【作者】 苏帅；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2013， 博士

【摘要】 马立克氏病毒(Marek′s disease virus, MDV)是α疱疹病毒的成员，基因组为180kb左右的双股DNA，可引起鸡的淋巴细胞瘤。GX0101株MDV是国内外从鸡体内分离到的第一株整合有网状内皮组织增生症病毒(Reticuloendotheliosis virus, REV)长末端重复序列(Long terminal repeat, LTR)的重组野毒株。本实验室前期研究中已经构建了GX0101的细菌人工染色体(Bacterial artificial chromosome, BAC)克隆以及敲除REV-LTR的突变株GX0101LTR，证实REV-LTR插入片段显著增强了GX0101的横向传播能力。敲除肿瘤基因meq的突变株GX0101meq病毒不但丧失对SPF鸡的致病性，而且不再引起肿瘤以及胸腺和法氏囊萎缩。本学位论文在上述研究的基础上，进一步构建不同的突变株，并以此深入研究MDV相关的生物学活性和研发MDV的基因缺失疫苗。1完成了GX0101株MDV的全基因组序列分析自然重组病毒GX0101在致病性、免疫原性、横向传播能力等方面具有独特的生物学活性。为了对GX0101进一步的分子病毒学研究，本研究利用GX0101的BAC克隆结合高通量测序技术完成了对其全基因组的序列分析。GX0101全基因组长178101bp,其基因组的TRL、UL、IRL、IRS、US和TRS区分别长12758bp、113572bp、12741bp、12700bp、11695bp、13134bp。GX0101全基因组仅含有一个REV-LTR重组片段，位于其基因组US区的SORF1中，SORF2基因前267bp碱基处。与rMd5不同，GX0101含有一个SORF2基因。通过与已发表的近10株MDV的比较,发现GX0101与英国分离株C12/130的两个不同毒力的感染性克隆pC12/130-10和pC12/130-15同源性最高。除了GX0101比其它毒株多出REV-LTR片段外，在经比较的190个开放阅读框(Open reading frame，ORF)中，不同毒株间发生不同程度变异的有77个。在这77个ORFs中， GX0101与pC12/130-10、pC12/130-15两个克隆呈100%同源的ORF分别有50个和47个，但与RB1B、Md5、GA、814和CVI988等毒株却只有7-27个ORFs呈现100%同源性。GX0101基因组TRS区的MDV97.3-97.6开放阅读框中，有5个连续的217bp碱基的重复，显著多于其它不同毒株的拷贝数。GX0101基因组的IRS区的86.2-86.4开放阅读框中，存在3个相同的217bp的重复序列，而其它MDV仅含有1-2拷贝的重复。GX0101的全基因组序列的比较分析，将有助于阐明与MDV致病性、传播性相关的基因，也有助于揭示不同地域间MDV的遗传变异和演化关系。2构建了meq基因缺失性疫苗候选株SC9-1，在SPF鸡实验中显示出比国内外最广泛应用的CVI988/Rispens有更好的保护性免疫力重组病毒GX0101meq表现出良好的保护性免疫作用，但基因组中带有卡纳霉素抗性基因，不符合农业转基因生物安全条例，因此不能作为疫苗使用。本研究设计的“对flp重组酶的部分诱导结合多重筛选”的方法，敲除掉GX0101meq中的卡那霉素抗性基因，筛选到具有良好复制能力的SC9-1(GX0101meq kana)株MDV。将SC9-1株MDV接种SPF鸡和含有MDV母源抗体的海兰褐蛋鸡，既没有致肿瘤性，也没有呈现免疫抑制、生长迟缓等任何致病性，而且能够为免疫鸡提供比CVI988/Rispens更好的保护性免疫。多次重复SC9-1株MDV对SPF鸡的免疫保护试验，以500PFU/只的剂量感染MDV超强毒rMd5，SC9-1均提供100%的免疫保护，显著高于CVI988/Rispens的免疫保护(保护率80-90%)。将SC9-1感染性克隆DNA通过肌肉注射1日龄SPF鸡，14天后能从鸡的体内分离到SC9-1病毒。为进一步探讨SC9-1的BAC质粒作为DNA疫苗提供思路。SC9-1株MDV基因缺失疫苗已获得农业部环境释放的审批书，并在3000只海兰褐蛋鸡完成了6个月的环境释放试验，并且持续观察6个月，不仅符合农业转基因生物安全，而且呈现很好的免疫保护效果，是一株理想的新型疫苗株，并且已经转让给北京翎羽生物科技有限公司进行后续相关的生产性试验。制备的中试产品证明SC9-1能提供比国内外最广泛应用的CVI988/Rispens株疫苗更好的保护性免疫力3对GX0101和GX0101LTR混合感染鸡群后连续病毒鉴别定量跟踪试验证明，REV-LTR插入片段使GX0101提高横向传播性，是其成为流行毒株的竞争性选择压优势为了模拟自然感染传播模式，比较并证实GX0101相对于其突变株GX0101LTR在鸡群横向传播上的竞争性选择压优势,本研究比较研究了能够对GX0101和GX0101LTR病毒基因组作鉴别性定量的双重荧光定量PCR的引物、探针和反应体系,并且成功的用于动态比较鸡群共感染以上两种病毒后在连续传代过程中不同病毒病毒血症。结果表明，将GX0101、GX0101LTR同等剂量感染SPF鸡后，第一代用于接触感染的空白SPF鸡在接触感染14天时即可检测到感染GX0101，而在接触感染28天时才能检测到感染GX0101LTR；而第二代用于接触感染的鸡在接触感染21、28天均能检测到感染GX0101，在28天时仅有一只鸡检测到GX0101LTR；同样第三代用于接触感染的鸡在与第二代鸡接触感染21、28天均能检测到感染GX0101，而没有检测到GX0101LTR。将GX0101显著低于GX0101LTR(100PFU/只的GX0101和2000PFU/只的GX0101LTR)的病毒共感染SPF鸡群，同一个隔离罩饲养进行接触感染传播。连续2次传代后，虽然在一部分接触感染病毒的鸡(6/10)中仍能够检出GX0101LTR，甚至在个别鸡仍为优势毒株，但是接触感染病毒的鸡全部能够检出带有REV-LTR的GX0101,并且部分鸡(4/10)仅能检出GX0101，不能检出GX0101LTR。以上结果证明了在鸡群内自然感染和传播过程中，带有REV-LTR的GX0101相对于敲除其REV-LTR的GX0101LTR株在横向传播性上显示出明显的竞争性选择压优势。这可以解释为什么在鸡群中发生率很低的MDV与REV间的基因重组产生的整合有REV-LTR的GX0101能从极低的比例演变为从鸡群中易于分离到的流行株的生物学机制。4敲除GX0101株SORF2基因的生物学活性比较表明SORF2基因表达产物与MDV不同株的横向传播性密切相关REV-LTR片段具有真核启动子和增强子活性，能够增强位于其插入位点后的MDV基因的表达。GX0101基因组中的REV-LTR片段位于SORF2基因上游,其对SORF2基因的转录表达都可能有很大影响，为了深入研究重组病毒GX0101的SORF2基因功能，本研究在感染性克隆GX0101-BAC的基础上构建了SORF2基因缺失株GX0101sorf2。试验结果表明SORF2基因并非MDV复制以及致肿瘤必需基因，敲除掉SORF2基因的GX0101sorf2在细胞培养与GX0101的复制能力相同。与GX0101相比，GX0101sorf2对鸡的致死率以及致肿瘤率下降，但是差异不显著。GX0101sorf2横向传播的感染率低于GX0101。GX0101中的REV-LTR片段插入增强SORF2蛋白的表达，表明GX0101具有较强的横向传播能力可能与SORF2蛋白的大量表达这一分子机制相关。5插入ALV-LTR的重组MDV的构建及其生物学活性的比较研究ALV与REV一样同属于禽反转录病毒，在我国鸡群中普遍存在MDV与ALV的共感染。