【作者】 李广明；

【导师】 姜平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2009， 博士

【摘要】 猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)为有囊膜的单链正股RNA病毒,属于尼多病毒目动脉炎病毒科,主要引起母猪早产、流产等繁殖障碍,仔猪、育肥猪呼吸困难及育成猪类流感疾病,并可持续性感染,引起免疫抑制,已经成为危害世界养猪业安全的重要病原之一。近年来,高致病性PRRSV的出现,对中国养猪业造成了巨大的损失。然而,目前使用的PRRSV疫苗只能够提供部分的保护力,阻止临床症状的出现,却不能防治PRRSV感染,另外不同毒株间的交叉保护力也较低,研究新型防控策略是分有必要。RNA干扰(RNAi)是由小干扰RNA (small interference RNA, siRNA)引发的信使RNA (mRNA)序列特异性消减现象,在真核生物细胞具有基因表达调控的作用,同时又是一种保守的抗病毒机制。siRNA是19-27nt的小分子双链RNA片段,可以由体外转录或载体DNA转录的发夹RNA (short hairpin RNAs, shRNAs)得到。siRNA可以抑制同源的内源或病原mRNA的表达。近年RNAi技术已被用于干扰抑制多种人或畜类病毒的复制和感染研究,提示RNAi作为一个有效的抗病毒策略,具有很大应用前景。本实验室利用pSUPER质粒介导PRRSV特异性shRNA可以有效抑制病毒在MARC-145中的复制增殖。本研究构建了表达靶向PRRSV不同基因和不同病毒受体基因shRNA的表达质粒及重组腺病毒,并在体外和体内对表达的shRNA抑制病毒复制和目的基因表达的效果进行了评价。本研究内容分为以下6个部分：1.PRRSV基因SYBR Green real-time PCR检测方法的建立针对PRRSV S1株和SY0608株的ORF1b基因设计了real-time PCR引物,通过优化反应条件,建立了real-time PCR检测ORF1b基因表达水平的方法。结果表明,建立的real-time PCR方法具有很好的敏感性、特异性和重复性,对PRRSV细胞培养物的检测下限为10～100TCID50。2.靶向ORF1b shRNA重组质粒在MARC-145细胞对PRRSV复制的抑制作用将靶向PRRSV ORF1b shRNA的重组质粒pSUPER-P2和pSUPER-P3分别转染MARC-145田胞,24h后再感染相同剂量PRRSV,感染PRRSV 48h或72h,采用TCID50, real-time PCR和IFA方法分别检测shRNA重组质粒对PRRSV复制的抑制效果,探明抑制机制。结果为,MARC-145细胞转染pSUPER-P2或pSUPER-P3后,接种PRRSV细胞病变(CPE)明显减轻,病毒TCIDso滴度比PRRSV对照组细胞中的病毒量降低了约100倍；PRRSV ORF1b mRNA和ORF1b蛋白水平比对照明显降低(p<0.05)。证明靶向ORF1基因的shRNA表达质粒能够在MARC-145细胞中有效抑制PRRSV的复制。3.靶向PRRSV不同基因的shRNA重组腺病毒的构建与鉴定将重组pSUPER (pSUPER-N3, pSUPER-G1, pSUPER-P2和pSUPER-P3)质粒中的PH1-shRNA表达框经PCR扩增后,隆入腺病毒穿梭载体pAdTrack-CMVPCMV启动子下游,重组穿梭载体测序鉴定后,与腺病毒骨架载体共转化BJ5183感受态细胞,获得shRNA腺病毒重组载体。再将重组腺病毒载体经PacI线性化后转染HEK-293A细胞,获得表达针对PRRSV不同阅读框shRNA的重组腺病毒rAd-N3 (ORF7), rAd-G1 (ORF5), rAd-P2 (ORF1b)和rAd-P3 (ORFlb)。同时构建了表达突变shRNA的重组腺病毒rAd-mN3和rAd-mP2,作为腺病毒对照。重组腺病毒接种HEK-293A细胞后72h出现CPE,荧光显微镜检查有GFP。重组病毒滴度均为1010～1011efu／mL。PCR检测重组腺病毒基因组内PH1-shRNA表达框均为阳性。4.靶向PRRSV不同基因的shRNA重组腺病毒在体外抑制PRRSV复制的效果的比较为验证上述构建的表达shRNA的重组腺病毒(rAd-shRNA)是否能够有效抑制PRRSV的复制,在MARC-145细胞上先别接种500MOI的rAd-shRNA,24小时后感染10MOI的PRRSV S1株；感染PRRSV 48小时后,观察PRRSV引起的CPE程度,并测定PRRSV的滴度；利用PRRSV N蛋白和ORF1b蛋白单克隆抗体及GP5蛋白多抗,采用间接免疫荧光(IFA)的方法检测蛋白含量,用real-time PCR的方法检测与shRNA对应的基因水平。结果表明：(1) shRNA重组腺病毒组CPE明显减轻,TCIDso滴度明显低于rAd-mN3对照组(p<0.05)；(2)与对照组相比PRRSV ORF7和ORFlb基因均被其特异性的shRNA抑制了至少100倍；(3)PRRSV N蛋白,GP5蛋白以及ORF1b蛋白的表达量与接种rAd-mN3及只感染PRRSV的对照组相比显著下降。该结果证明本研究构建的rAd-shRNA可以有效抑制PRRSV在MARC-145细胞中的复制。为了进一步研究rAd-shRNA是否能在PAM原代细胞中抑制PRRSV复制,在PAM细胞上,按照上述方法接种同样剂量的rAd-shRNA和PRRSV,感染PRRSV 48小时,先观察CPE,用N蛋白单抗检测病毒含量的同时采用real-timePCR的方法分别检测细胞中N蛋白基因的水平,并测定病毒滴度。结果证明：(1)与对照组比,4个靶向PRRSV不同基因shRNA的重组腺病毒,均可以在PAM中有效抑制PRRSV S1株的复制,抑制效力为100～1000倍左右；(2) shRNAs对PRRSV的复制抑制作用随着时间的延长而逐渐减弱；(3) shRNAs对PRRSV的复制抑制作用具有明显的剂量依赖性,即抑制效力随着rAd-shRNA接种剂量的增加而增强；(4)PAM感染PRRSV后再接种rAd-shRNA或同时感染两种病毒时,rAd-N3、rAd-G1、rAd-P2和rAd-P3仍然能够有效地抑制病毒复制。5.靶向PRRSV ORFlb的shRNA重组腺病毒在体内外抑制高致病性PRRSV复制抑制效果研究选取靶向PRRSV ORFlb的2个重组腺病毒,按上述同样方法在MARC-145细胞上观察其抑制PRRSV传统毒株S1和高致病性毒株SY0608复制效果。结果表明,与表达突变shRNA的rAd-mP2对照组以及PRRSV接毒对照组相比,rAd-P2对S1株和SY0608株的复制均具有明显的抑制作用；而rAd-P3只能有效抑制S1株复制,对SY0608株的抑制作用不明显。选取PRRSV阴性猪,制备PAM细胞,用上述同样方法进一步观察rAd-P2抑制SY0608株复制效果。结果显示,rAd-P2可以有效抑制PRRSV细胞病变的产生；与对照相比,PRRSV N蛋白以及细胞内ORFlb mRNA水平均明显降低(p<0.05),且该抑制作用具有剂量依赖性,并随时间的延长逐渐减弱。选取20头6周龄PRRSV阴性仔猪,分成4组,每组5头。