Broad spectrum antiviral BDGR-164 provides protection against lethal neurotropic alphavirus infection in mice

Abstract

The New World alphaviruses, eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and western equine encephalitis virus (WEEV), cause febrile illness that can progress to fatal disease in humans and equids. As there are no therapeutics approved for the treatment of neurotropic alphavirus disease in humans, we report a structurally novel, quinazolinone, BDGR-164, with nanomolar potency against VEEV, EEEV, and WEEV. Using an intranasal route of virus infection in a lethal BALB/c model, to model aerosol infection in a biodefense scenario, prophylactic subcutaneous administration of BDGR-164 conferred 100% (VEEV), 88% (EEEV), and 63% survival (WEEV) compared to uniform lethality in sham-treated animals. Using RNA-Seq and histopathology, we determined that viral RNA levels and antigen were reduced significantly in the brains of mice from the virus-infected, BDGR-164-treated group as compared to virus-infected, sham-treated group. Moreover, there was a significant reduction in the virus-infected, BDGR-164-treated mice in their host immune responses associated with inflammatory signaling, immune cell recruitment, and programmed cell death. Comparison of cytokines in sera from VEEV-, EEEV- or WEEV-infected mice with and without BDGR-164-treatment suggested that lower levels of IL-6, TNF-α, and RANTES levels correlated with protection and may serve as useful early biomarkers in treatment studies in animal models. In conclusion, our studies show broad and potent prophylactic efficacy of BDGR-164 against neurotropic alphaviruses.

1.0. Introduction

Venezuelan, eastern and western equine encephalitis viruses (VEEV, EEEV, WEEV) are mosquito-borne, New World alphaviruses that have caused several major epizootic and/or epidemic disease outbreaks in humans and equids in the Americas. The case fatality rate ranges from <1% in healthy adults to 35% in children for VEEV, 3–7% for WEEV and 30–75% for EEEV (Lindsey et al., 2018; Millet et al., 2021; Oliver et al., 2016; Rivas et al., 1997; Rocheleau et al., 2017; Ronca et al., 2016; Sherwood et al., 2020). Even with recovery from mosquito-borne illness, 4–14% (VEEV), 15–30% (WEEV), and 50–90% (EEEV) of cases may suffer from long-term neurological sequelae (Ronca et al., 2016). The development of VEEV as a bioweapon during the Cold War resulted in its designation as a select agent by the Center for Disease Control and Prevention, and along with EEEV, and as a Category B priority pathogen by the National Institutes of Health (Paessler et al., 2007; Zacks and Paessler, 2010). The threat of VEEV and EEEV as bioweapons rests in their ability to replicate to high titers and to be disseminated by aerosol exposure. Moreover, in aerosol or intranasal infections, VEEV reaches the olfactory nerve, and brain, much more rapidly compared to a natural infection via a mosquito bite (Ryzhikov et al., 1991). Hence, in addition to treatments that target systemic infection, the potential of these viruses to invade the brain from natural, lab or bioweapon-related exposures, requires therapeutics that effectively penetrate the brain barrier (BBB) to reduce viral replication and thereby mitigate neuronal damage caused by host responses to infection. Therefore, to evaluate the neurotropic aspect of disease, we evaluated the broad-spectrum efficacy of a brain penetrant, small molecule, called BDGR-164, using lethal BALB/c models of intranasal infection of VEEV, EEEV, or WEEV.

We have previously reported the discovery and efficacy of a novel brain-penetrant, antiviral, BDGR-49, against a lethal exposure to VEEV, WEEV or EEEV in BALB/c mice using subcutaneous (Cao et al., 2023) or intranasal routes of infection (Williams et al., 2025). BDGR-49 was discovered through iterative structure activity relationships with corresponding in vitro screening for antiviral activity to identify those molecules with improved activity. The BDGR-49 conferred 100% prophylactic and therapeutic efficacy when mice were challenged subcutaneously with VEEV strain TrD and treated at 4, 8 or 12 mg kg⁻¹ twice per day (Cao et al., 2023). For intranasal exposures of VEEV TrD, prophylactically administered BDGR-49 provided 100% efficacy when mice were treated at 6 mg kg⁻¹ twice per day (Williams et al., 2025). BDGR-49 prophylactic efficacy studies with subcutaneous EEEV infection in mice resulted in 70% survival (Cao et al., 2023) while intranasally challenged mice showed only a delay in mortality (Williams et al., 2025). A similar trend to EEEV was observed during treatment of intranasal challenge of mice with WEEV, where mortality was delayed by a day (Williams et al., 2025). Hence despite the promising antiviral potency and efficacy of BDGR-49 in vitro and in vivo, it lacked the desired broad spectrum efficacy with both routes of infection. Hence additional compounds in the BDRG library were screened in vitro. Of these, BDGR-164 had increased in vitro potency, solubility and biopharmaceutical properties that supported its testing in mice. We evaluated the broad-spectrum prophylactic efficacy of BDGR-164 for VEEV, EEEV, and WEEV in lethal BALB/c mouse models and correlates of protection. A comprehensive set of endpoints in mice (RNA-Seq, serum cytokines, histology, and immunohistochemistry) were used to evaluate efficacy in mice exposed to virus, or mice exposed to virus plus BDGR-164 treatment.

2.0. Materials and Methods

2.1. Cells and viruses

BHK clone 21 cells [BHK-21, American Type Culture Collection (ATCC) CCL-10)] and Vero 76 (ATCC CRL-1587) were maintained in modified Eagle’s medium with Earle’s balanced salt solution and L-glutamine supplemented with 10% fetal bovine serum (FBS). 293T cells (ATCC, CRL-3216) were maintained in Dulbecco’s Modified Eagle Medium with high glucose and GlutaMAX supplemented with 10% heat inactivated FBS. Cells were maintained at 37°C in humidified incubators with 5% CO₂. VEEV TrD was purchased from BEI (NR-332). EEEV strain FL93–939 and WEEV strain Fleming were obtained from the World Reference Center for Emerging Viruses and Arboviruses (University of Texas Medical Branch, Galveston, TX). VEEV TC-83 (lyophilized vaccine) was obtained as a gift from Dr. Connie Schmaljohn (United States Army Medical Research Institute of Infectious Diseases, Maryland). All viruses were amplified and stored at −80°C until use. All cell culture reagents were purchased from Thermo Fisher Scientific unless otherwise specified.

2.2. Selection and Sequencing of Resistant Mutants from Cell Culture

Prior to infection with VEEV TC-83, Vero 76 cells were seeded onto 6-well plates and grown to 90% confluence. Cells were infected at an initial MOI of 0.1. Resistant viruses were selected by serial passaging virus in two-fold increasing concentrations of BDGR-164, starting at 50 nM and increasing to 100, 200, 400, 800, 1,600, and lastly, 3,200 nM, for a total of 7 passages. We used 250 μl of supernatant from each passage to initiate the subsequent blind passage. Three biological replicates were conducted for each experiment. A control group, VEEV TC-83 without compound, was also passaged without compound in triplicate at the same time and in the same manner as the virus was passaged with compound.

