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Molecular analysis of binding region of an ACE2 as a receptor for SARS-CoV-2 between humans and mammals

10 Jul 2020-bioRxiv (Cold Spring Harbor Laboratory)-

TL;DR: This work investigated the structural homology of angiotensin-converting enzyme 2 (ACE2), a host-side receptor for SARS-CoV-2, between humans and other species including salmon and mink, and confirmed a highStructural homology between ACE2 and minks was confirmed, however, a non-high structuralHomology of ACE2 between salmon and humans was observed.
Abstract: In June 2020, a second wave of coronavirus disease-2019 (COVID-19) infections raised concern in Beijing, where salmon sold a fresh fish wholesale market was suspected of being the source of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. It has raised questions in the press and elsewhere about the scientific basis of salmon as a source of infection. With the number of cases growing, the surface of a salmon chopping board in the market was examined for the presence of SARS-CoV-2 and a positive reaction was observed. Following these test results, there has been debate over whether salmon can be infected with SARS-CoV-2. To find assess this, we investigated the structural homology of angiotensin-converting enzyme 2 (ACE2), a host-side receptor for SARS-CoV-2, between humans and other species including salmon and mink. As a result, a high structural homology between ACE2 and mink, which has reportedly transmitted SARS-CoV-2 to humans, was confirmed. However, a non-high structural homology of ACE2 between salmon and humans was observed. Further experiments are needed to find the source of SARS-CoV-2 transmission to the salmon.
Topics: Mink (53%), Coronavirus (51%)

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Hayashi et al. 7/2/2020 1:45 PM 1
Molecular analysis of binding region of an ACE2 as a receptor for SARS-CoV-2 between
humans and mammals
Takuma Hayashi
1, *
, Kaoru Aboko
1
, Masaki Mandan
2
, Nobuo Yaegashi
3
, Ikuo Konishi
1,2,4
1
National Hospital Organization Kyoto Medical Center, Kyoto, Kyoto, JAPAN
2
Department of Obstetrics and Gynecology, Kyoto University School of Medicine, Kyoto, JAPAN
3
Department of Obstetrics and Gynecology, Tohoku University School of Medicine, Miyagi, JAPAN
4
Immediate Past President of Asian Society of Gynecologic Oncology, Tokyo, JAPAN
Running Title:
A highly conserved binding region of an ACE2 as a receptor for SARS-CoV-2 between humans
and mammals
Keywords:
ACE2, SARS-CoV-2, COVID-19, binding region, mink, salmon
*
Corresponding Author
Takuma Hayashi D.M.Sci., GMRC, MS
National Hospital Organization, Kyoto Medical Center
Fukakusa Mukai-Cho, Fushimi-Ku, Kyoto-city, Kyoto, Japan.
E-mail: yoyoyo224@hotmail.com
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.09.196378doi: bioRxiv preprint

Hayashi et al. 7/2/2020 1:45 PM 2
Abstract
In June 2020, a second wave of coronavirus disease-2019 (COVID-19) infections raised concern in
Beijing, where salmon sold a fresh fish wholesale market was suspected of being the source of severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. It has raised questions in the
press and elsewhere about the scientific basis of salmon as a source of infection. With the number of
cases growing, the surface of a salmon chopping board in the market was examined for the presence
of SARS-CoV-2 and a positive reaction was observed. Following these test results, there has been
debate over whether salmon can be infected with SARS-CoV-2. To find assess this, we investigated
the structural homology of angiotensin-converting enzyme 2 (ACE2), a host-side receptor for SARS-
CoV-2, between humans and other species including salmon and mink. As a result, a high structural
homology between ACE2 and mink, which has reportedly transmitted SARS-CoV-2 to humans, was
confirmed. However, a non-high structural homology of ACE2 between salmon and humans was
observed. Further experiments are needed to find the source of SARS-CoV-2 transmission to the
salmon.
Introduction
After approximately two months during which no new cases of severe acute respiratory syndrome
coronavirus-type 2 (SARS-CoV-2) had been observed in Beijing, a new outbreak in the city was
identified in mid-June 2020. With more than 100 coronavirus disease 2019 (COVID-19) infections
reported in the days following June 12, Beijing was forced to raise its emergency response level (1,2).
The source of the infections is believed to have originated at a fresh fish market in Beijing.
Chairman Zhang Yuan, of the Beijing Fresh Fish Wholesale Market, reported that a sampling survey
conducted by China’s National Institute of Health had detected the presence of SARS-CoV-2 on a
chopping board used for cutting up imported salmon sourced from a deep seafood market in Beijing.
On June 13, a spokesman for the Beijing Health Commission reported that 40 samples had been
confirmed positive for SARS-CoV-2 at the Beijing Fresh Fish Wholesale Market. At the same time,
as the Chinese public became aware of the new cases, salmon was quickly removed from sale at
supermarkets and in Japanese restaurants in the city.
At this time, there is no scientific evidence that salmon can be infected with SARS-CoV-2 or carry
the pathogen; therefore, the likelihood of transmission to humans from fish contaminated with SARS-
CoV-2 is extremely low. Instead, it is highly possible that the surface of the fish or the cutting board
had been contaminated.
In April 2020, 16 international organizations, including the Food and Agriculture Organization of the
United Nations and the Chinese Fisheries Science Research Institute, jointly published a paper in
Asian Fisheries Science to assert that no evidence had indicated that marine products, including fish,
crustaceans, mollusks, and amphibians, could be infected with SARS-CoV-2.
It is known that the five genera of coronaviruses only infect birds and mammals, while the beta
coronaviruses to which SARS-CoV-2 belongs are believed to infect mammals alone. Therefore,
marine products are unlikely to be the source of the new infections.
On June 10, 2020, mink infected with SARS-CoV-2 were found in more than 10 farms in the
Netherlands (3). The following month, the Dutch Ministry of Agriculture reported the transmission
of SARS-CoV-2 from mink at one of these farms, to become the first recorded case of an animal
infecting a human (3).
In this COVID-19 pandemic, SARS-CoV-2 infections of species other than mammals and birds have
not been observed. A common amino acid locus with angiotensin-converting enzyme 2 (ACE2), a
host-side receptor for mammalian SARS-CoV-2, has been found in thermophilic animals (4,5,6).
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.09.196378doi: bioRxiv preprint

