key: cord-333703-1ku3jc9s
authors: Kraus, Aurora; Casadei, Elisa; Huertas, Mar; Ye, Chunyan; Bradfute, Steven; Boudinot, Pierre; Levraud, Jean-Pierre; Salinas, Irene
title: A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2
date: 2020-11-08
journal: bioRxiv
DOI: 10.1101/2020.11.06.368191
sha: 
doc_id: 333703
cord_uid: 1ku3jc9s

The COVID-19 pandemic has prompted the search for animal models that recapitulate the pathophysiology observed in humans infected with SARS-CoV-2 and allow rapid and high throughput testing of drugs and vaccines. Exposure of larvae to SARS-CoV-2 Spike (S) receptor binding domain (RBD) recombinant protein was sufficient to elevate larval heart rate and treatment with captopril, an ACE inhibitor, reverted this effect. Intranasal administration of SARS-CoV-2 S RBD in adult zebrafish recombinant protein caused severe olfactory and mild renal histopathology. Zebrafish intranasally treated with SARS-CoV-2 S RBD became hyposmic within minutes and completely anosmic by 1 day to a broad-spectrum of odorants including bile acids and food. Single cell RNA-Seq of the adult zebrafish olfactory organ indicated widespread loss of expression of olfactory receptors as well as inflammatory responses in sustentacular, endothelial, and myeloid cell clusters. Exposure of wildtype zebrafish larvae to SARS-CoV-2 in water did not support active viral replication but caused a sustained inhibition of ace2 expression, triggered type 1 cytokine responses and inhibited type 2 cytokine responses. Combined, our results establish adult and larval zebrafish as useful models to investigate pathophysiological effects of SARS-CoV-2 and perform pre-clinical drug testing and validation in an inexpensive, high throughput vertebrate model.

2 species that have been reported as naturally susceptible to SARS-CoV-2 include rhesus and 47 cynomolgus macaques (Munster et al., 2020; Rockx et al., 2020) , ferret (Kim et al., 2020) , cat 48 (Shi et al., 2020) , and Syrian hamster (Chan et al., 2020) . Mice, by contrast, are not 49 spontaneously permissive to the virus, but mice expressing the human ACE2 receptor provide a 50 useful animal model (Bao et al., 2020; Jia et al., 2020; Lutz et al., 2020) . All these mammalian 51 models have unique advantages and disadvantages for the study of immune responses to SARS-52

CoV-2 and other host-pathogen interactions but do not allow rapid, whole organismal, high 53 throughput, and low-cost preclinical testing of drugs and immunotherapies. 54 55

As model vertebrates, zebrafish are permissive to human viral pathogens including Influenza A 56 (Gabor et al., 2014) , Herpes Simplex virus type 1 (Burgos et al., 2008) , Chikungunya virus 57 (Palha et al., 2013) and human noroviruses GI and GII (Van Dycke et al., 2019). Zebrafish offer 58 many advantages over other animal models due to their high reproductive ability, rapid 59 development, low maintenance costs, and small transparent bodies. Importantly, zebrafish 60 olfactory, immune, and cardiovascular physiology share a significant degree of conservation 61 with humans (Postlethwait et al., 2020; Saraiva et al.) . Genetically, more than 80% of disease 62 related genes have a zebrafish orthologue (Howe et al., 2013) . The zebrafish innate immune 63 system is already developed in the transparent larval stages and members of all major groups of 64 mammalian cytokines have been identified in the zebrafish genome (Gomes and Mostowy, 2020; 65 Zou and Secombes, 2016) . Academic laboratories and the pharmaceutical industry use zebrafish 66 larvae in preclinical studies for assessing efficacy and toxicity of candidate drugs for several 67 diseases (Taylor et al., 2010) . Zebrafish is a proposed model for COVID-19 and has recently 68 been used in one vaccination study (Galindo-Villegas, 2020; Ventura-Fernandes et al., 2020). 69

This study aims to elucidate the physiopathology of wildtype zebrafish in response to SARS-70

CoV-2. 71 72 SARS-CoV-2 infection causes a wide litany of symptoms, ranging from asymptomatic to mild or 73 severe disease (Menni et al., 2020) . Apart from respiratory symptoms, multi-organ pathologies 74 are often reported with heterogeneous symptoms such as olfactory and taste loss, cardiac 75 dysfunction, renal pathologies, neurological damage, muscle and joint pain, gastrointestinal 76 symptoms, clotting disorders and others (Tabata et Mudd et al., 2020), and elevated type 2 cytokine levels (Lucas et al., 2020) . Importantly, this 84 cytokine pattern is in sharp contrast to that found in patients experiencing mild or moderate 85 symptoms, who are able to control exacerbated type 1 and type 3 cytokine responses (Lucas et  86 al., 2020). 87 88 SARS-CoV-2 enters the human host cells when SARS-CoV-2 Spike (S) protein receptor binding 89 domain (RBD) binds to angiotensin-converting enzyme 2 (ACE2) on a permissive host cell, then 90 a serine protease, such as TMPRSS2, cleaves the spike protein S1/S2 site to facilitate fusion of 91 the virion with the host cell membrane (Hoffmann et al., 2020a (Hoffmann et al., , 2020b . ACE2 is expressed in 92 many different cell types across many organs in the human body including lung, olfactory 93 sustentacular cells, enterocytes, and endothelial cells (Albini et ACE2, the use of ACE inhibitors is being considered as a therapeutic intervention in COVID-19 112 patients (Lopes et al., 2020) . Importantly, drugs currently used to treat the COVID-19 can be 113

pro-arrhythmic and therefore there is a need to incorporate cardiovascular damage into the list of 114 targets of therapeutic interventions in COVID-19 and for models that replicate human cardia 115 physiology (Kochi et al., 2020) . 116 117

A hallmark of SARS-CoV-2 infection is acute loss of smell (Cooper et al., 2020) . Viral-induced 118 anosmia is not unique to SARS-CoV-2 infections since viruses such as rhinoviruses, influenza, 119 parainfluenza and coronaviruses are known to be the main cause of olfactory deficits in humans 120 (Suzuki et al., 2007; Imam et al., 2020) . In mice and humans, ace2 expression is detected in 121 sustentacular cells, olfactory stem cells known as horizontal and globose basal cells in the 122 olfactory epithelium, and vascular cells (pericytes) in the olfactory bulb (Brann et al., 2020 The present study reports for the first time that zebrafish larvae exposed to SARS-CoV-2 appear 134 to mount innate immune responses that resemble cytokine responses of mild COVID-19 patients. 135