To sequence viral genomes from cell culture, 0.5 ml of supernatant from each passage was harvested, and tiled amplicons were made as described previously (Lee et al., 2020). PCR products were purified using the Wizard SV gel and PCR clean-up system (Promega). Libraries for NGS were prepared using the Nextera XT kit (Illumina) and sequenced on an Illumina MiSeq. Data was analyzed using CLC Genomics Workbench v.23 (Qiagen). Sequencing reads were filtered and trimmed to remove reads that were shorter than 50 nucleotides in length and trim sequences with a Phred score of 20 were removed. Remaining reads were mapped to the Jonsson laboratory VEEV TC-83 seed stock consensus sequence (Lee et al., 2020). Virus population variants were identified using the following parameters, minimum frequency ≥1%, minimum coverage ≥400, and variant count ≥10 (Table S1). Resulting variants were further analyzed to identify those that cause non-synonymous mutations. All raw sequencing reads are available under the BioProject ID PRJNA1077336.

2.3. General animal study information and compliance for mouse studies

Female BALB/c mice (5–6 weeks old) were purchased from the Charles River Laboratories. Mice were anesthetized with isoflurane prior to intranasal infection. The seed stock of each virus was diluted to the desired concentration in phosphate buffered saline (PBS) before use and kept on ice. Mice were challenged with a 10× LD₅₀ challenge dose of VEEV TrD (7500 PFU), EEEV FL-93–939 (7500 PFU) or WEEV Fleming (5000 PFU). BDGR-164 was administered by subcutaneous injection at 8 or 48 mg kg⁻¹ in a volume of 0.2 ml at 2 hours before the virus challenge and again at 10 hours after the first injection. Dosing was continued twice daily at 12-hour intervals for five additional days at 4 or 24 mg kg⁻¹ twice per day. Mice were monitored for mortality and clinical signs until the end of the study. All animal studies and methods were conducted under the protocol 18–044 or 21–0258 authorized by the Institutional Animal Care and Use Committee at the University of Tennessee Health Science Center.

2.4. Compound structure, purity, and formulation for mice studies

BDGR-164, provided in powder form from the lab of J.E.G at the University of Wisconsin-Madison, was synthesized by modifying the published procedure for BDGR-49 (Cao et al., 2023) which is the subject of a separate manuscript in preparation. BDGR-164 is structurally distinct from BDGR-49 and is characterized by the SMILES string: O=C1N(C2=CC=C(OC)C(F)=C2)C(C3NCCC3)=NC4=C(C5=CC=C(C)N=C5)C=C(C#N)C=C41 and IUPAC nomenclature 3-(3-fluoro-4-methoxyphenyl)-8-(6-methylpyridin-3-yl)-4-oxo-2-(pyrrolidin-2-yl)-3,4-dihydroquinazoline-6-carbonitrile. The compound was purified by column chromatography, certified for structural integrity by ¹H and ¹³C NMR, and analyzed for >95% purity by HPLC. The compound was formulated immediately prior to assessment in 25% PEG400/10% Kolliphor RH40/65% water to the appropriate concentration with intermediate vortexing. Kolliphor and PEG were purchased from Millipore-Sigma. Water was purchased from Henry Schein.

2.5. Measurement of virus titer from brain homogenate

For measurement of the viral loads, four mice from each group were sacrificed at 4 dpi, and the whole brain tissues were collected and homogenized. Homogenates were prepared and clarified to measure the amount of infectious virus using a crystal violet plaque assay as described previously (Cao et al., 2023).

2.6. Measurement of inflammatory cytokines in serum

Secreted cytokines, IFN-γ, IL-6, TNF-α, RANTES and IFN-β, were measured in serum samples using the Invitrogen ProcartaPlex multiplexed immunoassay (ThermoFisher) according to the manufacturer’s instructions. Cytokine standards were prepared to determine the concentration of cytokine in the samples, and the samples were analyzed on a MagPix (Luminex Corp.). Data analysis was performed using the xPONENT software. A curve fit was applied to the standards, and the sample concentrations were extrapolated from the standard curve using the five-parameter logistic method.

2.7. RNA-Seq and bioinformatic analysis

At 4 dpi mice from each group were humanely euthanized and their whole brain including the olfactory bulb collected and homogenized in DPBS (1x) in a tube containing ceramic beads using a Bead Mill 4 (Fisher Scientific). Total RNA was isolated from each brain using TRIzol Reagent and was further purified by means of DNase treatment using the TURBO DNA-free Kit (Invitrogen). Total RNA was quantified and qualified using the BioAnalyzer 2100 (Agilent) after which sequencing libraries were constructed by mRNA enrichment using Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina). Constructed libraries were sequenced by Novogene Co. on Illumina’s HiSeq platform. Sequencing results were processed with CLC Genomics Workbench (Qiagen, v23). Sequencing reads from each sample underwent trimming and filtering using default parameters. Read mapping was conducted using default parameters and performed to a concatenated genome reference genome consisting of Mus musculus (Ensembl, GRCm39) and sequenced lab strain of VEEV TrD, EEEV FL93–939, or WEEV Fleming. Gene count results were used to assess differential gene expression using DESeq2 and mock inoculated mice defined baseline gene expression. Determined differentially expressed genes (Log₂ fold change = ≤−1.5 and ≥1.5; false discovery rate = ≤ 0.05) were used to create Venn diagrams using BioVenn (Hulsen et al., 2008). Differentially expressed genes (Log₂ fold change = ≤−1.5 and ≥1.5; false discovery rate = ≤ 0.05) were used to assess canonical pathway activation as well as perform comparison analysis using Ingenuity Pathway Analysis (Qiagen). For the IPA analysis, a custom PANoptosis pathway was manually defined. The pathway included the following genes: ADAR, AIM2, MEFV, NLRC4, NLRP1, NLRP3, NLRP9a, NLRP9b, NLRP9c, TNFRSF1A, ZBP1 FADD, PYCARD, CASP1, CASP10, CASP12, CASP2, CASP3, CASP4, CASP6, CASP7, CASP8, ZBP1, GSDMD, MLKL, RIPK1, RIPK3, and TNF. The selection of these was based on publications on defining genes involved in PANoptosis. IPA’s Molecule Activity Predictor tool was used to assign activity states were all genes were assigned an activate state except for ADAR which was assigned an inhibit state. The activation z-score for this custom PANoptosis pathway was calculated in IPA alongside the canonical pathways. Unprocessed and processed RNA-Seq data is available on NIH Gene Expression Omnibus under the series GSE255795.