Hayashi et al. 7/2/2020 1:45 PM 3
Therefore, we investigated the molecular structural homology of ACE2 between humans and animals
including mink and salmon. As a result, a high structural homology of ACE2 was observed between
mink and humans. However, a non-high structural homology of ACE2 was observed between salmon
and humans. From these results, the infection of salmon with SARS-CoV-2 has not been
demonstrated. Further experiments are needed to examine the source of the new Beijing infections.
METHODS
Phylogenetic Analysis and Annotation
Reference genomes and amino acids of human, dog, cat, tiger, mink, salmon, bat, pangolin, and snake
ACE2 were obtained from the National Center for Biotechnology Information (NCBI) orthologs at
the National Library of Medicine. Amino acid homological analysis was performed using Align
Sequences Protein BLAST (algorithm protein–protein BLAST) with the protein accession numbers
of ACE2s listed in the NCBI Reference Sequence Database in order to determine the whole amino
acid homology of ACE2 between humans and other animals. Phylogenetic analyses of the complete
protein and major coding regions were performed with RAxML software (version 8.2.9) with 1000
bootstrap replicates using the general time-reversible nucleotide substitution model. Details of the
protein accession numbers of ACE2s are available in the supplementary materials.
Amino Acid Homology Analysis of the Binding Region of ACE2 for Interaction with SARS-
CoV-2 Spike Glycoprotein Between Humans and Other Animals
The binding region for interaction with the SARS-CoV-2 spike glycoprotein (82.aa-MYP-84.aa,
353.aa-KGDFR-357.aa) of the verified genome amino acid sequences of human ACE2 was predicted
using the NCBI protein database and Geneious software (version 11.1.5; Auckland, New Zealand),
and was annotated using the NCBI Conserved Domain Database. Amino acid homological analysis
was performed using Align Sequences Protein BLAST (algorithm protein–protein BLAST) with the
protein accession numbers of human and individual animal ACE2s listed in the NCBI Reference
Sequence Database. Details of the protein accession numbers of ACE2s are available in the
supplementary materials.
Analysis of the Three-Dimensional Structure of the Binding Site Between Mink, Salmon, and
Human ACE2
Spanner is a structural homology modeling pipeline that threads a query amino-acid sequence onto a
template protein structure. This is unique in that it handles gaps by spanning the region of interest
using fragments of known structures. To create a model, we must provide a template structure, as well
as an alignment of the query sequence to be modeled onto the template sequence. Spanner then
replaces mismatched residues and fills in any gaps caused by insertions or deletions. Details of the
analysis methods are available in the supplementary materials.
RESULTS
The outbreak of COVID-19 caused by the SARS-CoV-2 virus has become a pandemic, but there is
still little understanding about the zoonotic transmission and the antigenicity of the virus. We
therefore examined the homology of the whole ACE2 molecule, which is reported to be a cellular
receptor for the spike glycoprotein on the virion surface of SARS-CoV-2, between humans, dogs,
cats, tigers, mink, salmon, and other mammals. Our findings show that the ACE2 molecule
demonstrated 79%–92% homology between humans and dogs, 91%–92% homology between
humans and cats, 92% homology between humans and tigers, 93% homology between humans and
minks, and 74% homology between humans and salmon (Figure 1, Supplementary Materials). The
ACE2 molecule showed 80%–89% homology between humans and bats, 91% homology between
humans and pangolins, and 74%–75% homology between humans and snakes (Figure 1,
Supplementary Materials).
In addition, we examined the homology of the five amino acid residues KGDFR located in the binding
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.09.196378doi: bioRxiv preprint