为了研究ALV-LTR片段在GX0101基因组重组位点上的稳定性，以及ALV-LTR片段的插入对MDV生物学活性的影响，并以此为模型研究ALV与MDV的重组病毒。本研究利用基于链霉素负筛选系统的同源重组方法将ALV-J中国分离株NX0101的LTR片段完全等位取代bac-GX0101的REV-LTR片段，构建了含有ALV-LTR片段的重组病毒GX0101-ALV-LTR。GX0101-ALV-LTR在细胞培养上具有与GX0101相似的复制能力，在细胞上连续传代20代，通过PCR验证，ALV-LTR都能在GX0101-ALV-LTR的基因组中稳定存在。将GX0101-ALV-LTR接种1日龄SPF鸡，接种后6-8周，PCR验证ALV-LTR能在GX0101-ALV-LTR基因组中稳定存在。通过接触感染的病毒基因组中ALV-LTR依然能够稳定遗传。攻毒90天，GX0101-ALV-LTR的死亡率和肿瘤率分别为55%和20%。相比GX0101LTR，间接免疫荧光以及Western Blot试验显示，GX0101-ALV-LTR的SORF2蛋白表达量显著增加；Northern Blot试验显示GX0101-ALV-LTR的SORF2、US1、US10基因转录量显著增加。GX0101-ALV-LTR是国内外第一株整合进ALV来源LTR的重组MDV,证实整合进MDV同一位点的ALV-LTR片段能与插入的REV-LTR片段一样稳定遗传，并显示类似的生物学特性。6用基因芯片技术比较分析了LTR插入片段对重组MDV中不同基因表达水平的影响利用定制的Agilent表达谱芯片，分析比较了带有REV-LTR的GX0101及其LTR敲除株GX0101LTR和ALV-LTR替代株GX0101-ALV-LTR在感染CEF细胞上的病毒不同基因的表达水平。结果表明，在GX0101与GX0101LTR病毒差异表达显著的基因中(P<0.05)，75个MDV基因GX0101的转录水平显著增高，其中上调倍数大于10倍的共14个基因，而位于REV-LTR片段直接下游的SORF2、US1、US10基因上调倍数多达30倍以上，其中SORF2基因上调最为显著，高达399倍；GX0101的UL38基因转录降低，下调倍数为8.3倍。GX0101-ALV-LTR与GX0101非常类似，与GX0101LTR相比，同样位于ALV-LTR片段直接下游的SORF2、US1、US10基因上调倍数多达30倍以上，SORF2基因上调最为显著；GX0101-ALV-LTR的UL38基因转录下调倍数为6.7倍。利用Northern Blot以及Western Blot试验证实了插入LTR片段的重组病毒SORF2的转录子及表达蛋白均显著升高。这些比较研究结果将有助于进一步分析LTR插入片段赋予重组MDV显著升高的横向传播性究竟与MDV的那些基因表达产物相关。7用基因芯片技术比较分析了LTR插入片段对重组MDV感染的细胞基因组不同基因转录水平的影响利用定制的Agilent表达谱芯片，分析比较了带有REV-LTR的GX0101及其LTR敲除株GX0101LTR和ALV-LTR替代株GX0101-ALV-LTR对感染的CEF细胞基因组不同基因的表达水平的影响。对鸡41765个基因的表达差异进行了分析，结果显示，感染GX0101的CEF细胞基因与感染GX0101LTR相比，转录显著增高的有12个基因(差异倍数大于20倍)，其中ChEST1024a11(GeneBank NO. BU288902.1)、SKOR2(GeneBank NO. XM001233569.2)上调倍数分别为190倍、165倍；转录显著降低的基因有4个基因(差异倍数大于20倍)，其中ChEST445i1(GeneBank NO. CR390488.1)下调倍数为105倍。感染GX0101-ALV-LTR的CEF细胞基因与感染GX0101LTR相比，转录显著增高的有5个基因(差异倍数大于20倍)，其中ChEST1024a11(GeneBank NO. BU288902.1)、SKOR2(GeneBank NO. XM001233569.2)上调倍数分别为203倍、83倍；转录水平下降基因的下调倍数均小于20倍。通过基因芯片表达谱筛选出的显著差异基因有助于进一步研究插入LTR片段的重组MDV病毒不同的生物学活性与宿主基因转录水平的相关性。本研究不仅进一步通过模拟自然感染模式证明REV-LTR插入片段显著提高了病毒GX0101在鸡群中的横向传播性，是其从极少数的突变株变为流行株的流行病学机制，也部分阐明了REV-LTR提高GX0101横向传播性的分子机制。还利用BAC克隆技术将GX0101改造成了一株保护免疫力优于CVI988/Rispens的新的疫苗候选株。更多还原

【Abstract】 Marek’s disease virus （MDV） is a member of the Alphaherpesvirinae subfamily, and itsgenome contains a linear double-stranded DNA of about180kb. MDV causes Marek’s diseasein chickens with lymphomas. GX0101is the first recombinant MDV field strain containingthe reticuloendotheliosis virus （REV） long terminal repeat （LTR） insert isolated from chickenboth at home and abroad. In previous research in our laboratory, GX0101bacterial artificialchromosome and its mutant strain GX0101LTR with a deletion of the REV-LTR wereconstructed and demonstrated that REV-LTR insert increased the horizontal transmissionability of GX0101. Mutant strain GX0101meq with a deletion of meq oncogene wasdemonstrated to have lost its pathogenicity in SPF chicken, and no longer induced tumors andatrophy of thymus and bursa of fabricius. On the basis of the above research, we furtherconstructed different mutants to study MDV related biological activities in-depth anddeveloped MDV gene-deleted vaccine.1Completed the sequencing analysis of the whole-genome of MDV strain GX0101Recombinant field strain GX0101possesses unique biological activities in many aspects,for instance, pathogenicity, immunogenicity, horizontal transmission ability and so on. Inorder to further carry on molecular