【作者】 杨承槐；

【导师】 于康震； 杨汉春；

【作者基本信息】 中国农业大学 ， 预防兽医学， 2014， 博士

【摘要】 鸭病毒性肠炎(Duck viral enteritis, DVE),又名鸭瘟(Duck plague, DP),是鸭、鹅及其它雁形目禽类的一种急性、接触性传染病,其特征是血管损伤、组织出血、消化道粘膜损伤、淋巴器官受损和实质性器官退行性病变,死亡率高,造成重大经济损失。该病的病原为鸭肠炎病毒(Duck enteritis virus, DEV),又称为鸭瘟病毒,属于疱疹病毒科、甲型疱疹病毒亚科、马立克病毒属、鸭疱疹病毒1型。本研究利用鸡胚成纤维细胞(Chicken embryo fibroblast, CEF),将我国鸭肠炎病毒强毒参考株(DEV CSC)进行连续传代致弱,分析了强、弱毒株的全基因组序列特征以及致弱毒株的生物学特性,以期揭示DEV在体外传代致弱的分子基础；构建出DEV gE和gI基因的缺失毒株,分析了gE和gI基因对病毒毒力的影响,旨在为揭示DEV的分子致病机理奠定基础以及为开发DEV基因缺失疫苗提供科学依据。利用454测序平台,对DEV CSC进行了全基因组序列测定。结果表明,DEV CSC基因组全长162,131bp,共78个ORF,编码76个蛋白。全基因组比较发现,DEV CSC与2000年四川分离的DEV CHv株同源性高达99.99%,只有21个核苷酸替换(15个非同义和6个同义)和52个核苷酸缺失或插入,除了在UL41中有3个核苷酸连续插入外,其余核苷酸缺失或插入均在非编码区内；大部分非同义突变(10/15)位于基因组的5’末端。DEV CSC与2005年德国分离的DEV2085株的同源性为98.92%,主要差异是DEV2085株的UL区的5’端连续缺失1,170bp。与DEV CSC相比,鸡胚传代致弱株DEV K p63基因组短4,040bp,在UL区的5’端利3’端分别缺失3,513bp和528bp;76个ORF中有63个ORF的核苷酸序列完全一致,2个ORF(UL56和US10)在C端有移框变异,UL2连续缺失176个氨基酸。将DEV CSC经CEF连续传80代,进行全基因组序列测定,分析了基因组和生物学特性的变化。结果表明,前4代无明显细胞病变,第5代出现少量蚀斑样细胞病变,随代次增加,出现细胞病变时间提前；第25代蚀斑面积显著减小(p<0.01),随后蚀斑面积稳定,各代次间无明显差异；第80代毒(DEV p80)基因组全长为160,328bp。与亲本病毒DEV CSC基因组相比,DEV p80基因组的145,818-147,618nt连续缺失共1,801bp,导致US7(gI)基因3’端和US8(gE)基因5’端缺失,此外,还有12个点突变,其中8个位于预测的ORF内,导致LORF4、UL51、UL9、UL7、UL4和US3共6个蛋白存在单个氨基酸变异。经临床症状、体温、病毒血症、泄殖腔排毒、大体病变、组织学病变、免疫组化和荧光定量PCR检测,比较了传代前后DEV致病性的差异,结果显示,DEV p80接种鸭未出现体温明显升高或临床症状,对消化道和淋巴器官无明显病理学损伤,消化道和淋巴器官中病毒拷贝数明显下降,表明DEV p80对鸭无致病性。攻毒保护试验结果表明,DEV p80具有良好的免疫原性,鸭免疫后5d,即可100%抵抗致死性强毒的攻击。gI和gE基因是疱疹病毒重要的毒力决定因子。为了证实gI和gE基因对DEV毒力的影响,以DEV CSC为亲本病毒,应用同源重组技术,构建出一株缺失145,818～147,618nt的重组病毒DEV-△US78-GFP,并比较了重组病毒及其亲本病毒的体内体外生物学特性。结果表明,重组病毒DEV-△US78-GFP在DEF中产生的蚀斑明显小于亲本病毒(p<0.001)：一步生长曲线也有所不同,DEV-△US78-GFP在细胞和上清中病毒含量峰值均为亲本病毒1/80左右,且到达峰值的时间分别延迟了24h和36h；重组病毒DEV-AUS78-GFP接种4周龄鸭,未出现明显的临床症状和大体病变。这些结果表明,gI利/或gE对DEV在细胞间传播和细胞中的复制有重要影响,是DEV重要的毒力决定因子。综上所述,本研究完成了我国鸭肠炎病毒强毒参考株的全基因组序列测定与分析；发现DEV经鸡胚成纤维细胞连续传代可导致gI和gE基因的缺失,失去对鸭的致病力,并具有良好的免疫原性；构建了缺失gI和gE基因的DEV突变株,经体内、体外试验证实,与其他疱疹病毒一样,DEV gI和／或gE基因对病毒在细胞间传播和毒力具有重要影响。本研究结果为DEV的分子致病机理以及基因缺失疫苗开发提供了科学依据。更多还原

