Influenza-Infected Pigs Are Not Susceptible to SARS-CoV-2 Infection

Abstract

Since its emergence in 2019, SARS-CoV-2 has resulted in more than 7.1 million deaths worldwide. It has been shown that co-infection with influenza A virus (IAV) can worsen clinical symptoms in COVID-19 patients and small animal models have been used to elucidate the molecular mechanisms of co-infection between SARS-CoV-2 and IAV. This study aimed to establish a co-infection model in pigs, a natural reservoir for IAV but resistant to SARS-CoV-2 infection, by determining whether pigs become susceptible to SARS-CoV-2 following a primary IAV infection. Here, pigs were primarily infected with swine influenza A virus (SIV) via the intratracheal route and, 3 days later, secondarily challenged with SARS-CoV-2. SIV was isolated from nasal swabs, and pigs seroconverted to SIV. In contrast, SARS-CoV-2 RNA was detected in nasal and oropharyngeal swabs only at day 1 post-secondary challenge, with no evidence of seroconversion against SARS-CoV-2. These data indicate that pigs are not susceptible to SARS-CoV-2 following SIV infection and therefore are not a suitable model for IAV/SARS-CoV-2 co-infection research.

1. Introduction

The COVID-19 pandemic caused by SARS-CoV-2 has significant impacts on public health and socioeconomics, resulting in nearly 779 million infections and more than 7.1 million deaths worldwide since its first detection in late 2019 (as of December 2025). Infection with SARS-CoV-2 leads to acute respiratory distress with increased risk of severe COVID-19 in elderly or immunocompromised individuals [1]. In addition, underlying chronic medical conditions and co-infection with other pathogens increase the risk of severe COVID-19 outcomes [2].

Co-infections can be a factor associated with the development of severe clinical disease in COVID-19 patients. In particular, co-infection with influenza viruses increases the odds of receiving invasive mechanical ventilation and in-hospital mortality when compared to infections with SARS-CoV-2 alone [3]. In animal models, co-infection with influenza A virus (IAV) and SARS-CoV-2 results in more severe clinical disease and lung pathology in K18-hACE2 mice, Syrian hamsters, and ferrets compared to SARS-CoV-2 infection alone [4,5,6,7]. In particular, pre-infection with IAV results in higher SARS-CoV-2 replication compared to SARS-CoV-2 infection alone in K18-hACE2 mice; this may be due to elevated ACE2 expression induced by IAV infection [5]. In human pluripotent stem cell-induced lung organoids, IAV infection increased ACE2 and TMPRSS2 mRNA expression, whereas SARS-CoV-2 infection upregulated α2,3-linked sialic acid [8]. Consequently, infection with IAV significantly increased SARS-CoV-2 replication in human lung organoids when compared to SARS-CoV-2 infection alone [8]. In addition to the enhanced expression of viral receptors, the upregulated innate immune responses caused by IAV/SARS-CoV-2 co-infection led to more severe lung damage than in single SARS-CoV-2 infection [8,9]. Because the SARS-CoV-2 spike (S) protein binds to the ACE2 receptor to initiate virus entry into cells, increased expression of ACE2 following IAV infection could potentially enhance the ability of SARS-CoV-2 to infect host cells. SARS-CoV-2 utilizes the TMPRSS2 protease to cleave the S protein at the S1/S2 site, activating the S2 domain to fuse the viral membrane with the host cell membrane allowing the virus to enter the cell [10]; thus elevated expression of TMPRSS2 in the respiratory tract also facilitates SARS-CoV-2 entry into cells and could enhance SARS-CoV-2 susceptibility.

