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

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
Animals ; Chick Embryo ; Chickens ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; Herpesvirus 2, Gallid/genetics* ; Marek Disease

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

Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV (Marek's disease virus) encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq has been suggested as the MDV-associated oncogene. In this paper, based on the published sequence of Meq gene of GA strain of MDV, a pair of primers were designed and synthesized. Meq gene ORF (Open reading frame) of the four Chinese local MDV isolates, the reference strain J-1 and the vaccine strain 814 were amplified by using polymerase chain reaction(PCR). Then the PCR products were cloned and sequenced respectively. The results of sequence comparison indicated that the sequences of Meq gene in different strains are relatively conserved and homology of the amino acid sequences is 96.5%-99.7%. The proline-rich repeats of Meq gene of four MDV isolates have site mutations, and it is related to MDV's virulence. Two unique site mutations appear in Meq gene of Chinese local MDV isolates, but they aren't present in Meq gene of the published MDV strains from abroad and the early domestic strains. It seems that some regularities exist between such mutations in four Chinese local MDV isolates and the virulence of MDV, but the regularities need further research.
Amino Acid Sequence ; Animals ; Base Sequence ; Chickens ; Cloning, Molecular ; Herpesvirus 2, Gallid ; genetics ; Molecular Sequence Data ; Mutation ; Oncogene Proteins, Viral ; 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