【Abstract】 Duck viral enteritis, also known as duck plague, is an acute, contagious and lethal disease of ducks, geese and swans. The disease causes high mortality in domestic and wild waterfowl, resulting in significant economic losses. The causative agent of this disease is duck enteritis virus (DEV). DEV taxonomically belongs to the species Anatid herpesvirus1, in the genus Mardivirus, subfamily Alphaherpesvirinae, family Herpesviridae. In this paper, to explain the molecular basis of attenuation of DEV via in vitro serial passages, the virulent reference strain of DEV (DEV CSC) in China was serially passaged in chicken embryo fibroblast (CEF), then the genomic characteristics and biological properties were compared between the attenuated mutant and its parental virus. A DEV rtcombinant strain with a deletion in both gl and gE genes was constructed to identify the role of DEV gl and gE genes in determining viral virulence and their affect on virus growth in the tissue cell. The aim of this paper is to advance our understanding the molecular basis for the pathogenesis and develop a gene-deleted vaccine to control duck plague.DEV CSC genome was sequenced on a pyrosequencing platform, the Genome Sequencer20(GS20) system (454Life Sciences). The DEV CSC genome was162,131bp long and contained78predicted open reading frames (ORFs), which encoded76putative proteins. The DEV CSC genome sequence was compared with the published genome sequences of DEV. There was99.99%homology of genome sequence between CSC and CHv isolated in Sichuan in2000. Twenty-one nucleotide substitutions (15non-synonymous and6synonymous) and52nucleotide deletions/insertions were detected. Except one3-bp insertion in UL41, all other deletions/insertions were in non-coding regions. Most of the non-synonymous substitutions (n=10) were in the5’end of genome. There was98.92%gene sequence identity between DEV CSC and DEV2085isolated in German in2005, mainly because of1,170-bp deletion at5’end of the unique long (UL) in DEV2085. Compared with DEV CSC genome, DEV K p63, which was attenuated by serial passages in chicken embryo, was4,040bp shorter in length mainly because of3,513-bp and528-bp deletions at the5’and3’ends of UL, respectively. At the nucleotide level,63of the76ORFs in the DEV CSC genome were100%identical to the ORFs in the DEV K p63genome. Two ORFs (UL56and US10) had frame-shift mutations in the C-terminal regions, while UL2had a176-aa deletion in DEV K p63.To gain a better understanding of the genetic changes needed for attenuation, DEV CSC was attenuated by serial passages in CEF, then the complete genome sequence of the80th passage was determined and compared with its parental virus. Moreover, the in vitro and in vivo properties were described. Cytopathogenicity was not discernible during the first4serial passages. However, at the5th passage DEV p5, minute and discernible plaques were observed after72hours postinoculation. Cytopathogenicity increased with virus passage. The average plaques size of DEV p25was significantly smaller than DEV p20(p<0.01), however, there was not significantly different from DEV p25to DEV p80. The complete genome sequence of DEV p80was determined and compared with its parental virus. DEV p80genome is160,328bp in length. An1,801-bp deletion from145,818to147,618nt was identified in the genome of DEV p80, which affected two genes encoding gl and gE. Moreover, there were12base substitutions, which led to6ami no acid conversions existed in open reading frames LORF4, UL51, UL9, UL7, UL4and US3. DEV p80and its parental virus were inoculated intramuscularly to ducks to compare their pahogenicity with respect to induction of clininal sign, rectal temperature, viremia, virus shedding, gross and histopathologic lesion, immunohistochemical and real time PCR detection. None of the ducks inoculated with DEV p80showed either obvious elevation of rectal temperature or any clinical sign.There was no obviously gross and histopathologic lesion in disgestive tract and lymphoid organs, and the copies of DEV DNA were lower than ducks inoculated with DEV CSC. Moreover, DEV p80protected ducks from lethal DEV challenge as soon as5days postinoculation.In other herpesviruses, gI and gE genes have benn reported to be important virulence factors. To identify the role of DEV gI and gE genes in determining viral virulence, a DEV recombinant DEV-△US78-GFP with a deletion in both gI and gE genes was constructed. The recombinant was assayed in vitro and in vivo. The average sizes of DEV-AUS78-GFP plaques were significantly smaller than its parent(p<0.001). On the other hand, the growth curves were different. The virus titer in both cell and supernatant were less80-fold than its parent, and the time to reach a peak was late24hours and36hours, respectively. Four weeks ducks inoculated with DEV-AUS78-GFP did not show clininal sign and gross lesion. These results suggest intact DEV gI and gE genes are important factors in efficient cell-cell viral spread in vitro and for expression of DEV virulence.Taken together, we finished the complete genome sequence of DEV CSC. To our knowledge, It was first reported that there was an1,801-bp deletion in both gI and gE genes in the mutant attenuated by serial passages in CEF. The attenuated mutant was avirulent in natural host ducks and protected ducks from lethal DEV challenge. We constructed a DEV recombinant with a deletion in both gI and gE genes, and comfirmed DEV gI and gE genes are important factors in efficient cell-cell viral spread in vitro and for expression of DEV virulence as shown for other herpesviruses. The present study is contributed to our understanding the molecular basis for the pathogenesis and development a gene-deleted vaccine to control duck plague.更多还原