Porcine Respiratory Coronavirus Research Articles

Detection and differentiation of seven porcine respiratory pathogens using a multiplex ligation-dependent probe amplification assay

Respiratory diseases due to viral or bacterial agents, either alone or in combination, cause substantial economic burdens to the swine industry worldwide. Rapid and reliable detection of causal pathogens is crucial for effective epidemiological surveillance and disease management. This research aimed to employ the multiplex ligation-dependent probe amplification (MLPA) assay for simultaneous detection of seven distinct pathogens causing respiratory problems in swine, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV2), Pasteurella multocida, Actinobacillus pleuropneumoniae, and Glässerella parasuis.The results indicated no probe cross-reactivity among the seven target agents with other swine pathogens. The detection limits ranged from 5 to 34 copies per assay for the target organisms. The MLPA assay was evaluated with 88 samples and compared to real-time or multiplex PCR for the target pathogens. The MLPA assay demonstrated high relative test sensitivities (100 %) and reasonable to good relative specificities at 62.5 %, 95.1 %, 86.8 %, and 97.6 % for PRRSV, P. multocida, G. parasuis, and PCV2, respectively, relative to comparator PCR assays. In 71 samples where MLPA and comparator PCR assays matched exactly, infections were detected in 64 samples (90.1 %), with PRRSV being the most commonly found virus and 50.7 % of the samples showing co-infection with two to five of the pathogens. This approach serves as a valuable tool for conducting differential diagnoses and epidemiological investigations of pathogen prevalence within swine populations.

Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

Porcine respiratory coronavirus genome sequences; comparisons and relationships to transmissible gastroenteritis viruses

Porcine respiratory coronavirus (PRCV) was initially detected in Europe, and later in the United States of America (US), in the 1980s. In this study we obtained and compared PRCV sequences from Europe and the US, and investigated how these are related to transmissible gastroenteritis virus (TGEV) sequences.The whole genome sequences of Danish (1/90-DK), Italian (PRCV15087/12 III NPTV Parma), and Belgian PRCV (91V44) strains are presented. These sequences were aligned with nine other PRCV sequences from Europe and the US, and 43 TGEV sequences. Following alignment of the PRCV sequences, it was apparent that multiple amino acid variations in the structural proteins were distinct between the European and US strains. The alignments were used to build phylogenetic trees to infer the evolutionary relationships between the strains. In these trees, the European PRCV strains clustered as a separate group, whereas the US strains of PRCV all clustered with TGEVs.

Porcine Respiratory Coronavirus (PRCV): Isolation and Characterization of a Variant PRCV from USA Pigs.

Porcine respiratory coronavirus (PRCV), a mutant of the transmissible gastroenteritis virus (TGEV), was first reported in Belgium in 1984. PRCV typically replicates and induces mild lesions in the respiratory tract, distinct from the enteric tropism of TGEV. In the past 30 years, PRCV has rarely been studied, and most cited information is on traditional isolates obtained during the 1980s and 1990s. Little is known about the genetic makeup and pathogenicity of recent PRCV isolates. The objective of this study was to obtain a contemporary PRCV isolate from US pigs for genetic characterization. In total, 1245 lung homogenate samples from pigs in various US states were tested via real-time PCR targeting PRCV and TGEV RNA. Overall, PRCV RNA was detected in five samples, and a single isolate (ISU20-92330) was successfully cultured and sequenced for its full-length genome. The isolate clustered with a new group of variant TGEVs and differed in various genomic regions compared to traditional PRCV isolates. Pathogens, such as PRCV, commonly circulate in pig herds without causing major disease. There may be value in tracking genomic changes and regularly updating the diagnostic methods for such viruses to be better prepared for the emergence of variants in ecology and pathogenicity.

Experimental Infection of Pigs with a Traditional or a Variant Porcine Respiratory Coronavirus (PRCV) Strain and Impact on Subsequent Influenza A Infection.

Porcine respiratory coronavirus (PRCV) pathogenicity in pigs has been characterized using traditional PRCV isolates; however, information is lacking on pathogenicity of currently circulating PRCV isolates. Recently, a contemporary US PRCV variant was isolated. The infection dynamics of that strain (PRCV-var) and a traditional PRCV strain (PRCV-trad) were compared. In brief, 4-week-old pigs were divided into three groups with five pigs each. The pigs were inoculated with PRCV-trad or PRCV-var, or left uninfected. Nasal swabs were collected daily, and all pigs were necropsied at day (D) 3. PRCV nasal shedding was significantly higher in PRCV-var pigs compared to PRCV-trad pigs. To investigate the impact of trad and var PRCVs on subsequent infection with influenza A virus (IAV), four additional groups of five pigs were used: PRCV-trad-IAV (PRCV-trad at D0, co-infected with IAV at D5), PRCV-var-IAV, and IAV positive and negative controls. Significantly higher mean PRCV antibody titers and a significantly higher area under the curve (AUC) for PRCV shedding were observed in PRCV-var compared to PRCV-trad-pigs at D10. There was no impact on IAV infection. In conclusion, a 2020 PRCV variant isolate was similar in pathogenicity but more transmissible compared to a traditional 1989 isolate. These findings raise concerns about virus evolution towards more highly pathogenic and transmissible strains and the need to monitor such viruses.

Genetic comparison of transmissible gastroenteritis coronaviruses.

Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that threatens animal health and remains elusive despite years of research efforts. The systematical analysis of all available full-length genomes of TGEVs (a total of 43) and porcine respiratory coronaviruses PRCVs (a total of 7) showed that TGEVs fell into two independent evolutionary phylogenetic clades, GI and GII. Viruses circulating in China (until 2021) clustered with the traditional or attenuated vaccine strains within the same evolutionary clades (GI). In contrast, viruses latterly isolated in the USA fell into GII clade. The viruses circulating in China have a lower similarity with that isolated latterly in the USA all through the viral genome. In addition, at least four potential genomic recombination events were identified, three of which occurred in GI clade and one in GII clade. TGEVs circulating in China are distinct from the viruses latterly isolated in the USA at either genomic nucleotide or antigenic levels. Genomic recombination serves as a factor driving the expansion of TGEV genomic diversity.

Bioexposure assays to assess uptake and survival of viruses in mealworm (Tenebrio molitor) and black soldier fly (Hermetia illucens) larvae

Insects represent a sustainable source of food and feed. In order to increase the sustainability of insect production, new feed substrates should be explored including waste materials that contain animal by-products. However, the use of waste products does come with potential risks for transmission of pathogens. Experimental exposure studies of insects with selected pathogens can provide knowledge of some of these risks for future quantitative risk assessments. In this study, we have developed virus bioexposure assays to examine the uptake and survival of two different strains of porcine respiratory coronavirus (PRCV) in the larvae of two key insect species that are grown for the production of food and feed, namely the mealworm, Tenebrio molitor, and the black soldier fly, Hermetia illucens. T. molitor larvae were exposed to PRCV via oral uptake of virus suspensions while H. illucens larvae were exposed to PRCV via spiking of their feed. Using RT-qPCR assays, the amount of viral RNA extracted from individual larvae immediately after exposure varied from ~104.6 to 107.1 genome copies per insect. The PRCV RNA was detectable in the larvae of both T. molitor and H. illucens for up to 3 days post exposure but no longer. The assays applied to T. molitor and H. illucens for exposure to PRCV should be applicable for the exposure of further insect larvae to other viruses, and potentially also to other types of pathogens.

Metavirome Analysis Reveals a High Prevalence of Porcine Hemagglutination Encephalomyelitis Virus in Clinically Healthy Pigs in China.

Six swine coronaviruses (SCoVs), which include porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine hemagglutination encephalomyelitis virus (PHEV), porcine respiratory coronavirus (PRCV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV), have been reported as infecting and causing serious diseases in pigs. To investigate the genetic diversity and spatial distribution of SCoVs in clinically healthy pigs in China, we collected 6400 nasal swabs and 1245 serum samples from clinically healthy pigs at slaughterhouses in 13 provinces in 2017 and pooled them into 17 libraries by type and region for next-generation sequencing (NGS) and metavirome analyses. In total, we identified five species of SCoVs, including PEDV, PDCoV, PHEV, PRCV, and TGEV. Strikingly, PHEV was detected from all the samples in high abundance and its genome sequences accounted for 75.28% of all coronaviruses, while those belonging to TGEV (including PRCV), PEDV, and PDCoV were 20.4%, 2.66%, and 2.37%, respectively. The phylogenetic analysis showed that two lineages of PHEV have been circulating in pig populations in China. We also recognized two PRCVs which lack 672 nucleotides at the N-terminus of the S gene compared with that of TGEV. Together, we disclose preliminarily the genetic diversities of SCoVs in clinically healthy pigs in China and provide new insights into two SCoVs, PHEV and PRCV, that have been somewhat overlooked in previous studies in China.

