Animals ; Forecasting ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; genetics ; metabolism ; virology ; MicroRNAs ; metabolism

The pathogenicity of a field isolate of Marek's disease virus (MDV) named GXY2 integrated with retroviral long terminal repeat (LTR) sequence from a chicken with MD tumors was evaluated. Experimental chickens were divided into group A, B, C, D and E. The later four groups were vaccinated on one-day-old with CVI988/Rispens for group B and D, with HVT for group C and E, while group A was taken as no-vaccinated control. On 8-day-old, group A, B and C were challenged with GXY2 by intra-abdominal injection, group D and E were kept as un-challenged control. All the birds were raised routinely until 82 days post-challenge (PC), died birds during the experiment and the slaughtered birds at the end of the experiment were necropsied and examined for gross lesions of MD and further confirmed by a developed polymerase chain reaction (PCR) based differential diagnosis technique for avian neoplastic diseases. The results showed that time of onset of MD death of group A, B and C were PC 25, 77 and 29 days with the incidences of visible MD visceral tumors. On PC 82 days, tumor incidences and mortalities of group A, B and C were 72%, 34.8% and 50%, 84%, 21.7% and 20%, respectively. The vaccination protection of CVI988/Rispense and HVT were 51.67% and 30.56% respectively. Among all the visceral organs, heart had the highest tumor incidences (23.5%), and then followed by liver (14.7%) and gizzard (10.3%). The weight-gain of unvaccinated birds was significantly depressed and severe dystrophy of thymus and bursa of Fabricius were also found. The results of the study demonstrated that isolate GXY2 possessed the ability of causing acute tumors and overcoming the protection of the vaccinations of either CVI988/Rispense or HVT.
Animals ; Chickens ; Mardivirus ; genetics ; pathogenicity ; Marek Disease ; pathology ; virology ; Polymerase Chain Reaction ; Retroviridae ; genetics ; Terminal Repeat Sequences ; genetics

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect (CPE) was examined daily, and the MEQ expression in MDV-infected microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR (qPCR) and qRT-PCR, respectively. The transcripts of Toll-like receptor (TLR) mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the virus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro (P < or = 0.05/0.001). The transcriptions of TLR15 and TLR1LB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection (dpi), and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens (P < or = 0.01/0.001), while CVI988/Rispens induced more TLR1LB transcript than YL040920 (P < or = 0.001).
Animals ; Brain ; metabolism ; virology ; Chickens ; Gene Expression ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; genetics ; metabolism ; virology ; Microglia ; metabolism ; virology ; Poultry Diseases ; genetics ; metabolism ; virology ; Toll-Like Receptor 1 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Transcription, Genetic

Amino Acid Sequence ; Animals ; Birds ; Cell Transformation, Viral ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; virology ; Models, Biological ; Molecular Sequence Data ; Oncogene Proteins, Viral ; genetics ; physiology

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program.
Animals ; Baculoviridae/genetics/*immunology ; Chickens/*virology ; DNA Primers ; Gene Amplification ; HN Protein/genetics/*therapeutic use ; Korea ; Marek Disease/immunology/prevention & control ; Newcastle Disease/immunology/*prevention & control ; Spodoptera/virology ; Vaccines, Synthetic/genetics/therapeutic use ; Viral Vaccines/genetics/therapeutic use

