【作者】 白娟；

【导师】 姜平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2012， 博士

【摘要】 脑心肌炎病毒(Encephalomyocarditis virus, EMCV)是一种重要的人畜共患病病原,可以感染昆虫、爬行动物、啮齿类动物和灵长类动物等。本病毒可引起仔猪的致命性心肌炎和母猪的繁殖障碍,对世界养猪业造成了较大经济损失。目前,我国猪群已经存在该病毒感染,但其流行状况尚不十分清楚,也无有效预防和治疗方法。本研究从我国中东地区患病猪群分离获得EMCV NJ08毒株,人工感染试验证实我国存在该病,并完成该病毒全基因序列分析,阐明我国EMCV流行毒株分子特征；同时,设计构建了表达靶向EMCV不同基因shRNA表达质粒,发现其在体外和体内可以有效抑制EMCV复制及其机制,并利用腺病毒介导shRNA技术探讨该病防治新策略,为该病防治提供了新手段。1.猪源脑心肌炎病毒的分离与鉴定本研究采集临床发病仔猪心脏组织,匀浆处理后接种BHK-21细胞,盲传3代出现细胞病变,分离获得一株病毒,病毒传代至第15-25代,病毒滴度稳定,TCID50为10-100～10-10-25/mL;采用脑心肌炎病毒(EMCV)单克隆抗体进行间接免疫荧光试验,结果在感染细胞的胞浆中可见特异性荧光；EMCV VP1基因RT-PCR检测为阳性,扩增产物基因序列与GenBank中的EMCV VP1基因同源性为85.7%-99.8%；纯化病毒粒子负染电镜观察,大小约25-28nm,证明该分离病毒为EMCV,命名为EMCV NJ08株。该分离株接种MARC-145、HEK-293A和ST细胞系均出现明显CPE,病毒于56℃作用15min,感染滴度显著降低。将NJ08株接种BALB/c小鼠,可引起小鼠出现明显临床症状和心肌变性与脑炎等病变,免疫组织化学试验检测结果证明,脑组织含有EMCV抗原；商品仔猪人工感染NJ08株后未出现明显的临床症状及病理变化,但血清中出现EMCV特异性中和抗体,证明该分离株对小鼠具有较强的致病作用,表明我国猪群存在EMCV感染。2.猪脑心肌炎病毒NJ08分离株基因组序列测定与分析本研究根据EMCV基因序列,设计合成7对PCR引物,采用RT-PCR方法从EMCV NJ08分离株扩增获得5个基因片段,大小为1002-1580bp,同时,应用RACE方法,从该病毒株扩增获得其3’和5’基因末端两个基因,扩增产物经凝胶纯化后分别克隆于pMD18-T载体,进行基因测序,利用片段之间的重叠碱基序列顺序将这些片段序列进行拼接,获得EMCV NJ08株全基因组cDNA序列,并将全基因组及其推导的氨基酸序列与GenBank中登录的国内外参考病毒株进行同源性比较及系统进化分析。结果为：EMCV NJ08分离株基因组全长7724bp；属于Ⅰa亚群；与GXLC、 GX0602、BJC3、HB1等国内分离株以及BEL-2887A、CBNU、K3、K11、EMCV-R、PV21等国外分离株同源性达99%以上；EMCV流行株的非结构蛋白中3D最为保守,2A变异最大,结构蛋白中VP2最为保守,VP1变异最大,从而丰富了我国EMCV分子流行病学资料。3.脑心肌炎病毒SYBR Green Ⅰ实时PCR检测方法的建立本研究分别针对EMCV NJ08株的1D和3AB基因设计合成了特异性的引物,通过反应条件的优化,利用SYBR Green Ⅰ染料建立了快速定量检测脑心肌炎病毒的实时PCR方法。通过检测PCV2、PRRSV、CSFV、PRV和FMDV的基因检测及融解曲线检测分析,结果表明两对引物实时PCR反应的熔解曲线具有唯一的尖峰,证明其具有较好的特异性。采用EMCV cDNA产物进行梯度稀释后作为模板进行检测,结果为：该方法对EMCV细胞培养物的检测下限为0.01TCID50,敏感性比普通RT-PCR高100倍,并具有较好重复性。分别采用该法和普通RT-PCR方法检测10份EMCV人工感染的小鼠心肌、脑组织、5份EMCV人工感染仔猪血清样品及3份健康小鼠心脑组织和2份健康仔猪血清样品检测,其符合率为100%。表明该方法具有较高特异性和敏感性,同时具有更快速、准确、低污染等优点,可用于定量检测EMCV和该病诊断。4.靶向EMCV1D和3AB基因shRNA重组质粒构建及其体内外抑制EMCV复制作用本研究根据EMCV NJ08株基因序列(GenBank HM641897),采用计算机软件设计针对1D和3AB基因的4个siRNA靶序列,按pSUPER载体要求,合成了4对互补的寡核苷酸,分别体外退火形成双链,克隆至pSUPER载体,经Kpn Ⅰ/EcoR Ⅰ双酶切鉴定和基因测序,证明构建获得4个shRNA (short hairpin RNA)重组质粒(pSUPER-1D-1, pSUPER-1D-2, pSUPER-3AB-1和pSUPER-3AB-2).将其分别转染BHK-21细胞,观察其抑制EMCV NJ08复制及其机制,并以pSUPER-mN3(shRNA序列长度相同)为对照质粒,结果为：pSUPER-1D-1, pSUPER-3AB-1和pSUPER-3AB-2均能明显减轻EMCV引起的细胞病变(CPE). pSUPER-3AB-1和pSUPER-3AB-2转染细胞中EMCV>商度降低100-1000倍,pSUPER-1D-2或pSUPER-1D-1组病毒滴度降低10-100倍,EMCV VP1蛋白和3AB mRNA转录水平均明显降低(p<0.05)。选取90只4周龄BALB/c小鼠,随机分为6组,每组15只。第1-4组分别脑内接种重组质粒pSUPER-1D-1、pSUPER-3AB-1、pSUPER-3AB-2和pSUPER-mN3,第5组脑内接种ddH2O作为阴性对照,第6组为空白对照组。第1-5组接种后6h、12h和24h,分别每组随机选择5只小鼠,采用EMCV进行攻击,观察临床症状和死亡鼠病变,并于攻毒后14d,对存活小鼠全部剖杀,进行病理学观察,并用实时PCR方法检测小鼠脑组织中EMCV含量。结果为：与pSUPER-mN3组和攻毒对照组相比,pSUPER-VP1-1、pSUPER-3AB-1和pSUPER-3AB-2组小鼠临床症状、脑和心脏组织病变程度明显减轻,病死率明显降低,表明靶向EMCV1D和3AB基因的shRNA可以有效减低EMCV靶基因转录、表达和病毒复制,降低EMCV对小鼠致病作用。5.