Experimental inoculation of pigs with porcine respirovirus type 1 revealed pathological manifestations in the upper respiratory tract

Experimental inoculation of pigs with porcine respirovirus type 1 revealed pathological manifestations in the upper respiratory tract | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Experimental inoculation of pigs with porcine respirovirus type 1 revealed pathological manifestations in the upper respiratory tract Marianne Viuf Agerlin , Kasper Pedersen , Mathias Romar , Marta Canuti , Timm Harder , Nicole Bakkegård Goecke , Henrik Elvang Jensen , Lars Erik Larsen , Pia Ryt-Hansen , View ORCID Profile Charlotte Kristensen doi: https://doi.org/10.1101/2025.07.24.666573 Marianne Viuf Agerlin 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kasper Pedersen 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mathias Romar 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marta Canuti 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Timm Harder 2 Institute of Diagnostic Virology , Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nicole Bakkegård Goecke 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Henrik Elvang Jensen 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lars Erik Larsen 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pia Ryt-Hansen 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charlotte Kristensen 1 Department of Veterinary and Animal Sciences, University of Copenhagen , Frederiksberg C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Charlotte Kristensen Abstract Full Text Info/History Metrics Preview PDF Abstract Several pathogens are known to affect the respiratory tract of pigs resulting in decreased health and welfare. Virological investigations and the use of metagenomic sequencing of samples allowed the identification of new viruses in pigs, such as porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). The pathogenicity of PRV1 has been investigated experimentally by one research group, whereas SOV is yet to be studied. In this experimental trial, groups of weaners were inoculated with a European isolate of PRV1 ( n = 8), a pool of SOV RNA-positive clinical nasal swab material ( n = 8) or culture medium serving as controls ( n = 4). Four days post inoculation (DPI), two controls and four pigs from each of the PRV1 and SOV groups were euthanized and necropsied. All SOV inoculated pigs tested negative for SOV at DPI 4 and therefore four of these pigs were transferred to the stable with the PRV1- infected pigs to assess direct-contact transmission. Nasal swab samples were collected at regular intervals after challenge and blood samples were collected at DPI 0, 4, and 14. At 14 DPI, the remaining pigs ( n = 10) were euthanized and necropsied. Nasal swab samples and tissues from the respiratory tract were examined for PRV1 RNA by reverse transcriptase real- time PCR and blood samples were investigated for PRV1 antibodies by ELISA. Respiratory tissues were also evaluated macro- and microscopically and selected tissues were investigated for in situ detection of PRV1 mRNA by RNAscope. No clinical signs, except for nasal discharge, were observed in any of the pigs. PRV1-shedding was observed from DPI 2 to 11 with peaks between DPI 4 and 7, and PRV1 was transmitted horizontally to all direct-contact pigs. The highest viral load was detected in the upper respiratory tract, i.e. nose, upper and lower trachea compared to the lower respiratory tract, i.e., bronchioles, and alveoli. Generally, a chronic tracheitis at 4 DPI, developing into chronic, erosive tracheitis at 14 DPI was observed in the PRV1 groups and was supported by in situ detection of PRV1 by RNAscope. Three pigs also developed mild, bronchointerstitial pneumonia at 14 DPI. In conclusion, these results showed that PRV1 is a primary porcine respiratory pathogen. Author summary Respiratory diseases in pigs impair porcine health and welfare and are often caused by a complex interaction between pathogens. We investigated the pathogenesis of two recently discovered viruses: porcine respirovirus type 1 (PRV1) and swine orthopneumovirus (SOV). An experimental model in weaner pigs showed that PRV1 is capable of efficient replication, horizontal transmission and caused pathological manifestations mainly in the upper respiratory tract. The lesions consisted of chronic, erosive tracheitis in all PRV1 infected pigs, resulting in a breakage of the tracheal epithelial barrier, and a few pigs developed bronchointerstitial pneumonia. These findings confirm that PRV1 should be considered a primary porcine respiratory pathogen, but in contrast, SOV did not result in a successful infection, and the reasons why remain to be elucidated. Download figure Open in new tab Graphical abstract created with Biorender.com Introduction In intensive swine production, high stocking density makes respiratory pathogens a threat to swine health, also affecting negatively welfare, growth and, thus the sustainability of swine production ( 1 – 4 ). Porcine respirovirus type 1 (PRV1, formerly known as porcine parainfluenza 1 (PPIV1), species Respirovirus suis ) is an enveloped single-stranded negative-sense RNA (ssRNA) virus in the family Paramyxoviridae, genus Respirovirus ( 5 , 6 ). PRV1 genome consists of 15,298 nucleotides (nts) and encompasses six open reading frames (ORFs) coding for: nucleoprotein (N), phosphoprotein (with C/V editing), matrix (M) protein, fusion (F) protein, hemagglutinin-neuraminidase (HN) protein, and a large polymerase (L) ( 6 – 9 ). PRV1 was first identified among pigs in China in 2013 ( 10 ), and was isolated in cell culture in 2016 ( 11 ). It is widely present among conventional pigs globally. A passive surveillance study in Germany found PRV1 in 76/123 herds ( 12 ), in Poland it was detected in 23/30 herds ( 13 ), and in Hungary and Slovakia in 11/24 herds ( 14 ). Lower rates of detections have also been reported in Germany and the Netherlands where 