【作者】 黄耀伟；

【导师】 于涟；

【作者基本信息】 浙江大学 ， 生物医学工程， 2002， 博士

【摘要】 建立动物RNA病毒的反向遗传系统实现病毒的遗传拯救，从而可以在DNA水平上对RNA病毒进行遗传操作，为深入研究RNA病毒的分子生物学及病毒与宿主的相互作用提供了强有力的技术工具。对RNA病毒进行遗传拯救或构建感染性克隆已成为分子病毒学实验室进行病毒结构与功能深入研究的必经之路。而在国内开展这方面的研究，特别是动物RNA病毒不多。鸡传染性法氏囊病(IBD)是危害世界养禽业的严重传染性之一，本研究工作以该病的病原传染性法氏囊病病毒(IBDV)为模型，在克隆IBDV浙江分离株全基因组cDNA的基础上，在国内首次尝试构建IBDV反向遗传系统，拯救经过分子标记的IBDV，并初步进行IBDV功能学研究。 首先建立了长距离RT-PCR方案快速扩增并克隆IBDV基因组。IBDV是双链RNA病毒，属于双RNA病毒科(Biranviridae)禽双RNA病毒属(Avibimavirus)，其基因组由A、B两个节段组成。以IBDV浙江分离株HZ2细胞培养液为材料，比较了4种病毒纯化和基因组抽提方案，认为超速离心后用蛋白酶K消化病毒粒子是较好的方法。反转录合成单链cDNA，经RNase H除去杂交链的RNA后，使用经过计算机辅助设计的、位于病毒基因组非编码区(NCR)的一对引物直接扩增到A节段预期分子量的片段；进而对整个PCR过程进行了条件优化，建立了特异性强、省时省力、可重复的实验操作方案。使用T-A克隆法快速克隆PCR产物，序列分析证实获得了3259bp的A节段全长(GenBank登录号AF321054)。用类似的长距离RT-PCR和克隆方案获得HZ2株和JD1株含两端NCR的2827bp的B节段全长(GenBank登录号分别为AF493979和AY103464)。 在对IBDV浙江分离株双节段全基因组进行生物信息学分析时，提出了未见于已有文献报道的几种新猜测。三株IBDV浙江分离株HZ2、JD1、ZJ2000在核苷酸和推导的四种病毒蛋白VP2、VP3、VP4、VP5的氨基酸水平上高度同源，并具有位于VP2高变区的特征性氨基酸H253、N279、T284、R330，一般认为这些氨基酸是弱毒株的标志，但也出现于ZJ2000株和其它几个强毒株中；(新猜测①)对野毒株ZJ2000进行重新分析，发现其高强毒力可能与VP2前体蛋白(pVP2)剪切后的一种11个氨基酸的短肽有关；序列比较进一步支持IBDV主要宿主保护性抗原VP2并非是决定IBDV毒力的唯一因素；不同毒力表型毒株的两端NCR序列高度保守提示NCR可能与IBDV毒力并不直接相关；(新猜测②)另外，根据VP5在十多种不同表型毒株中高度保守，作者提出了一种VP5与病毒毒力关系的推测。 在B节段上，发现HZ2株、JD1株VP1基因(编码依赖RNA的RNA聚合酶，RdRp)上游的5′-NCR上都有一个与原来推定的起始密码同一阅读框(in-frame)的密码ATG，在国际上只有一株在GenBank上登录的加拿大毒株QC-2也有同一点突变，尚无任何公开文献报道。(新猜测⑧)对这一突变进行了RNA二级结构分析和理论意义评估；分析了IBDV VP1 基因上可能与病毒毒力有关的几个潜在位点。其中第 4、13、546氨基酸位点可能与病毒适 应细胞或致弱有关；（新猜测④）经比较，发现双NA病毒科的三个成员IBDV、IPNV和 DXV在其基因组 A节段或 B节段 5’．NCR上都有至少一个以上的额外起始密码，这可能与 该科病毒独特的翻译机制有关。 设计了一种以重组PCR（融合PCR）或常规PCR为介导的定点突变方案在IBDV基因 组上引入沉默突变的遗传标记。其中，A节段的置换突变为：A2368C（数字后面为突变后 的核昔酸，下同＊ A2371C，这两个点突变在A节段上产生了新的Nae酶切位点（GCE4 GGG人 同时，在 A节段 3’－NCR的特定位置上引进了 G或 C的插入突变，前者预期可以 改变 3’．NCR的H级茎环结构：B节段上的定点突变为：G2283A，C2286T，A2287C，这 三个点突变产生了新的 EcOR V酶切位点（GAT A匹人 以上突变测序证实。改变后的 A节 段和 B节段序列在 GenBank上的登录号分别为 AY163771，AY163772。 以 pCI真核表达载体为骨架，构建了几种突变或未突变的含 IBDV HZZ株基因组 CDNA 的重组子，命名为：pCIA、pCIB、PCIAA、PCI－InA、PCI－mAg、PCImB。其中，PCI－AA 是一种“ Head，to－tail” A节段双价重组表达载体。 脂质体介导共转染 pCI－A／pCI－mB、pClmA／pCI－mB、pCImAg／pCI－mB三组真核表达质 粒进入Vero细胞。KNA点杂交、免疫荧光分析表明重组子在Vero细胞得到了表达；显微镜 下观察到被转染质粒的细胞形态学发生变化，产生了类似野生IBDV感染细胞时出现的细胞 病变（Cvn），这种变化在用每次的细胞培养上清液接种新的 vero细胞时（类似病毒“传代”） 都可以观察到；电子显微镜下可以看到符合 IBDV病毒粒子结构的物质；在 PCI－ajCI－mB 转染组的细胞培养液中提取病毒样物质进行RT－PCR，可扩增出病毒双节段全长基因组。用 Nae酶切A节段PCR产物，用EcoR V酶切B节段PCR产物，获得与预期被引入的遗传 标记飓 Nae、EcoR V）一致的条带，证实 IBDV确实得到了拯救，而并非野生更多还原