靶向EMCV1D和3AB基因shRNA的重组腺病毒的构建与鉴定本研究以shRNA重组质粒pSUPER-1D-1、pSUPER-1D-2、pSUPER-3AB-1和pSUPER-3AB-2为模板,利用RT-PCR扩增获得4个PH1-shRNA表达框,分别克隆至重组腺病毒穿梭质粒pAdTrack-CMV中,然后与骨架质粒pAdEasy-1在大肠杆菌BJ5183内进行同源重组,再将重组质粒经转染HEK-293A细胞,获得表达针对EMCV1D或3AB基因的shRNA重组腺病毒,命名为rAd-1D-1、rAd-1D-2、rAd-3AB-1和rAd-3AB-2,重组腺病毒接种HEK-293A细胞后,出现明显CPE,呈变圆和脱落,同时在荧光显微镜下出现特异性荧光。提取HEK-293A细胞中的病毒DNA,利用PCR和基因序列分析,证明重组腺病毒基因组内PH1-shRNA表达框内基因序列正确,证明构建获得靶向EMCV1D或3AB shRNA的重组腺病毒,为EMCV感染防治研究奠定了基础。6.腺病毒介导的靶向EMCV1D和3AB基因shRNA体内外抑制EMCV复制作用本研究将4个靶向EMCV1D (rAd-1D-1和rAd-1D-2)和3AB (rAd-3AB-1和rAd-3AB-2) shRNA重组腺病毒(rAd5)分别接种MARC-145细胞,并以rAd-G1重组腺病毒为对照,观察其对EMCV复制作用,结果为：与对照组相比,该4个rAd5可以明显抑制EMCV靶基因mRNA转录和蛋白表达水平及病毒复制滴度,而且这种抗病毒抑制作用至少能持续72h,并有剂量依赖性。rAd5接种已经感染EMCV的MARC-145细胞,可以部分抑制EMCV复制。选取96只6周龄BALB/c小鼠,随机分为6组,每组16只。第1-4组腹腔接种rAd-1D-2、rAd-3AB-1、rAd-3AB-2和rAd-G1(阴性对照),其中8只小鼠接种剂量为107.0efu/mouse,另8只小鼠接种剂量为108.0efu/mouse,12h后在每只小鼠腹腔注射200TCID50的EMCV NJ08株病毒液,第5组作为EMCV攻毒对照组,第6组只注射PBS作为正常对照组。攻毒后观察临床症状和病理变化,第21d,对存活的小鼠全部剖杀,观察病变,并用实时PCR方法检测小鼠脑组织中EMCV含量。结果为：第1-5组小鼠均表现出不同程度临床症状和肉眼病变,与rAdG1阴性对照组和攻毒对照组相比,rAd-1D-2、rAd-3AB-1和rAd-3AB-2接种组临床症状明显减轻,且出现时间大大推迟,其中,rAd-1D-2(1080efu/mouse)接种组小鼠存活率为87.5%,而对照组小鼠全部死亡；脑组织中病毒病毒含量明显降低。该结果表明,由重组腺病毒介导的shRNA能在体内外有效抑制EMCV的复制,并有效提供小鼠对EMCV攻击,从而为该病防控提供了新策略。更多还原

【Abstract】 Encephalomyocarditis virus (EMCV) is a new kind of zoonoses virus. It can infect many host species including pigs, rodents, cattle, elephants, raccoons, marsupials, baboons, monkeys and human, and cause severe economic losses on pig production due to high mortality in piglets as a result of respiratory failure and in sows as a result of myocarditis and reproductive failure. EMCV has been isolated in the South and North of China. But the pathogenicity and molecular epidemiology of the virus have not been deeply understood. And there is no effective prevention and control method for the EMCV-associated diseases. In this study, EMCV strain NJ08were isolated and identified from clinical tissue samples originated from the suspected cases with encephalomyocarditis on a pig farm in Jiangsu Province. The viral complete genome was sequenced and compared with that of other isolates. Meanwhile, plasmids and recombinant adenoviruses expressing shRNA target to EMCV different genes were constructed and the suppression effect on target genes induced by shRNA was examined both in vitro and in vivo. The main content was as following:1. Isolation and identification of an EMCV strain from pigletsIn order to isolate EMCV from piglet, the clinical tissue samples were selected from the pig with clinical sings of encephalomyocarditis in Jiangsu Province. After homogenization, freeze-thawing and then filtrated with filter membrane, the supernatants were collected and inoculated into BHK-21cells for virus isolation. CPE in BHK-21cells could be observed after three passages and the virus titers were10-10.0～10-10.25TCID50/mL after passaged by15～25times. The specific fluorescence was observed in the cytoplasm of BHK-21cells infected with the strain by IF A with the molecular