ACEII gene analysis exposes SARS-CoV-2 as a potential threat to agricultural and national security

Coronavirus is now a significant human pathogen with the emergence of SARS-CoV-2. Until now there has been no data to support a threat to agricultural industries. Using a comparative genomic protein analysis, this study examined the angiotensin-converting enzyme II (ACEII) gene of 17 animal species with an emphasis on agriculture. To determine viral vulnerability the 20 known SARS-CoV-2 receptor-binding domain (RBD)/ACEII receptor interaction sites were compared to determine their potential susceptibility to the SARS-CoV-2 virus. With the known bat host’s (XP_032963186) number of binding sites as a threshold, we note that ALL animal species examined in this study contained significant numbers (≥10) of SARS-CoV-2 binding sites and could be at risk for SARS-CoV-2 infection. The data from this study suggest SARS-CoV-2 imposes a grave threat to the safety and security of the agricultural industry. Urgent studies are needed to determine if infected animals can transmit SARS-CoV-2 before and/or after processing. Introduction Until recently it was unknown whether animals could become infected by SARS-CoV-2. Despite the widespread suspicion that SARS-CoV-2 originated from a bat (RaTG13| MN996532.1), it remains unclear whether other animal species may be viable primary or Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 September 2020 doi:10.20944/preprints202009.0345.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. 2 secondary hosts. Preliminary data (Chen et al. 2020) suggest various animal groups contain SARS-CoV-2 interaction sites between the SARS-CoV-2 spike protein receptor-binding domain (RBD) and the angiotensin-converting enzyme II (ACEII) gene. This RBD has 16 amino acid residues capable of interacting with 20 ACEII amino acid sites (Li et al. 2005, Chen et al. 2020). Since the onset of the SARS-CoV-2 pandemic, we have identified and verified that tigers (Gollakner and Capua 2020, Mahdy 2020) and lions serve as SARS-CoV-2 hosts. To determine if SARS-CoV-2 poses a potential threat to agricultural and national security this study examines the SARS-CoV-2 spike protein RBD sites that are capable of interacting with the ACEII gene of 17 animals identified as having agricultural significance. Materials and Methods Human and animal ACEII gene sequences (Table 1) were queried on the NCBI gene database (Anjay 2012) and compiled to fasta format using Notepad++ (Ho 2020). The resulting fasta file of ACEII protein sequences was aligned using Muscle (Edgar 2004a, 2004b) with the default algorithm and parameters within UGENE v34 (Okonechnikov et al. 2012). A second alignment was performed using the Cobalt tool within the NCBI site (Papadopoulos and Agarwala 2007). The second alignment was necessary as UGENE lacks a nexus file format option when exporting alignments. Using UGENE a distance matrix was generated using the following parameters: Distance algorithm = Similarity, Profile mode = Percentage, Save profile to file= checked, File = Commaseparated (.CSV). The human reference sequence must be in the first position when generating a distance matrix. The distance matrix generation process was repeated after removing all amino acid residues other than the 20 known ACEII interactive sites. Using ACEII gene alignment, all sequences were examined for SARS-CoV-2/ACEII gene interaction sites using the 20 known human sites as the reference (Li et al. 2005, Chen et al. 2020). Amino acid changes were annotated and represented graphically using both MSWord and SnagIt Editor (Bragg 2002). The number of matching amino acid residue sites were denoted into a spreadsheet and exported as for visualization. Using human (NP_001358344) reference sequence, amino acids that differ from human SARS-CoV-2 interaction sites were queried manually and recorded in table format. The similarity value obtained from the lowest scoring known host was used as a threshold value to determine the possibility of COVID-19 infection. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 September 2020 doi:10.20944/preprints202009.0345.v1

secondary hosts. Preliminary data (Chen et al. 2020) suggest various animal groups contain SARS-CoV-2 interaction sites between the SARS-CoV-2 spike protein receptor-binding domain (RBD) and the angiotensin-converting enzyme II (ACEII) gene. This RBD has 16 amino acid residues capable of interacting with 20 ACEII amino acid sites (Li et al. 2005, Chen et al. 2020. Since the onset of the SARS-CoV-2 pandemic, we have identified and verified that tigers (Gollakner and Capua 2020, Mahdy 2020) and lions serve as SARS-CoV-2 hosts. To determine if SARS-CoV-2 poses a potential threat to agricultural and national security this study examines the SARS-CoV-2 spike protein RBD sites that are capable of interacting with the ACEII gene of 17 animals identified as having agricultural significance.

Materials and Methods
Human and animal ACEII gene sequences (Table 1) were queried on the NCBI gene database (Anjay 2012) and compiled to fasta format using Notepad++ (Ho 2020). The resulting fasta file of ACEII protein sequences was aligned using Muscle (Edgar 2004a(Edgar , 2004b  Using UGENE a distance matrix was generated using the following parameters: Distance algorithm = Similarity, Profile mode = Percentage, Save profile to file= checked, File = Commaseparated (.CSV). The human reference sequence must be in the first position when generating a distance matrix. The distance matrix generation process was repeated after removing all amino acid residues other than the 20 known ACEII interactive sites.
Using ACEII gene alignment, all sequences were examined for SARS-CoV-2/ACEII gene interaction sites using the 20 known human sites as the reference (Li et al. 2005, Chen et al. 2020). Amino acid changes were annotated and represented graphically using both MSWord and SnagIt Editor (Bragg 2002). The number of matching amino acid residue sites were denoted into a spreadsheet and exported as for visualization.
Using human (NP_001358344) reference sequence, amino acids that differ from human SARS-CoV-2 interaction sites were queried manually and recorded in table format. The similarity value obtained from the lowest scoring known host was used as a threshold value to determine the possibility of COVID-19 infection.