11/34 herds tested positive for PRV1 ( 15 ). In USA, 365/842 of routine diagnostic samples tested positive ( 16 ). Similar high detection rates were reported in Chile ( 17 ) and Italy ( 18 ). In Denmark, PRV1 was detected in 22/125 herds in an active surveillance project (submitted from pigs with a variety of symptoms such as coughing and diarrhea) performed in 2022 and in 33/176 herds in a passive surveillance project in 2023 ( 19 ). The pathogenesis and pathogenicity of the European PRV1 have yet to be elucidated, but experimental studies in pigs with the American PRV1 have been performed ( 6 – 8 ). No clinical signs were observed in a PRV1 experimental infection of caesarian derived-colostrum deprived (CDCD) pigs, whereas mild coughing was present later during the infection (6-16 days post inoculation (DPI)) in PRV1 experientially infected conventional pigs ( 6 ). These studies also revealed that PRV1 mainly affects epithelial cells in the upper respiratory tract (URT) of pigs causing mild transient tracheitis, and mild bronchointerstitial pneumonia in some of the experimentally infected pigs ( 6 – 8 ). Swine orthopneumovirus (SOV) is another virus recently discovered among pigs. This virus was discovered for the first time in the United States ( 20 ), and since then, it has been detected in France ( 21 ), Spain ( 22 ) and Asia ( 23 ). SOV, which did not yet receive an official species designation, belongs to the family Pneumoviridae, and genus Orthopneumonovirus , which includes huma n-, and bovine respiratory syncytial virus (HRSV and BRSV, respectively) ( 5 , 20 , 21 ). Like PRV1, SOV is an enveloped, single-stranded negative-sense RNA virus ( 20 ), and the pathogenesis and pathogenicity of this virus have not yet been assessed. Additionally, this virus has not been isolated in cell culture. The aim of the present study was to assess the pathogenesis of a European clade I isolate of PRV1 by experimental inoculation of conventional weaned pigs and to investigate horizontal transmission efficacy. Furthermore, another group of pigs was inoculated with nasal swab material from pigs that tested positive for SOV RNA in an attempt to study the pathogenesis of this virus, too. The study also allowed collection of PRV1-positive tissue samples for distribution to colleague laboratories throughout Europe enhancing collaborations regarding development and validation of robust diagnostic detection methods of PRV1 in the frame of the CoVetLab project PorParEU (2024–2025). Materials and methods Preparation of inoculum Two cell lines, LLC-MK2 (ATCC, CCL-7) and Calu-3 (ATCC, HTB-55) were maintained at 37 °C in a humidified 5% CO 2 incubator and grown in cultivation media consisting of Eagle’s Minimal Essential Media (MEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 U/ml penicillin-streptomycin (Invitrogen, Thermo Fisher Scientific), 2mM L-Glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (Gibco). Calu-3 cells were additionally supplemented with 1mM Sodium pyruvate (Gibco). A PRV1 isolate GER/2022AI03675/2022 was propagated in LLC-MK2 cells for three passages. The PRV1 isolate was inoculated into confluent monolayers of LLC-MK2 in either 48 flat- bottom Nunclon Delta Surface Plates (Thermo Fisher Scientific) or T75 tissue culture flasks (Thermo Fisher Scientific). After incubation for one hour, serum-free cultivation media supplemented with 2 μg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone treated trypsin (Sigma-Aldrich) was added. The supernatant was harvested after 72 – 96 hours, and the presence of viral nucleic acid was estimated by reverse-transcriptase real-time PCR (RT- qPCR). For the attempt to propagate SOV, four SOV RT-qPCR positive nasal swab samples, collected from pigs of 4 – 6 weeks of age in a field study, were pooled and filtered through a 0.45 µm membrane filter (Lab Logistics Group GmbH, Hamb, Meckenheim, Germany). One hundred µL of the pooled sample and 100 µL cultivation-media with only 2% fetal bovine serum (Thermo Fisher Scientific) was inoculated into either LLC-MK2 or Calu-3 cells in 48 flat-bottom Nunclon Delta Surface Plates (Thermo Fisher Scientific). After incubation for two hours, an additional 800 µL of cultivation media supplemented with 2% fetal bovine serum was added. For the final SOV inoculum, the resuspension fluid of 21 SOV-positive nasal swab samples from a previous study were all pooled and filtered. The presence of SOV RNA in the SOV inoculum was investigated using RT-qPCR analysis, and additionally it was evaluated for the presence of other relevant respiratory pathogens using a high-throughput RT-qPCR analysis (both methods described in further details below). Study design Twenty 4-week-old Norsvin Landsvin pigs were imported from a herd at NMBU (Norwegian University of Life Science) in Norway to obtain pigs without compromising agents, such as swine influenza A virus (IAV) and porcine reproductive and respiratory syndrome virus type 1 and 2 (PRRSV-1 and PRRSV-2). The pigs were confirmed negative for shedding of PRV1 and SOV by RT-qPCR nucleic acid detection on nasal swabs prior to inoculation, and serum was confirmed negative for antibodies against PRV1 by ELISA. The presence of SOV antibodies was not assessed due to lack of a SOV-antibody detection assay. The pigs were stratified by weight and randomly allocated into three groups (stratified randomization): PRV1 ( n = 8), SOV ( n = 8), and negative controls ( n = 4) (Gender and bodyweight information is available in Table S1). To reduce any respiratory bacteriological interference, the pigs were treated with 2.5 mg/kg tulathromycin at -7 days post inoculation (DPI). At 0 DPI, the pigs were anesthetized and intranasally inoculated in each nostril by MAD nasal intranasal mucosal atomization devices; PRV1 (a German isolate, GER/2022AI03675/2022-(SK398), PV767405): 1.5 mL of 1.19 × 10 4 50% tissue culture infectious dose (TCID 50 /mL, back-tirated) in Minimum Essential Medium Eagle (MEM), SOV