Swine testis (ST) cells were purchased from ATCC (ATCC CRL-1746) and were propagated using advanced minimum essential media (MEM; Gibco) supplemented with 5% foetal bovine serum (FBS), 2 mM L- glutamine and 10,000 units (U) per ml of penicillin and 10,000 µg/ml streptomycin (Life Technologies). LLC-PK1 cells, a porcine kidney epithelial cell line, were provided by the central services unit (CSU) at The Pirbright Institute and were propagated using 199 media (Sigma) supplemented with 10% FBS and 10,000 U/ml penicillin and 10,000 µg/ml streptomycin. HEK293T cells provided by CSU at The Pirbright Institute were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 1% sodium pyruvate solution (Sigma-Aldrich) and 10,000U/ml of penicillin and 10,000 µg/ml streptomycin, from here called cDMEM-10. All cells were maintained at 37°C, 5% CO₂.

PRCV strain AR310, abbreviated to 310, was purchased from ATCC (ATCC VR2384) and ISU-1 from Bei Resources (NR-43286). 310 was originally isolated from the small intestine of a pig within a TGEV endemically infected herd located in Arkansas, USA (24) ISU-1, GenBank accession number DQ811787.1, was isolated from nasal swabs collected from pigs in Indiana, USA (25). PRCV strains 86/137004 (abbreviated to 137) and 86/135308 (abbreviated to 135) were both isolated in 1986 from pigs in the UK and were gifts from S. Cartwright (26) (Animal and Plant Health Agency, APHA). Both 135 and 137 were isolated from infected respiratory tract tissue. The TGEV strain FS772/70 was a gift from S. Cartwright (27). All viruses were propagated on ST cells using PRCV medium (EMEM supplement with 0.02% yeast extract, 10% TPB, 2 mM L-glutamine, 10,000 U/ml penicillin and 10,000 µg/ml streptomycin) and quantified via titration, also in ST cells.

RNA was extracted from PRCV strains 135, 137, 310 and ISU-1 propagated on ST cells using the Qiagen viral RNA kit following the manufacturer’s instructions, including addition of carrier RNA. RNA quality was analysed using an RNA screentape on a Tapestation 4200 (Agilent, Santa Clara) and a Qubit RNA BR kit (Qiagen, Hilden). cDNA and library preparation were carried out in duplicate for each sample. Sequencing library preparation was performed with an input of 200 ng total RNA for each sample, using the NEBNext directional Ultra II RNA-Seq kit (NEB, Ipswich, MA). Library quality control was performed using the Tapestation DNA1000 kit and Qubit BR kit, prior to pooling. Sequencing pools were quantitated using the NEBNEXT Illumina library quantitation kit (NEB, Ipswich, MA) before dilution for sequencing. All libraries were run on an Illumina MiSeq, using a MiSeq reagent v3 600 cycle cartridge (Illumina, San Diego). The data generated were subjected to quality control checks on the raw fastq files using FastQC. Filtered reads for standard quality score threshold were aligned to the reference genome PRCV ISU1 (GenBank: DQ811787) using both BWA-MEM (version 0.7.12-r1039), and Bowtie2 (version 4.1.2) for comparison. A consensus sequence was generated for each sample and the duplicate samples compared. The consensuses generated by each duplicate were identical and therefore a single consensus was considered for further analysis and submitted to NCBI GenBank, accession numbers OM830318 (PRCV 135), OM830319 (PRCV 310), OM830320 (PRCV 137) and OM830321 (PRCV ISU-1). Sequence alignments of the S and 3b genes were performed with Snapgene, version 5.2.3 (Supplementary Figures 1,2).

ST cells were seeded in 12 well sterile tissue culture plates and incubated until confluent. Ten-fold serial dilutions were prepared using PRCV medium. Cells were washed with phosphate buffer saline (PBS) prior to inoculation with 250 µl per well of virus in either duplicate or triplicate. After 1 h incubation at 37°C, the inoculum was removed and 2 ml N,N-Bis(2-hydroxyethyl)-2aminoethanesulphonic acid) medium containing 1% agar (suitable for cell culture; Sigma) was added. Plaques were visualized 96 hpi using 0.1% crystal violet (H₂O) following fixation with 3.3% formaldehyde (PBS), and the number of plaque forming units (PFU) per ml calculated.

ST or LLC-PK1 cells were seeded in 6 well sterile tissue culture plates and incubated until confluent. Cells were washed twice with PBS prior to infection with 10⁴ PFU of either PRCV or TGEV strains in 500 µl PRCV medium. Mock infected cells were inoculated with medium only. Inoculated cells were incubated for 1 h, 37°C after which the inoculum was removed, the cells washed once with PBS, and 3 ml PRCV medium added per well. At defined intervals supernatant was harvested and titrated in either duplicate or triplicate in ST cells to determine the quantity of infectious progeny. RNA was extracted from the infected cells using RNeasy mini kit (Qiagen) including an on column DNAse treatment; viral RNA load was determined by qRT-PCR.

