Venezuelan equine encephalitis virus (VEEV) is a neurotropic arbovirus endemic to the Americas [1
]. VEEV is a New World (NW) alphavirus of the family Togaviridae
and is classified as a Group IV (+) ssRNA virus. VEEV is categorized as a select agent pathogen by the Centers for Disease Control and the United States Department of Agriculture due to its potential for being weaponized as a consequence of a very low infective dose and an ability to be aerosolized [2
]. The aerosol infective dose of VEEV TrD in a BALB/c mouse model has been shown to be less than one plaque forming unit (PFU) [3
]. Mosquito-transmitted infections can occur at doses as low as 10 to 1000 PFU [4
]. VEEV was previously developed into a biological weapon during the Cold War [5
]. Furthermore, as an RNA virus, VEEV has the potential to quickly generate novel mutations that may allow for epidemic spread by its mosquito vectors. Mutations in the E1 glycoprotein of Chikungunya virus (CHIKV), a related alphavirus, led to increased fitness in mosquitoes which caused a worldwide pandemic that still persists today [6
]. More than 1.5 million people have been infected in countries bordering the Indian Ocean since the outbreak began [7
]. Major epidemic outbreaks of VEEV in the 1960s resulted in the infection of as many as 200,000 humans in Columbia [2
]. VEEV has also been detected as far north as Texas and Florida [1
VEEV infection in humans presents with flu-like symptoms including high fever, headache, and malaise [8
]. Progression to an encephalitic phenotype can occur in 10–15% of cases and may result in long-term neurological complications and damage. The mortality rate following VEEV infection in humans is ~1% [1
]. Neurotropic viral infections cause nervous tissue damage principally through two mechanisms: direct neuronal cell death as a consequence of viral replication, and the associated tissue damage arising from the effects of high levels of inflammation [9
]. VEEV infection of the central nervous system (CNS) following subcutaneous infection occurs due to viral spread from replication sites in the periphery; however, the mechanism for CNS entry has not been definitively established [13
]. Recent studies have demonstrated that replication in mouse models occurs in the brain prior to blood-brain barrier disruption [9
], with the resulting inflammation damaging the blood-brain barrier and leading to increased permeability which may lead to neuroinvasion and subsequently cause permanent neurological sequelae [9
]. In addition, microglia, the resident macrophage cells of the CNS, react to the infection by releasing pro-inflammatory cytokines [14
]. This suggests that therapies targeting modulation of the inflammatory response following VEEV infection may be a promising avenue of investigation when compared to those directly targeting viral replication.
Currently, the only treatment available following VEEV infection is supportive intensive care. There are no FDA-approved commercially available vaccines or antiviral drugs to treat exposure to VEEV. In this study, we attempt to identify the efficacy and antiviral potential of three FDA-approved anti-inflammatory drugs against VEEV. The tested inhibitors are FDA-approved anti-inflammatory drugs that reduce inflammation by targeting a variety of pathways. Celecoxib was FDA-approved in 1998 and originally marketed as anti-arthritis drug with the trade name of Celebrex [15
]. Celecoxib is a cyclooxygenase-2 (COX-2) selective non-steroidal anti-inflammatory drug (NSAID). COX-2 is stimulated by inflammatory signals such as IL-1, IL-6, and IL-8, cytokines known to be induced by VEEV, and upregulates the production of potent pro-inflammatory cytokines, including prostaglandin E2 [14
]. Inhibition of this pathway may slow viral dissemination and reduce tissue damage that results from inflammation.
Rolipram inhibits the phosphodiesterase-4 (PDE4) pathway [17
]. It was discovered in the 1990s and initially investigated as an antidepressant [18
]. PDE4 modulates the cyclic AMP pathway by degradation cAMP and has pro-inflammatory effects on a range of inflammatory cells including macrophages, T cells, and B cells [19
]. Inhibition of the PDE4 pathway during VEEV infection may dampen the inflammatory response while still allowing adequate immune system activity against VEEV, thereby reducing tissue damage caused by excessive inflammation.
Tofacitinib is a Janus kinase (JAK) inhibitor, and specifically inhibits JAK1 and JAK3 activity. It was approved by the FDA in 2012 for the treatment of rheumatoid arthritis. Downstream signaling by the JAK-STAT signaling pathway mediates the upregulation of many pro-inflammatory cytokines, including IFNγ, IL-2, IL-4, and IL-10 [21
], and inhibiting this pathway may reduce inflammation-mediated host tissue damage at sites of infection.