Pigs are a natural reservoir for IAVs but are resistant to SARS-CoV-2 infection. We hypothesized that pigs infected previously with a swine IAV (SIV) would become more susceptible to SARS-CoV-2 infection. To test this hypothesis, we first evaluated ACE2 and TMPRSS2 mRNA expression in the respiratory tract of SIV-infected pigs. Next, pigs were primarily infected with SIV and secondarily challenged with SARS-CoV-2. Clinical samples were collected throughout the study to monitor virus shedding and seroconversion to ultimately determine whether prior SIV infection increases susceptibility of pigs to subsequent SARS-CoV-2 infection.

2. Materials and Methods

2.1. Cells and Viruses

Madin–Darby canine kidney cells (MDCK) and Vero-E6/TMPRSS2 cells [11] were used for virus propagation and titration. MDCK cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, Manassas, VA, USA), supplemented with 5% fetal bovine serum (FBS; R&D systems, Flower Branch, GA, USA) and antibiotics/antimycotics (Gibco, Grand Island, NY, USA). Vero-E6/TMPRSS2 cells were cultured in DMEM supplemented with 10% FBS, antibiotics/antimycotics and Geneticin (Gibco, Grand Island, NY, USA). The virus stock of A/Swine/Texas/4199-2/98 (TX98 H3N2) was grown and titrated in MDCK cells in influenza infection medium (DMEM supplemented with 0.3% bovine serum albumin, 1× MEM vitamin and 1% antibiotic–antimycotic). The SARS-CoV-2/human/USA/WA1/2020 lineage A (referred to as WA-1; BEI item #: NR-52281) and SARS-CoV-2/human/USA/CA_CDC_5574/2020 lineage B.1.1.7 (alpha variant of concern (VOC); NR-54011) strains were acquired from BEI Resources (Manassas, VA, USA) and grown and titrated in Vero-E6/TMPRSS2 cells [12].

2.2. Swine ACE2 and TMPRSS2 RNAscope™ In Situ Hybridization (RNAscope™ ISH)

Formalin-fixed paraffin-embedded tissue samples were retrieved from previous, independent animal studies [13]. SIV-infected samples (n = 4) originated from pigs which were infected with TX98 H3N2 and humanly euthanized at 3 or 5 days post-challenge for pathology and tissue collection. Mock samples (n = 3) were derived from control pigs from a previous SARS-CoV-2 challenge study [13].

For RNAscope™ ISH, an anti-sense probe targeting ACE2 and TMPRSS2 mRNA of Sus scrofa (865318, 568308-C2) were designed (Advanced Cell Diagnostics [ACD], Newark, CA, USA) in channels 1 and 2, respectively (C1 and C2). Four-micron sections of formalin-fixed paraffin-embedded tissues were mounted on positively charged Superfrost^® Plus Slides (VWR, Radnor, PA, USA). The RNAscope™ ISH assay was performed using the RNAscope 2.5 LS Duplex Reagent Kit (Advanced Cell Diagnostics, Newark, CA, USA) on the automated BOND RXm platform (Leica Biosystems, Buffalo Grove, IL, USA). Briefly, tissue sections were subjected to automated baking and deparaffinization followed by heat-induced epitope retrieval (HIER) using a ready-to-use EDTA-based solution (pH 9.0; Leica Biosystems) at 95 °C for 15 min. Subsequently, tissue sections were treated with a ready-to-use protease (RNAscope™ 2.5 LS Protease) for 15 min at 40 °C followed by a ready-to-use hydrogen peroxide solution for 10 min at room temperature. Slides were then incubated with a probe mixture containing Sus scrofa-specific ACE2 and TMPRSS2 probes at the concentration recommended by the manufacturer for 2 h at 40 °C. The signal from the C2 probe (TMPRSS2) was amplified using amplifiers 1 through 7 (AMP1 through AMP7) and the signal subsequently detected using a Fast-Red solution for 10 min at room temperature. The signal from the C1 probe (ACE2) was amplified following sequential incubation with amplifiers 8 through 10 (AMP8 through AMP10). The signal was finally detected by incubating 3,3′-diaminobenzidine (DAB) for 20 min and the BOND DAB Enhancer (Leica Biosystems) for an additional 20 min at room temperature. Slides were counterstained with hematoxylin for 5 min, followed by five washes with 1X BOND Wash Solution (Leica Biosystems) for bluing. Slides were finally rinsed in deionized water, dried in a 60 °C oven for 30 min, and mounted with Ecomount^® (Biocare, Concord, CA, USA). A negative control probe mixture was used as negative control, and a Sus scrofa-specific probe mixture targeting ubiquitin C (UBC) and peptidylprolyl isomerase B (PPIB) mRNA was used as a positive control to assess RNA integrity.

