【作者】 胡博；

【导师】 张改平；

【作者基本信息】 吉林大学 ， 预防兽医学， 2014， 硕士

【摘要】 日本乙型脑炎病毒(Japanese encephalitis virus, JEV)隶属黄病毒科、黄病毒属，可在人、蚊子与动物之间循环传播，是一种重要的人兽共患传染病病原。该病在临床上可简称为乙脑（Japanese encephalitis, JE)，具有病致死率较高，感染发病后大多留有神经性后遗症的特点。全球有超过十亿人生活居住在乙脑流行区域里，并且流行区域近年来仍在在不断扩大之中，已成为全球关注的公共卫生问题。亚洲是乙脑的主要流行区域，目前还没有特效药可于治疗该病，主要依靠疫苗免疫进行预防。通过对JEV全基因组序列分析能够有效获得该病毒的遗传信息。JEV的病毒基因组全长大约11kb，属于正链RNA病毒。其基因组5’端存在一个I型帽子结构，CU－OH结尾多出现在3’端，没有polyA尾。JEV基因组编码一个单独的开放性阅读框（96-10394位点之间），能够编码一个多聚蛋白前体（共计3432个氨基酸），经过蛋白酶剪切作用后可产生3个结构蛋白（C、M、E）与7个非结构蛋白（NS1-NS5）。其中结构蛋白E是JEV最为重要的结构蛋白，有多个抗原表位，在病毒入侵宿主过程发挥重要作用。E蛋白氨基酸的改变可能影响到病毒的毒力，其中最重要的是E176、E138等位点，其他位点的改变（如E177、E279、NS2b-63等）也将直接或间接的改变病毒的毒力。目前，国内已完成130个JEV分离株的全基因组测序工作，其中有63个基因组序列在NCBI GenBank上公布，但是猪源的分离株仅有5个，其余多是蚊源和人源分离株。为了解猪源JEV在细胞内连续传代后的基因组分子特征，本研究将两个猪源河南分离株BSF.ZZ-1和BSF.ZZ-3在BHK-21细胞上进行连续传代，并首先分析了传代中多个代次病毒的E基因序列，以期观察病毒E基因的稳定性与关键核苷酸和氨基酸位点变化情况。然后对两个细胞传代株BSF.ZZ-1-p60与BSF.ZZ-3-p60进行了全基因组序列测定，并对两个毒株的核苷酸与氨基酸序列进行了比对分析。通过对E基因序列分析表明，经连续传代60次后病毒E基因趋于稳定，BSF.ZZ-1毒株E蛋白氨基酸位点E21(A→V)、E200(T→A)、E244(G→E)、E279(M→K)和E426(G→D)，BSF.ZZ-3毒株的E蛋白氨基酸位点E244(G→E)、E255(F→L)、E285(M→L)、E368(L→S)和E497(N→S)传代后发生稳定点突变；通过对全基因组序列测定表明，BSF.ZZ-1-p60和BSF.ZZ-3-p60全基因组长度都由10977nt组成。BSF.ZZ-1-p60和BSF.ZZ-3-p60与SA14-14-2同源性较高，核苷酸同源性为99.6%和95.9%；氨基酸同源性为96.8%和85.0%。研究显示这些位点大多与弱毒株SA14-14-2一致，与强毒株SA14不一致。更多还原

【Abstract】 Japanese encephalitis virus (JEV) is a member of Flavivirus genus of theFlaviviridae family. The virus circulates in humans, mosquitoes, and animals to causeserious viral encephalitis in humans named as Japanese encephalitis (JE) with a highdeath rate and most patients leaving neurological sequelae. There are more than onebillion of people living in the endemic regions of the JEV, which are expandingconstantly in recent years. JE has become a public health problem of global concernand Asia is the main endemic region. There are no effective drugs for the therapy ofJEV infection and the most important controlment is to use vaccine immunization toprevent clinical cases.A lot of genetic information of JEV can be obtained by sequencing the viralgenomes. The complete genome of JEV is about11kb long, which belongs to thepositive-strand RNA viruses. There is a type I cap structure in5’-end of the viralgenome, a CU-OH structure in the3’-end while without poly A tail. JEV genomeencodes a single open reading frame (from sites96to10394) encoding a precursorpolyprotein (composed of3432amino acids) and can translates three structuralproteins (C, M&E) and seven non-structural proteins (NS1-NS5). The structuralprotein E is the most important structural protein with a plurality of epitopes and playsan important role in invading the host. Changing the gene sequences of protein E maylead to amino acid changes and further, among which the E176, E138and other sites(such as E177, E279and NS2b-63) may directly or indirectly relates to the virulenceof the virus. To date, genome sequencing of a total of130JEV Chinese isolates hasbeen completed, of which63genomes have been published in the GenBank data base.However, only five of them are isolated from swine, and the rest are most frommosquito and human. Thus, it is meaningful for understanding the JEV epidemiologyand the genome characteristics more directly through the genome-wide sequencing ofJEV isolates from pigs.In the present study, the genetic stability of E gene of JEV isolates BSF.ZZ-1andBSF.ZZ-3passaged in BHK-21cells was first studied. The results showed that the E gene was becoming stable after60passages in BHK-21cells. After serialsucultivation, stable amino acid mutation occured at the sites E21(F→L), E200(T→A), E244(G→E), E279(N→S) and E426(G→D) in isolate BSF.ZZ-1and sitesE244(G→E), E255(F→L), E285(M→L), E368(L→S) and E97(N→S) in isolateBSF.ZZ-3. No mutations were observed at part of the virulence correlated aa sites,such as E107, E138, E176, E177and E315, similar to those of the vaccine strainSA14-14-2. However, mutations at the virulence correlated amino acid sites of E279（M→K→M）of BSF.ZZ-3happened repeatedly. Whether these mutations werecorrelated with the virulence and adaptability of JEV to host cells needed to be furtherstudied. Sequencing of the viral genomes showed that the length of both of thecomplete genomes of BSF.ZZ-1-p60and BSF.ZZ-3-p60were10977nt. Compared tothe vaccine strain SA14-14-2, the homologies of nucleotide sequences ofBSF.ZZ-1-p60and BSF.ZZ-3-p60were99.6%and95.9%, and the homologies ofamino acid sequences were96.8%and85.0%, respectively. The phylogenetic analysiswith other30reference isolates that covered all the five JEV genotypes, indicated thatBSF.ZZ-1-p60and BSF.ZZ-3-p60located in a same evolutionary branch andbelonged to the genotype III.更多还原