Recombinant SARS-CoV-2 S RBD is sufficient to cause olfactory, renal and cardiovascular 136 pathologies in larvae and adult zebrafish. We also identify potential mechanisms of SARS-CoV-137 2 induced anosmia by scRNA-Seq. Our findings support the use of zebrafish as a novel 138 4 vertebrate model to elucidate SARS- CoV-2 pathophysiology and to screen drugs and other  139  therapies targeting COVID-19.  140  141  142  143 Results 144 145

Phylogenetic analyses of ACE2 molecules in vertebrates 146

Comparative analysis of ACE2 molecules in vertebrates indicated that ACE2 molecules are well 147 conserved in vertebrates with a 72%-73% similarity and 57.5%-58% identity between zebrafish 148 ACE2 and human ACE2, respectively (Table S1 ). Examination of ACE2 amino acid motifs in 149 the region involved in binding SARS-CoV-2 S protein revealed zebrafish ACE2 has 50%/64% 150 sequence similarity with the corresponding human ACE2 region compared to 71%/78% in 151 macaques ACE2 or 57%/71% in ferret ACE2 ( Systemic injection of recombinant SARS-CoV-2 protein into adult zebrafish has been shown to 159 induce some toxicity (Ventura-Fernandes et al., 2020). In order to determine whether 160 recombinant SARS-CoV-2 S RBD protein causes inflammatory responses in zebrafish larvae, we 161 exposed 5 dpf larvae to SARS-CoV-2 S RBD recombinant protein for 3 hours (h) and measured 162 cytokine responses by qPCR. As shown in Figure 1A , 3 h immersion with SARS-CoV-2 S RBD 163 protein induced a significant downregulation in ifnphi1 expression and significant increase in 164 expression of ccl20a.3, a pro-inflammatory chemokine. No changes in ace2, tnfα, il1b and 165

il17a/f3 expression were observed ( Figure 1A ). These results indicate that rapid immune 166 responses occur in zebrafish larvae exposed to SARS-CoV-2 S RBD. We next evaluated the 167 effects of SARS-CoV-2 RBD S on zebrafish larva heart function to validate zebrafish larvae as a 168 model for COVID-19 cardiac manifestations. We immersed 7-and 5-days post fertilization (dpf) 169 zebrafish larvae with SARS-CoV-2 S RBD, or with vehicle, and measured heart rate after 3 h. 170

As shown in Figure 1B , 7 dpf and 5 dpf zebrafish treated with SARS-CoV-2 S RBD had 171 significantly higher heart rates compared to vehicle treated controls. As a positive control for the 172 recombinant protein, we used animals treated with the same dose of recombinant infectious 173 hematopoietic necrosis virus (IHNV) glycoprotein (r-IHNVg), a rhabdovirus known to cause 174 severe endothelial damage in zebrafish (Ludwig et al., 2011) . r-IHNVg caused a severe decrease 175 in larval zebrafish heart rate compared to control treated animals ( Figure 1B -C). To determine if 176 increased heart rate induced by SARS-CoV-2 S RBD was dependent on Ace2 binding, we co-177 incubated 5 dpf larvae with captopril and reverted SARS-CoV-2 S RBD induced heart 178 dysfunction. Captopril had no effect on r-IHNVg induced bradycardia ( Figure 1B ). Ventricular 179 trace analyses showed marked differences in rhythm patterns in each treatment group ( Figure  180 1D). Importantly, the captopril and SARS-CoV-2 S RBD treated animals, despite having similar 181 heart rates to those of the vehicle treated controls, displayed a unique ventricular trace pattern, 182

warranting future studies regarding the potential cardioprotective role of captopril in Combined, these results indicate that zebrafish exposed to SARS-CoV-2 S RBD protein 184 5 experience tachycardia and suggest that zebrafish larvae constitute a valuable pre-clinical model 185

to test the effects of drugs for COVID-19 on cardiac activity in vivo. 186 187

zebrafish 189

Anosmia is one of the earliest manifestations of SARS-CoV-2 infection in humans (Cooper et 190 al., 2020) . We have previously shown that IHNV glycoprotein protein is sufficient to induce 191 rapid nasal immune responses as well as neuronal activation in teleost fish (Sepahi et ( Figure 2D ). Loss of the epithelial mosaic structure characteristic of the teleost olfactory 202 epithelium was observed on days 1, 3 and 5 post-treatment ( Figure 2E ). By day 5, loss of entire 203 apical lamellar areas due to severe necrosis was observed in the olfactory lamellae of all treated 204 animals compared to controls ( Figure 2F ). Significant loss of olfactory cilia was recorded in all 205 animals treated with SARS-CoV-2 S RBD at all time points ( Figure 2H ). These results indicate 206 the SARS-CoV-2 S RBD is sufficient to cause inflammation, edema, hemorrhages, ciliary loss, 207

and necrosis in the olfactory organ of zebrafish. Hence, olfactory damage can be caused by 208

indirect mechanisms and in the absence of active SARS-CoV-2 replication in this tissue. 209 210

Toxicity effects of intranasal SARS-CoV-2 S RBD delivery were also evaluated in distant tissues 211 such as the kidney, a target organ of SARS-CoV-2. Acute kidney injury (AKI) incidence varies 212 from 0.9% to 29% in COVID-19 patients (Su et al., 2020) . Renal damage, especially AKI, is also 213 common in patients with RAS dysfunction such as diabetic patients who suffer from 214 hypertension (Ribeiro-Oliveira et al., 2008; Advani, 2020). A recent study in zebrafish injected 215 with the N-terminal part of SARS-CoV-2 S protein reported inflammation and damage in several 216 tissues of adult zebrafish including kidney 7 and 14 d post-injection (Ventura-Fernandes et al., 217 2020). In the present study, histological examination of the head-kidney of zebrafish who 218 received SARS-CoV-2 S RBD intranasally revealed renal tubule pathology characteristic of AKI 219 3 h post-treatment ( Figure S1 ). Pathology was not as severe at later time points, but vacuolation 220 of the renal tubule epithelium was still visible 5 days post-treatment ( Figure S1 ). We did not 221 observe signs of glomerulopathology in treated animals compared to controls. Together, these 222 results indicate that intranasal delivery of SARS-CoV-2 S RBD is sufficient to cause 223 nephropathy in adult zebrafish but that pathology is not as severe as when the protein is delivered 224 by injection. 225 226

Intranasal delivery of SARS-CoV-2 S RBD causes anosmia in adult zebrafish 227 228 Adult zebrafish exposed to SARS-CoV-2 S RBD had a significant reduction of olfactory 229 responses to food extracts of ~50% of preexposure olfaction within minutes as measured by 230 6 electro-olfactogram (EOG), indicating an instant effect of the protein on olfactory function (Fig.  231 3A). Reduction of olfaction was sustained for least one hour of recording, but the olfactory organ 232 remained still semi functional. Zebrafish treated with PBS never lost olfaction at any time point. 233