2.3. Whole Slide Scanning and Quantitative Image Analysis

Duplex RNAscope™ ISH slides for ACE2 and TMPRSS2 were scanned at 40× magnification using a PhenoImager HT 2.0 whole slide scanner (Akoya Biosciences, Marlborough, MA, USA). Quantitative analysis was performed in QuPath 0.3.1 digital pathology image analysis software [14]. Guidelines established by ACD were followed with some modifications. Briefly, stain vectors were adjusted for each slide, respectively, before pursuing further analysis. Subsequently, 3 regions of each tissue compartment were selected to collect a minimum of 1000 cells (tracheal epithelium, bronchial epithelium, bronchioles, alveoli). Subsequently, the cell detection algorithm was performed with default values. After cell segmentation was performed, the subcellular detection algorithm was applied for detection of DAB (ACE2) and Fast Red (TMPRSS2) spots using default values except for minimum spot size of 0.1 and a DAB threshold between 0.4 and 0.8 based on the accuracy of the spot detection. Threshold for DAB and Residual (Fast Red) were assessed for each specific image to avoid erroneous detections. The estimated number of spots (ACE2 and TMPRSS2) were determined per cell per region and exported into an Excel file. Spots/cell for each tissue compartment were used to compute an H-score (range of 0 to 400) by binning cells with different levels of expression into separate bins (Bin 0 [0 dots/cell], Bin 1 [1–3 dots/cell], Bin 2 [4–9 dots/cell], Bin 3 [10–15 dots/cell], and Bin 4 [greater than or equal to 15 dots/cell]). The H-score was computed using the weighted formula: H–score = ∑ (Bin number × % cells per bin). Data distribution (H-scores for ACE2 and TMPRSS2 expression per tissue area) was evaluated using JMP16 Pro (Cary, NC, USA). Non-parametric data was analyzed using Wilcoxon test in JMP16 Pro. Graphics were subsequently generated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The level of significance was set at p-value < 0.05.

2.4. Animal Study

Nine conventional, high-health 3 week-old pigs were obtained from a commercial source. The number of animals used in this study was determined by the animal holding capacity of the BSL-3Ag facility, and negative control pigs could not be included in this study. Pigs were randomly assigned into two groups: four pigs in group 1 were infected with SIV and subsequently challenged with the SARS-CoV-2 Wuhan-like WA-1 strain, and five pigs in group 2 were infected with SIV and subsequently challenged with the SARS-CoV-2 alpha VOC. All nine pigs were infected with 10⁶ TCID₅₀ of SIV H3N2 TX98 in 2 mL inoculum via the intra-tracheal route. At 3 days post-primary infection (DP1I), four or five pigs were subjected to a secondary challenge with 10⁶ TCID₅₀ of either Wuhan-like WA-1 or alpha VOC, respectively, by inoculation through intra-tracheal (1 mL), intra-nasal (1 mL), and oral routes (1 mL). Pigs were humanely euthanized for post-mortem examination at 3, 7, and 14 days post-secondary challenge (DP2C). Nasal swabs were collected in 2 mL of DMEM at −1 and 1 DP1I, and nasal and oropharyngeal swabs were collected at 0, 1, 2, 3, 4, 5, 7, 10, 12, and 14 DP2C. Serum samples were collected at −1 DP1I, and at 3, 7, and 14 DP2C. At necropsy at 3, 7, and 14 DP2C, nasal wash, bronchioalveolar lavage fluid (BALF), and tissue samples were collected.

