坦布苏病毒的分离鉴定及重组腺病毒介导shRNA抑制坦布苏病毒在体外复制的研究
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摘要
坦布苏病毒（Tembusu virus，TMUV）是导致肉鸭神经症状及产蛋鸭出血性卵巢炎（duck hemorrhagic ovaritis，DHO）的主要病原，主要引起20日龄以内的雏鸭出现严重的神经症状，死淘率增高及产蛋鸭的产蛋大幅下降。发病雏鸭通常表现为采食量下降，腹泻，共济失调，瘫痪等症状。发病蛋鸭体温升高；四肢无力、瘫痪；食欲废绝；发病后不久开始产蛋大幅下降；发病后期排草绿色稀便，气味恶臭，出现神经症状。剖检可见卵泡破裂出血，卵黄性腹膜炎，脾脏出血，胰腺出血坏死，肝脏肿大、出血。
TMUV属于黄病毒科黄病毒属蚊媒病毒类的恩塔亚病毒群，是一种具有包膜的单股正链RNA病毒，通过节肢动物的叮咬进行传播而导致宿主发病。病毒的基因组全长约为11kb，包含5’和3’的非编码区，仅含有一个开放阅读框（Open reading frame，ORF），编码3种结构蛋白和8种非结构蛋白。其中包膜蛋白（Envelop protein，E）蛋白是病毒的主要结构蛋白，与病毒的吸附，融合，注入，血凝性及宿主范围和细胞嗜性有密切关系；NS5蛋白为TMUV分子量最大的蛋白，也是最保守的蛋白，它具有RNA依赖的RNA聚合酶活性，与病毒基因组的复制密切相关。在本研究中我们利用建立的多种TMUV核酸检测方法应来确定TMUV感染的宿主范围，以阐明该疾病的流行特点。并利用腺病毒介导shRNA技术靶向干扰E基因和NS5基因，探索TMUV防治的新策略，为我国有效防治该病奠定基础。
1. TMUV的半套式RT-PCR检测方法的建立
根据与TMUV同源性较高的Bagaza病毒的NS3基因的保守序列设计了3条引物，建立了一种适用于TMUV检测的半套式RT-PCR快速检测方法。采用该方法对TMUV山东分离株进行检测。结果显示,半套式RT-PCR对两株分离株均能扩增出277bp的特异性条带,而对照病原：鸭瘟病毒、禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合症病毒、扩增结果均为阴性;敏感性试验结果显示第一轮扩增的敏感度1×105copies/μL，第二轮扩增的敏感度为1×102copies/μL，第二次扩增的敏感性比第一次高103倍；该方法对山东省各地采集的24份疑似病料的检测出率可达87.5%。表明本试验所建立的套式PCR方法可用于TMUV感染的临床诊断和流行病学调查。
2. TMUV的RT-LAMP检测方法的建立及四种核酸检测方的比较
根据TMUV的NS5基因的保守序列设计一套引物，建立了一种适用于TMUV的逆转录环介导等温扩增快速检测方法。该方法检测的敏感性可达1×101拷贝/μL；对3株TMUV山东分离株的检测均为阳性，而对鸭瘟病毒、禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒、扩增结果均为阴性;应用该方法对山东省各地采集的76份疑似病料可检出68份阳性。表明本试验所建立的环介导等温扩增检测方法简便、快速、灵敏、特异性高，可用于TMUV感染的临床诊断和流行病学调查。
利用针对TMUV的NS5基因建立的RT-PCR,半套式RT-PCR,荧光定量RT-PCR及RT-LAMP四种检测方法对系列稀释的质粒DNA以及TMUV基因组RNA进行检测，评价4种检测方法的检测敏感度。结果显示四种检测方法的质粒检测敏感度分别为2×104拷贝/μL,20拷贝/μL,2拷贝/μL和20拷贝/μL,基因组RNA的检测敏感度分别为100pg,100fg,10fg和100fg。在针对采自疑似TMUV感染的鸭场的69份泄殖腔棉拭子的检测中，四种检测方法的检出率分别为38/69(55.1%),52/69(75.4%),57/69(82.6%)和55/69(79.7%)。综上所述，四中核酸检测方法中RT-LAMP以其在敏感度，特异性及检测时间等方面的优势，作为TMUV检测中的首选检测方法。
3.不同宿主来源的TMUV的分离鉴定
