ISGylation by HERCs facilitates STING activation.

Optimal activation of stimulator of interferon genes (STING) protein is crucial for host defenses against pathogens and avoiding detrimental effects. Various post-translational modifications control STING activity. However, the function of interferon (IFN)-stimulated gene (ISG) 15 modification (ISGylation) in controlling STING stability and activation is unclear. Here, we show that the E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice) facilitate STING activation by mediating ISGylation of STING at K150, preventing its K48-linked ubiquitination and degradation. Concordantly, Herc6 deficiency suppresses herpes simplex virus 1 infection-induced type I IFN responses and facilitates viral replication both in vitro and in vivo. Notably, severe acute respiratory syndrome coronavirus 2 protein papain-like protease cleaves HERC5-mediated ISGylation of STING, suppressing host antiviral responses. These data identify a mechanism by which HERCs-mediated ISGylation controls STING stability and activation and uncover the correlations and interactions of ISGylation and ubiquitination during STING activation.

A High MCT-Based Ketogenic Diet Suppresses Th1 and Th17 Responses to Ameliorate Experimental Autoimmune Encephalomyelitis in Mice by Inhibiting GSDMD and JAK2-STAT3/4 Pathways.

SCOPE: Inflammation and pyroptosis play important roles in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In this study, we evaluated the therapeutic potential of ketogenic diet (KD) in EAE. METHODS AND RESULTS: The administration of KD reduces demyelination and microglial activation in the spinal cord of EAE mice. Meanwhile, KD decreases the levels of Th1 and Th17 associated cytokines/transcription factors production (T-bet, IFN-γ, RORγt, and IL-17) and increases those of Th2 and Treg cytokines/transcription factors (GATA3, IL-4, Foxp3, and IL-10) in the spinal cord and spleen. Corresponding, KD reduces the expression of chemokines in EAE, which those chemokines associate with T-cell infiltration into central nervous system (CNS). In addition, KD inhibits the GSDMD activation in microglia, oligodendrocyte, CD31+ cells, CCR2+ cells, and T cells in the spinal cord. Moreover, KD significantly decreases the ratios of p-JAK2/JAK2, p-STAT3/STAT3, and p-STAT4/STAT4, as well as GSDMD in EAE mice. CONCLUSIONS: this study demonstrates that KD reduces the activation and differentiation of T cells in the spinal cord and spleen and prevents T cell infiltration into CNS of EAE via modulating the GSDMD and STAT3/4 pathways, suggesting that KD is a potentially effective strategy in the treatment of MS.

Over-expression of IL-33 enhances rabies virus early antigen presentations and cellular immune responses in mice.

Human inactivated rabies virus (RABV) vaccines have been widely used worldwide over 30 years. The mechanisms of humoral immunity elicited by previously reported rabies candidate vaccines have been fully investigated, but little is known about the cellular immunity profiles. Herein, the recombinant RABV rLBNSE-IL-33 overexpressing the mouse interleukin-33 (IL-33) proliferated well in Neuro-2a cells and had no effects with the parent virus on growth kinetic in vitro and viral pathogenicity in mice. The rLBNSE-IL-33 experienced more antigen presentations by MHC-II on DCs and activated more CD4+ T cells which helped recruit more CD19+CD40+ B cells in blood and promote rapid and robust IgG1 antibodies responses at initial infection stage compared with the parent rLBNSE strain. Simultaneously, the rLBNSE-IL-33 were also presented by MHC-I to CD8+ T cells which contributed to produce high levels of IgG2a. The rLBNSE-IL-33 elicited significantly high levels of RABV-specific IFN-γ secreting memory CD4+ T cells, more RABV-specific IL-4 and IFN-γ secreting memory CD8+ T cells in spleens at early infection stage in mice. Altogether, overexpression of IL-33 in rLBNSE-IL-33 enhanced early antigen presentation, markedly promote CD4+, memory CD4+ and CD8+ T cells-mediated responses and provided a 100 % protection from lethal RABV challenge in mice. These findings provided an alternative novel therapy and vaccine strategy in future.

Persistent and Severe Viral Replication in PBMCs with Moderate Immunosuppression Served an Alternative Novel Pathogenic Mechanism for Canine Morbillivirus.