2.5. Influenza Virus Titration

MDCK cells were seeded in a 96-well plate the day before virus titration. Nasal swab samples were filtered through a 0.45 µm syringe filter to remove bacterial/fungal contaminants. Ten-fold dilutions were prepared in influenza infection medium and transferred onto confluent MDCK cells. At 2 days post-inoculation, the presence of the infectious virus was confirmed by immunofluorescence assay. Briefly, the cells were fixed with cold methanol and incubated at −20 °C for 10 min. The plate was incubated with anti-NP monoclonal antibody (clone HB65, American Type Culture Collection, Manassas, VA, USA), followed by goat anti-mouse IgG conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA). The viral titer was subsequently determined using the Reed and Muench method.

2.6. Hemagglutination Inhibition Assay

The hemagglutination inhibition assay was used to detect antibody response to influenza virus infection [15]. Serum samples were treated with receptor-destroying enzyme II (Denka Seiken, Tokyo, Japan) according to the manufacturer’s instructions. Two-fold dilutions of treated sera were prepared in phosphate-buffered saline and mixed with 4 hemagglutinating (HA) units of the TX98 strain in a total volume of 25 μL in a V-bottomed 96-well plate. After incubation for 30 min, a total of 50 μL of a 0.5% chicken red blood cell suspension (Colorado Serum Company, Denver, CO, USA) was added. The reciprocal of the highest serum dilution inhibiting hemagglutination was considered the HI titer.

2.7. RNA Extraction and SARS-CoV-2 RT-qPCR

SARS-CoV-2 RNA detection was performed as previously described [16,17]. Briefly, swab samples, nasal wash, BALF, and 20% tissue homogenates were mixed with an equal volume of RLT lysis buffer (Qiagen, Germantown, MD, USA). A total of 200 µL of sample/RLT lysate was subjected to automatic extraction using a magnetic bead-based nucleic acid extraction kit (Taco™ mini nucleic acid extraction system, GeneReach USA, Lexington, MA, USA). SARS-CoV-2 RNA was amplified and detected using the CDC N2 RT-qPCR assay in duplicate reactions. The cut-off for a positive result was 38 cycles in duplicate RT-qPCR wells. The Ct value was converted to copy numbers/mL using the standard curve of quantified viral RNA (USA-WA1/2020 Wuhan-like isolate).

2.8. SARS-CoV-2 Serology

SARS-CoV-2 serology was performed using in-house RBD- and N-based indirect ELISAs and virus neutralization (VN) tests, as previously described [13,18,19]. Briefly, 100 ng of in-house-generated recombinant RBD and N proteins were coated onto a MaxiSorp 96-well plate (Thermo Scientific™, Waltham, MA, USA) and incubated overnight at 4 °C. The next day, the plate was washed three times with phosphate-buffered saline containing Tween 20 (PBS-T), and blocked with 200 µL per well of casein blocking buffer (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature (RT). After washing the plate three times with PBS-T, diluted serum samples (1:400 in casein blocking buffer) were added to the plate and incubated for 1 h at RT. The plate was washed and incubated for 1 h at RT with anti-pig IgG antibodies conjugated with horseradish peroxidase (Abcam, Waltham, MA, USA). After washing the plate with PBS-T five times, TMB substrate (Abcam, Waltham, MA, USA) was added, and the reaction was stopped by adding stop solution (Abcam, Waltham, MA, USA). The optical density (O.D.) of each well was measured at 450 nm. The cut-off for positive samples was the average O.D. plus 3 × the standard deviation (SD) of the four negative serum wells.

