Dynamic Properties Of Interaction Of Class Ⅰ Newcastle Disease Virus With Chicken Embryo Fibroblast Cells

Newcastle disease virus (NDV) is classified as a member of the newly defined genus Avulavirus in the family of Paramyxoviridae. It is the causative agent of Newcastle disease (ND), which is widely distributed in more than 200 types of birds and mammals and has inflicted substantial economic costs to poultry industry worldwide. Among the numerous strains, it can be divided into Classâ… (15198nt) and Classâ…¡(15192nt or 15186nt) by phylogentic analysis. Because classâ… NDV strains were avirulent and mainly isolated from waterfowls and birds, the epidemiology and pathogenicity of class I NDV in chickens were easy to be neglected. However, in our earlier research, we found a velogenic NDV was generated from a nonpathogenic waterfowl isolate by passaging in chickens. By sequencing the passages, the genomic length maintained with the size of 15198nt.In current study, to research the pathogenicity and evolution mechanism of this waterfowl origin NDV in chickens, Chicken Embryo Fibroblast cell ine (DF-1) was chosed to propagate the velogentic isolate. To systematically know the propagation process and pathogenic mechanism of class I NDV infecting chicken cells, the monolayer of DF1 cell was quantitatively inoculated with classâ… NDV velogenic strain. Cells and cultural supernatant were collected at intervals post infection. Virus titre was evaluated by determination of TCID50, localization of structure proteins in DF1 cells was identified by indirect immunofluorescence assay (IFA), mRNAs level of interest genes was quantitated by real-time RT-PCR and conformation change of cytoskeleton was stained by special dyes.1 Preparation of antibodies against structure proteins of NDVIn the study, several mice were immuned with the virus strain (Classâ… NDV virulent strain 9a5b) or recombinant proteins expressed in E. coli BL21 (DE3), including Nucleoprotein (NP), Phosphoprotein (P), Matirx (M), Hemagglutinin-neuraminidase (HN), and Fusion (F) proteins. Following by three immunation, the sera were collected. Monoclonal antibodies against NDV were developed by the hybridoma technology. And then the antibodies were identified by hemagglutination inhibition (HI), indirect immunofluorescence assay (IFA), cell neutralization test, ELISA and western-blot analysis. The results showed that, besides anti-sera against each protein, a panel of 12 hybridoma cell lines was successfully prepared. Among them, mAbs 2A8, 3H7 and 3H9 were specific to Class I NDV HN protein in the experiment. All of the other mAbs specific to P or NP protein had a cross-reaction with Classâ…¡NDV strains.2 Dynamic properties of interaction of Classâ… NDV with DF-1 cellsDF-1 cells were inoculated with 9a5b strains at a dose of 5 m.o.i, the cells and cultural supernatant were collected at intervals post infection. Virus titre was evaluated by determination of TCID50, localization of structure proteins in DF1 cells was identified by indirect immunofluorescence assay (IFA), mRNAs level of interest genes was quantitated by real-time RT-PCR and conformation change of cytoskeleton was stained by special dyes. The result shows that, the accumulation of mRNAs were in accordance with the growth curve of the virus. NP and P proteins could be observed in cytoplasm early at 1h post infection, while other proteins were accumulated later relatively. Otherwise, we found that the virus had little effect upon the cytoskeleton by stained of cell actin and Endoplasmic Reticulum with special dyes.3 Preliminary study on proteomics of avian cells infected with Classâ… NDV.DF-1 cells were inoculated with 9a5b and 1a strains at a dose of 0.001 m.o.i, and then the cells were collected at 12h post infection for proteomics research. Comparison of 2-DE image between 1a infection team and CEF control team revealed that 41 differentially expression protein spots, including 13 up-regulated protein spots, 28 down-regulated protein spots. The 11 protein spots were analyzed by matrixassociated laser dissociation /ionization time of flightmass spectrometry (MALDI-TOF-MS). 7 proteins were successfully identified, among which the protein spots with great importance were preliminary studied.In conclusion, the dynamic properties and proteomics of interaction of Classâ… NDV with chicken cells were firstly investigated. The propagation mechanism of NDV infection with chicken cells was systematically explained, which is benefit for further study on the mechanism of the virulence evolution and interaction of the class I NDV with avian cells.