Post-exposure intranasal IFNα suppresses replication and neuroinvasion of Venezuelan Equine Encephalitis virus within olfactory sensory neurons

C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed under pathogen-free conditions in Washington University School of Medicine animal facilities. All experiments were performed in compliance with Washington University animal studies guidelines.

8–10-week-old male mice were inoculated intranasally (10 µL per nostril) with VEEV strain ZPC-738 or ZPC-738-eGFP (10 or 50 pfu, respectively) under anesthesia. ZPC-738-eGFP was a generous gift of William Klimstra (Pittsburg, PA). ZPC-738-eGFP was generated by subgenomic insertion of GFP as a cleavable element between the capsid and PE2 structural proteins, described previously [17]. Parental ZPC-738 or ZPC-738-eGFP are uniformly lethal at 10 pfu. GFP genomic VEEV modification exhibited slight attenuation, delayed expansion/neuroinvasion during early infection (unpublished data), and extended MST [17]. To account for this, ZPC-738-eGFP studies were performed at 50 pfu. Mice were monitored daily for weight loss and scored daily for encephalitic sequelae. Moribund mice were sacrificed by CO₂ asphyxiation and recorded as dead the following day. Encephalitic score represents a progressive range of behaviors: (1) hunched, ruffled fur, (2) altered gait, slow movement, (3) not moving but responsive, (4) not moving, poorly responsive but upright, (5) moribund, (6) dead.

At various times post-infection, mice were anesthetized followed by extensive cardiac perfusion with PBS and perfusion fixation with 4% paraformaldehyde (PFA) in PBS. Tissue was immersion-fixed for an additional 24 h in 4% PFA. For slice preparations of mouse nasal cavities, skulls were decalcified by multiple exchanges 0.5 M EDTA (pH 7.4) in PBS over 7 days followed by PBS and cryoprotection (two-exchanges of 30% sucrose for at least 48 h) and embedding in OCT (Fisher). 10 μm-thick fixed-frozen sagittal sections were hydrated with PBS and blocked for 1 h in blocking solution, 5% normal donkey serum (Santa Cruz Biotechnology) with 0.1% Triton X-100 (Sigma-Aldrich). After block, slides were exposed to primary antibody at 4 °C overnight, washed with PBS and incubated with Alexa Fluor donkey secondary antibodies (Invitrogen) for 1 h at room temperature. Antibodies used: chicken anti-GFP (Abcam, 13970), goat anti-OMP (Wako Chemicals, 544-10001), rabbit anti-GAP43 (Novus Biologicals, NB300-143). Images were acquired using a Zeiss LSM 880 confocal laser scanning microscope and processed using Zen3.3 (Zeiss) and Image J. Quantification of immunofluorescence was performed using ImageJ.

In situ hybridization staining of decalcified sagittal skull section (described above) were performed using Advanced Cell Diagnostics (ACD) RNAscope system and probes. After rehydration of slides in PBS, slides were baked (30 min at 60 °C) and post-fixed in 4% PFA. Slides were dehydrated in progressive ethanol washes (50%, 70%, 100%, 100%, 5 min), air dried, treated with hydrogen peroxide (10 min). For in situ hybridization alone, Advanced Cell Diagnostics RNAscope 2.5 HD Detection Reagent—RED (322360) using standard manufactures protocol, RNAscope Target Retrieval Reagent (95–98 °C, 10 min), RNAscope Protease Plus (30 min), and standard hybridization with the Ldlrad3 probe (ACD,), signal amplification, and counter-staining with DAPI. For combined RNA–protein co-imaging, RNAscope Multiplex Fluorescent v2 Assay (323100) along with RNA–Protein Co-detection Ancillary Kit (323180) was used utilizing the Integrated Co-Detection Workflow (ICW). Following baking, post-fixation, dehydration, and hydrogen peroxide treatment, slides were immersed in Co-Detection Target Retrieval (95–98 °C, 5 min). Tissue was blocked and incubated overnight with GAP43 and OMP primary antibodies (see above) using Co-Detection Antibody Diluent. Samples were post-primary fixed using 10% neutral buffered formalin (30 min, RT) prior to RNAscope Protease Plus treatment, hybridization with the V-VEEV-ZPC-738 (ACD, 876381), Mm-Ldlrad3 (ACD, 872101), or dapB negative control probes (ACD, 310043), signal amplification with Opal 650 Dye (Akoya Biosciences, OP-001005) in RNAscope Multiplex TSA Buffer. Tissues were labeled with Alexa-conjugated secondary antibodies (see above) in Co-Detection Antibody Diluent, counter-stained with DAPI, and mounted in Prolong Gold (Invitrogen #P36930). Tissue were imaged as described above.

