The study findings showed that pig respiratory epithelial cells underwent apoptosis, or controlled cell death, in response to infection at a higher rate compared than to human epithelial cells and this could be the key clue that prevents pigs from getting sick.
The remarkable ability of SARS-CoV-2 virus to infect different species, including humans, dogs, cats, minks, ferrets, hamsters, tigers, and deer, pose a continuous threat to human and animal health.
Pigs, although closely related to humans and also possess ACE2 receptors, seem to be less susceptible to SARS-CoV-2.
Former in vivo studies failed to demonstrate clinical signs and transmission between pigs, while later attempts using a higher infectious dose reported viral shedding and seroconversion.
The study team investigated species-specific cell susceptibility, virus dose-dependent infectivity, and infection kinetics, using primary human (HRECs) and porcine (PRECs) respiratory epithelial cells. Despite higher ACE2 expression in HRECs compared to PRECs, SARS-CoV-2 infected, and replicated in both PRECs and HRECs in a dose-dependent manner. C
ytopathic effect was particularly more evident in PRECs than HRECs, showing the hallmark morphological signs of apoptosis. Further analysis confirmed an early and enhanced apoptotic mechanism driven through caspase 3/7 activation, limiting SARS-CoV-2 propagation in PRECs compared to HRECs.
The study findings shed light on a possible mechanism of resistance of pigs to SARS-CoV-2 infection, and it may hold therapeutic value for the treatment of COVID-19.
The study findings were published in the peer reviewed journal: Cell Death Discovery.
The ongoing global pandemic of SARS-CoV-2 has resulted in different clinical outcomes in two closely related mammalian species, humans and pigs. While in humans, the COVID-19 pandemic has resulted in >4.5 million deaths across the world (>223 million confirmed cases; as of 09/10/2021) , pigs, in contrast, and according to previous reports, seem to be either not susceptible to SARS-CoV-2 infection [14, 22, 23] or where the infection is mild and self-limited .
The successful reproduction of infection and clinical disease in vivo under experimental settings can be difficult in pigs, even with swine-restrictive viruses. Constraints include resource-intensive, susceptibility-related factors, inoculum dose and route of exposure, high variability, lack of sensitivity, interference with gut microbiome or secondary infections, and difficulty recording precise cell–viral interactions on a daily/hourly basis.
In contrast, in vitro, culture models based on cell lines are relatively easy to maintain, but often they are not the natural cell target of the virus, nor do they represent sufficient complexity (cell lineage, functionality) to mimic the natural infection process in vivo . For this reason, in vitro experimental data cannot often be extrapolated into clinical trials entirely, e.g., complicated cellular signals between cells and their matrix cannot be reproduced . This would justify using primary respiratory epithelial cell cultures to understand the immunopathogenesis of SARS-CoV-2.
Tracheobronchial-derived primary epithelial cells have been widely used to study early immune responses towards viral infections [29,30,31,32]. Thus, the first objective of this study was to confirm whether porcine respiratory epithelial cells were susceptible to infection by SARS-CoV-2 and comparing it with human respiratory epithelial cells.
Using ACE2 as entry receptor and proteases as entry activators , SARS-CoV-2 spike protein mediates virus entry into the respiratory epithelial cells of a susceptible host, where the virus primarily replicates [34, 35]. Previous studies reported the detection of viral antigens in the human trachea, bronchi, bronchiole, and pneumocytes  tracheal degeneration and necrosis in affected cats , alveolar damage and necrosis in minks , and detection of viral RNA in the bronchi of white tailed-deer .
First, the present study demonstrated the expression of ACE2 receptor and effective SARS-CoV-2 binding on the epithelial lining of both human and pig tracheal tissue sections, which contradicts a previous study that hypothesized that the lack of virus susceptibility or virus replication could be attributed to the absence of ACE2 receptors on the porcine respiratory tract epithelium . This finding is supported by a recent study showing that ACE2 is expressed, at different levels, in a wide range of porcine tissues, including the lungs .
In our study, a human ACE2 antibody was used for IHC analysis on both human and pig trachea tissue sections and primary respiratory cells. The expression levels of ACE2 were significantly low on pig tracheal epithelium tissue sections compared to their human counterparts (Fig. 1A, C). Further, flow cytometric analysis of isolated PRECs and HRECs quantified the amount of both pan-cytokeratin and ACE2 expressed on these cell types, confirming that human cells expressed more ACE2 than pig cells. Despite the paucity in ACE2 expression on pig trachea, heat-inactivated SARS-CoV-2 bound uniformly across the pig and human trachea tracheal epithelium (Fig. 2A, B).
The protein sequence homology studies between human ACE2 (NP_001358344) and pig ACE2 (NP_001116542) performed in this study and others  suggest that these closely related mammalian species share 81% identical amino acid residues. Further, a pair-wise alignment of porcine ACE2 protein with human ACE2 protein at the region targeted by the anti-ACE2 antibody (amino acids 631-805; sc-390851, Santa Cruz Biotechnology) used in this study shows a sequence similarity of 76% (Supplementary Fig. 1), suggesting the potential cross-reactivity and usefulness of anti-ACE2 antibody towards detection of porcine ACE2 receptor in tissues and cultures.
