鸡新城疫病毒强毒株的分离鉴定及其遗传变异分析
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摘要
本研究从河北、北京、天津等发病鸡群中分离到8株新城疫病毒，其代号分别为TJ／99、SJZ／99、YQ／01、TS／01、ZD-1／02、ZD-2／02、ZD-3／03、BT／03，经鸡胚终点稀释法克隆纯化后，对其进行了形态学观察和毒力指数(MDT、ICPI、IVPI)测定。电镜观察可见8株分离株呈圆形、线形和不规则形，病毒粒子表面有纤突样结构，各毒株大小不同，粒子直径或长轴在100～500nm之间。毒力指数测定结果显示，8株NDV的MDT在42.9～49.5h之间，ICPI在1.93-2.00之间，IVPI在2.68～2.89之间，表明8株NDV均为新城疫病毒强毒株。
对上述8株分离株以及La Sota、F_(48)E_8、SF、BJ、QH_8/79、SH_7/99等共14株NDV的F蛋白裂解位点附近的基因进行了克隆和同源性分析。结果发现：(1)疫苗用毒La Sota株同其它13种强毒株之间的同源性在85.0％～89.8％之间，普遍较低；国内标准强毒F_(48)E_8株与La Sota株及国内分离毒株的同源性在90％左右(89.1％～90.4％)；国内分离株之间的同源性在88.2％～99.4％之间，不同毒株之间的同源性有高有低。(2)F_(48)E_8株F蛋白的裂解位点为~(112)RRQRRF~(117)，而其它12株强毒株F蛋白的裂解位点为~(112)RRQKRF~(117)，符合强毒株裂解位点的特征，同毒力测定结果相一致。
应用DNAStar软件对上述克隆测序的14株NDV及从Genebank上下载的国内外有代表性的33株NDV等共47株NDV的F基因233bp(157～389bp)长度的核苷酸片段及其相应的氨基酸序列(53～130位氨基酸残基)进行了比较，建立了系统发育树，并确定了8个分离株及SF、BJ株的基因型。结果表明：(1)所有NDV分离毒株的F基因都没有碱基的缺失和插入，但有多处点突变，分散存在。(2)分离株的55～61、72、84～89及128～130之间的氨基酸均高度保守，突变的氨基酸位点多集中在101～111之间。(3)ZD-1／02、ZD-2／02、ZD-3／02、BJ、YQ／01、TJ／99同鹅源的NDV株JS01-5-GO、ZJ00-1-GO及JS98-3-GO株在系统发育树上的距离较近，说明当今鸡ND的流行同鹅的ND有着密切的关系。(4)BJ、SF、TJ／99、YQ／01、TS／01、ZD-1／02、ZD-2／02、ZD-3／03等8株为基因Ⅶ型，SJZ／99和BT／03为基因Ⅵ型，表明河北、北京、天津等地区ND的流行以新的基因Ⅶ型的为主，但同时又有旧基因Ⅵ型的存在，说明ND的流行情况比较复杂，给ND的防制工作带来了一定的难度。
在以上生物学试验及遗传变异分析的基础上，本研究对分离鉴定的8株NDV及La Sofa、F_(48)E_8、QH_8/79、SH_7／99等共12株NDV，采用固定病毒，稀释血清的方法，进行交叉血凝抑制试验和鸡胚交叉中和试验。结果表明，La Sota同强毒株之间的相关系数较低，在5.89％～48.41％之间。强毒株之间相关系数除去个别的几个相对较低外，大部份都在25.12％～186.19％之间。
将交叉中和试验和遗传变异分析的结果相结合，进行综合分析，结果说明疫苗毒La Sota株同现今流行毒株之间确实存在一定的抗原性差异性。另外可以看出同一基因型之间的相关系数较高，如属于基因Ⅵ型的SJZ／99和BT／03的相关性为114％，
属于基因Vn型的毒株之间的相关系数在25.12%~186.19%之间，从一定程度上表明
基因型同抗原性存在着一定的相关性，但没有明显的规律。若要彻底弄清基因型同抗
原性的相关关系，就必须进一步做交叉保护试验来验证。
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 been
used 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
对上述8株分离株以及La Sota、F_(48)E_8、SF、BJ、QH_8/79、SH_7/99等共14株NDV的F蛋白裂解位点附近的基因进行了克隆和同源性分析。结果发现：(1)疫苗用毒La Sota株同其它13种强毒株之间的同源性在85.0％～89.8％之间，普遍较低；国内标准强毒F_(48)E_8株与La Sota株及国内分离毒株的同源性在90％左右(89.1％～90.4％)；国内分离株之间的同源性在88.2％～99.4％之间，不同毒株之间的同源性有高有低。(2)F_(48)E_8株F蛋白的裂解位点为~(112)RRQRRF~(117)，而其它12株强毒株F蛋白的裂解位点为~(112)RRQKRF~(117)，符合强毒株裂解位点的特征，同毒力测定结果相一致。
应用DNAStar软件对上述克隆测序的14株NDV及从Genebank上下载的国内外有代表性的33株NDV等共47株NDV的F基因233bp(157～389bp)长度的核苷酸片段及其相应的氨基酸序列(53～130位氨基酸残基)进行了比较，建立了系统发育树，并确定了8个分离株及SF、BJ株的基因型。结果表明：(1)所有NDV分离毒株的F基因都没有碱基的缺失和插入，但有多处点突变，分散存在。(2)分离株的55～61、72、84～89及128～130之间的氨基酸均高度保守，突变的氨基酸位点多集中在101～111之间。(3)ZD-1／02、ZD-2／02、ZD-3／02、BJ、YQ／01、TJ／99同鹅源的NDV株JS01-5-GO、ZJ00-1-GO及JS98-3-GO株在系统发育树上的距离较近，说明当今鸡ND的流行同鹅的ND有着密切的关系。(4)BJ、SF、TJ／99、YQ／01、TS／01、ZD-1／02、ZD-2／02、ZD-3／03等8株为基因Ⅶ型，SJZ／99和BT／03为基因Ⅵ型，表明河北、北京、天津等地区ND的流行以新的基因Ⅶ型的为主，但同时又有旧基因Ⅵ型的存在，说明ND的流行情况比较复杂，给ND的防制工作带来了一定的难度。
在以上生物学试验及遗传变异分析的基础上，本研究对分离鉴定的8株NDV及La Sofa、F_(48)E_8、QH_8/79、SH_7／99等共12株NDV，采用固定病毒，稀释血清的方法，进行交叉血凝抑制试验和鸡胚交叉中和试验。结果表明，La Sota同强毒株之间的相关系数较低，在5.89％～48.41％之间。强毒株之间相关系数除去个别的几个相对较低外，大部份都在25.12％～186.19％之间。
将交叉中和试验和遗传变异分析的结果相结合，进行综合分析，结果说明疫苗毒La Sota株同现今流行毒株之间确实存在一定的抗原性差异性。另外可以看出同一基因型之间的相关系数较高，如属于基因Ⅵ型的SJZ／99和BT／03的相关性为114％，
属于基因Vn型的毒株之间的相关系数在25.12%~186.19%之间，从一定程度上表明
基因型同抗原性存在着一定的相关性，但没有明显的规律。若要彻底弄清基因型同抗
原性的相关关系，就必须进一步做交叉保护试验来验证。
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 been
used 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
引文
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