All serum samples were subject to the VN test against both Wuhan-like WA-1 and alpha VOC. Two-fold serial dilutions of heat-inactivated serum samples starting 1:8 were incubated with 100 TCID₅₀ of either the Wuhan-like WA-1 strain or the alpha VOC for 1 h at 37 °C. The mixture was transferred onto Vero-E6/TMPRSS2 cells and incubated for 72 h. The neutralizing antibody titer was recorded as the highest serum dilution at which at least 50% of wells showed virus neutralization as evidenced by cytopathic effect under a microscope.

3. Results

The level of expression of ACE2 and TMPRSS2 mRNA was analyzed in respiratory tract tissues (trachea, bronchi, bronchioles, and alveoli) from both mock (n = 3) and SIV-infected pigs (n = 4) using RNAscope™ duplex ISH. ACE2 was sporadically expressed in respiratory epithelial cells lining the trachea, bronchi, and bronchioles, while expression of TMPRSS2 was highly abundant within this same compartment (Figure 1 and Figure 2 and Supplementary Table S1). Quantitative analysis of ACE2 and TMPRSS2 signals between mock and SIV-infected pigs demonstrated that, while there was no difference in ACE2 expression, there was a significant increase in TMPRSS2 expression in SIV-infected pigs (p-value = 0.017), specifically in bronchial (p-value = 0.0339) and bronchiolar epithelia (p-value = 0.0339; Figure 2).

Two groups of pigs were primarily infected with SIV and secondarily challenged with either the SARS-CoV-2 Wuhan-like WA-1 strain (n = 4) or an alpha VOC (n = 5) strain on day 3 post-primary infection (DP1I). Infectious SIV was isolated in nasal swabs of infected pigs from 1 to 8 DP1I in both SARS-CoV-2-challenged groups. The SIV titers peaked at 3 to 4 DP1I and gradually declined from 5 DP1I. By 10 to 17 DP1I, SIV was no longer detected (Figure 3). Pigs developed detectable antibody responses against the homologous strain, TX98 H3N2, at 6 DP1I except for one animal (#1363), which seroconverted by 10 DP1I. HI titers ranged from 1:80 to 1:640 at 10 DP1I and reached 1:320 at 17 DP1I (

Table 1. Hemagglutination inhibition titers in co-infected pigs with SIV and SARS-CoV-2.

Group	Pig ID	-1 DP1I/-4 DP2C	6 DP1I/3 DP2C	10 DP1I/7 DP2C	17 DP1I/14 DP2C
SIV + SARS-CoV-2 (WA-1)	1361	<1:10	1:40	N/A	N/A
	1362	<1:10	1:40	1:160	N/A
	1363	<1:10	<1:10	1:640	1:320
	2095	<1:10	1:40	1:640	1:320
SIV + SARS-CoV-2 (alpha VOC)	1360	<1:10	1:40	N/A	N/A
	2092	<1:10	1:80	N/A	N/A
	2091	<1:10	1:20	1:80	N/A
	2093	<1:10	1:10	1:160	N/A
	2094	<1:10	1:80	1:320	1:320

DP1I: days post-primary infection with swine influenza virus (SIV); DP2C: days post-secondary challenge with SARS-CoV-2; N/A: not available.

).

Low levels of SARS-CoV-2 RNA were detected in nasal and oropharyngeal swabs at 1 DP2C (

Table 2. SARS-CoV-2 RT-qPCR results.

