Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model.

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.

Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations.

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research.

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory.

BACKGROUND: Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. RESULTS: S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. CONCLUSIONS: Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

Genetic and In Vitro Characteristics of a Porcine Circovirus Type 3 Isolate from Northeast China.

Porcine circovirus 3 (PCV3) is an emerging virus first discovered in the United States in 2015, and since then, PCV3 has been found in many regions of the world, including America, Asia, and Europe. Although several PCV3 investigations have been carried out, there is a lack of knowledge regarding the pathogenicity of PCV3, mostly due to the limited number of PCV3 isolates that are readily available. In this study, PCV3-DB-1 was isolated in PK-15 cells and characterized in vitro. Electron microscopy revealed the presence of PCV-like particles, and in situ hybridization RNA analysis demonstrated the replication of PCV3 in PK-15 cell culture. Based on phylogenetic analysis of PCV3 isolates from the Heilongjiang province of China, PCV3-DB-1 with 24 alanine and 27 lysine in the Cap protein was originally isolated and determined to belong to the clade PCV3a.

Pathogenesis of an experimental coinfection of porcine parainfluenza virus 1 and influenza A virus in commercial nursery swine.

Porcine parainfluenza virus 1 (PPIV-1) is a recently characterized swine respirovirus. Previous experimental studies reported PPIV-1 replicates in the porcine respiratory tract causing minimal clinical disease or lesions. However, it is unknown if PPIV-1 co-infections with viral respiratory pathogens would cause respiratory disease consistent with natural infections reported in the field. The objective of this study was to evaluate if PPIV-1 increases the severity of influenza A virus respiratory disease in swine. Fifty conventional, five-week-old pigs were assigned to one of three challenge groups (n = 15) or a negative control group (n = 5). Pigs were challenged with a γ-cluster H1N2 influenza A virus in swine (IAV-S; A/Swine/North Carolina/00169/2006), PPIV-1 (USA/MN25890NS/2016), inoculum that contained equivalent titers of IAV-S and PPIV-1 (CO-IN), or negative control. Clinical scores representing respiratory disease and nasal swabs were collected daily and all pigs were necropsied five days post inoculation (DPI). The CO-IN group demonstrated a significantly lower percentage of pigs showing respiratory clinical signs relative to the IAV-S challenge group from 2 to 4 DPI. The IAV-S and CO-IN groups had significantly lower microscopic composite lesion scores in the upper respiratory tract compared to the PPIV-1 group although the IAV-S and CO-IN groups had significantly higher microscopic composite lung lesion scores. Collectively, PPIV-1 did not appear to influence severity of clinical disease, macroscopic lesions, or alter viral loads detected in nasal swabs or necropsy tissues when administered as a coinfection with IAV-S. Studies evaluating PPIV-1 coinfections with different strains of IAV-S, different respiratory pathogens or sequential exposure of PPIV-1 and IAV-S are warranted.

Diagnostic investigation of Mycoplasma hyorhinis as a potential pathogen associated with neurological clinical signs and central nervous system lesions in pigs.

Mycoplasma hyorhinis (M. hyorhinis) is a commensal of the upper respiratory tract in swine with the typical clinical presentations of arthritis and polyserositis in postweaning pigs. However, it has also been associated with conjunctivitis and otitis media, and recently has been isolated from meningeal swabs and/or cerebrospinal fluid of piglets with neurological signs. The objective of this study is to evaluate the role of M. hyorhinis as a potential pathogen associated with neurological clinical signs and central nervous system lesions in pigs. The presence of M. hyorhinis was evaluated in a clinical outbreak and a six-year retrospective study by qPCR detection, bacteriological culture, in situ hybridization (RNAscope®), and phylogenetic analysis and with immunohistochemistry characterization of the inflammatory response associated with its infection. M. hyorhinis was confirmed by bacteriological culture and within central nervous system lesions by in situ hybridization on animals with neurological signs during the clinical outbreak. The isolates from the brain had close genetic similarities from those previously reported and isolated from eye, lung, or fibrin. Nevertheless, the retrospective study confirmed by qPCR the presence of M. hyorhinis in 9.9% of cases reported with neurological clinical signs and histological lesions of encephalitis or meningoencephalitis of unknown etiology. M. hyorhinis mRNA was confirmed within cerebrum, cerebellum, and choroid plexus lesions by in situ hybridization (RNAscope®) with a positive rate of 72.7%. Here we present strong evidence that M. hyorhinis should be included as a differential etiology in pigs with neurological signs and central nervous system inflammatory lesions.

Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

Co-infection of porcine deltacoronavirus and porcine epidemic diarrhea virus induces early TRAF6-mediated NF-κB and IRF7 signaling pathways through TLRs.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) infect the small intestine and cause swine enteric coronavirus disease. The mucosal innate immune system is the first line of defense against viral infection. The modulatory effect of PDCoV and PEDV coinfection on antiviral signaling cascades of the intestinal mucosa has not been reported. Here, we investigate the gene expression levels of pattern recognition receptors, downstream inflammatory signaling pathway molecules, and associated cytokines on the intestinal mucosa of neonatal piglets either infected with a single- or co-infected with PDCoV and PEDV using real-time PCR. The results demonstrate that single-PEDV regulates the noncanonical NF-κB signaling pathway through RIG-I regulation. In contrast, single-PDCoV and PDCoV/PEDV coinfection regulate proinflammatory and regulatory cytokines through TRAF6-mediated canonical NF-κB and IRF7 signaling pathways through TLRs. Although PDCoV/PEDV coinfection demonstrated an earlier modulatory effect in these signaling pathways, the regulation of proinflammatory and regulatory cytokines was observed simultaneously during single viral infection. These results suggested that PDCoV/PEDV coinfection may have synergistic effects that lead to enhanced viral evasion of the mucosal innate immune response.

Five years of porcine circovirus 3: What have we learned about the clinical disease, immune pathogenesis, and diagnosis.

Porcine circovirus type 3 (PCV3) is a non-enveloped, circular, single-stranded DNA virus in the family Circoviridae. This member of the genus Circovirus was initially described as affecting swine in 2016, and new research has provided further insight into its structural characteristics, disease presentations, pathogenesis, and immune response following infection. Therefore, this review aims to summarize advances in PCV3-related research about genomic characteristics epidemiology, pathogenesis, immune response, and the development of diagnostics. PCV3 has been detected globally and retrospectively in pigs of all ages and is associated with a range of clinical presentations, including multisystemic inflammatory syndrome, reproductive failure, porcine dermatitis and nephropathy syndrome, and subclinical infection. Experimental studies have successfully reproduced multisystemic inflammation but have not detected clinical disease. These findings, coupled with a large number of reports of coinfections coinciding with PCV3, may suggest that PCV3 infection alone may not be sufficient to cause evidenceable clinical disease. The pathogenesis of PCV3 has not been fully elucidated yet, and while receptors that facilitate cell-viral entry have not been identified, replication has been confirmed in a wide range of cell types, including trophoblasts, myocardiocytes, skin adipocytes, and neurons. PCV3 seems to evade the host immune response as evidenced by persistent viremia 42 days post-infection in experimental and longitudinal field studies despite a strong humoral response. Minimal differences in host cytokine profiles and peripheral cell-mediated responses have been observed, but certainly many questions still surround the mechanisms by which PCV3 evades the immune response. The epidemiology of PCV3 remains unclear, and the exact routes of transmission have not been described; but, PCV3 can be shed in oral fluids, nasal secretions, feces, colostrum, and semen, demonstrating the importance of lateral and vertical transmission. The detection of PCV3 in numerous domesticated and wild animal species, including cattle, dogs, mice, wild boar, chamois, roe deer, ticks, and mosquitoes, suggests the potential for multiple reservoirs and cross-species transmission. Current advances in PCV3 diagnostic tests have the ability to differentiate PCV3 from other PCVs and corroborate its presence within lesions. Given that the economic impact associated with PCV3 infection has not been assessed and the virus has the potential to emerge as a high-prevalence pathogen in the coming years, future research should focus on filling the knowledge gaps identified in this review.