To further quantify the degree of olfactory reduction due to SARS-CoV-2 S RBD, we took 234 advantage of the two easily accessible and isolated olfactory chambers present in zebrafish. We 235 exposed one naris to the SARS-CoV-2 S RBD protein and the other naris, from the same animal, 236

to PBS and waited 3 h or 1d before measuring olfaction by EOG. At 3 h we observed a 37-70% 237 reduction in food and bile olfactory responses between the treated and untreated naris and a 238 complete loss of olfactory function to both odorants 1 d post-treatment (Fig. 3B) . The reduction 239 of olfactory sensitivity for food extract was smaller than that found for bile, probably due to the 240 lower number of OSNs involved in bile acid detection compared to amino acids found in food 241 (Hansen et al., 2003) . Our results indicate that SARS-CoV-2 S RBD-induced-anosmia is not 242 specific for a subset of OSNs, since both food extracts and bile olfactory signals were suppressed 243

in SARS-CoV-2 S RBD treated zebrafish. This fact, together with the 1d time to develop 244 complete anosmia and disrupt olfactory epithelial structure, support the hypothesis that SARS-245

CoV-2 S RBD damage may occur first on sustentacular cells, with subsequent impacts on OSN 246 viability and function. 247

Single-cell analysis of the zebrafish olfactory organ 249 250

To understand the impact of SARS-CoV-2 S RBD on zebrafish OO, we performed single cell clusters, 3 endothelial cell (EC) clusters and 7 leucocyte (lymphoid and myeloid) clusters ( Figure  258 4A-B). 259

Of the 8 NCs, Neuron1 and Neuron2 corresponded to mature OSNs. Neuron1 expressed markers 260 of ciliated OSNs (ompa and ompb) in addition to several olfactory receptor (OR) genes 261 (Buiakova et al., 1996) . Neuron2, on the other hand, expressed markers of microvillus OSNs 262 (trpc2b, s100z, and gnao) and many vomeronasal receptors (VR) such as v2rh32 as well as OR 263 gene ( Figure S2 ) (Kraemer et al., 2008 Neuron7 and Neuron8 expressed cell cycle and early neuronal progenitor markers as well as 278 tmprss13b and tmprss4a, however the majority of cluster identifying genes in these two clusters 279 are undescribed ( Figure S2 ). 280

SCs are supporting cells that exist in the neuroepithelium around OSNs and in humans, they 281 express ACE2 (Bryche et al., 2020) . SCs in the olfactory epithelium can directly arise from 282 horizontal basal cells (HBCs) (Yu and Wu, 2017). We found 5 clusters of SCs in our datasets. subpopulation closely related to the Sustentacular4 cluster ( Figure S2 ). 292

We identified three clusters of ECs that all express tmprss13 and tmprss4. While we did not 293 detect ace2 expression in any cell clusters, we detected ace2 mRNA in adult zebrafish olfactory 294 organ, and at low levels in the olfactory bulb ( Figure S3 ). ace2 expression levels have previously 295

shown to be low in neuronal tissues and therefore may be hard to detect by scRNA-Seq (Song et  296 al., 2020). Endothelial1 and 2 clusters expressed the endothelial markers sox7 and tmp4a (Yao et  297 al., 2019). All three clusters broadly expressed genes associated with tight junctions (tjp3, jupa, 298 ppl, cldne, and cgnl1) as well as many keratin genes ( Figure S2 ). Interestingly, Endothelial3 299 cluster also expressed the calcium channel trpv6 and a slew of non-annotated genes. 300

There are copious amounts of immune cells in the teleost olfactory organ ( Intranasal delivery of SARS-CoV-2 S RBD induces inflammatory responses and 318 widespread loss of olfactory receptor expression in adult zebrafish olfactory organ 319 320

The cellular landscape of the zebrafish olfactory epithelium was affected by SARS-CoV-2 S 321 RBD treatment and time ( Figure 4A -D). This was especially evident in the proportions of 322 8 neuronal cell types 3 d post-treatment when the proportion of mature, omp + ciliated OSN was 323 much lower compared to controls and the 3 h treated group. In contrast, neuronal progenitors 324 expressing cell cycle markers (aubk, ecrg4, and mki67) and neuronal differentiation and 325 plasticity markers (neurod1, neurod4, gap43, sox11, and sox4) were expanded 3 d post-treatment 326

( Figure 4B -C). Further, we detected a noticeable decrease in the proportion of cells belonging to 327 the Lymphocyte2 cluster, a cluster that expressed markers of Treg cells (foxp3b) 3 h post 328

intranasal delivery of recombinant SARS-CoV-2 S RBD but this change was not noticeable at 329 day 3 ( Figure 4C ). At 3 d, we observed a third lymphocyte cluster, not detected at 3 h, highly 330 expressing the TCR subunit zap70 as well as plac8 onzin related protein (ponzr1), a molecule 331 that has immunoregulatory roles during Th1 type immune responses in mammals ( Figure S2 in olfactory neuronal clusters were enriched in processes such as neuron differentiation, sensory 367 system development, and sensory organ morphogenesis, whereas downregulated genes belonged 368 9 to sensory perception of smell, detection of chemical stimulus and GPCR signaling pathway 369 ( Figure 5F ). Functional enrichment analyses in Metascape showed that the top non-redundant 370 enriched clusters in both upregulated and downregulated genes in zebrafish OSNs 3 h post-371 treatment was sensory perception of smell ( Figure 5G ). The same was true 3 days post-treatment, 372 but processes such as regeneration, neuron development, neuron fate commitment and the p53 373 signaling pathway were also enriched within the upregulated genes ( Figure 5H ). Combined, 374

these results suggest that presence of SARS-CoV-2 S RBD in the olfactory organ instigates 375 harmful effects on OSNs within hours and that the magnitude of the OSN damage increases by 3 376 days post-treatment. Further, these analyses indicate that neuronal regeneration and 377 differentiation processes were initiated by day 3 in order to begin repair of olfactory damage. Our study allowed us to dissect how each cell type in the zebrafish olfactory organ responds to 387 SARS-CoV-2 S RBD. Our results indicated unique responses by SC clusters and EC clusters to 388 treatment (Figures 4 and 6) . At 3 h, we detected increased expression of apoeb, of transcription 389 factors foxq1a and id2b, the transcriptional regulator nfil3-5, two tumor necrosis factor receptor 390 superfamily members (tnfrsf11b and tnfrsf9a) as well as tcima (transcriptional and immune 391 response regulator), whose mammalian ortholog pcim regulates immune responses as well as 392 endothelial cell activation and expression of inflammatory genes ( Figure 6A ) (Kim et al., 2009 ). 393