Reverse transcription, using random primer, of 500 ng total RNA (cell lystate), 400 ng (BAL derived RNA), 500 ng (Nasal or trachea swab RNA) was performed using the Superscript IV. Reverse Transcriptase kit (Invitrogen, Waltham, MA, USA) as per the manufacturer’s protocol. Viral RNA load was quantified using quantitative real-time PCR (qRT-PCR) using primers and probes targeting the N gene as follows: forward primer 5’-ATAGACAAACTCGCTATCGC-3’, reverse primer 5’-CAACCCAGACAACTCCATC-3’, and probe FAM-TAGGCACTGGACCTCATGCA-TAMRA. Quantitative RT-PCR was performed with FastAdvance Mastermix (Applied Biosystems) using 500 nM forward and reverse primers, 300 nM probe and 100 ng of cDNA as normalized input. An applied Biosystems QuantStudio/7500 Fast real-time PCR instrument (Life Technologies) was used with the following cycling conditions: 10 min at 95°C, 35 cycles of 10 s at 95°C, 10 s at 60°C and 10 s at 75°C. Standard curves were generated to allow quantitation of RNA copy numbers based on cDNA levels using a pCR4-TOPO (Invitrogen) plasmid containing the sequence amplified by the set of primers listed above. The resulting Ct results were used to calculate the log of relative RNA copies using the linear equation from the standard curve.

Tracheal organ cultures (TOCs) were prepared from trachea extracted from a 4-week-old piglet. The trachea was washed with PBS and excess fat removed using a scalpel. The trachea was sectioned into rings of approximately 3 mm, and each ring placed in a glass test tube (10 mm by 16 mm) with 1 ml of medium (EMEM supplemented with 40 mM Hepes, 2 mM L-glutamine, 10,000 U/ml penicillin and 10,000 µg/ml streptomycin and 10,000 U/ml nystatin). The TOCs were incubated for 48 h prior to infection, in a non-CO₂ incubator (Incudrive-S, Life Technologies) at 37°C, 7 – 8 revolutions per h. Each TOC was inoculated with 500 µl PRCV medium only or 500 µl PRCV medium containing 1 x 10⁴ or 1 x 10⁵ PFU of either PRCV strains 135, 137, ISU-1, 310 or TGEV 772/70. TOCs were incubated for 1 h at 37°C, no rotation, after which the inoculum was removed and replaced with 1 ml 1 x PRCV media. TOCs were incubated for a further 23 h at 37°C, 7 - 8 rev per h. At 24 hpi the supernatant was harvested and the quantity of infectious virus determined by titration in ST cells. TOCs were either discarded or fixed for investigation by confocal microscopy.

Whole ex vivo TOCs were fixed in 4% formalin for 72 h, washed in PBS and frozen at -80°C in optimal cutting temperature compound (OCT compound). Frozen TOCs were sectioned using a cryostat (Leica Cryostat CM1860 UV) into 100 µm thick transverse sections. Individual sections were placed onto glass slides and allowed to air dry for 30 min before residual OCT compound was removed. Tissue sections were permeabilized with 0.1% Triton X-100 for 20 min and blocked with 0.5% PBS/BSA for 1 h. Tissue sections were labelled for 1 h with either the mouse monoclonal antibody, DA3, against the N protein of TGEV FS772/70 strain which targets the 23 terminal residues of the nucleoprotein (N) protein (28) or anti-dsRNA (Absolute Antibody, Ab00458-23.0) or co-labelled with anti-N (DA3) and anti-dsRNA together. The antibodies were diluted 1:400 and 1:1000 respectively in 0.5% BSA. Labelled tissues sections were washed three times in PBS prior to secondary antibody labelling for 1 h. Secondary antibodies were Alexa Fluor^® 488 Goat anti-mouse IgG2a (Invitrogen, A-21131) for the detection of anti-N and Alexa Fluor^® 568 Goat anti-Rabbit IgG (H+L) (Abcam, ab175471) for the detection of anti-dsRNA. Both were diluted 1:1000 in PBS/BSA. After three washes, nuclei were stained for 10 min with 4’,6-diamidino-2-phenylindole (DAPI) diluted 1:10,000 in deionised water. Tissue sections were washed with deionised water. GeneFrames, ~ 65 µl depth (1.5 x 1.6 cm - Thermo Scientific™) were placed around each labelled tracheal section and Vectashield mounting medium added followed by a coverslip. Tissue sections were imaged on a Leica confocal microscope.

All experiments were approved by the ethical review processes at the Pirbright Institute and Animal and Plant Health Agency and conducted according to the U.K. Government Animal (Scientific Procedures) Act 1986 under project license PP7764821. Both institutes conform to Animal Research: Reporting of In Vivo Experiments guidelines. Prior to infection, all pigs were screened for the absence of PRCV antibodies using the INgezim Corona Diferencial ELISA (Ingenasa, Spain). Clinical parameters including demeanour, appetite, respiratory signs, sneezing, coughing, nasal and eye discharge, faeces consistency and rectal temperature were assessed.