virology research of GX0101, this study competed thesequencing analysis of the whole-genome of GX0101using its BAC clone combined withhigh-throughput sequencing technologies.The whole-genome sequence of GX0101consists of178,101nucleotides （nt）, and theterminal repeat long （TRL）, unique long （UL）, internal repeat long （IRL）, internal repeat short（IRS）, unique short （US）, and terminal repeat short （TRS） regions with the lengths of12,758,113,572,12,741,12,700,11,695, and13,134nt, respectively. The GX0101genome containsonly one REV-LTR insert located within SORF1gene in US region, at a site267nt upstreamof the SORF2gene. GX0101contains only one SORF2gene, which is different from rMd5.After homology comparison of nearly10MDV strains, GX0101has the highest identity totwo BAC clones pC12/130-10and pC12/130-15of United Kingdom strain C12/130withdistinct virulence. Besides the accessorial REV-LTR insert in GX0101genome, there are77ORFs in the compared190ORFs between different strains occur different degrees ofmutation. Among the mutated77ORFs, GX0101has50and47ORFs100%identical to PC12/130-10and PC12/130-15but only7to27ORFs100%identical to RB1B, Md5, GA,814and CVI988strains respectively. There are5consecutive repeats of a217-nt fragment in97.3-97.6ORF of the TRS region of GX0101, which is significantly more than other strains.There are3same repeats of a217-nt fragment in86.2-86.4ORF of the IRS region of GX0101,but only1or2copies repeats in other MDV strains.The comparative analysis of whole-genome sequence of GX0101will contribute toclarify genes relative to pathogenicity and transmissibility of MDV. It will also be useful inimplying the genetic variation and evolutionary relationship of MDV between differentregions.2Constructed the meq gene-deleted candidate vaccine strain SC9-1with betterprotective immunity than the most widely used CVI988/Rispens at home and abroad inSPF chicken experimentsThe recombinant virus GX0101meq appeared good protective immunity, but it cannotbe used as a vaccine as residues of Kanamycin resistance genes in the genome which do notconform to Regulations on Administration of Agricultural Genetically Modified OrganismsSafety.We knocked off Kanamycin resistance genes in GX0101meq and selected MDV strainSC9-1（GX0101meq kana） with good replication ability using the method designed in thestudy that partial induction of flp recombinase combined with multiple filters. MDV strainSC9-1was inoculated in SPF chickens and Highland brown layers with MDV maternalantibodies, there were no tumorigenicity and any pathogenicity such as immunosuppressionand growth retardation, and it can provide better protective immunity than CVI988/Rispensfor vaccinated chickens. Many times over repeat of the immune protection test of MDV strainSC9-1to SPF chicken by inoculating with500PFU of very virulent MDV strain rMd5demonstrated that SC9-1can provide100%protective immunity, significantly higher thanCVI988/Rispens with protection rate of80%to90%. When inoculating DNA of SC9-1infectious clone into1-day-old SPF chickens by intramuscular injection, SC9-1virus could beisolated from chickens14days post-inoculation, which provides a way to further discussBAC plasmid of SC9-1as DNA vaccine.MDV gene-deleted vaccine strain of SC9-1has won the ministry of agricultureenvironmental