Proteomics analysis of PK-15 cells infected with porcine parvovirus and the effect of PCBP1 on PPV replication.

Porcine parvovirus (PPV) is one of the most important pathogens that cause reproductive failure in pigs. However, the pathogenesis of PPV infection remains unclear. Proteomics is a powerful tool to understand the interaction between virus and host cells. In the present study, we analyzed the proteomics of PPV-infected PK-15 cells. A total of 32 and 345 proteins were differentially expressed at the early and replication stages, respectively. Subsequent gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed these differentially expressed proteins were significantly enriched in pathways including toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and viral carcinogenesis. The expression of poly (rC) binding protein 1 (PCBP1) was observed to decrease after PPV infection. Overexpressed or silenced PCBP1 expression inhibited or promoted PPV infection. Our studies established a foundation for further exploration of the multiplication mechanism of PPV. IMPORTANCE: Porcine parvovirus (PPV) is a cause of reproductive failure in the swine industry. Our knowledge of PPV remains limited, and there is no effective treatment for PPV infection. Proteomics of PPV-infected PK-15 cells was conducted to identify differentially expressed proteins at 6 hours post-infection (hpi) and 36 hpi. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that various pathways participate in PPV infection. Poly (rC) binding protein 1 was confirmed to inhibit PPV replication, which provided potential targets for anti-PPV infection. Our findings improve the understanding of PPV infection and pave the way for future research in this area.

AMPK signaling pathway regulated the expression of the ApoA1 gene via the transcription factor Egr1 during G. parasuis stimulation.

Glaesserella parasuis (G. parasuis) is the causative agent of porcine Glässer's disease, resulting in high mortality rates in pigs due to excessive inflammation-induced tissue damage. Previous studies investigating the protective effects of G. parasuis vaccination indicated a possible role of ApoA1 in reflecting disease progression following G. parasuis infection. However, the mechanisms of ApoA1 expression and its role in these infections are not well understood. In this investigation, newborn porcine tracheal (NPTr) epithelial cells infected with G. parasuis were used to elucidate the molecular mechanism and role of ApoA1. The study revealed that the AMPK pathway activation inhibited ApoA1 expression in NPTr cells infected with G. parasuis for the first time. Furthermore, Egr1 was identified as a core transcription factor regulating ApoA1 expression using a CRISPR/Cas9-based system. Importantly, it was discovered that APOA1 protein significantly reduced apoptosis, pyroptosis, necroptosis, and inflammatory factors induced by G. parasuis in vivo. These findings not only enhance our understanding of ApoA1 in response to bacterial infections but also highlight its potential in mitigating tissue damage caused by G. parasuis infection.

Transcriptomic Analysis of PDCoV-Infected HIEC-6 Cells and Enrichment Pathways PI3K-Akt and P38 MAPK.

Porcine Deltacoronavirus (PDCoV) is a newly identified coronavirus that causes severe intestinal lesions in piglets. However, the understanding of how PDCoV interacts with human hosts is limited. In this study, we aimed to investigate the interactions between PDCoV and human intestinal cells (HIEC-6) by analyzing the transcriptome at different time points post-infection (12 h, 24 h, 48 h). Differential gene analysis revealed a total of 3560, 5193, and 4147 differentially expressed genes (DEGs) at 12 h, 24 h, and 48 h, respectively. The common genes among the DEGs at all three time points were enriched in biological processes related to cytokine production, extracellular matrix, and cytokine activity. KEGG pathway analysis showed enrichment of genes involved in the p53 signaling pathway, PI3K-Akt signaling pathway, and TNF signaling pathway. Further analysis of highly expressed genes among the DEGs identified significant changes in the expression levels of BUB1, DDIT4, ATF3, GBP2, and IRF1. Comparison of transcriptome data at 24 h with other time points revealed 298 DEGs out of a total of 6276 genes. KEGG analysis of these DEGs showed significant enrichment of pathways related to viral infection, specifically the PI3K-Akt and P38 MAPK pathways. Furthermore, the genes EFNA1 and KITLG, which are associated with viral infection, were found in both enriched pathways, suggesting their potential as therapeutic or preventive targets for PDCoV infection. The enhancement of PDCoV infection in HIEC-6 was observed upon inhibition of the PI3K-Akt and P38 MAPK signaling pathways using sophoridine. Overall, these findings contribute to our understanding of the molecular mechanisms underlying PDCoV infection in HIEC-6 cells and provide insights for developing preventive and therapeutic strategies against PDCoV infection.

Phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response.

Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.

Target silencing of porcine SPAG6 and PPP1CC by shRNA attenuated sperm motility.

The quality of sperm significantly influences the reproductive efficiency of pig herds. High-quality sperm is necessary for efficient fertilization and to maximize the litter numbers in commercial pig farming. However, the understanding of genes regulating porcine sperm motility and viability is limited. In this study, we validated porcine sperm/Sertoli-specific promoters through the luciferase reporter system and identified vital genes for sperm quality via loss-of-function means. Further, the shRNAs driven by the ACE and SP-10 promoters were used to knockdown the SPAG6 and PPP1CC genes which were provisionally important for sperm quality. We assessed the effects of SPAG6 and PPP1CC knockdown on sperm motility by using the sperm quality analyzer and flow cytometry. The results showed that the ACE promoter is active in both porcine Sertoli cells and sperms, whereas the SP-10 promoter is operating exclusively in sperm cells. Targeted interference with SPAG6 and PPP1CC expression in sperm cells decreases the motility and increases apoptosis rates in porcine sperms. These findings not only offer new genetic tools for targeting male germ cells but also highlight the crucial roles of SPAG6 and PPP1CC in porcine sperm function.

Porcine reproductive and respiratory syndrome virus infection induces microRNA novel-216 production to facilitate viral-replication by targeting MAVS 3´UTR.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses in the swine industry. In this study, the high-throughput sequencing, microRNAs (miRNAs) mimic, and lentivirus were used to screen for potential miRNAs that can promote PRRSV infection in porcine alveolar macrophages or Marc-145 cells. It was observed that novel-216, a previously unidentified miRNA, was upregulated through the p38 signaling pathway during PRRSV infection, and its overexpression significantly increased PRRSV replication. Further analysis revealed that novel-216 regulated PRRSV replication by directly targeting mitochondrial antiviral signaling protein (MAVS), an upstream molecule of type Ⅰ IFN that mediates the production and response of type Ⅰ IFN. The proviral function of novel-216 on PRRSV replication was abolished by MAVS overexpression, and this effect was reversed by the 3'UTR of MAVS, which served as the target site of novel-216. In conclusion, this study demonstrated that PRRSV-induced upregulation of novel-216 served to inhibit the production and response of typeⅠ IFN and facilitate viral replication, providing new insights into viral immune evasion and persistent infection.

Unveiling the Role of Protein Kinase C θ in Porcine Epidemic Diarrhea Virus Replication: Insights from Genome-Wide CRISPR/Cas9 Library Screening.

Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.

Towards identification of new genetic determinants for post-weaning diarrhea in piglets.

Post-weaning diarrhea in pigs is a considerable challenge in the pig farming industry due to its effect on animal welfare and production costs, as well as the large volume of antibiotics, which are used to treat diarrhea in pigs after weaning. Previous studies have revealed loci on SSC6 and SSC13 associated with susceptibility to specific diarrhea causing pathogens. This study aimed to identify new genetic loci for resistance to diarrhea based on phenotypic data. In depth clinical characterization of diarrhea was performed in 257 pigs belonging to two herds during the first 14 days post weaning. The daily diarrhea assessments were used for the classification of pigs into case and control groups. Pigs were assigned to case and control groups based only on the incidence of diarrhea in the second week of the study in order to differentiate between differences in etiology. Genome-wide association studies and metabolomics association analysis were performed in order to identify new biological determinants for diarrhea susceptibility. With the present work, we revealed a new locus for diarrhea resistance on SSC16. Furthermore, studies of metabolomics in the same pigs revealed one metabolite associated with diarrhea.

Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production.

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.

Survey on resistance occurrence for F4+ and F18+ enterotoxigenic Escherichia coli (ETEC) among pigs reared in Central Italy regions.