为研究TMUV的自然感染宿主范围及流行特点，我们从山东省各地采集麻雀样品68份，蚊子样品35组，蛋鸡样品73份，蛋鸭样品132份和雏鸭样品112份，应用建立的半套式RT-PCR进行TMUV的检测，并对阳性样品进行分离鉴定。结果显示5种样品的检出率分别为66%(45/68),60%(21/35),33%(24/73),62%(82/132)和69%(77/112)。对每类样品选取一株分离株进行NS5基因的序列分析，结果5株分离株的NS5基因同源性高达99.5%以上。代表毒株TMUV-SDHS与经典毒株YY5，BYD的相应基因的同源性在99%以上，与TMUV国外分离株的同源性在87%-91%之间。攻毒试验显示TMUV-SDHS及TMUV-SDMS可以引起55周龄的樱桃谷鸭种鸭发病且临床症状与剖检变化与自然感染病例一致。以上结果表明山东省TMUV感染现象已经十分普遍；山东省不同宿主来源的TMUV分离毒株为同一种病毒；TMUV不仅可以感染产蛋鸭及雏鸭也可以感染蛋鸡和麻雀，但无明显症状；麻雀及蚊子可携带TMUV并与TMUV在自然界中的传播有着密切的关系；麻雀携带TMUV与该病毒在冬季仍然流行有着密切关系。
4.TMUV山东分离株全基因序列的测定及分析
从山东潍坊发生产蛋下降的鸭群中分离到一株TMUV（SD1001），应用RT-PCR及RACE技术对该分离株的全基因序列进行测定并对获得的序列进行分析。结果显示该TMUV分离株为单股正链RNA病毒，基因组全长10990nt，仅含有一个独立的开放阅读框（Open reading frame，ORF），编码3410个氨基酸。基因组两端分别为5’和3’非编码区，长度分别为142nt和618nt。病毒基因组共编码11种蛋白，其中3种结构蛋白，8种非结构蛋白。通过将各编码蛋白的氨基酸序列与JEV、WNV、YFV、DEN2V、BAGV、TBEV的相应序列进行比较，结果TMUV与BAGV的氨基酸同源性最高（81.4%），与TBEV的同源性最低（41.8%）。同时TUMV的非编码区序列与BAGV具有高度的一致性，包含多个保守区域(conserved sequences, CS)，CS序列为RCS3-CS3-RCS2-CS2-CS1。以上研究结果为研究病毒宿主特点及组织嗜性提供了有力的数据支持。
5.腺病毒介导shRNA在体内外抑制TMUV复制研究.
RNA干扰（RNA interference, RNAi），又称RNA沉默，是指双链RNA特异性诱导mRNA降解的过程，是一种转录后基因表达沉默（post transcripitional gene silence,PTGS）现象。近年来RNAi在疾病治疗领域展现出极大的潜力，特别是抗病毒治疗等方面。
(1)靶向干扰TMUV E基因和NS5基因shRNA序列的筛选
本研究针对TMUV的E和NS5基因分别设计构建了三个特异性shRNA（short hairpinRNAs）表达载体pSilencer-E1、 pSilencer-E2、 pSilencer-E3及pSilencer-NS51、pSilencer-NS52、pSilencer-NS53。用RT-PCR扩增TMUV的E、NS5部分基因,纯化后将二者分别克隆到pEGFP-N1载体中,构建荧光报告重组质粒pEGFP-E和pEGFP-NS5。干扰载体和目的基因报告载体共转染293T细胞后的检测结果表明，6个干扰载体均不同程度的干扰了目的基因的表达，其中pSilencer-E1及pSilencer-NS52的抑制效果最佳，分别为75.8%和68.7%。将六种的shRNA表达载体分别转染293T细胞，转染24h后接种TMUV病毒，接种后48h应用相对定量荧光RT-PCR上述试验中病毒RNA的相对数量。结果显示两种干扰载体都能有效干扰病毒在细胞中的复制，抑制效率分别为74%及86%，因此选择pSilencer-NS52用于进一步研究。
(2)干扰TMUV复制的重组腺病毒的构建与鉴定
表达shRNA-NS52的腺病毒载体的构建采用AdMax载体系统，将shRNA-NS52表达盒从pSilencer-NS52转移到腺病毒穿梭载体pDC312上，构建重组穿梭载体pDC312-NS52。将腺病毒骨架质粒pBHGloxΔE1与pDC312-NS52穿梭质粒按1:3比例共转染HEK293细胞，进行同源重组,成功产生重组腺病毒pAd-NS52并通过HEK293细胞多次扩增后测定重组病毒滴度分别为2.1×109PFU/ml.
