Self-assembled nanoparticle with E protein domain III of DTMUV based on ferritin as carrier can induce a more comprehensive immune response and against DTMUV challenge in duck.

Duck Tembusu virus (DTMUV) causes severe reduction in egg production and neurological symptoms in ducklings. Vaccination is the primary measure used to prevent DTMUV infections. In this study, self-assembled nanoparticles with the E protein domain III of DTMUV, using ferritin as a carrier (EDⅢ-RFNp), were prepared using a prokaryotic expression system. Ducks were intramuscularly vaccinated with EDⅢ-RFNp, EDⅢ protein, an inactivated vaccine HB strain (InV-HB), and PBS. At 0, 4, and 6 weeks post-primary vaccination, the EDIII protein-specific antibody titre, IL-4, and IFN-γ concentrations in serum were determined by ELISA, and neutralising antibodies titres in sera were determined by virus neutralising assay. Peripheral blood lymphocytes proliferation was determined by CCK-8 kit. Following challenge with the virulent DTMUV strain, the clinical signals and survival rate of the vaccinated ducks were recorded, and DTMUV RNA levels in the blood and tissues of the surviving ducks were determined by real-time quantitative RT-PCR. The near-spherical EDⅢ-RFNp nanoparticles with 13.29 ± 1.43 nm diameter were observed by transmission electron microscope. At 4 and 6 weeks post-primary vaccination, special and Virus neutralisation (VN) antibodies, lymphocyte proliferation (stimulator index, SI), and concentrations of IL-4 and IFN-γ in the EDⅢ-RFNp group were significantly higher than in the EDⅢ and PBS groups. In the DTMUV virulent strain challenge test, the EDⅢ-RFNp-vaccinated ducks showed milder clinical signs and higher survival rates than EDⅢ- and PBS-vaccinated ducks. The DTMUV RNA levels in the blood and tissues of EDⅢ-RFNp-vaccinated ducks were significantly lower than those in EDⅢ- and PBS-vaccinated ducks. Additionally, the EDⅢ protein-special and VN antibodies, SI value, and concentration of IL-4 and IFN-γ in the InV-HB group was significantly higher than that of the PBS group at 4 and 6 weeks post-primary vaccination. InV-HB provided more efficient protection than PBS based on a higher survival rate, milder signals, and lower levels of the DTMUV virus in the blood and tissues. These results indicated that EDⅢ-RFNp effectively protected ducks against DTMUV challenge and could be a vaccine candidate to prevent DTMUV infection.

Self-assembled BPIV3 nanoparticles can induce comprehensive immune responses and protection against BPIV3 challenge by inducing dendritic cell maturation in mice.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory pathogens of cattle. No specific therapies are available for BPIV3 infection; vaccination is one of the most effective ways to prevent BPIV3 infection. We therefore prepared the self-assembled BPIV3 nanoparticles by genetically fusing the ectodomain of BPIV3 haemagglutinin-neuraminidase (HN) (HNex) to the NH2 terminus of ferritin (HNex-RFNp) using a baculovirus expression system. It was found that HNex-RFNp-induced bone marrow-derived dendritic cell (BMDC) maturation through the upregulated expression of surface molecules (MHC II, CD80, CD86, and CD40), increased the secretion of inflammatory cytokines (IL-6, IL-12, TNF-α, and IFN-γ), and reduced antigen phagocytosis and T cell activation capacity. HNex-RFNp positively regulated IκBα and NF-κB (p65) phosphorylation and facilitated NF-κB (p65) translocation into the nuclei of mature BMDCs. Incubating RFNp-treated BMDCs with TLR4 and NF-κB (p65) inhibitors, suppressed surface molecule expression as well as pro-inflammatory cytokine production and IκBα and NF-κB (p65) activities. The BPIV3 HNex protein induced BMDC maturation to some extent but was significantly weaker than HNex-RFNp. We found that HNex-RFNp induced a higher titre of specific antibodie, haemagglutinin inhibition (HI) antibody, and virus neutralisation (VN) antibody, and a comprehensive cellular immune response. We examined protection against BPIV3 challenge in a mouse model. Pathological changes were not observed in the lungs of HNex-RFNp-vaccinated mice. Levels of BPIV3 RNA and virus titres in the lungs and trachea were significantly lower in the HNex-RFNp, than HNex, inactivated BPIV3, and PBS groups. In summary, HNex-RFNp elicited better immunogenicity than HNex or inactivated BPIV3 and could be developed as an effective vaccine to protect against BPIV3 infection.

De novo transcriptome sequencing and comparative profiling of the ovary in partially engorged and fully engorged Haemaphysalis flava ticks.

Haemaphysalis flava is the vector of several pathogens and has medical and veterinary importance. Transcriptome information of the ovary of H. flava is unavailable and limits understanding of its molecular basis of reproduction. We studied the ovary transcriptome of partially engorged and fully engorged H. flava using high-throughput RNA sequencing technology. A total of 53,025,360 and 57,942,890 clean reads were obtained with 7.95 GB and 8.69 GB clean bases in partially engorged ticks (PETs) and fully engorged ticks (FETs), respectively. The clean reads were assembled into 138,711 unigenes. A total of 72,043 unigenes (51.93%) were annotated and 66,668 unigenes (48.07%) were unknown. A total of 38,487 differentially expressed genes (DEGs) were found between PET and FET with 19,031 upregulated genes and 19,456 downregulated genes. The RNA-seq results were validated by qRT-PCR, including six upregulated genes and three downregulated genes. Some unigenes coding for nutrient transporters, proteases, and protease inhibitors were found and analyzed. This study was the first time to perform the transcriptome sequences of the ovary of partially engorged and fully engorged H. flava. The results can benefit the understanding of the molecular basis of ovary maturation and oogenesis of the H. flava and boost the development of the strategies for control of H. flava.