Further, at 3 h we observed downregulation of the pro-inflammatory cytokine il17af/3 as well as 394 glutathione peroxidase gpx1b, the transcription factor notch1b, basal cell adhesion molecule 395

bcam, guanine nucleotide-binding protein subunit gamma gng8, and the calcium binding s100z 396 in SC and EC from SARS-CoV-2 S RBD treated olfactory organs relative to vehicle treated. At 3 397 d post-treatment, we observed significant increased expression of the gene that encodes brain 398 natriuretic peptide (nppc), a vasodilating hormone, the pro-inflammatory chemokine ccl19a.2, 399 the M2 macrophage marker arg2, the transcription factors foxq1a and sox11a, tubulin beta 5 400 tubb5, and the epithelial mitogen epgn, among others ( Figure 6B ). Downregulated genes at 3 d 401

post-treatment included hsp70.3, apoeb, the osteoblast specific factor b postb, the desmosomal 402 component periplakin (ppl), the vasoconstricting endothelin 2 (edn2), the heparin binding 403 molecule latexin (ltx) involved in pain and inflammation, and cd74b, a part of the MHC-II 404 complex ( Figure 6B ). Combined, these data indicated immune regulatory responses in SC and 405 EC clusters early after SARS-CoV-2 S RBD treatment, followed by transcriptional changes with 406 potential vasoactive effects by day 3. 407 408

GO and enrichment set analyses indicated that SC and EC clusters initially undergo 409

transcriptional changes enriched in metabolic responses, response to stress, and cell 410 differentiation ( Figure 6C ). Later on, at day 3, SC and EC responses were enriched in genes 411

involved not only in the stress response but also in immune responses and responses to wounding 412 ( Figure 6D ). Similar results were identified using Metascape, which showed that the 413 inflammatory response to wounding was moderately enriched in the downregulated genes at 3 h, 414 whereas by day 3, response to wounding became the top enriched set among the upregulated 415 genes ( Figure 6E -F). 416 417

Exposure of wildtype zebrafish larvae with SARS-CoV-2 does not support viral replication 418 419

Zebrafish larvae have been used as models to investigate several human viruses. Infecting 420 zebrafish larvae in a BSL-3 laboratory by immersion in contaminated water is comparable to 421 infecting a cell line. We first checked the stability of SARS-CoV-2 in zebrafish water overtime, 422

in the absence of any animals. We found that SARS-CoV-2 viral loads in the water remained 423 stable throughout the experiment ( Figure 7A -B). We exposed wildtype AB zebrafish larvae to 424 live SARS-CoV2 and examined viral mRNA abundance over time to determine if zebrafish 425 larvae can support viral replication. We detected no increases in the viral N copy numbers over 426 time and a steady decline in E gene copy numbers in both water from wells containing larvae and 427

virus as well as in the larval tissue ( Figure 7C -F). These results indicate that wild-type zebrafish 428 larvae cannot support efficient SARS-CoV-2 replication as suggested by the in silico 429 comparative sequence analyses of the zebrafish ace2 molecule. 430 431

Exposure of zebrafish larvae to SARS-CoV-2 decreases ace2 expression and triggers pro-432 inflammatory cytokine responses 433 434

In order to determine whether exposure of zebrafish larvae with live SARS-CoV-2 causes 435 changes in Ai, we measured ace2 mRNA levels in control and SARS-CoV-2 exposed larvae over 436 time. ace2 expression was significantly downregulated as early as 6 h post-infection. ace2 437 expression inhibition was sustained over the time course of the experiment with the greatest 438 decrease occurring 2 days post-infection (dpi) ( Figure 8A ). We next evaluated changes in 439 expression of cytokine and chemokine genes to establish whether zebrafish mount inflammatory 440 responses that resemble the patterns of mild or severe SARS-CoV-2 infection. Il1β expression 441

was significantly upregulated at 6h, 1 dpi (2-3 fold) and 2 dpi (10 fold) and significantly 442 downregulated at 4 dpi ( Figure 8B ). We detected a significant increase in tnfa expression in 443 SARS-CoV-2 exposed larvae 2 dpi ( Figure 8C ). ifnphi1 and ifnphi3 are the two main type I IFN 444

genes involved in larval zebrafish antiviral responses (Levraud et al., 2019) . We detected a 445 significant up-regulation of ifnphi1 at 1 and 2 dpi, whereas expression was inhibited at 4 dpi. 446

Interestingly, ifnphi3 expression followed a very different pattern compared to ifnphi1, which 447 was significantly downregulated 2 dpi but significantly upregulated at 4 dpi ( Figure 8D -E). Mxa 448 expression was significantly downregulated at all time points ( Figure 8F ). il17af/3 expression 449 was significantly elevated 1, 2 and 4 dpi ( Figure 8G ). Expression levels of il22, a member of the 450 IL10 family, were downregulated 6 hpi and 1 dpi ( Figure 8H ) whereas the type II cytokine 451

il4/il13b was downregulated at 6 hpi, 1 dpi and 2 dpi ( Figure 8I ). Further, a significant increase 452 in the expression of the chemokine ccl20a.3 was detected in infected larvae at 1 and 2 dpi 453 compared to controls ( Figure 8J) . A moderate increase in ccl19a.1 expression was observed at 2 454 dpi followed by a strong down-regulation (15 fold) at 4 dpi ( Figure 8K ). Taken together, these 455 data indicate that exposure to SARS-CoV-2 induces a significant antiviral and pro-inflammatory 456 immune response in wildtype zebrafish larvae. This response involved type I IFN, tnfa, il1b, il17 457

and ccl20, reminiscent of COVID-19 patients with mild disease. 458 459

The current COVID-19 pandemic has propelled the investigation of SARS-CoV-2 and the 461 development of animal models that help identify therapeutic interventions and vaccines for 462 COVID-19. Thus far, all animal models reported are mammals, and therefore breeding, genetic 463 manipulation, and animal housing in BSL-3 laboratories make these models costly and not 464 readily available in large numbers. Zebrafish can overcome many of the limitations of 465 mammalian models thanks to their transparent bodies, short life-span, low maintenance costs and 466

production of large numbers of embryos. We therefore performed the simplest infection 467 procedure, where SARS-CoV-2 was added to the water of zebrafish larvae. In this manner, BSL-468 3 trained personnel with no experience in zebrafish microinjection can readily expose larvae to 469 SARS-CoV-2 without the need of animal protocols in a similar fashion to in vitro cell culture 470

infections. Exposure of wildtype zebrafish larvae to SARS-CoV-2 in the water did not however 471 result in any detectable viral replication. downregulated in response to SARS-CoV-2 exposure. Combined, these data suggest the SARS-505

