Development and Evaluation of Antigen-Specific Dual Matrix Pestivirus K ELISAs Using Longitudinal Known Infectious Status Samples.

Pestivirus K, commonly known as atypical porcine pestivirus (APPV), is the most common cause of congenital tremor (CT) in pigs. Currently, there is limited information on the infection dynamics of and immune response against APPV and no robust serologic assay to assess the effectiveness of preventative measures. To that end, known infection status samples were generated using experimental inoculation of cesarean-derived, colostrum-deprived pigs. Pigs (2 per pen) were inoculated with minimum essential medium (n = 6; negative control) or APPV (n = 16). Serum, pen-based oral fluid samples, and nasal swabs were collected through 70 days postinoculation (dpi). The immune response to recombinant APPV Erns, E2, or NS3 antigens was evaluated using both serum and oral fluids via indirect enzyme-linked immunosorbent assays (ELISAs). APPV was detected by real-time reverse transcription-PCR (RT-qPCR) in all oral fluid and serum samples from APPV-inoculated animals by 24 and 35 dpi, respectively. All samples remained genome positive until 70 dpi. Detection of nasal shedding was less consistent, with APPV being detected by RT-qPCR in all inoculated animals at 42, 49, and 56 dpi. Antibodies were first detected in oral fluids at 14 dpi, 10 days before serum detection, and concurrently with the first oral fluids RT-qPCR detection. Across sample types and time points, the Erns ELISA outperformed the other targets. In conclusion, both oral fluid and serum APPV Erns ELISAs can be used to economically evaluate the individual and herd status prior to and following intervention strategies.

Porcine Hemagglutinating Encephalomyelitis Virus Infection In Vivo and Ex Vivo.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a betacoronavirus that causes vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study characterized PHEV infection, pathogenesis, and immune response in cesarean-derived, colostrum-deprived (CDCD) neonatal pigs. Infected animals developed mild respiratory, enteric, and neurological clinical signs between 2 to 13 days postoronasal inoculation (dpi). PHEV did not produce viremia, but virus shedding was detected in nasal secretions (1 to 10 dpi) and feces (2 to 7 dpi) by reverse transcriptase quantitative PCR (RT-qPCR). Viral RNA was detected in all tissues except liver, but the detection rate and RT-qPCR threshold cycle (CT ) values decreased over time. The highest concentration of virus was detected in inoculated piglets necropsied at 5 dpi in turbinate and trachea, followed by tonsils, lungs, tracheobronchial lymph nodes, and stomach. The most representative microscopic lesions were gastritis lymphoplasmacytic, moderate, multifocal, with perivasculitis, and neuritis with ganglia degeneration. A moderate inflammatory response, characterized by increased levels of interferon alpha (IFN-α) in plasma (5 dpi) and infiltration of T lymphocytes and macrophages were also observed. Increased plasma levels of interleukin-8 (IL-8) were detected at 10 and 15 dpi, coinciding with the progressive resolution of the infection. Moreover, a robust antibody response was detected by 10 dpi. An ex vivo air-liquid CDCD-derived porcine respiratory cells culture (ALI-PRECs) system showed virus replication in ALI-PRECs and cytopathic changes and disruption of ciliated columnar epithelia, thereby confirming the tracheal epithelia as a primary site of infection for PHEV.IMPORTANCE Among the ∼46 virus species in the family Coronaviridae, many of which are important pathogens of humans and 6 of which are commonly found in pigs, porcine hemagglutinating encephalomyelitis remains one of the least researched. The present study provided a comprehensive characterization of the PHEV infection process and immune responses using CDCD neonatal pigs. Moreover, we used an ex vivo ALI-PRECs system resembling the epithelial lining of the tracheobronchial region of the porcine respiratory tract to demonstrate that the upper respiratory tract is a primary site of PHEV infection. This study provides a platform for further multidisciplinary studies of coronavirus infections.

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

Introduction: The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Methods: Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24 h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). Results: No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. Discussion: This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

Characterization of the immunodominant regions of Senecavirus A-VP1 structural protein via ELISA epitope mapping.