scRNAseq data set of intranasal IFNα mice was originally generated as described previously. Briefly, 8–10-week-old C57BL6/J mice received either 200 ng of IFNα (Biolegend 752802, ~ 1 × 10⁴ U) or saline intranasally (N = 2), Respiratory and olfactory mucosa were isolated 12 h later. Single cells suspension were generated in media containing Liberase (Roche) and DNase I (Roche) and loaded on duplicate Seq-Well S3 arrays for sequencing using Illumina NextSeq. Raw expression counts for cells previously defined within Immature Olfactory Sensory Neurons and Olfactory Sensory Neurons (saline and IFNα treated) clusters were downloaded from published data set. https://​singlecell.​broadinstitute.​org/​single_​cell/​study/​SCP832?​scpbr=​the-alexandria-project#study-summary. Data were normalized and scaled using the Seurat R package (https://​satijalab.​org/​seurat/​). Differential expression tests between mature and immature OSNs within saline-treated group or between saline-treated or IFNα-treated OSNs were performed using Seurat FindAllMarkers function with default settings and Wilcoxon rank sum test (P value threshold = 0.05). GSEA analysis was performed using fgsea function from (fgsea, using the murine Gene Ontology gene sets (MSigDB). Genes were ordered by the Log2 fold change using Seurat FindMarkers function. Violin plots and heatmaps were generated using Seurat R package. Volcano plots were generated using the EnhancedVolcano package.

Recombinant mouse IFNα1 (Biolegend, 751806) was administered intranasally. Control mice were similarly administered vehicle solution of 0.1% bovine serum album (BSA) in PBS. Doses (8 × 10⁴ U, 10 µL/nostril) administered at time of infection were suspended in the inoculum under brief isoflurane anesthesia. Subsequent doses (8 × 10⁴ U, 5 µL/nostril) were administered at 1–3 h post-infection (hpi), as indicated, under brief isoflurane anesthesia.

CNS and nasal cavity tissue was collected isolated from cardiac-perfused mice at various timepoints after intranasal ZPC-738 (10 pfu i.n.) infection and/or IFNα treatment. Total nasal cavity tissue, including the nasal turbinates, was collected using forceps following removal of the nasal bone along the nasomaxillary suture. RNA was isolated from tissues using RNeasy kit (Qiagen) according to manufacturer’s instructions, and quantified using a NanoDrop (Thermo Scientific). Following DNAse I treatment (Invitrogen) of RNA samples (1 µg) was reverse transcribed using Taqman Reverse Transcriptase kit (Applied Biosystems). qRT-PCR was performed using Power SYBR Green (Applied Biosystems) on a CFX384 PCR Detection System (Bio-Rad) using manufacturer’s recommended cycle parameters. Values are reported as the Cq values for target genes normalized to Cq values of GAPDH (Cq_gene–Cq_GAPDH). Primers (5′–3′) used are reported in Additional file 1: Table S1.

At various post-infection intervals, nasal cavity and CNS tissue was collected from ZPC-738-infected mice after extensive cardiac perfusion with PBS. Viral titers were determined using standard plaque assay techniques by serial dilution of tissue homogenates over BHK cells, as described previously [18].

