摘要
新城疫(ND)是由新城疫病毒(Newcastle disease virus,NDV)引起的鸡的高度接触性传染病,是禽类重要的疫病之一。NDV是单股不分节的负链RNA病毒,属于副粘病毒科。全基因组长度约为15kb,分别编码核衣壳蛋白(NP)、磷蛋白(P)、基质蛋白(M)、融合蛋白(F)、血凝素-神经氨酸酶(HN)及聚合酶蛋白(L)等6个主要蛋白。
1、按照常规的病毒分离培养、血凝试验(HA)、血凝抑制试验(HI)等实验室诊断技术,对所采集的64份疑似ND病料进行了处理,初步鉴定其中17份病例感染了NDV,ND检出率约为26.6%。初步统计结果表明,不同品种不同日龄的鸡只,ND发病存在一定的差异。经蚀斑纯化分离出NDV HN09-68和HN09-83株,并对其致病指数MDT和ICPI进行了测定,HN09-68的MDT和ICPI值分别为53h和1.75,HN09-83株的MDT和ICPI值分别为59.5h和1.95,由此判定两分离株均为新城疫病毒强毒毒株。
2、依据GenBank中公布的新城疫病毒的相关全基因组序列,设计14对特异性引物,运用反转录-聚合酶链式反应(RT-PCR)扩增HN09-68和HN09-83两分离株的基因序列,运用DNASTARSeqMan软件进行全基因序列的拼接。经序列分析可知,两株分离株基因组长度均为15192个核苷酸,基因组长度符合“六碱基法则”,两毒株核苷酸同源性为98.6%。基因组5’端长度为55nt,3’端长度为114nt;各基因之间插入1～47nt;两毒株在NP基因非编码区比疫苗株LaSota株长6nt;F蛋白裂解位点推导序列均为“112R-R-Q-K-R-F117”,具备新城疫强毒的分子特征,与其MDT及ICPI毒力指数相符;HN基因ORF均编码571aa,其中347位点由E突变为K;两分离株其各基因在其编码区及非编码区均发生了一定的变异,大多突变属于无义突变。两分离株各基因核苷酸同源性对比发现,两分离株与国内部分NDV强毒分离株亲缘关系较近,而同国外及部分疫苗株同源性较低,说明我国NDV的流行趋于统一,并有着自身的地域特色。
Newcastle disease virus (NDV) is one of the most important infectious agents in poultry, affecting a wide variety of birds and causing impoutant economic losses.The virus belongs to the family paramxoviridae , subfamily paramyxovirinae, in the genus Avulavirus, and encompasses a diverse group of single-stranded, negative-sense, nonsegmented RNA virues of approximately 15kb. The genome encodes six major proteins in the 3’to 5’direction: nucleoprotein(NP), phosphoprotein (P), matrix protein (M),fusion protein (F), haemagglutinin- neuraminidase (HN) and large polymerase protein (L).
1 Isolation, Identified and The biological characteristics of Two NDV strains. After chicken embryos inoculation and serological identification, 17 NDV isolates were identified and collected from 64 ND suspected specimens. The isolation rate was about 26.6%. Two isolates were purified plaque-purification test, and characterized biologically. Based on its MDT(53h、59.5h) and ICPI(1.75、19.5),the isolates were predicted to be velogenic isolates. Statistics showed that ND occurred when the production of chicken was high, occurrence on Broiler was more than Layers. Moreover,it usually occurred on Chick. Hence, ND is still one of the dieases which hazard poultry industry. Different isolation strains had differences among regions.
2 Genome and genetic characterization analyses of Two NDV strains.14 pairs of primers were designed to amplify the specific gene fragment by RT-PCR and sequenced. The complete genome sequence was analysised by SeqMan. The genomic sequence consists of 15192nt, which agree with the“rule of six”, the two islotates , Homology between the two isolation was 98.6%. the leader sequence was 55 nt, and the trailer sequence was 114 nt in size. The additional six bases of two NDV strains lie in the noncoding region of the 3’terminus of the NP gene. The amino-acid sequence of the fusion protein cleavage site in two isolates was ,which is a typical sequence of velogenic strains and is in agreement with the results of in-vivo pathogenicity tests. A single amino acid change ,from glutamic acid (E) to lysine(K) at codon 347(E347K). The study showed that more non-sense mutation occurred in coding region and non-coding region of NDV protein gene. Homology between the two strains was high, however, homology between the two strains from abroad was low. Prevalence of NDV had the same tendency and had regional features.
引文
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