第1和2组分别肌肉注射rAd-P2和rAd-mP2,4×109TCID50／头,24小时后第1～3组分别肌肉注射PRRSV SY0608株(2×104.0TCID50／头),第4组作为正常对照；接种后隔离饲养观察,每日测量体温和采血。接种PRRSV后第9天,将所有猪只宰杀,进行病理学观察和PRRSV real-time PCR检测。结果为,(1)临床症状：rAd-mP2组和攻毒对照动物,攻毒后第3天起出现体温升高(40.0～42℃),并出现食欲不振,嗜睡,呼吸困难,皮肤发红,眼睑水肿,轻微腹泻和背毛杂乱等临床症状,而rAd-P2组的动物攻毒后第6天才出现体温升高,症状轻于对照组(40.0～41℃),在攻毒后第8天,3头动物出现轻微的临床症状；(2)病理学观察：]Ad-P2组动物的肺部病变明显轻于对照组动物,其中只有3头猪的肺有轻微的间质性肺炎,其余两头猪肺外观无明显病理损伤。肺组织病理切片观察显示,rAd-mP2组和攻毒对照组动物的肺组织出现明显的间质性肺炎,如肺泡壁增厚,单核巨噬淋巴细胞浸润和支气管渗出物增多,而rAd-P2组仅3头动物肺组织出现了轻微的微观病理损伤；(3)病毒血症：对攻毒后第1天和第5天的血清中病毒含量检测,结果显示,攻毒后第5天时rAd-P2组动物的病毒血症与对照组相比,被明显抑制(p<0.05)；(4)肺组织中PRRSV检测：rAd-P2组动物肺组织中病毒的平均含量同样明显低于对照组(p<0.05)。该结果表明,rAd-P2表达的shRNA可以在猪体内外有效抑制PRRSV的复制,并推迟动物发病至少3天时间。证明腺病毒载体介导的shRNA在体外和体内均能有效抑制PRRSV的复制,为控制PRRSV感染提供了一种可能的新策略。6.表达靶向PRRSV受体基因shRNA重组质粒和重组腺病毒的构建与鉴定根据CD151、CD163和CD169(唾液酸黏附素)的基因序列分别设计引物,将扩增的基因片断克隆于pEGFP-N1载体,获得3个EGFP融合表达质粒。并以CD151、CD163和CD169基因为靶基因,设计合成具有小发夹结构的寡核酸序列,经退火成互补双链后,克隆于pSUPER质粒中；利用PCR技术扩增PH1-shRNA表达框,再克隆入重组腺病毒载体,获得表达shRNA的重组腺病毒rAd-151-1和rAd-151-2, rAd-163-1和rAd-163-2、rAd-169-1和rAd-169-2.将shRNA重组质粒与构建的融合表达质粒两两共转染HEK-293A细胞,分别在转染后72小时观察荧光表达情况。结果shRNA质粒共转染细胞中的荧光表达与对照细胞相比均有不同程度的减弱,或表达荧光细胞数的减少。将表达shRNA的重组腺病毒接种PAM细胞,利用CD163和CD169的单克隆抗体检测PAM细胞中相应受体蛋白水平,结果显示实验组荧光细胞数和银光强度低于对照组；将重组腺病毒分别接种PAM后24h,再感染PRRSV S1毒株,24h后检测PRRSV TCID50滴度,结果与对照组无明显差异,针对PRRSV多细胞受体shRNA重组腺病毒联合作用,有待进一步研究。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single-stranded positive-sense RNA virus of the family Arteriviridae in the order Nidovirales. PRRSV is the causative agent of porcine reproductive and respiratory syndrome (PRRS) which characterized by reproductive problems in sows such as premature farrowings, poor farrowing rates, and increased stillbirths, as well as respiratory problems in piglets such as pneumonia and atrophic rhinitis. PRRSV can persist in pigs for a long period of time after initial infection and it may result in immunosuppression. Thus, PRRSV is one of the most economically significant viral diseases in the swine industry. Recently, a highly pathogenic PRRSV spread wildly in many swine herds in China and caused serious economic losses (Li et al.,2007b; Tian et al.,2007). The current commercial PRRSV vaccines are not sufficiently to protect pigs from PRRSV infection. Therefore, it is imperative to develop new antiviral strategies to prevent and control this viral infection.RNA interference (RNAi) is a sequence-specific mRNA degradation phenomenon that induced by small interference RNA (siRNA). RNAi is involved in the regulation of genetic functions and provides defense against virus at the posttranscriptional level in mammalian cells. siRNA is a 19 to 27 nucleotide (nt) RNA molecules homologous to the target genes. siRNA could be achieved by in vitro transcription or from short-hairpin RNA (shRNA) expressed by DNA vector. RNAi has been used to suppress a variety of viruses originated from human or animals, which implicated the potential of RNAi as an antiviral strategy.shRNAs expressd by pSUPER have been demonstrated could specifically inhibit he replication of PRRSV in MARC-145 cells. However, plasmid shRNA-expressing system is not suitable for in vivo application. In this