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猪源乙型脑炎病毒河南分离株的全基因测序及进化分析
Full-length Genomes and Phylogenetic Analysis of Japanese Encephalitis Virus Isolates from Swine in Henan
【作者】 禹乐乐；
【导师】 王爱萍；
【作者基本信息】 郑州大学 ， 动物学， 2013， 硕士
【摘要】 日本乙型脑炎病毒(Japanese encephalitis virus, JEV)是一种虫媒传播性病毒,归属于黄病毒科(Flaviridae)黄病毒属(Flavivitus)。乙脑病毒能够在一个人畜共患的循环圈内传播,涉及到猪,鹭科鸟类,库蚊属蚊子等。由乙型脑炎病毒引发的乙脑为自然疫源性的疾病,乙脑患者不能保持体内较高的病毒滴度,因此人常常出现急性的JEV感染,也通常是JEV的终末宿主。在乙型脑炎病毒的循环圈里,水禽为携带者,蚊子为其主要的传播媒介,猪为贮存宿主与增值宿主,给养猪业也带来了巨大的经济损失。乙脑病毒全基因组测序分析可以直观地了解JEV基因组整体信息与分子毒力特征,对乙脑的分子流行病学也具有直接意义。JEV的基因组为单股正链RNA,全长约为11Kb。有一个I型帽子结构(m7GpppAmp)存在于5’末端,3’末端多以CU—OH结尾,而没有polyA尾巴。氨基酸位点96至10394为全基因组的单一开放性阅读框,编码一个包含有3432个氨基酸残基的多聚蛋白前体。共包括3个结构蛋白：核衣壳蛋白(C)、囊膜糖蛋白(E)、前膜蛋白(prM/M),还有7个非结构蛋白：NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5。JEV基因组顺序为：5’—C—PrM/M—NS1—NS2—NS2b—NS3—NS4a—NS4b—NS5—3’,各基因区段间无重叠。目前,国内已完成全基因组测序的毒株约为130个,GenBank己公布全基因组序列的JEV毒株仅有63个,且多为人源和蚊源分离株,国内猪源JEV报道较少,已知全基因组序列信息的猪源毒株仅有5个,更多的对猪源JEV全基因组的测序及分析,不仅丰富国内猪源JEV的相关信息,对了解猪群JEV的流行动态及分子毒力特征有直接意义。为了全面了解猪源JEV的全基因组结构,丰富猪源毒株的全基因组信息以及分子毒力特征,本研究首先测定了3个JEV猪源河南分离株BSF.ZZ-1、BSF.ZZ-3和CSF.XZ-2D的全基因组序列,并对它们的核苷酸与氨基酸序列分别进行了遗传分析。结果表明,CSF.XZ-2D全长为10976nt,BSF.ZZ-1和BSF.ZZ-3全长均为10977nt,在后两个毒株基因组的1070Int位点处均有一个插入碱基“G”。BSF.ZZ-1与SA14-14-2的核苷酸同源性最高,为99.8%,氨基酸同源性为99.1%。CSF.XZ-2D、BSF.ZZ-3与SA14-14-2核苷酸同源性均为99.7%,氨基酸同源性分别为99.2%和99.1%。BSF.ZZ-1、BSF.ZZ-3、CSF.XZ-2D与疫苗株的亲本毒株SAl4株核苷酸和氨基酸同源性也较高,核苷酸同源性都为99.4%,氨基酸同源性均为99.0%,与猪源分离株HW (HW1)、HWe (HW2)和SH0601的核苷酸与氨基酸同源性均为98.0%以上。用涵盖全部5个基因型的30个JEV毒株的全基因组序列构建系统进化树,发现这3个猪源分离株位于同一进化分支,均属于基因Ⅲ型。
【Abstract】 Japanese encephalitis virus (JEV) is an arbovirus belonging to the family Flaviviridae. It is maintained in a zoonotic cycle involving pigs, ardeid birds and Culex species of mosquitoes. Humans are accidental dead end hosts of JEV infection because they cannot sustain high viraltiters. This cycle involves pigs as the major reservoir amplifying host, water birds as carriers and mosquitoes as vectors, causing serious economic losses to the pig industry.Sequence analysis of the whole genome can understand the overall information of genome and molecular virulence characteristics. It is also important for molecular epidemiology of JE. The genome is approximately11kb in length, containing short5′and3′non-coding regions, single-stranded, positive sense in polarity, capped at the5′-terminus and lacks a poly(A) tail at the3′-terminus. The genome order has been determined as5’-Cap-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′. The genome is translated into a single large polyprotein which is processed co-and post-translationally into three structural proteins:nucleocapsid protein (C), membrane protein (M), and envelope glycoprotein (E), as well as seven non-structural proteins: NS1through NS5. Up to now, the completed whole genome sequencing of strains is approximately130in China, GenBank published genome sequence of the JEV strains is only63. The paper about JEV strains isolated from swine is few, there are only five domestic strains. Sequence analysis of the genomic can learn more about the complete genome and molecular evolution regular of JEV strains isolated from swine.In order to learn more about the complete genome and molecular evolution regular of Japanese encephalitis virus (JEV) strains isolated from swine, the full-length genome sequences of three JEV isolates, namely as BSF.ZZ-1, BSF.ZZ-3and CSF.XZ-2D isolated from swine were analyzed in this paper. The results show that the length of the complete genomes of BSF.ZZ-1, BSF.ZZ-3and CSF.XZ-2D was10977nt,10977nt and10976nt, respectively. There is an insert base of "G" at the 10701nt site in the genomes of both BSF.ZZ-1and BSF.ZZ-3. Compared to the virulent strain SA14-14-2, the homologies of nucleotide and amino acid sequences of of BSF.ZZ-1is99.8%and99.1%, respectively. The homologies of nucleotide sequences of CSF.XZ-2D and BSF.ZZ-3are99.7%, and the homologies of amino acid sequences are99.2%and99.1%, respectively. However, compared to the virulent strain SA14, the homologies of nucleotide and amino acid sequences of BSF.ZZ-1, CSF.XZ-2D and BSF.ZZ-3are99.4%and99.0%, respectively. Compared to the isolates HW(HW1), HWe(HW2) and SH0601from swine, the homologies of the present three isolates are all more than98.0%. Phylogenetic analysises with other30reference isolates covering all five JEV genotypes indicated that BSF.ZZ-1, CSF.XZ-2D and BSF.ZZ-3located in a same evolutionary branch, belonging to the genotype Ⅲ.