Tissues were homogenised in 1 ml PBS containing 10,000 U/ml penicillin and 10,000 µg/ml streptomycin using 5 mm stainless steel beads and a Tissue Lyser II (Qiagen). 0.5 g of lung accessory lobe, 0.5 g of tracheal tissue, 0.5 g (from the pilot study) and 0.1 g of nasal turbinates were homogenised. The resulting supernatant was clarified by low-speed centrifugation. The quantity of infectious virus was determined by titration in ST cells.

Lungs harvested at necropsy were assessed blindly by a pathologist. Both dorsal and ventral surfaces were assessed for reddening and consolidation of lung lobes as previously described (31). Lung images taken at necropsy were analysed using Image J (Version 1.52a) and assessed blindly by a different operator to estimate percentage of lung lesions.

Formalin fixed tissues were processed by routine histology method. Hematoxylin and eosin staining and immunohistochemistry (IHC) against PRCV nucleoprotein (N) were performed on serially sectioned formalin-fixed paraffin-embedded tissues. Briefly, 4 μm sections were dewaxed and dehydrated through xylene and graded alcohol, endogenous peroxidase activity quenched with a methanol and hydrogen peroxide, followed by unmasking of epitope with heat-mediated antigen retrieval in pH 9 buffer at 100°C for 10 mins (Dako). Slides were assembled into Shandon Sequenza cover plates to facilitate IHC (Shandon, USA). Tissue sections were incubated with an anti-N mouse monoclonal antibody DA3 at 0.1 µg/ml or concentration matched isotype mouse monoclonal antibody for 1 hr at RT and then with Dako ENVISION™ polymer for 20 min at RT. Immunolabelling was performed at room temperature and sections were washed three times with Tris-buffered saline between incubations. Immunolabelling was visualized using 3,3-diaminobenzidine (Sigma Aldrich) and counterstained in Mayer’s haematoxylin (Surgipath, UK). Slides were dehydrated in absolute alcohol, cleared in xylene and mounted using Dibutyl Phthalate Xylene and glass coverslips. A serial section was stained with haematoxylin and eosin for histopathology evaluation. Positive control slides containing either TGEV or PRCV infected or uninfected cells prepared in agarose matrix were included during IHC experiment to confirm immunolabelling (32).

The spike sequence from PRCV ISU-1 (GenBank Accession number: DQ811787.1) was modified to replace the coding region for the C-terminal transmembrane and cytoplasmic domains, with a T4 foldon trimerization domain followed by a His⁸ tag. The sequence coding for amino acids E914 and L915 was mutated to two prolines in an attempt to stabilise the spike in a pre-fusion state similar to that described for other coronavirus spikes. The sequence was optimised to human codons and synthesised into the plasmid expression vector pTwist CMV BetaGlobin, to create vector pPRCV-ISU1-PP-Foldon. The spike was expressed in Expi293F cells (ThermoFisher) according to the manufacturer’s instructions. Culture supernatants were clarified by centrifugation and purified through 5 mL HisTrap FF column (GE Healthcare). Fractions containing spike were concentrated and the excess imidazole removed by using buffer exchange columns (Zeba, Merck).

The spike sequence from PRCV ISU-1 was optimised to human codons and synthesised in plasmid expression vector pTwist CMV BetaGlobin (Twist Bioscience), to create vector pPRCV-ISU1-PP. HEK293T cells were plated at 4 x 10⁵ cells per 6-well plate in cDMEM-10. The following day, the cells were transfected with 0.5 µg of pPRCV-ISU1-PP, along with 0.6 µg p8.91 (encoding HIV-1 gag-pol) and 0.6 µg CSFLW (the firefly luciferase reporter-expressing lentivirus-backbone) using 10 µL polyethylene imine (PEI; Sigma-Aldrich). Transfection mixtures were removed the next day and replaced with 3 ml cDMEM-10. Cells were incubated for 48 h with two harvests of supernatant taken at 24 and 48 h which were pooled, centrifuged at 1300 x g for 10 minutes at 4°C to remove cellular debris and then aliquoted and frozen at -80°C.

The sequence for porcine APN (UniProtKB/Swiss-Prot: P15145.4) was optimised to human codons and synthesised with an N-terminal HA-tag into the plasmid expression vector pTwist CMV BetaGlobin, to create vector pHA-pAPN. Supernatants containing pseudotyped PRCV (ISU1pp) were titrated on HEK293T cells that had been pre-transfected with pHA-APN. After 72 h, firefly luciferase activity was measured using the Bright-Glo system (Promega).

Fresh cells from TBLN and spleen were cultured as previously described (34). Briefly, cells were resuspended to 6 x 10⁷ cells/ml and plated, 100 µl/well, into Transwell^® permeable (0.4 µm pore size) membranes (12 mm diameter polyester membranes in standard 12-well plates Corning), with 1ml complete medium (RPMI with Glutamax, 10% FBS, 1x nonessential amino acids, 1 x sodium pyruvate, 1 x penicillin–streptomycin, 1 x Normocin (In vivoGen) and 1 x insulin/selenium/transferrin cocktail (Gibco)), in the lower chamber. PRCV (MOI 0.01) or medium control was added directly to the insert (cell-containing portion). Cultures were incubated at 37°C in a 5% CO₂ incubator and supplemented with additional medium to the lower wells if necessary. After 7 days, supernatants were removed for analysis of PRCV-specific antibodies by ELISA, neutralization or pseudo neutralization assays.

96-well Maxisorp ELISA plates (Biolegend, UK) were coated overnight at 4°C with pre-optimized concentrations of ISU-1 full length S protein (1 µg/ml). Two-fold dilutions of serum, either collected pre-inoculation (day 0) or collected post-mortem, starting from 1/20 in PBS-Tween plus Marvel (4%) were applied. Binding of antibodies was detected with goat anti-pig IgG (Fc-specific) conjugated to horseradish peroxidase (AA141P, Biorad, Watford, UK), used at optimal dilution. 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Scientific Pierce) was added and the optical density values were read for each well at dual wavelengths (450 nm and 630 nm) using a Labsystems Multiskan plate reader (Fisher Scientific, Loughborough, UK). The quantities of antibody were determined as the reciprocal value of the dilution that gave the first reading above the cut-off value (end-point titer). Cut-off values were determined as mean blank ODs plus 2-fold standard deviations. Starting dilutions were 1:20 for serum, 1:16 for BAL and 1:2 for nasal swab respectively.

MultiScreen™-HA ELISPOT plates (Millipore, Watford, UK) were coated overnight at 4°C with 0.5 µg/ml mouse anti-pig IFNγ (BD Biosciences, San Jose, CA) in carbonate buffer (0.1 M Na₂CO₃/NaHCO₃, pH 9.6). Plates were washed 3 times with PBS and subsequently incubated for two hours at 37°C with 200 µl per well of blocking buffer (RPMI, 10% FBS, supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. The blocking buffer was removed prior to plating stimuli which included medium alone (RPMI-1640 medium with glutamax-I and 25 mM Hepes (Invitrogen Ltd., Paisley, UK) supplemented with penicillin, streptomycin and 10% heat-inactivated FCS, PRCV viruses 135, 137, ISU-1 and 310, peptide pools covering the spike (S) and nucleoprotein (N) of PRCV 135 virus or 2.5 µg/mL Concanavalin A (Con A, Invitrogen). 16 mer peptides overlapping by 12 residues were designed and synthesized by Mimotopes, Melbourne, Australia (Supplementary Tables 2, 3). Three pools of peptides representing residues 1-100 (pool 1), 101-200 (pool 2) and 201-305 (pool 3) for the S protein and one pool of 94 for the N protein were used to stimulate T cells at a final concentration of 2 µg/ml.

For each culture condition, triplicate wells of cryopreserved cells (TBLN, BAL, PBMC) at 2 x 10⁶/ml were added to the coated ELISPOT plates (100 µl/well) and cultured for 48 h at 37°C, 5% CO₂. Cells were washed off the plates using PBS containing 0.05% Tween 20, then 100 µl per well biotinylated mouse anti-pig IFNγ (0.5 µg/ml, BD Biosciences) was added for 2 h at room temperature. Plates were washed five times with PBS containing 0.05% Tween 20 and 100 µl per well streptavidin conjugated to alkaline phosphatase (1/1000, Invitrogen Ltd.) was added for 1 h at room temperature. Plates were washed five times with PBS containing 0.05% Tween 20 and developed with 100 µl per well alkaline phosphatase substrate solution (Biorad laboratories, Hercules, CA) for 20 min at room temperature. Plates were rinsed with tap water and allowed to dry overnight at room temperature before counting the dark blue-coloured immunospots using the AID ELISPOT reader (AID Autoimmun Diagnostika GmbH, Strassberg, Germany). Results were expressed as IFNγ-producing cell number per 10⁶ stimulated cells after subtraction of the average number of spots in media stimulated control wells.