Porcine Post Weaning Diarrhoea (PWD) is one of the most important swine disease worldwide, caused by Enterotoxigenic Escherichia coli (ETEC) strains able to provoke management, welfare and sanitary issues. ETEC is determined by proteinaceous surface appendages. Numerous studies conducted by now in pigs have demonstrated, at the enterocytes level, that, the genes mucin 4 (MUC4) and fucosyltransferase (FUT1), coding for ETEC F4 and F18 receptors respectively, can be carriers of single nucleotide polymorphisms (SNPs) associated with natural resistance/susceptibility to PWD. The latter aspect was investigated in this study, evaluating the SNPs of the MUC4 and FUT1 genes in slaughtered pigs reared for the most in Central Italy. Genomic DNA was extracted from 362 swine diaphragmatic samples and then was subjected to the detection of known polymorphisms on MUC4 and FUT1candidate target genes by PCR-RFLP. Some of the identified SNPs were confirmed by sequencing analysis. Animals carrying the SNPs associated with resistance were 11% and 86% for the FUT1 and MUC4 genes respectively. Therefore, it can be assumed that the investigated animals may be an important resource and reservoir of favorable genetic traits for the breeding of pigs resistant to enterotoxigenic E.coli F4 variant.

Pathogenicity and horizontal transmission evaluation of a novel isolated African swine fever virus strain with a three-large-fragment-gene deletion.

African swine fever has caused substantial economic losses to China`s pig industry in recent years. Currently, the highly pathogenic African swine fever virus strain of genotype II is predominantly circulating in China, accompanied by a series of emerging isolates displaying unique genetic variations. The pathogenicity of these emerging strains is still unclear. Recently, a novel ASFV strain with a distinguishable three-large-fragment gene deletion was obtained from the field specimens, and its in vivo pathogenicity and transmission were evaluated in this study. The animal experiment involved inoculating a high dose of YNFN202103 and comparing its effects with those of the highly pathogenic strain GZ201801_2. Results showed that pigs infected by YNFN202103 exhibited significantly prolonged onset and survival time, lower viremia levels, and less severe histopathological lesions compared to GZ201801_2. These findings contributed valuable insights into the pathogenicity and transmission of ASFV and its prevention and eradication strategies in practical settings.

Cellular nuclear-localized U2AF2 protein is hijacked by the flavivirus 3'UTR for viral replication complex formation and RNA synthesis.

Japanese encephalitis virus (JEV) is a zoonotic pathogen belonging to the Flavivirus genus, causing viral encephalitis in humans and reproductive failure in swine. The 3' untranslated region (3'UTR) of JEV contains highly conservative secondary structures required for viral translation, RNA synthesis, and pathogenicity. Identification of host factors interacting with JEV 3'UTR is crucial for elucidating the underlying mechanism of flavivirus replication and pathogenesis. In this study, U2 snRNP auxiliary factor 2 (U2AF2) was identified as a novel cellular protein that interacts with the JEV genomic 3'UTR (the SL-I, SL-II, SL-III, and DB region) via its 1 to 148 amino acids. JEV infection or JEV 3' UTR on its own triggered the nuclear-localized U2AF2 redistributed to the cytoplasm and colocalized with viral replication complex. U2AF2 also interacts with JEV NS3 and NS5 protein, the downregulation of U2AF2 nearly abolished the formation of flavivirus replication vesicles. The production of JEV protein, RNA, and viral titers were all increased by U2AF2 overexpression and decreased by knockdown. U2AF2 also functioned as a pro-viral factor for Zika virus (ZIKV) and West Nile virus (WNV), but not for vesicular stomatitis virus (VSV). Mechanically, U2AF2 facilitated the synthesis of both positive- and negative-strand flavivirus RNA without affecting viral attachment, internalization or release process. Collectively, our work paves the way for developing U2AF2 as a potential flavivirus therapeutic target.

Whole genome resequencing reveals candidate genes for postaxial polydactyly in Large White pigs.