Measles virus and canine distemper virus (CDV) cause lethal infections in their respective hosts characterized by severe immunosuppression. To furtherly acknowledge the attenuated mechanisms of the regionally ongoing epidemic CDV isolates and provide novel perspectives for designing new vaccines and therapeutic drugs, a recombinant CDV rHBF-vacH was employed with a vaccine hemagglutinin (H) gene replacement by reverse genetics based on an infectious cDNA clone for the CDV wild-type HBF-1 strain. Interestingly, unlike previously published reports that a vaccine H protein completely changed a pathogenic wild-type CDV variant to be avirulent, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets with a prolonged period of disease. Further comparisons of pathogenic mechanisms proved that the weaker but necessary invasions into peripheral blood mononuclear cells (PBMCs) of rHBF-vacH, and subsequently persistent viral replications in PBMCs and multiple organs, together contributed to its 66.7% mortality. In addition, despite significantly higher titers than the parent viruses, rHBF-vacH would not be a suitable candidate for a live vaccine, with great invasion and infection potentials of PBMCs from 16 tested kinds of host species. Altogether, sustained and severe viral replication in PBMCs with moderate immunosuppression was first proven to be an alternative novel pathogenic mechanism for CDV, which might help us to understand possible reasons for CDV fatal infections among domestic dogs and the highly susceptible wild species during natural transmission. IMPORTANCE Despite widespread vaccine campaigns for domestic dogs, CDV remained an important infectious disease in vaccinated carnivores and wild species. In recent years, the regionally ongoing epidemic CDV isolates have emphasized conservation threats to, and potentially disastrous epidemics in, endangered species worldwide. However, little is known about how to deal with the CDV variants constantly regional epidemic. In this study, we employed a recombinant CDV rHBF-vacH with a vaccine H gene replacement in a CDV wild-type HBF-1 context to attenuate the epidemic CDV variant to design a new vaccine candidate. Interestingly, rHBF-vacH was only partially attenuated by alleviating the degree of viral immunosuppression, and still caused 66.7% lethality in ferrets by weaker but necessary invasions into PBMCs, and subsequently persistent and severe viral replications in PBMCs. Significantly higher virus titers of rHBF-vacH in vitro might indicate the rapid cell-to-cell spreads in vivo that indirectly contribute to fatal infections of rHBF-vacH in ferrets.

HDAC3 Inhibitor RGFP966 Ameliorated Neuroinflammation in the Cuprizone-Induced Demyelinating Mouse Model and LPS-Stimulated BV2 Cells by Downregulating the P2X7R/STAT3/NF-κB65/NLRP3 Activation.

Suppression of excessive microglial overactivation can prevent the progression of multiple sclerosis (MS). Histone deacetylases 3 inhibitor (HDAC3i) has been demonstrated to exert anti-inflammatory effects by suppressing microglia (M1-liked) activation. Here, we demonstrate that the RGFP966 (a selective inhibitor of HDAC3) protects white matter after cuprizone-induced demyelination, as shown by reductions in neurological behavioral deficits and increases in myelin basic protein. Moreover, in this study, we found that RGFP966 caused a significant reduction in the levels of inflammatory cytokines, including IL-1ß, TNF-α, as well as iNOS, and inhibited microglial (M1-liked) activation in the experimental cuprizone model and LPS-stimulated BV2 cells. Meanwhile, RGFP966 alleviated apoptosis of LPS-induced BV2 cells in vitro. Furthermore, RGFP966 suppressed the expression of P2X7R, NLRP3, ASC, IL-18, IL-1ß, and caspase-1, inhibited the ratio of phosphorylated-STAT3/STAT3 and phosphorylated NF-κB p65/NF-κB p65, as well as increased acetylated NF-κB p65 in vitro and in vivo. Furthermore, we confirmed that brilliant blue G (antagonists of P2X7R) suppressed the expression of microglial NLRP3, IL-18, IL-1ß, caspase-1, NF-κB p65 (including phosphorylated NF-κB p65), and STAT3 (including phosphorylated STAT3) in vitro. These findings demonstrated that RFFP966 alleviated the inflammatory response and exerted a neuroprotective effect possibly by modulating P2X7R/STAT3/NF-κB65/NLRP3 signaling pathways. Thus, HDAD3 might be considered a promising intervention target for neurodegenerative diseases, such as MS.