antibody to VP1of EMCV. And EMCV VP1gene was amplified by RT-PCR. The sequencing results of PCR product showed that the VP1gene shared85.7%to99.8%nucleotide identity with those reported in GenBank. It was further demonstrated by electron micrography after discontinuous sucrose gradient centrifugation. The results showed that this virus isolate represented EMCV and named as EMCV NJ08. An animal infection experiment showed that the isolate could cause severe clinical symptoms and pathological changes in the infected BALB/c mice. And EMCV antigen could be examined in the brain tissues of the infected mice. But no obvious clinical symptoms and pathological changes were observed in the piglets infected with the strain, even thought the antibodies to EMCV could be detected in the pig serum. It was confirmed that EMCV really exist in China and provided new epidemiologic data of this virus in our country.2. Complete genomic sequencing of an EMCV isolate NJ08from Mid-eastern ChinaBased on the sequence of EMCV in GenBank,7primers were designed for to amplify the7fragments with some overlapped between the upstream and downstream fragments, which covering the complete genomic sequence. And then five large overlapped fragments (fragments2-6) were amplified from EMCV NJ08isolate by RT-PCR method and cloned into the pMD18-T vector for sequencing. Authentic5’and3’terminal fragments of S1isolate were obtained by5’-Full RACE Core Set and3’-Full RACE Core Set and cloned into the pMD18-T vector and sequenced. Five overlapping genomic fragments of EMCV NJ08strain were amplified by RT-PCR and authentic5’ and31terminal fragments of NJ08strain were obtained by5’-and3’-full race core set. The PCR products were also cloned and sequenced. Then the complete nucleotide sequence of the viral genome was obtained by splicing of each fragment with overlapped gene sequence in order. The results of the sequence analysis showed that the complete genome of NJ08strain was7,724bp in length. It belonged to the sub-genotype la, with more than99%identity with the reference strains in GenBank. It was found that there were some variation of deduced amino acids in the non-structure protein2A and the structure protein VP1. These results could be helpful for understanding the molecular epidemiology information of EMCV in China.3. Establishment of SYBR Green I realtime PCR for detection of1D and3AB Genes of EMCVBased on the sequence of EMCV in GenBank, the primers specific to the1D and3AB of EMCV were designed, respectively. And two SYBR Green I real-time PCR methods were developed for the detection of the1D and3AB genes of EMCV, after a serial modification of the reaction condition and the reagents. The specificity of the primers was identified by melting curve analysis and there was no reaction in the presence of PCV2, PRRSV, CSFV, PRV and FMDV gene. Both