New insight into the mechanisms of Ginkgo biloba leaves in the treatment of cancer.

BACKGROUND: Ginkgo biloba leaves (GBLs), as an herbal dietary supplement and a traditional Chinese medicine, have been used in treating diseases for hundred years. Recently, increasing evidence reveals that the extracts and active ingredients of GBLs have anti-cancer (chemo-preventive) properties. However, the molecular mechanism of GBLs in anti-cancer has not been comprehensively summarized. PURPOSE: To systematically summarize the literatures for identifying the molecular mechanism of GBLs in cellular, animal models and clinical trials of cancers, as well as for critically evaluating the current evidence of efficacy and safety of GBLs for cancers. METHODS: Employing the search terms "Ginkgo biloba" and "cancer" till July 25, 2023, a comprehensive search was carried out in four electronic databases including Scopus, PubMed, Google Scholar and Web of Science. The articles not contained in the databases are performed by manual searches and all the literatures on anti-cancer research and mechanism of action of GBLs was extracted and summarized. The quality of methodology was assessed independently through PRISMA 2020. RESULTS: Among 84 records found in the database, 28 were systematic reviews related to GBLs, while the remaining 56 records were related to the anticancer effects of GBLs, which include studies on the anticancer activities and mechanisms of extracts or its components in GBLs at cellular, animal, and clinical levels. During these studies, the top six cancer types associated with GBLs are lung cancer, hepatocellular carcinoma, gastric cancer, breast cancer, colorectal cancer, and cervical cancer. Further analysis reveals that GBLs primarily exert their anticancer effects by stimulating cancer cell apoptosis, inhibiting cell proliferation, invasion and migration of cancers, exhibiting anti-inflammatory and antioxidant properties, and modulating signaling pathways. Besides, the pharmacology, toxicology, and clinical research on the anti-tumor activity of GBLs have also been discussed. CONCLUSIONS: This is the first paper to thoroughly investigate the pharmacology effect, toxicology, and the mechanisms of action of GBLs for anti-cancer properties. All the findings will reinforce the need to explore the new usage of GBLs in cancers and offer comprehensive reference data and recommendations for future research on this herbal medicine.

Identification of Breast Cancer Subtypes by Integrating Genomic Analysis with the Immune Microenvironment.

Objectives: We aim to identify the breast cancer (BC) subtype clusters and the crucial gene classifier prognostic signatures by integrating genomic analysis with the tumor immune microenvironment (TME). Methods: Data sets of BC were derived from the Cancer Genome Atlas (TCGA), METABRIC, and Gene Expression Omnibus (GEO) databases. Unsupervised consensus clustering was carried out to obtain the subtype clusters of BC patients. Weighted gene coexpression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO), and univariate and multivariate regression analysis were employed to obtain the gene classifier signatures and their biological functions, which were validated by the BC dataset from the METABRIC database. Additionally, to evaluate the overall survival rates of BC patients, Kaplan-Meier survival analysis was carried out. Moreover, to assess how BC subtype clusters are related to the TME, single-cell analysis was performed. Finally, the drug sensitivity and the immune cell infiltration for different phenotypes of BC patients were also calculated by the CIBERSORT and ESTIMATE algorithms. Results : TCGA-BC samples were divided into three subtype clusters, S1, S2, and S3, among which the prognosis of S2 was poor and that of S1 and S3 were better. Three key pathways and 10 crucial prognostic-related gene signatures are screened. Finally, single-cell analysis suggests that S1 samples have the most types of immune cells, S2 with more sensitivity to tumor treatment drugs are enriched with more neutrophils, and more multilymphoid progenitor cells are involved in subtype cluster S3. Conclusions: Our novelty was to identify the BC subtype clusters and the gene classifier signatures employing a large-amount dataset combined with multiple bioinformatics methods. All of the results provide a basis for clinical precision treatment of BC.

Lipid droplet targeting-guided hypoxic photodynamic therapy with curcumin analogs.

Two photosensitizers (CCOH and CCN) were designed and synthesized by introducing coumarin into the curcumin (CUR) structure. Compared with CUR, more reactive oxygen species (ROS) were generated by CCOH and CCN in type I and II synergy upon light irradiation. Cell experiments indicated that CCN with an excellent LD-targeting effect could be used to monitor the changes in the morphology and number of LDs in tumor cells during PDT.