Group	Pig ID	Swab	4 DP1I/ 1 DP2C	5 DP1I/ 2 DP2C	6 DP1I/ 3 DP2C	7 DP1I/ 4 DP2C	8 DP1I/ 5 DP2C	10 DP1I/7 DP2C	13 DP1I/10 DP2C	15 DP1I/12 DP2C	17 DP1I/14 DP2C
SIV + SARS-CoV-2 (WA-1)	1361	Nasal	5.45 × 103	Neg	Neg	N/A	N/A	N/A	N/A	N/A	N/A
		Oropharyngeal	4.38 × 103 *	Neg	Neg	N/A	N/A	N/A	N/A	N/A	N/A
	1362	Nasal	Neg	Neg	Neg	Neg	Neg	Neg	N/A	N/A	N/A
		Oropharyngeal	Neg	Neg	Neg	Neg	Neg	Neg	N/A	N/A	N/A
	1363	Nasal	5.39 × 103	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
		Oropharyngeal	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
	2095	Nasal	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
		Oropharyngeal	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
SIV + SARS-CoV-2 (alpha VOC)	1360	Nasal	6.27 × 103 *	Neg	Neg	N/A	N/A	N/A	N/A	N/A	N/A
		Oropharyngeal	Neg	Neg	Neg	N/A	N/A	N/A	N/A	N/A	N/A
	2092	Nasal	9.45 × 103 *	Neg	Neg	N/A	N/A	N/A	N/A	N/A	N/A
		Oropharyngeal	Neg	Neg	Neg	N/A	N/A	N/A	N/A	N/A	N/A
	2091	Nasal	Neg	Neg	Neg	Neg	Neg	Neg	N/A	N/A	N/A
		Oropharyngeal	Neg	Neg	Neg	Neg	Neg	Neg	N/A	N/A	N/A
	2093	Nasal	Neg	Neg	Neg	Neg	Neg	Neg	N/A	N/A	N/A
		Oropharyngeal	5.51 × 103 *	Neg	Neg	Neg	Neg	Neg	N/A	N/A	N/A
	2094	Nasal	5.06 × 103	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg
		Oropharyngeal	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg	Neg

DP1I: days post-primary infection with swine influenza virus (SIV); DP2C: days post-secondary challenge with SARS-CoV-2; positive (positive in both RT-qPCR duplicates) and suspect positive (positive in single RT-qPCR duplicate, labeled as an asterisk symbol in the table) are represented as copy number/mL; Neg: negative; N/A: not available.

) in both SARS-CoV-2-challenged groups. However, all nasal and oropharyngeal swab samples obtained at 2 DP2C and thereafter were negative for SARS-CoV-2 RNA by RT-qPCR. All tissue samples (nasal conchae, ethmoidal turbinates, soft palate, olfactory bulb, trachea, bronchi, lung, tonsil and tracheobronchial lymph node), BALF, and nasal washes collected on 3, 5 and 14 DP2C were also negative for SARS-CoV-2 RNA by RT-qPCR. Virus isolation was not performed since the amount of viral RNA in positive samples did not meet our criteria for virus isolation where samples with 10⁶ copy number or greater are subjected to virus isolation. In addition, all serum samples were sero-negative by SARS-CoV-2 RBD- and N-based ELISAs (below cut-off value) as well as by VN test (<1:8), with the latter using both Wuhan-like WA-1 and alpha VOC isolates.

4. Discussion

Some coronaviruses can bind to the swine ACE2 receptor and utilize the porcine cellular machinery for viral replication [20,21]. For example, pigs are susceptible to infection with SARS-CoV, a virus which caused the SARS epidemic in 2004, mainly in Asia. After experimental SARS-CoV infections, pigs developed neutralizing antibodies [20]. Furthermore, during field surveillance studies, two SARS-CoV-seropositive pigs were identified, and SARS-CoV was isolated from blood and fecal samples [21].

