An inactivated PDCoV vaccine induces robust neutralizing antibodies and immune protection in pigs lasting for three months.

Porcine deltacoronavirus (PDCoV), a novel enteropathogenic coronavirus, causes diarrhea mainly in suckling piglets and has the potential to infect humans. Whereas, there is no commercially available vaccine which can effectively prevent this disease. In this study, to ascertain the duration of immune protection of inactivated PDCoV vaccine, suckling piglets were injected subcutaneously with inactivated PDCoV vaccine using a prime/boost strategy at 3 and 17-day-old. Neutralizing antibody assay showed that the level of the inactivated PDCoV group was still ≥1:64 at three months after prime vaccination. The three-month-old pigs were orally challenged with PDCoV strain CZ2020. Two pigs in challenge control group showed mild to severe diarrhea at 10-11 day-post-challenge (DPC), while the inactivated PDCoV group had no diarrhea. High levels of viral shedding, substantial intestinal villus atrophy, and positive straining of viral antigens in ileum were detected in challenge control group, while the pigs in inactivated PDCoV group exhibited significantly reduced viral load, minor intestinal villi damage and negative straining of viral antigens. These results demonstrated that PDCoV was pathogenic against three-month-old pigs and inactivated PDCoV vaccine can provide effective protection in pigs lasting for three months.

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.

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.

Immune responses induced by a combined vaccination with a recombinant chimera of Mycoplasma hyopneumoniae antigens and capsid virus-like particles of porcine circovirus type 2.

BACKGROUND: Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are two important pathogens causing Mycoplasma pneumonia of swine (MPS) and porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs), respectively, and resulted in considerable economic loss to the swine industry worldwide. Currently, vaccination is one of the main measures to control these two diseases; however, there are few combination vaccines that can prevent these two diseases. To determine the effect of combination immunization, we developed capsid-derived (Cap) virus-like particles (VLPs) of PCV2 and a new recombinant chimera composed of the P97R1, P46, and P42 antigens of Mhp. Then we investigated the immune responses induced by the immunization with this combination vaccine in mice and piglets. RESULTS: The high level antibodies against three protein antigens (P97R1, P46, and P42 of Mhp) were produced after immunization, up to or higher than 1:400,000; the antibody levels in Pro group continuously increased throughout the 42 days for all the antigens tested. The lymphocyte proliferative response in PCV2 group was stronger than that in PBS, VP, Mhp CV in mice. The antibody levels for Cap remained stable and reached the peak at 35 DAI. The IFN-γ and IL-4 in sera were significantly enhanced in the Pro group than that in the negative control-VP group on Day 14 and 28 post-the first immunization in piglets. CONCLUSIONS: Above all, the combination immunization could induce humoral and cellular immune responses against all four antigens in mice and piglets. Therefore, our approach is a simple and effective vaccination strategy to protect pigs against MPS and PCVD/PCVAD.

Development of a Combined Genetic Engineering Vaccine for Porcine Circovirus Type 2 and Mycoplasma Hyopneumoniae by a Baculovirus Expression System.

Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are the main pathogens for mycoplasmal pneumonia of swine (MPS) and post-weaning multisystemic wasting syndrome (PMWS), respectively. Infection by these pathogens often happens together and causes great economic losses. In this study, a kind of recombinant baculovirus that can display P97R1P46P42 chimeric protein of Mhp and the capsid (Cap) protein of PCV2 was developed, and the protein location was identified. Another recombinant baculovirus was constructed without tag proteins (EGFP, mCherry) and was used to evaluate the immune effect in experiments with BALB/c mice and domestic piglets. Antigen proteins P97R1P46P42 and Cap were expressed successfully; both were anchored on the plasma membrane of cells and the viral envelope. It should be emphasized that in piglet immunization, the recombinant baculovirus vaccine achieved similar immunological effects as the mixed commercial vaccine. Both the piglet and mouse experiments showed that the recombinant baculovirus was able to induce humoral and cellular responses effectively. The results of this study indicate that this recombinant baculovirus is a potential candidate for the further development of more effective combined genetic engineering vaccines against MPS and PMWS. This experiment also provides ideas for vaccine development for other concomitant diseases using the baculovirus expression system.

Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae.

Recent studies have demonstrated that recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) produced by the silkworm pupae bioreactor is absorbed into blood through oral administration and functions as an active cytokine. The aim of this study was to further examine and identify synergetic protein factors in silkworm pupae that improve rhGM-CSF absorption via an oral route. The concentrations of rhGM-CSF in serum were evaluated in mice after oral administration of rhGM-CSF using different chemical compositions of silkworm pupae as pharmaceutical excipients. The experimental data revealed that the supernatant lyophilized powder (SLP) of a homogenized slurry of silkworm pupae caused a significant increase in the rhGM-CSF level in blood when rhGM-CSF was orally administered with SLP, suggesting that synergetic protein factors that improve the oral absorption of rhGM-CSF primarily exist in SLP. As shown by scanning electron microscopy, microspheres were formed when rhGM-CSF was coated with SLP. Animal experimental data showed that the absorption of orally administered rhGM-CSF through the gastrointestinal (GI) tract primarily resulted from protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration. Surface plasmon resonance spectroscopy analysis demonstrated that several protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration were bound to rhGM-CSF. Proteins bound to rhGM-CSF by liquid chromatography-mass spectrometry were identified as chymotrypsin inhibitor SCI-II precursor, cationic peptide CP8 precursor, Kazal-type proteinase inhibitor, and chymotrypsin inhibitor SCI-I. These findings indicate that these proteinase inhibitors play an important role in improving rhGM-CSF absorption in the GI tract.

A New Member of the TBC1D15 Family from Chiloscyllium plagiosum: Rab GTPase-Activating Protein Based on Rab7 as a Substrate.

APSL (active peptide from shark liver) is a hepatic stimulator cytokine from the liver of Chiloscyllium. It can effectively protect islet cells and improve complications in mice with alloxan-induced diabetes. Here, we demonstrate that the APSL sequence is present in the N-terminus of novel TBC (Tre-2, Bub2 and Cdc16) domain family, member 15 (TBC1D15) from Chiloscyllium plagiosum. This shark TBC1D15 gene, which contains an ORF of 2088 bp, was identified from a cDNA library of regenerating shark liver. Bioinformatic analysis showed that the gene is highly homologous to TBC1D15 genes from other species. Moreover, the N-terminus of shark TBC1D15 contains a motif of unknown function (DUF3548), which encompasses the APSL fragment. Rab-GAP activity analysis showed that shark TBC1D15 is a new member of the TBC1D15 family. These results demonstrated that shark TBC1D15 possesses Rab-GAP activity using Rab7 as a substrate, which is a common property of the TBC1D15 family. The involvement of APSL at the N-terminus of TBC1D15 also demonstrates that this protein might be involved in insulin signaling and may be associated with the development of type 2 diabetes. The current findings pave the way for further functional and clinical studies of these proteins from marine sources.

Wingless-type mammary tumor virus integration site family, member 5A (Wnt5a) regulates human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120)-induced expression of pro-inflammatory cytokines via the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK) signaling pathways.

BACKGROUND: HIV-1 infection causes chronic neuroinflammation in the central nervous system (CNS). RESULTS: The spinal cytokine up-regulation induced by HIV-1 gp120 protein depends on Wnt5a/CaMKII and/or Wnt5a/JNK pathways. CONCLUSION: gp120 stimulates cytokine expression in the spinal cord dorsal horn by activating Wnt5a signaling. SIGNIFICANCE: The finding reveals Wnt signaling-mediated novel mechanisms by which HIV-1 may cause neuroinflammation. Chronic expression of pro-inflammatory cytokines critically contributes to the pathogenesis of HIV-associated neurological disorders (HANDs), but the host mechanism that regulates the HIV-induced cytokine expression in the CNS remains elusive. Here, we present evidence for a crucial role of Wnt5a signaling in the expression of pro-inflammatory cytokines in the spinal cord induced by a major HIV-envelope protein, gp120. Wnt5a is mainly expressed in spinal neurons, and rapidly up-regulated by intrathecal injection (i.t.) of gp120. We show that inhibition of Wnt5a by specific antagonists blocks gp120-induced up-regulation of IL-1ß, IL-6, and TNF-α in the spinal cord. Conversely, injection (i.t.) of purified recombinant Wnt5a stimulates the expression of these cytokines. To elucidate the role of the Wnt5a-regulated signaling pathways in gp120-induced cytokine expression, we have focused on CaMKII and JNKs, the well characterized down-stream targets of Wnt5a signaling. We find that Wnt5a is required for gp120 to activate CaMKII and JNK signaling. Furthermore, we demonstrate that the Wnt5a/CaMKII pathway is critical for the gp120-induced expression of IL-1ß, whereas the Wnt5a/JNK pathway is for TNF-α expression. Meanwhile, the expression of IL-6 is co-regulated by both pathways. These results collectively suggest that Wnt5a signaling cascades play a crucial role in the regulation of gp120-induced expression of pro-inflammatory cytokines in the CNS.