included: 1.5 mL of pooled SOV from nasal swab samples (cycle threshold (Ct)-value of 20.72), and the control inoculum was 1.5 mL MEM. Nasal swab samples were collected from all pigs at 0, 1, 2, 3, 4, 7, 9, 11, and 14 DPI. Rectal temperatures were measured at 0, 1, 2, 4, 7 and 14 DPI. Blood samples and body weight measurements were collected at 0, 4 and 14 DPI. Clinical signs, including nasal discharge, coughing, and sneezing were recorded when observed. At 4 DPI, four PRV1, four SOV, and two control pigs were euthanized for macro- and microscopic evaluation and virological investigation of tissues. At 4 DPI, no shedding of SOV was detected in any of the SOV-inoculated pigs and therefore, the last four SOV- inoculated pigs were transferred to the stable of the four PRV1 pigs as “recipients” to investigate horizontal PRV1 transmission (0 days post contact, DPC). The study design is illustrated in Fig 1 . Download figure Open in new tab Fig 1: O v erview of the study design . All pigs were clinically monitored from admission to the experiment facilities one week before and until 14 days post inoculation (DPI). Control, PRV1 and SOV pigs were inoculated at 0 DPI. Serum samples were collected at 0, 4, and 14 DPI, and nasal swabs were collected at day 0, 1, 2, 3, 4, 7, 9, 11 and 14 DPI. PRV1-naïve recipient pigs (from the SOV group) were mixed with the PRV1 inoculated pigs at 4 DPI (0 days post contact, DPC). Tissue samples and bronchoalveolar lavage fluid (BALF) were collected during necropsy at 4 DPI and 14 DPI/10 DPC. The illustration was created with Biorender.com. The nasal swab samples were collected with flexible sterile rayon dryswabs (Medical Wire, Corsham, UK) inserted into the ventral meatus of each nostril of the pig, turned 360 degrees and placed into 2 mL Sigma Virocult medium (Medical Wire). Blood was collected from vena jugularis in a BD Vacutainer and 10 mL serum tubes (BD, Stockholm, Sweden). Necropsy and histopathology In anesthesia, the pigs were euthanized by an intracardiac injection of pentobarbital (400 mg/ml, ≥0.25 ml/kg). At necropsy, the lungs were photographed, and lung lesions were sketched manually. The percentage of affected lung tissue on the dorsal and ventral surface was measured from the lung photographs using the lung sketches and the area tool in Adobe Acrobat Reader. Pictures from pig number 550 were excluded from the analysis due to poor quality. Samples from the nose, upper and lower trachea and three different lung sections were collected from each pig ( Fig. 1 ). From all pigs euthanized 14 DPI, samples from Ln. tracheobronchialis cranialis were also collected. Tissue specimens for virus quantification were stored at −80°C until analysis. A post-mortem bronchoalveolar lavage (BAL) was performed by cutting the right lung from the right main bronchus and administering 5 ml phosphate-buffered saline (PBS) into the bronchi by a syringe, massaging the lungs, and the collected BAL fluid (BALF) was stored at −80°C. Specimens for histopathology were fixed in 10% neutral-buffered formalin for five days, embedded in paraffin wax (formalin-fixed and paraffin-embedded, FFPE), sliced into 3–4 μm sections, and stained with hematoxylin and eosin (H&E). FFPE tissue samples of the nose were not evaluated due to variable quality. Tissue culture infectious dose 50 (TCID50) To determine the viral titer of the inoculum, a TCID50 assay with RT-qPCR readout was performed. LLC-MK2 cells were seeded in 24-well plates and cultured until reaching confluency. The virus inoculum was serially diluted tenfold in serum-free cultivation media supplemented with 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich). Prior to inoculation, cells were washed with PBS and 200 μl of each virus dilution was added to the wells in five replicates. Plates were incubated for one hour at 37 °C in a humidified 5% CO₂ incubator. Following the incubation, the inoculum was removed, cells were washed with MEM and 1 mL of serum-free cultivation media supplemented with 1 μg/ml TPCK-treated trypsin was added to each well. The cells were incubated for three days under the same conditions. To assess viral replication a sample was collected before and after incubation and analyzed by RT-qPCR. The TCID50 was calculated using the Reed-Muench method. Cultivation of viral samples To assess if the viral samples collected during the animal study were infectious, samples were cultivated in LLC-MK2 cells. A total of four nasal swabs samples, five BALF samples, one serum sample, and twelve tissue samples were diluted and filtered through a 0.45 μm membrane filter. The samples were inoculated into LLC-MK2 cells as described above, and the presence of viral nucleic acid was monitored by RT-qPCR. Cultivation was performed in two independent attempts. Antibodies against PRV1 Serum samples collected from PRV1 and control (0 and 14 DPI) and recipient pigs (0 and 10 DPC) were tested for the presence of anti-PRV1 antibodies. First, the blood was centrifuged at 1550 × g for 10 minutes at 4°C and sera were stored at -20°C until analysis. Samples were tested for antibodies towards PRV1 by Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) with a whole virus (WV) indirect ELISA previously described ( 24 ). The true negative ratio (TNR) for cut-off 0.14 is evaluated to be 99% (CI 97-100%). RNA extraction and reverse transcription real-time PCR The nasal swab samples were stored at -80°C until RNA extraction. Each nasal swab samples was vortexed for 10 sec, centrifuged for 3 min at 9651 × g and 200 µL of the supernatant was mixed with 400 µL RTL-buffer (QIAGEN, Hilden, Germany) containing 2-mercaptoethanol (ME) (Sigma-Aldrich). In total, 70 mg of tissue were mixed with 1400 μL of RLT buffer, and lysed in a TissueLyser LT (QIAGEN) by bead beating for 3 min at 30 Hz, centrifuged for 3 min at 9651 × g , and 600 μL of the supernatant was used for RNA extraction. The RNA was extracted from the nasal swab and tissue samples using the