Recombinant adeno-associated virus serotype 9 AAV-RABVG expressing a Rabies Virus G protein confers long-lasting immune responses in mice and non-human primates.

Three or four intramuscular doses of the inactivated human rabies virus vaccines are needed for pre- or post-exposure prophylaxis in humans. This procedure has made a great contribution to prevent human rabies deaths, which bring huge economic burdens in developing countries. Herein, a recombinant adeno-associated virus serotype 9, AAV9-RABVG, harbouring a RABV G gene, was generated to serve as a single dose rabies vaccine candidate. The RABV G protein was stably expressed in the 293T cells infected with AAV9-RABVG. A single dose of 2 × 1011 v.p. of AAV9-RABVG induced robust and long-term positive seroconversions in BALB/c mice with a 100% survival from a lethal RABV challenge. In Cynomolgus Macaques vaccinated with a single dose of 1 × 1013 v.p. of AAV9-RABVG, the titres of rabies VNAs increased remarkably from 2 weeks after immunity, and maintained over 31.525 IU/ml at 52 weeks. More DCs were activated significantly for efficient antigen presentations of RABV G protein, and more B cells were activated to be responsible for antibody responses. Significantly more RABV G specific IFN-γ-secreting CD4+ and CD8+ T cells, and IL-4-secreting CD4+ T cells were activated, and significantly higher levels of IL-2, IFN-γ, IL-4, and IL-10 were secreted to aid immune responses. Overall, the AAV9-RABVG was a single dose rabies vaccine candidate with great promising by inducing robust, long-term humoral responses and both Th1 and Th2 cell-mediated immune responses in mice and non-human primates.

Preparation, Characterization, Evaluation of Neuroprotective Effect, and Related Mechanisms of Phosphatidylserine Emulsion in 5- and 12-Week Old Mice.

Phosphatidylserine (PS) improves learning and memory capacity. In this study, PS formulation was optimized by a response surface methodology. Moreover, we found that PS not only functions as a biologically active component in food preparations but also improves the emulsion's physical stability. Our results showed that the PS emulsions are characterized by a smaller particle size, higher ζ-potential (negative), higher viscosity, and lower surface tension and centrifugal stability constants than the emulsion without PS. Furthermore, we explored the neuroprotective effects of PS emulsion and its underlying mechanisms. Treatment with 2% (w/w) PS emulsion for three months enhanced spatial learning and memory in 5- and 12-week old mice in the Morris water maze test. Western-blotting analysis displayed that the 2% (w/w) PS emulsion treated group upregulated BDNF, TrkB, PSD95, mTOR, MBP, and ErbB4 expression in the hippocampus of 5- and 12-week old mice. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed elevated Nrg-1 and ErbB4 mRNA expression in the 2% (w/w) PS emulsion treated groups, and high Nrg-1 and ErbB4 expression levels were associated with better myelination. In conclusion, we reported PS emulsions with high stability and high bioavailability. Meanwhile, 2% (w/w) PS emulsion enhances learning, memory, and myelination in mice by activating the BDNF/TrkB and Nrg-1/ErbB4 signaling.

Research progress on neutralizing epitopes and antibodies for the Rabies virus.

Rabies is a zoonotic infectious disease with a high fatality rate. It is caused by a virus in the genus Lyssavirus and is a global public health threat. The rabies virus invades and infects cells mainly via a glycoprotein, which may involve multiple receptors. Neutralizing antibodies against the rabies virus function by blocking the binding of the glycoprotein to a receptor or preventing the membrane fusion process. Vaccination combined with anti-rabies virus neutralizing antibodies is essential for postexposure prophylaxis for category III exposure to the rabies virus. In this review, we discussed the neutralizing epitopes of the rabies virus and the neutralization mechanism of monoclonal antibodies. The neutralizing antibodies that have been commercialized or are under development are also summarized. Our review would provide a basis for the further development of safe and effective broad-spectrum neutralizing antibodies to replace the rabies virus immunoglobulin in rabies post-exposure prophylaxis.

A recombinant rabies virus expressing Echinococcus granulosus EG95 induces protective immunity in mice.