Polydactyly is a genetic abnormality that affects both pig welfare and industry profits. Despite efforts to explore the genetic basis of pig polydactyly, progress remains limited. In this study, we analyzed a group of Large White pigs with postaxial polydactyly, including 29 cases and 79 controls from 24 families. High-depth sequencing was performed on 20 pigs, while low-depth sequencing was improved through imputation for the remaining pigs. A genome-wide association study (GWAS) and genetic differentiation were conducted using the resequencing dataset, resulting in the identification of 48 significantly associated SNPs and 27 candidate regions. The genetic differentiation regions on chromosomes 5 and 18, which harbored GWAS-identified SNPs, were delineated as confidence regions. The confidence region at Chr18: 1.850-1.925 Mb covers the fifth intron of LMBR1, a gene that contains an important regulatory element for SHH, known as ZRS. Mutations in this ZRS have been found to cause polydactyly in animals and humans. Therefore, we propose LMBR1 as a prospective candidate gene for postaxial polydactyly. These findings emphasize the importance of exploring the role of ZRS within LMBR1 in the pathogenesis of polydactyly in pigs.

Metatranscriptomic assessment of diarrhoeic faeces reveals diverse RNA viruses in rotavirus group A infected piglets and calves from India.

RNA viruses are a major group contributing to emerging infectious diseases and neonatal diarrhoea, causing morbidity and mortality in humans and animals. Hence, the present study investigated the metatranscriptomic-derived faecal RNA virome in rotavirus group A (RVA)-infected diarrheic piglets and calves from India. The viral genomes retrieved belonged to Astroviridae in both species, while Reoviridae and Picornaviridae were found only in piglets. The nearly complete genomes of porcine RVA (2), astrovirus (AstV) (6), enterovirus G (EVG) (2), porcine sapelovirus (PSV) (2), Aichivirus C (1), and porcine teschovirus (PTV) (1) were identified and characterised. In the piglet, AstVs of PAstV2 (MAstV-26) and PAstV4 (MAstV-31) lineages were predominant, followed by porcine RVA, EVG, PSV, Aichivirus C, teschovirus (PTV-17) in decreasing order of sequence reads. In contrast, AstV accounted for the majority of reads in bovines and belonged to MAstV-28 and a proposed MAstV-35. Both RVA G4P[6] strains exhibited prototype Gottfried strains like a genotypic constellation of G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1. Ten out of eleven genes were of porcine origin, while the VP7 gene clustered with G4-lineage-1, consisting of human strains, suggesting a natural porcine-human reassortant. In the recombination analysis, multiple recombination events were detected in the PAstV4 and PAstV2 genomes, pointing out that these viruses were potential recombinants. Finally, the study finds diverse RNA virome in Indian piglets and calves for the first time, which may have contributed to diarrhoea. In the future, the investigation of RNA virome in animals will help in revealing pathogen diversity in multifactorial diseases, disease outbreaks, monitoring circulating viruses, viral discovery, and evaluation of their zoonotic potential.

Functional and evolutionary analysis of host Synaptogyrin-2 in porcine circovirus type 2 susceptibility.

Mammalian evolution has been influenced by viruses for millions of years, leaving signatures of adaptive evolution within genes encoding for viral interacting proteins. Synaptogyrin-2 (SYNGR2) is a transmembrane protein implicated in promoting bacterial and viral infections. A genome-wide association study of pigs experimentally infected with porcine circovirus type 2b (PCV2b) uncovered a missense mutation (SYNGR2 p.Arg63Cys) associated with viral load. In this study, CRISPR/Cas9-mediated gene editing of the porcine kidney 15 (PK15, wtSYNGR2+p.63Arg) cell line generated clones homozygous for the favorable SYNGR2 p.63Cys allele (emSYNGR2+p.63Cys). Infection of edited clones resulted in decreased PCV2 replication compared to wildtype PK15 (P<0.05), with consistent effects across genetically distinct PCV2b and PCV2d isolates. Sequence analyses of wild and domestic pigs (n>700) revealed the favorable SYNGR2 p.63Cys allele is unique to domestic pigs and more predominant in European than Asian breeds. A haplotype defined by the SYNGR2 p.63Cys allele was likely derived from an ancestral haplotype nearly fixed within European (0.977) but absent from Asian wild boar. We hypothesize that the SYNGR2 p.63Cys allele arose post-domestication in ancestral European swine. Decreased genetic diversity in homozygotes for the SYNGR2 p.63Cys allele compared to SYNGR2 p.63Arg, corroborates a rapid increase in frequency of SYGNR2 p.63Cys via positive selection. Signatures of adaptive evolution across mammalian species were also identified within SYNGR2 intraluminal loop domains, coinciding with the location of SYNGR2 p.Arg63Cys. Therefore, SYNGR2 may reflect a novel component of the host-virus evolutionary arms race across mammals with SYNGR2 p.Arg63Cys representing a species-specific example of putative adaptive evolution.

