Overview of the recent advances in porcine epidemic diarrhea vaccines.

Vaccination is the most effective means of preventing and controlling porcine epidemic diarrhea (PED). Conventional vaccines developed from porcine epidemic diarrhea virus (PEDV) GI-a subtypes (CV777 and SM98) have played a vital role in preventing classical PED. However, with the emergence of PEDV mutants in 2010, conventional PEDV GI-a subtype-targeting vaccines no longer provide adequate protection against PEDV GII mutants, thereby making novel-type PED vaccine development an urgent concern to be addressed. Novel vaccines, including nucleic acid vaccines, genetically engineered subunit vaccines, and live vector vaccines, are associated with several advantages, such as high safety and stability, clear targeting, high yield, low cost, and convenient usage. These vaccines can be combined with corresponding ELISA kits to differentiate infected from vaccinated animals, which is beneficial for disease confirmation. This review provides a detailed overview of the recent advancements in PED vaccines, emphasizing on the research and application evaluation of novel PED vaccines. It also considers the future directions and challenges in advancing these vaccines to widespread use in clinics.

Effect of feeding fermented distiller's grains diets on immune status and metabolomics of spleen and mesenteric lymph nodes in finishing cattle.

To explore the effect of feeding fermented distiller's grains (FDG) diets on spleen and mesenteric lymph nodes (MLN) immune status and metabolomics in finishing cattle, eighteen Guanling crossbred cattle (18 months old, 250.0 ± 25 kg) were randomly divided into 3 groups: a basal diet (Control) group, an FDG-15% group, and an FDG-30% group (containing 0%, 15% and 30% FDG to partially replace the concentrates, respectively). After 75 days, the spleens and MLN were collected for detection of relative spleen weight, immune parameters, and metabolomic analysis. Compared with the Control group, FDG-30% group significantly increased (P<0.05) the relative spleen weight. In addition, the level of IL-17A in the spleen of the FDG-30% group was significantly higher than that of the FDG-15% group. Metabolomic analysis showed that differential metabolites (VIP＞1, P＜0.05) of spleen and MLN in FDG-15% and FDG-30% groups are mostly lipids and lipid molecules. KEGG analysis illustrated that choline metabolism in cancer, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids and insulin resistance were metabolic pathways in spleen shared by FDG-15% group vs.Control group and FDG-30% group vs.Control group, and choline metabolism in cancer was a metabolic pathway in MLN shared by FDG-15% group vs.Control group and FDG-30% group vs.Control group. These results suggest that feeding FDG may promote spleen development by regulating choline metabolism in cancer, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids and insulin resistance. Additionally, it may affect MLN development by regulating choline metabolism in cancer. SIGNIFICANCE: Fermented distiller's grains (FDG) is a high quality alternative to feed because it is rich in beneficial microorganisms and nutrients. The spleen and mesenteric lymph nodes (MLN) are important peripheral immune organs in animals, whose status reflects the health of the animal. However, there are few reports on the effect of feeding FDG diets on spleen and MLN immune status and metabolomics in domestic animals. In this study, we found that feeding FDG may promote spleen development by regulating choline metabolism in cancer, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids and insulin resistance metabolic pathways, and may affect MLN development by regulating choline metabolism in cancer. This study extends our understanding of the metabolomics of the spleen and MLN in FDG and helps to further understand of the immunomodulatory effects of the FDG diet.

Probiotic-Fermented Distillers Grain Alters the Rumen Microbiome, Metabolome, and Enzyme Activity, Enhancing the Immune Status of Finishing Cattle.