RNeasy mini kit (QIAGEN) automated on the QIAcube Connect extraction robot (QIAGEN) according to the instructions from the manufacturer. For the blood samples collected 4 DPI, 140 μL serum was used for RNA extraction using the Viral kit (QIAGEN) and automated on the QIAcube Connect extraction robot (QIAGEN) according to the instructions from the manufacturer. For nucleic acid RT-qPCR detection of PRV1, already published primers targeting the fusion (F) gene were used. The primer-probe-mix consisted of 0.4 µL PRespiV-FF, 0.4 µl PRespiV-F-R, 0.1 µL PRespiV-F-FAM, and 3.1 RNase free water ( 12 ). For SOV detection, already published primers targeting the nucleoprotein (NP) gene were used, whereas a previously published probe (Pneumo-NP-FAM) was modified with a FAM in the 5’ end and BHQ3 in the 3’ end (FAM-CTG GGC TGC CTG ACA ATC GGA GGC-BHQ1) ( 12 ). The SOV primer-probe mix consisted of 0.4 µL Pneumo-NP-F, 4.0 µl Pneumo-NP-R, 0.1 µL Pneumo-NP-FAM and 3.1 RNase free water. In both RT-qPCR assays, the primer-probe-mix was combined with 2.5 µL RNase free water, 12.5 µL RT-PCR buffer, 1 µL of 25x RT-PCR enzyme mix from the AgPath-ID One-step RT-PCR kit (Applied Biosystems, Thermo Fisher Scientific), and 5 µL of extracted RNA. RT-qPCR reactions were run on a Rotorgene Q (QIAGEN) platform with the following thermal steps; 45° for 10 min, 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 sec, 56 °C for 20 sec, and 72 °C for 30 sec ( 12 ). High-throughput qPCR Extracted nucleic acids were initially reverse transcribed using a high-capacity cDNA RT Kit (Applied Biosystems). A final volume of 10 µL reaction mix was prepared by mixing 1 µL of 10X RT buffer, 0.4 µL dNTP mix (100 mM), 1 µL of 10X random hexamer, 0.5 µL of MultiScribe RT enzyme, 2.1 µL of nuclease free water and 5 µL of extracted nucleic acid. The cDNA synthesis was carried out in a PCRmax Alpha thermocycler (Cole-Parmer, Vernon Hills, IL, USA) with the following thermal conditions: 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. Afterwards, cDNA samples were pre-amplified using 2X TaqMan PreAmp master mix (Applied Biosystems). A total volume of 10 µL was prepared by mixing 2.5 µL of cDNA with 5 µL of 2X TaqMan PreAmp master mix (Applied Biosystems) and 2.5 µL of primer mix (200 nM, containing all sets of primers). The primers and probes used are published elsewhere and allowed the detection of: Mycoplasma hyorhinis , Streptococcus suis type 2, Haemophilus parasuis (Glaesserella parasuis) Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Bordetella bronchoseptica, Pasteurella multocida, IAV ((Inf M, Nagy2), porcine circovirus 2 and 3 (PCV2 and PCV3), porcine cytomegalovirus (PCMV), and porcine respiratory corona virus (PRCV) ( 25 , 26 ). The pre-amplification was carried out in a PCRmax Alpha thermocycler (Cole-Parmer) using the following thermal cycling program: 95°C for 10 min followed by 14 cycles of 95°C for 15 s and 60°C for 4 min. The pre-amplified products were stored at -20°C until further use. For high-throughput qPCR analysis, the BioMark HD (Standard BioTools, South San Francisco, CA, USA) and the 192.24 Dynamic array (DA) integrated fluidic circuit (IFC) chip (Standard BioTools) were used. A 4 µL sample mix was prepared for each of the samples by mixing 2.2 µL pre-sample mix (prepared by mixing 2 µL of 2X TaqMan Gene Expression Mastermix (Applied Biosystems) and 0.2 µL of 20X sample loading reagent (Standard BioTools) with 1.8 µL of the pre-amplified sample. Assay mix for each PCR assay was prepared by mixing 2 µL primer/probe stock (containing 33 µM of each primer and 10 µM of probe) with 2 µL of 2X assay loading reagent (Standard BioTools). Three µL of assay mix and 3 µL of sample mix were loaded into the respective inlets of the 192.24 DA IFC chip. The 192.24 DA IFC chip was placed in the IFC controller RX (Standard BioTools) for loading and mixing for approximately 30 min. Finally, the chip was inserted into the high- throughput qPCR platform BioMark HD (Standard BioTools) for thermal cycling with the following cycling condition: 50°C for 2 min, 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. In each chip run, positive and negative (nuclease-free water) controls were included. Amplification curves and Ct-values were obtained on the BioMark HD system and finally, analyzed using the high-throughput qPCR Analysis software 4.8.1 (Standard BioTools). Detection of PRV1 mRNA in situ by RNAscope To detect PRV1 mRNA in FFPE tissues, in situ hybridization (ISH) was performed using the RNAscope 2.5 HD BROWN kit (Advanced Cell Diagnostics, Bio-techne, Ireland, Dublin) according to the manufacturer’s instructions ( 27 ). Briefly, 9ZZ probe named V-PRV1-NP (catalog 1784821-C) targeting PRV1 mRNA of the nucleoprotein (NP) was designed and synthesized by Advanced Cell Diagnostics. Tissue sections were deparaffinized with xylene followed by two 99% ethanol washes and then hydrogen peroxidase blocking for 10 min. The slides were washed with Milli-Q water and pre-treated using kit-provided antigen retrieval buffer (boiled in water bath for 15 min) and protease plus (30 min). ISH signal was developed using the kit-provided pre-amplifier and amplifier conjugated to alkaline phosphatase and incubated with a DAB substrate solution for 10 min at room temperature. Sections were then counterstained with hematoxylin. This was performed on upper tracheal and lung tissues with PRV1 RT-qPCR Ct-values <35, i.e. upper tracheal tissues from all PRV1 pigs and three recipient pigs, lung tissues from two PRV1 pigs, and the cranial tracheobronchial lymph node from a PRV1 pig (two samples were missing from the recipient pigs). Sequencing The German PRV1 isolate used for inoculation was sequenced via the following protocol. After a centrifugation step of 10 min at 10,000 × g , 140 uL of the supernatant was used for total nucleic acid isolation, performed with the Qiamp viral RNA mini kit (QIAGEN). A DNAse treatment was then performed on 11 µl of NA