This study analyzed Porcine epidemic diarrhea virus (PEDV) spike protein sequences and structures in Thailand from 2008 to 2024 to provide predicted structural templates that could inform regional vaccine selection and planned exposure frameworks. Using an in-silico approach, the researchers reduced sequence redundancy via CD-HIT (v4.8.1), established evolutionary lineages with BEAST (v1.10.4), and reconstructed protein structures using SWISS-MODEL. Structural comparisons and clustering were performed using DALI Z-scores and DBSCAN (v1.2.2), while Discotope 3 (v3.0) and ElliPro mapped B-cell epitope landscapes against a G1 reference strain. The results revealed a major lineage shift from G2a to G2b strains around 2017, with the spike proteins categorized into 14 subtypes and 6 eigenvalue clusters. Notably, minor amino acid substitutions altered properties such as hydrophobicity without disrupting the core structure, and certain deletions caused minimal structural deviations, indicating that sequence data or predicted structures alone do not fully dictate viral virulence or immunogenicity. Furthermore, primitive TH2 strains shared evolutionary links with G1 or US-InDel strains despite their G2 classification, identifying Cluster 1 as a potential ancestral structural type. In conclusion, this updated analysis provides crucial baseline data to optimize regional PEDV preventative measures, though further rigorous structural investigations are needed to definitively link specific spike alterations to virulence and host immune response.

Recombinant DNA vaccines offer significant potential for disease prevention and therapy, but their clinical success is often limited by poor immunogenicity, low cellular uptake, instability, and inefficient delivery without proper carriers. To address these challenges, we developed a series of novel cationic delivery systems by modifying branched polyethyleneimine (PEI, 25 kDa) with biocompatible carboxylic acids either lactic acid or glycolic acid and incorporating choline-based ionic liquids (choline glycolate [CG] and choline lactate [CL]) to create advanced mPEI/ionic liquid (IL) formulations. These systems were designed to enhance DNA complexation, protect against enzymatic degradation, improve nanoparticle stability, and fine-tune physicochemical properties for optimal cellular interaction. The resulting polyplexes formed stable nanoparticles with diameters 100 -125 nm and surface charges of +24 to +29 mV, supporting efficient cellular uptake. Compared to unmodified PEI, the modified formulations showed markedly reduced cytotoxicity and significantly improved transfection performance. Among all tested combinations, the combination of lactic acid-modified PEI with choline glycolate (LA-mPEI + CG) was the most effective, achieving a 76% increase in transfection efficiency, a 66% improvement in cellular uptake, and 66% enhanced in cell viability. These findings highlight the synergistic advantage of combining carboxylic acid modification PEI with ionic liquid incorporation, providing a promising strategy for safer and more effective non-viral DNA delivery. This platform may serve as a foundation for future advancements in gene therapy and DNA vaccine development.