CoV-2 induces some type I IFN responses in zebrafish larvae while inhibits others. Future 506 12 studies are clearly needed to ascertain the role of teleost type I IFN in the anti SARS-CoV-2 507 immune response. 508 509 S protein is a structural protein of SARS-CoV-2 and therefore the target of several vaccine trials. 510

Therefore, we exposed zebrafish larvae to SARS-CoV-2 S RBD protein and investigated 511 transcriptional and physiological responses. Rapid changes in gene expression were detected in 512 treated larvae, including up-regulation of the chemokine ccl20a.3 and the down-regulation of 513

ifnphi3. The ccl19/ccl20 axis appears to be critical in teleost antiviral innate responses, as 514

previous studies have shown very rapid responses in larvae exposed to the rhabdovirus SVCV 515 (Sepahi et al., 2019) . This change was also detected in the larvae that were exposed to the live 516 SARS-CoV-2 virus in the present study. We further detected a significant down-regulation of 517 type I IFN ifnphi1 gene in larvae exposed to SARS-CoV-2 S RBD protein. 518 519

Examination of ace2 transcriptional changes in zebrafish larvae exposed to SARS-CoV-2 520 revealed a consistent down-regulation in expression throughout the course of infection. 521

Interestingly, we did not observe any changes in ace2 expression after 3h immersion with SARS-522

CoV-2 S RBD protein. Recently, enterocytes were found to be the main cell type expressing 523

Ace2 in 5 dpf-old zebrafish larvae (Postlethwait et al., 2020); and therefore it is possible that the 524 down-regulation in ace2 expression observed in our experiments was the result of enterocyte 525 responses to SARS-CoV-2. However, an olfactory epithelial cell cluster was not identified in this 526 dataset, probably because these cells constitute too small a fraction of the cells of an entire larva. 527

Importantly, we exposed larvae to the virus at 3 dpf, when the olfactory pit is already sampling 528 the surrounding water, while the gut fully opens only at 4 dpf. Thus, changes in ace2 expression 529 levels in the olfactory pit of the zebrafish larvae cannot be ruled out at this point. 530 531

Previous work has shown that ACE2 knockdown in mice protects from SARS-CoV infection 532 (Kuba et al., 2005) . Thus, down-regulation of zebrafish Ace2 expression may have protected 533 larvae from SARS-CoV-2 infection in our experiments. Our data agree with studies in mouse 534 lungs, where suppression of ace2 gene expression was consistently observed following SARS-535

CoV-2 infection (Chen et al., 2020). Interestingly, changes in ACE2 levels can occur in response 536 viruses that do not require ACE2 for host entry (Chen et al., 2020). Thus, although further 537 studies are warranted, our data suggest that Ace2 is involved in antiviral SARS-CoV-2 responses 538 in zebrafish. 539 540

We took advantage of the zebrafish fish model which allows for easy live imaging of heart beats 541 in transparent larvae. We detected in vivo cardiac/heart responses in larval zebrafish exposed to 542 SARS-CoV-2 S RBD protein characterized by tachycardia. Cardiac arrhythmia is a common 543 symptom among COVID-19 patients and current research efforts aim to understand how SARS-544

CoV-2 infection impacts cardiovascular function (Libby, 2020) . Our findings underscore that 545 SARS-CoV-2 S RBD is able to cause tachycardia in the zebrafish larval model and that this 546 model can be used for rapid evaluation of drug treatments for COVID-19. As a proof of concept, 547

we used captopril, an ACE inhibitor currently being evaluated in human clinical trials 548 (NCT04355429). Captopril treatment ameliorated tachycardia in zebrafish larvae exposed to 549 SARS-CoV-2 S RBD recombinant protein. Our studies therefore suggest the beneficial use of 550 captopril in COVID-19 patients undergoing cardiac arrhythmia, but clearly further studies are 551 13 required to fully translate these findings to the clinic and to determine the duration and timing of 552 captopril treatment in COVID-19 patients. 553 554

A recent report in zebrafish adults indicated that injection of recombinant SARS-CoV-2 S N 555

terminal protein caused histopathology of several tissues including the liver, kidney, brain and 556 ovary (Ventura-Fernandes et al., 2020). Additionally, some animals succumbed to injection with 557 the recombinant protein. We did not detect any mortalities neither in larvae nor in adults in any 558 of our experiments, perhaps suggesting that mortalities were due to the injection procedure rather 559 than the protein treatment itself. Of note, the dose used in the present study was considerably 560 lower than the dose delivered in the Ventura-Fernadez study, perhaps explaining the differences 561

in toxicity between both studies. We observed histological damage following a single intranasal 562 delivery of SARS-CoV-2 protein, specifically at the site of delivery, the olfactory organ, whereas 563 more transient and moderate damage was detected in the renal tubules. Renal damage may have 564 occurred by direct uptake of SARS-CoV-2 S RBD by the kidney once the protein reached the 565 bloodstream following intranasal administration or, alternatively, by activation of RAS or 566 inflammatory cascades at the olfactory organ. Thus, toxicity of SARS-CoV-2 S protein in adult 567 zebrafish may be less severe when delivered intranasally than by injection, and future studies 568

should evaluate whether current vaccine candidates also exert similar effects and whether 569 different administration routes cause the same side-effects or not. This is particularly important 570 as the intranasal route appears promising for some vaccine candidates study did not determine when zebrafish recover olfactory function following SARS-CoV-2 S 597 14 RBD intranasal treatment, but based on our histopathological observations, the olfactory organ 598 was still severely damaged 5 days after treatment, suggesting that recovery of olfactory function 599 may take several weeks in our model. Our findings therefore indicate that similar to humans, 600 zebrafish suffer from olfactory pathology and loss of smell in response to SARS-CoV-2 S RBD 601 protein. Thus, olfactory pathophysiology appears to occur even in the absence of viral replication 602 raising the possibility that nasal vaccines for COVID-19 may also cause transient anosmia in 603 humans. 604 605

In conclusion, the present study reports that both adult and larval wild-type zebrafish can be 606 useful models to advance our understanding of COVID zebrafish larvae in responses to SARS-CoV-2 S RBD. Animals were exposed to SARS-CoV-2 S 639 RBD protein (r-Spike, 2 ng/ml) for 3 h at 28.5°C or vehicle. Changes in gene expression were 640 measured by RT-qPCR using rps11 as the house-keeping gene. Each data point represents a pool 641 of 4 larvae/well. Data are expressed fold-change compared to vehicle controls using the Pffafl 642 method. 643 (B) Average heart beat per minute of 7 dpf (n=6) zebrafish larvae after 3 h of incubation with 644 vehicle, 2 ng/ml r-Spike, or 2 ng/ml r-IHNVg. 645