Recently, much work has been devoted to study MD-induced oncogenesis and the genes involved in this process. Among many genes in the MDV genome, several genes such as Meq, RLORF4, RLORF12, and 132bpr have been considered recently associated with virulence of MDV. In this paper, primers of Meq, RLORF4, RLORF12 and 132bpr genes were designed and synthesized, based on the published whole genome sequence of MDV strain GA. The genes of Meq, RLORF4 and RLORF12 from four Chinese epidemic MDV strains highly passaged on chicken embryo fibroblast (CEF), i. e. L-SYp85C, L-MSp75C, L-CZp75C, and L-ZYp75C, as well as their corresponding parent strains, i. e. L-SY, L-MS, L-CZ, and L-ZY, the reference virulent strain J-1 and the vaccine strain 814 were amplified by PCR respectively. Then the PCR products of interest were cloned and sequenced respectively. The results of sequence comparison and analysis of Meq genes in the study indicated that Meq genes from the two strains L-ZYp75C and L-CZp75C contained single nucleotide insertion and deletion. The Meq gene from strain L-ZYp75C contained an extra cytidine (C) insertion at nucleotide position 529 and a single thymidine (T) deletion at nucleotide position 602, resulting in a frameshift mutation. And this frameshift mutation could lead to changes in deduced amino acid sequence from position 177 to 200 of Meq gene. The extra C insertion at nucleotide position 625 in Meq gene of strain L-CZp75C was also predicted to cause frameshift mutation in three overlapping genes (Meq, RLORF6 and 23KD genes). The comparison of nucleotide sequences of RLORF4 genes in the study revealed that the RLORF4 gene of strain L-SYp85C contained a fragment deletion in Open Reading Frame (ORF) from nucleotide position 215 to 265, resulting in 17 amino acids deletions, which were not found in other sequenced strains. Comparison of nucleotide sequences of RLORF12 genes in the study revealed several mutations. The RLORF12 gene of strain L-MSp75C contained a single T deletion at nucleotide position 67 and of 814 vaccine strain a large fragment deletion from nucleotide position 18 to 86, both of the deletions located in Origin of replication site (Ori) of MDV genome. But strain L-ZYp75C possessed an unique "TGTTGGG" deletion in its RLORF12 gene. When the four Chinese epidemic MDV strains were serially passaged on CEF, the number of copies of the 132bp repeats increased from 2 to more than 10 copies. All of above results indicated that deletion and/or insertion mutation occurred in Meq, RLORF4, RLORF12 and 132bpr after serial passage of these four Chinese epidemic MDV strains on CEF.
Amino Acid Sequence ; Animals ; Cells, Cultured ; Chick Embryo ; Chickens ; DNA Mutational Analysis ; Fibroblasts ; Marek Disease ; genetics ; virology ; Molecular Sequence Data ; Mutation ; Open Reading Frames ; genetics ; Sequence Homology, Amino Acid ; Viral Proteins ; chemistry ; genetics

In this study, we investigated the effects of T-cell suppression on the pathogenesis of subgroup J avian leukosis virus (ALV-J). Chickens were treated with cyclosporin A (CSP) 50 mg/Kg body weight or a corresponding volume of olive oil per every three days after hatching until the end of experiment. Some of the chickens from each treatment group were infected with an isolate of ALV-J, ADOL-7501, at 2 weeks of age. The effects of viral infection were compared to uninfected birds in same treatment group. Intramuscular injection of CSP induced significant T-cell specific immunosuppression determined by decreased cutaneous basophilic hypersensitivity response and decreased lymphocyte mitogenic activity using concanavalin A. Most of the chickens examined had Marek's disease virus infection prior to 3 weeks of age. The percentage of antibody-positive birds and antibody titers were similar in infected chickens between both treatment groups. The ratio of viremic chickens was significantly higher in CSP treated group than that of the Oil treated group. Microscopically, one CSP treated chicken had a nephroblastoma at 10 weeks post infection. At 7 and 10 weeks post-infection, more chickens had myeloid cell infiltrations in multiple organs including heart, liver and occasionally lung. Expression of ALV-J viral antigen determined by immunohistochemical staining was significantly higher in CSP treated chickens than Oil treated chickens at 10 weeks post-infection. This study indicated that chemically-induced T-cell suppression may enhance pathogenicity of the AVL-J virus in broilers.
Animals ; Antibodies, Viral/blood ; Avian Leukosis/*immunology/virology ; Avian leukosis virus/genetics/*immunology ; Body Weight ; *Chickens ; Cyclosporine/*pharmacology ; Dermatitis, Contact/immunology/virology ; Flow Cytometry ; Immunocompromised Host ; Immunohistochemistry/veterinary ; Immunophenotyping ; Immunosuppressive Agents/*pharmacology ; Lymphocyte Activation/immunology ; Marek Disease/*immunology/virology ; RNA, Viral/chemistry/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; T-Lymphocytes/*immunology/virology ; Viremia/veterinary

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.
Animals ; Cell Line ; *Chickens ; DNA, Viral/chemistry/genetics ; Herpesvirus 3, Gallid/*genetics/*metabolism/pathogenicity ; Immunoblotting/veterinary ; Marek Disease/*virology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence/veterinary ; Open Reading Frames/*genetics ; Polymerase Chain Reaction/veterinary ; Recombinant Proteins/genetics ; Specific Pathogen-Free Organisms ; Transfection/veterinary ; Viral Proteins/genetics/*metabolism