Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.
Animals ; Chick Embryo ; Chickens ; Down-Regulation ; Fibroblasts ; virology ; Gene Targeting ; Lentivirus ; genetics ; metabolism ; Newcastle Disease ; virology ; Newcastle disease virus ; genetics ; physiology ; Phosphoproteins ; genetics ; metabolism ; Poultry Diseases ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

Newcastle disease viruses (NDVs) cause systemic diseases in chickens with high mortality. However, little is known about persistence of NDVs in contaminated tissues from infected birds. In this study, we examined viral replication in the feather pulp of chickens inoculated with viscerotropic velogenic NDV (vvNDV) genotype VII. Reverse transcription real-time PCR and immunohistochemistry were used to investigate viral persistence in the samples. vvNDV was detected in the oropharynx and cloaca and viral antigens were detected in the feathers, suggesting that feathers act as sources of viral transmission.
Animals ; Antigens, Viral/analysis ; Chickens ; Cloaca/virology ; Feathers/*virology ; Microbial Viability ; Newcastle Disease/transmission/*virology ; Newcastle disease virus/isolation & purification/*physiology ; Oropharynx/virology ; Poultry Diseases/transmission/*virology ; Virus Replication/*physiology

Toll-like receptor 5 (TLR5) is responsible for the recognition of bacterial flagellin in vertebrates. In the present study, the first TLR5 gene in duck was cloned. The open reading frame (ORF) of duck TLR5 (dTLR5) cDNA is 2580 bp and encodes a polypeptide of 859 amino acids. We also cloned partial sequences of myeloid differentiation factor 88, 2'-5'-oligoadenylate synthetase (OAS), and myxovirus resistance (Mx) genes from duck. dTLR5 mRNA was highly expressed in the bursa of Fabricius, spleen, trachea, lung, jejunum, rectum, and skin; moderately expressed in the muscular and glandular tissues, duodenum, ileum, caecum, and pancreas; and minimally expressed in the heart, liver, kidney, and muscle. DF-1 or HeLa cells transfected with DNA constructs encoding dTLR5 can activate NF-kappaB leading to the activation of interleukin-6 (IL-6) promoter. When we challenged ducks with a Herts33 Newcastle disease virus (NDV), mRNA transcription of the antiviral molecules Mx, Double stranded RNA activated protein kinase (PKR), and OAS was up-regulated in the liver, lung, and spleen 1 and 2 days post-inoculation.
2',5'-Oligoadenylate Synthetase/genetics/metabolism ; Animals ; Cell Line ; *Cloning, Molecular ; Ducks ; Gene Expression Regulation/*physiology ; Humans ; Immunity, Innate ; Myeloid Differentiation Factor 88/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/metabolism ; Newcastle Disease/metabolism ; Newcastle disease virus/classification ; RNA, Messenger/genetics/metabolism ; Species Specificity ; Toll-Like Receptor 5/genetics/*metabolism

Characteristic clinical manifestations of Newcastle disease include leukopenia and immunosuppression. Peripheral blood mononuclear cells (PBMCs) are the main targets of Newcastle disease virus (NDV) infection. To survey changes in proteomic expression in chicken PBMCs following NDV infection, PBMC proteins from 30 chickens were separated using two-dimensional electrophoresis (2-DE) and subjected to mass spectrometry analysis. Quantitative intensity analysis showed that the expression of 78 proteins increased more than two-fold. Thirty-five proteins exhibited consistent changes in expression and 13 were identified as unique proteins by matrix assisted laser desorption ionization-time of flight mass spectrometer/mass spectrometer including three that were down-regulated and 10 that were up-regulated. These proteins were sorted into five groups based on function: macromolecular biosynthesis, cytoskeleton organization, metabolism, stress responses, and signal transduction. Furthermore, Western blot analysis confirmed the down-regulation of integrin-linked kinase expression and up-regulation of lamin A production. These data provide insight into the in vivo response of target cells to NDV infection at the molecular level. Additionally, results from this study have helped elucidate the molecular pathogenesis of NDV and may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.
Animals ; Avian Proteins/*genetics/metabolism ; *Chickens ; *Gene Expression Regulation ; Leukocytes, Mononuclear/enzymology/virology ; Newcastle Disease/*genetics/virology ; Newcastle disease virus/*physiology ; Poultry Diseases/*genetics/virology ; *Proteome ; Specific Pathogen-Free Organisms ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/veterinary ; Tandem Mass Spectrometry/veterinary

OBJECTIVESome research has shown that Newcastle disease virus (NDV) is effective in the treatment of various tumors, including transferred melanoma and well differentiated renal cell carcinoma. This study aimed to evaluate the effect of NDV against human acute monocytic leukemia SHI-1 cells in vitro and in vivo.

METHODSIn vitro, the density and morphologic changes between wild SHI-1 cells (control) and NDV-infected SHI-1 cells were observed. MTT assay was utilized to observe the effect of NDV on the proliferation of SHI-1 cells. In vivo, the effect of NDV on the tumor inhibition was assessed using SHI-1 xenografts subcutaneously established in CD-1 nude mice. NDV was given by intra-tumor injections, and the tumor inhibition rate and toxic effects were evaluated.

RESULTSIn the control group, the SHI-1 cells were observed using an inverted microscope to be regular in morphology and intensive in distribution. In the NDV-infected group, the SHI-1 cells were irregular and sparsate, and the aggregate and fused cells were common. MTT assay showed that the proliferation of SHI-1 cells were significantly inhibited by NDV at different concentrations (P<0.01) and in a time- and concentration-dependent manner. The tumor inhibition rate in the NDV group was 84.7%, which was significantly higher than that in the control group (P<0.01). No toxic effects were observed in the nude mice.

CONCLUSIONSNDV can suppress the proliferation of human acute monocytic leukemic cells both in vitro and in vivo. The safety of NDV is reliable.

Animals ; Cell Proliferation ; Humans ; Immunotherapy, Active ; Leukemia, Monocytic, Acute ; therapy ; Mice ; Mice, Nude ; Newcastle disease virus ; physiology ; Xenograft Model Antitumor Assays

Naturally existing Newcastle disease virus (NDV) can specifically execute oncolytic ability in clinical studies. Reports from clinical trials using NDV as oncolytic agents showed promise and warrant results in cancer therapy. In recent years, reverse genetics technology has been used widely in the studies of NDV virology. More recently, the technology was applied to optimize the oncolytic efficacy of NDV, for instance, modification of the F gene, and expression of GM-CSF, IFN-gamma, IL-2 or TNF-alpha. NDV is widely investigated in cancer therapy and will definitely offer a prosperous future for clinical cancer therapeutics. We reviewed the developments of cancer therapy by recombinant NDV using reverse genetics technology, as well as our own experience in this domain.
Animals ; Humans ; Neoplasms ; pathology ; therapy ; Newcastle disease virus ; genetics ; physiology ; Oncolytic Virotherapy ; methods ; Oncolytic Viruses ; genetics ; physiology ; Recombination, Genetic

The plaque-forming characteristics of Newcastle disease viruses of chickens and geese source were compared on various cells. The result showed that there were obvious differences of plaque formation between F48E9 and NA-1 on Vero cells, chicken embryo fibroblast cells (CEF) and goose embryo fibroblast cells (GEF). The plaque-forming ability of NA-1 was higher than F48E9 on GEF, but lower than F48E9 on CEF. On Vero cells, the plaque-forming ability of NA-1 was slightly stronger than F48E9. It demonstrated that the plaque-forming characteristics were consistent with host tropism of virus. The morphogenesis of F48E9 and NA-1 on Vero cells was observed with transmission electron microscope. There were different replication processes between F48E9 and NA-1 on cells at different stages. NA-1 had stronger adaptability to host than F48E9 according to budding processes and envelope integrity.
Animals ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Geese ; Host-Pathogen Interactions ; Newcastle Disease ; virology ; Newcastle disease virus ; growth & development ; isolation & purification ; physiology ; ultrastructure ; Poultry Diseases ; virology ; Vero Cells ; Viral Plaque Assay

OBJECTIVETo study the anti-tumor effects of Newcastle disease virus (NDV) strain D817 on human colon carcinoma model in nude mice.