study, recombinant adenoviruses and plasmids expressing shRNA target to PRRSV genes and viral receptor genes were constructed and the suppression effect on target genes induced by shRNA was examined both in vitro and in vivo The content of this thesis consists of seven parts as the following:1. Development of SYBR Green real-time PCR method for detection of PRRSV genesPrimers specific to the ORFlb of PRRSV S1 and SY0608 isolates were designed, respectively. And a rapid and sensitive method of real-time PCR detection of ORF1b gene was developed after a serial modification of reaction system. The results showed that the real-time PCR detection methods developed in this study were sensitive, specific and repetitive, and the lowest range of the sensitivity of the primers both were 10～100TCID502. Inhibition of PRRSV replication induced by shRNA plasmids targeting ORF1bTwo shRNA expression vectors, pSUPER-P2 and pSUPER-P3, targeting ORF1b gene of PRRSV were transfected into MARC-145 cells, and infected with identical amount of PRRSV 24h later. At 48h or 72h post-infection, the inhibition effect on PRRSV replication induced by shRNA plasmids and the mechanism were investigated TCID50, real-time PCR and IFA test. The results showed that PRRSV induced cytopathic effect (CPE) could be inhibited in the cells transfected with pSUPER-P2 and pSUPER-P3, and the virus titers were reduced by approximately 100-fold compared to those control cells. The expression of PRRSV ORFlb gene was significantly decreased both at RNA and protein levels in the cells comparing to the controls. It indicated that vector based shRNA targeting ORF1 region could effectively inhibit PRRSV replication in MARC-145 cells.3. Construction of recombinant adenoviruses expressing shRNA targeting different PRRSV genesRNA-expression cassettes in recombinant pSUPER (pSUPER-N3, pSUPER-G1, pSUPER-P2 and pSUPER-P3) were amplified by PCR and coloned into adenovirus shuttle vector pAdTrack-CMV, under the control of Pcmv promotor. The recombinant shuttle vectors were identified by sequencing and cotransformed with the pAdEasy-1 into E.coli BJ5183 by electroporation and the recombinant adenoviral vectors were generated by homologous recombination. The resultant adenoviral plasmids were linearized by Pac I, and were transfected into HEK-293A cells. The production of recombinant adenoviruses rAd-N3 (ORF7), rAd-G1 (ORF5), rAd-P2 (ORFlb) and rAd-P3 (ORF1b) were obtained. The mutant shRNA expressing adenoviruses rAd-mN3 and rAd-mP2 were constructed as controls. All the recombinant adenoviruses could express GFP and induce CPE 72h after infecton in HEK-293A cells, and all the adenoviruses could reach a titer of 1010～1011efu/mL. Detection of PH1-shRNA in the genome of the recombinant adenoviruses was all positive.4. Comparison of inhibition effect on PRRSV replication induced by rAd-shRNA targeting different viral genes in vitroTo examine whether the shRNA expressing adenovirus could suppress PRRSV replication effectively, MARC-145 cells were first inoculated with 500MOI rAd-shRNAs individually; 24h later, the cells were challenged withlOMOI