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鸡新城疫(Newcastle Disease,ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起禽的一种高度接触性、急性败血性传染病。新城疫是威胁世界养禽业的主要疾病之一,被世界动物卫生组织(OIE)归为A类传染病。新城疫病毒几乎可感染全部鸟类,并可随侯鸟的迁徙而广泛传播,NDV属于副粘病毒科、副粘病毒亚科的腮腺炎病毒属,单股负链RNA病毒。我国长期以来普遍开展了以接种疫苗为主的新城疫综合防制措施,很好控制了ND的暴发,但近年来ND的流行又出现了新的特点:非典型新城疫日益增多,甚至高抗体鸡群也发生该病,这些流行特点很可能是由变异或超强毒株引起。近年来鹅源病毒新城疫变异株的出现使人们对于非典型新城疫发病原因的尤为感兴趣。本研究在2008年从新乡某鸡场不表现典型ND症状且高抗体蛋鸡中分离到的一株新城疫野毒(NDV ND-xx08株),在对其进行生物学特性研究的基础上,扩增出其F和HN基因,并做了免疫保护试验,为探讨新城疫是否发生变异和其预防提供理论依据,为河南省NDV分子流行病学研究提供有益参考,丰富了我国ND分子流行病学特征,为制定控制ND方法提供重要依据。首先对NDV ND-xx08株进行了一系列生物学特性研究。SPF鸡胚接种培养后,经过试验鉴定,该分离株具有血凝性,且可被ND标准阳性血清所抑制和中和,不能被禽流感(H5 、H9亚型)标准阳性血清和EDS-76标准阳性血清所抑制,因此该分离株为新城疫病毒。该病毒的最小致死量病毒致死鸡胚的平均时间(MDT)为50h;1日龄SPF鸡脑内接种分离病毒致病指数(ICPI)为1.71;6周龄SPF鸡静脉接种致病指数(IVPI)为2.38;由此NDV ND-xx08株确定属于NDV强毒型。其次分别设计了一对引物,Trizol法提取了其RNA,RT-PCR扩增出NDV ND-xx08株F基因和HN基因的全基因序列,并分别两条基因的核苷酸序列进行测定和分析,并对氨基酸序列进行了分析。用DNAStar软件对本研究毒株与国内外发表的一些有代表性的46株参考毒株的NDV F基因47nt-420nt区域进行同源性比较,绘制了基因进化树,分析其遗传变异关系。经测定NDV ND-xx08 毒株的F基因开放性阅读框架全长1662bp, 共编码553个氨基酸,F蛋白的裂解位点为112R-R-Q-K-R-F117。系统发育分析得出该分离株属于基因Ⅶe型,将其与国内外发表的部分新城疫病毒毒株之间F基因核苷酸和氨基酸序列进行比较,其核苷酸序列的同源性在82.7%-97.8%之间,氨基酸同源性在87.5%-97.7%之间,与F48E9株的氨基酸同源性为91.5%,与 Lasota,Clone30和V4株的同源性分别为88.1%,88.6%和88.3%。从分子水平上说明NDV ND-xx08 株为发生了一定程度变异的新城疫强毒株。NDV ND-xx08 毒株的HN基因开放性阅读框架全长1 716 bp,编码571个氨基酸,属于C群。它与其他24株的 HN 基因核苷酸序列同源性在80%-98.4%。它与北京毒株(SRZ03)和江苏毒株(zj1)同源性较高,分别经测定为98.1%和98.4%。从基因进化树来看,该毒株与北京和江苏所分离的毒株同源性较近,基本上属于同一基因群;与 Lasota 和Clone30等疫苗株的同源性较远,属不同的基因群。ND-xx08株 HN 蛋白氨基酸序列与其他24株同源性在 87.2%-98.2%,与 Lasota 和Clone30 的同源性分别为88.2 %和89.3 %,同源性较低。最后用该毒株做了免疫保护性试验,分别将NDV ND-xx08株尿囊液稀释10、100、1000倍,将原尿囊液和3种稀释倍数的尿囊液按照常规方法做成油苗。将四种疫苗对鸡群免疫了2次,研究其抗体增长规律。组后对免疫鸡群进行攻毒,看油苗的保护率,筛选出效果最好的油苗和免疫剂量。 [ Last edited by jwk3029 on 2010-5-19 at 17:29 ] |
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2楼2010-05-20 03:59:21
★ ★ ★
jwk3029(金币+90, 翻译EPI+1): 2010-05-20 17:16:48
sirljz(金币+3):谢谢交流 2010-05-20 17:33:41
jwk3029(金币+90, 翻译EPI+1): 2010-05-20 17:16:48
sirljz(金币+3):谢谢交流 2010-05-20 17:33:41
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仅供LZ参考。 Chicken Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious, acute septic infectious disease. ND is among the most serious disease threats to the poultry industry worldwide. It’s included in the list A infectious disease by OIE. NDV, is a negative-sense single-stranded RNA virus, a member of rubulavirus, paramyxoviridae family, affects most avian species, and spread as the immigration of birds. For a long time, ND had been well prevented by a comprehensive measure mainly by vaccination in China. But, ND has new features in recent years, such as more and more non-typical ND, even in chicken flocks have high antibodies concentration, which might induced by variant or highly virulent strains. Newly identified geese-derived NDV variant attracted researchers’ attention on the pathogenesis of inapparent ND. In this study, we isolated a wild NDV strain (strain NDV ND-xx08) from layers with no apparent ND symptoms, in a hennery of Xinxiang. After basic assay on its biological characteristic, the F and HN gene were amplified, immune protection efficiency was evaluated, which would provide theoretical foundation for investigation on ND