Systematic investigation of the mechanism of herbal medicines for the treatment of prostate cancer.

Due to various unpleasant side effects and general ineffectiveness of current treatments for prostate cancer (PCa), more and more people with PCa try to look for complementary and alternative medicine such as herbal medicine. However, since herbal medicine has multi-components, multi-targets and multi-pathways features, its underlying molecular mechanism of action is not yet known and still needs to be systematically explored. Presently, a comprehensive approach consisting of bibliometric analysis, pharmacokinetic assessment, target prediction and network construction is firstly performed to obtain PCa-related herbal medicines and their corresponding candidate compounds and potential targets. Subsequently, a total of 20 overlapping genes between DEGs in PCa patients and the target genes of the PCa-related herbs, as well as five hub genes, i.e., CCNA2, CDK2, CTH, DPP4 and SRC were determined employing bioinformatics analysis. Further, the roles of these hub genes in PCa were also investigated through survival analysis and tumour immunity analysis. Moreover, to validate the reliability of the C-T interactions and to further explore the binding modes between ingredients and their targets, the molecular dynamics (MD) simulations were carried out. Finally, based on the modularization of the biological network, four signaling pathways, i.e., PI3K-Akt, MAPK, p53 and cell cycle were integrated to further analyze the therapeutic mechanism of PCa-related herbal medicine. All the results show the mechanism of action of herbal medicines on treating PCa from the molecular to systematic levels, providing a reference for the treatment of complex diseases using TCM.

RBCK1 is an endogenous inhibitor for triple negative breast cancer via hippo/YAP axis.

BACKGROUND: Triple negative breast cancer (TNBC) is one of the most lethal breast cancer subtypes. Due to a lack of effective therapeutic targets, chemotherapy is still the main medical treatment for TNBC patients. Thus, it is important and necessary to find new therapeutic targets for TNBC. Recent genomic studies implicated the Hippo / Yap signal is over activated in TNBC, manifesting it plays a key role in TNBC carcinogenesis and cancer progression. RBCK1 was firstly identified as an important component for linear ubiquitin assembly complex (LUBAC) and facilitates NFKB signaling in immune response. Further studies showed RBCK1 also facilitated luminal type breast cancer growth and endocrine resistance via trans-activation estrogen receptor alpha. METHODS: RBCK1 and YAP protein expression levels were measured by western blotting, while the mRNA levels of YAP target genes were measured by RT-PCR. RNA sequencing data were analyzed by Ingenuity Pathway Analysis. Identification of Hippo signaling activity was accomplished with luciferase assays, RT-PCR and western blotting. Protein stability assays and ubiquitin assays were used to detect YAP protein degradation. Ubiquitin-based immunoprecipitation assays were used to detect the specific ubiquitination modification on the YAP protein. RESULTS: In our current study, our data revealed an opposite function for RBCK1 in TNBC progression. RBCK1 over-expression inhibited TNBC cell progression in vitro and in vivo, while RBCK1 depletion promoted TNBC cell invasion. The whole genomic expression profiling showed that RBCK1 depletion activated Hippo/YAP axis. RBCK1 depletion increased YAP protein level and Hippo target gene expression in TNBC. The molecular biology studies confirmed that RBCK1 could bind to YAP protein and enhance the stability of YAP protein by promoting YAP K48-linked poly-ubiquitination at several YAP lysine sites (K76, K204 and K321). CONCLUSION: Our study revealed the multi-faced RBCK1 function in different subtypes of breast cancer patients and a promising therapeutic target for TNBC treatment. Video abstract.

Systematic investigation of the clinical significance and prognostic value of the CBXs in esophageal cancer.