Senecavirus A (SVA) is an RNA virus in the family Picornaviridae that has been detected in swine-production systems and is associated with vesicular disease and neonate mortality. The viral capsid is composed of four structural proteins: VP1-VP4. Although the VP1 protein has been reported to be the most immunogenic protein in vivo, no information on the immunodominant regions of the SVA polyprotein is available. The objective of this study was to identify the immunodominant regions of SVA polyprotein using an enzyme-linked immunosorbent assay (ELISA) epitope-mapping approach. The binding effect of SVA polyclonal antibody (SVA-pAb), SVA-VP1 monoclonal antibodies (SVA-mAb), and SVA-positive sera from clinically affected animals were characterized using a set of 18 overlapping SVA VP1-derived peptides by indirect and blocking ELISAs. All VP1 peptides yielded significant signal against SVA-pAb and SVA-VP1-mAb upon indirect ELISA. One peptide (aa 1-20) showed significantly high optical density on SVA recombinant VP1 protein (rVP1) and whole-virus-based indirect ELISAs. The blocking ELISA results demonstrated that peptides spanning aa 165-185 and 225-245 had a 50 % or greater inhibitory effect on SVA-pAb, while six groups of overlapping peptides spanning aa 1-35, 45-80, 90-140, 150-170, 195-230, and 240-264 and two groups of overlapping peptides spanning aa 1-50 and 60-264 showed a 50 % inhibitory effect or greater on swine VP1-mAb and SVA-seropositive swine serum, respectively, against SVA rVP1. Three-dimensional protein homology modeling showed that the peptides binding SVA-pAb are located on the outer surface of the viral capsid, while SVA mAbs and swine-positive sere can bind to epitopes located in both the inner and outer surfaces of the capsid. These linear epitopes showed differential binding and inhibitory activity on mAb and pAb; however, further studies will be necessary to evaluate whether they can act as decoy or neutralizing epitopes. Because mAb antibodies demonstrated a high binding affinity for this set of peptides, this information could lay the foundation for generating and screening specific antibodies for therapeutic potential.

Differential antigenicity of individual Mycoplasma hyorhinis variable lipoproteins.

The Mycoplasma hyorhinis (Mhr) variable lipoprotein (Vlp) family, comprising Vlps A, B, C, D, E, F, and G, are highly variable in expression, size, and cytoadhesion capabilities across Mhr strains. The 'Vlp system' plays a crucial role in cytoadhesion, immune evasion, and in eliciting a host immunologic response. This pilot study described the development of Vlp peptide-based ELISAs to evaluate the antigenic reactivity of individual Vlps against Mhr antisera collected throughout a longitudinal study focused on Mhr strain 38983, reproducing Mhr-associated disease under experimental conditions. Specifically, serum samples were collected at day post-inoculation 0, 7, 10, 14, 17, 21, 24, 28, 35, 42, 49, and 56 from Mhr- and mock (Friis medium)-inoculated cesarean-derived, colostrum-deprived pigs. Significant Mhr-specific IgG responses were detected at specific time points throughout the infection, with some variations for each Vlp. Overall, individual Vlp ELISAs showed consistently high accuracy rates, except for VlpD, which would likely be associated with its expression levels or the anti-Vlp humoral immune response specific to the Mhr strain used in this study. This study provides the basis and tools for a more refined understanding of these Vlp- and Mhr strain-specific variations, which is foundational in understanding the host immune response to Mhr.

Detection and Monitoring of Highly Pathogenic Influenza A Virus 2.3.4.4b Outbreak in Dairy Cattle in the United States.

The emergence and spread of highly pathogenic avian influenza virus A subtype H5N1 (HP H5N1-IAV), particularly clade H5N1 2.3.4.4b, pose a severe global health threat, affecting various species, including mammals. Historically, cattle have been considered less susceptible to IAV, but recent outbreaks of H5N1-IAV 2.3.4.4b in dairy farms suggest a shift in host tropism, underscoring the urgency of expanded surveillance and the need for adaptable diagnostic tools in outbreak management. This study investigated the presence of anti-nucleoprotein (NP) antibodies in serum and milk and viral RNA in milk on dairy farms affected by outbreaks in Texas, Kansas, and Michigan using a multi-species IAV ELISA and RT-qPCR. The analysis of ELISA results from a Michigan dairy farm outbreak demonstrated a positive correlation between paired serum and milk sample results, confirming the reliability of both specimen types. Our findings also revealed high diagnostic performance during the convalescent phase (up to 96%), further improving sensitivity through serial sampling. Additionally, the evaluation of diagnostic specificity using serum and milk samples from IAV-free farms showed an excellent performance (99.6%). This study underscores the efficacy of the IAV NP-blocking ELISA for detecting and monitoring H5N1-IAV 2.3.4.4b circulation in dairy farms, whose recent emergence raises significant animal welfare and zoonotic concerns, necessitating expanded surveillance efforts.