A total of 30 Simmental crossbred cattle (6.50 months old, 265.0 ± 22.48 kg) were randomly divided into three groups, with 10 heads per group, and fed for 45 days. The diet treatments consisted of the Control group without PFDG supplementation, the PFDG-15% group with 15% PFDG substituting for 15% concentrate, and PFDG-30% group with 30% PFDG substituting for 30% concentrate. The results showed that compared with the Control group, the average daily gain (ADG) of the cattle in the PFDG-30% group decreased significantly (0.890 vs. 0.768 kg/d, p = 0.005). The serum malondialdehyde content of cattle in the PFDG-15% and PFDG-30% groups decreased significantly (p = 0.047) compared to that of the Control group. However, the serum superoxide dismutase activity of cattle in the PFDG-30% group was significantly higher than that of the Control group (p = 0.047). Meanwhile, both the PFDG-15% and PFDG-30% groups (1758.47 vs. 2061.30 µg/mL) showed higher serum levels of immunoglobulin G, while the interleukin-10 concentration was lower in the PFDG-30% group (p = 0.027). In addition, the PFDG-15% and PFDG-30% groups shifted the rumen microbiota by improving the abundances of F082 (related to propionic acid production) and fiber-degrading bacteria (Lachnospiraceae_UGG-009 and Prevotellaceae_UCG-001) and reducing the abundance of the disease-associated bacteria Selenomonas. A Kyoto encyclopedia of genes and genomes (KEGG) analysis illustrated that three key metabolic pathways, including phenylalanine metabolism, pyrimidine metabolism, and tryptophan metabolism, were enriched in the PFDG-15% group, but eight key metabolic pathways, including arachidonic acid metabolism, were enriched in the PFDG-30% group. Importantly, both the PFDG-15% and PFDG-30% groups increased (p < 0.01) the activities of cellulase, lipase, and protease in the rumen. Finally, the different bacterial abundance in the rumen was associated with changes in the ADG, serum antioxidant capacity, immune status, rumen enzyme activity, and metabolites. These results suggest that PFDG alters rumen microbiome abundance, metabolome, and enzyme activity for enhancing serum antioxidant capacity and the immune status, but when the supplemental level reaches 30%, it has a negative effect on ADG and the anti-inflammatory factors in finishing cattle.

The effect of feeding fermented distillers' grains diet on the intestinal metabolic profile of Guanling crossbred cattle.

Fermented distiller's grains (FDG)-based diets are nutritious and can improve the growth and intestinal immunity in livestock. However, there is limited research examining the effect of feeding FDG-based diets on changes in intestinal metabolites and related pathways in livestock. In this study, nine Guanling crossbred cattle (Guizhou Guanling Yellow cattle × Simmental cattle) were selected and randomly divided into a basal diet (BD) group and two experimental groups fed with FDG replacing 15% and 30% of the daily ration concentrates (FDG-Case A and FDG-Case B), respectively, with three cattle in each group. Fresh jejunum (J) and cecum (C) tissues were collected for metabolomic analysis. Differential metabolites and metabolic pathways were explored by means of univariate and multivariate statistical analysis. Compared with the J-BD group, 30 and 100 differential metabolites (VIP > 1, p < 0.05) were obtained in the J-FDG-Case A group and J-FDG-Case B group, respectively, and the J-FDG-Case B vs. J-FDG-Case A comparison revealed 63 significantly differential metabolites, which were mainly divided into superclasses including lipids and lipid-like molecules, organoheterocyclic compounds, and organic acids and derivatives. Compared with the C-BD, 3 and 26 differential metabolites (VIP > 1, p < 0.05) were found in the C-FDG-Case A group and C-FDG-Case B group, respectively, and the C-FDG-Case B vs. C-FDG-Case A comparison revealed 21 significantly different metabolites, which were also mainly divided into superclasses including lipids and lipid-like molecules, organoheterocyclic compounds, and organic acids and derivatives. A total of 40 metabolic pathways were identified, with a significance threshold set at p < 0.05. Among them, 2, 14, and 18 metabolic pathways were significantly enriched in the J-FDG-Case A vs. J-BD, J-FDG-Case B vs. J-BD, and J-FDG-Case B vs. J-FDG-Case A comparisons, respectively. Meanwhile, 1, 2, and 3 metabolic pathways were obtained in the C-FDG-Case A vs. C-BD, C-FDG-Case B vs. C-BD, and C-FDG-Case B vs. C-FDG-Case A comparisons, respectively. Furthermore, four significant metabolic pathways, namely insulin resistance, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and primary bile acid biosynthesis, were significantly enriched in Guanling crossbred cattle fed FDG diets. These results suggest that feeding FDG diets may promote the growth and intestinal immunity of Guanling crossbred cattle by regulating metabolic patterns of lipid compounds and related metabolic pathways. This study sheds light on the potential metabolic regulatory mechanisms of FDG diets and offers some references for their use in livestock feed.

Partially Alternative Feeding with Fermented Distillers' Grains Modulates Gastrointestinal Flora and Metabolic Profile in Guanling Cattle.

