So far, the origin and intermediate host of the new coronavirus remains a mystery, and its ability to infect pets and livestock remains unclear. Recently, the Ministry of Education”, “Animal Health and Food Safety” joint laboratory for international cooperation and other teams found that the known infection of the new coronavirus species ACE2 (angiotensin conversion enzyme 2) can withstand many amino acid changes, indicating that the barrier of their species may be low, that is, cross-species transmission risk is greater. At the same time, the new coronavirus may not be specifically adapted to any of its assumed intermediate hosts.
The team analyzed the X-ray structure of the newly released human ACE2 binding domain (RBD) of the S-protein receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) to predict the binding of ACE2 to viral proteins in different pets, farm animals, the hypothetical intermediate host of the virus, etc.
Effective binding to host receptor ACE2 is the primary prerequisite for the new coronavirus infection of cells, which determines the range of virus hosts. The researchers found that ACE2, bats, psychics, etc. had a large number of amino acid changes compared to human ACE2, but was still able to bind to the new coronavirus S protein, in which bat ACE2 protein contained at least five amino acid changes.
The study noted that pets (cats) and domestic animals are at risk of being infected by SARS-CoV-2 because they have less change in amino acids at the ACE2-S protein interface than other animals.
At the same time, pangolin ACE2 shows seven variations in relation to human ACE2, with a similar number of replacements in ACE2 for bats, raccoons, and lingua del sacs, suggesting that SARS-CoV-2 may not be particularly adapted to ace2 of a hypothetical intermediate host, i.e. the mechanism of new coronavirus infected animals may differ from those of infected humans.
These analyses provide new insights into SARS-COV-2 intrusion receptors and their animal sources and natural origin. The study was published online recently on bioRxiv, a bioprenomy platform entitled “Comparison of SARS-CoV-2 spike protein to human, pet, farm animals, and putative intermediate hosts ACE2 and ACE2 tions”.
The research team includes the Joint Laboratory for International Cooperation on Animal Health and Food Safety of the Ministry of Education of Nanjing Agricultural University, the Institute of Virology of the Faculty of Veterinary Medicine of the Free University of Berlin, the Changchun Institute of Veterinary Medicine of the Chinese Academy of Agricultural Sciences, and the authors of the study are Professor and Doctoral Mentor of the School of Animal Medicine of Nanjing Agricultural University, And Michael Veit of the Free University of Berlin.
ACE2, such as bats and cats, can withstand a large number of amino acid changes and is still bound to the new coronal S protein
Studies have shown that SARS-CoV-2 may originate in bats, and pangolins may be potential intermediate hosts. Specifically, SARS-CoV-2 is consistent with the bat coronavirus RaTG13, but the middle part of its genome, the gene that encodes the virus S protein, may have been recombined with pangolin-like coronavirus.
The S protein of the new coronavirus is responsible for binding to the receptor to invade the host, and during infection, the S protein is lysed by the host protease to S1 subunit and S2 subunit, respectively, mediated receptor binding and membrane fusion. S1 contains the receptor binding domain (RBD), which is critical in determining the tissue-to-host and host range.
When the virus invades the human body, RBD binds to the human receptor ACE2 (angiotensin conversion enzyme 2). ACE2 is a human receptor membrane protein common to the new coronavirus and SARS-CoV (Severe Acute Respiratory Syndrome coronavirus), which has homologous nature with angiotensin-converting enzyme 1 and shows protective effects in the human cardiovascular system and many other organs.
Previous studies have shown that SARS-CoV-2 replicates poorly in dogs and pigs, but cats can be infected. Using comparative bioinformatics and structural methods, the researchers analyzed the X-ray structure of HUMAN ACE2 and the viral S protein structure in humans bound to the S-protein receptor binding domain of SARS-CoV-2.
ACE2 in humans, pigs, psychiccats and bats is susceptible to SARS-CoV-2, but ACE2 in mice is not sensitive due to different transfections in the Hela cell lines containing encoded ACE2 receptors.
To assess which interacting amino acids in ACE2 are essential for their binding to the virus S protein, the researchers compared the human ACE2 sequence with the ACE2 sequence of other species, and then identified amino acid sites that are critical to the binding of the virus to ACE2.
Studies have shown that 17 residues of the S protein were in contact with 20 amino acids in ACE2 when the new coronavirus invaded, of which 8 amino acids formed a hydrogen bond with 13 residual bases in the S protein.
The interaction surface of SARS-CoV-2 S protein (green) and human ACE2 (blue)
According to previous studies, the S protein of SARS-CoV-2 has a higher binding affinity to its receptors than the S protein of SARS-CoV. Amino acids in SARS-CoV-2 form more hydrogen bonds than when interacting with ACE2.
The researchers found that ace2 in pigs had five amino acids on the surface of the interaction with SARS-CoV-2’s S protein compared to human ACE2, three of which were located at the periphery of the binding site.
Compared to human ACE2, the pig’s ACE2 is replaced by amino acids present on the surface of the S protein interaction with SARS-CoV-2, 5 replacing positionred in red
The researchers extracted three groups of Chinese chrysanthemum manta rays from the database, from Guangxi, Hubei and Yunnan. Surprisingly, although their overall amino acid homogeneity was high (99%), large differences were shown in N-end amino acids in contact with the virus S protein.
