OBJECTIVE: To investigate the anti-tumor effects and the mechanism of the recombinant fowlpox virus expressing Apoptin gene on human laryngeal carcinoma Hep-2. METHOD: Hep-2 cells cultured in vitro were infected with vFVApoptin. The anti-tumor effects on Hep-2 cells were measured through MTT staining and, the mitochondrial trans-membrane potential (delta psi m) and reactive oxygen species (ROS) were analyzed by flow cytometry. Western blot was used to detect the release of cytochrome c (Cyto c). Caspase-3/9 activities were measured by colorimetric assay. RESULT: vFVApoptin could restrain Hep-2 cells significantly and, had the function of down-regulating delta psi m, up-regulating ROS, promoting Cyto c release and activating Caspase-3/9. CONCLUSION Cyto c were released from mitochondria by the function of up-regulating ROS of vFVApoptin. Cyto c triggered Caspase-9 and, after the activation of Caspase-9, downstream apoptotic factors, such as caspase-3, were activated. Eventually, Hep-2 cells were suppressed by mitochondrial pathway apoptosis induced by vFVApoptin.
Animals ; Apoptosis ; drug effects ; Capsid Proteins ; genetics ; pharmacology ; Chicken anemia virus ; genetics ; Fowlpox virus ; genetics ; Humans ; Tumor Cells, Cultured

One-day-old SPF chicks were inoculated with the Cux-l strain of chicken infectious anemia virus (CIAV), and the clinical development of disease and its macroscopic and microscopic alterations in the thymus and bone marrow, were observed. Tissue sections of thymus and bone marrow were stained using the streptavidin-biotin peroxidase method and examined under light microscope for evaluation of antigenic intensities in tissues. Those findings were then compared with blood parameters and ELISA results obtained through collected sera during sacrifice procedures. We sought to determine: the localization of viral antigens in thymus and bone marrow tissues after inoculation, the correlation between antigen intensities and hematologic, serologic and histopathologic findings, definitive diagnostic criteria using histopathologic and immunoperoxidase methods, and the reliability of these methods in the diagnosis of CIAV infection. For this purpose, 83, one-day-old SPF chicks were used. The birds were divided into experimental (n = 52) and control (n = 26) groups. A virus dose of TCID50 of 100,000/ml was administered intramuscularly to every bird in the experimental group. Based on the results of this study, we have suggested that clinical examination, along with macroscopic and microscopic evaluation of the thymus and bone marrow, maybe undertaken starting from day 7 post-inoculation (PI). ELISA, might be of value, as it might give consistent results starting from day 14 PI. However, the most reliable results were obtained through examination of thymus and bone marrow sections from infected birds stained by immunoperoxidase technique, as early as day 4 PI.
Animals ; Bone Marrow/*pathology ; *Chicken anemia virus ; Chickens ; Circoviridae Infections/pathology/*veterinary ; Immunoenzyme Techniques ; Poultry Diseases/*pathology ; Specific Pathogen-Free Organisms ; Thymus Gland/*pathology

Capsid Proteins ; genetics ; therapeutic use ; Chicken anemia virus ; genetics ; Gene Transfer Techniques ; Genes, Viral ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; genetics

In order to observe the effect of the immune and weight of chickens after use the attenuated vaccine with low dose of chicken infectious anemia virus (CIAV). In this study, the effects of low dose of CIAV on the weight of SPF chickens and NDV antibody production were observed by simulated experiments. The results showed that 10 EID50 and 5 EID50 CIAV per plume attenuated NDV vaccines were used to cause the weight loss of SPF chickens. Compared with the use of the non contaminated vaccine group, it has significant difference. And NDV antibody levels compared with the use of the non contaminated groups also decreased after use the vaccine with two doses of CIAV contaminated. It has significant difference. A certain proportion of CIAV antibody positive was detected at the beginning of the second week after use the NDV vaccine with two doses of CIAV contaminated. The detection of a high proportion of CIAV nucleic acid was detected in the first week after the use of a contaminated vaccine. The results of the study demonstrate the effects of CIAV pollution on the production and immune function of SPF chickens, and it is suggested that increasing the detection of viral nucleic acid can help save time and improve the detection rate in the detection of exogenous virus contamination by SPF chicken test method.
Animals ; Antibodies, Viral ; immunology ; Chicken anemia virus ; genetics ; immunology ; physiology ; Chickens ; Circoviridae Infections ; immunology ; veterinary ; virology ; Poultry Diseases ; immunology ; virology ; Specific Pathogen-Free Organisms ; Vaccines, Attenuated ; administration & dosage ; genetics ; immunology