Genome characteristics of atypical porcine pestivirus from abortion cases in Shandong Province, China.

BACKGROUND: Atypical porcine pestivirus (APPV) is a novel, highly variable porcine pestivirus. Previous reports have suggested that the virus is associated with congenital tremor (CT) type A-II in piglets, and little information is available about the correlation between the virus and sow abortion, or on coinfection with other viruses. In China, reported APPV strains were mainly isolated from South China and Central China, and data about the APPV genome from northern China are relatively scarce. METHODS: Eleven umbilical cords, one placenta, and one aborted piglet, were collected from aborted sows of the same farm in Shandong Province of northern China. Nucleic acids were extracted from the above samples, and subsequently pooled for viral metagenomics sequencing and bioinformatics analysis. The viral coexistence status and complete genome characteristics of APPV in Shandong Province were determined. RESULTS: In abortion cases, APPV was present with Getah virus, porcine picobirnavirus, porcine kobuvirus, porcine sapovirus, Po-Circo-like virus, porcine serum-associated circular virus, porcine bocavirus 1, porcine parvovirus 1, porcine parvovirus 3 and porcine circovirus 3, etc. The first complete genome sequence(11,556 nt) of APPV in Shandong Province of northern China, was obtained using viral metagenomics and designated APPV-SDHY-2022. Comparison with Chinese reference strains revealed that the polyprotein of APPV-SDHY-2022 shared 82.6-84.2%, 93.2-93.6%, and 80.7-85% nucleotide identity and 91.4-92.4%, 96.4-97.7%, and 90.6-92.2% amino acid identity with those of the Clade I, Clade II and Clade III strains, respectively. Phylogenetic analysis based on the complete polyprotein CDS and NS5A sequences concluded that APPV-SDHY-2022 belongs to Clade II. Analysis of the NS5A nucleotide sequences revealed homology of greater than 94.6% for the same isoform, 84.7-94.5% for different isoforms of the same clade and 76.8-81.1% for different clades. Therefore, Clade II was further divided into three subclades, and APPV-SDHY-2022 belonged to subclade 2.3. Members of Clade II have 20 unique amino acids in individual proteins, distinguishing them from Clade I and Clade III members. The E2 protein showed the greatest diversity of putative N-glycosylation sites with 9 patterns, and APPV-SDHY-2022 along with other Chinese APPV strains shared the conserved B-cell conformational epitope residues 39E, 70R, 173R, 190K and 191N of the E2 protein. CONCLUSIONS: We reported viral coexistence and the first complete genome sequence of APPV from abortion cases and from Shandong Province. The new APPV isolate belongs to an independent branch of Clade II. Our results increase the molecular and epidemiological understanding of APPV in China.

Toxoplasma gondii infection regulates apoptosis of host cells via miR-185/ARAF axis.