Inactivation of SARS-CoV-2 and Other Enveloped and Non-Enveloped Viruses with Non-Thermal Plasma for Hospital Disinfection.

As recently highlighted by the SARS-CoV-2 pandemic, viruses have become an increasing burden for health, global economy, and environment. The control of transmission by contact with contaminated materials represents a major challenge, particularly in hospital environments. However, the current disinfection methods in hospital settings suffer from numerous drawbacks. As a result, several medical supplies that cannot be properly disinfected are not reused, leading to severe shortages and increasing amounts of waste, thus prompting the search for alternative solutions. In this work, we report that non-thermal plasma (NTP) can effectively inactivate SARS-CoV-2 from non-porous and porous materials commonly found in healthcare facilities. We demonstrated that 5 min treatment with a dielectric barrier discharge NTP can inactivate 100% of SARS-CoV-2 (Wuhan and Omicron strains) from plastic material. Using porcine respiratory coronavirus (surrogate for SARS-CoV-2) and coxsackievirus B3 (highly resistant non-enveloped virus), we tested the NTP virucidal activity on hospital materials and obtained complete inactivation after 5 and 10 min, respectively. We hypothesize that the produced reactive species and local acidification contribute to the overall virucidal effect of NTP. Our results demonstrate the potential of dielectric barrier discharge NTPs for the rapid, efficient, and low-cost disinfection of healthcare materials.

A Serological Investigation of Porcine Reproductive and Respiratory Syndrome and Three Coronaviruses in the Campania Region, Southern Italy.

Porcine coronaviruses and reproductive and respiratory syndrome (PRRS) are responsible for severe outbreaks that cause huge economic losses worldwide. In Italy, three coronaviruses have been reported historically: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV). Although repeated outbreaks have been described, especially in northern Italy, where intensive pig farming is common, there is a worrying lack of information on the spread of these pathogens in Europe. In this work, we determined the seroprevalence of three porcine coronaviruses and PRRSV in the Campania region, southern Italy. A total of 443 samples were tested for the presence of antibodies against porcine coronaviruses and PRRSV using four different commercial ELISAs. Our results indicated that PEDV is the most prevalent among porcine coronaviruses, followed by TGEV, and finally PRCV. PRRSV appeared to be the most prevalent virus (16.7%). For coronaviruses, seroprevalence was higher in pigs raised in intensive farming systems. In terms of distribution, TGEV is more widespread in the province of Avellino, while PEDV and PRRSV are more prevalent in the province of Naples, emphasizing the epidemic nature of both infections. Interestingly, TGEV-positive animals are more common among growers, while seropositivity for PEDV and PRRSV was higher in adults. Our research provides new insights into the spread of swine coronaviruses and PRRSV in southern Italy, as well as a warning about the need for viral surveillance.

Here Comes the Sun—Methylene Blue in Combination with Sunlight Sanitises Surgical Masks Contaminated with a Coronavirus and a Tenacious Small Non-Enveloped Virus

In the context of the SARS-CoV-2 pandemic, the reuse of personal protective equipment, specifically face coverings, has been recommended. Reuse of such items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of face coverings contaminated with either infectious SARS-CoV-2 and animal coronaviruses or a highly resistant, non-enveloped norovirus via a novel photochemical treatment. Contaminated materials were coated with photosensitive methylene blue dye and were subsequently exposed to a visible bright light source (LED-equipped light boxes) to trigger the generation of virucidal singlet oxygen. A possible factor restricting the widespread use of such photochemical decontamination is its reliance on the availability of electricity to power light sources. Here, we show that natural sunlight can be used in lieu of artificial light. We demonstrate efficient inactivation of a SARS-CoV-2 surrogate, porcine respiratory coronavirus, via 10 µM dye coating in conjunction with short outdoor exposures of 5–30 min (blue sky to cloudy day; mean 46,578 lx). A tenacious human norovirus surrogate, murine norovirus, is inactivated via methylene blue solar decontamination involving 100 µM dye concentrations and 30 min of high-illuminance sunlight (blue sky; mean 93,445 lx) or 2 h of mid- to low-illuminance (cloudy day; mean 28,558 lx). The protocol developed here thus solidifies the position of methylene blue solar decontamination as an important equitable tool in the package of practical pandemic preparedness.