Reported values are mean values ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 7 software. Survival curves were analyzed by Mantel–Cox test. Cytokine and ISG expression in infected mice were analyzed via one-way analysis of variance (ANOVA), Bonferroni’s post hoc test was subsequently used for comparison of individual means. ISG expression following IFNα treatment were analyzed by unpaired Welch’s t test with Welch’s correction, as appropriate for samples with different variances. Weight loss and encephalitic sequelae scores were compared via two-way repeated measure ANOVA, followed by Bonferroni’s post hoc test. P values P < 0.05 were considered significant. Statistical values are indicated as follows *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001 unless otherwise stated.

Intranasal (i.n.) infection with an enzootic strain of VEEV (ID, ZPC-738; herein VEEV) results in rapid progression of weight loss and onset of encephalitic symptoms, with a mean survival time (MST) of 6.5 dpi (Additional file 2: Fig. S1A). In previous studies we demonstrated i.n. VEEV infection rapidly disseminates into the OB and CTX within 24 h, with viral loads peaking at 2–3 dpi in the OB and at 4–6 dpi in CTX, hindbrain regions, and spinal cord, highlighting early infection to the OB as a critical period to control VEEV dissemination along the olfactory route after i.n. exposure [3]. To define cellular tropism within the ONE we utilized a reporter strain of VEEV, ZPC-738-GFP, which exhibits similar virulence as the parent strain and labels infected cells green, in conjunction with OSN markers, all detected via double-label confocal microscopy [17]. Within the infected ONE, GAP43+ iOSNs are the earliest site of infection at 1 dpi [Fig. 1A, B (top) white arrowhead]. OMP+ mOSN were also infected at this timepoint [Fig. 1A (open arrowheads)]. GFP is also detected within GAP43+ and OMP+ OSN axons traversing the cribriform plate and within the olfactory nerve layer (ONL) of the OB (Fig. 1B, bottom). VEEV–RNA, as assessed via fluorescent in situ hybridization, is also detected within GAP43+ and OMP+ axons within the ONE and OB ONL (Fig. 1C, Additional file 2: Fig. S1B). By 3 dpi, GFP+ cells are observed throughout the ONE and OB, including the glomerular, mitral cell, and ganglion cell layers (Fig. 1D, E). At this timepoint, VEEV infection continues to spread into the forebrain along the olfactory tract and piriform CTX (Fig. 1E). Together, these data indicate that VEEV may utilize both immature and mature OSNs for anterograde transport into the OB.

Despite the knowledge that peripheral neurons, including OSNs, are targets for many neurotropic viruses, there are few studies reporting their differential expression of viral entry receptors and innate immune responses [19‐21]. To determine whether the SARS-CoV-2 entry receptor angiotensin converting enzyme (ACE2) is an ISG within cells of the ONE, Ziegler et al. performed single-cell RNA sequencing of murine nasal epithelium derived from mice 12 h after i.n. administration of saline versus IFNα (10⁴ U) [22]. While they found little to no ACE2 expression in iOSN or mOSN (with our without IFNα exposure), they provided a large data set for investigation of the differential expression of other viral entry receptor mRNAs and overall innate immune response networks in iOSN and mOSN [23]. To define differences between transcriptomic signatures of iOSN and mOSN that would underlie the observed enhanced infectivity of VEEV to iOSN, we analyzed differentially expressed genes (DEG) between the two cell types under saline treatment. As expected, the top DEG included genes involved in olfactory sensory perception, cilium development, and ion channel/transport protein expression (Adcy3, Omp, Pde1c, Cngb1, Cnga4 (Additional file 3: Fig. S2A) [24, 25]. Similarly, gene set enrichment analysis (GSEA) using murine gene ontology (GO) pathways identified key differences in pathways associated with neuronal differentiation, axonal growth and synapse formation between iOSN and mOSN (Additional file 3: Fig. S2B). While mRNAs of genes relating to innate immunity or control of virus infection were not among the top DEG, some genes associated with GO pathways, including Innate Immune Response, Response to Virus, Response to Type 1 Interferon, Response to Interferon Alpha, and Response to Interferon Beta, were differentially expressed between iOSN and mOSN (Fig. 2A). For example, mRNA levels of Interferon Induced Protein with Tetratricopeptide Repeats 1 (Ifit1) was significantly higher in mOSN (Fig. 2B). However, none of these pathways were more significantly enriched by GSEA in either OSN population (Table 1), consistent with lack of differences in mRNA expression levels of IFNαβ receptor (Ifnar1) (Fig. 2B, Additional file 3: Fig. S2B). Together, broad differences in innate immunity do not explain the observed early VEEV tropism and infection of iOSN compared with mOSN. However, mRNA levels of a VEEV receptor, Ldlrad3 [4], are significantly higher in iOSN versus mOSN (Fig. 2B). While detection of Ldlrad3 mRNA via fluorescent in situ hybridization (FISH) was observed in both iOSN and mOSN (Fig. 2C), it is likely that overall difference in levels of expression of Ldlrad3 underlie earlier infection of iOSN.