Cystic echinococcosis (CE), caused by Echinococcus granulosus (E, is a zoonosis with a worldwide distribution, resulting in heavy impact to public health and social economics. In this study, we generated a recombinant rabies virus (RABV) expressing EG95 protein of E. granulosus (LBNSE-EG95) as a bivalent candidate vaccine for use in sheep and cattle against CE and rabies, which is another severe health threat in CE-endemic areas. It was found that EG95 was successfully expressed without altering the pathogenicity of parent LBNSE vector. Further study showed that LBNSE-EG95 immunization in mice elicited activation of dendric cells (DCs) and B cells and induced Th1-/Th2-mediated cellular immune responses, leading to robust production of RABV neutralizing antibodies and high level of EG95-sepecific antibodies with more than 90% protection against CE. In addition, single dose of LBNSE-EG95 conferred full protection against lethal RABV challenge in mice. Collectively, these results suggest that the recombinant LBNSE-EG95 has the potential to be developed as an efficient bivalent vaccine for sheep and cattle use in endemic areas of CE and rabies.

Western equine encephalitis virus virus-like particles from an insect cell-baculovirus system elicit the strong immune responses in mice.

Western equine encephalitis virus (WEEV) causes lethal encephalitis in humans and equines, and it poses a serious public health threat in many countries. Therefore, the development of an efficient vaccine remains an important challenge for the prevention of WEEV infection. This study presents the first description of WEEV virus-like particles (VLPs) generated from insect cells using recombinant baculoviruses. WEEV VLPs with 206 adjuvant could trigger a strong cellular immune response; increase the levels of IL-2, IL-4 and IFN-γ; and induce a high level of neutralizing antibodies against WEEV in mice. These data showed that the insect cell-baculovirus system is suitable for the production of WEEV VLPs and that these VLPs could elicit the strong immunogenicity in mice. These results suggest a new, nonreplicating, and effective vaccine candidate against WEEV infection.

A rabies virus vectored severe fever with thrombocytopenia syndrome (SFTS) bivalent candidate vaccine confers protective immune responses in mice.

The Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic zoonotic disease, which is potentially fatal in human with mortality rates ranging from 16.2%-32%. The rabies virus (RABV) LBNSE vector expressing foreign antigens have shown considerable promise as vaccines against viral diseases, which is effective and safe. In the present study, we generated a recombinant RABV rLBNSE-Gn expressing a SFTSV glycoprotein Gn by reverse genetic technology to control rabies and SFTS in animals. An extra insertion of Gn gene did not impact replication of the recombinant virus rLBNSE-Gn in NA and BHK-21 cells compared to the parent rLBNSE strain. The SFTSV Gn gene together with RABV N and G genes were efficiently expressed in rLBNSE-infected Vero cells by immunostaining and immune blots. A single dose of 107 FFU of the rLBNSE-Gn intramuscularly inoculated in BALB/c mice induced rapid and robust humoral responses against both RABV and SFTSV without any signs of disease or weight loss. Compared to the rLBNSE and DMEM groups, the extra Gn expression contributed to the recruitments and/or activations of the dendritic cells and B cells from inguinal lymph nodes of BALB/c mice vaccinated with rLBNSE-Gn. The protective efficacy of rLBNSE-Gn against SFTSV in C57BL/6 mice was evaluated, and the virus loading in the spleens reduced to 10 TCID50/mg at 7 days post SFTSV infections, which indicated that the rLBNSE-Gn conferred efficacious protective immune responses from SFTSV in C57BL/6 mice. All the mice immunization with rLBNSE-Gn and rLBNSE survived after a lethal RABV challenge, suggesting a 100 % protection from RABV. Therefore, the rLBNSE-Gn would be a promising bivalent candidate vaccine against SFTS and rabies in animals.

Genetically modified rabies virus vector-based bovine ephemeral fever virus vaccine induces protective immune responses against BEFV and RABV in mice.

Bovine ephemeral fever (BEF), caused by the bovine ephemeral fever virus (BEFV), is associated with an acute febrile infection in cattle and widespread in tropical and subtropical areas, leading to great economic losses to cattle and milk industry. However, no efficacious BEF vaccine is currently available in China. Herein, we generated a recombinant rabies virus (RABV) expressing BEFV glycoprotein (LBNSE-BG), utilizing a reverse genetics system based on the recombinant rabies virus strain LBNSE. It was found that mice immunized with LBNSE-BG produced robust neutralizing antibodies against both BEFV and RABV, and developed complete protection from lethal RABV challenge. Further studies showed that LBNSE-BG activated more dendritic cells (DCs), B cells and T cells in immunized mice than the parent virus LBNSE. Collectively, these findings demonstrate that the recombinant LBNSE-BG described here has the potential to be developed as a cost-effective and efficacious bivalent vaccine for cattle use in endemic areas of BEF and rabies.