variation and prevention, provide important basis for formulation of ND prevention methods, and also be a useful reference for NDV molecular epidemiology in Henan, enrichment for ND molecular epidemiology features in China. Firstly, biological characteristics of NDV ND-xx08 were examined. Cultured by inoculating SPF chicken embryo, this new strain was able of inhibiting blood coagulation, and was neutralized by NDV standard positive serum, can’t be inhibited by bird flu (subtype H5, H9) and EDS-76 standard positive serum. So we identified this isolation as NDV. It took average 50h (MDT) for minimum lethal dose of this virus to kill chick embryo. Pathogenicity index for 1-day SPF chicken inoculated the isolation in brain (ICPI) was 1.71, Pathogenicity index for 6-week chicken inoculated the virus through vein (IVPI) was 2.38. These data revealed NDV ND-xx08 was a highly virulent strain. Secondly, the F and HN gene were amplified by RT-PCR from RNA (extracted by Trizol method) using primers we designed. After sequencing, we analyzed the nucleotide sequences and the protein sequences. DNAstar software was used to compare the NDVF gene 47nt-420nt region of this strain with other 46 typical strains published in China and other countries, draw the gene phylogenetic tree and analyze the genetic variation. The open reading frame of NDV ND-xx08 strain F gene is 1662 bp, encodes 553 amino acids. The lysis site locates at 112R-R-Q-K-R-F117. Phylogenetic analysis shows this strain belongs to VIIe genotype. Comparison of the nucleotide sequence for this strain and other published strains, revealed 82.7%-97.8% homology, 87.5%-97.7% homology in amino acid sequence. A 91.5% amino acids sequence homology was found when compared to F48E9, 88.1%, 88.6% and 88.3% to Lasota, Clone30 and V4 respectively. This means NDV ND-xx08 is a highly virulent NDV strain has some variation in the molecular level. The open reading frame of NDV ND-xx08 strain HN gene is 1716 bp, encodes 571 amino acids, belonging to C group. The nucleotide sequence of this strain has 80-98.4% homology to other 24 strains. High homology was found to Beijing strain (SRZ03) and Jiangsu strain (zj1), 98.1% and 98.4% respectively. On the gene phylogenetic tree, ND-xx08 strain is near to Beijing strain (SRZ03) and Jiangsu strain (zj1), they are basically in the same gene group; but far away from Lasota and Clone30, belonging to different groups. The amino acid sequence of ND-xx08 has 87.2%-98.2% homology with other 24 strains, a low homology of 88.2% and 89.3% to Lasota and Clone30 respectively. At last, immune protection experiments were carried out using this strain. Inactivated oil emulsified vaccine were prepared by original amniotic fluid and amniotic fluid diluted 10, 100 and 1000 times according to traditional method. Chicken flocks were inoculated twice with these four oil-emulsified vaccines, following with examination of antibody concentrations. Subsequently, we evaluated the protection efficiency of chicken flocks to the challenge of the virus strain, found the vaccine with high efficiency and best dose. 顺便说一句,一个摘要,这么多车轱辘话。 |
3楼2010-05-20 10:30:37

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