MicroRNA-452 promotes tumorigenesis in hepatocellular carcinoma by targeting cyclin-dependent kinase inhibitor 1B.

Dysregulation of miR-452 has been observed in many tumors, but its biological function in hepatocellular carcinoma (HCC) is still unknown. Our results showed that miR-452 expression is significantly increased in HCC tissues and HCC cell lines. We also found that overexpression of miR-452 dramatically accelerated proliferation, induced cell cycle from G1 to S transition, and blocked apoptosis of HCC cells. Migration and matrigel invasion assays indicated that miR-452 significantly promotes HepG2 and QGY-7703 cells migration and invasion in vitro. Further studies showed that miR-452 directly targets the 3'-untranslated region of cyclin-dependent kinase inhibitor 1B (CDKN1B), ectopic miR-452 expression suppressed CDKN1B expression on mRNA and protein level. Silencing CDKN1B by small interfering RNA resembled the phenotype resulting from ectopic miR-452 expression. This study provides new insights into the potential molecular mechanisms that miRNA-452 contributed to HCC.

Anti-diabetic effects of CTB-APSL fusion protein in type 2 diabetic mice.

To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.

The Chinese Hamster Ovary Cell-Based H9 HA Subunit Avian Influenza Vaccine Provides Complete Protection against the H9N2 Virus Challenge in Chickens.

(1) Background: Avian influenza has attracted widespread attention because of its severe effect on the poultry industry and potential threat to human health. The H9N2 subtype of avian influenza viruses was the most prevalent in chickens, and there are several commercial vaccines available for the prevention of the H9N2 subtype of avian influenza viruses. However, due to the prompt antigenic drift and antigenic shift of influenza viruses, outbreaks of H9N2 viruses still continuously occur, so surveillance and vaccine updates for H9N2 subtype avian influenza viruses are particularly important. (2) Methods: In this study, we constructed a stable Chinese hamster ovary cell line (CHO) to express the H9 hemagglutinin (HA) protein of the major prevalent H9N2 strain A/chicken/Daye/DY0602/2017 with genetic engineering technology, and then a subunit H9 avian influenza vaccine was prepared using the purified HA protein with a water-in-oil adjuvant. (3) Results: The results showed that the HI antibodies significantly increased after vaccination with the H9 subunit vaccine in specific-pathogen-free (SPF) chickens with a dose-dependent potency of the immunized HA protein, and the 50 µg or more per dose HA protein could provide complete protection against the H9N2 virus challenge. (4) Conclusions: These results indicate that the CHO expression system could be a platform used to develop the subunit vaccine against H9 influenza viruses in chickens.

RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori.

BACKGROUND: Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm. RESULTS: Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm. CONCLUSIONS: Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect development and evolution.

Progress on innate immune evasion and live attenuated vaccine of pseudorabies virus.

Pseudorabies virus (PRV) is a highly infectious disease that can infect most mammals, with pigs as the only natural host, has caused considerable economic losses to the pig husbandry of the world. Innate immunity is the first defense line of the host against the attack of pathogens and is essential for the proper establishment of adaptive immunity. The host uses the innate immune response to against the invasion of PRV; however PRV makes use of various strategies to inhibit the innate immunity to promote the virus replication. Currently, live attenuated vaccine is used to prevent pig from infection with the PRV worldwide, such as Bartha K61. However, a growing number of data indicates that these vaccines do not provide complete protection against new PRV variants that have emerged since late 2011. Here we summarized the interactions between PRV and host innate immunity and the current status of live attenuated PRV vaccines to promote the development of novel and more effective PRV vaccines.

