【作者】 周静；

【导师】 孙继国；

【作者基本信息】 河北农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 从河北省不同地区的发病鸡群中，分离到12株具有血凝性的病毒，经血凝试验及和多种阳性血清的血凝抑制试验，确定12个分离株均为新城疫病毒，分别命名为EBB、HBBa、HBC、HBCh、HBH、HBHa、HBL、HBQ、HBS、HBT、HBX和HBZ。对各毒株进行了毒力鉴定和生物学特性的研究，结果表明：12个NDV地方分离毒株均呈现良好的血凝活性和特异的血凝抑制活性，对鸡胚的致病作用可被新城疫标准阳性血清所抑制。通过最小致死量致死鸡胚平均死亡时间(MDT)、1日龄雏鸡脑内接种致病指数(ICPI)和6周龄鸡静脉接种致病指数(IVPI)的测定证实：12个NDV分离株中除HBC和HBQ为弱毒株外，其余10株均为强毒株。利用RT-PCR技术，对12个NDV分离毒株的F基因主要功能区片段进行了扩增，经鉴定、回收纯化后，克隆到PBS-T载体上，经PCR鉴定和酶切鉴定筛选出阳性重组子，进行核苷酸序列测定及遗传变异分析。将所测得的各分离株F基因序列与GeneBank中国内外已发表的NDV不同基因型代表株的F基因389bp(1bp～389bp)长度的核苷酸片段及其相应的氨基酸序列(1aa～130aa)进行比较，构建系统发育进化树，并确定了各分离毒株的毒力强弱及其所属的基因型。通过遗传进化分析表明，所有毒株在该区段没有碱基缺失和插入，但有多处点突变，分散存在。12个NDV分离株F基因核苷酸序列同源性为81.2％～99.7％，氨基酸序列同源性为77.5％～100％，12个NDV分离株F基因核苷酸序列与国内外已发表毒株的相应序列同源性为80.5％～98.5％，所推导氨基酸序列同源性为77.5％～97.7％。依据核苷酸及其推导的氨基酸同源性的不同可将其分为明显的三群，群内各毒株的同源性较高，而群间差异则较大，其中第一群包括7株(HBB、HBBa、HBHa、HBL、HBS、HBT和HBX)，属于典型的基因Ⅶ型强毒株，分布于进化树的基因Ⅶ型区；第二群包括3株(HBCh、HBH和HBZ)，属于传统的基因Ⅵ型强毒株，分布于进化树的基因Ⅵ型区；第三群包括2株(HBC和HBQ)，属于古典基因Ⅱ型毒株，分布于进化树的基因Ⅱ型区。研究表明河北省目前新城疫的流行情况比较复杂，流行的NDV以基因Ⅶ型最为普遍，同时兼有传统的基因Ⅵ型和古老的基因Ⅱ型。本研究旨在从分子水平分析河北省NDV的流行趋势，从而为新城疫的防制提供科学依据。更多还原

【Abstract】 Twelve isolates of virus which could agglutinate chicken red blood cell were isolated from sick or dead chickens in different parts of Hebei province. These agglutinations were inhibited and neutralized by Newcastle disease virus standard antiserum. These isolates were confirmed to be Newcastle disease virus based on HA and HI test and were named by HBB, HBBa, HBC, HBCh, HBH, HBHa, HBL, HBQ, HBS, HBT, HBX and HBZ. The isolates were identified and studied on their biological characteristics. The results showed that the twelve NDV isolates are hemagglutination (HA) active and hemagglutination inhibition (HI) active, and the pathogenicity to chicken embryo could be inhibited by ND positive serum. Pathogenicity of the isolates was identified. By means of the test of mean death time (MDT) of chicken embryo, intracerebral pathogenicity index (ICPI) of 1-day-old chicken and intravenous pathogenicity index (IVPI) in 6-week-old chicken, HBC and HBQ strains were verified to be lentogenic strains, and the other strains were all virulent.The Main function fragment of Fusion gene of 12 NDV isolates were amplified by revers transcription-polymerase chain reaction (RT-PCR) and sequenced after being cloned into PBS-T plasmids. Gene F of 389bp (1bp～389bp) and their corresponding amino acid sequence (1～130 amino acid residues) of the NDV strains, including the 12 isolates and 22 typical strains from Genebank, were compared and analyzed. The phylogenetic tree system was constructed based on the obtained nucleotide (from 1 to 389bp), and the virulence and genotype of 12 isolates were confirmed. The results showed that none of the F genes of the isolates suffered any base deletion and insertion, but there are mutations in many points scattering about. The homologous rate of F gene nucleotides sequences was 81.2%～99.7%, and of the deduced amino acid sequences was 77.5%～100%. The F gene sequences from 12 isolates shared a high identity between 80.5%～98.5% with that of related sequences reported home and abroad. The amino acid residues predicted, shared an identity between 77.5%～97.7% with that of other strains. The results showed that the 12 isolates can be divided obviously into three groups. The nucleotide sequence and deduced amino acid sequences of this fragment of 12 NDV isolates were compared with that of published NDV F genes respectively. Their homology was higher in the same group and lower between the groups. There were 7 strains in the first group, 3 strains in the second group and 2 strains in the third group. The NDV isolates in group 1, 2, 3 belonged to genotypeⅦ,ⅥandⅡrespectively.The results revealed that the NDV epidemic in Hebei province was complicated and all the isolates belonged to 3 genotypes (Ⅱ,ⅥandⅦ). Most of the isolates were classified into genotypeⅦ. It indicated that the genotypeⅦNDV was the major pathogeny causing the epizootic of ND in Hebei province.更多还原