Fermented distillers' grains (FDG) are commonly used to enhance the health and metabolic processes of livestock and poultry by regulating the composition and activity of the intestinal microbiota. Nevertheless, there is a scarcity of research on the effects of the FDG diet on the gastrointestinal microbiota and its metabolites in cattle. This study examines the impact of FDG dietary supplements on the gastrointestinal flora and metabolic profile of Guanling cattle. Eighteen cattle were randomly assigned to three treatment groups with six replicates per group. The treatments included a basal diet (BD), a 15% concentrate replaced by FDG (15% FDG) in the basal diet, and a 30% concentrate replaced by FDG (30% FDG) in the basal diet. Each group was fed for a duration of 60 days. At the conclusion of the experimental period, three cattle were randomly chosen from each group for slaughter and the microbial community structure and metabolic mapping of their abomasal and cecal contents were analyzed, utilizing 16S rDNA sequencing and LC-MS technology, respectively. At the phylum level, there was a significant increase in Bacteroidetes in both the abomasum and cecum for the 30%FDG group (p < 0.05). Additionally, there was a significant reduction in potential pathogenic bacteria such as Spirochetes and Proteobacteria for both the 15%FDG and 30%FDG groups (p < 0.05). At the genus level, there was a significant increase (p < 0.05) in Ruminococcaceae_UCG-010, Prevotellaceae_UCG-001, and Ruminococcaceae_UCG-005 fiber degradation bacteria. Non-target metabolomics analysis indicated that the FDG diet significantly impacted primary bile acid biosynthesis, bile secretion, choline metabolism in cancer, and other metabolic pathways (p < 0.05). There is a noteworthy correlation between the diverse bacterial genera and metabolites found in the abomasal and cecal contents of Guanling cattle, as demonstrated by correlation analysis. In conclusion, our findings suggest that partially substituting FDG for conventional feed leads to beneficial effects on both the structure of the gastrointestinal microbial community and the metabolism of its contents in Guanling cattle. These findings offer a scientific point of reference for the further use of FDG as a cattle feed resource.

Impact of feeding dried distillers' grains with solubles diet on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle.

Dried distillers' grains with solubles (DDGS) are rich in nutrients, and partially alternative feeding of DDGS effectively reduces cost of feed and improves animals' growth. We used 16S rDNA gene sequencing and LC/MS-based metabolomics to explore the effect of feeding cattle with a basal diet (BD) and a Jiang-flavor DDGS diet (replaces 25% concentrate of the diet) on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle. The results showed that the ruminal and cecal contents shared the same dominance of Bacteroidetes, Firmicutes and Proteobacteria in two groups. The ruminal dominant genera were Prevotella_1, Rikenellaceae_RC9_gut_group, and Ruminococcaceae_UCG-010; and the cecal dominant genera were Ruminococcaceae_UCG-005, Ruminococcaceae_UCG-010, and Rikenellaceae_RC9_gut_group. Linear discriminant analysis effect size analysis (LDA > 2, P < 0.05) revealed the significantly differential bacteria enriched in the DDGS group, including Ruminococcaceae_UCG_012, Prevotellaceae_UCG_004 and Anaerococcus in the ruminal contents, which was associated with degradation of plant polysaccharides. Besides, Anaerosporobacter, Anaerovibrio, and Caproiciproducens in the cecal contents were involved in fatty acid metabolism. Compared with the BD group, 20 significantly different metabolites obtained in the ruminal contents of DDGS group were down-regulated (P < 0.05), and based on them, 4 significantly different metabolic pathways (P < 0.05) were enriched including "Linoleic acid metabolism," "Biosynthesis of unsaturated fatty acids," "Taste transduction," and "Carbohydrate digestion and absorption." There were 65 significantly different metabolites (47 were upregulated, 18 were downregulated) in the cecal contents of DDGS group when compared with the BD group, and 4 significantly different metabolic pathways (P < 0.05) were enriched including "Longevity regulating pathway," "Bile secretion," "Choline metabolism in cancer," and "HIF-1 signaling pathway." Spearman analysis revealed close negative relationships between the top 20 significantly differential metabolites and Anaerococcus in the ruminal contents. Bacteria with high relevance to cecal differential metabolites were Erysipelotrichaceae_UCG-003, Dielma, and Solobacterium that affect specific metabolic pathways in cattle. Collectively, our results suggest that feeding cattle with a DDGS diet improves the microbial structure and the metabolic patterns of lipids and carbohydrates, thus contributing to the utilization efficiency of nutrients and physical health to some extent. Our findings will provide scientific reference for the utilization of DDGS as feed in cattle industry.

