
46]. Differences also exist in the structure and function of human and murine parenchymal cells. For example, astrocytes are much larger and perform more complex signaling in humans than in mice [
47]. Human primary CNS cells are relevant models, but they are difficult to obtain and introduce donor variability. Immortalized cell lines offer a useful alternative for in vitro studies, but the interpretation of results is limited by alterations in specific pathways and differences among different cell lines. Here, we report the use of a primary hNSC-derived neuron/astrocyte cell co-culture system as a more accurate and reproducible in vitro model to study encephalitic viruses. In addition to allowing for the repeated study of human primary cells without donor variability, co-culturing neurons and astrocytes allows for a more physiologically relevant model than neuronal monoculture. While the lack of donor variability is useful for the identification of pathogenic mechanisms, it may also obscure different aspects of disease as seen in other studies where different hNSC strains responded differently to infection [
30]. Astrocytes play a key role in the physiologic support of neuronal health and function such as synaptic development, protection from glutamate toxicity and oxidative stress, and metabolic support [
48]. Astrocytes also are increasingly recognized as major contributors to inflammatory responses during CNS infection. In addition to pathogenesis studies, neuron/astrocyte co-cultures may provide a relevant in vitro system for the evaluation of antiviral therapies against neurotropic viruses.
Neuron/astrocyte co-cultures were susceptible to LACV infection (Fig.
1) and high viral titers were reached regardless of initial viral inoculum (Fig.
1b). However, there was a drop in viral RNA measured at 48 HPI (Fig.
1c), for which the reasons are unknown, but the timing coincides with the peak IFN-β response suggesting that cells may become less susceptible to infection during this period (Fig.
4a). Future studies should evaluate the role of IFNs and the viral IFN antagonist NSs during LACV neuron/astrocyte infection. CPE was also observed at later time points consisting of cell rounding in detachment (Fig.
1a). Indeed, cytotoxicity and apoptosis assays indicate increased numbers of apoptotic cells beginning at 48 HPI (Fig.
3). Additionally, it seems that neurons are more susceptible to apoptosis than astrocytes in response to LACV infection (Fig.
3d). These data mirror those seen in previous work using murine models and the human NT2N cell line [
9,
12,
33,
34]. Of note, while cytotoxicity and apoptosis were noted, large amounts of viable cells remain as late as 96 HPI. This contrasts reports of LACV as a highly cytopathic virus in mouse primary neurons and human NT2N cells and are likely due to intact IFN responses and the protective effects of astrocytic co-culture resulting in a more physiologic response and greater resilience to infection [
12,
33].
The overall percentage of cells infected during this study was lower than expected, reaching maximums of 64% neuronal infection and 50% astrocytic infection (Fig.
2b). Again, this is likely due to intact antiviral sensing pathways and IFN responses. Multiple studies have indicated that both neurons and astrocytes are important producers of type I IFN during LACV infection, but astrocytes appear to be responsible for greater responses [
11,
15]. Therefore, astrocytic IFN production may be responsible for limiting the magnitude of infection. The current study also demonstrates neurons as astrocytes are both highly susceptible to LACV infection. Neurons have long been recognized as a target of infection, but astrocytic infection has been largely ignored [
8,
9,
11,
15]. One study demonstrated that in the weanling mouse model of LACV encephalitis, less than 1% of infected cells were astrocytes [
11]. However, in the same study, deleting the LACV NSs gene (an IFN antagonist) resulted in large increases of astrocytic IFN-β production suggesting nonproductive infections or infections below the detection limit of utilized assays may be higher than previously thought [
11]. Another group has proposed in a review that astrocytic infections are common and that the rapid death of infected astrocytes leads to a lack of detection in vivo [
1]. Here, we have shown that human astrocytes are indeed highly susceptible to LACV infection in vitro (Fig.
2). At early time points, neurons and astrocytes are infected at a nearly 1:1 ratio, and at an MOI of 10, the infection in astrocytes is significantly higher than in neurons at 12 HPI (Additional file
2: Figure S2c). However, by 72 HPI, neurons became the predominant cell type infected in the 0.01 MOI infections. Despite the shifting tropism of viral infection, no changes were detected in the overall ratio of neurons to astrocytes, and astrocytes seemed to be less susceptible to apoptosis compared to neurons (Additional file
1: Figure S1, Fig.
