Multifaceted Effects and Mechanisms of N-Acetylcysteine on Intestinal Injury in a Porcine Epidemic Diarrhea Virus-Infected Porcine Model.

SCOPE: This study investigates the potential effects of N-acetylcysteine (NAC) on intestinal injury in a porcine epidemic diarrhea virus (PEDV)-infected porcine model. METHODS AND RESULTS: Thirty-two piglets are randomly assigned to one of four groups: the control, PEDV, NAC, and NAC+PEDV. Piglets in the NAC+PEDV group are orally administrated with NAC (100 mg (kg·BW)-1  day-1 ) for 4 consecutive days after 2 days of PEDV infection. The results show that NAC administration decreases the diarrhea rate and improves intestinal morphology. The concentration of diamine oxidase and intestinal fatty-acid binding protein, as well as IL-1ß, IL-8, and TNF-α in the plasma, is decreased by NAC. Intriguingly, NAC administration significantly increases the viral load in the jejunum and ileum and down-regulates the expression of interferon-related genes. Microarray and proteomic analyses show that the differentially expressed genes/proteins between NAC+PEDV and PEDV groups are highly enriched in substance transport. Furthermore, aquaporin 8/10 expression is significantly increased by NAC upon PEDV infection. CONCLUSION: NAC administration alleviates PEDV-induced intestinal injury by inhibiting inflammatory responses and improving substance transport, but promotes viral replication by inhibiting interferon signaling. These results suggest NAC exhibits multifaceted effects upon PEDV infection, and thus caution is required when using NAC as a dietary supplement to prevent viral infection.

Chemokines induced by PEDV infection and chemotactic effects on monocyte, T and B cells.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging pathogen that causes severe economic loss in the pig industry. The host's innate immune system is the first line of defense on virus invasion of the small intestinal epithelial cells. Chemokines, as a part of the innate immune system, play an important role in host immunity against infection, however, and their expression and chemotactic effect on key immune cells in PEDV infection remains unclear. In this study, cDNA microarray was firstly performed to analyzed ileum tissue of piglets on the third day after PEDV infection. The differentially expressed genes mainly involved in multiple biological processes, chemokine signaling pathway and cytokine receptor interaction signaling pathway had the highest enrichment according to GO and KEGG enrichment analysis. The expression levels of chemokines MCP-1, MIP-1ß, IL-8, CXCL9, CXCL10 and CXCL13 in ileum of PEDV- infected piglets were significantly higher than those in the control group. The expression of chemokines in vivo experiment was further verified by RT-qPCR and ELISA using PEDV-infected IPEC-J2 cells. The results showed that the PEDV-infected IPEC-J2 cells had significantly induced protein expression of MCP-1, MIP-1ß, IL-8, CXCL9, CXCL-10 and CXCL13. These results indicated that the changes of chemokines expressed in the ileum of piglets (in vivo) were consistent with those in IPEC-J2 cells (in vitro) after PEDV infection. Finally, the role of chemokines in immune cell migration during PEDV infection was illustrated by siRNA-mediated knock down method and the co-culture model of IPEC-J2 cells with peripheral blood leukocyte cells (PBLCs). The FACS analysis showed that MCP-1 induced by PEDV infection played a chemotactic effect on CD14+ cells, CXCL9 on CD3+CD4-CD8-γδ T, CD3+CD4-CD8+ Tc, CD3+CD4+CD8- Th and CD3+CD4+CD8+ Tm subsets, and CXCL13 on CD19+ B cells. Collectively, our findings first indicate that PEDV-induced chemokines MCP-1, CXCL-9 and CXCL-13 attracted CD14+ cells, T cells and B cells, respectively. These results provide a theoretical basis for studying the mechanism of anti-PEDV infection in piglets.

