【作者】 吴昊；

【导师】 丁壮；

【作者基本信息】 吉林大学 ， 预防兽医学， 2009， 硕士

【摘要】 近几年,在我国吉林、上海、浙江、安徽及福建等省市在发病猪体内相继分离出副黏病毒病,经研究初步表明分离株为新城疫病毒。新城疫病毒(NDV)的感染宿主主要是鸡,但经过世界范围内的几次大流行后,其宿主范围已经明显扩大。迄今已知能自然或人工感染的鸟类超过250余种,而且可能有更多的易感宿主还没有被发现。但1997年以后鹅新城疫病毒病的出现并持续流行,则打破了不引起水禽发病的这一规则。目前,研究表明新城疫病毒已跨越种间屏障感染哺乳动物(猪)。这种现象揭示,新城疫病毒的感染和致病宿主范围正在不断扩大,给新城疫的防控带来新的挑战。因此对猪源副黏病毒的进化关系分析及寻找来源是非常必要的。本研究从发病猪体内分离出一株病毒,电镜观察初步鉴定为副黏病毒,分子鉴定确定该病毒为新城疫病毒变异株,暂命名为JL-1株。该病毒可在SPF鸡胚尿囊腔和猪肾细胞中增殖,并能导致鸡胚发生病变。为进一步鉴定及明确该病毒的起源及所处的分类地位,本研究采用RT-PCR和RACE法对该株病毒含有6个蛋白及末端序列的全基因组进行了克隆和测序。对F、HN蛋白氨基酸序列疏水性和抗原表位预测及对比,并将该病毒的结构和非结构蛋白与同属的其它病毒进行了同源性比较和进化关系分析,对猪源副黏病毒的来源进行了推测,明确了猪源副黏病毒JL-1株的病毒起源分类地位。本研究为阐明导致新城疫病毒感染宿主特性改变的原因,决定NDV宿主特异性改变的分子机理奠定了基础。更多还原

【Abstract】 Avian Paramyxovirus consisted of nine serotypes, APMV 1-9, but APMV-1 only included Newcastle disease virus. There existed tremendous virulence diversity among different hosts in which chicken was the most sensitive, pigeon and parrot also could be infected and falled ill. Waterfowls such as geese or ducks constantly were not considered to be infected by NDV. For the past few years, uninterruptly expanding of the host infection spectrum about NDV, chicken, pigeon, parrot and some rare birds could be infected, as well as waterfowls. The outbreak of goose-host paramyxovirus disease broken traditionary theory which said waterfowls could not be infected by NDV. However, since 1999, researchers isolated paramyxovirus from swine. After studying the virus is the variation of NDV strains that are infected the mammals (swine).In this study, our lab isolated NDV from illed swines in 2005, named JL-1. Electron microscopy revealed the morphology of a paramyxovirus. Hemagglutination and hemagglutination inhibition tests confirmed the JL-1 and APMV-1 have the relationship between the serum. Through MDT, EID50, ICPI, IVPI tests determind that JL-1 is avirulent. After molecular identification confirm the porcine paramyxovirus strain JL-1 is the Newcastle disease virus variant. Cloning and sequence analysis of the F protein found that the encoding F protein cleavage site amino acid composition for the 112-G-R-Q-G-R-L-117, which is characteristics of avirulent, that confirm the JL-1 is avirulent in molecular level again, LaSota, and homology of 99.5%, belong to F typeⅡgene. To confirmed virus origin taxonomic status and the source of the porcine paramyxovirus strain JL-1. A total of ten overlapping cDNA clones, covering the entire genome of JL-1, were obtained by either target gene walking RT-PCR or 5’- and 3’-RACE. The genomes of nine NDV strains including Aus/32, Beaudette C, Herts/33, LaSota, Ulster, HB92 V4, SF02, ZJ1 and NA-1 were compared to the nucleotide sequence of the gene of strain JL-1. JL-1 was isolated from swine; NA-1, SF02 and ZJ1 were all isolated from geese, others from chicken. The results showed that NP nucleotide homology of 85.37% - 99.32%; P nucleotide homology of 82.66% - 99.92%; M nucleotide homology of 84.84% - 99.82%; F nucleotide homology 84.60 % - 99.52; HN nucleotide homology of 81.66% - 100%; L nucleotide homology of 86.20% - 99.83%; 5’nucleotide homology of 60.2% - 99.1%; 3’nucleotide homology of 85.5% - 100%. The results showed that JL-1 strains was closely related to the LaSota, Beaudette C strain, with higher nucleotide homology, but low nucleotide homology with geese NDV。Sequence compiled from these clones showed that the genome of strain JL-1 was 15,021 nt long (GenBank accession # EU546165). Analysis of sequence shown that JL-1 had changed in 1590-1798 nt, 1821-1894 nt, 2298-2418 nt, 3091-3263 nt, 4418-4670 nt, 15010-15090 nt. That confirmed infect swine is the Newcastle disease virus variant first. Phylogeny of JL-1 showed that it is the same evolutionary branch with LaSota, Beaudette C, and Clone 30. It revealed NDV has infected mammal (swine). This study provide a theoretical basis for the origin and prevalence characteristics of porcine paramyxovirus, and had founded basic for the reasons for change that Characteristics of infected host and the molecular mechanism change of NDV specificity host.更多还原