(C) Average zebrafish heart beats per minute in 5 dpf zebrafish larvae (n=8) after 3 h of 646 incubation with vehicle, 2 ng/ml r-Spike, or 2 ng/ml r-IHNVg with and without treatment with 647 12mM of captopril. Heart beats were recorded for 3 min at 20 under a Nikon Ti microscope 648

and (C-D) Mean viral loads quantified as log of SARS-CoV-2 N gene and SARS-CoV-2 E gene copy 726 numbers in control supernatants from well with larvae not exposed to virus, and supernatants 727 from wells with larvae that were exposed to 10 4 pfu of SARS-CoV-2 for 6 h, 1 d, 2 d and 4 d. 728

Each sample represents the supernatant of one well containing 12 larvae. 729 (E-F) Mean viral loads quantified as log of SARS-CoV-2 N gene and SARS-CoV-2 E gene copy 730 numbers in control larvae and larvae exposed to 10 4 pfu of SARS-CoV-2 for 6 h, 1 d, 2 d and 4 731 d. Each sample point represents one well containing 12 larvae. 732

Larval infections began at 2 dpf after mechanical dechorionation at 1 dpf. * P-value<0.05; ** P-733 value<0.01 *** P-value<0.001. Results are representative of two independent experiments. 734 735 For all experiments, wild type AB zebrafish were obtained from ZIRC (Oregon, USA). For the 764 intranasal delivery of SARS-CoV-2 S RBD protein into adult zebrafish, female and male adult 765 zebrafish were obtained from Dr. Wong's laboratory at the University of Nebraska due to 766 lockdown of ZIRC during the pandemic. All fish were maintained in a filtered aquarium system 767 at 28℃ with a 14 h light and 10 h dark cycle at the University of New Mexico Aquatics Animal 768

Facility. All experiments with adults utilized a mix of male and female animals, and the larvae 769 sex is indeterminable. Animals were fed ad libitum Gemma complete nutrition (Skretting). AB 770 larvae were obtained by batch-crossing AB adults allowing for natural fertilization. The morning 771 of fertilization, larvae were collected at n=50 per Petri dish and kept in E3 medium containing 772 0.002% methylene blue. In the afternoon, larvae were placed in fresh E3 medium without 773 methylene blue and non-surviving embryos were removed. Larvae were maintained at 28.5℃ in 774 E3 medium until 4dpf when they are slowly changed to system water. 775 776 SARS-CoV-2 777

The SARS-CoV-2 isolate, a CDC isolate from a US patient (USA-WA1/2020), was obtained 778 from BEI Resources. The strain was grown at a low MOI to minimize generation of 779 noninfectious particles and low passaged virus was used in all experiments described. The virus 780 was propagated on Vero E6 cells and viral loads quantitated by RT-qPCR and by plaque forming 781 assays as we have described previously (Bradfute et al, 2020). 782 783

Intranasal delivery of SARS-CoV-2 S RBD recombinant protein to adult zebrafish 785

Adult zebrafish were anesthetized for 1 min in 0.04 mg/ml Tricaine-S (Syndel) solution and then 786 moved to an absorbent boat where their gills were still covered with anesthetic solution for 787 administration of solutions to nares. Using a microloader tip (Eppendorf, 930001007), 5 μl of 20 788 ng/μl SARS-CoV-2 S RBD (kindly provided by Dr. F. Krammer) was directly pipetted into each 789 naris, while 5 μl of sterile PBS was applied in control fish. After inoculation, animals were 790 recovered in a separate tank supplemented with O2 before returning to their rearing tank until the 791 end of the experiment. Euthanasia was performed on ice to ensure rapid death without perturbing 792

the combined supernatants were centrifuged for 10 min at 400g in supplemented neurobasal medium 820 and cells were counted with a hemocytometer. Viability was estimated by trypan blue staining. 821

Cells were then strained twice through Flowmi 10 μm strainers and loaded onto the chromium 822

controller with a viability of > 85%. Cell libraries were generated according to 10x Genomics 823 protocols at the University of New Mexico Cancer Center Genomics core facility and sequenced 824 on an Illumina NovaSeq 6000 at the University of Colorado Genomics and Microarray core 825 facility. Sequencing depth and statistics of the scRNA-Seq run are shown in Figure S2 . SRAs for 826 this project can be found on NCBI under bioproject #PRJNA668529. 827

Fastqs were run through the Cell Ranger v3.0 pipeline with default settings using the GRCz11 828 zebrafish genome. Output matrices were loaded to R (v1.2.5001) as a Seurat object (Package 829

Seurat v3.1.1). First, cells with less than 200 or greater than 2500 features, and greater than 5% 830 mitochondrial features were removed. After counts were normalized using the "lognormalize" 831 method and a scale factor of 10000, 2000 variable genes were selected using the 'vst" method. 832

Data was scaled, and PCA dimensional reduction was run. Jackstraw analysis determined the 833 vehicle control to have 38 significant principal components (PCs) and the treated samples to 834 have 40 significant PCs which were used for clustering analysis. SARS-CoV-2 S RBD treated 835 samples were integrated with the vehicle treated sample and clustered together using 30 836 significant PCs and a resolution of 0.5. Cluster markers were identified with "FindAllMarkers" 837

in Seurat and exported for cluster identification. Differential expression analysis was done with 838 seurat "FindMarkers" in default settings for each cluster and exported for gene ontology 839

analysis. 840

Gene ontology (GO) analysis was done with web-based GUIs Metascape and ShinyGO v0.61 841 which draw multiple currently maintained databases (ensembl, ENTRZ, KEGG among others). 842

Biological process webs were created using biological process output from ShinyGO v0.61 in 843

Prism GraphPad. Biological processes bar graphs were produced by Metascape. 844 845

Electro-olfactogram recordings 846

Adult AB zebrafish were anesthetized and received 2 µl of recombinant SARS-CoV-2 S RBD 847 protein (50 ng/µl) in PBS or PBS alone. After 3 h or 1 day, zebrafish were anaesthetized (0.1 g 848 MS222/L), placed in a v-shape stand and supplied with aerated water containing MS222 849 anesthetic (0.05 g/L). The nasal flap was removed with sterile fine forceps to expose the 850 olfactory rosettes to a continuous tank water source. Olfactory responses to zebrafish food 851 extract or goldfish bile were measured by electrical recordings as detailed in Sepahi et al., 2019. 852 The food extract was prepared as a filtered solution of 1L tap water and 0.1 g of dry food pellets. 853

