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A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2

Aurora Kraus, Elisa Casadei, Mar Huertas, Chunyan Ye, Steven Bradfute, Pierre Boudinot, Jean-Pierre Levraud, Irene Salinas

Preprint posted on November 08, 2020 https://www.biorxiv.org/content/10.1101/2020.11.06.368191v1

Zebrafish- a low cost, high-throughput choice to study COVID-19-related cardiac and olfactory pathology.

Selected by Mariana De Niz

Categories: immunology, microbiology

Background

Development of laboratory animal models that recapitulate the pathophysiology of SARS-CoV-2 infection in humans would be a significant step to accelerate drug and vaccine testing. Animals that have shown susceptibility to SARS-CoV-2 include rhesus and cynomolgus macaques, ferrets, cats, and Syrian hamsters. Moreover, mice expressing the human ACE2 receptor are also being explored as a potentially useful animal model. While these model organisms pose advantages and disadvantages in the study of host-pathogen interactions, they do not allow rapid, whole organism, high-throughput and low-cost preclinical testing of drugs and immunotherapies. Zebrafish have been used as model organisms that are permissive to human viral pathogens, as they offer many advantages over other animal models due to their high reproductive ability, rapid development, low maintenance costs, and small transparent bodies. Zebrafish has already been used to study vaccine development in the context of COVID-19. In their work, Kraus et al (1) explore the physiopathology of wildtype larval and adult zebrafish in response to SARS-CoV-2 and perform pre-clinical drug testing and validation in this inexpensive, high throughput vertebrate model.

Figure 1. H&E stains of zebrafish olfactory organ paraffin sections of zebrafish that received intranasal delivery of SARS-CoV-2 S RBD protein (From Ref. 1).

 

Key findings and developments

SARS-CoV-2 enters the human host cells when SARS-CoV-2 Spike (S) protein receptor binding domain (RBD) binds to angiotensin-converting enzyme 2 (ACE2) on a permissive host cell. ACE2 is expressed in many different cell types across many organs in the human body. Kraus et al began by performing phylogenetic analyses of ACE2 molecules in vertebrates. They found around 70% conservation between vertebrate ACE2 and human ACE2, and around 60% conservation between zebrafish ACE2 and human ACE2. Further examination of the ACE2 amino acid motifs in the region involved in SARS-CoV-2 S protein binding showed that zebrafish have 50/64% similarity with the corresponding human ACE2 region. This is lower similarity than that observed between other mammals and humans.

Cardiac effects of SARS-CoV-2 exposure in larval zebrafish

Next, they went on to investigate the effects of SARS-CoV-2 spike (S) receptor binding domain on zebrafish larvae. Previous studies had shown that SARS-CoV-2 induce toxicity in zebrafish adults (2). Measurement of cytokine responses in zebrafish larvae showed that ifnphi1 expression, the main type I IFN gene in zebrafish, is significantly downregulated, while the pro-inflammatory cytokine ccl20a.3 was significantly upregulated, even after short exposure to SARS-CoV-2. Upon analysis of larva heart function upon exposure to SARS-CoV-2 S receptor binding domain (RBD) to determine if cardiac manifestations are observed, the authors showed that exposure results in significantly higher heart rates. To determine if the increased heart rate was dependent on ACE2 binding, they went on to explore the effects of captopril, an inhibitor of ACE, used as treatment for various cardiac disorders. Their observations point towards a potential cardioprotective role of captopril in COVID-19, as well as presenting zebrafish as potentially useful models for clinical testing.

Olfactory effects of SARS-CoV-2 intranasal exposure in adult zebrafish

The authors began by exploring the effect of SARS-CoV-2 exposure on the olfactory epithelium of zebrafish. They delivered SARS-CoV-2 S RBD to the nasal cavity of adult zebrafish and sampled the olfactory organ at various time points post-exposure. They found that SARS-CoV-2 S RBD caused severe damage to the olfactory organ at all time points analysed. This severe damage included oedema and endothelial inflammation shortly after exposure, while hemorrhages, and loss of structures due to necrosis were observed after more prolonged exposure. At all time points, loss of olfactory cilia was observed. Importantly, they conclude that this severe damage can be caused even in the absence of viral replication in the tissue.

Renal effects of SARS-CoV-2 intranasal exposure in adult zebrafish

The kidney is also a target organ of SARS-CoV-2, and acute kidney damage has been shown in up to 29% of patients with COVID-19. Kidneys of fish exposed to SARS-CoV-2 by intranasal delivery showed features of acute kidney injury. The authors conclude that intranasal delivery of SARS-CoV-2 S RBD is sufficient to cause nephropathy in adult zebrafish but that pathology is not as severe as when the protein is delivered by injection.