2.8. Assessment of virus mutations during in vivo infections

Sequencing reads (described above) that mapped to the genome sequence of our lab seed stock viruses were used to assess virus mutations using CLC Genomics Workbench (Qiagen, v23). Viral SNPs were identified using Low Frequency Variant Detection with the following parameters: minimum coverage = 100, minimum count = 50, and a minimum frequency = 1.0%. Ns mutations were determined by using the Amino Acid Change tool to filter away SNPs that did not result in nonsynonymous changes.

2.9. Antiviral resistance testing using a trans-replicon assay

To determine antiviral resistance profiles of resistant mutants observed in this study (Table S1, Fig. S1), a Trans-replicon system based on VEEV TrD, a kind gift by the Dr. Andres Merits, was utilized(Lello et al., 2021) and performed as previously described (Williams et al., 2025). The system has two plasmids that express the nsP1234 (pCMV-1234) and the reporters firefly luciferase (Fluc) and Gaussia luciferase (Gluc) (pHSPolI-FG). For the negative control, a plasmid (pCMV-1234^GAA) was constructed where motif C in nsP4 was mutated from GDD to GAA, resulting in an inactive replication complex. Mutations were constructed in nsP2 and nsP4 genes of the plasmid pCMV-1234 to create: pCMV-1234_NSP2:Y102C, pCMV-1234_NSP4:Y87F, and pCMV-1234_NSP4:Q210R. Site-directed mutagenesis was performed by GenScript and amplified and purified to their Research Grade to limit endotoxin levels. For the assay, 293T cells were seeded in 96-well plates at a density of 35,000 cells/well. After 16 hours, BDGR-164 diluted 2-fold between 3000–0.006 nM for a total of 10 concentrations (n=4) was added to the pre-seeded plates. Following the addition of BDGR-164, cells were transfected using FuGene 6 (Promega), following manufactures guidelines. 18-h post-transfection expression of Fluc and Gluc was measured using the Dual-Luciferase Reporter Assay System (Promega) on a GloMax Navigator Microplate Luminometer (Promega).

2.10. Immunohistochemistry

Brain immunohistochemistry was processed as previously reported (Williams et al., 2023). Briefly, mouse brains were harvested and fixed with 10% buffered neutral formalin solution and sectioned to a thickness of 40 μm. For immunohistochemistry labeling, brain sections were incubated with goat anti VEEV glycoprotein (1:12000 dilution, AlphaVax, NC, USA), or rabbit anti-GFAP antibody (1: 2000, NB300–141, Novus Biologicals) or rabbit AIF-1/Iba-1 antibody (NBP2–19019, Novus Biologicals) separately. Followed with biotinylated anti-goat IgG or anti-rabbit IgG antibody (1:200 with BA-5000, or BA1100, Vector Laboratories. USA) and followed with Avidin-biotin complex (Vectastain ABC Elite Kit PK-6100 Vector Laboratories (USA) and developed with DAB solution. Some sections were counterstained with methyl green.

2.11. Statistical analyses

Survival data from VEEV, EEEV or WEEV mouse studies were analyzed using the Log-rank (Mantel-Cox) survival analysis and P values were generated from the comparisons between the survival curve of each treatment group and the survival curve of the virus only group (Prism 10, GraphPad Software, Inc).

2.12. Data Availability

All NGS data from the studies herein are publicly available. Viral passaging raw sequencing reads are available under BioProject PRJNA1077336 and the RNA-seq results are available under the Gene Expression Omnibus series GSE255795.

3.0. Results

3.1. BDGR-164 showed broad spectrum, in vitro antiviral activity against the New World Alphaviruses

We tested the potency of a novel, quinazolinone, BDGR-164 (see Material and Methods for structure) against VEEV, EEEV, WEEV, and chikungunya virus (CHIKV) in Vero 76 primate cell-based assays that measured the cytopathic effect (CPE) induced from viral infection (CPE-protection assay) in the presence of escalating concentrations of BDGR-164 administered prior to infection (Chung et al., 2014). BDGR-164 demonstrated a strong, specific potency against the three neurotropic New World alphaviruses with EC₅₀ values ranging from 3–10 nM (Table 1). No cytotoxicity was observed at any concentration ≤50 μM BDGR-164. No protection from CPE was noted against CHIKV at the highest concentration used.

Table 1.

Antiviral activity of BDGR-164 in CPE-protection assays

Virus	Strain	EC50 ± S.D. nM	No. of replicates
VEEV	TC-83	4.6±3.1	12
	TrD	3.5±0.8	2
	INH9813	5.3±1.1	4
EEEV	V105	9.6±1.5	2
	FL93–939	4.9±2.6	2
WEEV	California	4.1±2.1	9
CHIKV	S27	>50,000	2

S.D.=standard deviation

Antiviral efficacy against various strains of VEEV, EEEV and WEEV were further validated in virus yield reduction assays using Vero 76 cells treated with 1 μM (Fig. 1). Reduction of viral titer ranged from 4.4–5 Log₁₀-fold for EEEV and WEEV, respectively, to 7 to 8.1 Logs for EEEV strain V105 and VEEV vaccine strain TC-83, respectively (Fig. 1). As noted in the CPE assay, BDGR-164 showed a range of potencies against different strains of EEEV.

Figure 1. Inhibition of infectious virus production by BDGR-164 in cell culture.

The antiviral activity of BDGR-164 was tested in cell culture against a number of neurotropic alphaviruses. The figure shows the amount of virus produced after 48 hours in the presence or absence of 1 μM BDGR-164. Viral titer was measured by plaque assay as described in the material and methods.

3.2. Prophylactic treatment of BALB/c mice with BDGR-164 provided complete protection against lethal intranasal challenge with VEEV TrD

We evaluated the prophylactic efficacy of BDGR-164 in BALB/c mice challenged intranasally with ten times the median lethal dose (LD₅₀) of VEEV TrD (Fig. 2A). On the first day of infection (0 dpi), a two-times higher bolus of BDGR-164 was administered subcutaneously at 8 (low) or 48 (high) mg kg⁻¹ dose given twelve hours apart. Thereafter, BDGR-164 was administered subcutaneously at 4 (low dosing) or 24 (high dosing) mg kg⁻¹ doses twice a day for a total of five additional days. The treatment regimens will be referred to as ‘low dosing’ or ‘high dosing’ herein. Clinical signs were assessed through 14 dpi for all groups. The PBS mock-inoculated, sham control group of mice were treated with only the high dose regimen of BDGR-164. As the brain is the main site of intranasal VEEV TrD infection, viral titer in whole brain were measured on 4 dpi by plaque assay.

Figure 2. Experimental design, morbidity, survival, and viral brain titer data from prophylactic BDGR-164 treatment in BALB/c mice infected with VEEV TrD.