In this study, we tested the toxicity and efficacy of these drugs in the context of VEEV infection in the human microglial (HMC3s) and astrocyte (U87 MG) cell lines. In the nontoxic concentration of all inhibitors, we observed a significant decrease in viral titers following pre-treatment of cells with Celecoxib, Tofacitinib, or Rolipram with either the TC-83 or the virulent Trinidad Donkey (TrD) strains. In addition, post-exposure efficacy against the TC-83 strain was also observed following treatment of infected cells with all three tested drugs at 2, 4, and 6 hpi (hours post infection). Celecoxib exhibited the highest anti-VEEV activity among the tested inhibitors, with treatment inhibiting replication up to six hours after exposure. Furthermore, cytokine gene levels in infected cells were reduced after treatment with Celecoxib, most notably IL1A, IL17F and TNFα. Overall, Celecoxib demonstrated functionality and effectivity by delaying viral replication and infection-induced inflammation in VEEV-infected cells.
2. Material and Methods
2.1. Cell Lines, Viruses, and Reagents
Celecoxib (S1261), Rolipram (S2127), and Tofacitinib (S5001) were obtained from SelleckChem. The live-attenuated TC-83 vaccine strain of VEEV was acquired from BEI Resources. HMC3 human microglial cells (ATCC CRL-3304), U87 MG human brain astrocytoma cells (ATCC HTB-14), and VERO African green monkey kidney cells (ATCC CCL-81) were obtained from the American Type Culture Collection. U87 MG cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (VWR Life Science VWRL0102-500) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco 10,437,028), 1% Penicillin/Streptomycin (Gibco 15-140-122), and 1% L-Glutamine (VWR Life Science VWRL0131-0100) at 37 °C and 5% CO2. HMC3 cells were maintained in Eagle’s minimal essential medium (EMEM) (VWR Life Sciences 10128-214) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37 °C and 5% CO2. African green monkey kidney cells (VEROs) were maintained in DMEM supplemented with 5% heat-inactivated fetal bovine essence (FBE) (VWR Life Sciences 10803-034), 1% Penicillin/Streptomycin, and 1% L-Glutamine at 37 °C and 5% CO2. All reagents for cell maintenance were pre-warmed to 37 °C before use.
2.2. Viral Infections and Inhibitor Studies
In 96-well plates, HMC3 or U87 MG cells were seeded at a density of 3E4 or 1E4 cells/well, respectively. The inhibitors were resuspended in dimethyl sulfoxide (DMSO) prior to dilution to the indicated concentrations in culture media and cells were pre-treated before infection for 2 h. After 2 h, media was removed and saved, and is thereafter referred to as conditioned media. The virus was diluted in media and cells were then infected for 1 h at indicated multiplicities of infection at 37 °C, 5% CO2 to allow for the uptake of the virus. Viral inoculum was removed, cells were gently washed three times with Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium (Gibco 14190144), and conditioned media was placed onto the cells. Cells were incubated at 37 °C and supernatants were collected at indicated times post infection and stored at –80 °C.
2.3. Viral Plaque Assays
Plaque assays were performed using VERO cells grown to a concentration of 2E5 cells/well in a 12-well plate. Supernatants from infected cells were serially diluted in media and used to infect VERO cells for one hour. After infection, a 1:1 overlay consisting of EMEM (without phenol red, supplemented with 10% FBS, non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 20 U/mL of penicillin, and 20 μg/mL of streptomycin) and 0.6% agarose was added to each well. Plates were incubated at 37 °C for 48 h. Cells were fixed with 10% formaldehyde for 1 h at room temperature. Formaldehyde was aspirated and the agarose overlay was removed. Cells were stained with crystal violet (1% CV w/v in a 20% ethanol solution). Viral titer (PFU/mL) of VEEV infection was determined by plaque count.
2.4. Cell Viability Assay
The viability of HMC3 and U87 MG cells treated with indicated inhibitors was determined using the CellTiterGlo assay (Promega). Inhibitors were resuspended in DMSO prior to dilution to tested concentrations in culture media. U87 MGs were seeded at 1E4 and HMC3s at 3E4. Inhibitors were overlaid on cells grown in a white-walled 96-well plate, which was incubated at 37 °C, 5% CO2 for 24 h. CellTiterGlo substrate was used according to the manufacturer’s instructions. Luminescence was determined using a DTX 880 multimode detector (Beckman Coulter) with an integration time of 100 ms/well. Cell viability was determined to be percent cell viability normalized to a DMSO-treated control.