Hayashi et al. 7/2/2020 1:45 PM 4
region of the ACE2 molecule, which directly recognize and bind the spike glycoprotein on the virion
surface of SARS-CoV-2 in humans, dogs, cats, and other mammals. Our findings show that these five
amino acid residues have 100% homology among humans, dogs, cats, tigers, and bats, 80% homology
between humans and pangolins, and 60% homology between humans and snakes (Figure 1,
Supplementary Materials). These five amino acid residues have 80% homology between humans and
mink (Figure 1, Supplementary Materials). Our findings also show that these five amino acid residues
have 60% homology between humans and salmon (Figure 1, Supplementary Materials). These results
suggest that SARS-CoV-2 may transmit from humans to dogs, cats, tigers, and minks.
We analyzed the three-dimensional structure of the binding region in mink and human ACE2s for the
spike glycoprotein of SARS-CoV-2 using Spanner, a structural homology modeling pipeline method
(Supplementary Materials). The three-dimensional structure of the binding region containing five
amino acids of mink ACE2 for the spike glycoprotein of SARS-CoV-2 was highly conserved in
comparison with the structure of human ACE2 (Figure 2A). However, the three-dimensional structure
of the binding region containing five amino acids of salmon ACE2 for the spike glycoprotein of
SARS-CoV-2 was incompletely conserved in comparison with the structure of human ACE2 (Figure
2A). The analysis revealed that the three-dimensional structure of the binding region of salmon ACE2
is an imperfect match with the structure of the receptor binding domain (RBD) of the spike
glycoprotein of SARS-CoV-2 (Figure 2B). In the case of salmon, the key (RBD) and the keyhole
(ACE2-binding residues) do not completely match (Figure 2B). From these results, there is no
medical or scientific evidence that SARS-CoV-2 infects salmon. Our research also suggests that
SARS-CoV-2 zoonotic disease may spread worldwide (Figure 3). Our findings, alongside recent
studies, may provide important insights into animal models for SARS-CoV-2 and animal management
for COVID-19 control. The World Health Organization considers zoonotic infections to be a special
case; however, it has called for individuals to also avoid cross-transmission of SARS-CoV-2 between
humans, their pets, and other animals. The information provided from our examinations will support
precision vaccine design and the discovery of antiviral therapeutics which will, in turn, accelerate the
development of medical countermeasures.
Discussion
A history of visits to the Beijing Fresh Fish Wholesale Market was confirmed in four patients with a
confirmed diagnosis of SARS-CoV-2 infection on June 13, 2020. As a result, employees working at
the market were voluntarily screened for SARS-CoV-2, with a positive reaction observed in 45 people.
Normally, SARS-CoV-2 infects the respiratory and intestinal tracts of homeotherms such as mammals
and birds (7,8,9). The results of previous studies have not demonstrated that poikilotherms can be
infected with the coronavirus. Fish are poikilotherms, and humans are homeotherms (10,11). In
general, species that are closer in genetic classification are more susceptible to common infectious
diseases. Due to the thermoregulatory mechanism of the species, the probability of transmission of a
common infectious disease among homeotherms is higher than the rate of infection between
homeotherms and poikilotherms animals (10,11). On the genetic classification, the difference in
genetic background between humans and other poikilotherms such as fish, snakes, turtles, and soft-
shelled turtles is much larger than that between humans and homeothermic animals such as birds and
beasts (12).
However, in March 2020, the possibility of coronavirus infection in fish was reported. Sequences of
coronavirus genes were confirmed in two separate cDNA pools (13). The initial cDNA sample pool
was obtained from the Carassius auratus cell line (13). The second cDNA pool was obtained from
Ctenopharyngodon idella kidney tissue (13). BLAST analysis revealed a DNA sequence very similar
to SARS-CoV-2 in these cDNA pools (13), though these sequencing results might also be artifacts of
the bioinformatics pipeline used in this study. However, it is possible that the aquatic habitat is an
environmental pathogen of common coronaviruses such as SARS.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.09.196378doi: bioRxiv preprint