The US poultry industry suffers significant economic losses due to Avian Reovirus (ARV) infections, which mainly cause arthritis/tenosynovitis in turkeys and chickens. The emergence of outbreaks since 2012 highlights the urgent need for improved epidemiological tools. Given the distinct evolutionary history of each segment of the virus and limited resolution of existing typing methods for ARV based on a single gene, a novel genotyping scheme was developed utilizing a constellation-based genotyping approach to enhance source tracing and control strategies especially for ARV in turkeys. A dataset of 199 ARV sequences from turkey hosts was curated and organized based on branch distances from maximum likelihood phylogenetic trees using TreeCluster. The grouping performance was evaluated and optimized according to established criteria described in this study. The proposed methods selected the M2, S1 σC-encoding region, and L3 genomic segments due to their non-random reassortment and biological significance. The novel scheme identified 8 major genotypes and revealed clear epidemiological links between turkey breeder and meat-type farms, as well as common shared sources among different meat-type farms, suggesting both vertical and horizontal transmission pathways. Additionally, reassortment events were detected using our novel typing scheme, highlighting the complex evolutionary dynamics of ARV. By correlating genotypic patterns with epidemiological data, this study provides a foundation for improved ARV monitoring and disease management.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus Betacoronavirus, known for its impact on the central and peripheral nervous systems in pigs. Traditionally associated with vomiting and wasting disease (VWD) and encephalomyelitis, PHEV was first reported in Canada in the late 1950s and has since been identified in numerous countries. Although serologic studies indicate global dissemination, the prevalence of PHEV remains unclear due to sporadic reporting and lack of active surveillance. Neonatal pigs are particularly vulnerable, with outbreaks resulting in high morbidity and mortality. Histopathological findings typically include non-suppurative encephalomyelitis and lymphoplasmacytic perivascular cuffs, gliosis, and neuronal degeneration. Recent observations have suggested a potential role for PHEV in respiratory disease, a hypothesis prompted by cases of influenza-like symptoms in pigs in Michigan in 2015 and corroborated by subsequent reports. This study aims to explore this possibility through a combination of clinical outbreak analysis and retrospective investigation. PHEV was confirmed via qPCR in 83.33 % of pigs examined for respiratory disease, with histological lesions such as necrotizing bronchitis and bronchiolitis. In-situ hybridization (ISH) confirmed the presence of PHEV mRNA in respiratory epithelium, and immunohistochemical analysis revealed significant macrophage infiltration in affected lung. Phylogenetic analysis indicated that PHEV strains from respiratory cases cluster closely with historical respiratory strains, though distinct from neurologic strains. This genetic differentiation suggests possible phenotypic variation contributing to respiratory tropism. The retrospective study identified PHEV in 7.62 % of cases with necrotizing bronchitis or bronchiolitis, reinforcing the virus's potential role in respiratory disease. Notably, PHEV co-infection with other respiratory pathogens such as PRRSV was observed, suggesting it may contribute to the porcine respiratory disease complex (PRDC). These findings suggest that PHEV is a significant respiratory pathogen in swine, warranting its inclusion in the differential diagnosis for respiratory disease in nursery pigs. Future research should focus on elucidating the pathogenesis of PHEV in respiratory disease, host-virus interactions, and the virus's impact on immune response and secondary infections. Understanding these factors will be crucial in developing effective preventive and therapeutic strategies against PHEV in swine.

Avian reovirus (ARV) has emerged as an important pathogen in turkeys, causing economic losses through tenosynovitis, necrotizing hepatitis, immunosuppression, and enteric disease. Despite its ubiquity, the evolutionary history of ARV cross-species transmission among chickens, turkeys, and wild birds remains poorly understood, hindering effective control and surveillance. This study investigates ARV temporal phylogenetics with an emphasis on interspecies transmission in turkeys. Whole genome sequences (WGSs) from seventy-seven turkey cases and one quail case at the Iowa State University Veterinary Diagnostic Laboratory, along with 74–136 segment sequences per gene from GenBank (1970–2023), were analyzed. Temporal phylogenetic analyses identified chickens as the ancestral host, with spillover into turkeys beginning in the mid-20th century, followed by stable transmission within turkey populations. Migration analyses revealed predominantly unidirectional transmission from chickens to turkeys. WGS analyses showed high variability in the M2 and σC-encoding region of the S1 segment, suggesting selective pressure on outer capsid proteins. M2, S1 σC, and L3 had the highest substitution rates, implicating their role in adaptation and antigenic diversity. These findings highlight the complexity of ARV evolution across hosts and underscore the need for robust genotyping schemes and surveillance strategies to mitigate outbreaks in poultry.

Torque Teno viruses (TTVs) are ubiquitous, small DNA viruses which are highly epidemiologically associated with respiratory infections, hepatitis, neurological disease and autoimmune disorders in humans and animals. Swine TTVs (TTSuVs) can be considered opportunistic pathogens as they exacerbate clinical signs due to coinfecting agents. While further understanding of how TTVs contribute to disease is crucial, there is a notable lack of animal models and tools to study the in vivo infection patterns of TTV. RNA in situ hybridization (RNA-ISH) with multiple probe amplification has recently gained popularity due to its high levels of specificity and sensitivity and ability to detect agent specific RNA or mRNA. Currently there are no commercial TTSuV1 antibodies that allow viral antigen detection by immunohistochemistry assay that can be used to advance the understanding of TTSuV1 pathogenicity. Therefore, the goal of this study was to develop an RNA-ISH assay for TTSuV1. To generate positive control, PK-15 cells grown in chamber slides were either infected with TTSuV1 or transfected with the TTSuV1 genome. A cocktail of TTSuV1 ORF1-specific RNA probes was hybridized to the cells, and specific binding was successfully visualized using a chromogenic reaction. Liver, kidney, heart, spleen and intestines were collected from mice infected with TTSuV1 at 15- and 30-days post infection. Finally, the RNA-ISH was optimized for TTSuV1 mRNA detection in tissues. TTSuV1-specific signal was detected in the hepatocytes and renal tubular epithelium of infected mice at a detection rate of 33 % 15- and 30-days post infection. In summary, the described RNA ISH assay is a useful tool to visualizeTTSuV1 viral replication in tissues and has potential application to clinical specimens in the future.