Plasma Metabolomic Profiling after Feeding Dried Distiller's Grains with Solubles in Different Cattle Breeds.

Dried distiller's grains with solubles (DDGS) are rich in nutrients and can enhance animals' growth and immunity. However, there are few reports on the effects of a diet of DDGS on plasma metabolism and the related action pathways in domestic animals. In this study, groups of Guanling yellow cattle (GY) and Guanling crossbred cattle (GC) having a basal diet served as the control groups (GY-CG and GC-CG), and DDGS replacing 25% of the diet of GY and GC served as the replacement groups (GY-RG and GC-RG), with three cattle in each group. Plasma samples were prepared for metabolomic analysis. Based on multivariate statistical and univariate analyses, differential metabolites and metabolic pathways were explored. Twenty-nine significantly different metabolites (p < 0.05) were screened in GY-RG compared with those in GY-CG and were found to be enriched in the metabolic pathways, including choline metabolism in cancer, linolenic acid metabolism, and amino acid metabolism. Nine metabolites showed significant differences (p < 0.05) between GC-RG and GC-CG and were mainly distributed in the metabolic pathways of choline metabolism in cancer, glycerophospholipid metabolism, prostate cancer metabolism, and gonadotropin-releasing hormone (GnRH) secretion. These results suggest that a DDGS diet may promote healthy growth and development of experimental cattle by modulating these metabolic pathways. Our findings not only shed light on the nutritional effects of the DDGS diet and its underlying mechanisms related to metabolism but also provide scientific reference for the feed utilization of DDGS.

Hydroxytyrosol mitigates Mycoplasma gallisepticum-induced pulmonary injury through downregulation of the NF-κB/NLRP3/IL-1ß signaling pathway in chicken.

In this study, the anti-inflammatory and antiapoptotic effects of hydroxytyrosol (HT) in Mycoplasma gallisepticum (MG)-infected chicken were investigated, and the underlying molecular mechanisms were explored. The results revealed severe ultrastructural pathological changes after MG infection in the lung tissue of chicken, including inflammatory cell infiltration, thickening of the lung chamber wall, visible cell swelling, mitochondrial cristae rupture, and ribosome shedding. MG possibly activated the nuclear factor κB (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin (IL)-1ß signaling pathway in the lung. However, HT treatment significantly ameliorated MG-induced pathological damage of the lung. HT reduced the magnitude of pulmonary injury after MG infection by reducing apoptosis and releasing the proinflammatory factors. Compared with the MG-infected group, the HT-treated group exhibited significant inhibition of the expression of NF-κB/NLRP3/IL-1ß signaling-pathway-related genes; for example, the expressions of NF-κB, NLRP3, caspase-1, IL-1ß, IL-2, IL-6, IL-18, and TNF-α significantly decreased (P < 0.01 or <0.05). In conclusion, HT effectively inhibited MG-induced inflammatory response and apoptosis and protected the lung by blocking the activation of NF-κB/NLRP3/IL-1ß signaling pathway and reducing the damage caused by MG infection in chicken. This study revealed that HT may be a suitable and effective anti-inflammatory drug against MG infection in chicken.

Effect of feeding a dried distillers' grains with solubles diet on the metabolism of the intestinal wall in Guanling crossbred cattle: a preliminary assessment.