Cryopreserved cells (TBLN, BAL, PBMC) were thawed and seeded at a conc. of 1 x 10⁶ cells/well and stimulated overnight with 135 virus (MOI=0.5) or medium control at 37 °C, 5% CO₂ before the addition of Brefeldin A (GolgiPlug™, BD Biosciences) allowing accumulation of the cytokine inside the cell. A cocktail of phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Biolegend) was added as a positive control at the same time as the GolgiPlug to separate control wells. Duplicate wells, each containing 1 x 10⁶ cells, were set up for each stimulation condition. After 4 h incubation at 37°C, replicate wells for each stimulation were pooled, centrifuged for 4 min, washed twice with PBS and analyzed for cytokine production using the antibodies listed in Supplementary Table 4. Briefly, cells were stained with the primary Abs for surface staining and with Near-Infrared Fixable LIVE/DEAD stain (Invitrogen), for identification of live cells. Following a 20 min incubation at 4°C, cells were washed twice, fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) as per the manufacturer’s instructions. Cells were incubated for 30 min at 4°C with the directly conjugated cytokine antibodies, washed, re-suspended in PBS, and analyzed using a MACSquant analyser10 (Miltenyi). In the case of the IL-2 cytokine antibody, a further incubation step of 20 min at 4°C with the corresponding secondary antibody was necessary since it was not directly conjugated. Thereafter, cells were washed and resuspended in PBS before analysis. Downstream analysis was performed using FlowJo software.

We characterised the in vitro replication of four PRCV strains: 135 and 137, both isolated from the respiratory tract of pigs in the UK; 310, isolated in the USA from the small intestine (24) and the previously characterised ISU-1 strain (5, 25). We initially assessed their replication and associated cytopathic effects (CPE) in ST and LLC-PK1 porcine derived cells lines, routinely used for assessment of PRCV and TGEV (Figures 1A, B). The quantity of infectious progeny was determined by titration in ST cells. Over a 96-h growth period using ST cells, ISU-1 generated higher quantities of infectious progeny than 135, 137 and 310, however statistical significance was only reached in comparison to 137 from 24 – 72 hours post infection (hpi), to 135 from 48 and 96 hpi and to 310 from 72 hpi (p<0.05). In LLC-PK1 cells, 135 generated the lowest titer of virus and was statistically different from ISU-1 from 48 - 96 hpi (p<0.05). Despite these differences, the pattern of replication over 96 h is comparable, with similar peak titers observed at the same time point in both cell types ( Figure 1A).

No major differences in the quantity of infectious progeny were identified between the four strains in the first 12 h, in either ST or LLC-PK1 cells further indicating that in vitro replication was comparable (Figure 1B). Infectious progeny present in the cell supernatant was determined by titration. RNA replication in the harvested cells was analysed by quantitative real time PCR (qRT-PCR) using primers and probes targeting the N gene. The quantity of 135 and 137 RNA in LLC-PK1 cells was higher compared to 310 and ISU-1 indicating an increased rate of RNA replication. This however was not observed in ST cells and did not translate into higher numbers of infectious progeny.

Previous work had shown that high titers of PRCV strain TLM83 were isolated from tracheal tissue from experimentally infected pigs, with viral antigen detected in epithelial cells, suggesting active PRCV replication in tracheal epithelial cells (35). To establish whether the four PRCV strains used in this study could replicate in the trachea, primary ex vivo TOCs were infected with 1 x 10⁴ PFU of either 135, 137, ISU-1 or 310. The titers of infectious progeny virus from TOCs infected with ISU-1 and 310 were lower than those observed for 135 and 137 in two independent experiments (Figures 1C, D). Each experiment used TOCs prepared from the trachea harvested from an individual animal and therefore differences may be the result of a host or virus-host interaction effect. In another separate experiment TOCs were inoculated with a log higher titer of 1 x 10⁵ PFU to investigate whether a higher dose was required for the establishment of productive infection for both ISU-1 and 310. Less infectious progeny from ISU-1 and 310 infection was detected in comparison to 135 (p<0.05) (Figure 1E). Interestingly the observed titers were comparable between 137 and ISU-1 indicating either ISU-1 requires a higher quantity of input virus to initiate productive replication, or there is a differential host effect, with perhaps trachea extracted from some pigs that are more susceptible to infection than others. TGEV was included in the experiment as previous reports have shown that it is able to replicate in the trachea (36, 37). Titers of TGEV were comparable to those of 135 and 137 strains, confirming the previous observations that TGEV can replicate in the trachea (Figure 1E).

To investigate RNA and protein production, the TOCs from the high dose experiment were analysed by confocal microscopy using DA3 and dsRNA antibodies (Figure 1F). DA3 was generated from the TGEV nucleoprotein (N) protein and recognizes the four PRCV strains as confirmed by Western blot analysis (28, 38) (Supplementary Figure 3). The second antibody targets double stranded RNA (dsRNA) and is a well-established marker for coronavirus RNA replication (32, 39). Both dsRNA and N protein were detected in the mucosal epithelium in TOCs infected with TGEV and PRCV strains 135 and 137 (Figure 1F). Small, localized regions of signal for N protein and dsRNA could be detected in TOCs infected with PRCV ISU-1 (Figure 1F). No dsRNA or N protein were detected in mock infected TOCs nor in TOCs infected with 310, suggesting that 310 may not be able to replicate in tracheal epithelial cells in vivo.

Overall, these data indicated that although the four strains showed comparable replication in ST and LLC-PK1 cells, the 135 and 137 strains replicated more efficiently than ISU-1 and 310 in the tracheal epithelial cells of ex vivo TOCs.

