Zebrafish models of COVID-19

Abstract

Although COVID-19 has only recently appeared, research studies have already developed and implemented many animal models for deciphering the secrets of the disease and provided insights into the biology of SARS-CoV-2. However, there are several major factors that complicate the study of this virus in model organisms, such as the poor infectivity of clinical isolates of SARS-CoV-2 in some model species, and the absence of persistent infection, immunopathology, severe acute respiratory distress syndrome, and, in general, all the systemic complications which characterize COVID-19 clinically. Another important limitation is that SARS-CoV-2 mainly causes severe COVID-19 in older people with comorbidities, which represents a serious problem when attempting to use young and immunologically naïve laboratory animals in COVID-19 testing. We review here the main animal models developed so far to study COVID-19 and the unique advantages of the zebrafish model that may help to contribute to understand this disease, in particular to the identification and repurposing of drugs to treat COVID-19, to reveal the mechanism of action and side-effects of Spike-based vaccines, and to decipher the high susceptibility of aged people to COVID-19.

Keywords: SARS-CoV-2; animal models; drug repurposing; senescence; telomeres; zebrafish.

Figure 1.

SARS-CoV-2 structure. (A) Structure of single virion of SARS-CoV-2 including structural proteins S, M, E, and N as well as the single stranded RNA. (B) Schematic representation of the genomic structure of SARS-CoV-2 genome with individual components of S protein (not to scale). E—envelope protein gene; M—membrane protein gene; N—nucleocapsid protein gene, S—spike protein gene; SP—signal peptide; NTD—N-terminal domain; RBD—receptor binding domain; FP—fusion protein; HR1- heptad repeat 1; CH—central helix; CR—connecting region; HR2–heptad repeat 2; TD—transmembrane domain; * protease cleavage sites (including furin cleavage site).

Figure 2.

Multiple sequence alignment of different ACE2 sequences. Sequences of human ACE2 (Q9BYF1), rhesus macaque (F7AH40), mice (Q8R0I0), hamster (A0A1U7QTA1), cat (Q56H28), European domestic ferret (Q2WG88), and zebrafish (E7F9E5) were obtained from UniProt, aligned using CLUTAL OMEGA and represented using ESPript (Gouet et al. 1999). Strictly conserved residues have a red background. Symbols above the human sequence blocks represent secondary structure, springs represent helices and arrows represent β-strands.

Figure 3.

Analysis of COVID-19 vaccines and immunopathology in zebrafish larvae. Normal and aged larvae are obtained from wild type (wt, tert^+/+) and telomerase-deficient (tert^−/−) lines, respectively. Larvae of 2 days post-fertilization are then injected with recombinant spike variants or infected with SARS-COV-2, and several phenotypes analyzed with the indicated reporter lines at different times post-inoculation.

Figure 4.

Chemical screening in zebrafish larvae to identify antiviral compounds to treat COVID-19. Normal and aged larvae are obtained from transgenic fish expressing hACE2 driven by the ubiquitin promoter [Tg(ubi:hACE2)] and an NF-κB reporter [Tg(NFκB-ER:eGFP)]. Larvae of 2 days post-fertilization (dpf) are then infected with SARS-COV-2, distributed in 96-well plates, treated with FDA-approved or natural chemical libraries, and NF-kB analyzed at 1–3 days-post-infection (dpi). Positive hits can reduce NF-kB activation, easily assayed as decreased green fluorescence. Three classes of compounds will be identified: group 1 reduce inflammation in normal and aged larvae (violet), group 2 reduce inflammation in normal larvae (blue), and group 3 reduce inflammation in aged larvae (orange).