The dose-titration study of inactivated vaccine of porcine epidemic diarrhea virus

This study aims to investigate the protective effect of infected piglets which were immunized with different dose of inactivated porcine epidemic diarrhea virus (PEDV) vaccines. The number of infective virus particles and total virus particles of PEDV with different concentrations were determined, and the mice were immunized with different concentration vaccine prepared as antigen, respectively. The humoral and cellular immune production were determined by ELISA antibody detection method, neutralization test and ELISPOT method. Vaccine with appropriate antigen content was selected to immunize piglets, then the antibody was determined. The relationship between concentrated vaccine and protective effect was studied by challenge experiment. The results showed that, when the antigen dose was equal or greater than 8x106 pfu.mL-1, the inactive vaccine could effectively stimulate mice to produce humoral and cellular immunity. The piglets immunized with 2 mL inactivated PEDV vaccine containing 8x106 pfu.mL-1 antigen could resist diarrhea and continuous viral shedding caused by PEDV challenge. Compared with the total number of virus particles, the number of infectious virus particles was significantly correlated with antibody production (r=0.998 1), and neutralization titer was significantly correlated with piglet protection (r=0.974 7). PEDV inactivated vaccine can provide good immune protection, in which the number of infectious virus particles is the key factor to improve the antibody level. Antibody titer, as an index of humoral immunity, is an important reference for judging immune protection.

Comparison of pathogenicity of porcine deltacoronavirus CZ2020 from cell culture and intestinal contents in 27-day-old piglets.

Porcine deltacoronavirus (PDCoV) is an emenging swine enteropathogenic coronavirus that can cause high mortality rate. It affects pigs of all ages, but most several in neonatal piglets. Little is known regarding the pathogenicity of PDCoV against 27-day-old piglets. In this study, 27-day-old piglets were experimentally infected with PDCoV CZ2020 from cell culture, the challenged piglets do not have obvious symptoms from 1 to 7 days post-challenge (DPC), while viral shedding was detected in rectal swab at 1 DPC. Tissues of small intestines displayed slight macroscopic and microscopic lesions with no viral antigen detection. On the other hand, 27-day-old piglets were infected with PDCoV from intestinal contents, the piglets developed mild to severe diarrhea, shedding increasing from 2 to 7 DPC, and developed macroscopic and microscopic lesions in small intestines with clear viral antigen confirmed by immunohistochemistry staining. Indicating the small intestine was still the major target organ in PDCoV-challenged pigs at the age of 27-day-old. Diarrhea caused by PDCoV from intestinal contents in 27-day-old piglets is less reported. Thus, our results might provide new insights into the pathogenesis of PDCoV.

Effects of niacin on intestinal epithelial Barrier, intestinal Immunity, and microbial community in weaned piglets challenged by PDCoV.

The objective was to evaluate effects of niacin on the intestinal epithelial barrier, intestinal immunity, and microbial community in weaned piglets challenged by Porcine Deltacoronavirus (PDCoV). In this study, fifteen weaned piglets were randomly assigned to 1 of 3 groups, (1) control group, normal diet; (2) PDCoV group, infected with 1 × 107 TCID50 of the PDCoV CHN-HN-17 strain by oral administration; (3) NA + PDCoV group, infected with 1 × 107 TCID50 of the PDCoV CHN-HN-17 strain by oral administration following administration of 40 mg of niacin for three days. The results showed that PDCoV infection induced diarrhea and other clinical symptoms with intestinal villi shedding and atrophy in weaned piglets. Niacin alleviated the symptoms of diarrhea and intestinal damage of PDCoV-infected weaned piglets. Additionally, PDCoV increased (P < 0.05) the mRNA expression of tight junction proteins [zonula occludens-1 (ZO-1) and Claudin] and antimicrobial peptides [porcine ß defensin 1 (pBD1), pBD2, proline-arginine rich 39-amino acid peptide (PR39) and protegrin 1-5 (PG1-5) in the jejunum and ileum of weaned piglets, while niacin increased (P < 0.05) the expression of PG1-5 compared with PDCoV. PDCoV increased (P < 0.05) the contents of serum interleukin-1ß (IL-1ß), IL-8 and intestinal IL-8, and up-regulated the mRNA expression of tumor necrosis factor-α (TNF-α), IL-1ß, IL-6, IL-10, IL-12, and IL-18 in ileum of weaned piglets compared with control. However, niacin decreased (P < 0.05) the contents of serum IL-1ß, IL-6 and intestinal IL-10 and IL-8, and also reduced (P < 0.05) the mRNA expression of ileal TNF-α, IL-10 and IL-12 in the PDCoV-infected piglets. Compared with control, PDCoV up-regulated (P < 0.05) the mRNA expression of key genes related to innate immune and antiviral molecules [toll-like receptor 4 (TLR4), NOD1, NOD2, DDX58, CCL2, STAT2, Mx1, IFN-γ, and protein kinase R (PKR) in the ileum of weaned piglets. Niacin decreased (P < 0.05) the mRNA expression of NOD1, NOD2, STAT2, IFN-γ, and PKR in PDCoV-infected weaned piglets. Moreover, the mRNA expression of IL-6 decreased (P < 0.05) and 2'-5'-oligoadenylate synthetase (OAS), IFN-α, and PKR increased (P < 0.05) in PDCoV-infected IPEC-J2 cells treated with niacin in vitro. Furthermore, niacin decreased (P < 0.05) the elevation of protein expression including inducible NOS (iNOS), nuclear factor-κB (NF-κB p65), inhibitor kappa B (IKKß), histone deacetylase [Sirtuin 1 (SIRT1) and histone deacetylase 7 (HDAC7) and phosphorylation of histone H3 at serine s10 (pH3s10) in the ileum of PDCoV-infected piglets, and increased (P < 0.05) the expression of G protein-coupled receptor (GPR109A). PDCoV disrupted the composition and structure of microflora in the colon of weaned piglets, and reduced the relative abundance of the beneficial bacteria Spirobacterium, but niacin could improve the intestinal microbial flora of the PDCoV-infected piglets associated with increasing the relative abundance of Lactobacillus. Overall, niacin could alleviate diarrhea, intestinal barrier damages, intestinal immune response and colonic microflora disfunction in PDCoV-infected weaned piglets.