Diversity of respiratory viruses present in nasal swabs under influenza suspicion in respiratory disease cases of weaned pigs

Respiratory diseases in weaned pigs are a common problem, with a complex etiology involving both viruses and bacteria. In the present study, we investigated the presence of eleven viruses in nasal swabs, collected from nurseries (55 cases) under the suspicion of swine influenza A virus (swIAV) and submitted by swine veterinarians for diagnosis. The other ten viruses included in the study were influenza B (IBV) and D (IDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine respiratory coronavirus (PRCV), Porcine cytomegalovirus (PCMV), Porcine circovirus 2 (PCV2), 3 (PCV3) and 4 (PCV), Porcine parainfluenza 1 (PPIV1) and Swine orthopneumovirus (SOV). Twenty-six swIAV-positive cases and twenty-nine cases of swIAV-negative respiratory disease were primarily established. While IBV, IDV, PCV4 and PPIV1 were not found in any of the cases, PRCV, SOV, and PCMV were more likely to be found in swIAV-positive nurseries with respiratory disease (p < 0.05). Overall, PCV3, PRRSV, and PCMV were the most frequently detected agents at herd level. Taken individually, virus prevalence was: swIAV, 48.6%; PRCV, 48.0%; PRRSV, 31.6%; SOV, 33.8%; PCMV, 48.3%, PCV2, 36.0%; and PCV3, 33.0%. Moreover, low Ct values (<30) were common for all agents, except PCV2 and PCV3. When the correlation between pathogens was individually examined, the presence of PRRSV was negatively correlated with swIAV and PRCV, while was positively associated to PCMV (p < 0.05). Also, PRCV and SOV were positively correlated between them and negatively with PCMV. Besides, the analysis of suckling pig samples, collected in subclinically infected farrowing units under an influenza monitoring program, showed that circulation of PRCV, PCMV, SOV, and PCV3 started during the early weeks of life. Interestingly, in those subclinically infected units, none of the pathogens was found to be correlated to any other. Overall, our data may contribute to a better understanding of the complex etiology and epidemiology of respiratory diseases in weaners. This is the first report of SOV in Spain and shows, for the first time, the dynamics of this pathogen in swine farms.

Characterization and cross-protection of experimental infections with SeCoV and two PEDV variants.

The aim of this study was to characterize the infection of weaned pigs with swine enteric coronavirus (SeCoV) - a chimeric virus most likely originated from a recombination event between porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus, or its mutant porcine respiratory coronavirus - and two PEDV G1b variants, including a recently described recombinant PEDV-SeCoV (rPEDV-SeCoV), as well as to determine the degree of cross-protection achieved against the rPEDV-SeCoV. For this purpose, forty-eight 4-week-old weaned pigs were randomly allocated into four groups of 12 animals. Piglets within each group were primary inoculated with one of the investigated viral strains (B: PEDV; C: SeCoV and D: rPEDV-SeCoV) or mock-inoculated (A), and exposed to rPEDV-SeCOV at day 20 post-infection; thus, group A was primary challenged (-/rPEDV-SeCoV), groups B and C were subjected to a heterologous re-challenge (PEDV/rPEDV-SeCoV and SeCoV/rPEDV-SeCoV, respectively), and group D to a homologous re-challenge (rPEDV-SeCoV/rPEDV-SeCoV), Clinical signs, viral shedding, microscopic lesions and specific humoral and cellular immune responses (IgG, IgA, neutralizing antibodies and IgA and IFN-γ-secreting cells) were monitored. After primo-infection, all three viral strains induced an undistinguishable mild-to-moderate clinical disease with diarrhoea as the main sign and villus shortening lesions in the small intestine. In homologous re-challenged pigs, no clinical signs or lesions were observed, and viral shedding was only detected in a single animal. This fact may be explained by the significant high level of rPEDV-SeCoV-specific neutralizing antibodies found in these pigs before the challenge. In contrast, prior exposure to a different PEDV G1b variant or SeCoV only provided partial cross-protection, allowing rPEDV-SeCoV replication and shedding in faeces.