Lipopolysaccharide promotes NLRP3 inflammasome activation by inhibiting TFEB-mediated autophagy in NRK-52E cells.

The NLRP3 inflammasome is involved in many inflammatory diseases. Its activity must be strictly controlled to alleviate the inflammatory process. Autophagy plays a protective role in the negative regulation of NLRP3 inflammasome activation. However, the regulatory mechanism of autophagy controlling NLRP3 inflammasome activation remains to be further investigated. Here, we showed that in NRK-52E cells, lipopolysaccharide (LPS) and ATP stimulation significantly decreased mitochondrial membrane potential, increased ROS production and mtDNA copy number in cytosol. Moreover, autophagic flux was blocked when challenged with LPS and ATP as evidenced by increased LC3 II and p62 expression, reduced TFEB and CTSD expression, and impaired lysosomal acid environment. Furthermore, TFEB deficiency increased cytosolic mtDNA and enhanced LPS and ATP induced NLRP3 inflammasome activation and proinflammatory cytokine expression. Taken together, these findings reveal that LPS and ATP stimulation promoted NLRP3 inflammasome activation through inhibiting TFEB-mediated autophagy in NRK-52E cells, and TFEB could be a potential therapeutic target for the treatment of NLRP3 inflammasome-related kidney diseases.

Chitosan-Based Nanomaterial as Immune Adjuvant and Delivery Carrier for Vaccines.

With the support of modern biotechnology, vaccine technology continues to iterate. The safety and efficacy of vaccines are some of the most important areas of development in the field. As a natural substance, chitosan is widely used in numerous fields-such as immune stimulation, drug delivery, wound healing, and antibacterial procedures-due to its good biocompatibility, low toxicity, biodegradability, and adhesion. Chitosan-based nanoparticles (NPs) have attracted extensive attention with respect to vaccine adjuvants and delivery systems due to their excellent properties, which can effectively enhance immune responses. Here, we list the classifications and mechanisms of action of vaccine adjuvants. At the same time, the preparation methods of chitosan, its NPs, and their mechanism of action in the delivery system are introduced. The extensive applications of chitosan and its NPs in protein vaccines and nucleic acid vaccines are also introduced. This paper reviewed the latest research progress of chitosan-based NPs in vaccine adjuvant and drug delivery systems.

Protective Effect of Phloretin against Hydrogen Peroxide-Induced Oxidative Damage by Enhancing Autophagic Flux in DF-1 Cells.

Phloretin (PHL) is a dihydrochalcone flavonoid isolated from the peel and root bark of apples, strawberries, and other plants with antioxidative characteristic. In this study, we aimed to investigate the protective effect and the potential mechanism of PHL on hydrogen peroxide (H2O2)-induced oxidative damage in DF-1 cells. The results showed that PHL exhibited no cytotoxic effect on DF-1 cells at concentration below 20 µM. PHL markedly increased H2O2-reduced cell viability, decreased H2O2-induced apoptosis, as evidenced by reduced apoptosis rate, the upregulation of gene and protein level of Bcl-2, and the downregulation of gene and protein level of Bax and Cleaved caspase3. In addition, PHL reduced H2O2-induced reactive oxygen species (ROS) production and restored antioxidant enzymes activities as well as mitochondrial membrane potential in a dose-dependent manner. Moreover, PHL prior to H2O2 further increased LC3-II level, promoted p62 turnover and improved lysosomal function. Importantly, autophagy inhibitor chloroquine (CQ) reversed the protective effect of PHL, and increased H2O2-induced apoptosis. Furthermore, PHL inhibited the phosphorylation levels of ERK, p38, and JNK. Collectively, these results indicate that PHL could attenuate H2O2-induced oxidative injury and apoptosis by maintaining lysosomal function and promoting autophagic flux, and MAPKs pathway may be involved in this process. Our study provides evidence that PHL could as a new strategy to against oxidative damage in poultry industry.