Cuprizone-Induced Demyelination in Mouse Hippocampus Is Alleviated by Ketogenic Diet.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Recently, ketogenic diet (KD) supplementation has attracted great interest. Therefore, we established the cuprizone (CPZ)-induced demyelination mouse model to investigate the possible neuroprotective effect of KD on the hippocampus of mice. We found that KD significantly elevated the level of serum ß-hydroxybutyric acid, improved behavioral and motor abnormalities, and impaired the spatial learning and memory of CPZ-induced demyelination mice. Meanwhile, KD lessened the hippocampal demyelination by enhancing the expression of mature oligodendrocytes (OLs), which was revealed by the elevated expression of MBP and CNPase, as well as the luxol fast blue-staining intensity. Furthermore, KD inhibits the activation of microglia (especially M1-like microglia) and reactive astrocytes. Interestingly, KD attenuated the CPZ-induced oxidative stress by decreasing the malondialdehyde (MDA) content and restoring the glutathione (GSH) levels. In addition, the double immunofluorescence staining revealed that KD enhanced the expression of SIRT1 in astrocytes, microglia, and mature oligodendrocytes. Concomitantly, Western blot demonstrated that KD increased the expression of SIRT1, phosphorylated-AKT, mTOR, and PPAR-γ. In conclusion, KD exerted a neuroprotective effect on CPZ-induced demyelination mice, and this activity was associated with the modulation of the SIRT1/PPAR-γ and SIRT1/P-Akt/mTOR pathways.

Ketogenic Diet Elicits Antitumor Properties through Inducing Oxidative Stress, Inhibiting MMP-9 Expression, and Rebalancing M1/M2 Tumor-Associated Macrophage Phenotype in a Mouse Model of Colon Cancer.

Many advanced cancers are characterized by metabolic disorders. A dietary therapeutic strategy was proposed to inhibit tumor growth through administration of low-carbohydrate, average-protein, and high-fat diet, which is also known as ketogenic diet (KD). In vivo antitumor efficacy of KD on transplanted CT26+ tumor cells in BALB/c mice was investigated. The results showed that the KD group had significantly higher blood ß-hydroxybutyrate and lower blood glucose levels when compared with the normal diet group. Meanwhile, KD increased intratumor oxidative stress, and TUNEL staining showed KD-induced apoptosis against tumor cells. Interestingly, the distribution of CD16/32+ and iNOS+ M1 tumor-associated macrophages (TAMs) increased in the KD-treated group, with concomitantly less arginase-1+ M2 TAMs. Moreover, KD treatment downregulated the protein expression of matrix metalloproteinase-9 in CT26+ tumor-bearing mice. Western blot analysis demonstrated that the expression levels of HDAC3/PKM2/NF-κB 65/p-Stat3 proteins were reduced in the KD-treated group. Taken together, our results indicated that KD can prevent the progression of colon tumor via inducing intratumor oxidative stress, inhibiting the expression of the MMP-9, and enhancing M2 to M1 TAM polarization. A novel potential mechanism was identified that KD can prevent the progression of colon cancer by regulating the expression of HDAC3/PKM2/NF-κB65/p-Stat3 axis.

A Bivalent Human Adenovirus Type 5 Vaccine Expressing the Rabies Virus Glycoprotein and Canine Distemper Virus Hemagglutinin Protein Confers Protective Immunity in Mice and Foxes.

