【作者】 马光刚；

【导师】 陆承平； Nikolaus Osterrieder；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2009， 博士

【摘要】 马疱疹病毒1型(equine herpesvirus type 1, EHV-1)是马科动物重要的病原,马最为易感,通常会引起呼吸道疾病和病毒性流产,严重时引发神经性疾病。该病毒在世界范围内流行,对养马业和赛马业的危害很大。目前市场上广泛使用的主要为灭活疫苗,无法产生有效的细胞免疫反应,免疫保护性较差。EHV-1所有毒株都可引起流产,但只有个别毒株可以引起神经症状。近些年来,由EHV-1引发的神经性疾病出现的频率越来越高。目前已经证实,EHV-1 DNA聚合酶第752位氨基酸的点突变决定其神经致病性。本研究将EHV-1强毒RacL11株的DNA聚合酶第752位氨基酸用同源重组方法从神经致病型(D752)突变为非神经致病型(N752),同时将其gC蛋白基因敲除,构建了基因改造活疫苗L11_D752N△gC。在用绿色荧光蛋白基因EYFP取代聚合酶基因后,RacL11只能在表达聚合酶蛋白的细胞系RK13中生长,首次证实DNA聚合酶对EHV-1的体外复制是必需的。L11_D752N△gC在体外对RK13细胞的吸附能力相对于野毒显著下降,病毒滴度下降约20倍,从细胞中胞吐的效率降低,但形成的空斑却比野毒和gC蛋白修复株大10%(p<0.0001)。进一步用流式细胞仪分析了不同突变株对马成纤维细胞NBL6表面MHC-1分子的下调作用,首次发现gC蛋白可下调MHC-1表达水平,表明gC蛋白可能参与了EHV-1在宿主体内的免疫调控。用小鼠做动物模型对Lll_D752NAgC进行了毒力和攻毒试验发现,L11_D752N△gC在体内复制能力显著下降,接毒后第2天肺组织中病毒滴度小于野毒滴度的0.01%,未造成明显病理损伤,在接毒后28天用野毒进行攻毒可有效阻止病毒复制,在攻毒后第2天肺组织中病毒滴度与PBS对照组相比下降约100倍。上述结果表明,构建的重组病毒L11_D752N AgC毒力显著减弱,但具有良好的免疫保护效果,有望成为安全有效的减毒活疫苗。在自然流行的EHV-1毒株中,其DNA聚合酶的第752位氨基酸主要存在天冬酰胺(N752)和天冬氨酸(D752)两种形式,该位点的突变可影响聚合酶的活性并足以决定病毒对宿主的神经致病性。DNA聚合酶对EHV-1的复制是必需的,而第752位氨基酸位于聚合酶高度保守的结构域内,该位点对DNA聚合酶的功能是否也是必需并不清楚。本研究通过细菌人工染色体和Red重组技术将该位点进行了敲除,并对突变株L11△752gp2进行了N752和D752两种修复。L11△752gp2在体外可以正常增殖,形成的空斑大小和生长曲线与母源病毒以及两个修复株没有显著性差异,但对靶向聚合酶的抗病毒药Aphidicolin的敏感性显著降低。上述结果表明,EHV-1 DNA聚合酶第752位氨基酸对病毒的体外复制是非必需的,但该位点的突变可以改变病毒对抗病毒药Aphidicolin的敏感性。蓝舌病毒为呼肠孤病毒科成员,是引起牛羊等反刍动物蓝舌病的病原,为虫媒病毒,主要依靠库蠓传播。通常认为该病毒只存在于热带和亚热带地区,但在2006年8月,蓝舌病毒8型首次登陆欧洲北部,并大规模流行,造成了巨大的经济损失。蓝舌病毒各血清型间交叉保护性很差,目前针对8型的疫苗主要为灭活疫苗,无法区分免疫和自然感染。马疱疹病毒1型作为活病毒载体具有感染谱广,容纳外源基因能力强,机体缺少抗载体免疫等优势,作为活病毒载体已经得到了初步的应用。本研究首先将克隆为细菌人工染色体的马疱疹病毒1型RacH疫苗株进行了启动子改造,并以此为载体构建了可表达蓝舌病毒8型VP2蛋白的重组病毒rH_VP2。在体外,rH_VP2形成的空斑显著小于野毒RacH,但可达到与野毒相同的滴度,并可稳定的表达VP2蛋白。该重组活载体疫苗有望成为蓝舌病毒8型的新型标记性疫苗。更多还原

【Abstract】 Equine Herpesvirus 1 (EHV-1) is the causative agent of respiratory disease, abortions and neurological disorders in horses. Sequence analysis as well as reverse genetic studies shown that a single amino acid variation in the EHV-1 DNA polymerase (D752/N752) determines the virus’neuropathogenic potential. Glycoprotein C (gC) of EHV-1 was known to play important roles in the early steps of infection and in release of virions and contribute to viral virulence. Here, a gC-negative EHV-1 strain RacLll mutant with a D752 to N752 mutation in the polymerase was constructed. A complementing rabbit kidney cell line designated RK13_Pol expressing the non-neurological form of the polymerase (N752) was generated. A Pol-negative RacLl 1 mutant was then constructed by inserting EYFP in lieu of the authentic polymerase gene via co-transfection of RacLl 1 DNA and a shuttle plasmid into the RK13_Pol cell line. After a homogenous Pol-negative virus population was obtained that was unable to grow on non-complementing cells, homologous recombination was used again to introduce the N752 Pol variant, which was amplified from non-neurologic EHV-1 strain NY03. To delete the gC open reading frame from the L11_D752N mutant, the EYFP gene flanked with LoxP sites was used as a positive selection marker. By expressing Cre upon co-transfection, EYFP was finally excised and a gC-negative, non-neurological RacLll mutant, termed L11_D752N AgC, was engineered. L11_D752N AgC was shown to replicate less effectively in vitro with virus titer that was 20-fold reduced when compared to wild type and gC revertant, and impaired in case of attachment to and release from RK13 cells. Interestingly, plaque sizes induced by L11_D752N AgC were increased and 10% larger than those of wild type. Using FACS analysis, it was demonstrated for the first time that, gC is responsible for the MHC-1 down-regulation by EHV-1. In vivo, infection with this modified virus did not cause apparent bodyweight loss and clinic signs. Importantly, L11_D752N AgC can only be recovered at day 2 post infection from murine lungs. Upon challenge with wild type virus, apparent protection by L11_D752N AgC was observed with virus titers that was more than 100-fold decreased in lung tissues.Equine herpesvirus type 1 (EHV-1), a member of the subfamily Alphaherpesviridae, infects horses worldwide. Althogh all EHV-1 strains are capable of causing abortions and respiratory disease, only a few sets of strains can induce neurological disease. It is known that a single amino acid variation ar position 752 in EHV-1 DNA polymerase (D752/N752) determines the virus’neuropathogenic potential. While the D to N752 mutation affects the function of polymerase, which is believed to be indispensable for virus growth, the effect of a deletion of residue 752 on virus growth and pathogenicity is not clear. Here, an EHV-1 strain RacLll mutant with a deletion of residue 752 of the viral polymerase was constructed using two-step (en passant) Red mutagenesis. The mutant virus was then repaired to code for D752 or N752, respectively, with the same mutagenesis strategy. The correct genotypes of the final mutant viruses were confirmed by sequencing and restriction fragment length polymorphisms (RFLPs) analysis. After transfection of the A752 clone into a rabbit kidney (RK13) cell line, mutant virus could be recovered and was shown to grow with kinetics that were indistinguishable from those of parental, wild-type RacLll. The results clearly show that the absence of the residue 752 in the essential enzyme is not required for virus growth in vitro. In addition, biochemial assays of the△752 mutant addressed that the mutant is more resistance to Aphidicolin, a Pol-targeting drug, than either D752 or N752 gene type, indicating that the deletion of residue 752 does alter the function of EHV-1 DNA polymerase. The effect of the single amino acid deletion on virus pathogenicity will be tested in a murine infection model and ultimately in horses in the futher.Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economical losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) in northern Europe started and has caused significant disease in cattle and sheep before comprehensive vaccination was introduced. Equine herpesvirus type 1 (EHV-1), a member of subfamily Alphaherpesviridae, has been shown to be an efficient live virus vector. Here, the construction and characterization of a recombinant vectored vaccine that expresses VP2 of BTV-8 using EHV-1 as the delivery vehicle is described. The VP2 gene was synthesized after codon optimization and cloned into a generic transfer plasmid. Two-step (en passant) Red mutagenesis was employed to manipulate a bacterial artificial chromosome (BAC) of EHV-1 vaccine strain RacH and generate the recombinant vaccine. To avoid any unwanted recombination, the HCMV promoter upstream of the egfp gene in mini-F sequence of pRacHrv was replaced with the EF-la promoter. Afterwards, the vp2 gene was inserted in lieu of ORF1 and ORF2 in the RacH genome. Southern blot and restriction fragment length polymorphism analyses (RFLPs) confirmed the correct insertion of the transgene. The recombinant virus rH-VP2 was finally achieved by restoring gp2 encoding gene71, and shown to grow with smaller plaque size but comparable growth kinetics when compared with parental virus rRacH. Stable expression of VP2 by the recombinant EHV-1 was demonstrated using indirect immunofluorescence assay and western blotting. Vaccination/challenge experiments in ruminants are planned.更多还原