Given that OSN exhibit low levels of expression of innate immune molecules at baseline, we analyzed the scRNAseq data set deposited by Ziegler et al. for DEG in iOSN and mOSN following intranasal IFNα (10⁴ U,12 h) treatment [22, 23]. Both OSN cell types exhibit similar DEG (Fig. 3A, B). As expected DEGs and GSEA indicate strong enrichment of ISGs relating to Type 1 interferon responses following treatment, which was broader for mOSN (Additional file 3: Fig. S2C, D). Overall, this indicates that these cells, critical to early ONE replication and neuroinvasion into the OB, are responsive to such treatment. To validate ISG expression in the ONE and OB in a separate cohort of mice, we examined candidate ISG expression in total nasal cavity (NC) and OB following similar i.n. administration of recombinant murine IFNα (8 × 10⁴ U) followed by quantitative (q)PCR. Robust upregulation of ISG, including Ifit1, Irf7, Ifitm3, Isg20, and cGas, was observed in both the nasal cavity and OB at 24 h post-treatment with IFNα compared with vehicle-treated animals (Fig. 3C). To determine if IFNα also altered expression of Ldlrad3, we quantified Ldlrad3 expression as assessed by FISH within the ONE following IFNα treatment at 12 hpi during VEEV infection (Fig. 3D, Additional file 2: Fig. S1C). While Ldlrad3 expression was enhanced by VEEV infection, IFNα treatment did not synergistically impact its level of expression. Overall, these data indicate that IFNα treatment elicits rapid IFN response in both the ONE and OB, suggesting a potential therapeutic approach for limiting infection and neuroinvasion along this route.

While endogenous IFN signaling is critical for controlling VEEV infection in the periphery [7], the extent to which it controls VEEV infection and dissemination along the olfactory route is unknown. Knowledge of the kinetics of this response is also important for determining if exogenous administration of IFNα would be expected to limit VEEV neuroinvasion. To address this, IFN mRNA expression within the nasal cavity and OB was assessed in uninfected animals and at various timepoints (12, 24, 48 h) post-infection (hpi). IFNα mRNA is not significantly upregulated in the nasal cavity or OB until 48 hpi (Fig. 4A), while IFNβ mRNA induction is observed at 12 and 24 hpi within the nasal cavity, with significant induction at 24 hpi in the OB (Fig. 4B). Separate analyses of ISG mRNAs linked to inhibition of alphavirus infection showed similarly delayed onset of expression in the nasal cavity and OB (Fig. 4C–H) [26‐31]. Only IRF7 was upregulated within 24 hpi (Fig. 4D), with all other candidates not exhibiting expression in both the NC and OB until 48 hpi (Fig. 4C–H). These data indicate potential windows of intervention with i.n. administered IFNα after i.n. exposure to VEEV.