The Effects of Swine Coronaviruses on ER Stress, Autophagy, Apoptosis, and Alterations in Cell Morphology

Swine coronaviruses include the following six members, namely porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta coronavirus (PDCoV), swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine hemagglutinating encephalomyelitis virus (PHEV), and porcine respiratory coronavirus (PRCV). Clinically, PEDV, TGEV, PDCoV, and SADS-CoV cause enteritis, whereas PHEV induces encephalomyelitis, and PRCV causes respiratory disease. Years of studies reveal that swine coronaviruses replicate in the cellular cytoplasm exerting a wide variety of effects on cells. Some of these effects are particularly pertinent to cell pathology, including endoplasmic reticulum (ER) stress, unfolded protein response (UPR), autophagy, and apoptosis. In addition, swine coronaviruses are able to induce cellular changes, such as cytoskeletal rearrangement, alterations of junctional complexes, and epithelial-mesenchymal transition (EMT), that render enterocytes unable to absorb nutrients normally, resulting in the loss of water, ions, and protein into the intestinal lumen. This review aims to describe the cellular changes in swine coronavirus-infected cells and to aid in understanding the pathogenesis of swine coronavirus infections. This review also explores how the virus exerted subcellular and molecular changes culminating in the clinical and pathological findings observed in the field.

SEROPREVALENCE OF PORCINE RESPIRATORY CORONAVIRUS AND TRANSMISSIBLE GASTROENTERITIS VIRUS INFECTIONS ON COMMERCIAL PIG FARMS IN SERBIA

Porcine respiratory coronavirus is an enzootic, viral, respiratory disease of pigs, which manifests with mild clinical signs, but it takes part in the etiopathogenesis of the porcine respiratory disease complex. The virus was first discovered in Belgium in 1984 as a deletion mutant of the transmissible gastroenteritis virus. The two viruses are strongly antigenically related which is why they cross-react in serological tests. In this study, we tested 276 serum samples from different categories of pigs using ELISA test, which allows differentiation between the porcine respiratory coronavirus infection and transmissible gastroenteritis. The seroconversion for coronavirus infection was determined in 80.4% of tested samples. Out of 222 positive samples, 219 samples (98.6%) were positive for porcine respiratory coronavirus antibodies, while 3 (1.01%) samples were positive for transmissible gastroenteritis virus antibodies. Depending on the production category, 97.7% of piglets, 83% of sows, and 35% of gilts tested positive for porcine respiratory coronavirus antibodies. In total, 2.3% of piglets tested positive for transmissible gastroenteritis virus antibodies. Taking into account the characteristics of the ELISA test, its sensitivity and specificity, this result can be considered a false positive, because of a cross-reaction between the porcine respiratory coronavirus antibodies and the transmissible gastroenteritis virus. Specific antibodies in other swine production categories against the transmissible gastroenteritis virus were not determined.

Single-pass wind tunnel testing for recirculating virus aerosol control technologies

A number of recirculating flow aerosol control technologies have been commercialized to mitigate aerosol-transmitted virus infections. Many of these technologies incorporate filters for particle collection and some may also incorporate technologies for virus inactivation. Given the wide variety of commercially available aerosol control technologies to consumers, it is extremely important to develop standardized methods to characterize their performance in bioaerosol removal and inactivation, such that technologies can be compared on an “equivalent-test” basis. However, no standard procedures have been established to evaluate the effectiveness of bioaerosol removal and inactivation in recirculating aerosol control technologies. We propose the use of a single-pass tunnel to assess the performance of bioaerosol control technologies, as single-pass wind tunnels can be sealed with well-controlled velocity and particle concentration profiles. Here, we specifically describe the construction of a single-pass wind tunnel and apply it to three recirculating aerosol control technologies, incorporating UV-C sources, filters, and electrostatic precipitators, respectively. We utilize a porcine respiratory coronavirus (PRCV) challenge aerosol, generated via pneumatic nebulization of a high titer (∼107 TCID50 mL−1) viral suspension. Following guidelines similar to those used in the ANSI/ASHRAE Standard 52-2 test procedure for HVAC filters, in single-pass wind tunnel tests, velocity uniformity and particle uniformity are first monitored across the cross-section of the tunnel. The size distribution of viable particles is additionally determined in advance of tests by the collection of particles in the wind tunnel using a cascade impactor, with both RT-qPCR and titration used to quantify viruses collected on each impaction stage. We show that the viable particle size distribution follows the volumetric size distribution of the nebulized virus-laden suspension, and that this distribution can be tuned to be similar in shape to the observed distribution of aerosol from human respiratory activities. Following tunnel and virus aerosol characterization, for each tested technology, using triplicate tests, the single-pass log reduction based on RT-qPCR and viable virus titration is determined by simultaneously collecting virus aerosol particles upstream and downstream of the control technology. The tested technologies in this study have titration-based single-pass log reductions in the 1.5–4.0 range.Overall, design and testing suggest that the single-pass wind tunnel approach is a tractable method to examine the efficacy of aerosol control technologies in removing and inactivating viruses in aerosols, and suggest that such technologies should be described by their single-pass log reduction and operating flow rate, with the test virus size distribution reported alongside test results. In addition, we examine the limits of detection in single-pass wind tunnel tests in comparison to chamber tests, and in doing so find that for most control technologies, the wind tunnel test will yield higher concentrations downstream or during sampling, and hence clearer results for the log reduction.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