3d). This suggests that rapid astrocytic death is not a major driver in the shift in tropism in vitro. This shift may be due to the type I IFN response which begins at 48 HPI. Astrocytes may be more sensitive to type I IFN signaling and induce strong antiviral states, protecting this cell type while leaving neurons more vulnerable. This shift is less prominent at higher MOIs, likely because too many cells are infected early to adequately demonstrate changes in tropism and/or IFN responses were mounted too late to counter the heavy infection. However, when comparing only infected cells, neurons still tend to dominate at later timepoints even at late MOIs (Additional file
2: Figure S2b). Future studies will attempt to determine the relative roles of IFN on neurons and astrocytes during viral infection.
Neurons and astrocytes are increasingly recognized as important components of CNS immune responses. We tested the responsiveness of hNSC-derived neuron/astrocyte co-cultures by stimulating with the inactivated virus and a dsRNA analogue, Poly I:C (Additional file
3: Figure S3). The co-cultures are highly responsive to Poly I:C, responding with large increases in IFNs and proinflammatory cytokines and chemokines at both the RNA and protein level. It is reasonable to conclude that these cells have intact antiviral sensing pathways and are capable of initiating inflammatory responses. No responses to inactivated LACV were detected, suggesting that viral replication is necessary for such cellular responses. Alternatively, these results may indicate that viral entry is necessary as heat inactivation may denature attachment and entry glycoproteins. Another interesting finding was that while CCL2 mRNA is induced following Poly I:C treatment, the levels of CCL2 secretion remain unchanged. This suggests additional levels of regulation of CCL2 at the translational level.
LACV infection generated similar, but more limited, proinflammatory responses in these neuron/astrocyte co-cultures with mRNA levels peaking around 48 HPI and protein levels closely mirroring (Fig.
4 and Additional file
4: Figure S4). Interestingly, LACV, but not Poly I:C induced an increase in IFN-α mRNA similar to previous studies [
35,
36]. At the same time, unlike Poly I:C, LACV failed to induce IL-1β, IL-2, IL-5, IL-15, and IL-17. Additionally, LACV only resulted in modest increases in IL-10, IL-12, IL-9, and IL-13 only at late time points with high MOIs. This may be explained as either viral inhibition of select responses, lower levels of stimulation relative to Poly I:C, or differences in signaling pathways used to detect Poly I:C versus LACV. Indeed, Poly I:C was added to the supernatant without transfection, which typically stimulates TLR3 while LACV infection likely stimulates a larger range of endosomal and cytosolic RNA sensing receptors. These results clearly show that neurons and astrocytes are likely important factors in shaping the immune response to LACV encephalitis. This has been demonstrated for the type I IFN responses, but not yet the proinflammatory chemokines [
11,
15].
Human pathology reports and animal models note inflammation primarily composed of macrophages and lymphocytes [
8,
9,
14]. A recent study by Winkler et al. revealed that lymphocytes do not appear to be important in the development of neuropathology [
13]. The same group has also shown inflammatory monocytes to be the primary infiltrating cell type, although their role in immune-mediated neuropathology was not addressed [
14]. In the same study, it was noted that CCR2 was necessary for monocytes to migrate to lesions in the CNS, but not for recruitment to the CNS. Interestingly, while changes in mRNA expression for CCL2 were noted, protein expression in the cell culture supernatant was unchanged. Neurons and astrocytes constitutively express low levels of CCL2 in the healthy brain, as seen in our results, but some studies have noted a microglial requirement for upregulation after infection [
17,
49]. Microglia are missing from this system and should be further evaluated for their effect on responses after LACV infection. Their response is potentially via bystander effects, as preliminary experiments infecting primary human microglia did not yield productive infection or chemokine responses (data not shown). Future studies should address the role of inflammatory monocytes during LACV encephalitis.