The replication differences in the ex vivo TOCs suggested altered tissue tropism between the four strains. To investigate further, next generation sequencing analysis was performed. As a major determinant of tropism is the S glycoprotein, we compared the amino acid sequence of the four PRCV strains ( Supplementary Figure 1). PRCV 135 and 137 differed by 7 amino acids and 310 and ISU-1 by 9 amino acids. There were a larger number of differences between both 135 and 137 compared to 310- and ISU-1. Interestingly, there is an insertion of amino acids TSV between residue P20 and C21 in 135 and 137, not present in either ISU-1 or 310.

Following the ex vivo analysis we characterized the clinical, virological and pathological parameters of PRCV strains 135, 137, 310 and ISU-1 in vivo. Initially a pilot study was carried out using two pigs inoculated with 2 x 10⁷ PFU of each PRCV strain by the intra-nasal/intra-tracheal (IN/IT) route and observed for 5 days (Figure 2A). The rectal temperatures of the animals were taken daily and all were within the normal range. At 5 days post-infection (DPI), one PRCV 135 inoculated animal exhibited nasal discharge and coughing, and one PRCV 137 inoculated animal was observed sneezing. Both PRCV 135 animals and the PRCV 137 animal without respiratory signs, displayed signs of lethargy, as did the second animal infected with 137. No clinical signs were observed in PRCV ISU-1 or 310 inoculated animals.

Nasal swabs were taken daily and assessed for presence of infectious virus by titration and PRCV RNA by qRT-PCR of the N gene (Figure 2B). The titers of infectious ISU-1 and 310 viruses were lower in the first 2 DPI than 135 and 137 viruses detected in the nasal swabs, suggesting a decrease in productive viral replication. However, the quantities of PRCV derived RNA detected in the nasal swabs were largely similar for all strains. Virus load and the quantity of RNA were comparable between the four strains in nasal swabs, nasal turbinates and BAL fluid taken at post-mortem (5 DPI) (Figures 2C–E).

In contrast, the titers of infectious virus and the quantity of PRCV RNA differed between the PRCV strains in the tracheal swab and tracheal tissues taken at post-mortem (5 DPI) (Figure 2F). Three segments of trachea, one from the top, middle and bottom of the trachea, were harvested, with no observable difference in virus titer and therefore the mean titer of the three tracheal segments was presented. Less infectious virus was detected in ISU-1 and 310 inoculated animals suggesting that these viruses replicated to a lower level than 135 and 137 in the trachea. No infectious virus for ISU-1 and 310 were detected in the accessory lung lobe, in contrast to 135 and 137 which had titers of ~10⁶ PFU/ml per 0.5 g of tissue (Figure 2G). This indicates that no productive replication of 310 and ISU-1 was occurring in the accessory lung lobe, supported by the lack of RNA. No RNA was detected in the blood from any of the four PRCV strains.

These data indicated that ISU-1 and 310 did not replicate efficiently in the trachea and lung of the experimentally inoculated pigs, although they were detected at comparable levels to 135 and 137 strains in nasal swabs, nasal turbinates and BAL at 5 DPI. The patterns of replication for the four PRCV strains in vivo in the trachea were comparable to those observed in ex vivo TOCs.

Infected pigs were subjected to necropsy at 5 DPI. On gross examination, infection of the ISU-1 and 310 strains resulted in less than 5% of pulmonary consolidation across all lobes. In contrast, 135 and 137 produced a median of 21% and 13.5%, respectively, of areas of consolidation (Figure 3A). The distribution of lesions were characterized by multifocal to coalescing, and were predominantly in middle, caudal, and accessory lung lobes (Figure 3B). There were no observable changes within the nasal cavity or the trachea grossly.

The nasal turbinates, trachea and right lung lobes were subjected to histopathology and PRCV N antigen immunohistochemistry (IHC) using DA3 antibody. The changes observed within the nasal turbinates were generally mild, with multifocal lymphoplasmacytic inflammation of the submucosa and occasional intra-epithelial neutrophils (Figure 3C). Epithelial necrosis was occasionally observed in pigs infected with 135 or 137, co-localising with N labelling of the mucosa epithelium within areas of lesion. N labelling within the nasal turbinates in pigs infected with ISU-1 or 310 strains were rare. Similarly, in the trachea, microscopic changes were limited to 135 and 137 infected pigs, which exhibited small areas of attenuated epithelium, loss of cilia, which colocalized to areas of virus antigens of the mucosa epithelium. No N labelling was observed in the ISU-1 infected pigs.

The pulmonary changes in ISU-1 and 310 infected pigs were mild, with presence of bronchiole-associated lymphoid tissues (BALT) and occasional expansion of alveolar septum with mononuclear inflammatory cells, representative of antigenic stimulation from viral infection (Figure 3C). Only one ISU-1 pig (P21-3414) showed minimal N antigen labelling in bronchiolar epithelium and alveoli.