Hayashi et al. 7/2/2020 1:45 PM 5
Amino acid loci similar to ACE2, which is a host-side receptor for mammalian SARS-CoV-2, have
been found in poikilotherms animals. However, amphibians, reptiles, and mammals are not very
closely genetically related. Our findings indicate that the homology of ACE2 between fish and
humans is not high enough for coronavirus to infect fish (Figure 1). On the other hand, the homology
of ACE2 between mammals including mink and humans is high, and cross-infection of coronavirus
between different species is observed (Figure 1). Generally, it is considered that infectious diseases
do not cross between terrestrial organisms and aquatic organisms (14). Therefore, it is considered that
SARS-CoV-2 is unlikely to infect salmon.
It is possible that the surfaces of marine products and other foods were contaminated with SARS-
CoV-2 in the new Beijing outbreak. In other words, it is possible that workers in food factories
infected with SARS-CoV-2 were in contact with the marine products, allowing SARS-CoV-2 to
adhere to their surface. It is known that virus can survive for long periods in a low-temperature
environment, with previous studies having shown that coronaviruses can survive for years at minus
60 degrees (15,16). Therefore, SARS-CoV-2 may have been transported to the seafood market via
the cold chain.
All cases of SARS-CoV-2 animal infections reported to date worldwide have been in mammals.
Moreover, the transmission route of SARS-CoV-2 from humans to animals has been confirmed. In
the United States, cases of infection from human to 5 dogs and lions three animals and pets 2 cats of
the tiger have been reported (17-22). In Hong Kong, cases of infection of SARS-CoV-2 from human
to two dogs and one cat have been reported. Medical evidence has been reported to suggest that
COVID-19 is a zoonotic disease.
From the experiments to date, there has been no evidence to show the transmission of SARS-CoV-2
to salmon. Further research is needed to obtain medical evidence of this.
Footnote
ACE2-Angiotensin-Converting Enzyme 2
The protein encoded by this gene belongs to the angiotensin-converting enzyme family of dipeptidyl
carboxypeptidases and has considerable homology with the human angiotensin-converting enzyme 1.
This secreted protein catalyzes the cleavage of angiotensin I into angiotensin, and angiotensin II into
the vasodilator angiotensin. The organ- and cell-specific expression of this gene suggests that it may
play a role in the regulation of cardiovascular and renal functions, as well as fertility. In addition, the
encoded protein is a functional receptor for the spike glycoprotein of the human coronaviruses SARS
and HCoV-NL63. [Provided by RefSeq, Jul 2008: www.ncbi.nlm.nih.gov/gene/59272/
ortholog/?scope=7776].
Abbreviations
ACE2: Angiotensin-converting enzyme 2; COVID-19: Coronavirus disease 2019; NCBI: National
Center for Biotechnology Information; SARS-CoV-2: Severe acute respiratory syndrome coronavirus
2
Data Sharing
Data are available on various websites and have been made publicly available (more information can
be found in the first paragraph of the Results section).
Disclosure
The authors declare no potential conflicts of interest. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.09.196378doi: bioRxiv preprint

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Abstract: COVID‐19 has brought speculations on potential transmission routes of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the causal agent of the pandemic. It is reported that the main route of virus transmission to be person‐to‐person by respiratory droplets;however, people have raised concerns on the possible transmission of SARS‐CoV‐2 to humans via food and packaging and its potential effects on food safety. This review discusses food safety issues in the COVID‐19 pandemic and reveals its possible transmission in cold‐chain food. The first outbreak of COVID‐19 in late 2019 was associated with a seafood market in Wuhan, China, while the second outbreak of COVID‐19 in June 2020 was also related to a seafood market in Beijing, China. As of 2020, several frozen seafood products linked with SARS‐CoV‐2 have been reported in China. According to the current survey and scientific studies, the risk of infection by SARS‐CoV‐2 from cold‐chain food, food products, and food packaging is thought to be very low. However, studies on food cold chain contamination have shown that SARS‐CoV‐2 remained highly stable under refrigerated (4°C) and even in freezing conditions (−10 to −80°C). Since one mode of SARS‐CoV‐2 transmission appears to be touching contaminated surfaces, it is important to clean and sanitize food contact surfaces properly. Understanding food safety hazard risks is essential to avoid potential negative health effects and SARS‐CoV‐2 transmission in the food supply chain during the COVID‐19 pandemic. [ABSTRACT FROM AUTHOR] Copyright of Journal of Food Safety is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)