Dynamics of antibody response and bacterial shedding of Mycoplasma hyorhinis and M. hyosynoviae in oral fluids from experimentally inoculated pigs.

Mycoplasma hyorhinis (Mhr) and M. hyosynoviae (Mhs) are commensal organisms of the upper respiratory tract and tonsils but may also cause arthritis in pigs. In this study, 8-week-old cesarean-derived colostrum-deprived (CDCD) pigs (n = 30; 3 groups, 10 pigs per group, 2 pigs per pen) were inoculated with Mhr, Mhs, or mock-inoculated with culture medium and then pen-based oral fluids were collected at different time points over the 56 days of the experimental study. Oral fluids tested by Mhr and Mhs quantitative real-time PCRs revealed Mhr DNA between day post inoculation (DPI) 5-52 and Mhs DNA between DPI 5-15. Oral fluids were likewise tested for antibody using isotype-specific (IgG, IgA, IgM) indirect ELISAs based on a recombinant chimeric polypeptide of variable lipoproteins (A-G) for Mhr and Tween 20-extracted surface proteins for Mhs. Mhr IgA was detected at DPI 7 and, relative to the control group, significant (p < 0.05) antibody responses were detected in the Mhr group between DPI 12-15 for IgM and DPI 36-56 for both IgA and IgG. In the Mhs group, IgM was detected at DPI 10 and significant (p < 0.05) IgG and IgA responses were detected at DPI 32-56 and DPI 44-56, respectively. This study demonstrated that oral fluid could serve as an effective and convenient antemortem sample for monitoring Mhr and Mhs in swine populations.

Self-administration of a Salmonella vaccine by domestic pigs.

Hand vaccinating is time consuming and inefficient. Oral vaccines delivered by drenching are less likely to be used due to a lack of labor on farms. Current environmental enrichment (EE) technologies do not allow pigs to express certain natural behaviors such as rooting and getting a reward. We developed a sprayer so that domestic pigs can self-apply any liquid. By adding an attractant (pig maternal pheromone), the use of EE devices by individual pigs can be increased. In this study, we used a Salmonella oral vaccine to evaluate efficacy of three delivery methods: (1) Control, no vaccine, (2) hand drenching as labeled, and (3) self-administration by this EE rooting device. All pigs sprayed themselves within 80 min of exposure to the EE device. While control pigs had little or no Salmonella serum and oral fluid IgG or IgA, hand-drenched and self-vaccinated pigs built similar levels of both serum and oral fluid IgA and IgG. We conclude we were able to significantly reduce human labor needed and achieved 100% efficacy in eliciting a serologic response when pigs self-administered a Salmonella vaccine. This technology could benefit commercial pig production while providing an enriched behavioral environment. Self-vaccination could also assist in control or immunization of feral swine and improve domestic pig health and food safety.

SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

Molecular mechanisms of human coronavirus NL63 infection and replication.

Human coronavirus NL63 (HCoV-NL63) is spread globally, causing upper and lower respiratory tract infections mainly in young children. HCoV-NL63 shares a host receptor (ACE2) with severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 but, unlike them, HCoV-NL63 primarily develops into self-limiting mild to moderate respiratory disease. Although with different efficiency, both HCoV-NL63 and SARS-like CoVs infect ciliated respiratory cells using ACE2 as receptor for binding and cell entry. Working with SARS-like CoVs require access to BSL-3 facilities, while HCoV-NL63 research can be performed at BSL-2 laboratories. Thus, HCoV-NL63 could be used as a safer surrogate for comparative studies on receptor dynamics, infectivity and virus replication, disease mechanism, and potential therapeutic interventions against SARS-like CoVs. This prompted us to review the current knowledge on the infection mechanism and replication of HCoV-NL63. Specifically, after a brief overview on the taxonomy, genomic organization and virus structure, this review compiles the current HCoV-NL63-related research in virus entry and replication mechanism, including virus attachment, endocytosis, genome translation, and replication and transcription. Furthermore, we reviewed cumulative knowledge on the susceptibility of different cells to HCoV-NL63 infection in vitro, which is essential for successful virus isolation and propagation, and contribute to address different scientific questions from basic science to the development and assessment of diagnostic tools, and antiviral therapies. Finally, we discussed different antiviral strategies that have been explored to suppress replication of HCoV-NL63, and other related human coronaviruses, by either targeting the virus or enhancing host antiviral mechanisms.