Parainfluenza viruses are a common cause of respiratory illness in many species. In this study, experimental, alphavirus-derived RNA particle vaccines either with or without adjuvant were evaluated against porcine parainfluenza virus 1 (PPIV1) challenge and compared to live virus exposure. Groups of ten, three-week-old pigs were vaccinated intramuscularly with an adjuvanted RNA particle (RPAdj/C) or non-adjuvanted RP (RP/C) or administered an intranasal live exposure (LE/C) dose of PPIV1 at 0- and 21-days post vaccination (DPV) followed by challenge with PPIV1 at 40 DPV. In addition, two groups were included as non-vaccinated, non-challenged (NV/NC) and non-vaccinated, challenged (NV/C) controls. Intranasal virus exposure and RP vaccination, regardless of adjuvant, reduced PPIV1 shedding in nasal swabs by 5 days post inoculation (DPI). All vaccinated or exposed pigs seroconverted as shown by enzyme-linked immunosorbent assay and serum virus neutralization. The antibody isotype detected in bronchoalveolar lavage fluid (BALF) LE/C was predominantly IgA while RP vaccination induced an IgG response. Reduced PPIV1 antigen was observed in the LE/C, RP/C and RPAdj/C groups in lung, trachea, or nasal turbinate epithelium. Additionally, the RPAdj vaccine significantly reduced nasal shedding compared to NV/C pigs although not as much as LE/C pigs. These results suggest vaccination could mitigate PPIV1 infection in commercial systems.

IntroductionOrganoids are 3-dimensional (3D) stem cell-derived cultures that offer a variety of technical advantages compared to traditional 2-dimensional (2D) cell cultures. Although murine models have proved useful in biomedical research, rodent models often fail to adequately mimic human physiology and disease progression, resulting in poor preclinical prediction of therapeutic drug efficacy and toxicity. An interesting alternative is to use the canine model in research, due to its numerous similarities to humans (shared environment, intact immune system, and development of civilization diseases). The use of canine organoids in drug testing and disease modeling has been limited by the number of models as well as the depth of characterization. Therefore, we believe these types of models can expedite drug testing and create a platform for personalized medicine.MethodsHere, we report the establishment, maintenance, and molecular characterization of six adult-stem cell-derived canine organoid cell lines including endometrium, pancreas, urinary bladder, kidney, lung, and liver from two genetically related canines (B816 and B818). Characterization of these lines was done using multiple techniques including immunohistochemistry (UPKIII, TTF-1) and bulk RNA-seq. Furthermore, scRNA-seq was utilized on a subset of the organoids to identify organoid specific transcriptomic signatures including lung, pancreas, kidney, and bladder.ResultsIn total, six tissues and organoid lines from each donor were characterized, allowing for a unique, multi-organ comparison between these two individuals and identification of specific cell types within the organoids. Bulk RNA-seq revealed tissue-specific transcriptomic profiles, with organoids enriched in proliferation-related genes and tissues enriched in inflammation-related genes. Principal component analysis showed organ-based clustering, while scRNA-seq identified diverse epithelial subtypes.ConclusionThese organoids begin to establish a platform for reverse translational research, reducing reliance on live animal testing. By leveraging genetically related donors, it highlights tissue-specific variations, facilitating applications in personalized medicine, disease modeling, and pharmacology to bridge veterinary and human research gaps.