Detection of porcine parainfluenza virus type-1 antibody in swine serum using whole-virus ELISA, indirect fluorescence antibody and virus neutralizing assays.

BACKGROUND: Porcine parainfluenza virus 1 (PPIV-1) is a respiratory virus in the family Paramyxoviridae and genus Respirovirus. It is closely related to bovine parainfluenza virus 3, human parainfluenza virus 1, and Sendai virus. Recent reports suggest PPIV-1 is widespread in swine herds in the United States and abroad. However, seroprevalence studies and the ability to evaluate cross neutralization between heterologous strains is not possible without validated antibody assays. This study describes the development of an indirect fluorescence antibody (IFA) assay, a whole virus enzyme-linked immunosorbent assay (wv-ELISA) and a serum virus neutralization (SVN) assay for the detection of PPIV-1 antibodies using 521 serum samples collected from three longitudinal studies and two different challenge strains in swine. RESULTS: The area under the curve (AUC) of the wv-ELISA (95% CI, 0.93-0.98) was significantly higher (p = 0.03) compared to the IFA (95% CI, 0.90-0.96). However, no significant difference was observed between the IFA and wv-ELISA when compared to the SVN (95% CI, 0.92-0.97). All three assays demonstrated relatively uniform results at a 99% true negative rate, with only 11 disagreements observed between the IFA, wv-ELISA and SVN. CONCLUSIONS: All three serology assays detected PPIV-1 antibody in swine serum of known status that was collected from experimental studies. The SVN detected seroconversion earlier compared to the IFA and the wv-ELISA. Both the wv-ELISA and the SVN had similar diagnostic performance, while the IFA was not as sensitive as the wv-ELISA. All three assays are considered valid for routine diagnostic use. These assays will be important for future studies to screen seronegative swine for research, determine PPIV-1 seroprevalence, and to evaluate vaccine efficacy against PPIV-1 under experimental and field conditions.

Streptococcus gallolyticus and Bacterial Endocarditis in Swine, United States, 2015-2020.

To evaluate trends in bacterial causes of valvular endocarditis in swine, we retrospectively analyzed 321 cases diagnosed at Iowa State University Veterinary Diagnostic Laboratory (Ames, IA, USA) during May 2015--April 2020. Streptococcus gallolyticus was the causative agent for 7.59% of cases. This emerging infection in swine could aid study of endocarditis in humans.

Characterization of Two Porcine Parainfluenza Virus 1 Isolates and Human Parainfluenza Virus 1 Infection in Weaned Nursery Pigs.

Porcine parainfluenza virus 1 (PPIV1) is a newly characterized porcine respiratory virus. Recent experimental challenge studies in three-week-old nursery pigs failed to cause disease. However, it remains unclear how genetic differences contribute to viral pathogenesis. To characterize the pathogenesis of different PPIV1 isolates, three-week-old nursery pigs were challenged with either PPIV1 isolate USA/MN25890NS/2016 (MN16) or USA/IA84915LG/2017 (IA17). A human parainfluenza virus 1 (HPIV1) strain C35 ATCC® VR-94™ was included to evaluate swine as a model for human parainfluenza. All viruses were successfully re-isolated from bronchoalveolar lavage fluid and detected by RT-qPCR at necropsy. Microscopic lung lesions were more severe in the IA17 group compared to the non-challenged negative control (Ctrl) group whereas differences were not found between the MN16 and Ctrl groups. Immunohistochemistry staining in respiratory samples showed a consistent trend of higher levels of PPIV1 signal in the IA17 group followed by the MN16 group, and no PPIV1 signal observed in the HPIV1 or Ctrl groups. This study suggests potential pathogenesis differences between PPIV1 isolates. Additionally, these results indicate that HPIV1 is capable of replicating in nursery pigs after experimental inoculation. However, clinical disease or gross lung lesions were not observed in any of the challenge groups.

Protective Efficacy of an Orf Virus-Vector Encoding the Hemagglutinin and the Nucleoprotein of Influenza A Virus in Swine.

Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses challenges to public health given its zoonotic potential. Thus effective IAV-S vaccines are needed and highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs, with higher levels of neutralizing antibodies (NA) being detected in OV-HA-NP-immunized animals pre-challenge infection. Although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S-specific proliferating CD8+ T cells upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in incremental protection against challenge infection as evidenced by a reduced secondary antibody response (NA and HI antibodies) following IAV-S challenge and reduced virus shedding in nasal secretions (lower viral RNA loads and frequency of animals shedding viral RNA and infectious virus), when compared to animals in the OV-HA group. Interestingly, broader cross neutralization activity was also observed in serum of OV-HA-NP-immunized animals against a panel of contemporary IAV-S isolates representing the major genetic clades circulating in swine. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine.

Pathogenesis of a novel porcine parainfluenza virus type 1 isolate in conventional and colostrum deprived/caesarean derived pigs.

Two experimental challenge studies were conducted to evaluate the pathogenesis of a porcine parainfluenza virus type 1 (PPIV-1) isolate. Four-week-old conventional (CON) pigs were challenged in Study 1 and six-week-old caesarean derived/colostrum deprived (CDCD) pigs were challenged in Study 2. Results indicate that PPIV-1 shedding and replication occur in the upper and lower respiratory tracts of CON and CDCD pigs as detected by RT-qPCR and immunohistochemistry. Mild macroscopic lung lesions were observed in CON pigs but not in CDCD pigs. Microscopic lung lesions were mild and consisted of peribronchiolar lymphocytic cuffing and epithelial proliferation in CON and CDCD pigs. Serum neutralizing antibodies were detected in the CON and CDCD pigs by 14 and 7 days post inoculation, respectively. This study provides evidence that in spite of PPIV-1 infection and replication in challenged swine, significant clinical respiratory disease was not observed.

Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication.

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

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.

Coinfection of porcine deltacoronavirus and porcine epidemic diarrhea virus increases disease severity, cell trophism and earlier upregulation of IFN-α and IL12.

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause an enteric disease characterized by diarrhea clinically indistinguishable. Both viruses are simultaneously detected in clinical cases, but a study involving the co-infection has not been reported. The study was therefore conducted to investigate the disease severity following a co-infection with PEDV and PDCoV. In the study, 4-day-old pigs were orally inoculated with PEDV and PDCoV, either alone or in combination. Following challenge, fecal score was monitored on a daily basis. Fecal swabs were collected and assayed for the presence of viruses. Three pigs per group were necropsied at 3 and 5 days post inoculation (dpi). Microscopic lesions and villous height to crypt depth (VH:CD) ratio, together with the presence of PEDV and PDCoV antigens, were evaluated in small intestinal tissues. Expressions of interferon alpha (IFN-α) and interleukin 12 (IL12) were investigated in small intestinal mucosa. The findings indicated that coinoculation increased the disease severity, demonstrated by significantly prolonged fecal score and virus shedding and decreasing VH:CD ratio in the jejunum compared with pigs inoculated with either PEDV or PDCoV alone. Notably, in single-inoculated groups, PEDV and PDCoV antigens were detected only in villous enterocytes wile in the coinoculated group, PDCoV antigen was detected in both villous enterocytes and crypts. IFN-α and IL12 were significantly up-regulated in coinoculated groups in comparison with single-inoculated groups. In conclusion, co-infection with PEDV and PDCoV exacerbate clinical signs and have a synergetic on the regulatory effect inflammatory cytokines compared to a single infection with either virus.

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.