2.5. Quantitative Real-Time Polymerase Chain Reaction
For QRT-PCR, samples were collected using Trysol reagent (ThermoFisher 15596026). RNA was extracted using the Zymo Research Direct-zol RNA Miniprep Kit (R2104) as per manufacturer’s instructions. Extracellular samples were collected from supernatants. Intracellular RNA was collected by direct cell lysis. The pre-cycling conditions were adapted from Verso 1-step RT-qPCR kit (ThermoFisher AB4101C) manufacturer’s instructions: 1 cycle at 50 °C for 20 min, 1 cycle at 95 °C for 15 min, 40 cycles at 95 °C for 15 s and at 51 °C for 1 min using a StepOnePlus™ Real Time PCR system (Applied Biosystems 4376600). VEEV TC-83 primers and probes targeted nucleotides 7931-8005 in VEEV capsid: forward primer (5’-TCTGACAAGACGTTCCCAATCA-3’) and reverse primer (5’-GAATAACTTCCCTCCGACCACA-3’) [22
]. The probe utilized different tags (5ʹ6-FAM/TGTTGGAAG/ZEN/GGAAGATAAACGGCTACGC/3ʹIABkFQ) to improve sensitivity. Primers and probe were designed by and obtained from Integrated DNA Technologies (Skokie, IL). A standard curve was used to quantify levels of RNA based on threshold cycle (Ct) counts.
2.6. Statistical Analysis
Statistical analyses were performed using the software Prism 5 (Graph Pad). Data are presented as mean ± standard deviation (SD) after analysis with an unpaired, two-tailed t-test. Differences in statistical significance are indicated with asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Number of replicates per experiment is indicated in each figure legend.
VEEV is a category B select agent and an emerging infectious agent that is capable of causing systemic and neuronal disease upon exposure. There is currently no FDA-approved countermeasure strategy available to address VEEV infection and disease. VEEV-infection dependent neurological disease is characterized by a strong inflammatory component that may play a critical role in the onset of neurological manifestations. An ideal treatment strategy to deal with VEEV infections should therefore be capable of controlling not only the viral load, but also the inflammatory output and resulting damage to the tissue microenvironment.
With the intention of identifying countermeasure strategies, three anti-inflammatory drugs namely, Celecoxib, Tofacitinib, and Rolipram, were evaluated to determine their efficacy in controlling VEEV infection in an in vitro model of infection. We determined that Celecoxib was the most effective drug out of the three that were evaluated. Celecoxib inhibited VEEV TC-83 replication in multiple human cell lines, HMC3 microglia and U87 MG astrocytes, as well as the wild-type strain Trinidad Donkey in HMC3 cells. We observed lower levels of inhibition in U87 MG cells when compared to that observed in HMC3 cells. This may be due to the use of a lower dose of drug treatment in these cells due to toxicity observed at higher doses. Previous studies have shown that alphavirus infection activates microglia and astrocytes in vivo [26
], and both cell types have been identified as mediators in the development of Venezuelan equine encephalitis through the production of pro-inflammatory cytokines following infection [14
]. Astrocytes and microglia are both activated in vivo; however, it is unknown whether or not they are infected in vivo following infection. Both cell types have been shown to contribute to inflammation following viral infection by producing cytokines such as TNFα and inducible nitrous oxide synthase [7
Previous in vivo studies using VEEV have shown that non-selective COX inhibitors can delay the onset of symptoms in a mouse model. In one study, mice were treated once per day with naproxen during the course of infection to evaluate the importance of inflammation in a VEEV infection model. Overall mortality in the study was not reduced for the treated cohort, but the onset of symptoms was delayed following naproxen treatment of infected mice [10
]. Naproxen was not evaluated for any anti-viral properties and was chosen since it is a known anti-inflammatory drug. For these reasons, Celecoxib may be a viable candidate for an in vivo study testing its efficacy against VEEV.
We also evaluated the efficacy of Celecoxib against VEEV when administered to cells post-infection. Treatment reduced viral titers up to six hours after exposure to VEEV had occurred. This post-exposure efficacy of Celecoxib may be beneficial as a countermeasure strategy for treating VEEV infections and should be investigated in an animal model mimicking exposure to an intentional release or a lab-acquired infection.
Viral infections are known to induce pro-inflammatory cytokines [14
]. Specifically, activation of microglia during a NW alphavirus infection causes activation of many inflammatory pathways, including IL-1, IL-6, and TNFα [12
]. The inflammatory response can lead to an encephalitic phenotype that can cause permanent neurological damage or death [9
]. The effect of Celecoxib treatment on these pathways was evaluated and Celecoxib treatment reduced the upregulation of several critical cytokines including IL-1α and TNFα following infection. Furthermore, cells primed with supernatants from previous VEEV exposure have also been shown to produce higher viral titers and cytokine concentrations [14
]. Treatment with Celecoxib lowers both viral load and cytokine concentrations following indirect inflammatory exposure. Controlling the inflammatory environment during a neurotropic viral infection may be beneficial to preventing long-term neurological complications.
Overall, our data demonstrates that Celecoxib, an FDA-approved COX-2 inhibitor, is capable of slowing the replication of both TC-83 and a wild-type strain of VEEV in an in vitro model of infection. Furthermore, Celecoxib treatment reduces the upregulation of pro-inflammatory cytokines that can contribute to VEEV-mediated neurological damage following infection.