release approval book and finished six months of environmental release test in3000Highland brown layers with continuous observation for six months. SC9-1not onlyconforms to the agricultural genetically modified organisms safety, and renders good immuneprotection effect, making it an ideal new type of vaccine strain. In addition, it has been transferred to Beijing Lingyu Bio-tech for subsequent related productive experiment.Preparation of pilot product certificates that SC9-1can provide better protective immunitythan the most widely used vaccine CVI988/Rispens at home and abroad.3Tracking test of consecutive virus identification and quantification of GX0101andGX0101LTR after the mixed infection in chickens demonstrated that REV-LTR insertimproved the horizontal transmission ability of GX0101and was the competitiveselection pressure advantage of making it a popular strainIn order to simulate natural infection transmission mode to compare and confirm thecompetitive selection pressure of GX0101with its mutant strain GX0101LTR in horizontaltransmission ability in chickens, comparative study on the primers, probes and reactionsystem of dual fluorescence quantitative PCR was carry out to differentially quantify genomeof GX0101and GX0101LTR virus, which were further successfully used to compare thevirus viremia dynamic of different viruses in the process of continuously passage afterco-infection of the above two viruses in chickens.The results show that after inoculating SPF chicken with the same dose of GX0101andGX0101LTR, GX0101can be detected from blank SPF chickens used for contacting withchallenged chickens14days post contact infection, while GX0101LTR can be detected28days post contact infection. GX0101can be detected from the second generation used tocontact with the first generation chickens both21and28days post contact infection, whileGX0101LTR can be detected from only one chicken28days post contact infection.Similarly, GX0101can be detected from the third generation used to contact with the secondgeneration chickens both21and28days post contact infection, while GX0101LTR can notbe detected. When part of SPF chickens were co-infected with GX0101significantly lowerthan GX0101LTR（100PFU GX0101and2000PFU GX0101LTR per chicken）and kept inthe same isolator to infect blank chickens by horizontal transmission. After two consecutivepassages, although GX0101LTR can be detected from part of chickens （6in10） used forcontacting with challenged chickens and is still the dominant strain in individual chicken,GX0101containing REV-LTR can be checked out from all of the chickens used for contactingwith challenged chickens. Also, GX0101can be detected, while GX0101LTR can not, frompart of chickens （4in10）.The above results demonstrated that GX0101containing REV-LTR shows a significantcompetitive selection pressure advantage compared with GX0101LTR with a deletion of itsREV-LTR in natural infection and transmission process in chickens. This can explain thebiological mechanisms why GX0101containing REV-LTR with a low rate of recombination between MDV and REV in chickens can evolve from a very low proportion into a popularstrain easily isolated from chickens.4Comparison of biological activity of GX0101SORF2gene-deleted strain demonstratethat SORF2gene expression product is closely