Intranasal delivery of SARS-CoV-2 S RBD causes anosmia in adult zebrafish

Adult zebrafish exposed to SARS-CoV-2 S RBD were shown to have a significant reduction (of up to 50%) of olfactory responses to food extracts within minutes. The authors then studied in detail the effects of exposure, in two isolated olfactory chambers, on food extracts and bile. They found that anosmia (loss of smell) is not specific for a subset of olfactory sensory neurons as both, food and bile extracts were suppressed upon treatment with SARS-CoV-2 S RBD. The time lapse study the authors performed suggests that SARS-CoV-2 S RBD damage may occur first on sustentacular cells, with subsequent impacts on OSN viability and function.

Single-cell analysis of the zebrafish olfactory organ

To understand the impact of SARS-CoV-2 S RBD on the zebrafish olfactory organ, the authors performed single cell RNA-Seq of adult zebrafish olfactory organ from control or SARS-CoV-2 S RBD intranasally-treated fish, at 3 hours and 3 days following intranasal administration. They identified 4 main groups of clusters, namely, 8 neuronal cell clusters, 5 sustentacular cell clusters, 3 endothelial cell clusters, and 7 leukocyte clusters.

Induction of inflammatory responses and widespread loss of olfactory receptor expression

The single cell RNA-Seq analysis of zebrafish receiving intranasal delivery of SARS-CoV-2 S RBD identified a range of changes across clusters, indicating altogether important cellular responses in neuronal and immune cell subsets of the zebrafish olfactory organ. These changes are related to induction of inflammatory responses and widespread loss of olfactory receptor expression in the adult zebrafish olfactory organ. Examples include significantly increased expression in the endothelial cell cluster, of several cytokine and prostaglandin genes, and increased expression in the myeloid cluster of interferon-related genes, and specific inflammatory markers and chemokine receptors. Moreover, the authors found significant changes in genes related to endothelial integrity, vasoconstriction and clotting factors.  Overall, the data suggested that inflammatory responses and endothelial disruption are both hallmarks of SARS-CoV-2 induced damage in the olfactory epithelium. Gene ontology analyses and functional enrichment analyses of genes up- or down-regulated at 3 hours or 3 days post-exposure to SARS-CoV-2 S RBD point towards harmful effects on the olfactory sensory neurons already within hours, and that the magnitude of the damage to these neurons increases with time. Moreover, the findings also suggest that neuronal regeneration and differentiation processes are initiated by day 3, to begin repair of olfactory damage.

Altered responses in sustentacular cells and endothelial cell clusters

Previous work by others has shown that co-expression of ACE2 and TMPRSS2 by olfactory sustentacular cells and basal cells in the human olfactory epithelium and subsequent onset of damage and inflammation in these cells, may explain olfactory loss in COVID-19 patients (3,4). In their present study, the authors dissected how each cell type in the zebrafish olfactory organ responds to SARS-CoV-2 S RBD. They describe the main changes occurring after 3 hours or 3 days of exposure. After 3 hours, the GO and enrichment analyses suggest pathways altered include metabolic responses, response to stress, and cell differentiation. This is followed by transcriptional changes with potential vasoactive effects by day 3, whereby enriched genes are not only those involved in innate responses, but also those involved in response to wounding.

Effects of exposure of zebrafish larvae to SARS-CoV-2

Zebrafish larvae do not support viral replication

After determining SARS-CoV-2 survival in zebrafish water, the authors exposed zebrafish larvae to water containing live SARS-CoV-2 and examined viral mRNA abundance over time to determine if zebrafish larva can support viral replication.  They detected no increases in viral copy number over time. The results indicate that wild-type zebrafish larvae cannot support efficient SARS-CoV-2 replication as suggested by the in silico comparative sequence analyses of the zebrafish ace2 molecule.

Decrease of ace2 expression and trigger of pro-inflammatory cytokine responses

The authors found that in exposed zebrafish larvae, ace2 expression was significantly downregulated as early as 6 h post-infection, and was sustained for various days post-infection. Examination chemokine and cytokine responses suggest that exposure to SARS-CoV-2 induces a significant antiviral and pro-inflammatory immune response in zebrafish larvae involvingtype I IFN, tnfa, il1b, il17 and ccl20, reminiscent of COVID-19 patients with mild disease.

