Isolation, Identification And Analysis Of Genetic Variation Of High Virulent Strains Of Newcastle Disease Virus

The research is based on the experimental treatment of 8 strains of Newcastle disease virus respectively isolated from the disease-infected poultry farms of Hebei, Beijing, Tianjin, etc, with their respective labels of TJ/99, SJZ/99, YQ/01, TS/01, ZD/02, ZD-2/02, ZD-3/03, and BT/03. After being cloned and purified by the method of limited-point dilution with egg embryos, morphologic observation and testing of pathogenicity index (including MDT, ICPI and IVPI) have been done on these 8 isolates. Round, filamentous and irregular virus particles are seen through electron microscope, and the surfaces of the strains are observed to have spike-like structures. Different strains have different sizes with diameters or long axis ranging from 100 to 500 nm. The result obtained through measuring their pathogenicity index shows that the 8 strains of NDV claim these data: MDT, 42.9~49.5h; ICPI, 1.93-2.00; IVPI, 2.68-2.89, which indicate that these strains are all velogenic Newcastle disease virus.Included in this research are also 14 strains of NDV, among which are F48E8, La Sota, SF, BJ, QH8/79, SH7/99, etc, together with the above mentioned 8 strains, whose genes in the protein F cleavage site region are cloned and their homogeneity analyzed. It can be found in the result that, a) there is a low level of homology among the vaccinal strain of La Sota and other 13 strong virulent strains, with homogeneity ranging from 85.0% to 89.8%; The homogeneity between domestic standard velogenic strain-F48E8 and La Sota, as well as another domestic isolates is about 90% (89.1%~90.4%); While the homogeneity among the domestic isolates ranges from 88.2% to 99.4%. The homogeneity between different NDV strains is different, b) the cleavage site of F protein of F48E8 is 112RRQRRF 117, while another 12 strains isolates have the cleavage site-112RRQR/KRF117, which is in agreement with the characteristics of high virulent strains and the result of pathogenicity test is similar.Comparison has been made on the nucleotides of the gene F with a length of 233 bp (157 ~389 bp) and their corresponding amino acid sequence (53-130 amino acid residues), which include the 8 strains of NDV used for the test and the 33 other typical strains downloaded from Genebank, totaling 47 strains altogether. Software DNAStar has beenused for the purpose of analysis, phylogenetic tree system has been set up, and the genotypes of the 8 isolated strains and the strains of SF and BJ have been established. Result shows that a) none of the F genes of the isolated NDV virulent strains suffer any base deletion and insertion, but there are many points mutation scattering about; b) ammo acids at 55 to 61, 72, 84 to 89 and 128 to 130 of the isolated strains remain highly conservative and their mutation points locate between 101 and 111; c) the distance is close on the phylogenetic trees of the NDV strains of ZD-1/02, ZD-2/02, ZD-3/02, BJ, YQ/01 and TJ/99 and that of the JS01-5-GO, ZJ00-1-GO and JS98-3-GO of Goose NDV, which shows that the ND of the chickens and that of the geese's are closely related; d) the NDV strains of BJ, SF, TJ/99, YQ/01, TS/01, ZD-2/02 and ZD-3/03 belong to the typical genotype of VII, SJZ/99 and BT/03 fall into the genotype of VI, which shows that the epidemic ND in Hebei, Beijing and Tianjin are mainly of the genotype VII, and at the same time there is also the genotype of VI. Thus we can see the complicated situation of Newcastle disease epidemiology in our country, which brings no small difficulty to our work of prevention and treatment of the disease.On basis of biological experiment and genetic variation analysis, cross-HI inhibition and cross-neutralization experiment in egg embryos has been conducted on the 12 strains of NDV (8 self-isolated NDV strains and vaccine virus La Sota, F48E8, QH8/79 and SH7/99), with the method of constant virus and variable serum. Result shows that there is low correlation coefficient between La Sota and the high virulent virus, with the rates ranging from 5.8% to 48.41%. The experiment also shows, apart from a f...