(A) Female BALB/c mice, 5–6 weeks old (n = 12 per group), were treated by subcutaneous administration with a low dose (8 mg kg⁻¹) or a high dose (48 mg kg⁻¹) of BDGR-164 two hours before intranasal challenge with VEEV TrD. A second dose of 8 or 48 mg kg⁻¹ was given 10 hours after VEEV TrD infection. Dosing at one-half the initial dose was continued twice daily at 12-hour intervals for an additional five days. (B) Virus titers from homogenized whole brains at 4 dpi (n = 4 per group) with intranasal virus challenge and receiving BDGR-164 at 0, low or high doses. ** = P ≤ 0.01. LOD = limit of detection. (C) Morbidity curves (n = 8 per group on 0 dpi) determined by average percent weight change. (D) Survival curves (n = 8 per group) comparing the same cohorts of mice shown in C.

All the VEEV TrD-challenged mice that did not receive BDGR-164 or the low BDGR-164 treatment had viral titers greater than 10⁸ PFU/mL (Fig. 2B), lost weight (Fig. 2C) and met clinical criteria for euthanasia by 6 dpi. Clinical signs were scored in appearance, behavior, and neurological symptom categories. In untreated VEEV-TrD-infected mice, appearance, reduced grooming, hunched posture, began on 2 dpi while behavior, the level of responsiveness, began on 3 dpi. Mice that reached 25% weight loss were euthanized regardless of low scores in other categories. Animals treated with the low dose of BDGR-164 reached euthanasia criteria by 9 dpi, although one mouse recovered and regained normal body weight by 14 dpi. Mice treated with the high dose of BDGR-164 had reduced viral load in the brain (Fig. 2B), and did not lose body weight and were comparable to the PBS control group (Fig. 2B,C). Mice treated with the high dose of BDGR-164 showed 100% survival, while the low dose treatment had only 12.5% (1/8) survival (Fig. 2D). The treatment with the high dose regimen provided the greatest reduction of infectious virus (P = 0.0033) in the brain, while there was no statistical difference (P = 0.3663) in viral titer between the sham and the low dose regimen groups (Fig. 2D).

3.3. Prophylactic treatment of BDGR-164 confers survival to BALB/c mice given lethal challenge of EEEV FL-93–939 or WEEV Fleming

Next, we evaluated the antiviral efficacy of BDGR-164 using the same treatment regimen and experimental design as stated above for VEEV (Fig. 2A) to assess the level of protection against intranasal challenge with a 10xLD₅₀ of EEEV strain FL93–939 or WEEV strain Fleming in BALB/c mice. On the first day of infection (0 dpi), a higher bolus was administered subcutaneously at 8 mg kg⁻¹ and 48 mg kg⁻¹ dose twelve hours apart. Thereafter, BDGR-164 was administered subcutaneously at 4 mg kg⁻¹ or 24 mg kg⁻¹ doses twice a day, respectively, for an additional five days. Again, the dosing regimens are referred to as ‘low dose’ and ‘high dose’ herein. Body weights and clinical signs were assessed through 14 dpi. One group of mice mock-challenged with PBS were given the high dose BDGR-164 regimen as a drug control.

Sham-treated mice infected with EEEV or WEEV met clinical criteria for euthanasia by 4 dpi (Fig. 3A). Eighty-eight percent (7/8) of the EEEV mice treated with the high dose of BDGR-164 survived through 14 dpi while the mice treated at the low dose survived through 7 dpi and only 12.5% of these mice survived to 14 dpi. The brain viral titer in EEEV-infected mice was reduced at the low dose (P = 0.0002) and the high dose (P < 0.0001) by BDGR-164 treatment (Fig. 3B). Sixty-three percent (5/8) of the WEEV mice treated with the high dose of BDGR-164 survived through 14 dpi while 12.5% (1/8) of these mice treated at the low dose survived though 14 dpi (Fig. 3C). Animals challenged with WEEV displayed significant reductions in their brain viral loads at the high dose (P < 0.0001) of BDGR-164 treatment, but there was no statistical difference (P = 0.0559) between the sham and low dose treatment groups (Fig. 3D).

Figure 3. Survival and viral titer (brain) from prophylactic BDGR-164 treatment in BALB/c mice infected with EEEV FL-93-939 or WEEV-Fleming.

Female BALB/c mice at 5 to 6 weeks of age were treated by subcutaneous administration of 8 (low dose) or 48 (high dose) mg kg⁻¹ of BDGR-164 at two hours before intranasal challenge with lethal doses of EEEV FL93–939 (7500 PFU) or WEEV Fleming (5000 PFU). A second dose of 8 or 48 mg kg⁻¹ BDGR-164 was given 10 hours after infection. Treatment with one-half the dose (4 or 24 mg kg⁻¹) was continued twice daily at 12-hour intervals for an additional five days. (A, C) Survival curves of mice (n=8 per group) intranasally infected with PBS or virus and treated with sham, low or high dose of ±BDGR-164. (B, D) Viral titers from homogenized whole brains of mice (n = 4 per group). *** = P < 0.001, **** = P < 0.0001.

3.4. BDGR-164 effectively mitigates inflammatory responses to infection by VEEV, EEEV or WEEV

To further evaluate the efficacy of BDGR-164, we measured the host innate immune responses by RNA-Seq within whole brain at 4 dpi for each virus and compared the high dose treatment regimen (96 mg kg⁻¹ day⁻¹ on the first day of infection followed by 48 mg kg⁻¹ day⁻¹ for 3 days) to the sham-treated group. Mice were not treated on the day of euthanasia, i.e., 4 dpi. The Venn diagram of the RNA-Seq data revealed that 996 genes were differentially expressed and shared among the three viruses (Fig. 4A). WEEV-infected mice showed the greatest number of uniquely differentially expressed genes with 744. A total of 503 differentially expressed genes were shared between EEEV- and WEEV-infected mice, while only 75 were shared between VEEV and WEEV infected mice. In the BDGR-164 treated, virus-infected mice, low numbers of genes were differentially expressed as compared to each of the untreated, virus only groups. Additionally, more differentially expressed genes were unique to each virus infection than were shared amongst the viruses with only six differentially expressed genes being shared among mice infected with either of the three viruses. In both the treated and untreated animals, WEEV-infected mice had the greatest number of differentially expressed genes.

Figure 4. Venn diagram of differentially expressed genes and heat map of activated canonical pathways in the brains of mice infected with VEEV TrD, EEEV FL93–939, or WEEV Fleming with BDGR-164-treatment or with no treatment.

Five- to six-week-old, female BALB/c mice (n = 4 per group) were treated by subcutaneous administration with sham (PBS) or with the high dose treatment of BDGR-164 and intranasally challenged with VEEV TrD, EEEV FL93–939, or WEEV Fleming. At 4 dpi brains were harvested and processed for RNA-Seq as described in Material and Methods. Differential gene expression was calculated using DESeq2 comparing virus-infected mice to mock-inoculated mice for both treatment groups. (A) Differentially expressed genes (Log₂-fold change ≤ −1.5 and ≥ 1.5; false discovery rate ≤ 0.05) were used to create Venn diagrams depicting unique and shared genes following infection by each virus for both treatment groups. (B) Activated pathways within the brains of infected mice were determined based on differentially expressed genes (Log₂-fold change ≤ −1.5 and ≥ 1.5; false discovery rate ≤ 0.05) using Ingenuity Pathway Analyses. The color bar indicates the activation score for each pathway.