Dynamics of Infection of Atypical Porcine Pestivirus in Commercial Pigs from Birth to Market: A Longitudinal Study.

Atypical porcine pestivirus (APPV) was found to be associated with pigs demonstrating congenital tremors (CT), and clinical signs in pigs have been reproduced after experimental challenge. Subsequently, APPV has been identified in both symptomatic and asymptomatic swine of all ages globally. The objective of this research was to perform a longitudinal study following two cohorts of pigs, those born in litters with pigs exhibiting CT and those born in litters without CT, to analyze the virus and antibody dynamics of APPV infection in serum from birth to market. There was a wide range in the percentage of affected pigs (8-75%) within CT-positive litters. After co-mingling with CT-positive litters at weaning, pigs from CT-negative litters developed viremia that was cleared after approximately 2 months, with the majority seroconverting by the end of the study. In contrast, a greater percentage of pigs exhibiting CT remained PCR positive throughout the growing phase, with less than one-third of these animals seroconverting. APPV RNA was present in multiple tissues from pigs in both groups at the time of marketing. This study improved our understanding of the infection dynamics of APPV in swine and the impact that the immune status and timing of infection have on the persistence of APPV in serum and tissues.

Modeling the accuracy of a novel PCR and antibody ELISA for African swine fever virus detection using Bayesian latent class analysis.

Introduction: Diagnostic test evaluation for African swine fever (ASF) in field settings like Vietnam is critical to understanding test application in intended populations for surveillance and control strategies. Bayesian latent class analysis (BLCA) uses the results of multiple imperfect tests applied to an individual of unknown disease status to estimate the diagnostic sensitivity and specificity of each test, forgoing the need for a reference test. Methods: Here, we estimated and compared the diagnostic sensitivity and specificity of a novel indirect ELISA (iELISA) for ASF virus p30 antibody (Innoceleris LLC.) and the VetAlert™ ASF virus DNA Test Kit (qPCR, Tetracore Inc.) in field samples from Vietnam by assuming that disease status 1) is known and 2) is unknown using a BLCA model. In this cross-sectional study, 398 paired, individual swine serum/oral fluid (OF) samples were collected from 30 acutely ASF-affected farms, 37 chronically ASF-affected farms, and 20 ASF-unaffected farms in Vietnam. Samples were tested using both diagnostic assays. Diagnostic sensitivity was calculated assuming samples from ASF-affected farms were true positives and diagnostic sensitivity by assuming samples from unaffected farms were true negatives. ROC curves were plotted and AUC calculated for each test/sample combination. For comparison, a conditionally dependent, four test/sample combination, three population BLCA model was fit. Results: When considering all assumed ASF-affected samples, qPCR sensitivity was higher for serum (65.2%, 95% Confidence Interval [CI] 58.1-71.8) and OF (52%, 95%CI 44.8-59.2) compared to the iELISA (serum: 42.9%, 95%CI 35.9-50.1; OF: 33.3%, 95%CI 26.8-40.4). qPCR-serum had the highest AUC (0.895, 95%CI 0.863-0.928). BLCA estimates were nearly identical to those obtained when assuming disease status and were robust to changes in priors. qPCR sensitivity was considerably higher than ELISA in the acutely-affected population, while ELISA sensitivity was higher in the chronically-affected population. Specificity was nearly perfect for all test/sample types. Discussion: The effect of disease chronicity on sensitivity and specificity could not be well characterized here due to limited data, but future studies should aim to elucidate these trends to understand the best use of virus and antibody detection methods for ASF. Results presented here will help the design of surveillance and control strategies in Vietnam and other countries affected by ASF.

Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells.

Human coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies. IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.