Development of a Multi-Epitope Vaccine for Mycoplasma hyopneumoniae and Evaluation of Its Immune Responses in Mice and Piglets.

Mycoplasma hyopneumoniae (Mhp), the primary pathogen causing Mycoplasma pneumonia of swine (MPS), brings massive economic losses worldwide. Genomic variability and post-translational protein modification can enhance the immune evasion of Mhp, which makes MPS prone to recurrent outbreaks on farms, even with vaccination or other treatments. The reverse vaccinology pipeline has been developed as an attractive potential method for vaccine development due to its high efficiency and applicability. In this study, a multi-epitope vaccine for Mhp was developed, and its immune responses were evaluated in mice and piglets. Genomic core proteins of Mhp were retrieved through pan-genome analysis, and four immunodominant antigens were screened by host homologous protein removal, membrane protein screening, and virulence factor identification. One immunodominant antigen, AAV27984.1 (membrane nuclease), was expressed by E. coli and named rMhp597. For epitope prioritization, 35 B-cell-derived epitopes were identified from the four immunodominant antigens, and 10 MHC-I and 6 MHC-II binding epitopes were further identified. The MHC-I/II binding epitopes were merged and combined to produce recombinant proteins MhpMEV and MhpMEVC6His, which were used for animal immunization and structural analysis, respectively. Immunization of mice and piglets demonstrated that MhpMEV could induce humoral and cellular immune responses. The mouse serum antibodies could detect all 11 synthetic epitopes, and the piglet antiserum suppressed the nuclease activity of rMhp597. Moreover, piglet serum antibodies could also detect cultured Mhp strain 168. In summary, this study provides immunoassay results for a multi-epitope vaccine derived from the reverse vaccinology pipeline, and offers an alternative vaccine for MPS.

A Novel Pathway for Porcine Epidemic Diarrhea Virus Transmission from Sows to Neonatal Piglets Mediated by Colostrum.

