The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Animals ; Antibodies, Viral ; Chickens ; HN Protein/metabolism* ; Newcastle Disease/prevention & control* ; Newcastle disease virus/metabolism* ; Oryza/genetics*

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

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

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.
Animals ; Body Weight ; Cell Line, Tumor ; Cell Proliferation ; Chick Embryo ; Cytotoxicity, Immunologic ; Female ; Genetic Therapy ; Interleukin-15 ; genetics ; metabolism ; Melanoma, Experimental ; pathology ; therapy ; Mice ; Neoplasm Transplantation ; Newcastle disease virus ; genetics ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.
Animals ; Antibodies, Viral/*blood ; Antigens, Viral/*diagnostic use/genetics/metabolism ; Baculoviridae/genetics ; Chickens ; HN Protein/*diagnostic use/genetics/metabolism ; Hemagglutination Inhibition Tests/*methods/veterinary ; Newcastle Disease/*diagnosis/immunology/virology ; Newcastle disease virus/genetics/*immunology/metabolism ; Poultry Diseases/*diagnosis/immunology/virology ; Recombinant Proteins/diagnostic use/genetics/metabolism ; Sf9 Cells ; Spodoptera

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.
Animals ; Antimetabolites, Antineoplastic ; metabolism ; pharmacology ; Cell Death ; drug effects ; Chick Embryo ; Cytosine Deaminase ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Flucytosine ; metabolism ; pharmacology ; Fluorouracil ; metabolism ; pharmacology ; Genetic Vectors ; Hep G2 Cells ; Humans ; Lethal Dose 50 ; Liver Neoplasms, Experimental ; pathology ; Mice ; Newcastle disease virus ; genetics ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; drug effects

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

The resistance effect on Newcastle disease virus (NDV) and Infectious Bursal Disease Virus(IBDV) in vitro of a new antimicrobial substance (AS), which produced by a Bacillus subtilis strain named B. subtilis fmbJ. Results showed that the TD50 and TD0 value of this AS on Chicken Embryo Fibroblasts cell (CEF) were 128.95mg/L and 25.79mg/L, respectively. This AS could strongly inhibit the cytopathic effects of cell induced by NDV as well as IBDV, and increase the survival rate of cell remarkably. This AS could inhibit the function of NDV and IBDV, and it could defend against the infection and inhibit multiplication of NDV and IBDV, and the effect was the same as the antiviral medicine Ribavirin. It had lower toxicity to CEF cell, therefore we would study it further that it was as antiviral medicine.
Animals ; Antiviral Agents ; metabolism ; toxicity ; Bacillus subtilis ; metabolism ; Chick Embryo ; cytology ; Fibroblasts ; cytology ; drug effects ; Infectious bursal disease virus ; drug effects ; Newcastle disease virus ; drug effects