In in vitro experiments, SARS-CoV-2 caused cytopathic effects and replicated in several swine-derived cell lines [13,22]. However, early studies failed to detect productive infection with SARS-CoV-2 in pigs upon experimental infection via intranasal, intraoral, intratracheal, intravenous, and/or intramuscular routes [13,23,24,25]. One study reported the presence of infectious virus in the submandibular lymph node of one out of 16 experimentally SARS-CoV-2-infected pigs at 13 days post-infection [26]. Overall, this indicates that pigs are rather resistant to SARS-CoV-2 infection. In the present study, pigs were primarily infected with SIV and, 3 days later, secondarily challenged with SARS-CoV-2. This timepoint was chosen based on our previous studies indicating that robust replication of influenza A virus, as well as enhanced expression of TMPRRS2, is observed in the respiratory tracts of pigs at day 3 post SIV infection [27,28]. Upon experimental challenge of SIV-infected pigs with SARS-CoV-2, low levels of SARS-CoV-2 RNA were detected at 1 DP2C. This could be due to residual inoculum and/or abortive local replication; however, none of these pigs developed detectable SARS-CoV-2-specific antibody responses, an indicator for efficient SARS-CoV-2 replication. These results demonstrate that pigs are not permissive to infection with either the original SARS-CoV-2 Wuhan-like WA-1 strain or an alpha VOC strain following primary SIV infection; thus, prior IAV infection had no effect on the susceptibility of pigs to these different SARS-CoV-2 strains.

Co-infection with IAV isa crucial factor contributing to the increased severity of COVID-19 in human patients. In this context, efforts have been made to elucidate the molecular mechanisms of increased clinical severity after co-infection with SARS-CoV-2 and IAV. A recent study showed that IAV infection increased ACE2 mRNA levels and regulated post-translational modification of ACE2 in human small airway epithelial cells (SAECs) [28]. ACE2 is an interferon-stimulated gene (ISG) in human airway epithelial cells [29], suggesting that IAV infection-induced antiviral responses may contribute to the altered expression pattern of ACE2n. In contrast, ACE2 mRNA expression decreased in murine lungs after H1N1 infection [30] and also after SARS-CoV-2 infection [31]. In addition, infection with either the H5N1 or H7N9 virus led to a downregulation of ACE2 protein expression in murine lungs [32,33]. Our study showed no significant changes in ACE2 mRNA expression across respiratory tract compartments following SIV infection. In contrast, infection with SIV led to elevated expression of swine TMPRSS2 mRNA in the lower conductive portions of the respiratory tract, specifically in bronchial and bronchiolar epithelia, consistent with previous findings in human SAECs [28]. These results might support the notion that elevated expression of the SARS-CoV-2 receptor and/or cellular proteases involved in viral entry can enhance the susceptibility of animals to SARS-CoV-2 infection; however, based on this study, such a scenario does not seem sufficient to increase SARS-CoV-2 susceptibility in pigs. Viral infection of cells induces host antiviral defense mechanisms and stimulates the release of cytokines such as type I interferons. Therefore, it is plausible that the antiviral response to SIV protected pigs from subsequent SARS-CoV-2 infection. However, in-depth immune profiling, including cytokines and ISGs, was not conducted in this study.

There are several limitations to this study. First, only early SARS-CoV-2 isolates were evaluated. Since then, SARS-CoV-2 has undergone substantial genetic evolution, accumulating mutations across its genome, resulting in the emergence of novel variants. It is therefore possible that other virus variants may exhibit altered host susceptibility and/or improved adaptation, leading to outcomes that differ from those observed here. Another limitation of this study is that we evaluated pigs challenged with SARS-CoV-2 at 3 days post-SIV infection only; thus, we cannot rule out that challenging pigs with SARS-CoV-2 at an earlier or later timepoint post-SIV infection could influence the results observed. Therefore, experimental challenge with SARS-CoV-2 at different timepoints post-SIV infection could provide additional insights into the susceptibility of influenza-infected pigs to SARS-CoV-2. Lastly, the presence of SARS-CoV-2-specific RNA loads and antibody responses in the clinical samples was evaluated by RT-qPCR, VN, and ELISA. A more extensive analysis of samples using newer, more sensitive and specific methods could be valuable for corroborating our findings.

In conclusion, this study evaluated whether pre-infection with SIV increases susceptibility of pigs to subsequent SARS-CoV-2 infection. Our findings showed no productive replication of either SARS-CoV-2 strain in SIV-infected pigs. Overall, we conclude that pigs are not a suitable animal model for SARS-CoV-2 and IAV co-infection studies.