Esophageal cancer (ESCA), one of the most aggressive malignant tumors, has been announced to be the ninth most common cancer and the sixth leading cause of cancer-related death in the world. Chromobox family members (CBXs) are important epigenetic regulators which are related with the transcription of target genes. The role of CBXs in carcinomas has been reported in many studies. However, the function and prognostic value of different CBXs in ESCA are still largely unknown. In this article, we first performed differential expression analysis through several methods including Oncomine and Gene Expression Profiling Interactive Analysis. The results led us to determine the differential expression of CBXs in pan-cancer, especially ESCA. Then we evaluated the prognostic value of different CBX messenger RNA (mRNA) expression in patients with ESCA through the Kaplan-Meier plotter and the Human Protein Atlas database. In addition, we used cBioPortal to explore all genetic alterations and mutations in the CBXs in ESCA. Simultaneously, the correlation between its expression and the level of immune infiltration of ESCA was visualized by TIMER. Finally, the biological function of CBXs in ESCA is obtained through Biological Enrichment Analysis including gene ontology and Kyoto Encyclopedia of Genes and Genomes. The expression levels of CBX3/4/5 and CBX8 in ESCA tissues increased significantly and the expression level of CBX7 decreased through differential expression analysis. Additionally, CBX1 is significantly related to the clinical cancer stage and disease-free survival of ESCA patients. The high mRNA expression of CBX4 is related to the short overall survival of patients with esophageal squamous cell carcinoma, and the high mRNA expression of CBX3/7/8 is related to the short overall survival of patients with esophageal adenocarcinoma, indicating that CBX1/3/4/7/8 may be a potential prognostic biomarker for the survival of ESCA patients. Besides, the expression of CBXs is significantly related to the infiltration of a variety of immune cells, including six types of CD4-positive T-lymphocytes, macrophages, neutrophils, bursindependentlymphocyte, CD8-positive T-lymphocytes cells and dendritic cells in ESCA. Moreover, we found that CBXs are mainly associated with the inhibition of cell cycle and apoptosis pathway. Further, enrichment analysis indicated that CBXs and correlated genes were enriched in mismatch repair, DNA replication, cancer pathways, and spliceosomes. Our research may provide new insights into the choice of prognosis biomarkers of the CBXs in ESCA.

Interleukin-24 Limits Tumor-Infiltrating T Helper 17 Cell Response in Patients with Hepatitis B Virus-Related Hepatocellular Carcinoma.

Interleukin (IL)-24 is a multifunction cytokine in infectious diseases and cancers. IL-17-secreting CD4+ T (Th17) and CD8+ T (Tc17) cells promotes pathogenesis of hepatitis B virus (HBV)-associated liver disorders. However, the regulatory role of IL-24 to Th17/Tc17 cell response was not fully elucidated during HBV infection and HBV-hepatocellular carcinoma (HCC). In this study, plasma and peripheral blood mononuclear cells were isolated from 27 chronic hepatitis B (CHB) patients, 42 HBV-HCC patients and 17 normal controls (NC). Liver-infiltrating lymphocytes (LILs) were prepared from tumor and para-tumor tissues of 17 HBV-HCC patients, whereas CD4+ T cells in LILs were purified. LILs were stimulated with recombinant human IL-24. CD3+CD4+IL-17+ Th17 cells and CD3+CD8+IL-17+ Tc17 cells were investigated by flow cytometry. IL-24 level was measured by enzyme-linked immunosorbent assay. Purified CD4+ T cells were polarized for Th17 cells, and the regulatory role of IL-24 to liver-infiltrating Th17 polarization was assessed. There were no significant differences of peripheral Th17 or Tc17 cell percentage among NC, CHB, and HBV-HCC patients. Liver-infiltrating Th17 and Tc17 cell proportion was reduced in tumor tissues compared with para-tumor tissues. In contrast, plasma IL-24 level was increased in CHB and HBV-HCC patients. Exogenous IL-24 stimulation (10 or 100 ng/mL) in vitro downregulated of Th17 frequency and IL-17 secretion in LILs from both para-tumor and tumor tissues without affecting cellular proliferation or Tc17 percentage. Only 100 ng/mL of IL-24 inhibited tumor-infiltrating Th17 polarization, and this process was accompanied by suppression of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase phosphorylation. In conclusion, IL-24 might dampen Th17 cell response through NF-κB pathway in HBV-HCC tumor microenvironment. Elevated IL-24 might enhance anti-tumor immune response in HBV-HCC patients.

Comprehensive Analysis of CDCAs Methylation and Immune Infiltrates in Hepatocellular Carcinoma.

BACKGROUND: As essential components of cycle growth, the cell division cycle-associated family genes (CDCAs) have crucial roles in tumor development and progression, especially in hepatocellular carcinoma (HCC). However, due to the tumor heterogeneity of HCC, little is known about the methylation variability of CDCAs in mediating phenotypic changes (e.g., immune infiltrates) in HCC. Presently, we aim to comprehensively explore the expression and prognosis of CDCAs methylation with regard to immune infiltrates of HCC. METHODS: We first identified the correlating differentially expressed genes (co-DEGs) among 19 different types of cancer cohorts (a total of 7,783 patients) and then constructed the weighted gene co-expressed and co-methylated networks. Applying the clustering analysis, significant modules of DEGs including CDCAs were selected and their functional bioinformatics analyses were performed. Besides, using DiseaseMeth and TIMER, the correlation between the methylation levels of CDCAs and tumor immune infiltrates was also analyzed. In final, to assess the influence of CDCAs methylation on clinical prognosis, Kaplan-Meier and Cox regression analysis were carried out. RESULT: A total of 473 co-DEGs are successfully identified, while seven genes of CDCAs (CDCA1-3 and CDCA5-8) have significant over-expression in HCC. Co-expressed and co-methylated networks reveal the strong positive correlations in mRNA expression and methylation levels of CDCAs. Besides, the biological enrichment analysis of CDCAs demonstrates that they are significantly related to the immune function regulation of infiltrating immune cells in HCC. Also, the methylation analysis of CDCAs depicts the strong association with the tumor immunogenicity, i.e., low-methylation of CDCA1, CDCA2, and CDCA8 dramatically reduced the immune infiltrate levels of T cells and cytotoxic lymphocytes. Additionally, CDCA1-6 and CDCA8 with low-methylation levels significantly deteriorate the overall survival of patients in HCC. CONCLUSIONS: The co-expressed and co-methylated gene networks of CDCAs show a powerful association with immune function regulation. And the methylation levels of CDCAs suggesting the prognostic value and infiltrating immune differences could be a novel and predictive biomarker for the response of immunotherapy.

The down-regulation of casein kinase 1 alpha as a host defense response against infectious bursal disease virus infection.

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus of chickens. Although the gene functions of IBDV have been well characterized, the host responses during IBDV infection remain much poor. In the present study, casein kinase 1 alpha (CK1α), a novel VP2-associated protein, was down-regulated during IBDV replication in DF1 cells. Further experiments showed that siRNA-mediated knockdown of CK1α inhibited IBDV replication, while overexpression of CK1α promoted IBDV growth. Finally, we revealed that the effects of CK1α expression level on IBDV replication were involved in the negative regulation of CK1α on type I interferon receptor (IFNAR1), because ubiquitination assay analyses demonstrated that CK1α could promote the ubiquitination of IFNAR1, thereby affecting the stability of this receptor. In conclusion, down-regulation of CK1α during IBDV infection as a host defense response increased abundance of IFNAR1, which in turn enhanced an inhibitory effect on IBDV replication.

Assessment of Crohn's disease activity by Doppler sonography.

OBJECTIVES: To investigate the diagnostic accuracy of ultrasound for evaluation of inflammatory activity in patients with Crohn's disease (CD). Methods: Fifty-six patients with histologically proven CD (39 with active, 17 with inactive disease) and 30 healthy volunteers as a control group were enrolled in the study at WeiFang People's Hospital, Weifang Province, China from October 2012 to December 2014. Bowel wall thickness, and vascularity pattern were measured by Doppler ultrasound. Results: There was a significant difference in flow volume of the superior mesenteric artery (585 ± 235 ml/min) in the patients with active disease, compared with those with inactive disease (401 ± 238 ml/min) and the control group (390 ± 189 ml/min, p less than 0.001). Wall thickness was 5.1 ± 1.5 mm in the active CD group, 3.3 ± 1.6 mm in the inactive disease group (p less than 0.001) and  less than 3 mm in the control group. Resistance index in the thickened bowel wall showed some differences: 0.68 ± 0.05 in the active disease group, 0.78 ± 0.08 in the inactive disease group, and 0.85 ± 0.07 in the control group (p less than 0.05). Conclusion: Doppler ultrasound is a useful diagnostic tool in detecting CD and assessing inflammatory activity.

C-terminal region of apoptin affects chicken anemia virus replication and virulence.