To determine if i.n. administration of IFNα during early VEEV infection would improve outcomes following VEEV infection, IFNα treatment (8 × 10⁴ U) was administered concomitantly or at 1 and 3 h after i.n. VEEV infection (ZPC-738, 10 pfu) of wild-type mice. Pre-treatment (0 hpi) with IFNα delays morbidity, as assessed via encephalitic scoring and weight loss, compared with similarly infected vehicle-treated mice (Fig. 5A). Specifically, weight loss was significantly lower in IFNα-treated mice, and onset lagged approximately 2 days behind that of vehicle-treated VEEV-infected mice (Fig. 5A, left). While VEEV encephalitic signs were similar between vehicle- and IFNα-treated animals, scores were significantly lower and delayed by approximately 2 days in IFNα-treated mice (Fig. 5A, middle, Additional file 4: Fig. S3A). IFNα-treatment at the time of VEEV infection extended mean survival time (+ 2 dpi MST) but was ultimately insufficient to improve overall mortality (Fig. 5A, right). As knowledge of exposure to VEEV may be delayed, we determined whether post-infectious IFNα treatment at 1 or 3 hpi impacts disease and survival after i.n. infection with VEEV. Both treatment paradigms significantly delayed onset of encephalitic sequelae and weight loss compared with vehicle-treated VEEV-infected mice (Fig. 5B, left and middle, Additional file 4: Fig. S3B). However, weight was not as well-maintained as observed during concomitant IFNα and VEEV i.n. exposure compared with similarly infected vehicle-treated mice, especially when IFNα was administered at 3 hpi. Similar to concomitant treatment, IFNα treatment extended mean survival time (+ 2 dpi and + 1.6 dpi, respectively), without reducing overall mortality (Fig. 5B, right).

To determine if i.n. administration of IFNα limited VEEV replication within the ONE and/or CNS dissemination, viral titers were assessed in IFNα-treated animals. For mice administered IFNα at the time of VEEV infection (0 hpi), viral titers were assessed at 1, 3, and 5 dpi via standard plaque assays. In contrast with vehicle-treated animals, viral titers were undetectable in the brain (OB, CTX) or sera in IFNα-treated mice at 1 dpi (Fig. 5C). By 3 dpi, VEEV was detectable in the majority of CNS tissues derived from vehicle- and IFNα-treated mice (VEEV OB: 5/6; CTX: 4/6). However, overall VEEV titers in OBs and cortices derived from IFNα-treated mice were significantly reduced at this timepoint compared with similarly infected vehicle-treated animals (Fig. 5C). IFNα treatment, however, failed to control viremia beyond 1 dpi, as viral titers were equivalent in both treatment groups of VEEV-infected mice by 3 dpi. Similarly, VEEV viral titers in IFNα-treated mice reached equivalency to vehicle-treated mice by 5 dpi in all CNS regions (Fig. 5C). Direct observation of i.n. ZPC-738-GFP infection (50 pfu) in fixed whole skull mounts revealed strong suppression of infection in the ONE and OB at 3 dpi in IFNα-treated compared to vehicle-treated mice, which displayed robust ZPC-738-GFP throughout the ONE, OB, and olfactory tract (Fig. 5D). When infected cells were observed in IFNα-treated ONE, infected iOSNs were similarly observed earlier than mOSNs, suggesting that IFNα does not alter VEEV tropism beyond the period of suppression.

Endogenous IFNα and IFNβ expression correlated with the presence of VEEV during the time course of infection in the nasal cavity, OB, and cortices (Additional file 4: Fig. S3C, D). In IFNα-treated mice, endogenous IFNα and IFNβ expression was not as robustly induced until later in infection, correlating with the early suppression of VEEV in these mice. ISGs Ifit1 and IRF7, which rapidly responded to i.n. IFNα previously, were induced by IFNα treatment in the nasal cavity and OB at 1 dpi, despite reduced endogenous Type I interferon expression (Additional file 4: Fig. S3E, F). Expression was similar, but significantly reduced, compared to vehicle control tissues through 3 dpi, suggesting that the effect of IFNα treatment on ISG expression may have begun to wane to levels that allow for VEEV replication during this period.

Finally, similar to concurrent (0 hpi) administration, i.n. IFNα treatment after infection (1 hpi) suppressed early infection, as VEEV viral titers at 1 dpi within the nasal cavity (NC), OB, and sera were undetectable compared with vehicle-treated mice (Fig. 5E). By 3 dpi, infection in the nasal cavity and OB remained significantly reduced in VEEV-infected mice treated with IFNα compared with vehicle-treated animals but was detectable in a majority of animals (VEEV+ NC: 4/6, VEEV+ OB: 4/6, VEEV+ Sera: 5/6).