solution (DNAse I, New England Biolabs (NEB), Ipswich, MA, USA) (according to manufacturer’s specifications) and the remaining RNA was subjected to reverse transcription (ProtoScript® II First Strand cDNA Synthesis Kit, NEB) and second strand synthesis (NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module, NEB). Prepared dsDNA was finally purified with MAGBIO magnetic beads 1:1:v:v (HighPrep PCR-DX, MAGBIO, Gaithersburg, MD, USA) and outsourced to Novogene (Munich, Germany) for Illumina sequencing. Obtained reads were quality-controlled and trimmed using BBDuck in Geneious Prime (Dotmatics, Boston, MA, USA), and surviving reads were mapped to a reference PRV1 sequence using Bowtie. Finally, the obtained contig was visually inspected, manually polished, and ambiguities were inserted where appropriate. Phylogenetic analysis and genomic comparison A maximum-likelihood phylogenetic tree of the PRV1 contained in the inoculum used in this study, together with 13 other PRV1 available in GenBank and including the virus in the inoculum used in Welch et al. 2021, was constructed by IQ-TREE version 3.0 ( 6 , 28 ) using a 15,140 nts MAFFT-generated alignment. Mid-point rooting was conducted. The Modelfinder function in IQTREE 3.0 ( 29 ) was used to determine the best fitting substitution model ( 30 – 34 ). Tree topology reliability evaluation was performed by bootstrap tests with 1000 pseudo- replicates ( 35 ). The same alignment was used for direct nt and amino acid (aa) comparison of the sequences of the different isolates. Results Presence of other relevant respiratory pathogens at inoculation At 0 DPI, nasal swab samples were investigated for the presence of other relevant respiratory pathogens by high-throughput qPCR. The analysis revealed that five pigs were positive for M. hyorhinis (one control, one PRV1, one SOV and two recipient pigs), two were positive for S. suis type 2 (two PRV1 pigs), and one was positive for G. parasuis (PRV1 pig). The specific pigs and Ct-values are listed in Table S2. Nasal swab samples from all the pigs were negative for M. hyopneumoniae , A. pleuropneumoniae , B. bronchoseptica, P. multocida, IAV, PCV2, PCV3, PCMV, and PRCV. Unsuccessful SOV inoculation The isolation of SOV was unsuccessful after two passages in both cell lines (LLC-MK2 and Calu-3 cells). No cytopathic effect was observed in the cells, and the cell suspension was negative for SOV RNA. The SOV inoculum showed a Ct-value of 20.72 in the RT-qPCR analysis, but no SOV shedding was observed from the SOV-pigs at 4 DPI, and therefore, these four pigs were used as PRV1 recipients. A high-throughput qPCR analysis of the SOV inoculum revealed the presence of IAV (Ct-value of 23), PCV3 (Ct-value of 26), PCMV (Ct-value of 11), S. suis type 2 (Ct-value of 20), M. hyorhinis (Ct-value of 19), B. bronchiseptica (Ct-value of 21) and G. parasuis (Ct-value of 13). Serous nasal discharge was observed in five pigs (546, 550, 576, 659, 666) at 2 days post inoculation (DPI). Successful PRV1 inoculation One PRV1 pig (512) showed serous nasal discharge at 4 DPI, and two PRV1 pigs (512, 612) plus one recipient pig (659) showed mucoserous nasal discharge at 11 DPI and 7 DPC, respectively. Otherwise, no clinical signs were observed during the study. No PRV1 or SOV nucleic acid was detected in nasal swabs by RT-qPCR nor in the tissues of the control group at any time point during the study ( Fig 2A ). Download figure Open in new tab Fig 2. Scatterplot of PRV1 RT-qPCR nucleic acid detection (A, C, D, E) with Ct-values indicated on the y-axis and WV-ELISA (B) with sample/positive (S/P) ratio on the y-axis and mean with standard deviation presented. In A) and B), days post inoculation (DPI) or days post contact (DPC) are on the x-axis. In C), d) and E), the x-axis represents the tissue type at different time points. The dots indicate the value for each pig: A) ventral meatus nasal swab, C) tissue samples at 4 DPI from PRV1 pigs, D) tissue samples at 10 DPC from recipients, and E) tissue samples at 14 DPI from PRV1 pigs. Ln. Tracheobronch. Cr.= Ln. tracheobronchialis cranialis , BALF= bronchoalveolar lavage fluid. The graphs were made using GraphPad Prism. The PRV1 inoculated pigs tested positive for PRV1 RNA from 1 DPI until 11 DPI, with the lowest Ct-value (highest viral load) observed between 4 and 7 DPI ( Fig. 2A ). The recipient pigs were moved into the pen at 0 DPC (4 DPI) and PRV1 RNA shedding was detected at 3 DPC and continued until euthanasia at 10 DPC ( Fig 2A ). The highest viral shedding in the recipient pigs was observed at 7 DPC. In the PRV1 pigs, the highest viral load was observed in tissues of the upper and lower trachea at 4 DPI, followed by the ventral meatus swab and BALF, as only two pigs were PRV1 RNA positive in selected lung tissues ( Fig 2C ). At 10 DPC all recipients were PRV1 RNA negative in lung tissue samples, and samples from other tissues presented with Ct-values close to the limit of detection, except for one pig (507) in whose trachea and BALF samples PRV1 was detected with Ct-values of ∼20. At 14 DPI, PRV1 RNA was detected in the lymph node and BALF of only one PRV1 inoculated pig (658). Furthermore, one pig (505) tested positive for PRV1 RNA in serum at 4 DPI with a Ct-value of 28.44. PRV1 was re-isolated from a sample collected from a recipient pig (659) at 7 DPC (nasal swab). Otherwise PRV1 was not re-isolated in the selected samples including the PRV1 RNA-positive serum sample. PRV1 pigs seroconverted at 14 DPI At 14 DPI, all the PRV1 pigs ( n = 4) seroconverted, and one control pig had a sample/positive (S/P) value at 0.15 which is 0.01 S/P above the cut-off, but PRV1 nucleic acid was never detected in this pig ( Fig 2B ). None-to-mild macroscopic lung lesions At 4 DPI, no macroscopic lesions were observed in the PRV1 pigs, except for one pig (532) showing pulmonary rib impressions ( Fig 3A ). At 14 DPI, three pigs, one PRV1 pig and two recipients (10 DPC), showed lesions consistent with