In the 135 and 137 infected pigs, there was moderate bronchiolar exudation, variably hyperplastic and occasionally attenuated bronchiolar epithelium, and the surrounding airways loosely cuffed by lympho-plasmacytic cells. In the alveoli, there were multifocal to coalescing areas of intra-alveolar oedema, alveolar macrophages, and neutrophils (Figure 3D), type II pneumocyte hyperplasia, and the alveolar wall expanded by mononuclear inflammatory cells. Alveolar wall damage was only observed in 135 infected pig (P21-3415). The level of N antigen immunolabelling was moderate within bronchiolar epithelium, and small amounts in type II pneumocytes, intravascular macrophages, alveolar macrophages and occasionally in type I pneumocytes. Overall, the pilot experiment demonstrated that PRCV strains 135 and 137 induced moderate broncho-interstitial pneumonia, while the pathological changes in ISU-1 and 310 were very mild.

No PRCV specific Ab responses were detected in serum or BAL as measured by ELISA at 5 DPI using recombinant full-length Spike protein or pseudo-neutralization. We enumerated IFNγ secreting cells by ELISpot following re-stimulation with the four PRCV strains or three pools of peptides covering the S protein and one pool of peptides for the N protein of PRCV 135 (Supplementary Figure 4, Supplementary Tables 2, 3). IFNγ secreting cells were detected by ELISpot in tracheobronchial lymph nodes (TBLN) and peripheral blood mononuclear cells (PBMC). The summed response to the S pool of peptides is shown (Supplementary Figures 4C, D). The responses to virus stimulation were low whilst there was a greater response to the peptides in TBLN and peripheral blood monocular cells (PBMC) (Supplementary Figures 4A, B). The responses to the peptides in PBMC were lower compared to TBLN. Overall, it was difficult to establish whether there were differences in the IFNγ responses between the animals infected with different PRCV strains, because too few animals were used, however it was clear that there was a good cross-reactive T cell response at the early time point of 5 DPI.

As the observed pathology induced by 135 virus was most severe among the four PRCV strains investigated, we further evaluated the production of infectious virus, pathology and immune responses up to 16 DPI. In addition, we compared two different routes of inoculation – aerosol (Aer) and intra-nasal/intra-tracheal (IN/IT) to determine if this might affect clinical disease, pathology and host responses. Two groups of nine pigs were inoculated with 1 x 10⁷ PFU of PRCV 135 by the IT/IN or by the Aer route using a face mask with Aerogen Solo nebuliser (Figure 4A). Three animals from each group were culled on 4, 9 and 16 DPI and blood, nasal turbinates, trachea, BAL, mesenteric, retro-pharyngeal and TBLN collected for analysis. Temperatures of above 40°C were recorded in several pigs from both groups as early as 2 DPI, sometimes accompanied with reduced activity, loose faeces and respiratory signs. On only one occasion was a temperature above 41°C recorded (pig 12512, IN/IT group, 3 DPI) and this was accompanied with reduced activity. Six animals from the IN/IT group had loose faeces starting at 4 DPI. Five pigs (one from the IN/IT group and four from the Aer group) showed transient coughing/sneezing between 5 DPI and 8 DPI, in each case resolving within 24 h (Supplementary Figure 5). Overall, the clinical signs were mild, detected only in some of the animals, resolved rapidly and never exceeded a score of 5 out of maximum 19 (Clinical signs scoring in Supplementary Table 1).

The presence of infectious 135 virus was assessed by plaque assay from daily nasal swabs. No differences in the amount of virus detected were observed between the IN/IT and Aer groups, with no virus detected after 10 DPI (Figure 4B). Analysis of nasal swabs ( Figure 4C), nasal turbinates ( Figure 4D), tracheal swab ( Figure 4E), tracheal tissue ( Figure 4F), BAL fluid ( Figure 4G) and lung accessory lobe ( Figure 4H) also showed there were no differences in the amount of infectious virus detected in the samples between the IN/IT and Aer groups. Although low amounts of virus were detected in the nasal swabs and nasal turbinates in the IN/IT group at 9 DPI no virus was detected in the samples obtained from the Aer group at 9 DPI. The quantity of PRCV derived RNA was not measured in this experiment as the pilot study did not show any difference between the RNA load and infectious viral load determined by titration (Figure 2).

Overall the route of inoculation did not affect the amount of infectious virus in any respiratory tissues with no virus detected on or after 9 DPI, although from previous work studying influenza viruses infection of pigs, aerosol delivers only 20-30% of the input dose with uniform distribution throughout the lung compared to IN/IT which delivers a higher proportion to the lung although unevenly, concentrated in a smaller area (40).