Water food extracts were separated in 200 ml aliquots and kept frozen until the recording day. A 854 0.5 ml mix of bile fluid from 50 adult goldfish was aliquoted in 10 µl and kept frozen until the 855 day of the recording. Before each recording, bile aliquots were diluted 1:1000 in water from the 856 EOG system. There were no significant differences in olfactory responses between males and 857 females, hence responses of both sexes were averaged together. The percentage reduction in 858 olfactory activity was calculated by dividing the amplitude of the olfactory signal at time x by 859 amplitude of the olfactory signal at time 0 *100. Percentage of olfactory signal reduction 860 between control and treated naris was calculated as follows (Amplitude response to odorant in 861 control naris (mV) -Amplitude response to odorant in treated naris (mV))/Amplitude response to 862 odorant in control naris (mV)) * 100. 863 (Sigma) at 42℃, stabilized for 5 min at RT and then imaged to record heart-beat activity. 872 873

Zebrafish larvae heart-beat recordings and analysis 874

As the agarose solidified, animals were adjusted to the microscope stage (approx. 5 min) then 875 hearts were recorded using brightfield AVI for 3 min at 16.667 frames/s at RT. AVI images were 876 then opened in ImageJ with the Time series analyzer V3 plugin. Circular ROIs were drawn in 877 either the atrium or ventricle and average intensity was extracted. The maximum average 878 intensity peaks were identified and counted per 60s as bpm. Data were analyzed by one-way 879 ANOVA with Tukey's post hoc. 880 881

Infection of zebrafish fish larvae with SARS-CoV-2 882

Ten animals were placed in each well in 12-well plates containing 2 ml of tank water and 883 transferred to BSL3 facility the day before infection. Gene expression analyses by RT-qPCR 903

Whole larvae RNA was extracted using trizol. For tissue homogenization bead beater tubes are 904 preloaded with 1.5 g 1.0 mm dia Zirconia beads, 1.5 g 2.0 mm Zirconia beads and 500 µl Trizol. 905

Samples were loaded into the tubes and bead beat at 4350 rpm for 45 s. Tubes were then 906 centrifuged at 7,000 rpm for 7 min. The homogenate/lysates were transferred to clean 1.5 ml 907 microfuge tubes and spun at 7,000 rpm for 7 min to pellet debris. Supernatants were then 908 processed to extract the total RNA using a standard chloroform/phenol extraction protocol. RNA 909 was quantified by Nanodrop and samples were normalized and 1 µg of RNA was used to 910 synthesize cDNA using the Superscript III first strand system (Thermofisher, 18080051). qPCR 911 was performed using SSOadvanced supermix (Biorad, 1725270) and primers listed in Table S3  912 (Supplemental Methods). Gene expression changes were quantified using the Pfaffl method 913 (Pfaffl, 2001 Glycerophospholipid biosynthesis Apoptosis microtubule polymerization or depolymerization GPCR signaling, coupled to cyclic nucleotide 2nd messenger negative regulation of cell differentiation Iron uptake and transport circadian regulation of gene expression sensory perception of smell Developmental process

Anatomical structure development

Response to wounding

Response to stress

Immune system response

Cell chemotaxis

Multicellular organism development 

Acute Kidney Injury: A Bona Fide Complication of Diabetes

The SARS-CoV-2 990 receptor, ACE-2, is expressed on many different cell types: implications for ACE-inhibitor-and 991 angiotensin II receptor blocker-based cardiovascular therapies

Myocardial injury and COVID-19: Possible mechanisms Savalan

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

The establishment 1000 of neuronal properties is controlled by Sox4 and Sox11

Imbalanced Host Response to SARS-CoV-2 1007 Drives Development of COVID-19

Macrophage-mediated neuroprotection and neurogenesis in the olfactory 1010 epithelium

CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-1015 associated anosmia

Massive transient damage of the olfactory 1019 epithelium associated with infection of sustentacular cells by SARS-CoV-2 in golden Syrian 1020 hamsters

Olfactory marker protein (OMP) gene 1024 deletion causes altered physiological activity of olfactory sensory neurons

Zebrafish as a New Model for Herpes Simplex Virus Type 1 Infection

Integrating single-cell 1031 transcriptomic data across different conditions, technologies, and species

Simulation of the clinical and pathological 1036 manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: 1037 implications for disease pathogenesis and transmissibility

Individual variation of the SARS-CoV-2 receptor ACE2 gene expression and regulation

Acute inflammation regulates neuroregeneration 1044 through the NF-κB pathway in olfactory epithelium

Chronic Inflammation Directs an Olfactory Stem 1047

Cell Functional Switch from Neuroregeneration to Immune Defense

COVID-19 and the Chemical 1052 Senses: Supporting Players Take Center Stage Keiland

Broad host range of SARS-CoV-2 1056 predicted by comparative and structural analysis of ACE2 in vertebrates

Smell 1060 and taste disorders during COVID-19 outbreak: Cross-sectional study on 355 patients

An inflammatory cytokine 1065 signature predicts COVID-19 severity and survival

Promoter Transgenes Direct Macrophage-Lineage Expression in Zebrafish

Expressions and significances of the angiotensin-converting enzyme 2 gene, the receptor of 1072 SARS-CoV-2 for COVID-19

Influenza A virus infection in zebrafish recapitulates mammalian 1076 infection and sensitivity to anti-influenza drug treatment

COVID-19: Real-time dissemination of scientific information to 1079 fight a public health emergency of international concern

A mechanistic model and therapeutic 1083 interventions for COVID-19 involving a RAS-mediated bradykinin storm

ShinyGO: a graphical gene-set enrichment tool for 1086 animals and plants

Epigenetic Contribution of High-1089

Mobility Group A Proteins to Stem Cell Properties

Differential Downregulation of ACE2 by the 1093

Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus and Human Coronavirus 1094 NL63

The Case for Modeling Human Infection in Zebrafish

COVID-19 and the 1101 cardiovascular system: Implications for risk assessment, diagnosis, and treatment options

Impaired type I interferon activity and 1106 exacerbated inflammatory responses in severe Covid-19 patients

Crystal Structure of 1109

Zebrafish Interferons I and II Reveals Conservation of Type I Interferon Structure in Vertebrates

Correlation between Olfactory Receptor Cell Type and Function in the Channel Catfish

A Multibasic Cleavage Site in the 1117

Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

SARS-CoV-2 Cell Entry 1122 Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

The zebrafish reference genome 1127 sequence and its relationship to the human genome

Angiotensin-converting enzyme 2 protects from severe acute lung failure

Is SARS-CoV-2 1134 (COVID-19) postviral olfactory dysfunction (PVOD) different from other PVOD?