chronic, lobular bronchopneumonia (658 ( Fig 3B ), 550 and 659), which affected 1.4 – 2.9% of the lung tissue. Pig 658 also showed mild hyperplasia of ln. tracheobronchialis cranialis . Three pigs (two PRV1 and one recipient) showed pulmonary rib impressions at 14 DPI/10 DPC (546, 612, 687). No macroscopic changes were observed in the SOV group. Download figure Open in new tab Fig 3. Macroscopic changes observed in PRV1 infected pigs. A) Arrows indicate pulmonary rib impressions observed in a PRV1 pig at 4 DPI (532). B) Chronic, lobular bronchopneumonia (arrows) observed in a PRV1 pig at 14 DPI (658). Microscopic changes observed in the trachea Microscopic tissue evaluation at 4 DPI, 2/4 PRV1 pigs (505 and 532) showed mild exudation of neutrophils, single cell necrosis, multifocal areas with loss of cilia and infiltration of mononuclear cells in lamina propria ( Fig 4 ). One PRV1 pig (499) showed similar lesions but with exudation consisting of mixed inflammatory cells and no infiltration of mononuclear cells in the lamina propria, also comparable to chronic tracheitis and one pig (688) had a focal infiltration of mononuclear cells with multifocal areas of cilia loss. Download figure Open in new tab Fig 4. Chronic tracheitis from a PRV1-inoculated pig (505) at 4 days post inoculation (DPI). Histopathological changes consisted of exudation of neutrophils (arrowheads), single cell necrosis (thick arrow), loss of cilia (thin arrow) and infiltration of mononuclear cells in lamina propria (asterisks). At 14 DPI, all PRV1 pigs and two recipient pigs (10 DPC) showed varying degrees of exudation with mixed inflammatory cells, epithelial erosion, loose tight junctions, and infiltration of mononuclear cells in the lamina propria (chronic, erosive tracheitis) ( Fig 5 ). One of the remaining recipient pigs (546) showed chronic tracheitis (as defined for the PRV1 pigs at 4 DPI), and the other (659) severe erosive changes (only the basal epithelial layer remaining) with suppurative exudation (Fig S2). Download figure Open in new tab Fig 5. Chronic, erosive tracheitis from a PRV1-inoculated pig (658) at 14 days post infection (DPI). Histopathological changes consisted of loose tight junctions (arrowhead), erosion of the epithelial layer (arrow) and infiltration of mononuclear cells in the lamina propria (asterisks). Varying degrees of acute (no infiltration of mononuclear cells and exudation of neutrophils) to chronic tracheitis (like the PRV1 pigs 4 DPI) was observed in the tracheal tissues of the SOV pigs at 14 DPI, but otherwise no lesions were observed. No lesions were observed in the controls (Fig S1), except for one pig (616) that showed multifocal areas in the tracheal epithelium with pustules. In the lungs, two PRV1 pigs at 14 DPI (pigs 512, 658) and one recipient pig at 10 DPC (pig 550) showed chronic, bronchointerstitial pneumonia in one or two of the cranial lung samples at 14 DPI ( Fig 6 ), otherwise no lesions were observed except for one control pig (616) that showed interstitial pneumonia at 4 DPI. Download figure Open in new tab Fig 6. Microscopic changes in the lungs of PRV1-infected pigs. Lungs from a PRV1 pig (658) at 14 DPI showing exudation of neutrophils in a terminal bronchiole that exceeds into the lumen of the alveoli and infiltration of mononuclear cells in the interstitium. These findings are consistent with chronic, bronchointerstitial pneumonia. In situ detection of mRNA PRV1 in trachea All PRV1 pigs showed a high number of PRV1 mRNA-positive cells in the epithelial cells of the upper tracheal tissues at 4 DPI ( Fig 7A ). One PRV1 pig (532) also showed a high amount of PRV1-positive bronchiolar epithelial cells with a few positive cells in the alveoli ( Fig 7B ), whereas another PRV1 pig (505) only showed a few positive cells in the alveoli. One recipient pig (507) showed PRV1 mRNA-positive cells in the epithelial cells of the upper tracheal tissues at 10 DPC. Download figure Open in new tab Fig 7. PRV1 positive epithelial cells in the upper trachea, bronchioles and alveoli. A) A high number of PRV1 mRNA-positive epithelial cells (brown) of the upper trachea in a PRV1 pig at 4 DPI (499). B) A high amount of PRV1-positive bronchiolar epithelial cells (close-up showing a PRV1 mRNA-positive terminal bronchiole) and a few in the alveoli of a PRV1 pig at 4 DPI (532). Genomic distance to the previously evaluated clade II PRV1 Phylogenetic analysis revealed that the 14 included PRV1 sequences separate into the two pre- defined PRV1 clades, I and II ( Fig 8 ). The German PRV1 GER/2022AI03675/2022(SK398) (GenBank: PV767405 ) isolate evaluated in this study appeared in clade I, whereas the PRV1 isolate evaluated experimentally by studies from Welch et al. (2021, 2022a, 2023) appeared in clade II. Evaluation of nucleotide (nt) and amino acid (aa) similarity between the two isolates used for two experimental PRV1 inoculations (DE, clade I and US, clade II) disclosed that the highest difference appeared in the phosphoprotein (pairwise identity of 88.9% and 84.5% at nt and aa level respectively) and partly also the surface proteins (90.0% and 92.4% nt and aa pairwise identity respectively in the hemagglutinin/neuraminidase region and 90.4% and 91.4% nt and aa identity in the fusion protein region respectively; Table S3). The nucleoprotein, matrix protein, and polymerase of the German isolate were all 92.0-92.6% and 96.0-96.1% identical to the PRV1/US/2016 isolate at nt and aa levels, respectively. Download figure Open in new tab Fig 8. A phylogenetic tree of 14 PRV1 genomes (nt 120-12,260). The inoculum from this study (red) and an experimental inoculum from Welch et al., 2021 (blue) are included. The maximum-likelihood phylogenetic tree was built with IQ-TREE 3.0 according to the general time reversible model with empirical base frequencies and four discrete rate category gamma- distributed rate heterogenity (GTR+F+G4). The outcome of the bootstrap analysis (1000 replicates) used to test branch