Porcine circovirus type 3 (PCV3) was identified in 2016 and has since been associated with reproductive failure, multisystemic inflammation, and subclinical infection in swine. Numerous countries have retrospectively detected the presence of PCV3 before its first clinical description in 2016. The reported detection rate of PCV3 has varied from 6.5 to 84 % in pigs with various coinfections. Today, PCV3/PCV2 coinfection is commonly observed. However, the PCV3 prevalence and coinfection rate with PCV2 in the US swine industry had not been reported before 2016. The present study used serum samples from US grower finisher farms from 2000, 2006, and 2012 to determine the PCV3 and PCV3/PCV2 farm prevalence and geographical distribution, to evaluate the PCV3 evolutionary rate and selection pressure forces, and to identify structural and morphological changes in the Cap protein. Our findings revealed that PCV3 was endemic in the US swine industry before its first description in 2016. The PCV3 farm prevalence decreased from 47 % in 2000 to 22 % in 2012. The PCV3/PCV2 coinfection rate at the farm level was 47 % in 2000 and 39 % in 2006. After the introduction of PCV2 vaccines in 2006, the PCV3/PCV2 coinfection rate drastically declined to 3 % and 59 % of farms were negative for both PCV3 and PCV2 in 2012. From 13 PCV3 whole genome sequences, 12 sequences were clustered with PCV3a reference strain and one with the PCV3c subtype. From 28 PCV3 ORF2 sequences, PCV3a1 (1/28), PCV3a2 (4/28), PCV3a3 (19/28), PCV3b (2/28), and PCV3c (2/28) subtypes were obtained. ORF2 nucleotide identity ranged from 97.8 to 100 %. Diversifying selection occurred at amino acids 24 and 150 in 2006 and at amino acids 24 and 27 in 2012. Mutations A24V and R27K were common among all PCV3 subtypes in sequences identified in the present study and in reference sequences. Both the S77T and I150L mutation was common among sequences within the PCV3a2 subtype. The F104Y mutation lies within a predicted T-cell epitope and was present in sequences of the PCV3c, PCV3b, and PCV3a3 subtypes. Cap molecular modeling revealed that the structural folding of amino acids 24 and 27 changed from alpha helix to coiled in 2012 sequences. Thus, this study broadens current knowledge of PCV3's prevalence and molecular evolution in the US swine herd from 2000 to 2012.

While the vast majority of the US swine population is concentrated in 5 states (Iowa, Minnesota, North Carolina, Illinois, and Indiana), the rest is spread out across the US including pork production, hobby farms, pet pigs, and research facilities. The number of novel diseases and laboratory tests that have been established in recent years can be intimidating or overwhelming for practitioners who do not routinely work with this species. This review aims to help clinicians across the country that may not have an in-depth experience in swine medicine become more familiar with both common and novel pathogens, formulate a differential diagnosis based on the age of the animals and affected system (eg, respiratory, systemic, nervous, and enteric), select proper samples and laboratory testing, and interpret laboratory data to achieve a disease diagnosis in porcine patients.

This study was conducted to evaluate cellular and humoral mucosal immune responses of pigs infected with porcine epidemic diarrhea virus (PEDV) genogroups 1 (G1) or 2 (G2). Anamnestic response following homologous or heterologous reinfection were also investigated. Forty-five PEDV-negative, 3-week-old pigs were allocated into 3 groups of 15 pigs each. Pigs were orally inoculated with EAS1 (G1) or CBR1 (G2) or cell culture supernatant and serially monitored for PEDV-specific IFN-γ producing cells (IFN-γ PC) and IgA antibody secreting cells (ASC). Three pigs served as baseline at day 0. The CD4+IFN-γ+ PC in G1 group was detected at 3 days post infection (dpi) in both EAS1 and CBR1 recall antigen. In contrast, CD4+IFN-γ+ PC in G2 group detected only when EAS1 was used. Similar findings were found with CD8+IFN-γ+ PC. After reinfection, only G2 exhibited a booster effect of CD4+IFN-γ+ and CD8+IFN-γ+ cells by heterologous antigen recall. Regarding CD4+CD8+IFN-γ+cells, G1 showed significantly higher levels at 3 dpi and demonstrated a secondary boost at 14 dpi following heterologous recall stimulation. In contrast, G2 showed a slight increase in response to both homologous and heterologous recall at 3 dpi. PEDV-specific IgA ASC were detected at 14 dpi. However, anamnestic response after reinfection was variant-dependent. Our results indicate that the G1 variant specific cellular response is triggered earlier or simultaneously than G2 variant. Meanwhile, the timing of humoral response was similar, and anamnestic response was driven by the variant involved during reinfection.

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.