 

What I like about this preprint

Coming from a field where many main findings have come from mammalian animal models, I find work done in other animal models very interesting. Sometimes the simpler models are overlooked based on the assumption that they might not serve as good surrogates of human pathology. On the other hand, zebrafish has already proven to be an outstanding model for many devastating human infectious diseases and therefore, it is not surprising that COVID-19 is now on that list. Together, I like this work because it explores an alternative animal model for the current pandemic which can accelerate preclinical drug testing with in vivo physiologically relevant readouts. The preprint also successfully identifies the strengths and limitations of the model and pinpoints how this model can continue to be refined.

References

  1. Kraus et al, A zebrafish model for COVID-19 recapitulates olfactory and cardiovascular pathophysiologies caused by SARS-CoV-2, bioRxiv,
  2. Ventura Fernandes et al, Zebrafish studies on the vaccine candidate to COVID-19, the Spike protein: Production of antibody and adverse reaction, bioRxiv, 2020.
  3. Brann et al, Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia, Adv. 2020.
  4. Cooper et al, COVID-19 and the chemical senses: supporting players take center stage, Neuron, 2020.

 

 

 

Posted on: 4th December 2020

doi: https://doi.org/10.1242/prelights.26132

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Author's response

Irene Salinas shared

Open questions 

1.You mention throughout your work, the use of larvae zebrafish, and adult zebrafish. This might be a naïve question: in some animal models, the age of the animal during experimentation may or may not reproduce pathology specific to certain ages in humans, for example malaria in children (modeled in young rodents) as opposed to adult malaria (modeled in adult mice). Do you think the age of the zebrafish might represent different findings of pathology of COVID-19 in young versus adult patients?

Yes, this is one thing I was always very intrigued about our work. The larval model is useful to investigate innate immune responses only because larvae do not have a fully developed adaptive immune system. Children, however, already have an adaptive immune system so the direct comparison is not quite perfect there. In adults, B and T cells will respond to the virus and therefore I anticipate that the immune response of infected adults would be quite different to what we have described in larvae.

 

2.You mention in your results that examination of the ACE2 amino acid motifs in the region involved in SARS-CoV-2 S protein binding showed that zebrafish have 50/64% similarity with the corresponding human ACE2 region. Another naïve question: what are the limitations of such similarity levels?

This level of similarity suggests the low probability of SARS-CoV-2 S protein binding zebrafish ACE2. This may be the reason why we do not see any active viral replication in our infections and should motivate the generation of transgenic animals that express human ACE2, in a similar way to what has been done in mice.

 

3.You go into detail in the analysis of the olfactory cavity and renal pathology upon nasal exposure to SARS-CoV-2 S RBD. Are these changes reversible? Did you/will you study the long term consequences of exposure? For instance, the effects on the olfactory organs seem very severe. Moreover, similar to your experiments with captopril, are there any protective drugs for the olfactory organs in specific?

This is certainly something we are currently working on. The recovery of olfaction experiments are underway and we should have an answer in a couple of weeks. Testing drugs or other interventions that accelerate recovery of smell is something we haven’t started exploring yet but we would obviously love to do.

 

4.You discuss in your work the results of single cell RNA-sequencing. Among the main findings you cover in more detail are cytokine and chemokine responses. You briefly mentioned across your results, that the finding on chemokines is interesting given that this might be mediating directed motility of cells. This is a very interesting topic. Can you expand a bit more on how this can be further studied to understand COVID-19-related pathology?

Chemokines along with cytokines have been shown to be associated with disease severity in covid-19 patients since they are part of the inflammatory response. Further, chemokines as you mention guide cells (both neurons and immune cells) during development and during the response to any insult. Thus, we think that chemokine responses in the olfactory organ are very important in the context of COVID-19 as shown in our single cell data set. My guess is that they have multiple functions ranging from recruitment of immune cells to tissue repair and even neuronal regeneration.

 

4. A big question currently in this pandemic is why some people are relatively asymptomatic, while others develop mild symptoms, others, severe symptoms yet recover, and yet others develop symptoms and do not survive. Did you observe high variation between fish in all the various parameters you studied, including survival? Are there any clues that zebrafish might provide on the severity of symptoms developed?

Our zebrafish were all genetically similar (larvae came from crosses of 2-3 males and females in one batch crossing). We did see some variability in the innate immune responses but not a huge amount, in part because we did perform experiments in pools of 5 larvae, for instance. In the case of the adults, the damage was also very consistent in every individual examined. I believe that we will observe differential disease severity once we start making the humanized ace2 animals. We can drive human ace2 expression under different promoters which will determine how much virus infects the zebrafish host. As an example, expression of human ace2 under a ubiquitous promoter will likely result in severe disease whereas doing it using a tissue specific promoter may result in moderate or mild disease, similar to disease severities reported in humans. As a parallelism, it is thought that children may have less symptoms because ace2 levels in the children nasal mucosa are lower compared to humans.

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