pairs of the primers have a threshold range between10-100TCID50of EMCV as well as good repetitive. The result of detecting of the tissues or sera of mice and pigs showed that these methods had highly sensitivity and specificity for the viral detection. It should be useful for diagnosis and investigation of EMCV1D and3AB genes expression both in vitro and in vivo.4. Inhibition of EMCV replication by shRNA targeting to1D and3AB genes in vitro and in vivoBased on the genome sequence of encephalomyocarditis virus (EMCV) NJ08strain, four siRNA sequences targeting to1D and3AB genes of EMCV were selected with aid of web-based tool. And the oligonucleotides with overhang ends with64-nt long were synthesized, annealed and cloned into pSUPER plasmid. And four short hairpin RNA (shRNA) expression plasmids were constructed and identified with Kpn I and EcoR I restriction enzymes digestion and sequencing, and named as pSUPER-1D-1, pSUPER-1D-2, pSUPER-3AB-1and pSUPER-3AB-2. After transfection of these plasmids into BHK-21cells, EMCV replication were examined by virus titers, IFA, and real-time PCR, respectively. The results showed that EMCV induced cytopathic effect (CPE) could be inhibited in the cells transfected with pSUPER-1D-1, pSUPER-3AB-1and pSUPER-3AB-2, and the virus titers were reduced by approximately100-1000fold compared to those control cells. The expression of EMCV3AB gene was significantly decreased both at RNA and protein levels in the cells comparing to the controls. Ninety BALB/c mice were randomly assigned into six groups. And pSUPER-1D-1, pSUPER-3AB-1, pSUPER-3AB-2and pSUPER-mN3were interperitoneally inoculated to mice in group1to4, respectively. Meanwhile, mice in group5were injected with ddF2O as negative control, and mice in group6served as positive control. At6,12and24h after infection, five mice were randomly selected from each group were challenged with EMCV NJ08interperitoneally. At14dpc, all mice were killed and submitted to pathological and real-time PCR examinations for EMCV. The results showed that the clinical signs and pathological lesions of the mice in the groups inoculated with the shRNA constructs were milder obviously, compared with those in pSUPER-mN3and challenge control groups. The loads of EMCV in the brain tissue of the mice pretreated with the constructs were significantly lower than those in other control groups. It indicated that the vector-based shRN A targeting to3AB and1D gene might be a potential anti-EMCV strategy.5. Constrcution and identification of recombinant adenoviruses expressing shRN A tarteting to EMCV1D and3AB genesThe PH1-shRNA expression frames in the recombinant pSUPRE vectors pSUPER-1D-1, pSUPER-1D-2, pSUPER-3AB-land pSUPER-3AB-2were amplified by PCR and cloned into shuttle vector pAdTrack-CMV, respectively. After the homologous recombination of the recombinant shuttle vectors with pAdEasy-1in BJ5183, the recombinant adenoviruses vectors containing the shRNA were obtained, and identified by Pac I digestion. After transfection of the linearized plasmids with Pac I into HEK-293A cells, and4recombinant adenoviruses expressing shRNA (rAd-shRNA) specific to1D and3AB were produced and named rAd-1D-1、rAd-1D-2、rAd-3AB-1and rAd-3AB-2, respectively. Forty-eight hours after inoculation with these rAd-shRNAs,293A cells developed cytopathogenic effect (CPE) as rounding and detached as well as expressed green fluorescence (GFP) observed under fluorescence microscopy. The adenoviruses genome DNA was extracted from the cell culture and the PH1-shRNA expression frames were detected by PCR. The results showed that all the rAd-shRNAs were positive, which confirmed the existence of PH1-shRN A expression frames in adenovirus genome. This study successfully constructed adenovirus expressing shRNA targeting to EMCV different genes that will be useful for evaluating the antiviral effect induced by shRNA both in vitro and in vivo.6. Adenovirus-mediated shRNA inhibit EMCV replication both in vitro and in vivoIn order to evaluate the inhibition efficiency of EMCV replication induced by the recombinant adenovirus-medated shRNA targeting to the ID and3AB genes of EMCV, the four recombinant adenoviruses expressing shRN As were used to inoculate MARC-145cells and their inhibition efficiency on the replication of EMCV was evaluated firstly. The results showed that delivery of these shRNAs rAd-1D-2, rAd-3AB-1, rAd-3AB-2could induce a significant inhibition of EMCV replication in viral RNA and protein levels in MARC-145cells. And the antiviral effect was dose-dependent and sustained for at least72h. Moreover, 96BALB/c mice were randomly assigned into six groups. And rAd-1D-2, rAd-3AB-1, rAd-3AB-2and control rAd-G1were inoculated to mice in group1to4with dose of107efu or108efu per mouse, respectively. Meanwhile, mice in group5were injected with ddH2O as negative control, and mice in group6served as mock control. At12h after infection, the mice in group1-5were challenged with200TCID50EMCV NJ08. At21dpc, all mice were killed and submitted to pathological and real-time PCR examinations for EMCV. The results showed that mice injected with those shRNAs before EMCV infection had low viral load in the brain during the period of21days post-EMCV infection. The clinical signs and pathological lesions in the mice inoculated with the shRNA were milder than those in rAd-G1negative and EMCV control groups. The survival rates of mice inoculated with108efu rAds were up to87.5%, while, in the control groups all mice died. These results indicated that shRNAs mediated by the adenovirus could provide protective efficacy against EMCV challenge in mice. It might be a potential new tool for controlling EMCV infection.更多还原