related with horizontal transmissionability of different MDV strainsREV-LTR fragment possesses eukaryotic promoter and enhancer activity, which canenhance expression of genes downstream of insertion site in MDV. REV-LTR locatesupstream of SORF2gene in GX0101genome and it is likely to have a significant impact ontranscription and expression of SORF2gene. To study the function of SORF2gene inrecombinant virus GX0101in-depth, SORF2gene-deleted strain GX0101sorf2wasconstructed on the basis of infectious clone GX0101-BAC.The results show that SORF2is not the essential gene for MDV replication andtumorigenicity. SORF2gene-deleted strain GX0101sorf2exhibited the same replicationdynamics as GX0101on cell culture. Compared with GX0101, the mortality and tumor rate ofGX0101sorf2in chickens were lower but not significantly different. The horizontaltransmission rate of GX0101sorf2was lower than GX0101. The REV-LTR insert in GX0101enhances the expression of SORF2protein, demonstrated that the relative strong horizontaltransmission ability of GX0101may be related with the molecular mechanism of a greatamount of SORF2expression.5Construction of recombinant MDV with ALV-LTR insert and comparative study on itsbiological activityReticuloendotheliosis virus （REV） and avian leukosis virus （ALV） are avian retroviruses.Co-infection of ALV-J and MDV in chickens is very common. To study on the stability ofALV-LTR insert at the recombinant site of GX0101genome and the influence of ALV-LTRinsert on biological activity of MDV, recombinant virus GX0101-ALV-LTR containingALV-LTR fragment was constructed by completely replacing REV-LTR of bac-GX0101withLTR fragment of ALV-J China field strain NX0101at the same site using homologousrecombination, based on Streptomycin negative screening system. GX0101-ALV-LTR can beused as a model for further study of recombination between ALV and MDV.GX0101-ALV-LTR exhibited the same replication dynamics as GX0101at the cellularlevel. PCR proved that the ALV-LTR insert remained stable in GX0101-ALV-LTR genomefollowing20passages on cell culture. GX0101-ALV-LTR was inoculated in1-day-old SPFchickens and ALV-LTR was proved to remain stable in GX0101-ALV-LTR genome6-8weekspost-inoculation by PCR analysis. ALV-LTR still remains stable inheritance in the genome of virus by contact infection. Mortality and tumor rate of GX0101-ALV-LTR were55%and20%respectively90days post-inoculation. Compared with GX0101LTR, IFA and Western blotresults demonstrated that SORF2protein expression was increased significantly inGX0101-ALV-LTR, and Northern blot results demonstrated that the amount of transcripts ofSORF2, US1, US10were increased significantly.GX0101-ALV-LTR is the first recombinant MDV containing LTR derived from ALV athome and abroad. It has been demonstrated that ALV-LTR fragment integrated at the samesite as REV-LTR in MDV also inherits stably and displays similar biological characteristics.6Comparative analysis of the influence of LTR insert fragment on different geneexpression level of recombinant virus using gene chipCompare and analyze virus gene expression differences on CEF infected with GX0101containing REV-LTR, its LTR deletion strain GX0101LTR and ALV-LTR alternative strainGX0101-ALV-LTR using the customized Agilent expression profile chip. The results