ACE2 mouse models: a toolbox for cardiovascular 1139 and pulmonary research

TC1(C8orf4) Is a Novel Endothelial Inflammatory Regulator Enhancing NF-κB Activity

Infection and Rapid Transmission of SARS-CoV-2 in Ferrets

Cardiac and 1150 arrhythmic complications in patients with COVID-19

Structural and functional 1154 diversification in the teleost S100 family of calcium-binding proteins

Intranasal Vaccination with a Lentiviral Vector Strongly 1158 Protects against SARS-CoV-2 in Mouse and Golden Hamster Preclinical Models

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS 1163 coronavirus-induced lung injury

Arrhythmias and sudden cardiac death in the COVID-19 pandemic

SARS-CoV-2 1170 productively infects human gut enterocytes

Genes in Zebrafish and Humans Define an Ancient Arsenal of Antiviral Immunity

Angiopoietin-like 4 increases pulmonary tissue leakiness and 1178 damage during influenza pneumonia

The Heart in COVID-19: Primary Target or Secondary Bystander? JACC Basic 1181 to

Composition and divergence of coronavirus spike proteins and host ACE2 receptors 1185 predict potential intermediate hosts of SARS-CoV-2

Continuing versus suspending angiotensin-converting enzyme inhibitors and angiotensin 1190 receptor blockers : Impact on adverse outcomes in hospitalized patients with severe acute 1191 respiratory syndrome coronavirus 2 ( SARS-CoV-2 ) -The BRACE CORONA Trial

Longitudinal analyses reveal immunological misfiring in 1196 severe COVID-19

Whole-Body Analysis of a Viral Infection: Vascular 1200 Endothelium is a Primary Target of Infectious Hematopoietic Necrosis Virus in Zebrafish 1201 Larvae

COVID-19 preclinical models: Human 1204 angiotensin-converting enzyme 2 transgenic mice

Characterization of the 1207 immune barrier in human olfactory mucosa

Real-time tracking of self-1211 reported symptoms to predict potential COVID-19

Targeted Immunosuppression 1215 Distinguishes COVID-19 from Influenza in Moderate and Severe Disease

Respiratory disease 1220 in rhesus macaques inoculated with SARS-CoV-2

Real-Time Whole-Body Visualization 1224 of Chikungunya Virus Infection and Host Interferon Response in Zebrafish

CC Chemokine Receptor 9 Expression Defines a 1229 Subset of Peripheral Blood Lymphocytes with Mucosal T Cell Phenotype and Th1 or T-1230 Regulatory 1 Cytokine Profile

Type I and Type III Interferons -Induction

Evasion, and Application to Combat COVID-19

Angiotensin II induced proteolytic cleavage of myocardial ACE2 is 1237 mediated by TACE/ADAM-17: A positive feedback mechanism in the

Activating a Reserve Neural Stem Cell Population In Vitro 1242

A new mathematical model for relative quantification in real-time RT-1245 PCR

An intestinal cell type in zebrafish 1248 is the nexus for the SARS-CoV-2 receptor and the Renin Angiotensin-Aldosterone System that 1249 contributes to COVID-19 comorbidities

The renin-angiotensin system and diabetes: An update. Vasc. 1253 Health Risk Manag

Comparative 1257 pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model

Innate immune signaling in the 1262 olfactory epithelium reduces odorant receptor levels: modeling transient smell loss in COVID-19 1263 patients

Interplay between SARS-CoV-2 1266 and the type I interferon response

Molecular and neuronal homology between the olfactory systems of zebrafish and mouse. Sci. 1270 Rep

Tissue 1273 Microenvironments in the Nasal Epithelium of Rainbow Trout ( Oncorhynchus mykiss

Two Distinct CD8α + Cell Populations and Establish Regional Immunity

Olfactory sensory neurons 1279 mediate ultrarapid antiviral immune responses in a TrkA-dependent manner

coronavirus 2

Placenta-specific 8 limits IFNγ production by CD4 T cells in vitro and promotes establishment of 1288 influenza-specific CD8 T cells in vivo

Neuroinvasion of SARS-CoV-2 in human and mouse brain

Alterations in Smell or Taste in Mildly Symptomatic Outpatients With SARS-1296

CoV-2 Infection

Renal histopathological analysis of 26 postmortem findings of patients with 1300 COVID-19 in China

Identification of viruses in patients with postviral olfactory dysfunction

Clinical characteristics of COVID-19 in 104 people with 1309 SARS-CoV-2 infection on the Diamond Princess cruise ship: a retrospective analysis

Small molecule screening in 1313 zebrafish: An in vivo approach to identifying new chemical tools and drug leads

Proinflammatory Cytokines in the Olfactory Mucosa Result in COVID-19 Induced Anosmia

A robust human norovirus 1323 replication model in zebrafish larvae

Zebrafish 1327 studies on the vaccine candidate to COVID-19, the Spike protein: Production of antibody and 1328 adverse reaction

A rampage through the body

Neuronal Wiskott-Aldrich syndrome protein regulates 1335 TGF-β1-mediated lung vascular permeability

Receptor Recognition by the 1338 Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of 1339 SARS Coronavirus

Clinical Characteristics of 138 Hospitalized Patients with

Coronavirus-Infected Pneumonia in Wuhan, China

Remdesivir and chloroquine effectively inhibit the recently emerged novel 1348 coronavirus (2019-nCoV) in vitro

The zebrafish activating immune receptor Nitr9 signals via 1352 Dap12

SARS and MERS: 1355 Recent insights into emerging coronaviruses

SOX Transcription Factors in Endothelial 1358 Differentiation and Endothelial-Mesenchymal Transitions

Spatiotemporal photolabeling of neutrophil trafficking 1361 during inflammation in live zebrafish

Regeneration and rewiring of rodent olfactory sensory neurons

Severe Acute Respiratory Syndrome Coronavirus 2 Infects and 1368 Damages the Mature and Immature Olfactory Sensory Neurons of Hamsters

Angiomotin-like protein 1 controls endothelial 1374 polarity and junction stability during sprouting angiogenesis

Metascape provides a biologist-oriented resource for the analysis of 1378 systems-level datasets

Clinical 1381 characteristics of 3062 COVID-19 patients: A meta-analysis

SARS-CoV-2 Receptor ACE2 Is an

Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell 1386 Subsets across Tissues

The function of fish cytokines

Single-cell RNA-seq data 1391 analysis on the receptor ACE2 expression reveals the potential risk of different human organs 1392 vulnerable to 2019-nCoV infection