We down-selected the most significant, activated canonical pathways in the brains of mice infected with VEEV TrD, EEEV FL93–939, or WEEV Fleming and compared those to the three groups that were treated with BDGR-164 (Fig. 4B). As illustrated in the heatmap, most of the activated pathways in the untreated, virus-infected groups were associated with proinflammatory immune responses of the brain, neurons, and host immune cells. In addition, several pathways were associated with cell death. The majority of these pathways were not upregulated in the virus-infected-BDGR-164 treatment groups (Fig. 4B). In contrast to VEEV and EEEV, the activation scores for pathways observed after WEEV-infection were higher in the BDGR-164 treated group as compared to the untreated, WEEV alone group. This agrees with the survival data (67%) that suggested that the treatment regimen was not yet optimal for WEEV (Fig. 3C). Also, given the greater levels of WEEV in the brains of mice (Fig. 3D), we would anticipate a greater host response (Fig. 4). However, the EEEV-infected mice had similarly high virus in the whole brain at this timepoint suggesting key differences of host responses between EEEV and WEEV in the brain. Review of pathways using Qiagen’s Ingenuity Pathway Analysis (IPA) software show that cell death and neuroinflammatory signaling pathways were much higher for WEEV alone suggesting that its pathogenesis may be more aggressive than EEEV.

In figure 5, we present the fold-change in expression levels of a few of the key genes from activated, inflammatory pathways in the brains of mice. The proinflammatory genes IFNB1, TNF, CCL5, and IL6 were similar across all virus-infected groups. Additionally, AIF1 and GFAP, which indicates activation of astrocytes and microglia, were observed in all virus-infected, untreated groups as compared to virus groups with treatment. IL10 levels were only significantly upregulated for WEEV-infected mice, again highlighting a distinction of this model compared to VEEV and EEEV. TGFB1 expression was at baseline for all groups. In summary, the virus-infected BDGR-164 high dose regimen group showed significantly reduced changes of IFNB1, TNF, CCL5, IL6, IL10, TGFB1, AIF, and GFAP in the brain, in support of its potent antiviral efficacy, i.e., demonstrating that the reduction in viral load is accompanied by a reduction in the host inflammatory response.

Figure 5. Fold change in expression of selected genes from activated canonical pathways in the brains of mice infected with VEEV TrD, EEEV FL93–939, or WEEV Fleming with or without BDGR-164-treatment.

Five- to six-week-old, female BALB/c mice (n = 4 per group) were treated by subcutaneous administration with PBS or with high treatment regimen of BDGR-164 prophylactically and challenge with VEEV TrD, EEEV FL93–939, or WEEV Fleming. At four dpi brains were harvested and processed for RNA-Seq as described in Material and Methods. Differential gene expression was calculated using DESeq2 comparing virus-infected mice to mock-infected mice. The dotted line indicates the Log₂-fold change value of 1.5. All Log₂-fold change values above 1.5 have an FDR-value ≤ 0.05 unless indicated by ns (not significant).

3.5. Levels of selected proinflammatory proteins in sera

On 4 dpi, blood was drawn from all groups of mice infected with VEEV, EEEV or WEEV with or without BDGR-164 treatment (Fig. 2 and 3) to evaluate serum levels of IL-6, TNF-α, RANTES, and IFN-β (Fig. 6). A comparison of virus versus the mock (PBS) infected showed that IL-6, TNFα, RANTES, and IFN-β, were elevated in all virus-infected groups of mice. Levels of IFN-γ between virus-infected mice for VEEV, EEEV or WEEV with or without BDGR-164 treatment were similar (data not shown). IL-6, TNF-α, RANTES, and IFN-β, were reduced to background levels with the high dose treatment regimen for all virus-infected groups. As anticipated from the survival data, the inflammatory responses of the virus-infected, low dose BDGR-164 treatment animals were variable, suggesting dosing was suboptimal. In summary, the results suggest that treatment at the higher dosing regimen mitigates inflammation and that IL-6, TNF-α, RANTES (CCL-5), and IFN-β represent potential biomarkers for evaluation of treatment.

Figure 6. Levels of serum cytokines in mice at four days post VEEV TrD, EEEV FL93–939, or WEEV Fleming infection with sham, low or high dose BDGR-164 treatment.

Blood from the groups of BALB/c mice presented in Figures 1 and 2 were collected at 4 dpi. Sera were evaluated by multiplexing for levels of (A) TNF-α, (B) RANTES and (C) IL-6, (D) IFN-β. Error bars indicate mean±SD. Cytokine levels were compared by Two-Way ANOVA and Šídák multiple comparison. Statistical values are indicated as follows: *P<0.05, **P<0.005, ***P<0.005, ****P<0.0005.

3.6. BDGR-164 treatment of alphavirus infection in mice effectively mitigates upregulation of cell death pathways

We further investigated the expression of lytic cell death genes based on upregulation of both necroptosis, pyroptosis and PANoptosis pathways (Fig. 4). PANoptosis incorporates genes associated with apoptosis, necroptosis and pyroptosis (reviewed in (Karki and Kanneganti, 2023; Pandeya and Kanneganti, 2024)). Therefore, we included the PANoptosis pathway gene set to select these specific genes for assessment (Mall et al., 2022). Evaluation of the gene expression profile from each virus group without treatment revealed some subtle differences in the fold change of genes based on the virus species (Fig. 7), however the three viruses shared similar patterns of upregulation of key genes such as ZBP1, MLKL, NLRP3, MEFV, and IL1B. Virus-infected, sham-treated groups showed upregulation of BCL2A1A which can act as an inhibitor of apoptosis and may explain the absence of pro-apoptosis genes BAK1 and BAX. A comparison of the virus-infected-high dose regimen to the virus-infected, sham-treated group on 4 dpi, showed that cell death was significantly abrogated at the transcriptional level for each virus.

Figure 7. Expression of selected genes from PCD pathways in the brains of mice infected with VEEV TrD, EEEV FL93–939, or WEEV Fleming with sham or a high dose of BDGR-164.

At 4 dpi, whole brains (including the olfactory bulb) from five- to six-week-old, female BALB/c mice (n = 4 per group) were harvested and processed for RNA-Seq as described in Material and Methods. The key signatures of each of the major PCD pathways are represented in panels A-D. Differential gene expression was calculated using DESeq2 comparing virus-infected mice to mock-infected mice. Each of the graphs depicts the Log₂-fold change values of genes and their corresponding FDR. The size of the circle indicates the Log₂-fold change and the color bar the FDR of the gene. All genes that have a Log₂-fold change ≥ 1.5 have an FDR-value ≤ 0.05 unless indicated by grey dots.

