Without viral translocation to the brain I.N. IHNV changes adult behavior at 1 d.

(A) schematic of intranasal administration of aquatic rhabdovirus infectious hematopoietic necrosis virus (IHNV) or vehicle (VeH). (B) quantification of IHNV N-protein presence in the olfactory organ and olfactory bulb 15 m and 1 d after I.N. delivery of virus. N = 4-6, each replicate is a pool of 4 animals. (C) Average velocity of the animal over the test for 15 m (C) or 1 d (D) after IHNV or media control. Test was run for 5 m after IHNV or control odorant wa added to the flow cell. Statistics are Student’s T test. Error bars are SEM. Male (blue) and female (pink) animals do not show a difference in behavior.

Neuronal activation of the zebrafish OO and OB in response to nasal viral delivery.

(A) Median speed of larva in a lateral flow chamber exposed to IHNV for 4 seconds. Speed adjusted for the flow rate. (B) Turning angle of larval zebrafish in lateral flow cell. Test was run for 5 m after IHNV or control odorant was added to the flow cell. Statistics are Student’s T test. (C) Maximum intensity projection of olfactory system of a Tg(elavl3:GCaMP6S) larva imaged by 2-P microscopy. Odorants were applied to the olfactory pit randomly every 2 minutes and animals imaged for 6 h., OB = olfactory bulb. Indicated in blue are ROIs activated by to IHNV virus but do not respond to the other stimuli. (D) Stimulus triggered average changes in fluorescence (F/F0) following each stimulus (indicated by color) for cells that show showing neuronal activation specific to the IHNV (red) and not the positive (KCl, green) or negative (Media, blue) controls. Mean response across IHNV-selective cells is indicated in bold. (E) F/F0 traces of the highlighted ROI in C. Vertical lines color coding indicates the type of stimuli delivered at each interval.

sc-RNA seq of the olfactory bulb reveals changes to the cellular landscape after I.N. IHNV.

tSNE plots of single cells isolated from the olfactory bulb at 15 m (A) and 1 d (B). (C) tSNE plots showing Vehicle (VeH) (black), 15 m IHNV (yellow), and 1 d IHNV (red). (D) Bar plots of proportions of cells from each cluster for each treatment. Statistical proportional analysis for changes to the proportions following IHNV treatment is in supplemental figure X.

Transcriptional profiles of neurons reveal increased development at 1 d afte intranasal delivery of virus.

(A) Bar plots showing fold change expression of significantly differentially expressed genes in neurons 1 d after I.N. IHNV relative to control. Bars are colored according to the cluster in which they are differentially expressed. (B) tSNE highlighting only the neuronal clusters (C) violin plots of differentially expressed genes in neuronal clusters 1 d after intranasal IHNV delivery shows many genes related to plasticity and development of neurons are upregulated compared to vehicle treated animals. (C) gene ontology of all differentially expressed genes in neuronal clusters 1 d I.N. IHNV compared to 1 d I.N. vehicle treatment. Gene ontology was run on Metascape (Zhou et al., 2019).(E) Feature plots for genes enriched in the top GO:0007218 Neuropeptide signaling pathway (pacap, npb, penkb, scg5, and tac1) and pacap receptor pac1.

Neuropeptide PACAP-38 expression increases in the zebrafish OB 1 d after I.N. IHNV but not 1 d after I.P. IHNV treatment.

(A) Representative confocal image of the zebrafish OB stained with anti-PACAP-38 antibody after receiving vehicle by I.N. delivery. (B) Representative confocal image of the zebrafish OB stained with anti-PACAP-38 antibody after receiving IHNV by I.N. delivery. (C) Quantification of the mean fluorescent intensity values for A and B. (D) Representative confocal image of the zebrafish OB stained with anti-PACAP-38 antibody after receiving vehicle by I.P. delivery. (E) Representative confocal image of the zebrafish OB stained with anti-PACAP-38 antibody after receiving IHNV by I.P. delivery. (F) Quantification of the mean fluorescent intensity values for D and E. P-values were calculated by a student’s T test, with error bars indicating standard error of the mean.

Amidated PACAP-96% protects EPC cells from IHNV-induced cell death.

Relative percent viability of EPC cells that were either pretreated with amidated PACAP-38 prior to infection (pretreatment), treated with amidated PACAP-38 at the time of the infection (competition) or infected with IHNV and then treated with different concentrations of amidated PACAP-38 (postinfection). Results are representative of two independent experiments with N=4 for each treatment group. Different letters indicate significant differences among treatments by one-way ANOVA (P<0.05).