Recombinant ferritin nanoparticles can induce dendritic cell maturation through TLR4/NF-κB pathway.

OBJECTIVE: Immune response initiation and regulation require activation of dendritic cells (DCs). However, the mechanism by which ferritin, a carrier for immunogen, induces DCs maturation remains unclear. RESULTS: Recombinant ferritin nanoparticle (RFNp), were prepared through the baculovirus expression vector system, formed spherical and hollow cage-liked proteins with a diameter of approximately 12.17 ± 0.87 nm. They induced bone marrow-derived DC (BMDC) maturation via surface molecules up-regulation of (MHC II, CD80, CD86 and CD40), increased pro-inflammatory cytokines production (IL-6, IL-12, TNF-α, and IFN-γ), and decreased antigen capturing capacity. They positively regulated IκBα and NF-κB (p65) phosphorylation, and facilitate NF-κB (p65) translocation into mature BMDCs nuclei. Following pre-treatment of RFNp-treated BMDCs with TLR4 and NF-κB (p65) inhibitors, respectively, surface molecule expression, pro-inflammatory cytokines production, and IκBα and NF-κB (p65) activities were suppressed. RFNp-treated BMDCs can also facilitate T-cell proliferation and differentiation into Th1 and Th2. CONCLUSION: RFNps induced DCs maturation lends the potential application of RFNps as carrier platforms in DC-based vaccine.

Expression, purification, and characterisation of recombinant ferritin in insect cells using the baculovirus expression system.

OBJECTIVE: Ferritin is an attractive vector for the delivery of drug molecules and antigen proteins because of its unique structural and biochemical features. In this study, recombinant ferritin from Helicobacter pylori was expressed in the soluble form employing the baculovirus expression system. RESULTS: The optimum conditions for producing recombinant ferritin comprised MOI 5 of rBV-ferritin for 96 h of infection. The recombinant ferritin was purified by Ni Sepharose™ 6 Fast Flow, with a purity and yield of 92.5% and 11.25 mg/L, respectively. In addition, the recombinant ferritin showed a multimeric structure under non-denaturing conditions, as well as self-assembled spherical cage architecture with a diameter of approximately 12 nm. Dot-ELISA results suggested that the His-tag at the N-terminus likely existed on the surface of the recombinant ferritin. CONCLUSION: Recombinant ferritin was produced by the baculovirus expression system, which has the potential to display exogenous proteins, and may aid in the delivery of drugs for disease prevention and treatment.

BICP0 Negatively Regulates TRAF6-Mediated NF-κB and Interferon Activation by Promoting K48-Linked Polyubiquitination of TRAF6.

The infected cell protein 0 (BICP0) is an immediate early protein encoded by BHV-1, and its RING finger domain, which endows BICP0 with intrinsic E3 ubiquitin ligase activity, is common in all ICP0 proteins. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the TRAF family members and is ubiquitously expressed in mammalian tissues. TRAF6 forms the MyD88-TRAF6-IRF7 complex and activates interferon induction in the TLR (Toll-like receptors) and the RLR (RIG-I-like receptor) pathway. Previous studies showed that BICP0 reduced IFN-ß promoter activity by interacting with IRF7. In this study, we found that BICP0 promoted the K48-ubiquitination and degradation of TRAF6 through the ubiquitin proteasome system. The interaction between BICP0 and TRAF6 is a prerequisite for ubiquitination modification, and the 346-PAERQY-351 of BICP0 is indispensable. The motif mutation experiments showed that the tyrosine 351 of BICP0 is the key amino acid involved. Further studies demonstrated that BICP0 suppressed the NF-κB pathway via the interference of TRAF6. Moreover, degradation of TRAF6 protein influenced the K63-linked ubiquitination of IRF7 and activation of interferon promoter. Collectively, these findings indicate that the BICP0 protein suppresses the inflammation signaling and IFN production by K48-linked polyubiquitination of TRAF6 and may further clarify the immune evasion function of BICP0.

Pathogenicity of duck hepatitis A virus type 3 and innate immune responses of the ducklings to virulent DHAV-3.

Duck virus hepatitis caused by duck hepatitis A virus (DHAV) is an acute and contagious disease. To better understand the pathogenic mechanism of DHAV-3 in ducklings, an infection experiment was performed. Our results showed that typical symptoms were observed in the infected ducklings. DHAV-3 could infect many tissues, leading to pathological lesions, especially on the livers and spleen, and the host immune responses are activated in infection. Real-time quantitative PCR demonstrated that expression of many innate immune-related genes was mostly up-regulated in the livers and spleen, and antiviral innate immune response was established, but not sufficient to restrict the virus replication of lethal dose. Many major pattern recognition receptors (PRRs) (RIG-1, MDA5, and TLR7) are involved in the host immune response to DHAV-3, and the expression of interferon (IFNα, IFNß and IFNγ) and antiviral proteins (MX, OAS and PKR) are also up-regulated in the liver and spleen. The expression of most cytokines (IL-1ß, IL-2 and IL-6) was also up-regulated to different degrees and was various; the expression of IL-2 increased most significantly in liver. Our data provide a foundation for further study of the pathogenicity of duck virus hepatitis and extend our understanding of the immune responses of ducklings to DHAV-3 infection.