3.7. Viral genome levels and evaluation of viral genomes for nonsynonymous mutations

The RNA-Seq dataset from brain tissue on 4 dpi was further scrutinized for relative levels of viral RNA, depth of coverage and nonsynonymous mutations (Fig. 8). The apparent viral RNA levels in the brains of mice were measured as transcripts per million (TPM) for mice infected with VEEV TrD, EEEV FL93–939, or WEEV Fleming with the sham treatment or the high dose regimen of BDGR-164. The reduction in viral genome level was highest for VEEV (Fig. 8A, ten-thousandfold reduction), followed by EEEV (Fig. 8B, one thousand-fold reduction), and WEEV (Fig. 8C, one hundred-fold reduction). The depth of coverage of each of the genomes for each virus within each brain tissue is presented in the illustrations in Fig. 8D-F. There was one mouse infected with VEEV TrD that had higher TPM values and level of depth of coverage. We assessed all the viral sequences for single nucleotide polymorphisms and potential nonsynonymous mutations and found that the VEEV TrD genome from this particular mouse brain was the only sample that had a nonsynonymous mutation in the nsP2 gene, Y102C, at a penetrance of 99.2%.

Figure 8. Viral load in the brains after prophylactic BDGR-164 treatment in BALB/c mice infected with VEEV-TrD, EEEV-FL82–939, or WEEV-Fleming.

We evaluated the viral RNA levels at 4 dpi for each of the groups of female BALB/c mice (Figures 2, 3) using the RNA-Seq data set. The apparent viral RNA levels in the brains of mice were measured as transcripts per million for mice infected with (A) VEEV TrD, (B) EEEV FL93–939, or (C) WEEV Fleming and prophylactically treated with no BDGR-164 (red diamonds) or high dose regimen (orange circles) of BDGR-164. RNA-Seq results were used to determine the depth of coverage in the brains of mice infected with (D) VEEV TrD, (E) EEEV FL93–939, or (F) WEEV Fleming and prophylactically treated with sham (red lines) or the high dose regimen (orange lines) of BDGR-164. Symbols: Φ - one individual mouse with a nonsynonymous mutation indicating resistance. Evaluation of the genome revealed a Y102C mutation in the nsP2 protein. Viral genome TPMs were compared by Unpaired T-test. Statistical values are indicated as follows; ***P<0.005, ****P<0.0005.

3.8. Spatial distribution of viral antigen and microglial response in brains of treated and untreated, VEEV TC-83-infected C3H/HeN mice

We employed the lethal model of intranasal infection with VEEV TC-83 in C3H/HeN mice (Williams et al., 2023) to evaluate the distribution of viral glycoprotein antigen in the brain (Fig. 9) and glial activation (Fig. 10) at 6 dpi with and without BDGR-164 treatment. Because VEEV TC-83 is a BSL-2 virus, this facilitated easier assessment of tissues than with VEEV TrD, which is a select agent. VEEV TC-83-infected mice met euthanasia criteria at 7–9 dpi (Williams et al., 2023). Three groups (n=4) were evaluated; (1) mock-inoculated, sham-treated, (2) virus-infected, sham-treated and (3) virus-infected, high dose BDGR-164. Sham or BDGR-164 were administered subcutaneously twice per day for 6 days prior to euthanasia.

Fig 9. Representative immunohistochemistry images of VEEV glycoprotein distribution within mouse brains infected with mock or VEEV TC-83 and treated with sham or BDGR 164 at dpi 6.

(A) Mock-infected brain (treated with Vehicle) in low magnification; M-S: moto somatosensory cortex; Pir: piriform cortex. (B) Enlarged images from moto-somatosensory area of cerebral cortex. (C) Piriform-olfactory-tubercle area of the cerebral cortex. (D) VEEV TC-83-infected brain section at dpi 6. (E) Enlarged high magnification images from D. (F) High magnification images from piriform area. (G) Brain sections with low magnification of TC-83-infected and BDGR-164-treated mice. (H) Enlarged images from moto-somatosensory area of cerebral cortex. (I) Piriform-olfactory-tubercle area of the cerebral cortex. VEEV-TC 83 infected mice at 6 dpi showed intense VEE GP signal in the cerebral cortex (CTX), corpus callosum(cc), medial striatum (CPu) and anterior commissure (aco) (in panel D). Higher magnification of somatosensory area showed virus glycoprotein in soma of pyramidal neurons in layer 5 and in the processes in layers 1,2/3,4,5 and 6. The morphology of neurons was preserved (E). Figure F showed VEEV GP was in all 3 layers of piriform cortex and there was more cell debris presenting more neuronal damage in this area (F). Panels A, B, C and G, H, I, showed VEE GP signal was not detected in sham-inoculated, BDGR-164-treated mice. Scale bar: A, D, and G have the same magnification=1mm; B,C,E,F,H, and I have the same magnification=100 micrometer.

Fig 10. Inflammation at dpi 6 in the piriform cortex of TC83-infected and BDGR-164-treated mice.

To assess the presence of glial cells, astrocytes, and microglia, we probed sagittal sections of the mouse brain by staining for GFAP (A,C,E) and Iba-1 (B,D,F), respectively. Representative images of the piriform cortex are shown that were taken from mock-inoculated, mock treated mice (A, B), TC83-infected mice (C,D), and TC-83-infected mice treated with BDGR-164 (E,F) on 6 dpi. (A) Image shows normal astrocytes distribution via GFAP expression in a mock-inoculated, sham-treated mouse specimen. (B) Image shows normal microglial/Iba-1 expression in piriform cortex with ramified microglial morphology with thin/fine processes, distributed evenly and normal blood vessels (BV) and pia mater (P). (C) TC83-infected mouse section showed increased GFAP expression in layer 1 (1) and layer 2 (2) and increased cellular components around blood vessels (BV) and pia mater (P). (D) VEEV TC83-infected mouse section showed activated microglia with thick and shorter processes, amoeboid nucleus counter stain showed prominent cellular damages with nucleus pyknosis and karyorrhexis and increased cellular components in gray mater, perivascular area (BV) and pia mater (P). (E, F) Image of TC83-infected mouse treated with BDGR-164, which showed no visible difference presence of GFAP or Iba-1 as compared to sham, mock-inoculated mice. Scale bar: all images with the identical magnification= 50 micrometer.

To evaluate viral antigen distribution in the brain, coronal sections were stained for glycoprotein (Fig. 9A, D,G). Two sections of the brain are highlighted, the frontal cortex (Fig. 9B, E, H) and the piriform cortex (Fig. 9C, F, I). No glycoprotein staining was observed in the mock-inoculated, sham-treated (Fig. 9B, C) or the virus-infected, high dose of BDGR-164 (Fig. 9H, I) groups. Staining was prominent in the neurons in the virus-infected, sham-treated group (Fig. 9E, F).