ARNT Inhibits H5N1 Influenza A Virus Replication by Interacting with the PA Protein.

Increasing evidence suggests that the polymerase acidic (PA) protein of influenza A viruses plays an important role in viral replication and pathogenicity. However, information regarding the interaction(s) of host factors with PA is scarce. By using a yeast two-hybrid screen, we identified a novel host factor, aryl hydrocarbon receptor nuclear translocator (ARNT), that interacts with the PA protein of the H5N1 virus. The interaction between PA and human ARNT was confirmed by co-immunoprecipitation and immunofluorescence microscopy. Moreover, overexpression of ARNT downregulated the polymerase activity and inhibited virus propagation, whereas knockdown of ARNT significantly increased the polymerase activity and virus replication. Mechanistically, overexpression of ARNT resulted in the accumulation of PA protein in the nucleus and inhibited both the replication and transcription of the viral genome. Interaction domain mapping revealed that the bHLH/PAS domain of ARNT mainly interacted with the C-terminal domain of PA. Together, our results demonstrate that ARNT inhibits the replication of the H5N1 virus and could be a target for the development of therapeutic strategies against H5N1 influenza viruses.

KAP1 Positively Modulates Influenza A Virus Replication by Interacting with PB2 and NS1 Proteins in Human Lung Epithelial Cells.

Influenza virus only encodes a dozen of viral proteins, which need to use host machinery to complete the viral life cycle. Previously, KAP1 was identified as one host protein that potentially interacts with influenza viral proteins in HEK 293 cells. However, the role of KAP1 in influenza virus replication in human lung alveolar epithelial cells and the underlying mechanism remains unclear. In this study, we first generated KAP1 KO A549 cells by CRISPR/Cas9 gene editing. KAP1 deletion had no significant effect on the cell viability and lack of KAP1 expression significantly reduced the influenza A virus replication. Moreover, we demonstrated that KAP1 is involved in the influenza virus entry, transcription/replication of viral genome, and viral protein synthesis in human lung epithelial cells and confirmed that KAP1 interacted with PB2 and NS1 viral proteins during the virus infection. Further study showed that KAP1 inhibited the production of type I IFN and overexpression of KAP1 significantly reduced the IFN-ß production. In addition, influenza virus infection induces the deSUMOylation and enhanced phosphorylation of KAP1. Our results suggested that KAP1 is required for the replication of influenza A virus and mediates the replication of influenza A virus by facilitating viral infectivity and synthesis of viral proteins, enhancing viral polymerase activity, and inhibiting the type I IFN production.

A Glycolipid α-GalCer Derivative, 7DW8-5 as a Novel Mucosal Adjuvant for the Split Inactivated Influenza Vaccine.

Influenza virus infects the host and transmits through the respiratory tract (i.e., the mouth and nose); therefore, the development of intranasal influenza vaccines that mimic the natural infection, coupled with an efficient mucosal adjuvant, is an attractive alternative to current parenteral vaccines. However, with the withdrawal of cholera toxin and Escherichia coli heat-labile endotoxin from clinical use due to side effects, there are no approved adjuvants for intranasal vaccines. Therefore, safe and effective mucosal adjuvants are urgently needed. Previously, we reported that one derivative of α-Galactosylceramide (α-GalCer), 7DW8-5, could enhance the protective efficacy of split influenza vaccine by injection administration. However, the mucosal adjuvanticity of 7DW8-5 is still unclear. In this study, we found that 7DW8-5 promotes the production of secret IgA antibodies and IgG antibodies and enhances the protective efficacy of the split influenza vaccine by intranasal administration. Furthermore, co-administration of 7DW8-5 with the split influenza vaccine significantly reduces the virus shedding in the upper and lower respiratory tract after lethal challenge. Our results demonstrate that 7DW8-5 is a novel mucosal adjuvant for the split influenza vaccine.

Porcine Epidemic Diarrhea Virus Induces Vero Cell Apoptosis via the p53-PUMA Signaling Pathway.

Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.