Porcine Respiratory Coronavirus as a Model for Acute Respiratory Coronavirus Disease.

In the light of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we have developed a porcine respiratory coronavirus (PRCV) model for in depth mechanistic evaluation of the pathogenesis, virology and immune responses of this important family of viruses. Pigs are a large animal with similar physiology and immunology to humans and are a natural host for PRCV. Four PRCV strains were investigated and shown to induce different degrees of lung pathology. Importantly, although all four strains replicated equally well in porcine cell lines in vitro and in the upper respiratory tract in vivo, PRCV strains causing more severe lung pathology were also able to replicate in ex vivo tracheal organ cultures as well as in vivo in the trachea and lung. The time course of infection of PRCV 135, which caused the most severe pulmonary pathology, was investigated. Virus was shed from the upper respiratory tract until day 10 post infection, with infection of the respiratory mucosa, as well as olfactory and sustentacular cells, providing an excellent model to study upper respiratory tract disease in addition to the commonly known lower respiratory tract disease from PRCV. Infected animals made antibody and T cell responses that cross reacted with the four PRCV strains and Transmissible Gastroenteritis Virus. The antibody response was reproduced in vitro in organ cultures. Comparison of mechanisms of infection and immune control in pigs infected with PRCVs of differing pathogenicity with human data from SARS-CoV-2 infection and from our in vitro organ cultures, will enable key events in coronavirus infection and disease pathogenesis to be identified.

Structures of a deltacoronavirus spike protein bound to porcine and human receptors

Porcine deltacoronavirus (PDCoV) can experimentally infect a variety of animals. Human infection by PDCoV has also been reported. Consistently, PDCoV can use aminopeptidase N (APN) from different host species as receptors to enter cells. To understand this broad receptor usage and interspecies transmission of PDCoV, we determined the crystal structures of the receptor binding domain (RBD) of PDCoV spike protein bound to human APN (hAPN) and porcine APN (pAPN), respectively. The structures of the two complexes exhibit high similarity. PDCoV RBD binds to common regions on hAPN and pAPN, which are different from the sites engaged by two alphacoronaviruses: HCoV-229E and porcine respiratory coronavirus (PRCoV). Based on structure guided mutagenesis, we identified conserved residues on hAPN and pAPN that are essential for PDCoV binding and infection. We report the detailed mechanism for how a deltacoronavirus recognizes homologous receptors and provide insights into the cross-species transmission of PDCoV.

Electroceutical fabric lowers zeta potential and eradicates coronavirus infectivity upon contact

Coronavirus with intact infectivity attached to PPE surfaces pose significant threat to the spread of COVID-19. We tested the hypothesis that an electroceutical fabric, generating weak potential difference of 0.5 V, disrupts the infectivity of coronavirus upon contact by destabilizing the electrokinetic properties of the virion. Porcine respiratory coronavirus AR310 particles (105) were placed in direct contact with the fabric for 1 or 5 min. Following one minute of contact, zeta potential of the porcine coronavirus was significantly lowered indicating destabilization of its electrokinetic properties. Size-distribution plot showed appearance of aggregation of the virus. Testing of the cytopathic effects of the virus showed eradication of infectivity as quantitatively assessed by PI-calcein and MTT cell viability tests. This work provides the rationale to consider the studied electroceutical fabric, or other materials with comparable property, as material of choice for the development of PPE in the fight against COVID-19.