Given the elevated levels of glial markers in the RNA-Seq datasets (Fig. 4), we wished to qualitatively explore the magnitude of activation in the brain of the astrocytes and the microglial cell populations using immunohistochemistry. Sagittal sections were made of brains from the three cohorts and probed for GFAP (Fig. 10A, C, E) or Iba-1 (Fig. 10B, D, F). Minimal GFAP staining was observed in the mock-inoculated, sham-treated (Fig. 10A) or the virus-infected, high dose of BDGR-164 (Fig. 10E) groups. Staining was prominent in the astrocytes in the virus infected, sham-treated group (Fig. 10C). Similarly, the level of Iba-1 staining, and hence microglial cell activation, was high in the in the virus infected, sham-treated group (Fig. 10D) as compared to the mock-inoculated, sham-treated (Fig. 10B) or the virus infected, BDGR-164-treated (Fig. 10F) groups.

4.0. Discussion

The identification of broad-spectrum, direct acting antivirals has been a major challenge in drug discovery research and development for most viruses causing acute infections. To face these challenges, scientists increasingly rely upon trans-disciplinary efforts in the development of new tools and approaches and academic-industrial partnerships. Despite these efforts, the number of FDA licensed antiviral drugs stands at less than 100 with most targeting only a handful of viruses that cause persistent infections (i.e., human immunodeficiency, hepatitis B, C, and herpes simplex viruses) and only a couple of viruses responsible for acute infections (i.e., influenza A virus, SARS-CoV-2). The lack of drug pipelines for most acute viral infections has only recently drawn global attention from foundations, government, and the biopharmaceutical sectors during the COVID pandemic (Jonsson et al., 2021). To meet this challenge for the neurotrophic alphaviruses discussed herein, we began with a cell-based, high throughput screening program that resulted in a promising hit against VEEV (Chung et al., 2014). Through a campaign of iterative SAR and testing in vitro and in vivo in mice, we have continued to advance two chemical series with the goal of reaching a lead with broad spectrum potency to other alphaviruses (Cao et al., 2023; Jonsson et al., 2019; Ryan et al., 2022; Schroeder et al., 2014). Herein we report the broad-spectrum activity, potency, and efficacy of BDGR-164 against intranasal challenge of VEEV, EEEV or WEEV in a lethal BALB/c model.

In our prior antiviral discovery efforts for the neurotropic alphaviruses, we have reported on the in vitro and in vivo efficacy of benzamidine ML366 (Chung et al., 2010; Jonsson et al., 2019; Lee et al., 2020; Ogorek and Golden, 2023; Schroeder et al., 2014; Skidmore et al., 2020), benzamidine BDGR-4 (Jonsson et al., 2019), and quinazolinone BDGR-49 (Cao et al., 2023). The benzamidine antivirals, ML-336 and BDGR-4, show nanomolar potency in vitro and 100% prophylactic protection at 12.5 mg kg⁻¹ per day given intraperitoneally, twice daily for 8 days in mice challenged subcutaneously with VEEV TrD (Jonsson et al., 2019). In our assessment of therapeutic administration at 24 and 48 hours post-subcutaneously (hpi) with VEEV TrD showed 100% and 90% protection. In addition, we demonstrated 90% prophylactic protection for BDGR-4 in mice against EEEV (Jonsson et al., 2019). Lastly, we reported on the efficacy of BDGR-49, which differs structurally from the ML336 and BDGR-4 as it is a 2-pyrrolidinoquinazolinone scaffold. Prophylactically or therapeutically (48 hpi) administered BDGR-49 at 25 mg kg⁻¹ per day fully protected against subcutaneous administered VEEV TrD in BALB/c mice (Cao et al., 2023). Herein, we focused on protection of BALB/c mice treated with BDGR-164 at 48 mg kg⁻¹ per day against intranasal challenge VEEV, EEEV and WEEV. In contrast to mouse models of lethal subcutaneous infection with VEEV, EEEV, and WEEV, the progression of neurological disease in an intranasal challenge occurs rapidly resulting in a shorter time period to humane euthanasia in mice (Cao et al., 2023; Honnold et al., 2015b; Williams et al., 2025).

To our knowledge, BDGR-164 is the first reported broad-spectrum antiviral with prophylactic efficacy against VEEV, EEEV and WEEV. While many small molecules have been tested in vitro, the follow-on studies in vivo are limited (Ogorek and Golden, 2023), and those that have been tested show limited efficacy in lethal mouse models. One study tested the importance of RNA-induced silencing complex to support VEEV, EEEV and WEEV replication by assessment of the Ago2 inhibitor, acriflavine (ACF). While ACF decreased replication of these viruses in vitro, in vivo studies had mixed results. In a 16-day survival study involving intranasally infected VEEV TC-83 C3H/HeN mice, treatment with 10 mg kg⁻¹ ACF, resulted in a 60% survival rate compared to a 10% survival rate observed in sham-treated mice, while antiviral efficacy was not observed in BALB/c mice infected with VEEV TrD and treated with 5 mg kg⁻¹ ACF (Madsen et al., 2014). In a second study, favipiravir (Furuta et al., 2013), a pyrazinecarboxamide derivative, exhibited an EC₉₀ against WEEV of 312 μM; however, there was no efficacy in vivo in mice (Julander et al., 2009). Mice treated with 200 mg kg⁻¹ BID of favipiravir had a survival rate of 40% compared to the 20% survival observed in placebo-treated mice and treatment reduced WEEV titer by 1-log in mouse brains harvested at 4 dpi (Julander et al., 2009). A third and more promising study with EIDD-1931, aka β-D-N4-hydroxycytidine 5′-triphosphate, showed potent antiviral activity in mice intranasally challenged with VEEV TrD (Painter et al., 2019). Treatment with 1,000 mg kg⁻¹ day⁻¹ of EIDD-1931 from 6 to 12 hpi produced 100% survival (Painter et al., 2019). However, initiating treatment at 24- and 48-hpi decreased survival rates to 90% and 40%, respectively. Plaque assay of the brain of the virus-infected, EIDD-1931-treated mice euthanized at the end of the study (14 dpi) showed no detectable virus for treatments starting at 6 or 12 hpi (Painter et al., 2019).