After confirming the expression of ACE2 receptors in human and pig tracheobronchial-derived tissue sections and cells, we performed a comparative in vitro infection study to investigate possible factors related to possible differences in the susceptibility of primary porcine and human tracheobronchial epithelial cells to SARS-CoV-2 infection. Firstly, the optimal viral dose in PRECs and HRECs cultures was established for subsequent infection studies.
As to the question of whether SARS-CoV-2 can infect and replicate in PRECs, SARS-CoV-2 replicated in both PRECs and HRECs in a dose-dependent manner, as evidenced by RT-qPCR and IHC assays (Fig. 3). Virus replication was monitored in PRECs and HRECs cultures inoculated with three different infectious doses (MOI 5.0, 5.0 × 10−2, and 5.0 × 10−4) over 120 hpi period. SARS-CoV-2 N protein was gradually accumulated in both PRECs and HRECs cultures as infection progressed, particularly at the higher infectious dose (MOI 5.0) used in this study. The CPE was particularly evident in PRECs compared to HRECs or the corresponding mock-inoculated controls.
This CPE was dose- and time-dependent, dramatically enhanced in PRECs at MOI 5.0 infectious dose and 96 hpi (Fig. 3E–G). This strongly indicates that virus dose is a potential factor in the outcome of SARS-CoV-2 infection in these primary cells, as it was previously hypothesized . Previous in vivo studies in pigs demonstrated that only using a high infectious dose (2 mL of approximately 106 TCID50/mL intranasally and intratracheally) triggered the production of anti-SARS-CoV-2 neutralizing antibodies even in the absence of clinical signs [24, 25].
Subsequently, we investigated the overall mechanism behind the CPE and massive cell death particularly observed in PRECs cultures inoculated with the higher infectious dose (MOI 5.0) evaluated in this study. In general, CPE and cell death could be either cell-associated (i.e., cells died because of their inability to reproduce) or virus-induced (i.e., lysis and dissolution caused by virus infection). This can be elucidated on the basis of general morphological, biochemical, and functional features .
Specifically, morphological analysis of SARS-CoV-2 infected PRECs cultures revealed all the hallmark morphological signs of apoptosis, a controlled form of cell death , including cell shrinkage and detachment, plasma membrane blebbing, the formation of apoptotic bodies, chromatin condensation (pyknosis), and nuclear fragmentation (karyorrhexis) leading to cell death (Fig. 5C). In contrast, in HREC cultures, most cells were attached to the plate and appeared viable with no notable differences in the morphology of the nucleus between SARS-CoV-2- and mock-inoculated cultures (Fig. 5A, B).
On the other hand, the genetic and biochemical cell-death analysis includes activation of cysteine aspartate-specific proteinases (caspases) and releasing mitochondrial factors as crucial features of the apoptotic process . Using the biochemical ApoTox-Glo triplex assay, we further demonstrated an early and enhanced apoptotic mechanism mediated through caspase 3/7 activation in response to SARS-CoV-2 infection in PRECs compared to HRECs. The decrease in cell viability was particularly high in SARS-CoV-2 infected PRECs after 48 hpi (Fig. 4G).
Contrary, the expression levels of caspase 3/7 in HRECs infected cultures remained stable throughout the infection period. Additional SARS-CoV-2 infection studies on human bronchial epithelial cells (BEAS-2B) also reported no induction of apoptosis . Interestingly, SARS-CoV-2 induced enhanced cytotoxicity in HRECs compared to PRECs cultures after 48 hpi (Fig. 4F).
In addition, the supernatants collected from HRECs infected with SARS-CoV-2 contained infectious virions that were able to infect Vero-E6 cells, while the corresponding supernatants from PRECs undergo apoptosis lacked viable virus and were, therefore, non-infectious. In 2012, Nelli and others reported comparable findings in primary human and porcine respiratory epithelial cells infected with highly pathogenic H5N1 influenza A virus (IAV) [31, 44].
The results of the present study demonstrated that an early onset of apoptosis via caspase 3/7 activation is a crucial event to limit SARS-CoV-2 propagation in PRECs. Thus, further research on modulation of apoptosis and the effect of caspase inhibitors is needed. The early apoptotic cell death observed in PRECs may favor the host cell, while the delayed cell death observed in HRECs may favor the virus. Previous experimental studies in vivo observed complete virus (RNA) clearance one week after virus inoculation in pigs . This, together with additional studies reporting absence of clinical signs and effective virus transmission between animals [14, 22,23,24,25], led to conclude that pigs are more resistant to SARS-CoV-2 infection than humans and other animal species such as cats, mink and deer.
Taken together, our findings shed light on the possible molecular mechanism of resistance of pigs to SARS-CoV-2 infection and/or virus propagation, and it may hold therapeutic value for the treatment of COVID-19.