The strongest responses in our study were in the monocytic and lymphocytic chemokines, CCL5 and CXCL10. These chemokines have been associated with a wide range of viral CNS infections and are important for lymphocyte recruitment [
50]. As previously mentioned, lymphocytes do not appear to play a role in immunopathology, but it is likely that during human infection their role is critical in the recovery from LACV encephalitis [
13]. The receptors for these CCL5 and CXCL10 (CCR5 and CXCR3, respectively) have been shown to be critical for T cell recruitment for several encephalitic viruses such as West Nile virus (WNV), murine hepatitis virus (MHV), and herpes simplex virus (HSV) [
51‐
53]. CCR5 has also been shown to be important for the control of WNV [
54]. Our data therefore suggest that neurons and/or astrocytes are important for the recruitment of T cells to the CNS during LACV encephalitis, although in vivo studies will be required to confirm this hypothesis. Additionally, we show strong IFN-γ responses which have been shown to be critical for the control of several viral CNS infections such as HSV, Sindbis virus, measles virus, and Theiler’s murine encephalomyelitis virus [
55‐
58].
One limitation of the current study is the difficulty in determining which cell types are responsible for signaling. Predictions can be made using other models, but as cells behave differently in co-culture, simple assessment of primary astrocytes and neurons may not be accurate. Both cell types appear to be important for cytokine responses in WNV infection, with astrocytes producing CXCL10, CCL2, and CCL5, but not IL-1β or IL-6 [
59]. Neuronal cells were then shown to produce IL-1β, IL-6, IL-8, and TNF-α [
60]. Both neurons and astrocytes appear to be important producers of CXCL10 during viral infection [
59,
61]. However, these differences are often virus-specific, with H7N9 influenza inducing IL-6 and IL-8 in both neuronal and astrocytic cells [
62]. Another layer of complexity is that host species differences are known to exist in cytokine expression and IFN responses [
63]. These conflicting data highlight the need for species-specific models for viral infection. However, immunofluorescent or immunohistochemical staining for cytokines and chemokines is often problematic due to low intercellular levels of protein, and separation via FACS is difficult due to the fragility of the cells. Future effort needs to focus on better understanding cell-specific responses.
The neuron/astrocyte co-cultures in this system also demonstrated a potential to disrupt the BBB. While initial LACV neuroinvasion is thought to occur via hematogenous spread through capillaries in the olfactory bulb, further disruption of the BBB after neuroinvasion may contribute to greater viral neuroinvasion or increased inflammatory responses leading to greater damage [
7]. Rift Valley fever virus, a related bunyavirus, is likely to also use the olfactory bulb for CNS entry, but generally maintains BBB integrity during infection in contrast to LACV [
64,
65]. TNF-α, IL-6, and VEGF had modest upregulation in this study and have long been associated with increased BBB permeability (Fig.
5 and Additional file
4: Figure S4) [
18,
19]. We additionally assessed MMPs and TIMPs (Fig.
5). MMP9 and MMP2 are typically the primary MMPs associated with BBB disruption [
19]. Initial BioPlex screens and RT-PCR did not detect MMP9 after virus infection (data not shown). We did however observe modest increases in MMP2 at 6 HPI and large increases in MMP7. MMP7 has not been commonly studied in the context of viral encephalitis and its relevance is unknown. However, MMP7 is important for leukocyte infiltration during experimental autoimmune encephalomyelitis and is found in the CSF during AIDS dementia [
40,
41]. TIMP-2 is constitutively expressed in the brain while TIMP-1 is inducible [
42]. The current study demonstrates that TIMP-1 is induced following LACV infection and that TIMP-2 appears to be downregulated (Fig.
5a). As both MMPs and TIMPs are upregulated, the functional status of MMP activity required further study. An MMP substrate cleavage assay revealed that high MOI LACV infection could induce an increased MMP response at 96 HPI. However, this is very late in infection, and the MMP activity was not high (Fig.
5b). This suggests that TIMP upregulation may be limiting BBB breakdown by MMP upregulation during LACV infection, but the functional activity of these enzymes on the BBB during LACV infection in vivo remains to be assessed.