BACKGROUND: Toxoplasmosis is a zoonosis with a worldwide presence that is caused by the intracellular parasite Toxoplasma gondii. Active regulation of apoptosis is an important immune mechanism by which host cells resist the growth of T. gondii or avoid excessive pathological damage induced by this parasite. Previous studies found that upregulated expression of microRNA-185 (miR-185) during T. gondii infection has a potential role in regulating the expression of the ARAF gene, which is reported to be associated with cell proliferation and apoptosis. METHODS: The expression levels of miR-185 and the ARAF gene were evaluated by qPCR and Western blot, respectively, in mice tissues, porcine kidney epithelial cells (PK-15) and porcine alveolar macrophages (3D4/21) following infection with the T. gondii ToxoDB#9 and RH strains. The dual luciferase reporter assay was then used to verify the relationship between miR-185 and ARAF targets in PK-15 cells. PK-15 and 3D4/21 cell lines with stable knockout of the ARAF gene were established by CRISPR, and then the apoptosis rates of the cells following T. gondii infection were detected using cell flow cytometry assays. Simultaneously, the activities of cleaved caspase-3, as a key apoptosis executive protein, were detected by Western blot to evaluate the apoptosis levels of cells. RESULTS: Infection with both the T. gondii ToxoDB#9 and RH strains induced an increased expression of miR-185 and a decreased expression of ARAF in mice tissues, PK-15 and 3D4/21 cells. MiR-185 mimic transfections showed a significantly negative correlation in expression levels between miR-185 and the ARAF gene. The dual luciferase reporter assay confirmed that ARAF was a target of miR-185. Functional investigation revealed that T. gondii infection induced the apoptosis of PK-15 and 3D4/21 cells, which could be inhibited by ARAF knockout or overexpression of miR-185. The expression levels of cleaved caspase-3 protein were significantly lower in cells with ARAF knockout than in normal cells, which were consistent with the results of the cell flow cytometry assays. CONCLUSIONS: Toxoplasma gondii infection could lead to the upregulation of miR-185 and the downregulation of ARAF, which was not related to the strain of T. gondii and the host cells. Toxoplasma gondii infection could regulate the apoptosis of host cells via the miR-185/ARAF axis, which represents an additional strategy used by T. gondii to counteract host-cell apoptosis in order to maintain survival and reproduce in the host cells.

Searching for new signals for susceptibility to umbilical hernia through genome-wide association analysis in three pig breeds.

Umbilical hernia (UH) is a prevalent congenital disorder in pigs, resulting in considerable economic losses and severe animal welfare issues. In the present study, we conducted a genome-wide association study (GWAS) using the GeneSeek 50K Chip in 2777 pigs (Duroc, n = 1267; Landrace, n = 696; and Yorkshire, n = 814) to explore the candidate genes underlying the risk of umbilical hernia in pigs. After quality control analyses, 2748 animals and 48 524 single nucleotide polymorphisms (SNPs) were retained for subsequent GWAS analysis using the FarmCPU model. The heritability of umbilical hernias was estimated to 0.51 ± 0.04, indicating a reasonable basis for investigating genetic markers associated with this disorder. We identified 54 SNPs and 517 candidate genes that showed significant associations with susceptibility to umbilical hernia across the combined population of the three pig breeds. Gene enrichment analyses highlighted several crucial pathways for platelet degranulation, inflammatory mediator regulation of TRP channels and ion transport. These findings provide further insights into the underlying genetic architecture of umbilical hernias in pigs.

The pattern of stability of African swine fever virus in leeches.

African swine fever virus (ASFV) can accumulate and survive in leeches for a long time. The reasons for the survival of ASFV in leeches are not entirely clear. Here, we elucidate the virus survival pathway in infected leeches. One of the questions reported previously is addressed in this article. How the virus concentration in the body of the leech is equal to or higher than in the water infected with ASFV? Examination of blood swallowed by leeches reveals that the blood cells retain their morphological characteristics for several weeks. It can explain the long-term persistence of the high levels of ASFV in the leeches that ingested ASFV-infected pig blood. qRT-PCR assay showed the transcription of ASFV genes in infected leeches. However, the infectious particles of the virus measured by HADU haven't increased. Quantitative studies of the ASFV revealed a high content of both viral genes and infectious particles in the skin of leeches compared with other body parts. Electron microscopy analysis revealed the ability of the ASFV to effectively bind to the skin surface of the leeches, which explained the high concentrations of ASFV in the leeches' skin. A significant difference in the transcriptional activity between early and late viral genes indicates that the virus entered the initial stage of replication, but for some reason failed to complete it, which is typical of abortive infections.

PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production.

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication.