The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has proved to be a deadly and rapidly spreading plague, with the death count nearing three million so far. A new preprint research paper posted to the bioRxiv* server describes the conservation of an important receptor gene for the virus among several animal species that live in close contact or proximity to humans.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
The ACE2 receptor
This virus enters host cells via the angiotensin-converting enzyme 2 (ACE2) receptor using its spike protein. The virus shows extensive similarity or near-identity with coronaviruses (CoVs) isolated from multiple other species. This has led to the hypothesis that it probably originated from the bat CoVs and jumped over the species barrier to infect humans via an intermediate host as yet unknown.
The ACE2 receptor was first reported in 2000, with an ~800 amino acid sequence. It is found in various animal species and in multiple animal tissues, including the small and large intestines, the kidneys, testes, and heart. The viral spike engages the receptor through its receptor-binding domain (RBD).
The ACE2 receptor is part of the cardiovascular and osmoregulatory system, involving electrolyte and water homeostasis and blood pressure regulation. The viral RBD binds to the peptidase domain of the receptor from different animals, providing an important method of tracking the origin of this virus.
Moreover, identifying structural and sequence differences in the ACE2 found in different animals may affect the host susceptibility and the immune response to the virus.
Study aims
The study, therefore, aimed to uncover the conservation of this gene in 11 animal species, as well as to describe its expression at ribonucleic acid (RNA) or transcription level, and at protein synthesis (translation) level in three species – wild bats (Hipposideros pomona), mice (Rattus norvegicus) and tree shrew (Tupaia belangeri).
Wild mice and bats are found together in the forest habitat near PuEr and Kunming in China, which led to their use in this study.
ACE2 gene conservation
The researchers found that the ACE2 genes were conserved among the 11 selected animal species, at levels between 85% between wild mice or bats and humans, to 88% between wild rabbits and humans, with the others falling within this range.
Phylogenetically, the ACE2 gene sequence was suggestive of the clustering of wild bat and tree shrew receptors near the human receptor, while the wild mice, C57 and BALB/C formed a close bunch by themselves. Wild mice and Sprague-Dawley Rat formed another tight cluster. Rabbits were in a separate clade.
Differences in RBD-binding polar ACE2 residues
There were 18 residues on all species of ACE2 that take part in binding with the SARS-CoV-2 RBD. Of these, 14 were polar, and four non-polar. Nine of them were highly conserved and may be crucial to the high binding affinity with this virus.
Five residues were not conserved, which may have an even more significant impact on binding affinity. For all species, the ACE2 proteins had similar sequences to humans, from 80% for wild bat ACE2 to 85% for the rabbit ACE2.
The 14 key polar residues formed different sequences at the binding interface in different species. The binding free energy of each virus-receptor protein complex and the stability were
Distribution of ACE2 mRNA
The phylogenetic tree showed that the wild bat and the tree shrew had the closest ACE2 sequence to humans. Wild mice were added to this group, as they were found in the same area as the bats.
RNA from various tissues showed different ACE2 expressions in each tissue and each animal. The stomach of wild bats had high levels of ACE2 mRNA, followed by the kidney, liver, colon and duodenum, trachea, heart, and finally, the spleen and brain.
In the tree shrew, ACE2 mRNA was highest in the colon and then the duodenum, with the kidney, heart, stomach, and trachea following these organs
With wild mice, the highest levels were in the stomach, then the duodenum and colon, followed by the kidney, trachea, heart, lung. In both tree shrews and wild mice, the brain, liver, and spleen had the lowest levels.
Distribution of ACE2 protein
High ACE2 protein levels were seen in the liver in wild bats, as well as kidneys, large and small bowel, and stomach, with the trachea, indicating increased susceptibility to infection by this virus.
In wild mice, the highest levels were in the kidney, colon, duodenum, stomach, and trachea, and the most vulnerable organs were the colon, duodenum, and kidney.
What are the implications?
This study used actual samples from domestic, wild and laboratory animals to explore the conservation of this gene in different animal species, as well as the level of expression of this protein. The results showed high levels of conservation of the ACE2 sequences across the tested mammalian species, congruous with earlier studies.
This implies the existence of multiple different animal hosts for SARS-CoV-2.
The closest phylogenetic relationship was between humans, wild bats and tree shrews. The tree shrew has been used as a useful animal model of many different viral infections, such as herpes simplex, influenza, Zika virus, and hepatitis C virus, and shows potential to be a model for SARS-CoV-2.
The study also shows the length of amino acid sequences unrelated to the presence or sequence of the 14 key polar amino acids forming the binding interface in different species. The greatest similarity among these residues was with the golden hamster.
According to these findings, the expression of ACE2 at both mRNA and protein level shows significant tissue specificity but are not necessarily consistent with each other. Further study will be necessary to understand how these are related to species susceptibility to the virus.
*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
New study challenges traditional view of how the genetic code evolved