| Newcastle disease is a contagious bird disease affecting many domestic and wild avian species. Its effects are most notable in domestic poultry due to their high susceptibility and the potential for severe impacts of an epidemic on the poultry industries. It is endemic to many countries. Since 1997, goose flocks in South and East of China have suffered severe outbreaks of Newcastle disease virus (NDV) infection. Although measures have been taken to control the disease, it is still a big problem around of the world. The pathogenesis and biology of NDV is still unclear.In this study,30 samples from clinical chickens seemed to be infected with NDV and 3 clocal swabs from wild ducks were isolated the virus by chicken embryos. The isolated virus was identified with heamoagglutination assay (HA), heamoagglutination inhibit assay (HI) and immunoflurenscent assay (IFA) with HN monoclonal antibody to NDV. The results showed that 18 isolates were NDV. F genes and HN genes of the isolates were amplified by RT-PCR. The sequence data indicated that the DTC-050508 from chicken and 3 isolates from wild duck have characteristic of very virulent NDV and DTC-050408 from chicken is an avirulent NDV according to the amino acid at 112,115 and 117 sites. Further analysis results indicated that the sequences of NDV isolates from wild ducks have high homologue to the sequences of NDV from goose.The whole genome of JS01/06/wd isolate from wild duck was sequenced. The whole gene length is 15192bp, which is the same length as the sequences of ZJ1,NA-1,SF02,IT-227,ITdove published in GenBank. Compared different gene open read fragment (ORF) among JS01/06/wd and other NDV strains, the genomic structure of NDV is conservative.6 proteins coded by 6 genes of JS01/06/wd has the exact length of ZJ1,NA-1,SF02 isolates, which is different from the avirulent virus. Interesting is that there is a lack of 6 amino acid in JS01/06/wd and the isolates from goose. These results suggested that NDVs from goose or wild duck may have the same original resource.A panel of monoclonal antibodies (mAbs) to NDV were developed by fusions between SP2/0 cells and spleen cells from the Blab/c mice immunized with the recombinant NDV F protein expressed by Bac-To-Bac system, which were named as 1B4,4B11,5F3,4C1,2H10,6F8,4H10,1D10,2F12,4D9,5C6,6C4. The titers of these mAbs were 1:1600 to 1:12800 in IFA. All mAbs could recognize 55KD-fusion protein of NDV in Western blot.3 of 12 mAbs showed neutralization activity in CEF infected with NDV. However all twelve mAbs showed different reactions with NDV isolates from different poultry species such as goose, pigeon, chicken in IFA, Western blot and ELISA.A method for rapid detecting NDV by IFA was established based on the monoclonal antibody. The result demonstrated that NDV could be detected 18hrs post infection without any cross reaction with other virus when CEF were infected with 100TCID50 NDV. This method is more convenient, quickly and accurately than traditional chicken embryo inoculation and RT-PCR assay. It will be helpful to the rapid diagnosis and control NDV in the future. |
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