The N-terminal Subunit of the Porcine Deltacoronavirus Spike Recombinant Protein (S1) Does Not Serologically Cross-react with Other Porcine Coronaviruses.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus, is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) -7 to 42, from pigs (n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples (n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples (n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs (n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.

Characterization of the Subclinical Infection of Porcine Deltacoronavirus in Grower Pigs under Experimental Conditions.

This study characterized the susceptibility and dynamic of porcine deltacoronavirus infection in grower pigs under experimental conditions using a combination of syndromic and laboratory assessments. Seven-week-old conventional pigs (n = 24) were randomly distributed into PDCoV- (n = 12) and mock-inoculated (n = 12) groups. Serum was collected at -7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42 days post-inoculation (DPI) to evaluate viremia (RT-qPCR) and antibody response (S1-based ELISA). Viral shedding and potential infectivity were determined using pen-based oral fluids and feces collected every other day between DPI 0 and 42. Pigs showed no clinical signs or viremia throughout the study. Active virus shedding was detected in feces (6-22 DPI) and oral fluids (2-30 DPI), peaking at DPI 10. IgG was first detected at DPI 10, being statistically significant after DPI 14 and increasing thereafter, coinciding with the progressive resolution of the infection. Likewise, a significant increase in proinflammatory IL-12 was detected between DPI 10 and 21 in PDCoV-inoculated pigs, which could enhance innate resistance to PDCoV infection. This study demonstrated that active surveillance based on systematic sampling and laboratory testing combining molecular and serological tools is critical for the accurate detection of subclinical circulation of PDCoV in pigs after weaning.

The Betacoronavirus PHEV Replicates and Disrupts the Respiratory Epithelia and Upregulates Key Pattern Recognition Receptor Genes and Downstream Mediators, Including IL-8 and IFN-λ.

The upper respiratory tract is the primary site of infection by porcine hemagglutinating encephalomyelitis virus (PHEV). In this study, primary porcine respiratory epithelial cells (PRECs) were cultured in an air-liquid interface (ALI) to differentiate into a pseudostratified columnar epithelium, proliferative basal cells, M cells, ciliated cells, and mucus-secreting goblet cells. ALI-PRECs recreates a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. PHEV replicated actively in this environment, inducing cytopathic changes and progressive disruption of the mucociliary apparatus. The innate immunity against PHEV was comparatively evaluated in ALI-PREC cultures and tracheal tissue sections derived from the same cesarean-derived, colostrum-deprived (CDCD) neonatal donor pigs. Increased expression levels of TLR3 and/or TLR7, RIG1, and MyD88 genes were detected in response to infection, resulting in the transcriptional upregulation of IFN-λ1 in both ALI-PREC cultures and tracheal epithelia. IFN-λ1 triggered the upregulation of the transcription factor STAT1, which in turn induced the expression of the antiviral IFN-stimulated genes OAS1 and Mx1. No significant modulation of the major proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α) was detected in response to PHEV infection. However, a significant upregulation of different chemokines was observed in ALI-PREC cultures (CCL2, CCL5, CXCL8, and CXCL10) and tracheal epithelium (CXCL8 and CXCL10). This study shed light on the molecular mechanisms driving the innate immune response to PHEV at the airway epithelium, underscoring the important role of respiratory epithelial cells in the maintenance of respiratory homeostasis and on the initiation, resolution, and outcome of the infectious process. IMPORTANCE The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) primarily infects and replicates in the swine upper respiratory tract, causing vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study investigated the modulation of key early innate immune genes at the respiratory epithelia in vivo, on tracheal tissue sections from experimentally infected pigs, and in vitro, on air-liquid interface porcine respiratory cell cultures. The results from the study underscore the important role of respiratory epithelial cells in maintaining respiratory homeostasis and on the initiation, resolution, and outcome of the PHEV infectious process.