Dried distillers' grains with solubles (DDGS)-based diets are nutritious and can improve the inflammations and intestinal immunity in livestock. However, there is limited research examining the effect of feeding DDGS-based diets on changes in intestinal metabolites and related pathways in livestock. In this study, six Guanling crossbred cattle (Guizhou Guanling Yellow cattle × Simmental cattle) were selected and divided into a basal diet (BD) group and an experimental group fed with DDGS replacing 25% of the daily ration concentrates (DDGS) (n=3), respectively. Fresh jejunum (J), ileum (I) and cecum (C) tissues were collected for metabolomic analysis. Differential metabolites and metabolic pathways were explored by means of univariate and multivariate statistical analysis. In comparison to the J-BD group, 123 differential metabolites (VIP > 1, p < 0.05) were identified in the J-DDGS group, which (top 20) were mainly divided into superclasses, including lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compounds. Compared with the I-BD group, 47 differential metabolites were obtained in the I-DDGS group, which were mainly divided into superclasses, including lipids and lipid-like molecules and organic acids and derivatives. The C-DDGS vs. C-BD comparison revealed 88 differential metabolites, which were mainly divided into superclasses, including lipids and lipid-like molecules, organic oxygen compounds, and nucleosides. A total of 34 significant metabolic pathways were found (p < 0.05, -log(p) > 1.3). Among them, 3 significant pathways were significantly enriched in the J-DDGS group, 11 significant pathways were significantly enriched in the I-DDGS group, and 20 significant pathways were significantly enriched in the C-DDGS group. Importantly, primary bile acid biosynthesis, linoleic acid metabolism, and arachidonic acid metabolism correlated with intestinal inflammation and immunity by regulating gut microbiota, prostaglandin synthesis, and cell signaling. The data suggest that DDGS-fed cattle unregulated three metabolic pathways mentioned above and that a DDGS-based diet was able to maintain a balance of these three metabolic pathways, thus resulting in improvement of intestinal inflammation and enhanced immunity in cattle. In conclusion, the DDGS diet has the potential to improve intestinal inflammation and enhance the immunity of Guanling crossbred cattle by regulating the metabolic patterns of lipids and lipid-like molecules, organic acids and derivatives, and related metabolic pathways. These results allude to potential metabolic regulatory mechanisms of DDGS diets and also provide a theoretical basis for the application of DDGS in livestock feed.

Mycoplasma ovipneumoniae induces caspase-8-dependent extrinsic apoptosis and p53- and ROS-dependent intrinsic apoptosis in murine alveolar macrophages.

Mycoplasma ovipneumoniae (MO) is a principle causative agent of chronic respiratory disease in ruminants, including sheep, goats, and deer, posing a great threat to the ruminant industry worldwide. However, the pathogenesis of MO infection still remains not well understood and needs further clarification. Here we report a time-dependent apoptosis in cultured murine alveolar macrophage (MH-S) cell lines in response to MO infection in vitro. Mechanistically, MO infection activated apoptosis in MH-S cells through caspase-8-dependent extrinsic pathway and through tumor protein 53 (p53)- and reactive oxygen species (ROS)-dependent intrinsic mitochondrial pathways. Moreover, MO infection promoted both transcription and translation of proinflammatory cytokine genes including interleukin-1ß (IL-1ß), IL-18, and tumor necrosis factor-α (TNF-α), in a caspase-8-, p53-, and ROS-dependent manner, implying a potential link between MO-induced inflammation and apoptotic cell death. Collectively, our results suggest that MO infection induces the activation of extrinsic and intrinsic apoptotic pathways in cultured MH-S cells, which is related to upregulated expression of proinflammatory cytokines. Our findings will contribute to the elucidation of pathogenesis in MO infection and provide valuable reference for the development of new strategies for controlling MO infection.

A fowl adenovirus serotype 4 (FAdV-4) Fiber2 subunit vaccine candidate provides complete protection against challenge with virulent FAdV-4 strain in chickens.

Hypervirulent fowl adenovirus serotype 4 (FAdV-4)-induced hepatitis-hydropericardium syndrome (HHS) with high mortality causes huge economic losses to the poultry industry worldwide. However, commercially available vaccines against FAdV-4 infection remain scarce. Here, we prepared a subunit vaccine candidate derived from the bacterially expressed recombinant Fiber2 protein (termed as rFiber2 subunit vaccine) of FAdV-4 GZ-QL strain (a hypervirulent strain isolated in Guizhou province) and a recombinant plasmid pVAX1-Fiber2 as DNA vaccine candidate (termed as Fiber2 DNA vaccine). The immune effects of different dosages (50, 100, and 150 µg) of these were evaluated through immunization and challenge studies in chickens. Three injections of the rFiber2 subunit vaccine or the Fiber2 DNA vaccine induced robust humoral and cellular immune responses in chickens, which was assessed based on the secretion of high-level neutralizing antibodies, Th1- (IL-2, IFN-γ) and Th2-type cytokines (IL-4, IL-6). Importantly, the efficacy of the rFiber2 subunit vaccine was significantly higher (80 %-100 %) compared with the Fiber2 DNA vaccine (50 %-60 %) and a commercial inactivated vaccine (80 %). Collectively, these results suggest that the rFiber2 subunit and Fiber2 DNA vaccine candidate induced remarkable humoral and cellular immune responses, while the rFiber2 subunit vaccine candidate possesses better potential in the fight against FAdV-4 infection, laying foundations for the effective control of HHS in chickens.

Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013.

BACKGROUND: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry. RESULTS: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013. CONCLUSIONS: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

Corrigendum: Dual NDP52 Function in Persistent CSFV Infection.

[This corrects the article DOI: 10.3389/fmicb.2019.02962.].

LDHB inhibition induces mitophagy and facilitates the progression of CSFV infection.

Cellular metabolism caters to the energy and metabolite needs of cells. Although the role of the terminal metabolic enzyme LDHB (lactate dehydrogenase B) in the glycolysis pathway has been widely studied in cancer cells, its role in viral infection is relatively unknown. In this study, we found that CSFV (classical swine fever virus) infection reduces pyruvate levels while promotes lactate release in pigs and in PK-15 cells. Moreover, using a yeast two-hybrid screening system, we identified LDHB as a novel interacting partner of CSFV non-structural protein NS3. These results were confirmed via co-immunoprecipitation, glutathione S-transferase and confocal assays. Furthermore, knockdown of LDHB via interfering RNA induced mitochondrial fission and mitophagy, as detected reduced mitochondrial mass. Upon inhibition of LDHB, expression of the mitophagy proteins TOMM20 and VDAC1 decreased and the ubiquitination of MFN2, a mitochondrial fusion mediator, was promoted. In addition, a sensitive dual fluorescence reporter (mito-mRFP-EGFP) was utilized to analyze the delivery of autophagosomes to lysosomes in LDHB inhibition cells. Furthermore, LDHB inhibition promoted NFKB signaling, which was regulated by mitophagy; meanwhile, infection with CSFV negated these NFKB anti-viral responses. Inhibition of LDHB also inhibited apoptosis, providing an environment conducive to persistent viral infection. Finally, we demonstrated that LDHB inhibition promoted CSFV growth via mitophagy, whereas its overexpression decreased CSFV replication. Our data revealed a novel mechanism through which LDHB, a metabolic enzyme, mediates CSFV infection, and provides new avenues for the development of anti-viral strategies.Abbreviations: 3-MA:3-methyladenine; CCCP:carbonyl cyanide 3-chlorophenylhydrazone; CCK-8:cell counting kit-8; CSFV:classical swine fever virus; DAPI:4',6-diamidino-2-phenylindole; DMSO:dimethyl sulfoxide; EGFP:enhanced green fluorescent protein; FBS:fetal bovine serum; FITC:fluorescein isothiocyanate; GST:glutathione-S-transferase; HCV:hepatitis C virus; IFN:interferon; LDH:lactate dehydrogenase; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MFN2:mitofusin 2; MOI:multiplicity of infection; NFKB:nuclear factor kappa B subunit 1; NFKBIA:nuclear factor inhibitor alpha; NS3:nonstructural protein 3; NKIRAS2:NFKB inhibitor interacting Ras like 2; PRKN:parkin E3 ubiquitin protein ligase; PBS:phosphate-buffered saline; qRT-PCR:real-time quantitative reverse transcriptase polymerase chain reaction; RELA:RELA proto-oncogene, NF-kB subunit; shRNA: short hairpin RNA; siRNA: small interfering RNA; TCID50:50% tissue culture infectious doses; TEM:transmission electron microscopy; TNF:tumor necrosis factor; TOMM20:translocase of outer mitochondrial membrane 20; VDAC1:voltage dependent anion channel 1.

Classical swine fever virus employs the PERK- and IRE1-dependent autophagy for viral replication in cultured cells.

Endoplasmic reticulum stress (ERS)-mediated autophagy is indispensable for modulation of replication and pathogenesis of numerous mammalian viruses. We have previously shown that classical swine fever virus (CSFV) infection induces ERS-mediated autophagy for maintaining viral replication both in vivo and in vitro, however, the underlying mechanism remains unclarified. Here we found that CSFV infection activates the PERK pathway-dependent complete autophagy to promote viral replication in cultured PK-15 and 3D4/2 cells. Likewise, our results also suggested the essential roles of the IRE1/GRP78-mediated complete autophagy in CSFV replication in vitro. Furthermore, we suggested that CSFV infection induces activation of the PERK and IRE1 pathway for potential immunoregulation via promoting transcription of proinflammatory cytokine (IFN-γ and TNF-α) genes in the CSFV-infected cells. Finally, pharmacological treatment of PERK- or IRE1-pathway regulators, and the corresponding SiRNAs interventions did not affect the viabilities of the cells, excluding the potential interference elicited by altered cell viabilities. Taken together, our results suggest that CSFV infection induces complete autophagy through activation of the PERK and IRE1 pathway to facilitate viral replication in cultured cells, and modulation of proinflammatory cytokines may be a potential mechanism involved in this event. Our findings will open new horizons for molecular mechanisms of sustainable replication and pathogenesis of CSFV, and lay a theoretical foundation for the development of ERS-autophagy-targeting therapeutic strategies for clinical control of CSF.