Pigs infected with PRCV 135 IN/IT or by Aer were necropsied at 4, 9 and 16 DPI (Figure 4A). At 4 DPI animals inoculated by IN/IT or Aer inoculation showed group mean lung consolidation of 32% or 27% respectively, which reduced to 8% and 2% respectively by 16 DPI (Figures 5A, B). Histological lesions were mainly detected at 4 and 9 DPI, but N antigen only at 4 DPI (Figures 5C–E). The lesions at 4 DPI were predominantly bronchiolar exudation, and within the alveoli were variably oedematous air spaces with neutrophils and alveolar macrophages infiltration. By 9 DPI, there was mild bronchiolar exudation with epithelial attenuation and infrequently necrosis. Occasionally this led to the formation of bronchiolitis obliterans. The alveolar walls were expanded with lymphocytic, plasmacytic and histiocytic cells, and loss of air spaces. By 16 DPI, some residual changes were noted including mild hyperplasia of bronchiole epithelium, and loose perivascular or peri-bronchiolar cuffing with lymphocytes and plasma cells.

The upper respiratory tracts were unremarkable at post-mortem. Histological changes in nasal turbinates were mild, with multifocal inflammation of the submucosa and occasional transmigration of neutrophils across the epithelium in both groups of pigs. However, there was occasional mild epithelial attenuation and exudation in the olfactory nasal mucosa, particularly in the Aer animals at 4 DPI and this frequently colocalized with areas of N labelling. N-labelled cells included olfactory receptors and sustentacular cells. No N immunolabelling was detected at 9 and 16 DPI.

In the trachea, similar lesions and progression comprising mild multifocal lymphoplasmacytic inflammation of the submucosa, occasional epithelial attenuation and exudation were observed (Figures 5C, D) Lesions were more commonly detected and scored higher in the caudal component of the trachea in the Aer group in contrast to the higher scoring in the cranial trachea in the IN/IT group.

Overall, both inoculation methods produced similar moderate pulmonary pathology, with acute lesions and maximal viral infection at 4 DPI and resolution by 16 DPI, similar to known porcine viral pneumonia. In addition, we demonstrated a diversity of cellular tropism within the upper respiratory tract.

The titers of PRCV neutralizing antibodies increased over time in serum and BAL (Figure 6A). Sera and BAL from both IN/IT and Aer groups cross neutralized against the four PRCV strains and the TGEV strain equally well (Figure 6A), indicating that highly cross-reactive antibody responses were generated by both routes of inoculation. A pseudo-virus-based neutralization assay, commonly used for studying SARS-CoV-2 induced responses, was also used to measure PRCV Ab responses and gave similar results to virus neutralization (Figure 6B). PRCV specific antibodies were also detected by ELISA using recombinant full-length Spike (S) protein from PRCV ISU-1 strain (Figure 6C). S-specific IgG responses were detected at 9 DPI and increased by 16 DPI in serum, broncho-alveolar lavage (BAL) and nasal swabs with no differences between the IN/IT and Aer groups. In addition, we have developed in vitro organ cultures with spleen and TBLN harvested at 16 DPI (Figures 6D, E) (34). Cultures were stimulated with PRCV 135 or the polyclonal activator R848. After 7 days S-specific IgG was detected in the supernatant in the virus stimulated cultures, which also exhibited both pseudo-virus neutralizing and virus neutralizing activities.

Overall, the data indicate that high titers of cross neutralizing antibodies were produced against the S protein in pigs and in vitro systems were developed for the first time in pigs, and used to dissect these responses.

We enumerated IFNγ secreting cells by ELISpot following stimulation with the four PRCV strains, three pools of peptides covering the S protein and one pool of peptides for the N protein of PRCV 135. Because the IFNγ responses to the stimuli were very similar in the IN/IT and Aer groups, we have shown the grouped responses for all six animals for 4, 9 and 16 DPI. The individual responses by groups are shown in Supplementary Figures 6, 7.

The TBLN, BAL and PBMC responses to live virus stimulation were lower compared to the peptide pools, especially to the S protein (Figures 7A–C). The summed responses to the three S pools and N peptide pool were highest at 9 and 16 DPI. The responses to the three S peptide pools are shown in Supplementary Figures 6, 7. There was animal inter-variability, but it appears that the BAL response, at least to whole virus, was lower reaching a peak at 16 DPI. A similar delayed kinetic of response was observed in BAL of influenza infected animals (30, 41). A response to peptide pools in the spleen, mesenteric and retropharyngeal lymph nodes was detected (Figures 7D–F). We also analysed IFNγ, TNF and IL-2 production by CD8β and CD4 T cells in BAL, TBLN and PBMC by intracellular staining (ICS) following stimulation with PRCV 135 virus (Supplementary Figure 8). The PRCV 135 specific CD4 and CD8 responses were dominated by IFNγ. The PBMC responses were weak compared to TBLN and BAL.

These data demonstrated that the strongest IFNγ ELISpot response for both virus and S and N peptides was detected in BAL, followed by TBLN and PBMC. There was a good S and N IFNγ ELISpot response in spleen and retropharyngeal lymph nodes, which drain the upper respiratory tract, and a much weaker responses in the distant mesenteric lymph node.