Infection and immune response to porcine hemagglutinating encephalomyelitis virus in grower pigs.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is the cause of acute outbreaks of vomiting and wasting disease and/or encephalomyelitis in neonatal pigs, with naïve herds particularly vulnerable to clinical episodes. PHEV infections in older pigs are generally considered to be subclinical, but are poorly characterized in the refereed literature. In this study, twelve 7-week-old pigs were oronasally inoculated with 0.5 mL (1:128 HA titer) PHEV (Mengeling strain) and then followed through 42 days post inoculation (dpi). Fecal and oral fluid specimens were collected daily to evaluate viral shedding. Serum samples were tested for viremia, isotype-specific antibody responses, cytokine, and chemokine responses. Peripheral blood mononuclear cells were isolated to evaluate phenotype changes in immune cell subpopulations. No clinical signs were observed in PHEV inoculated pigs, but virus was detected in oral fluid (1-28 dpi) and feces (1-10 dpi). No viremia was detected, but a significant IFN-α response was observed in serum at 3 dpi, followed by the detection of IgM (dpi 7), and IgA/IgG (dpi 10). Flow cytometry revealed a one-off increase in cytotoxic T cells at 21 dpi. This study demonstrated that exposure of grower pigs to PHEV results in subclinical infection characterized by active viral replication and shedding followed by an active humoral and cell-mediated immune response that attenuates the course of the infection and results in viral clearance.

Characterization of the Humoral Immune Response to Porcine Epidemic Diarrhea Virus Infection under Experimental and Field Conditions Using an AlphaLISA Platform.

Coronavirus infections are a continuous threat raised time and again. With the recent emergence of novel virulent strains, these viruses can have a large impact on human and animal health. Porcine epidemic diarrhea (PED) is considered to be a reemerging pig disease caused by the enteropathogenic alphacoronavirus PED virus (PEDV). In the absence of effective vaccines, infection prevention and control through diagnostic testing and quarantine are critical. Early detection and differential diagnosis of PEDV infections increase the chance of successful control of the disease. Therefore, there is a continuous need for development of reduced assay-step protocols, no-wash, high-throughput immunoassays. This study described the characterization of the humoral immune response against PEDV under experimental and field conditions using a rapid, sensitive, luminescent proximity homogenous assay (AlphaLISA). PEDV IgG and IgA antibodies were developed toward the beginning of the second week of infection. PEDV IgG antibodies were detected for at least 16 weeks post-exposure. Remarkably, the serum IgA levels remained high and relatively stable throughout the study, lasting longer than the serum IgG response. Overall, AlphaLISA allows the detection and characterization of pathogen-specific antibodies with new speed, sensitivity, and simplicity of use. Particularly, the bridge assay constitutes a rapid diagnostic that substantially improves upon the "time to result" metric of currently available immunoassays.

Detecting and Monitoring Porcine Hemagglutinating Encephalomyelitis Virus, an Underresearched Betacoronavirus.

Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

Porcine Hemagglutinating Encephalomyelitis Virus: A Review.

The porcine hemagglutinating encephalomyelitis virus (PHEV) is classified as a member of genus Betacoronavirus, family Coronaviridae, sub-family Cornavirinae, and order Nidovirales. PHEV shares the same genomic organization, replication strategy, and expression of viral proteins as other nidoviruses. PHEV produces vomiting and wasting disease (VWD) and/or encephalomyelitis, being the only known neurotropic coronavirus affecting pigs. First clinical outbreak was reported in 1957 in Ontario, Canada. Although pigs are the only species susceptible to natural PHEV infections, the virus displays neurotropism in mice and Wistar rats. Clinical disease, morbidity, and mortality is age-dependent and generally reported only in piglets under 4 weeks old. The primary site of replication of PHEV in pigs is the respiratory tract, and it can be further spread to the central nervous system through the peripheral nervous system via different pathways. The diagnosis of PHEV can be made using a combination of direct and indirect detection methods. The virus can be isolated from different tissues within the acute phase of the clinical signs using primary and secondary pig-derived cell lines. PHEV agglutinates the erythrocytes of mice, rats, chickens, and several other animals. PCR-based methods are useful to identify and subsequently isolate animals that are actively shedding the virus. The ability to detect antibodies allows producers to know the status of first-litter gilts and evaluate their risk of tier offspring to infection. PHEV is highly prevalent and circulates subclinically in most swine herds worldwide. PHEV-related disease is not clinically relevant in most of the swine-producing countries, most likely because of dams are immune to PHEV which may confer passive immunity to their offspring. However, PHEV should be considered a major source of economic loss because of the high mortality on farms with high gilt replacement rates, specific pathogen-free animals, and gnotobiotic swine herds. Thus, in the absence of current PHEV vaccines, promoting virus circulation on farms with early exposure to gilts and young sows could induce maternal immunity and prevent disease in piglets.