To confirm the effectiveness of BDGR-164 on reduction of viral-induced proinflammatory responses, we investigated host gene expression in the brain using RNA-Seq for each of the three lethal, encephalitic alphaviruses. Our prior studies show that BDGR-49 when administered by itself in mice does not induce or reduce inflammation (Cao et al., 2023). Neurotropic alphavirus infection of mice significantly increases the levels of proinflammatory cytokines and chemokines and immune cells in the sera and brain (Grieder et al., 1997; Hollidge et al., 2021; Honnold et al., 2015a). Gene expression analysis of brains from CD-1 mice infected with VEEV V3000 (recombinant strain of VEEV TrD) reveals peak gene modulation on 4 dpi and so we chose this day to evaluate transcript levels (Gupta et al., 2017; Sharma et al., 2008; Sharma and Maheshwari, 2009). In prior published studies, the genes upregulated from viral infection include IFNB1, IFNA CCL2, CCL5 IL6, IL12B, CXCL10, and CXCL11 which resulted in the upregulation of TLR and JAK/STAT signaling as well as activation of inflammatory pathways associated with macrophages, DCs, NK cells, and T cells (Gupta et al., 2017; Sharma et al., 2008; Sharma and Maheshwari, 2009). In agreement with these studies, we observed similar activation of proinflammatory pathways such as neuroinflammation, interferon signaling, and NK cells in virus-infected-untreated BALB/c mice. BDGR-164 treatment of virus-infected mice resulted in a dramatic reduction of these pathways in EEEV-infected and BDGR-164-treated mice. In contrast, some pathways remained upregulated for VEEV/WEEV-infected and in BDGR-164-treated animals with WEEV-infected BDGR-164-treated mice having more (Fig. 4). The differences noted in pathway activation of VEEV/WEEV as compared to EEEV may be attributed to the restricted cell tropism of EEEV for myeloid cells and subsequent viral load, which correlates with greater neurovirulence as compared to VEEV (Gardner et al., 2008). TNF-α, CCL-2, and CCL-5 in the brains of VEEV TrD-infected BALB/c correlates with disease severity, even more so than viral load, but this is not yet known for EEEV or WEEV (Phelps et al., 2023). Similar elevation of proinflammatory cytokines (IFN-γ, RANTES (CCL-5), MIP-1β (CCL-4), MIG (CXCL9), and G-CSF) occur in sera of BALB/c mice intranasally infected with EEEV FL93–939 (Honnold et al., 2015a). Here, we observed elevated levels of TNF-α and RANTES in sera from each of the virus-infected, sham treated BALB/c mice (Fig. 6). Notably, BDGR-164 treatment of virus-infected BALB/c mice significantly reduced TNF-α and RANTES to that of the mock-inoculated control mice. Importantly, our studies of cytokines in sera from VEEV-, EEEV- or WEEV-infected mice suggest that IL-6, TNF-α, and RANTES levels correlated with protection and may serve as useful early biomarkers in treatment studies in neurotropic alphavirus animal models.

To further confirm the effectiveness of BDGR-164 on reduction of viral-induced disease, we investigated PCD markers in the RNA-Seq datasets in the brain for each of the three lethal, encephalitic alphaviruses in BDGR-164-treated and untreated mice (Fig. 7). In the brains from VEEV, EEEV, and WEEV-infected mice, we noted greater upregulation of lytic cell death genes than nonlytic cell death genes (Lu et al., 2022; Seo et al., 2021; Singh et al., 2019). Although we did not observe upregulation of apoptosis-associated transcripts in our models, it has previously been reported that EEEV and VEEV infection leads to apoptosis of cells in the brain tissues from CD-1 mice (Bocan et al., 2019; Gupta et al., 2017; Jackson and Rossiter, 1997; Schoneboom et al., 2000; Sharma et al., 2008; Sharma and Maheshwari, 2009). In two of these studies, VEEV driven apoptosis of cells in the CNS was measured by TUNEL staining, which is unable to distinguish among the apoptosis, necroptosis, and PANoptosis pathways (Charriaut-Marlangue and Ben-Ari, 1995; Grasl-Kraupp et al., 1995). Sharma et al. reported the upregulation of over 40 genes in VEEV-infected mice brains that are associated with apoptosis with the number of genes increasing each day up until 4 dpi (Sharma et al., 2008). The PCD genes reported in this current study for the VEEV/EEEV/WEEV-infected-untreated BALB/c mice were similar to those we reported previously in C3H/HeN mice intranasally infected by VEEV TC-83; in which we observed a strong induction of necroptotic and pyroptotic genes but not apoptotic genes (Williams et al., 2023). In addition to the lack of proapoptotic genes such as BAK1 and BAX, we also noted the presence of BCL2A1A which inhibits apoptosis (Hamasaki et al., 1998). Evaluation of the key genes that are involved in necroptosis, pyroptosis and PANoptosis by RNA-Seq did not give any clear insight into which of these pathways were dominant, but did suggest potential pathways (Fig. 7). The lack of RIPK1 but high expression levels of MLKL and RIPK3 suggest that necroptosis occurs through a RIPK1-independent manner (Karki et al., 2022; Kuriakose et al., 2016; Lin et al., 2016; Newton et al., 2016); however, the RIPK1 may still be expressed at the protein level. Secondly, pyroptosis was suggested in VEEV/EEEV/WEEV-infected-untreated mice with the upregulation of GSDMD and IL1B, but confirmation of cleaved CASP-3 protein remains (He et al., 2015). GSDMD functions by forming pores in cells releasing inflammatory proteins (Aglietti et al., 2016; Ding et al., 2016; Liu et al., 2016; Sborgi et al., 2016). Alternatively, the lack of CASP3 upregulation suggests that pyroptosis occurred through a CASP3-independent manner as CASP3 inhibits GSDMD activity, but this awaits further confirmation (Taabazuing et al., 2017). Lastly, the evaluation of the PANoptosis signatures suggests a ZBP1-PANoptosome, but this too requires further evaluation at the protein level in future studies (Karki and Kanneganti, 2023).

The discovery of the prophylactic, broad-spectrum antiviral, BDGR-164, for the neurotrophic alphaviruses offers promise to mitigate the devastating acute and chronic disease caused by these agents; however, we recognize that additional research is required to fully evaluate the potential of BDGR-164. The additional questions that need to be addressed to advance this compound into preclinical studies include elucidation of the PK-PD profiles to optimize treatment regimens following infection. As these studies require inactivation of select agent material, one must generate processes that inactivate the virus and RNA but leave enough compound intact for measurements using LCMS/MS methods. While this may be feasible, our future efforts will avoid the destruction of samples in favor of running the LCMS/MS within BSL-3, select agent-registered space to assess the PK-PD profile. Once the optimal dosing regimen is identified from the PK-PD studies, we are also interested in furthering the temporal assessment of the viral load and host response with and without treatment in mice, using RNA-Seq. Lastly, it is important to further assess and understand the pathogenesis of these viruses in animal models to appreciate how this type of antiviral can treat systemic acute infection and abrogate acute and chronic CNS disease.

Highlights.

• The New World alphaviruses, eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and western equine encephalitis virus (WEEV), cause febrile illness that can progress to fatal disease in humans and equids.

• The structurally novel, quinazolinone, BDGR-164, conferred 100% (VEEV), 88% (EEEV), and 63% survival (WEEV) in a lethal BALB/c mouse model.

• BDGR-164 reduced viral RNA levels and antigen in the brains of infected mice.

• BDGR-164-treated mice showed reduced levels of genes associated with infection and pathogenesis.

• IL-6, TNF-α, and RANTES levels correlated with protection conferred by BDGR-164.