METHODSThe nude mouse model of human colon carcinoma was established by subcutaneous inoculation of human colon cancer LOVO cells. The tumor-bearing mice were given PBS, 5-Fu, high-dose NDV D817, moderate-dose NDV D817 or low-dose NDV D817 via caudal vein injection. The tumor size and weight of mice were measured. The liver damages were examined by histopathology. Apoptosis and necrosis of tumor cells were detected by flow cytometry. The endotumoral content of TNF-alpha was detected using a mouse TNF-alpha ELISA kit. The live virus was detected by hemagglutination (HA) test.

RESULTSThe moderate-dose NDV D817 inhibited the tumor growth more apparently than 5-Fu. The tumor growth inhibition rate reached to 48.1%. The liver damage and the weight change caused by NDV were less severe. NDV D817 made an increased apoptosis index and induced production of TNF-alpha. Live virus was not detected in important organs except in the tumor of nude mice by HA test.

CONCLUSIONIn the anti-tumor process in nude mice bearing xenografts of human colon carcinoma, a suitable dose of NDV D817 is more safe and effective.

Animals ; Apoptosis ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; therapy ; Humans ; Liver ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Newcastle disease virus ; physiology ; Random Allocation ; Tumor Burden ; Tumor Necrosis Factor-alpha ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effects of Newcastle disease virus (NDV) infection on the expression of survivin and cell cycle in human tongue squamous carcinoma TSCCa cells.

METHODSThe proliferation of TSCCa cells infected with NDV in vitro was evaluated by means of MTT assay, and survivin expression in the infected cells was detected using RT-PCR and Western blotting. Flow cytometry was performed to assess the changes in the cell apoptosis, cell cycle and cell proliferation index (PI) of the cells.

RESULTSNDV infection resulted in decreased survivin expression and increased apoptosis of TSCCa cells, with reduced cell percentage in G2/M and S phases and lowered PI of the cells, showing significant differences from those of the negative control cells (P<0.05).

CONCLUSIONNDV infection can inhibit survivin expression, affect the cell cycle of TSCCa cells and induce their apoptosis.

Apoptosis ; physiology ; Blotting, Western ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cell Cycle ; physiology ; Cell Line, Tumor ; Host-Pathogen Interactions ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Newcastle disease virus ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Tongue Neoplasms ; metabolism ; pathology ; virology

Raw white rice has not been considered a good carrierfor oral vaccination, probably because of its antiviralactivity. Methods are required to overcome antiviralactivity in raw white rice. This study was carried out todetermine the effects of various treatments of raw whiterice on the survival of strain I-2 of Newcastle diseasevirus. These included cooking and baking the rice ormixing the rice with vegetable oil prior to coating withvaccine virus. The vaccine-coated rice was then stored for30min and 24h, followed by quantitative recovery of thevirus. Thirty min after mixing, uncooked, cooked, andbaked rice, and rice mixed with vegetable oil showed titersof 10(6.2), 10(7.2), 10(6.6), and 10(7.0) EID50/0.1ml, respectively.After storage for 24h at 22-25oC, the titers dropped to10(5.0), 10(6.5), 10(5.0), and 10(6.0) EID50/0.1ml for uncooked,cooked, baked, and oiled rice, respectively.
Animals ; Chick Embryo ; Chickens ; Cookery ; Newcastle Disease/*virology ; Newcastle disease virus/growth & development/*physiology ; Oryza sativa/chemistry/*virology ; Viral Vaccines/chemistry