The development of a safe and efficient multivalent vaccine has great prospects for application. Both rabies virus (RABV) and canine distemper virus (CDV) are highly infectious antigens, causing lethal diseases in domestic dogs and other carnivores worldwide. In this study, a replication-deficient human adenovirus 5 (Ad5)-vectored vaccine, rAd5-G-H, expressing RABV glycoprotein (G) and CDV hemagglutinin (H) protein was constructed. The RABV G and CDV H protein of rAd5-G-H were expressed and confirmed in infected HEK-293 cells by indirect immunofluorescence assay. The rAd5-G-H retained a homogeneous icosahedral morphology similar to rAd5-GFP under an electron microscope. A single dose of 108 GFU of rAd5-G-H administered to mice by intramuscular injection elicited rapid and robust neutralizing antibodies against RABV and CDV. Flow cytometry assays indicated that the dendritic cells and B cells in inguinal lymph nodes were significantly recruited in rAd5-G-H-immunized mice in comparison with the mock and rAd5-GFP groups. rAd5-G-H also activated the Th1- and Th2-mediated cell immune responses against RABV and CDV in mice, which contributed to 100% survival of a lethal-dose RABV challenge without any clinical signs. In foxes, a single dose of 109 GFU of rAd5-G-H could elicit high levels of neutralizing antibodies against both RABV and CDV in comparison with the mock and rAd5-GFP groups. All foxes in the rAd5-GFP and mock groups died, while the foxes inoculated with rAd5-G-H all survived and showed no clinical signs of disease after being challenged with a lethal wild-type CDV strain. These results suggested that rAd5-G-H has great potential as a bivalent vaccine against rabies and canine distemper in highly susceptible dogs and wildlife animals.

Recombinant Human Adenovirus Type 5 Co-expressing RABV G and SFTSV Gn Induces Protective Immunity Against Rabies Virus and Severe Fever With Thrombocytopenia Syndrome Virus in Mice.

Both severe fever with thrombocytopenia syndrome (SFTS) and rabies are severe zoonotic diseases. As co-hosts of rabies virus (RABV) and SFTS virus (SFTSV), dogs and cats could not only be infected but also transmit the virus to human. Hence, developing a bivalent vaccine against both SFTS and rabies is urgently needed. In this study, we generated a recombinant replication-deficient human adenovirus type 5 (Ad5) co-expressing RABV G and SFTSV Gn (Ad5-G-Gn) and evaluated its immunogenicity and efficacy in mice. Ad5-G-Gn immunization activated more dendritic cells (DCs) and B cells in lymph nodes (LNs) and induced Th1-/Th2-mediated responses in splenocytes, leading to robust production of neutralizing antibodies against SFTSV and RABV. In addition, single dose of Ad5-G-Gn conferred mice complete protection against lethal RABV challenge and significantly reduced splenic SFTS viral load. Therefore, our data support further development of Ad5-G-Gn as a potential bivalent vaccine candidate against SFTS and rabies for dog and cat use.

Amelioration of clinical course and demyelination in the cuprizone mouse model in relation to ketogenic diet.

Ketogenic diet (KD) is defined as a high-fat, low-carbohydrate diet with appropriate amounts of protein, which has broad neuroprotective effects. However, the mechanisms of ameliorating the demyelination and of the neuroprotective effects of KD have not yet been completely elucidated. Therefore, the present study investigated the protection mechanism of KD treatment in the cuprizone (bis-cyclohexanone oxalydihydrazone, CPZ)-induced demyelination mice model, with special emphasis on neuroinflammation. After the KD treatment, an increased ketone body level in the blood of mice was detected, and a significant increase in the distance traveled within the central area was observed in the open field test, which reflected the increased exploration and decreased anxiety of mice that received CPZ. The results of Luxol fast blue and myelin basic protein (MBP) immunohistochemistry staining for the evaluation of the myelin content within the corpus callosum revealed a noticeable increase in the number of myelinated fibers and myelin score after KD administration in these animals. Concomitant, the protein expressions of glial fibrillary acidic protein (GFAP, an astrocyte marker), ionized calcium-binding adaptor molecule 1 (Iba-1, a microglial marker), CD68 (an activated microglia marker) and CD16/32 (a M1 microglial marker) were down-regulated, while the expression of oligodendrocyte lineage transcription factor 2 (OLIG2, an oligodendrocyte precursor cells marker) was up-regulated by the KD treatment. In addition, the KD treatment not only reduced the level of the C-X-C motif chemokine 10 (CXCL10), which is correlated to the recruitment of activated microglia, but also inhibited the production of proinflammatory cytokines, including interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), which are closely correlated to the M1 phenotype microglia. It is noteworthy, that the expression levels of histone deacetylase 3 (HADC3) and nod-like receptor pyrin domain containing 3 (NLRP3) significantly decreased after KD administration. In conclusion, these data demonstrate that KD decreased the reactive astrocytes and activated the microglia in the corpus callosum, and that KD inhibited the HADC3 and NLRP3 inflammasome signaling pathway in CPZ-treated mice. This suggests that the inhibition of the HADC3 and NLRP3 signaling pathway may be a novel mechanism by which KD exerts its protective actions for the treatment of demyelinating diseases.