PRRSV; At 48h after infection with PRRSV, the cells were examined for GFP expression and CPE, and the culture supernatants were collected and virus yields were detected by TCID50. PRRSV N, ORFlb and GP5 protein were investigated by indirect immunofluorescence (IFA) using specific antibody accordingly, and the shRNA targeting genes were detected by real-time PCR using specific primers. It showed that (1) PRRSV induced CPE and viral titers were decreased in the four rAd-shRNA incoculated cells as compared to rAd-mN3 control (p<0.05); (2) PRRSV ORF7 and ORF1b genes were suppressed by 100-fold relative to control; (3) The level of N, GP5 and ORF1b protein were significantly reduced compared to rAd-mN3 and PRRSV challenge control groups. It indicated that adenoviruses expressing shRNA could inhibit PRRSV replication in MARC-145 cells.To determine whether rAd-shRNAs could inhibit PRRSV replication in primary PAM cells, PAM cells were submitted to the same treatment as in MARC-145 cells; At 48h after PRR.SV infection, the cells were examined for CPE, and PRRSV ORF7 gene expression in the cells was investigated by IFA using N protein monoclonal antibody and real-time PCR. The results demonstrated that (1) the four shRNAs targeting different PRRSV genes could all suppress viral replication in PAM cells by 100～1000 fold;(2)The suppression effect induced by shRNA was reduced over time; (3) The suppression effect was dose-dependent on shRNA quantity as inoculation of more rAd-shRNA could induce stronger antiviral effect; (4)rAd-N3、rAd-G1、rAd-P2 and rAd-P3 could inhibit PRRSV replication in PAM previously or simultaneously infected with PRRSV5. The inhibition of high pathogenic PRRSV replication induced by rAd-shRNA targeting ORF lb gene both in vitro and in vivoThe inhibition effect induced by rAd-P2 and rAd-P3 on the replication of PRRSV traditional S1 strain and high pathogenic SY0608 strain was investigated first in MARC-145 cells. It showed that PRRSV S1 and SY0608 could be both suppressed by rAd-P2, whereas rAd-P3 could not inhibit SY0608 strain as effective as S1 strain.PAM cells were isolated from PRRSV negative pig, and the antiviral effect induced by rAd-P2 was further examined on PAM cells. The results showed that rAd-P2 was competent to inhibit PRRSV CPE in PAM and the level of N protein and ORF1b mRNA were significantly reduced as compared to control groups. And the inhibition effect was dose-dependent on shRNA and reduced over time.Twenty 6-week old PRRSV negative pigs were devided into four groups,5 for each. Group 1 and 2 were intramuscularly inoculated with 4×109TCID50 of rAd-P2 each pig,24h later, pigs in group 1～3 were challenged with 2×1040 TCID50 of PRRSV SY0608, and group 4 was treated as a control. Pigs were observed separately, and body temperature data and blood were collected everyday. At 9d post-challenge, all the pigs were euthanized and necropsied, and submitted to pathological examination and real-time PCR detection. The results showed that (1) clinical signs: The rAd-mP2 inoculated group and virus control group developed high fever (40.0～42℃) from 3 dpc, and