Coronaviruses (CoVs) are enveloped single-stranded, positive-strand RNA viruses belonging to the Coronaviridae family, which includes four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus).. Several viruses of this family constantly and silently circulate or emerge and re-emerge in the human and animal populations causing, in many cases, mild to severe diseases [1–8]. The most recent dramatic example of a novel human coronavirus is the severe acute respiratory syndrome- coronavirus 2 (SARS-CoV–2), detected in the city of Wuhan, China, in December 2019, and that caused the severe pandemic of Coronavirus Disease 2019 (COVID–19) in this Asian country and then worldwide, critically threatening the public health at the global level [9–13].
Several animal species can act as reservoirs of coronaviruses and different mechanisms have been suggested for host cell and cross-species transmission of coronaviruses infections [14–19].
Viral entry, that starts from the receptor recognition, is an essential step determining host range and cross-species infection. Coronaviruses encode a spike (S) glycoprotein, which recognizes and binds to the host receptor on the cell surface . The region of the spike protein that mediates the interaction with the host-cell receptor is called receptor-binding domain (RBD). This domain is constituted by the ectodomain subunit S1 which, in turn, has two main domains: the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD; ). The S1-NTDs are usually responsible for binding sugar components of the receptors [22–25] whereas the S1-CTDs are responsible for recognizing protein receptors [26–31]. Subsequently, nearby host proteases cleave the spike glycoprotein, which releases the spike fusion peptide S2. The cleaved S2 peptide allows fusion of viral and cellular membranes facilitating virus entry into the host cell . The infection process has two critical and general issues that should be considered: i) the diversity of the host receptor usage from different coronaviruses and ii) the different level of sequence similarity of the S1 subunit of the spike from different genera, whereas those from the same genus have significant sequence similarity of this subunit .
A few host receptors, that could be specific or less specific for different coronavirus groups, have been identified: i) angiotensin-converting enzyme 2 (ACE2) is specific for the alphacoronavirus HcoV-NL63 and the betacoronaviruses SARS-CoV and SARS-CoV–2 [32–36], ii) aminopeptidase N (APN or ANPEP), described to be the receptor of the human coronavirus NL63 (HcoV-NL63) and other alphacoronaviruses, like the porcine epidemic diarrhea virus or PEDV, the porcine respiratory coronavirus or PRCV and the transmissible gastroenteritis virus or TGEV [25, 37–38] and iii) dipeptidyl peptidase–4 (DPP4), the receptor of the SARS-CoV and other betacoronaviruses, such as Middle-East respiratory syndrome coronavirus (MERS-CoV) and Tylonycteris bat coronavirus HKU4 or Bat-CoV HKU4 [39–40]. All these coronavirus receptors also play their own additional physiological functions in the host other than their role in the viral surface recognition step. The most studied host protease for S protein priming is the transmembrane serine protease 2 (TMPRSS2) which is mainly involved in SARS-CoV and SARS-CoV–2 infections [36, 41–43].
Crystal structures resolved for a number of S1 domains of different coronaviruses complexed with their respective receptor, along with functional studies and in silico comparative analyses of receptor sequences across host species, have identified several critical receptor domains and structures that are relevant for the interactions between the host and the infecting viruses [44–45]. These studies also suggested the utilizing capability of receptors from different animal species by coronaviruses, indicating potential cross-species transmission according to the structural compatibility between the spike domains and the host receptors [46–47].
Structural variations and different expression levels of the receptors and S protein priming proteases could potentially affect the spike/receptor interactions and subsequent spike cleavage efficiency which might cause differences of susceptibility of the host for the coronavirus infection capability and disease progression. A few studies in humans that investigated the ACE2 and TMPRSS2 genes reported variants segregating in different cohorts that might confer resistance against SARS-CoV–2 infection or modulate COVID–19 severity [48–53].
Several coronaviruses (PEDV, PDCV, SADS-CoV and TGEV), that originated from interspecies transmission, infect the pig (Sus scrofa) and cause acute gastroenteritis in neonatal piglets and death of the animals, leading to economically relevant problems to the pig industry [7, 54]. Genetic resistance to the infection of these coronaviruses might be present within and among pig populations and breeds . Only few studies have evaluated if pigs can become infected with other coronaviruses causing human diseases, such as SARS-CoV or MERS-CoV. These studies challenged the pigs with the two viruses and the obtained results indicated that a small fraction of the challenged animals were SARS-CoV or MERS-CoV antibody positives without any clinical signs or lesions, indicating that, even if remote, transmission of these viruses to the pigs and other animals cannot be excluded [56–58]. Shi et al.  reported that SARS-CoV–2 replicates poorly in pigs but other animals such as ferrets and cats are permissive to infection. Still, Zhou et al.  reported that SARS-CoV–2 could use ACE2 from four animal species including the porcine ACE2 as the receptor to enter the cell in vitro, suggesting that pigs might be potentially susceptible to SARS-CoV–2 infection and could be a potential intermediate host. In other studies, however, pigs did not result to have developed antibodies against SARS-Cov–2 and were negative for viral RNA after intranasal infection [60–61].
Epidemiological, biological and virological characteristics of coronaviruses, including their demonstrated ability to easily cross species barriers, suggest that pets and livestock should be considered as part of a global control and of a “One Health” approach to evaluate if animals that are close to human contacts could represent a risk source of infections for humans and vice versa [62–63]. Based on the mentioned preliminary evidences on the potential relationships between SARS-CoV–2 and pigs (even if contrasting) and considering i) the relevance of the pig production systems for meat supply, ii) that several other coronaviruses circulate in pigs and cause diseases in this livestock species [7–8, 40], iii) that receptor variants may confer different susceptibility to infections within species [48–53], iv) that coronaviruses may jump the species barriers easily [5,18,46,57] and v) that variability of the RBD region of the spike protein might determine a quite large host spectrum for every coronaviruses [45, 64], as part of a “One Health” approach , it is needed to evaluate the genetic variability segregating in pig populations potentially conferring differences of sensitivity to coronavirus-related diseases.
In this study, we investigated the variability in several pig genes (ACE2, ANPEP, DPP4 and TMPRSS2)that can serve as receptors or protease for priming the infection of coronaviruses. We also evaluated their relevance in conferring potential differences in susceptibility to coronavirus diseases, also considering a comparative analysis between the corresponding human genes and the information available in other species. Analysis of variability included a total of 22 European pig breeds and wild boars and two Asian pig populations using next generation sequencing data (NGS). This dataset covered a broad number of pig genetic resources raised in Europe [65–66] in comparison with a few Asian populations. The obtained results could be useful i) to establish a risk evaluation system in a “One Health” approach, including information on the diversity of pig populations, ii) to define cross species evolutionary analyses of genes involved in coronavirus infections and iii) to identify natural genetic variability within the Sus scrofa species that could help to design genetic improvement strategies to increase genetic resistance in commercial and autochthonous pig populations against emerging and re-emerging coronavirus diseases.