Immunogenicity Assessment of Rift Valley Fever Virus Virus-Like Particles in BALB/c Mice.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and is spread by arthropod vectors. Virus-like particle (VLP) vaccines, which have the advantages of strong immunogenicity and safety, play an important role in the prevention of this disease. VLPs for RVFV were successfully prepared by our research group using a baculovirus-insect cell expression system. To study the immunogenicity of these RVFV VLPs, a correct 3rd or 4th generation recombinant baculovirus, rBac-N-G, was identified and used to infect Sf9 cells, which were cultured in suspension at a large scale. Subsequently, cell debris was removed by centrifugation, and the VLPs were concentrated by ultracentrifugation and purified using a sucrose gradient, after which they were used to immunize BALB/c mice by intramuscular injection. The results showed that the RVFV VLPs prepared by our research group could effectively induce mice to produce RVFV neutralizing antibodies and that the prepared VLPs could stimulate mouse spleen cells to produce high levels of the cytokines IL-4 and IFN-γ. Moreover, the proportion of lymphocytes producing IL-4 and IFN-γ in the spleen of mice immunized with RVFV VLPs was significantly increased. Therefore, the RVFV VLPs prepared in this study had strong immunogenicity and could effectively activate humoral and cellular immunity in mice. This study lays a solid foundation for the development of RVFV VLP vaccine candidates and promotes the healthy development of animal husbandry and human public health.

RNA sequencing analyses of gene expressions in a canine macrophages cell line DH82 infected with canine distemper virus.

Virulent morbillivirus infections, including Meals Virus (MeV) and Canine Distemper Virus (CDV), caused severe immune suppression and leukopenia, while attenuated vaccine strains developed protective host immune responses. However, the detailed molecular foundations of host antiviral responses were poorly characterized. In order to better understand the interactions between attenuated vaccine and host antiviral responses, the global gene expression changes in CDV-11-infected DH82 cells, a macrophage-derived cell line from canine, were investigated by transcriptomic analysis, and portions of results were confirmed with quantitative RT-PCR. The results exhibited that 372 genes significantly up-regulated (p < .01) and 119 genes were significantly down-regulated (p < .01) in CDV-infected macrophages DH82 at 48 h p.i.. The enriched functions of the significantly up-regulated (p < .01) genes were closely associated with interferon stimulated genes (ISGs), chemokine genes and pro-inflammatory factor genes. Gene ontology and pathway analysis of differentially expressed genes (DEGs) revealed that the most significantly involved pathways in CDV-infected DH82 cells were NF-κB and TNF signaling pathway, cytokine-cytokine receptor interaction, and pathogen associated molecular patterns (PAMPs), such as Toll-like, RIG-I-like and NOD-like receptor signalings. Thus, the findings indicated that pattern recognition receptors (PRRs) possibly mediated host innate and protective antiviral immune responses in CDV-11 infected DH82 cells.

Assembly of pigeon circovirus-like particles using baculovirus expression system.

Pigeon circovirus (PiCV) is able to infect racing and meat pigeons of all ages and is a key factor that triggers young pigeon disease syndrome (YPDS). PiCV vaccine research has been impeded because PiCV cannot be grown or propagated in cell cultures. Virus-like particles (VLPs), which can be generated by a wide range of expression systems, have been shown to have outstanding immunogenicity and constitute promising vaccines against a wide range of pathogens. Cap protein, which contains neutralizing antibody epitopes, is the only capsid protein of PiCV. In this study, the baculovirus expression system was utilized to express the PiCV Cap protein, which was self-assembled into VLPs with a spherical morphology and diameters of 15-18 nm. Specific antibodies against the Cap protein were induced after BALB/c mice immunized intramuscularly (i.m.) with VLPs combined with adjuvant. Based on these findings, PiCV VLPs may be a promising candidate vaccine against PiCV.