displayed a range of clinical signs, including inappetence, lethargy, dyspnoea, red skin, eyelid oedema, lightly diarrhea and rough hair coats. However, piglets inoculated with rAd-P2 showed fever which only started from 6 dpc and clinical sighs were milder (40.0～41℃). At 8 dpc, three pigs in rAd-P2 group also showed light inappetence and lethargy. (2) pathological examination:the extent of lung lesions in rAd-P2 group was significant low than controls, three pigs developed milder interstitial pneumonia and the other two have no clear pathological lesions. Lung tissue chips observation showed that pigs in rAd-mP2 and virus control group developed clear interstitial pneumonia as thickened alveolar walls, infiltration with intensive macrophage lymphomononuclear cells, and increased amounts of bronchiole exudates. But only three piglets in rAd-P2 group showed minor microscopic lesions of lung. (3) viremia:PRRSV in serum samples collected on 1d and 5d post-chanllenge were detected and it demonstrated that the level of viremia in rAd-P2 pigs was much lower than control groups on 5d post-chanllenge (p<0.05); (4) PRRSV quantity in lung:the average amount of PRRSV in lung from rAd-P2 inoculated pigs was significantly lower than control group (p<0.05). The results proved that shRNA expressed by rAd-P2 could suppress PRRSV replication in vivo, and delay the onset of disease for at least 3 days. It demonstrated that shRNA directed by adenovirus could inhibit PRRSV replication both in vitra and in vivo and provide a novel potential anti-PRRSV strategy.6. Construction and identification of plasmids and recombinant adenoviruses expressing shRNA targeting PRRSV receptor genesPrimers were designed according to the sequences of CD151、CD 163 and CD 169 (sialoadhesin) genes. The genes were cloned into pEGFP-N1. Three EGFP-fusion expression plasmids were constructed. siRNAs were designed according to the genes of CD151、CD 163 and CD 169, respectively. Short-hairpin oligos were synthesized and coloned into pSUPER after annealing. PH1-shRNA cassettes were amplified from positive recombinant plasmids by PCR and coloned into recombinant adenovirus vector. The recombinant adenoviruses expressing shRNA (rAd-151-1、rAd-151-2、rAd-163-1、rAd-163-2、rAd-169-1 and rAd-169-2) were obtainedEGFP fusion-expression plasmids were cotransfected with shRNA-expressing plasmid accordingly into HEK-293A cells. The EGFP expression was observed at 72h post-transfection. The results showed that EGFP in cells transfected with shRNA-expressing plasmids was decreased as compared to control cells. CD 163 and CD 169 on PAM inoculated with rAd-shRNA were detected by monoclonal antibody accordingly, and it showed that the amount and intensity of fluorescences were relatively lower than control cells. Furtherly, PAM cells were inoculated with rAd-shRNAs individually before PRRSV infection. At 24h after PRRSV challenge, viral titers showed that there was no significant difference between rAd-shRNA inoculated cells and control cells. The cooperation of shRNA targeting to multiple PRRSV receptor genes is needed to be investigated.更多还原