BACKGROUND: Chicken anemia virus (CAV) causes anemia and immune suppression, which are important diseases in the poultry industry. CAV VP3, also referred as 'apoptin', has been shown to selectively kill tumor cells, raising great hopes for its utilization as an anticancer therapy. The ability of apoptin to induce apoptosis is closely related to its nuclear localization. The C-terminal region of apoptin contains a bipartite nuclear localization signals (NLS), and a nuclear export signal (NES) is located between the arms of the NLS. Most previous studies have expressed apoptin of different lengths in vitro to understand the relationship between its localization and its induction of apoptosis. METHODS: In this study, we investigated the replication of CAV and its induction of apoptosis in vitro and in vivo with VP3-truncated infectious virus. Quantitative PCR was used to detect viral replication in MDCC-MSB1 cells, and the viral localization was observed by confocal microscopy. Flow cytometry was uesed to analyze virus-induced apoptosis in MDCC-MSB1 cells. Additionally, chickens infected with the rescued viruses compared with the parental virus rM9905 to evaluate the viral replication in vivo and virulence. RESULTS: Based on the infectious clone, we rescued two viruses in which were deleted NES-NLS2 (rCAV-VP3N88) or NLS1-NES-NLS2 (rCAV-VP3N80) in the C-terminal region of apoptin. The viral load of rCAV-VP3N88 decreased significantly between 60 and 108 hpi, and was always 10-100-fold lower than that of the parental virus rM9905. The levels of rCAV-VP3N80 were also 10-100-fold lower than that of rM9905 and declined significantly at three time points. There was almost no difference in the viral loads of rCAV-VP3N88 and rCAV-VP3N80. Additionally, rM9905 induced 85.39 ± 2.18% apoptosis at 96 hpi, whereas rCAV-VP3N88 and rCAV-VP3N80 induced 63.08 ± 4.78% and 62.56 ± 7.35% apoptosis, respectively, which were significantly (about 20%) lower than that induced by the parental virus. The rescued viruses altered the nuclear localization in MDCC-MSB1 cells. Moreover, deletion of C-terminal region of apoptin impaired viral replication in vivo and reduced the virulence of CAV in chickens. CONCLUSIONS: In summary, we have demonstrated that the C-terminal deletion of apoptin in infectious CAV affected the replication of the virus. The deletion of the C-terminal region of apoptin not only significantly reduced viral replication in vitro but also reduced its induction of apoptosis, which correlated with the loss of its nuclear localization. The deletion of the C-terminal region of apoptin also impaired the replication of CAV and attenuated its virulence in chickens.

Avian leukosis virus subgroup J induces VEGF expression via NF-κB/PI3K-dependent IL-6 production.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic virus causing hemangiomas and myeloid tumors in chickens. Interleukin-6 (IL-6) is a multifunctional pro-inflammatory interleukin involved in many types of cancer. We previously demonstrated that IL-6 expression was induced following ALV-J infection in chickens. The aim of this study is to characterize the mechanism by which ALV-J induces IL-6 expression, and the role of IL-6 in tumor development. Our results demonstrate that ALV-J infection increases IL-6 expression in chicken splenocytes, peripheral blood lymphocytes, and vascular endothelial cells. IL-6 production is induced by the ALV-J envelope protein gp85 and capsid protein p27 via PI3K- and NF-κB-mediated signaling. IL-6 in turn induced expression of vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR-2, in vascular endothelial cells and embryonic vascular tissues. Suppression of IL-6 using siRNA inhibited the ALV-J induced VEGF-A and VEGFR-2 expression in vascular endothelial cells, indicating that the ALV-J-induced VEGF-A/VEGFR-2 expression is mediated by IL-6. As VEGF-A and VEGFR-2 are important factors in oncogenesis, our findings suggest that ALV-J hijacks IL-6 to promote tumorigenesis, and indicate that IL-6 could potentially serve as a therapeutic target in ALV-J infections.

TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. IMPORTANCE: Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

Identification of a linear B-cell epitope on the avian leukosis virus P27 protein using monoclonal antibodies.

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can induce various clinical tumors. The capsid protein P27 is the group-specific antigen of ALV and has many viral antigen sites that are easy to detect. In this study, we produced a monoclonal antibody (mAb), 3A9, that is specific for the P27 protein. A series of partially overlapping peptides were screened to define (181)PPSAR(185) as the minimal linear epitope recognized by mAb 3A9. The identified epitope could be recognized by chicken anti-ALV and mouse anti-ALV P27 sera. The epitope was highly conserved among a number of ALV-A, ALV-B and ALV-J strains. MAb 3A9 might be a valuable tool for the development of new immunodiagnostic approaches for ALV, and the defined linear epitope might help further our understanding of the antigenic structure of the P27 protein.

