UL56基因下游3513bp对鸭肠炎病毒生物特性的影响

The Effects of Downstream 3513bp of UL56 on Characterization of Duck Enteritis Virus

【目的】鸭肠炎病毒(duck enteritis virus,DEV)属于疱疹病毒,能感染鸭、鹅等雁形目禽类,可引起产蛋下降及高死亡率,已报道了多株DEV的全基因组序列,DEV基因组大,长度约158-162 kb。与疫苗株相比,DEV疫苗检验用参考强毒株基因组UL区5′端连续插入3513 bp(2716-6228位核苷酸)导致UL56基因移框突变。目前关于DEV基因功能、致病机理等报道较少。本研究通过构建了重组病毒,以研究连续3513 bp插入对DEV生物特性的影响;【方法】以提取的DEV基因组DNA为模板,分别扩增UL56基因上下游两端同源臂UL56-u、UL56-d。将两同源臂片段克隆到pMD18T-Simple载体,获得重组质粒pT-UL56ud。将重组质粒pT-UL56ud用Mlu I酶切,电泳回收去磷酸化后,将红色荧光蛋白(RFP)表达盒插入到pT-UL56ud中,获得重组质粒pT-UL56-RFP。DEV接种鸭胚成纤维细胞(DEF)(MOI=0.1),吸附1-2 h后,按Lipofectamine 2000说明书转染高纯质粒pT-UL56-RFP,通过同源重组的方法,以红色荧光蛋白(RFP)基因为报告基因,构建了缺失DEV UL56下游3513bp的重组病毒DEV△3513及其回复株DEVΔ3513(R)。将重组病毒及其亲本病毒分别接种25 cm^2的细胞瓶中(MOI=0.01),测量其病毒含量,绘制一步生长曲线;将重组病毒及其亲本病毒分别作适当稀释,接种已长成单层DEF,加入M199营养琼脂糖覆盖,培养5-7 d,随机选取20个蚀斑,测量蚀斑面积;将重组病毒、回复株及其亲本病毒分别以10^5.0TCID50肌肉注射6周龄易感麻鸭进行致病性试验,观察10 d,每天记录发病死亡情况。试验结束后剖检所有存活鸭。对全部动物取肝脏组织,固定于4%多聚甲醛溶液,按常规程序制备病理切片并进行H.E染色;将重组病毒及鸭瘟商品化活疫苗株(CVCC AV1222)分别以10^3.0TCID50肌肉注射6周龄易感麻鸭进行免疫原性试验,在免疫后14 d,腿部肌肉注射接种DEV强毒(CVCC AV1221),每只10^3.0MLD。观察10 d,每天记录发病死亡情况。【结果】通过同源重组的方法,获得携带RFP的重组病毒DEV△3513-RFP、缺失DEV UL56下游3513 bp的重组病毒DEV△3513及其回复株DEVΔ3513(R)。重组病毒DEVΔ3513、DEVΔ3513(R)及其亲本毒均在接种DEF后72 h,病毒含量达到峰值(10^6.2-6.5TCID50/0.1 mL),病毒含量无明显差异;均能在DEF细胞中形成清晰蚀斑,大小无明显差异;DEVΔ3513对6周龄易感麻鸭毒力明显减弱,未出现任何临床症状和死亡;DEVΔ3513以10^3.0TCID50/只免疫麻鸭,能抵抗DEV强毒株的攻击;【结论】成功构建了缺失UL56基因下游3513 bp的重组病毒,首次证实UL56基因下游3513 bp对DEV在细胞中的复制是非必需的;缺失UL56基因下游3513 bp后病毒仍保留良好的免疫原性;UL56基因下游3513 bp是DEV的一个毒力决定因子。

【Objective】Duck enteritis virus(DEV)taxonomically belongs to family Herpesviridae and infects ducks,geese,and swans,which results in high mortality and decreased egg production in domestic and wild waterfowl.Several DEV whole genomic sequences were published,which contained 158-162 kb.Compared with DEV vaccine strain,the virulent DEV strain for vaccine evaluation had a 3513-bp insertion,resulting to UL56 frameshift mutation.At present,there are few papers about DEV gene function and pathogenic mechanism.To study the effect of the 3513-bp insertion on DEV characterization,a recombinant DEV with the 3513-bp deletion was constructed.【Method】The extracted DEV genomic DNA was used as template to amplify the UL56-u and UL56-d of the upstream and downstream ends of UL56 gene,respectively.The two homologous arm fragments were cloned into pMD18 T-Simple vector,and the recombinant plasmid pT-UL56 ud was obtained.The recombinant plasmid pT-UL56 ud was cut by Mlu I enzyme,and after electrophoretic recovery and dephosphorylation,the recombinant plasmid pT-UL56-RFP was obtained by inserting RFP expression box into pT-UL56 ud.After DEV was inoculated with duck embryo fibroblast(DEF)(MOI=0.1)for 1 to 2 h,the purified plasmid pT-UL56-RFP was transfected according to Lipofectamine 2000 instructions,and then purified by plaque.A 3513-bp deletion mutant,DEVΔ3513-RFP,was generated by targeted homologous recombination,in which red fluorescent protein(RFP)as a reporter replaced the 3513 bp.The RFP was then removed to generate DEV△3513.A rescue mutant,DEVΔ3513(R),was constructed by reinserting the 3513 bp into the genome of DEVΔ3513-RFP.The recombinant virus and its parent virus were inoculated in DEF(MOI=0.01),the virus titer was measured and the growth curve was plotted.The recombinant virus and its parent virus were diluted properly and inoculated into monolayers DEF,covered with M199 agarose and cultured 5-7 days.Twenty plaques were selected randomly and the area of plaques was assayed.Six-week old susceptible ducks were inoculated with 10^5.0 TCID50 of the 3513-bp deletion mutant,rescue mutant and its parental virus,respectively.After 10 days,all the surviving ducks were euthanized.The liver tissues were taken from all the animals and fixed to 4%poly Formaldehyde Solution;the pathological sections were prepared by routine procedures and stained with HE.The recombinant virus and the Duck Plague Live Vaccine(CVCC AV1222)were injected with 10^3.0 TCID50 in muscles for 6 weeks of age susceptible ducks to be tested for immunogenicity.After 14 days,all vaccinated ducks were challenged with 10^3.0 MLD of lethal DEV(CVCC AV1221).【Result】The recombinant viruses,DEVΔ3513 and DEVΔ3513(R),and their parental virus possessed similar growth kinetics,and their titer peaked at 72 hours with the peak titer 10^6.2-6.5 TCID50/0.1 mL.Their average plaques sizes were not significantly different;DEVΔ3513 was avirulent in 6-week ducks;the ducks vaccinated with 103 TCID50 were protected against subsequent challenge with lethal DEV.【Conclusion】We successfully constructed a DEV mutant with 3513 bp deletion,and firstly confirmed that the deletion of the 3513 bp had no effect on virus replication in cells and immunogenicity in ducks.Moreover,the 3513 bp was associated with DEV virulence.