Antigenic Comparative Analysis Of Newcastle Disease Viruses With Evolutional Mutations In HN And F Genes Under Antibody Immune Pressures

The Newcastle disease virus (NDV) strain TZ060107was proliferated in chicken fibroblast cell (CEF). Then the cDNA of HN gene and F gene was amplified, cloned and sequenced. The result of sequence analysis shows that cDNA of HN gene is composed of1801bp nucleotides, the coding region is composed of1716bp nucleotides, encoding for571amino acids, and F gene is composed of1725bp nucleotides, the coding region is composed of1662bp nucleotides, encoding for553amino acids.First round, in chicken fibroblast cell (CEF) cultures with antiserum against Newcastle disease virus (NDV) strain TZ060107, the virus was passed serially for50passages in3independent lineages. At the same time, set up3independent lineages without antiserum. HN and F genes were amplified and sequenced every10passages.The starting strain in the second round is strain A1-50which has the largest variation of virus in the50th generation. SPF chickens were inoculated in muscle with oil emulsion inactivated vaccine prepared by NDV field strain A1-50. Two weeks later the chickens were vaccinated again,then after ten days, chickens were bled and serum samples were collected as antiserum against NDV strain A1-50. According to the virus neutralization test the right concentration of antiserum for the next serial passages is1:20. This concentration is used as the immune selective pressure of antibody. in chicken fibroblast cell (CEF) cultures with antiserum against Newcastle disease virus (NDV) strain Al-50, the strain A1-50was passed serially for50passages in3independent lineages. Respectively for al, a2, a3. At the same time, set up3independent lineages without antiserum. Respectively for b1, b2, b3. HN and F genes were amplified and sequenced for the60th,70th,80th,90th,100th generations of each passage serial. The sequencing results indicated that the ratio of nonsynonymous mutations (NS) vs synonymous mutations (S) for HN genes in the passed lineages with antiserum against A1-50was5.25, which was obviously higher than2.375of NS/S in the lineages without the antiserum. The stable NS mutations occurred in the first round with the antiserum against the original TZ060107were still maintained and one more new stable NS mutation appeared. For the F genes,3new stable NS mutations occurred during the second round in lineages with antiserum against A1-50when the original NS mutations obtained in the first round with antiserum against TZ060107still existed. It illustrates that after the amino acids associated with antigen epitope is replaced by the new amino acids,it can form new antigen epitope associated with neutralization of virus.Take average value of five times test of Cross hemagglutination inhibition(HI) between original virus TZ060107and its derivative viruses A1-50,A2-50,A3-50. the results showed that the antigenic homology between original virus and its derivative viruses is smaller than the antigenic homology between A1-5-,A2-50,A3-50. Set up another set of experiments, Take average value of five times test of Cross hemagglutination inhibition(HI) between virus TZ060107,A1-50, al-100. the results showed that the antigenic homology between strain A1-50and strain TZ060107is higher than the antigenic homology between strain al-100and strain TZ060107. This indicated that the more continuous passages in cell culture with antiserum passed, the bigger difference of antigenicity between the virus and the original virus had. But the antigenicity didn’t differ significantly between derivative viruses in same generation.The newcastle disease virus (NDV) strain was passed continuously in chicken fibroblast cell(CEF) cultures with antiserum against specific NDV strain, genes have taken place of change or deviation in a certain degree,according to this inference,genes can mutate under the influence of the immune selection pressure such as Multiple vaccinations, Inappropriate immune program in the process of raising chickens, Causing the immune escape strains appear constantly,it will lead to the immune prevention effect of the existing vaccine failed. Therefore, the results of this study can provide a theoretical reference for the clinical practice to better develop immunization program.