Antiviral Role of Serine Incorporator 5 (SERINC5) Proteins in Classical Swine Fever Virus Infection.

Serine incorporator 5 (SERINC5), a multipass transmembrane protein, protects cells from viral infections. The mechanism by which SERINC5 protects against classical swine fever virus (CSFV) infection is unknown. In this study, overexpression of SERINC5 in PK-15 and 3D4/2 cells significantly inhibited the growth of CSFV, whereas SERINC5 silencing enhanced CSFV growth. Additionally, CSFV infection reduced SERINC5 production in cells and tissues. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and analyze protein and peptide molecules that potentially interact with SERINC5. A total of 33 cellular protein candidates were identified. Next, SERINC5 was shown to interact with melanoma differentiation-associated protein 5 (MDA5) by yeast two-hybrid, protein co-localization and co-immunoprecipitation assays. Furthermore, SERINC5 enhanced MDA5-mediated type I interferon (IFN) signaling in a dose-dependent manner. Our results suggest that the anti-CSFV effect of SERINC5 is dependent on the activation of the type I IFN, which may function along with MDA5. The inhibitory effect of SERINC5 on CSFV was disappeared when the endogenous expression of MDA5 was silenced using siRNA, suggesting that SERINC5 exerts an anti-CSFV effect in an MDA5-dependent manner. Our study demonstrated a novel link between SERINC5 and MDA5 in the inhibition of CSFV replication via the type I IFN signaling pathway.

MG132 Attenuates the Replication of Classical Swine Fever Virus in vitro.

The 26S proteasome, in charge of intracellular protein degradation, plays significant roles in the modulation of various cellular activities as well as in the interplay between virus and host. However, studies about the relationship between 26S proteasome and classical swine fever virus (CSFV) is limited up to now. MG132 is a proteasome inhibitor and has been extensively used in studies about replication of many viruses. Herein, we investigated the role of MG132 in CSFV replication and results showed that MG132 significantly decreased virus titers and viral RNA copies in CSFV-infected PK-15 cells. Further studies demonstrated that MG132 upregulated the expression of several interferon-stimulated genes (ISGs), in CSFV-infected cells. Since the activation of ISGs is controlled by the JAK-STAT signal pathway, we next examined the effect of MG132 on the expression and localization of key molecular STAT1 in the infected cells using Western blot and confocal laser scanning microscopy, respectively. Results showed that CSFV infection and viral NS4A protein decreased the protein level of STAT1, and MG132 promoted the accumulation of STAT1 in the nucleus of cells adjacent to the CSFV-infected cells. Besides, MG132 did not affect the expressions of IFN-α, STAT1, Mx1, OAS1, and PKR genes in cells without CSFV. In conclusion, we identify that MG132 significantly inhibits CSFV replication in vitro, in which the activation of the JAK-STAT pathway and the subsequent upregulation of expressions of ISGs might play significant roles, providing a potential preventive method for CSF.

The Role of Autophagy and Autophagy Receptor NDP52 in Microbial Infections.

Autophagy is a general protective mechanism for maintaining homeostasis in eukaryotic cells, regulating cellular metabolism, and promoting cell survival by degrading and recycling cellular components under stress conditions. The degradation pathway that is mediated by autophagy receptors is called selective autophagy, also named as xenophagy. Autophagy receptor NDP52 acts as a 'bridge' between autophagy and the ubiquitin-proteasome system, and it also plays an important role in the process of selective autophagy. Pathogenic microbial infections cause various diseases in both humans and animals, posing a great threat to public health. Increasing evidence has revealed that autophagy and autophagy receptors are involved in the life cycle of pathogenic microbial infections. The interaction between autophagy receptor and pathogenic microorganism not only affects the replication of these microorganisms in the host cell, but it also affects the host's immune system. This review aims to discuss the effects of autophagy on pathogenic microbial infection and replication, and summarizes the mechanisms by which autophagy receptors interact with microorganisms. While considering the role of autophagy receptors in microbial infection, NDP52 might be a potential target for developing effective therapies to treat pathogenic microbial infections.