ofmicroarray analysis indicate: among differentially expressed gene（sP<0.05）between GX0101and GX0101LTR virus, the expression level of75MDV genes in GX0101are significantlyup-regulated, there are14genes with an up-regulation fold change more than10, and theup-regulation fold change of SORF2, US1and US10genes directly downstream of REV-LTRis more than30, in which SORF2gene with the highest up-regulation fold change of399; thetranscription level of UL38in GX0101lower with a down-regulation fold change of8.3.GX0101-ALV-LTR is much similar to GX0101. Compared with GX0101LTR, theup-regulation fold change of SORF2, US1and US10gene also located directly downstreamof ALV-LTR is more than30, in which SORF2gene with the highest up-regulation foldchange; the down-regulation fold change of UL38in GX0101-ALV-LTR is6.7. Northern blotand Western blot results demonstrated that the amount of transcripts and protein expression ofSORF2in recombinant virus with LTR insert are both increased significantly.The results of comparative study will contribute to further analyze what gene expressionproducts are exactly associated with the increased horizontal transmission ability ofrecombinant MDV that the LTR insert confers.7Comparative analysis of the influence of LTR insert fragment on different geneexpression level of cell genome infected by recombinant MDV using gene chipCompare and analyze cell genome gene expression differences of CEF infected withGX0101containing REV-LTR, its LTR deletion strain GX0101LTR and ALV-LTRalternative strain GX0101-ALV-LTR using the customized Agilent expression profile chip.Analysis of expression differences between41,765chicken genes indicate: compared with GX0101LTR, transcription level of12genes are significantly higher in cell genome ofCEF infected by GX0101with a fold-change of more than20, in which the up-regulation foldchange of ChEST1024a11（GenBank:BU288902.1） and SKOR2（NCBI Reference Sequence:XM₀01233569.2）is190and165respectively; transcription level of4genes are significantlylower with a fold-change of more than20, in which the down-regulation fold change ofChEST445i1（GenBank: CR390488.1） is105. Compared with GX0101LTR, transcriptionlevel of5genes are significantly higher in cell genome of CEF infected byGX0101-ALV-LTR with a fold-change of more than20, in which the up-regulation foldchange of ChEST1024a11（GenBank:BU288902.1） and SKOR2（NCBI Reference Sequence:XM₀01233569.2）is203and83respectively; the fold-change of down-regulation genes areall less than20.The significantly different gene screened from gene chip expression profile willcontribute to further explore the relationship between different biological activity ofrecombinant MDV containing LTR insert with the host gene transcription level.This study not only demonstrates that REV-LTR insert improves the horizontaltransmission ability of GX0101virus in chickens significantly by simulating natural infectiontransmission mode, which is the epidemiology mechanism of GX0101evolving from ahandful of mutant strains into a popular strain, but also partly illustrates the molecularmechanism of REV-LTR improving the horizontal transmission ability of GX0101. Inaddition, GX0101is converted into a new candidate vaccine strain with better protectiveimmunity than CVI988/Rispens using BAC clone technology.更多还原