robustness, is shown for each branch. Discussion In this experimental trial, we demonstrated that five-week-old, PRV1-seronegative pigs directly exposed to PRV1 got infected and excreted PRV1 RNA from the day after exposure and for 11 days. The inoculated pigs successfully transmitted the virus through direct contact to recipient pigs, resulting in sustained shedding of infectious virus. Although none of the exposed pigs exhibited respiratory clinical signs (other than nasal discharge), histopathology examination revealed chronic tracheitis at 4 DPI, which progressed to chronic, erosive tracheitis by 14 DPI. These findings appeared both in intranasally-inoculated pigs and in direct- contact pigs (horizontally infected pigs). The role of PRV1 in causing histopathological lesions was supported by in situ viral mRNA detection showing a strong signal in the upper tracheal epithelial cells of all pigs. PRV1 mRNA positive cells were not completely restricted to epithelial cells in the URT, but the progression to bronchiolar epithelial cells and pneumocytes was limited. Observed histopathological changes (tracheitis and chronic, bronchointerstitial pneumonia) did not lead to any observed macroscopic changes apart from pulmonary rib impressions or mild lobular bronchopneumonia in seven pigs, five of which showed nasal discharge as their only clinical manifestation. Overall, these results, combined with the findings of Welch et al. ( 6 – 8 ), prove that PRV1 is a primary porcine respiratory pathogen. PRV1 is frequently detected in pigs with respiratory signs, but often in combination with other pathogens ( 12 , 13 ). PRV1 has an extensive effect on the epithelial lining, which is regarded as a significant part of the innate immune defense ( 36 , 37 ), thereby compromising significantly this mechanical barrier against pathogens. It has also been proven how the breakage of epithelial integrity provide opportunity for further infection with A. pleuropneumoniae ( 38 ). The degeneration of the epithelial integrity by PRV1 may therefore be highly considered to facilitate secondary bacterial infections, thus contributing to the porcine respiratory disease complex (PRDC) ( 39 ). This is also indirectly proven by the conventional group of pigs included in the study of Welch et al. 2021, because their PRV1-associated lesions in the presence of co-infecting bacteria were different compared to pigs without bacterial detections. Interestingly, experimental co-infection with another virus, IAV, did not worsen PRV1 symptoms ( 8 ). On the contrary, the authors found that co-infections with IAV reduced PRV1 nucleic acid load in the URT. The two viruses also showed different tissue tropisms in the respiratory tract of pigs. PRV1 primarily showed tropism for the URT, whereas IAV also showed tropism for the lower respiratory tract (LRT, bronchi, bronchioles and alveoli) with multiple studies showing IAV-induced pneumonia ( 40 – 42 ). The host receptor of PRV1 remains unknown, but human respiroviruses bind to sialic acids (SA) linked to N- Acetyllactosamine (Galβ1-4GlcNAc) by either α2,3 or α2,6 linkages (SA-α2,3/6-Galβ1- 4GlcNAc) and swine IAVs mainly bind to SA-α2,6-Gal ( 43 , 44 ). Therefore, a potential competition of host receptor between PRV1 and IAV could explain the reduced PRV1 antigen detection in co-infected pigs from Welch et al., 2023 . The findings observed in this study are comparable to the findings of the CDCD group of pigs in Welch et al. 2021 (US, clade II)( 6 ) and those described for other viruses in the same family as PRV1, such as bovine parainfluenza and human parainfluenza viruses, which cause pharyngitis, croup (laryngotracheobronchitis), and pneumonia ( 6 , 45 – 47 ). However, in our study, PRV1 was sparsely detected in the LRT and only a few pigs developed pneumonia. This difference in involvement of the LRT could be explained by the CDCD origin of the pigs in the study of Welch et al. 2021 ( 6 ) contrary to conventional Norwegian SPF pigs with none-to- few influential secondary pathogens ( M. hyorhinis, S. suis type 2, G. parasuis ), recognized by high-throughput qPCR in our study. Genomic difference (9,1% nt difference) between the isolates used in the two studies could also be relevant. Indeed, the PRV1 isolate used in this study is from clade I, which is different from the clade II PRV1 isolate used in the studies of Welch et al., ( 6 – 8 ). The aa variation observed between clades I and II isolates could impact the virulence of these viruses since the primary variation appears in the ORFs for the phosphoprotein and the surface protein (hemagglutinin/neuraminidase and the fusion genes). Nonetheless, the knowledge about this virus is still limited, and additional sequencing efforts and pathogenicity studies will be required to identify molecular determinants of disease severity. Serous nasal discharge was reported in 5/8 pigs at 2 DPI and lesions were observed in the SOV pigs at 4 DPI, whereas no clinical signs or lesions were observed in the controls at 4 and 14 DPI (except one control pig showing distinct lesions at 4 DPI), indicating a potential effect of other pathogens present in the SOV inoculum. This could also explain why the two PRV1 recipient pigs (which received the SOV inoculum at 0 DPI) showed different lesions compared to the PRV1 pigs. However, it is worth mentioning that, due to the pre-amplification step, the Ct-values reported for the high-throughput qPCR are, in general, lower (around 8-12 Ct-values) compared to Ct-values obtained by traditional RT-qPCR platforms. The presence of other respiratory pathogens was also detected in BALF samples from conventional pigs in the study from Welch et al. 2021 ( 6 ). Therefore, some of the lesions observed in this study might be due to the presence of opportunistic pathogens or the synergistic effect of multiple pathogen infections. The serum of one control pig was mildly reactive in PRV1 wv-ELISA but since no PRV1 RNA-positive were