Serum Lipidomics Analysis of Classical Swine Fever Virus Infection in Piglets and Emerging Role of Free Fatty Acids in Virus Replication in vitro.

Lipids metabolism plays a significant role in cellular responses to virus pathogens. However, the impact of lipids metabolism in CSFV infection is not yet confirmed. In the present study, for the fist time, we performed serum lipidomics analysis of piglets infected with CSFV based on ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and identified 167 differentially expressed lipid metabolites. Interestingly, free fatty acids (FFAs) accumulated significantly in these metabolites, accompanied by an increase in sphingolipids and a decrease in glycerolipids and glycerophospholipids, suggesting that CSFV infection markedly changed the serum lipid metabolism of piglets. FFAs are the principal constituents of many complex lipids and are essential substrates for energy metabolism. Based on this, we focused on whether FFAs play a prominent role in CSFV infection. We found that CSFV infection induced FFAs accumulation in vivo and in vitro, which is due to increased fatty acid biosynthesis. Meanwhile, we discovered that alteration of cellular FFAs accumulation by a mixture of FFAs or inhibitors of fatty acid biosynthesis affects progeny virus production in vitro. Furthermore, in the absence of glucose or glutamine, CSFV still has replication capacity, which is significantly reduced with the addition of fatty acid beta oxidation inhibitors, suggesting that the process of FFAs enter the mitochondria for beta oxidation to produce ATP is necessary for virus replication. Finally, we demonstrated CSFV induced FFAs accumulation results in impaired type I IFN signaling-mediated antiviral responses by down-regulating RIG-I-like receptors (RLRs) signaling molecules, which may represent a mechanism of CSFV replication. Taken together, these findings provide the first data on lipid metabolites during CSFV infection and reveal a new view that CSFV infection requires FFAs to enhance viral replication.

Classical Swine Fever Virus Infection Induces Endoplasmic Reticulum Stress-Mediated Autophagy to Sustain Viral Replication in vivo and in vitro.

Endoplasmic reticulum (ER) stress-mediated autophagy plays significant roles in replication and pathogenesis of many animal viruses. However, the relationship between ER stress, autophagy, and viral replication during in vivo and in vitro infection of classical swine fever virus (CSFV) remains unclear. In this study, we established a pig model for CSFV infection and found that viral loads of CSFV differed in 10 kinds of infected organs, and that the degree of tissue lesions was to some extent positively correlated with CSFV replication in vivo. Next, we found that CSFV infection not only induced ER stress and subsequently activated three unfolded protein responses (UPR) pathways including protein kinase R-like ER kinase (PERK), inositol requiring enzyme 1 (IRE1), and activating transcription factor-6 (ATF-6) pathways, but also triggered complete autophagy in main immune organs and partial nonimmune organs exhibiting severer pathological injuries and higher viral loads. However, only the IRE1 pathway and no autophagy were activated in some other nonimmune organs with slighter pathologies and lower viral loads. These results indicate a potential link between CSFV-induced ER stress and autophagy, both of which are associated with the CSFV replication in vivo. We further performed in vitro experiments and found that CSFV infection activates the PERK and IRE1 pathways and autophagy in cultured porcine kidney cell lines (PK-15) and macrophage cell lines (3D4/2), and pharmacological regulation of ER stress remarkably changed autophagic activities induced by CSFV, suggesting that CSFV-induced autophagy can be mediated by ER stress possibly via the PERK and IRE1 pathway. Furthermore, treatment with ER stress regulators significantly altered copy numbers of NS5B genes, expression of Npro proteins, and viral titers in CSFV-infected cells or in cells treated with autophagy regulators prior to CSFV infection, suggesting the requirement of ER stress-mediated autophagy for CSFV replication in vitro. Collectively, our data demonstrate that CSFV induces ER stress-mediated autophagy to sustain its replication in vivo and in vitro, which may be one of the potential strategies exploited by CSFV for immune evasion. This finding will provide new insights into mechanisms of replication and pathogenesis of CSFV, and development of new strategies for controlling CSF.