The mechanisms of colostrum-mediated virus transmission are difficult to elucidate because of the absence of experimental animal models and the difficulties in tissue sample collection from mothers in the peripartum period. Porcine epidemic diarrhea virus (PEDV) is a reemerging enteropathogenic coronavirus that has catastrophic impacts on the global pig industry. PEDV primarily infects neonatal piglets by multiple routes, especially 1- to 2-day-old neonatal piglets. Here, our epidemiological investigation and animal challenge experiments revealed that PEDV could be vertically transmitted from sows to neonatal piglets via colostrum, and CD3+ T cells in the colostrum play an important role in this process. The results showed that PEDV colonizing the intestinal epithelial cells (IECs) of orally immunized infected sows could be transferred to CD3+ T cells located just beneath the IECs. Next, PEDV-carrying CD3+ T cells, with the expression of integrin α4ß7 and CCR10, migrate from the intestine to the mammary gland through blood circulation. Arriving in the mammary gland, PEDV-carrying CD3+ T cells could be transported across mammary epithelial cells (MECs) into the lumen (colostrum), as illustrated by an autotransfusion assay and an MECs/T coculture system. The PEDV-carrying CD3+ T cells in colostrum could be interspersed between IECs of neonatal piglets, causing intestinal infection via cell-to-cell contact. Our study demonstrates for the first time that colostrum-derived CD3+ T cells comprise a potential route for the vertical transmission of PEDV. IMPORTANCE The colostrum represents an important infection route for many viruses. Here, we demonstrate the vertical transmission of porcine epidemic diarrhea virus (PEDV) from sows to neonatal piglets via colostrum. PEDV colonizing the intestinal epithelial cells could transfer the virus to CD3+ T cells located in the sow intestine. The PEDV-carrying CD3+ T cells in the sow intestine, with the expression of integrin α4ß7 and CCR10, arrive at the mammary gland through blood circulation and are transported across mammary epithelial cells into the lumen, finally leading to intestinal infection via cell-to-cell contact in neonatal piglets. Our study not only demonstrates an alternative route of PEDV infection but also provides an animal model of vertical transmission of human infectious disease.

Coronaviruses in swine

This work aims to review about coronaviruses in swine, emblazoning there's differences. Transmissible gastroenteritis and epidemic diarrhea are imported diseases, as they generate a high mortality rate in lactating piglets and economic losses. These viruses reach enterocytes, causing villous atrophy;affected animals present watery diarrhea, vomiting, hypoxia and dehydration. Two other viruses have recently emerged, the swine deltacoronavirus and the swine acute diarrhea syndrome virus, which have pathogenicity and clinical signs similar to those described in previous diseases. In the other hand, the porcine respiratory coronavirus and the hemagglutinating encephalomyelitis virus have distinct clinical signs. The first causes mild or subclinical respiratory manifestations and the second neurological signs, vomiting and emaciation, mostly affecting piglets younger than 4 weeks. As there is no specific treatment for these diseases, and vaccines do not provide full protection, a good biosecurity program is the main way to control and prevent them.

Effects of recombinant porcine Lactobacillus reuteri secreting expression of bovine lactoferrin peptides on growth performance of newborn piglets and protective effect against diarrhea virus infection

This experiment was conducted to investigate the effects of recombinant porcine Lactobacillus reuteri secreting bovine lactoferrin peptide (LFCA) on growth performance of newborn piglets and the protective effect on porcine transmissible gastroenteritis virus (TGEV)infection which caused piglet diarrhea. Experiment 1:thirty-six one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 3 groups, which were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group and control group, each group with 12 piglets. Piglets in each group were orally administered recombinant porcine Lactobacillus reuteri expressing LFCA pPG-LFCA/LR-CO21, containing empty vector plasmid PPG/LR-CO21 and equal volume phosphate buffer (PBS);oral administration continued for 3 days, and the observation time after oral administration was 14 d. During the period, piglets were fed freely, and the changes of body weight and diarrhea were recorded. Experiment 2:thirty one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 5 groups and given TGEV with a half tissue culture infection dose (TCID50) of 10-7.50/mL by oral administration of 1, 3, 6, 9 and 12 mL, respectively. The observation period of 7 d was set to analyze the conditions of half lethal dose. Experiment 3:another thirty-two newborn piglets with an average body weight of about 1.5 kg were selected as experimental animals and randomly divided into 4 groups, with 8 piglets in each group. The groups were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group, control group and TGEV infect group. There were 8 replicates in each group and 1 piglet in each replicate. Each head of the experimental group was orally fed ppG-LFCA/LR-CO21, pPG/LR-CO21 and equal volume of PBS at a dose of 2..1010 CFU per day for 1 consecutive week. At 8 days of age, TGEV was infected by oral administration at half lethal dose, and samples were collected after 7 days of infection. The weight change and diarrhea of each group of piglets were recorded;hematoxylin-eosin staining was used to detect the length of intestinal villi and the depth of crypts;enzyme linked immunosorbent assay (ELISA) was used to determine total serum total immunoglobulin G (IgG) and total secretory immunoglobulin A (sIgA) antibody contents. RT-qPCR was used to detect the mRNA relative expression levels of Claudin-1, Occludin, tight junction protein-1 (ZO-1), inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-P (IFN-P), tumor necrosis factor-a (TNF-a) and Toll-like receptor 2 (TLR2). The flora structure of the contents of the piglet's cecum was analyzed. After oral recombinant porcine Lactobacillusreuteri, compared with the control group, the average daily gain of newborn piglets in the pPG-LFCA/LR-CO21 group was significantly increased (P < 0.01), while the diarrhea rate was significantly decreased (P < 0.01). Compared with TGEV infection group, the average daily gain of piglets in pPG-LFCA/LR-CO21 group was increased and diarrhea rate was decreased, and the differences were significant (P < 0.05). Villus height and the ratio of villus height to crypt depth in jejunum and ileum were significantly increased (P < 0.05). The contents of total IgG and intestinal mucosal total sIgA antibody in serum of piglets were significantly increased (P < 0.05);the mRNA relative expression levels of tight junction protein-related genes Claudin-1, Occludin and ZO-1 in intestinal mucosal tissue were extremely significantly increased (P < 0.01), and the serum TNF-a content was extremely significantly decreased (P < 0.01). Serum IFN-P, IL-6, IL-8 and TLR2 contents were significantly increased (P < 0.01), and the survival rate of piglets was improved. The analysis of the bacterial diversity in the contents of the piglets' cecum showed that the proportion of normal intestinal flora of piglets decreased after TGEV infection. Compared with the TGEV infect group, the proportion of pathogenic bacteria Bacteroides in piglet's intestinal flora decreased by o

Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026.

We report the generation of a full-length infectious cDNA clone for porcine deltacoronavirus strain USA/IL/2014/026. Similar to the parental strain, the infectious clone virus (icPDCoV) replicated efficiently in cell culture and caused mild clinical symptoms in piglets. To investigate putative viral interferon (IFN) antagonists, we generated two mutant viruses: a nonstructural protein 15 mutant virus that encodes a catalytically-inactive endoribonuclease (icEnUmut), and an accessory gene NS6-deletion virus in which the NS6 gene was replaced with the mNeonGreen sequence (icDelNS6/nG). By infecting PK1 cells with these recombinant PDCoVs, we found that icDelNS6/nG elicited similar levels of type I IFN responses as icPDCoV, however icEnUmut stimulated robust type I IFN responses, demonstrating that the deltacoronavirus endoribonuclease, but not NS6, functions as an IFN antagonist in PK1 cells. Collectively, the construction of a full-length infectious clone and the identification of an IFN-antagonistic endoribonuclease will aid in the development of live-attenuated deltacoronavirus vaccines.

A strain of porcine deltacoronavirus: Genomic characterization, pathogenicity and its full-length cDNA infectious clone.

As a novel enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) warrants further investigation. In this study, a Chinese PDCoV strain, designated CHN-HN-1601, was isolated from the faeces of a diarrhoeic piglet. After plaque purification, the genome was determined which shared 97.5%-99.5% nucleotide identities with 71 representative PDCoV strains available in the GenBank. The pathogenic properties of CHN-HN-1601 were evaluated using 5-day-old piglets. All inoculated piglets developed severe diarrhoea from 2 days post-infection (dpi) onwards. To our surprise, two periods of diarrhoea starting from 2 to 7 dpi and from 13 to 19 dpi were observed in affected piglets during the experiment. Faecal viral shedding of the inoculated piglets was detected by real-time RT-PCR, with viral shedding peaked at 4 and 16 dpi, respectively. At necropsy at 5 dpi, the main gross lesions included transparent, thin-walled and gas-distended intestines containing yellow watery contents. Further histopathological examinations, including haematoxylin and eosin staining, immunohistochemistry and RNAscope in situ hybridization, revealed that the virus infection caused severe villous atrophy of the small intestines, with PDCoV antigen and RNA mainly distributed in the cytoplasm of the villous epithelial cells of jejunum and ileum in piglets. The dynamic production of PDCoV-specific IgG and neutralizing antibodies in serum of the affected piglets was also assessed using a whole virus-based ELISA and an immunofluorescence assay-based neutralization test, respectively. Furthermore, a full-length cDNA infectious clone of CHN-HN-1601 was constructed using a bacterial artificial chromosome system. The rescued virus exhibited in vitro growth and pathogenic properties similar to the parental virus. Taken together, our study not only enriches the information of PDCoV, but also provides a useful reverse genetics platform for further pathogenesis exploration of the virus.