Results
Amino acid alignment of the ACEII binding domain (Appendix 1) reveals a highly homologous binding domain between species. Most SARS-CoV-2/ACE II interactive sites in this binding domain lie within the α-helices and β-sheets of the ACEII complex structure.
Our study noted the following AA residue differences in the hydrophobic pockets created by F28, L79, Y83, and L97: Birds p.L79N, Pig p.L79I, and all others except donkey and horse p.L79M; birds and bat p.Y83F; and birds p.L97I in birds (Appendix 1). F28 was conserved in all species.
The known host species with the least number of SARS-CoV-2/ACEII complex structure interaction sites were identified as being horseshoe bat (XP_032963186) with 10 interaction sites. The known host tiger, as well as cattle, sheep, goat, bison, and deer, contained the most SARS-CoV-2 interaction sites outside of humans (Table 2). Other than birds, all animals in this study contained at least 15 SARS-CoV-2/ACEII complex structure interaction sites. This number of interaction sites is well above our threshold value established by the know bat host at 10. This may or may not be cause for concern but it certainly leads to a high index suspicion of their SARS-CoV-2 host viability.

Historical coronavirus evidence in agricultural species
It is important to understand that ALL species in this study have been previously  Infected animals and humans may also contaminate a water supply if in direct contact.
Studies have shown that coronavirus inactivation in water can take over 500 days at 4 ο C or 10 days at 23 ο C (Gundy et al. 2009). This study also indicates the possibility of human to animal spread through an open, contaminated water source such as a water trough. Due to the extreme importance of this viral capacity, the water study should be repeated with SARS-CoV-2 for comparative results.
Until now there has been no data to support a threat to the global food supply. However, existing control measures are not adequate to mitigate SARS-CoV-2 propagation. Food poverty, hunger, and food inequality lead to instability at the local, national, and international levels (Pinstrup-Andersen 2003). These factors highlight the importance of agricultural industries to the security of a nation (Tweeten 1999, Falcon and Naylor 2005, Etim et al. 2017. One needs only to look at recent events in Venezuela for an example of this concept. Preparing and mitigating any potential impact of SARS-CoV-2 on animals is paramount to agricultural and national security (Batie and Healy 1980, Winters 1990, HORN and BREEZE 1999, Casagrande 2000.
Agricultural industries should begin preparation for a worst-case-scenario before they are forced to respond to one.

Mitigation and control
Coronavirus is now a significant human pathogen with the emergence of SARS-CoV-2.
This pathogen has caused severe illness and death in humans and until recently no evidence Currently, social isolation, respiratory barrier masks, and personal sanitation measures are the only available forms of SARS-CoV-2 mitigation. This is not a possibility for agricultural industry animals where animals are typically kept in large numbers and close proximity to each other. As a result, the risk of one animal infecting another would appear to be extremely high.
Even with free-range animals, their natural herd tendencies sustain a viral transmission risk.
While numerous studies have shown that viruses can spread quickly with a high degree of population penetrance, no studies are found to document coronavirus transmission to humans or other animals through food/water consumption or handling during processing. The absence of this data does not eliminate this prospect. The rate of raw meat processing plant worker infections is alarming and should raise concern as to the root source of spread since this phenomena is not observed in other industries of similar scale.
While the ACEII gene is highly conserved between species, this study was limited by using the human ACEII gene sequence as a reference. Actual SARS-CoV-2/ACEII animal complex structure interactions could differ in animals. Another limitation of this study was that SARS-CoV-2 survivability in tissues and water must be inferred from other human coronaviruses. SARS-CoV-2 survivability in these media may differ under identical environmental conditions. Lack of animal testing precludes a definitive viral susceptibility analysis. Our work establishes the framework for a SARS-CoV-2 infection risk amongst animals.
Future studies should test living and processed animal samples to determine primary and secondary host viability.

Summary
Using a comparative genomic protein analysis this study found that all animal species in this study contained significant numbers of SARS-CoV-2/ACEII complex structure interaction sites and could be considered at risk for SARS-CoV-2 infection. Data from this study suggest SARS-CoV-2 imposes a grave threat to agricultural and national security. Urgent studies are needed to determine if infected animals can transmit SARS-CoV-2 before and/or after processing.

Acknowledgments:
We gratefully acknowledge the authors, originating and submitting laboratories of the sequences from the NCBI Database on which this research is based. I would like to thank the University of Georgia-Griffin faculty for their valuable guidance in molecular processes. We also recognize the reviewers for their valuable time and insight. Funding: no outside funding sources. Author contributions: Michael Ruhl performed data query, protein analysis, and developed the initial manuscript. Tracie Jenkins was the primary editor and contributed to genomic analysis. Competing interests: Authors have no competing interests to declare. Data and materials availability: all accessions are publicly available and listed in Table 1. All software used is publicly available with citations provided in the manuscript text.