【Abstract】 The study of viruses and their interactions with host cells and organisms has benefited greatly from the ability to engineer specific mutations into viral genomes,a technique known as reverse genetics.To develop the reverse genetics system,or so-called genetic rescue techniques for infectious bursal disease virus (IBDV),Birnaviridae family,the full-length cDNA of the double segment of the chicken IBDV was amplified and cloned by long RT-PCR in the first-step.A comparison of four purification and extraction methods of RNA from IBDV( HZ2 strain,isolated originally in Zhejiang Province)-infected chicken embryoid fibroblasts (CEF) showed that the ultracentrifugation followed by proteinase K digestion extracted dsRNA more effectively. Then reverse transcription was carried out at 50C using Superscipt II enzyme,followed by RNase H digestion. Amplification of single stranded cDNA in a single step resulted in the synthesis of the full-length segment A of 3259 bp and segment B of 2827 bp respectively. The amplified products were cloned and sequenced,identifying that it was an IBDV. This method is superior to other methods based on amplifying different parts of the genome many times,therefore the cloning procedure was simplified.Analysis of the full-length of segment A of IBDV isolatedin Zhejiang Province,including an attenuated strain HZ2,an attenuated vaccine strain JD1 and a virulent field isolate ZJ2000,revealed the identity of IBDV with two overlapping open reading frames( ORF ) flanked by 5’ -and 3’ -noncoding regions( NCR ) in 3259bp long. The strains shared high identity with each other at nucleotide or deduced amino acid level,and also had four unique sites H253,N279,T284,R330 which are common in other attenuated and some classic or highly virulent strains. The virulent strain ZJ2000 had several key amino acid mutations located in hypervariant region of VP2 and near the VP2-VP4 cleavage site of polypeptide,one of which is in the new identified 11 aasmall peptide. It may be related to the virulence. Sequence comparison supported that VP2 is not the sole determinant of the virulence. The highly conservation in 5’ - and 3’ - NCR of different strains indicated the NCR may be not responsible for the virulence. But the same conservation appeared in VPS revealed another complex relationship between VP5 and the virulence.The full-length nucleotide sequence including the 5’ - and 3’ -noncoding regions (NCR) of IBDV strain HZ2 and JD1 was established. The nucleotide sequence encoding VP1,an RNA-dependent RNA polymerase (RdRp),of HZ2 and JD1 were aligned with that of all other IBDV strains. Surprisingly,An in-frame start codon (ATG) is presented in the 5’ -NCR upstream of usual VP1 genes which are coded by all the compared strains except an Canadian strain QC-2. According to the sequences information of other two birnaviruses including IPNV and DXV,we suppose the advanced in-frame start codons presented generally in the first ORFs of birnaviruses do not come by accident and play an important role in the initiation of virus proteins translation. The ACG may be the start codon in other IBDV strains in the same position. For IBDV,it is possible that the VP1 gene started from the first or second in-frame start codon respectively results in two different size products. The potential sites responsible for IBDV virulence were also searched and listed. The results showed that substitution at positions 4,13,256 may be the key amino acids related to the virus adaption or attenuation.The genetic tags were introduced to two segments of HZ2 strain respectively that were distinguished from the parent virus sequences by site-directed silent mutagenesis. The cDNA clone of segment A was engineered to contain two silent nucleotide changes to create a Nae I site (A to C and A to C at nt 2368 and 2371,respectively). At the same time,the 3’-NCR of coding strand of segment A was inserted a G at nt 3243 or a C at 3235. The former is expected to change the stem-loop secondary structure at the 3’ terminus of segment A. The cDNA cl更多还原