In order to testify the antitumor effect, especially its effect against liver carcinoma in vivo, of VP3 protein, one kind of protein coded by chicken anemia virus, recombinants pcDNA-vp3 containing chicken anemia virus vp3 gene, and control vector pcDNA3 were mixed with murine liver carcinoma cell lines H22 respectively. The mixture was injected subcutaneously into Balb/C mice. Some days later, the mice were killed and the solid tumor weighed. The antitumor efficiency was evaluated. The manners of VP3 protein in vivo inducing tumor cell death were identified by using TUNEL assay. All the results suggested that the injection of pcDNA-vp3 and H22 mixture resulted in a significant reduction of tumor growth in mice when compared with the results of control groups. TUNEL assay revealed that VP3 induced apoptosis in vivo. All these indicated that CAV vp3 might be a potential new gene in reducing the growth rate of tumor cells in liver carcinoma or in other kind of solid tumors in vivo.
Animals ; Apoptosis ; drug effects ; Capsid Proteins ; biosynthesis ; genetics ; pharmacology ; Chicken anemia virus ; genetics ; metabolism ; Female ; Genetic Therapy ; Liver Neoplasms, Experimental ; genetics ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-beta-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.
Animals ; Antibodies, Monoclonal/biosynthesis/genetics/*immunology ; Antigens, Viral/analysis ; Capsid Proteins/genetics/*immunology ; Chicken anemia virus/genetics/*immunology ; *Chickens ; Circoviridae Infections/blood/immunology/*veterinary/virology ; Escherichia coli/genetics ; Immunohistochemistry/veterinary ; Liver/virology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence/veterinary ; Poultry Diseases/blood/immunology/*virology ; Specific Pathogen-Free Organisms ; Thymus Gland/virology

Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA-vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE-mediated transfection in vitro with pcDNA-vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L-02, and RT-PCR was performed afterward. The results of RT-PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA-vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.
Apoptosis/*drug effects ; Capsid Proteins/*genetics ; Carcinoma, Hepatocellular/pathology ; Cell Line, Tumor ; Chicken anemia virus ; Chickens ; Cloning, Molecular ; Eukaryotic Cells ; Genetic Vectors ; Lipids ; Liver Neoplasms/*pathology ; Polymerase Chain Reaction ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Recombinant Proteins/pharmacology ; Sequence Analysis, DNA ; *Transfection ; Viral Proteins/genetics

Using PCR technique, the vp3 gene of chicken anemia virus (CAV) was cloned into the eukaryotic expression vector pcDNA3 to construct a recombinant pcDNA-vp3. Restriction enzyme digestion and sequencing analysis revealed that CAV vp3 gene was correctly inserted into the blank vector pcDNA3. After LipofectAMINE-mediated transfection in vitro with pcDNA-vp3 and pcDNA3 respectively, the total mRNA was extracted from liver carcinoma cell lines HepG2 and diploid cell line L-02, and RT-PCR was performed afterward. The results of RT-PCR suggested that vp3 gene was expressed in these two cell lines. At the same time, using in situ apoptotic detection assay, TUNEL kits, the apoptotic cells were found in pcDNA-vp3 transfected HepG2, but not in mock transfected cell lines. VP3 could induce cell death by apoptosis in cancer cell lines, but not in diploid cell lines. All the results indicated that CAV vp3 gene, a potential therapeutic agents, has the potential of being used for cancer treatment.
Animals ; Apoptosis ; drug effects ; Capsid Proteins ; genetics ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Chicken anemia virus ; Chickens ; Cloning, Molecular ; Eukaryotic Cells ; Genetic Vectors ; Humans ; Lipids ; Liver Neoplasms ; pathology ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Sequence Analysis, DNA ; Transfection ; Viral Proteins ; genetics