The researchers found that bat ACE2 protein compared to human ACE2 contained at least five amino acid variations, three of which were replaced in all bat ACE2 sequences and had the same residue as in pig ACE2, but were replaced by other amino acids. The team believes that the ACE2 gene between chinese chrysanthemum bats in different regions, which is widely regarded as the natural host of the new coronavirus, is surprising and requires further study. However, one of the more easily speculated reasons is that local coevolution between bats and neo-coronaviruses drives these amino acid changes.
Next, the researchers analyzed ACE2 in the cat, which has been shown to be a receptor for SARS-CoV-2. The ace 2 of the lingus has 7 amino acid variations compared to human ACE2, three of which are the same as the replacements in ACE2 in pigs.
Compared to pigs, bats, and psychic cats, ACE2 in mice that do not support SARS-CoV-2 cell infection has eight amino acids on the surface that interact with the SARS-CoV-2 S protein.
In short, some animalACE2 compared with human ACE2, and the new coronavirus S protein interaction surface of a large number of amino acid changes do not affect its binding, of which pig ACE2 has 5 amino acids replaced, while the cat ACE2 has 7 amino acid changes.
Even if Bat ACE2 acquires a new and important N-glycosylation site during multiple amino acid substitutions, it will not prevent SARS-CoV-2 from using bat ACE2 as a receptor in transfection cells.
Can the new crown cross the species barrier? Cross-species transmission risk is high and requires continuous monitoring
Earlier, on April 3, Professor Jin Meilin of Huazhong Agricultural University and Professor Shi Zhengli of the Wuhan Virus Institute of the Chinese Academy of Sciences, in collaboration with the team of professor Shi Zhengli of the Chinese Academy of Sciences, published a research paper on bioRxiv, saying that serum ELISA tests from 102 cats showed that serum from 15 cats (14.7%) tested positive for the receptor binding domain (RB) of the neo-coronavirus. But so far, there is no evidence that cats can infect people.
In this study, the researchers also looked at the condition sedation of species disorder (cross-species transmission) in pets when the new coronavirus S protein is combined with receptor ACE2. Compared to humans, the amino acids in which ACE2 in dogs come into contact with the virus S protein contain 5 amino acid variations, three of which were replaced in ACE2 in pigs.
Amino acid changes in dog ACE2 in contact with virus S protein
Compared to humans, cat ACE2 has only four amino acid variations, and they are also present in ACE2 in dogs. The researchers previously analyzed the ability of ACE2 from pigs, dogs and cats to bind to the virus S protein, and found that SARS-CoV-2 replicates less in dogs, but is effective lysing in cats and spread ingested to other cats.
It was previously reported that tigers and lions at the Bronx Zoo in New York City were infected with SARS-CoV-2. So the researchers analyzed their ACE2 genes. In ACE2 in cats and tigers, researchers detected one amino acid difference, but they were the same as the residuals of the virus S protein exposure, which explains why tigers are also susceptible to SARS-CoV-2 infection.
Compared to Cat ACE2, the lion’s ACE2 has another more conservative change, and like dog ACE2, one of its N-glycosylation sites is missing.
The researchers then analyzed animal models showing several animals with a higher risk of spreading the new crown. Recent studies have shown that ferrets are more sensitive to SARS-CoV-2 infections and, although less efficient, can spread the virus through droplets. The results showed that ferret ACE2 showed the exact same 5 amino acid variations as ace2 in the dog.
In addition, the Syrian hamster’s ACE2 protein contains only two amino acid substitutions compared to human ACE2, and as an animal model of higher neo-coronavirus susceptibility, and as a pet of part of the family, the guinea pig ACE2 has seven amino acids to replace.
In farm animals, chicken’s ACE2 protein contains 10 amino acid variations and loses an N-glycosylation site at a level of 90. Ducks’ ACE2 also has 10 amino acids to replace, and studies suggest that the lack of sensitivity to experimental SARS-CoV-2 appears to be due to
Viruses do not bind to poultry ACE2 receptors. Ace2 in cattle and sheep has only two amino acid variations compared to humans, and retains the N-glycosylation site at the 90 position of human ACE2. As a result, both species of ACE2 proteins are likely to act as SARS-CoV-2 receptors and are likely to be highly susceptible animal models, and the researchers note that enhanced monitoring is needed to show whether they are susceptible to SARS-CoV-2.
Overall, almost all known mammalian species (cats and ferrets) known to be susceptible to SARS-CoV-2 infection have mutations in many amino acids of their ACE2 proteins. This suggests that these species, especially those in close contact with humans, are at risk of contracting the virus, and SARS-CoV-2 may establish an additional reservoir of viruses in one of the animals.
The researchers also found that pigs were not infected with new coronaviruses and dogs were not effectively infected, and that the combination of ACE2 in the two animals was only a small change from those in humans, but that ace2 levels in their respiratory tracts were relatively low.
It also suggests that the binding of the virus’s S protein to the ACE2 receptor is only the first step in the virus’s invasion, and that the level of ACE2 in different tissues of the same species may also play an important role in viral transmission, for example, ACE2 is less expressive in the upper respiratory tract of some pets and livestock. Therefore, the team believes that the risk of infection in animals with the neo-coronavirus needs to be continuously monitored to explore the source of infection in animals and the potential risk of cross-species transmission.