(A) Olfactory system of a Tg(elavl3:GCaMP6S) larva imaged by 2-P microscopy. Odorants were applied to the olfactory pit randomly every 2 minutes and animals imaged for 6 h., OB = olfactory bulb. Indicated in green are ROIs selective to KCl that do not respond to the other stimuli. (B) Stimulus triggered average changes in fluorescence (F/F0) following each stimulus for cells that show neuronal activation specific to the positive (KCl) control (green line) but not the IHNV (red line) virus or negative control (Media, blue line). Mean response across KCl-selective cells. (C) F/F0 traces of the highlighted ROI in A. Vertical lines color coding indicates the type of stimuli delivered at each interval.

Transcriptional landscape of the adult zebrafish olfactory bulb 15 m and 1 d after intranasal viral delivery.

(A) Metrics from each sample collected by 10x Genomics Chromium platform after sequencing on Illumina MiSeq. (B) Heat map of cluster markers for lymphocyte, microglial, and neuronal clusters from CCA integrated Vehicle and IHNV treated cells from the olfactory bulb 15 m after intranasal treatment. (C) Heat map of cluster markers for lymphocyte, microglial, and neuronal clusters from CCA integrated Vehicle and IHNV treated cells from the olfactory bulb 1 d after intranasal treatment.

Cellular landscape of the olfactory bulb 15 m and 1 d after intranasal delivery of vehicle (PBS) and virus.

(A&B) proportional analysis of cells in each cluster in each treatment for 15 m with statistical analysis in (C). (D&E) proportions analysis of cells in each cluster in each treatment for 15 m with statistical analysis in (F) When the P-value<0.05, the null hypothesis that population sizes are the same in both treatment groups is rejected.

Microglia and macrophage populations in the adult zebrafish OB are diverse.

(A) tSNE of the whole OB 15 m after I.N. IHNV to represent the microglial cell types that were subclustered in (B) and represented as either vehicle (VeH) control (blue) or IHNV treated (pink) (C). (D) Feature plots showing markers of microglia (marco, csf3r, mfap, apoeb) and ameboid like microglia (ccl34b.1) as well as macrophages (mpeg1.1, ccl19a.1, grn2). (E) Gene ontology of differentially regulated genes from microglial clusters 15 min after I.N. IHNV compared to vehicle. Gene ontology was run on Metascape (Zhou et al., 2019).

Diverse lymphocyte clusters are found in the adult zebrafish OB.

(A) Proportions of clusters from figure 6 of only control cells shows innate-like lymphocytes are 20% of cells captured from the control olfactory bulb and T lymphocytes2 are 15% of cell captured from the control olfactory bulb. (B) t-SNE of innate/T lymphocyte subsets from the olfactory bulb show 6 unique phenotypes. (C) Violin plots of markers of natural killer (ccl38.6, ccl34b.4, ccl38a.5, nitr), cytotoxic (gzm3.4, lyz, ifng1-2, tbx21), type II innate-like lymphocytes (il13, il4), type III innate like lymphocytes (il2, rorc) and Tregs (foxp3a, cd4). (D) Gene ontology of significantly differentially expressed genes from lymphocytes in the olfactory bulb 15 m after I.N. IHNV compared to 1d I.N. vehicle treated lymphocytes. (E) Gene ontology of significantly differentially expressed genes from lymphocytes in the olfactory bulb 1 d after I.N. IHNV compared to 1d I.N. vehicle treated lymphocytes. Gene ontology was run on Metascape (Zhou et al., 2019).

I.N. IHNV does not induce proliferation in the zebrafish olfactory organ or the subventricular zone of the telencephalon.

Proliferation detected using Edu (pink) in control (A) 3 d I.N. Poly(I:C) (C) or 1 d I.N. IHNV (E) in the olfactory organ (OO) quantified in (G). Proliferation detected using Edu in control (B) 3 d I.N. Poly(I:C) (D) or 1 d I.N. IHNV (F) in the subventricular zone (SVZ) quantified in (H). scale = 20mm. N= 3-5. Each color indicates a separate animal and each dot a field of view at 60x. Statistics are Student’s T test between control and each treatment and not significant for the IHNV treatment.

Isotype control staining of zebrafish OB cryosections for anti-PACAP-38 staining. Scale bar = 20um.

Alignment of PACAP-38 sequences from D. rerio (NP_001259010.1:96-133), Homo sapiens mature PACAP-38 peptide (NP_001093203.1:132-169), and C. gariepinus from (Semple et al., 2019). Percent identity is compared to D. rerio.

Cytotoxicity of PACAP in EPC cells. (Left, amidated PACAP-96%; right, PACAP-85%;). Different letters indicate statistically significant differences by one-way ANOVA. Results are indicative of two independent experiments.

Enrichment table of the top gene ontology terms and the gene differentially expressed in neurons of the OB at 1 d after I.N. IHNV.