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus.

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

An optimized, highly efficient, self-assembled, subvirus-like particle of infectious bursal disease virus (IBDV).

Infectious bursal disease virus (IBDV) causes immunosuppression in young chickens, leading to increased susceptibility to other diseases and a reduction in the immune response to other vaccines. Thus, IBDV results in great economic losses to the poultry industry. The most effective method of prevention is vaccination. However, medium-virulence vaccines can cause bursal pathological damage and immunosuppression. Here, we describe a safer, self-assembled, subvirus-like particle (sVP) vaccine without a complex purification process. The IBD-VP2 gene was cloned into Pichia pastoris, and the expressed protein self-assembled into T=1 sVPs (∼23nm). Immunization experiments showed that the sVP vaccine elicited high IBDV-neutralizing antibodies in each group, and all birds survived challenge with very virulent IBDV (vvIBDV). Additionally, IBDV RNA was not detected, and sterile immunity was achieved. In conclusion, the IBD-sVP is a suitable candidate for a recombinant subunit vaccine against IBDV.

Integrin αvß1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection.

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin αv and/or ß1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin αv and ß1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin αvß1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV.

Ribosomal protein L4 interacts with viral protein VP3 and regulates the replication of infectious bursal disease virus.

VP3 protein is a structural protein which plays important roles in the virus assembly and the inhibition of antiviral innate immunity of infectious bursal disease virus (IBDV). To explore the potential roles of VP3 in the interplay of IBDV with the host cell, an immunoprecipitation (IP)-coupled mass spectra (MS) screening was performed and the host cellular ribosomal protein L4 (RPL4) was identified as a putative interacting partner of VP3 protein. The interaction of RPL4 with VP3 was further confirmed by co-immunoprecipitation (co-IP) and their colocalization in DF1 cells were observed by confocal microscopy. In addition, knockdown of RPL4 in DF1 cells resulted in reductions of the viral protein pVP2 expression and the virus titers, which reveals a significant role of RPL4 in IBDV replication. Taken together, we indicated for the first time that ribosomal protein L4 (RPL4) was an interacting partner of VP3 and involved in the modulation of IBDV replication. The present study contributes to further understanding the pathogenic mechanism of IBDV.

Molecular and pathogenicity characterization of Gallid herpesvirus 2 newly isolated in China from 2009 to 2013.

During the course of our continuous surveillance of Gallid herpesvirus 2 (GaHV-2), 44 isolates were obtained from GaHV-2-positive chickens of different flocks in China from 2009 to 2013. The meq gene, considered as a major GaHV-2 oncogene, was sequenced and was found to contain an open reading frame of 1020 nucleotides encoding a 339 amino acid (aa) polypeptide in all isolates. Compared with the GaHV-2 GA strain, the meq genes in 15.9 % (7/44) of the isolates analyzed in this study contained an aa substitution mutation at position 88 (A to T) of which is the first report. The main characteristics of Chinese GaHV-2 isolates meq genes included the substitutions K77E, D80Y, V115A, T139A, P176R, and P217A, and the aa substitution frequency at positions 139 and 176 showed an increase. To test the pathogenicity of the isolates, a pathogenicity study and a vaccination-challenge test were performed on three selected isolates (ZY/1203, WC/1203, and WC/1110) and reference strain GA. The results showed that the three isolates induced gross Marek's disease (MD) lesions in 95.0-100 % cases, which was a higher rate than that obtained for strain GA (82.4 %). Three isolates induced mortality in 10-21.1 % of specific-pathogen-free chickens, which was similar to results with strain GA (23.5 %). The commercially available CVI988 vaccine induced lower protective indices (PIs) against ZY/1203 (82.4) and WC/1110 (83.3) as compared to those against WC/1203 (100) and GA (100). These results showed an evolving trend in the meq genes of the isolates; three isolates exhibited higher morbidity as compared to the reference strain and the vaccine induced lower PIs against two isolates as compared to that against the reference strain.