. In 2022, a new epornitic of H5N1 highly pathogenic avian influenza (HPAI) virus clade 2.3.4.4b emerged in U.S. domestic poultry with high prevalence in wild bird populations. We describe pathological fi ndings of HPAI H5N1 in nine wild birds encompassing eight different species, including Accipitriformes (red-tailed hawk, bald eagle), Cathartiforme (turkey vulture), Falconiforme (peregrine falcon), Strigiforme (one adult great-horned owl, one juvenile great-horned owl), Pelecaniforme (American white pelican), and Anseriformes (American green-winged teal, trumpeter swan). All these birds died naturally (found dead, or died in transit to or within a rehabilitation center), except for the bald eagle and American green-winged teal, which were euthanized. Gross lesions were subtle, characterized by meningeal congestion observed in the turkey vulture, bald eagle, and adult great-horned owl. Histologically, encephalitis was observed in all cases (9/9, 100%). Leukocytoclastic and fi brinoid vasculitis with necrotizing encephalitis was observed in the red-tailed hawk, great-horned owls, and American white pelican (5/9, 55.6%), and perivascular lymphohistiocytic encephalitis was seen in the turkey vulture, peregrine falcon, green-winged teal, and bald eagle (4/9, 44.4%). Coagulative necrosis or lymphohistiocytic/lymphoplasmacytic inflammation was identified in the kidney (6/8, 75%), liver (6/9, 66.7%), heart (5/9, 55.6%), and lung (2/9, 22.2%). Immunopositive signals against Influenza virus A nucleoprotein were predominantly detected within the brain (9/9, 100%), air sac (7/9, 77.8%), lung (7/9, 77.8%), kidney (6/8, 75%), heart (6/9, 66.7%), and liver (5/9, 55.6%). Additionally, other organs, such as the pancreas, spleen, intestines, gonads, and adrenals occasionally exhibited positive viral protein signals. In these organs, in addition to parenchymal cells, viral protein signals were often identified in endothelial cells. Our results suggest that the 2022-2023 HPAIV H5N1 clade 2.3.4.4b replicated systemically in all examined birds, with brain lesions being the most prevalent and associated with a subset of birds displaying clinical signs observed perimortem.

Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions.Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant.Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.

Pigs are affected by various parvoviruses (PPVs); eight have been reported to date (PPV1-PPV8). Porcine parvovirus 1 is considered a primary agent of porcine reproductive failure (PRF), while it is unknown whether other PPVs impact porcine health. Recently, the presence of PPV2 has been confirmed in the lung, either as a single agent or in the form of coinfection with other respiratory; therefore, it has been proposed as a potential participant in the porcine respiratory disease complex (PRDC). In the present study, the presence of PPV2 alone and coinfection with other viruses (PCV2, PCV3, and PRRSV) was evaluated in lung samples obtained from pigs with respiratory signs (respiratory group: RG) (n = 146) and stillborn lungs (stillborn group: SG) (n = 19) from 82 farms in the five regions with the highest swine production in Colombia. The overall PPV2 prevalence was 37.6% (62/165), with the highest proportion mainly detected in grow-finisher pigs (62.5%), while its herd prevalence was 51.2% (42/82). The most prevalent virus was PRRSV in both groups, while PPV2 alone was found only in the RG group. The most common dual coinfection in the RG and SG was PCV2/PRRSV (17.8% and 10.5%), while the most frequent coinfections involving PPV2 in the RG were PPV2/PCV2 (7.5%) and PPV2/PRRSV (4%) and PPV2/PCV2 (5.3%) in the SG. The most common triple coinfection was PPV2/PCV2/PRRSV at 15% in the RG and 21% in the SG, while quadruple coinfection PVV2/PCV2/PCV3/PRRSV was detected only in the RG (5.5%). Histopathological evaluation of 21 PPV2-positive lungs showed variable degrees of histiocytic or lymphohistiocytic interstitial pneumonia (9%) in the RG, while no significant changes were observed in SG; in addition, neutrophilic bronchopneumonia was observed in 73.7% if cases evaluated. In situ hybridization-RNAScope® confirmed the presence of PPV2 within pulmonary lesions in 2/19 RG pigs, while no in situ detection was observed in the SG pigs. The phylogenetic evaluation of seven PPV2 sequences detected in Colombia was compared with another 102 reported sequences, indicating that the Colombian strains are located in clade 2. Our results confirm the presence of PPV2 in pigs with PRDC alone and pigs coinfected with PCV2, PCV3, and PRRSV. Likewise, its presence alone or in coinfection in stillbirths suggests that PPV2 is also involved in PRF.