detected in the control group, the sample should be regarded as an ELISA false positive. A productive PRV1 infection in the inoculated pigs was confirmed by the rapid (1-2 days) horizontal infection to recipient pigs confirmed by PRV1, nasal shedding and seroconversion at 14 DPI. The pigs had direct contact, and therefore, it is not possible to determine the exact route of transmission. PRV1 was re-isolated from one nasal swab from a recipient pig at 7 DPC confirming a successful transmission. PRV1 was not re-isolated from the rest of the samples, which might be due to sample handlings (freeze/thaw cycles), as these samples were not intended for culturing. The finding of PRV1 RNA in serum from only one pig and the lack of ability to re-isolate indicates that PRV1 causes a local infection in the respiratory tract with likely oronasal transmission. At 10 DPC, it was no longer possible to isolate viable viruses from nasal swabs, and only high Ct values were detected by RT-qPCR. This suggests that the virus is transmissible for approximately up to nine days after exposure, with a possible peak at 4 DPI. This conclusion is also supported by the overall RT-qPCR detections in respiratory tissues. Similar to handling of the samples for PRV1 re-isolation, the PRV1 inoculation isolate was also frozen/thawed before performing the RT-qPCR-based TCID50/ml, potentially resulting in an underestimation of the inoculation titer. More knowledge about the immunological response against PRV1, as well as the implications of co-infections, should be investigated. Understanding these interactions could shed light on factors that could lead to systemic infections and/or more severe tissue damage and that could have a greater clinical impact on the overall piglet health. Until possible vaccines against PRV1 becomes available, or autogenous vaccines can be provided, management practices limiting respiratory pathogens in general should be implemented to reduce PRV1 influence on porcine health. These include sectioning, increased air exchange, and uncompromised stocking density. The attempt to propagate SOV in LLC-MK2 or Calu-3 cells was unsuccessful, as it was the case in another study by Graaf-Rau et al. ( 12 ). The competence of other cell lines for SOV should be investigated since using cell culture supernatant, instead of pooled SOV positive nasal swabs, could potentially improve the success with SOV experimental infections and reduce the changes of acquiring additional infections. Either the prepared pool did not contain sufficient viable replicative virus for successful infection, or the pigs had antibodies capable of neutralizing SOV. Further research on SOV is needed, including investigations focused on understanding the clinical relevance and distribution of this virus in pigs. Conclusion PRV1 was confirmed to be a primary porcine respiratory pathogen without causing clinical signs, except for nasal discharge, by experimental mono-infection. PRV1 primarily affected epithelial cells in the URT resulting in chronic, erosive tracheitis. PRV1 RNA was detected from 1 to 11 DPI and was transmitted horizontally to direct-contact pigs. Given the pathological manifestations, PRV1 should be considered a pathogen that facilitates the way for other potential pathogens. Ethics statement The pig experiment was performed at the animal experimental facilities at University of Copenhagen from May to June 2024. The experiment was approved by the Danish Animal Experimentation council under license number 2024-15-0201-01620 and performed in biosafety level 2 conditions. Supporting information Supplementary file S1. Raw data presented in Fig. 2. Figure S1. Trachea from a control pig at 4 days post inoculation (DPI) with no histopathological changes observed. Figure S2. Trachea from a recipient pig at 14 days post inoculation (DPI) chronic, severe, erosive tracheitis. The histopathological changes observed was suppurative exudation, with disseminated epithelial erosion leaving only the basal cell layer and with infiltration of mononuclear cells in lamina propria. Table S1. Overview of the pigs that were used in the experiment including group name, ID, gender, date of euthanization, weight (W) at -7 days post inoculation (DPI), 0, 4 and 14 DPI. Table S2. Presence of other relevant respiratory microbes in nasal swabs collected at 0 DPI investigated by high-throughput qPCR. Table S3. Pairwise sequence identities comparing the 15.320 nt long genome and single ORFs of PRV1-isolates used in this study (clade I) and in studies from Welch et al., 2021, 2022a, 2023 (clade II). 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Share Experimental inoculation of pigs with porcine respirovirus type 1 revealed pathological manifestations in the upper respiratory tract Marianne Viuf Agerlin , Kasper Pedersen , Mathias Romar , Marta Canuti , Timm Harder , Nicole Bakkegård Goecke , Henrik Elvang Jensen , Lars Erik Larsen , Pia Ryt-Hansen , Charlotte Kristensen bioRxiv 2025.07.24.666573; doi: https://doi.org/10.1101/2025.07.24.666573 Share This Article: Copy Citation Tools Experimental inoculation of pigs with porcine respirovirus type 1 revealed pathological manifestations in the upper respiratory tract Marianne Viuf Agerlin , Kasper Pedersen , Mathias Romar , Marta Canuti , Timm Harder , Nicole Bakkegård Goecke , Henrik Elvang Jensen , Lars Erik Larsen , Pia Ryt-Hansen , Charlotte Kristensen bioRxiv 2025.07.24.666573; doi: https://doi.org/10.1101/2025.07.24.666573 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13894) Bioinformatics (41951) Biophysics (21455) Cancer Biology (18592) Cell Biology (25507) Clinical Trials (138) Developmental Biology (13380) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24321) Genetics (15610) Genomics (22509) Immunology (17737) Microbiology (40398) Molecular Biology (17182) Neuroscience (88618) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7641) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)