(3)重组腺病毒介导的shRNA抑制TMUV在HEK293细胞中复制的研究
为评价重组腺病毒pAd-NS52对TMUV的体外干扰效果。将重组病毒pAd-NS52接种HEK293细胞，24h后再接种TMUV病毒，继续培养48h后用相对荧光定量RT-PCR检测TMUV。结果显示pAd-NS52可以显著抑制TMUV在HEK293细胞中的复制。试验中发现pAd-NS52的抑制效果与感染剂量有关，当感染剂量500个MOI时，抑制效果为88%。
Tembusu virus (TMUV), the causative agent of duck hemorrhagic ovaritis (DHO)disease or encephalitis in ducklings, mainly infects20day old aged causes increasingmortality, neurological symptoms, and a severe drop in egg production within1to2weeksafter the ducks were affected. Hyperemia, hemorrhage, degeneration, distortion andlymphocyte infiltration in the ovaries, portal area interstitial inflammation in the livers werethe main pathological changes observed consistently in almost all diseased ducks. Othersymptoms include yolk peritonitis, diarrhea, severe hemorrhages in spleen and pancreas.
TMUV which belong to mosquito-borne flavivirus of the Ntaya virus group was knownas disease agents causing severe clinical symptoms in vertebrates and are transmitted bymosquitoes. TMUV share several common aspects like other flaviviruses: common size(40-65nm), symmetry (enveloped, icosahedral nucleocapsid), nucleic acid (positive-sense,single-stranded RNA of approximately11,000bases), one long open reading frame thatincludes three5’ structural genes and eight3’ non-structural genes, and appearance in theelectron microscope. As the major surface protein of TMUV, the envelope (E) protein isinvolved in many events, such as viral attachment, fusion, penetration, hemagglutination, hostrange and cell tropism. NS5is the largest TMUV protein; NS5is also the most conserved oneacross the genus. Early on a motif of AdoMet-dependent MTases was identified within theN-terminal domain of NS5whereas RdRp motifs were identified in the C-terminal domain ofprotein NS5. In this study, we used different detection methods to determine the TMUV hostrange and the spread of the virus. Moreover, the way of the inhibition of TMUV replicationwere established by recombinant adenovirus expressing shRNA targeting on E and NS5genesof TMUV in HEK293cells.
1. Development and application of a semi-nested RT-PCR assay for detection of TMUV
According to the sequence of NS3gene of flavivirus Bagaza strain published in GenBank,three primers were designed and synthesized. A semi-nested RT-PCR assay for rapiddetection of TMUV was established. A specific277bp fragment was amplified from RNAtemplates of TMUV of Shandong isolates, but no bands were amplified with templatesextracted respectively from duck plagues virus（DPV）, avian influenza virus (AIV) subtype H9, Newcastle disease virus(NDV), infectious bursal disease virus (IBDV)，egg dropsyndrome virus(EDS). Sensitivity of the amplifications by the semi-nested RT-PCR assay was1×105copies/μL and1×102copies/μL, respectively. The sensitivity of the2nd amplificationswas increased by103times and had a high detection rate of clinical samples. These resultssuggested that the semi-nested RT-PCR assay could be used as a method for the diagnosis anddetection of clinical cases, and for molecular epidemiological investigation of TMUV.
2. Rapid detection of TMUV by reverse-transcription, Loop-mediated isothermalamplification (RT-LAMP) and the comparison of four nucleic acid detectionmethods of Tembusu virus
A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP)assay was developed for the rapid detection of TMUV infection.The reaction was performedin one step in a single tube at64℃for45min, with SYBR GreenⅠdye added prior toamplification. The detection limit of the RT-LAMP assay was approximately10copies/μl,and no cross reaction with other avian viruses was observed. The assay was evaluated furtherfor the diagnosis of TMUV in field samples and compared with conventional RT-PCR,demonstrating that results of the RT-LAMP assay corresponded to those of conventionalRT-PCR. In conclusion, RT-LAMP with SYBR GreenⅠdye was shown to be a sensitive,simple assay for the rapid diagnosis of TMUV infection in ducks.
For the detection of TMUV, we investigated the performances of conventional RT-PCR(C-RT-PCR), semi-nested PCR (SN-RT-PCR), reverse-transcriptase real-time quantitativePCR (Q-RT-PCR), and reverse-transcription loop-mediated isothermal amplification(RT-LAMP) targeting the TMUV-specific NS5gene. To compare the detection sensitivitiesof the four techniques, we used two template systems that used plasmid DNA (plasmiddetection sensitivity), including a partial region of the NS5gene, and genomic RNA (genomicdetection sensitivity) from TMUV-positive cell culture supernatants. The plasmid detectionsensitivities of C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were2×104copies/μl,20copies/μl,2copies/μl, and20copies/μl, respectively. The genomic RNA detectionsensitivities of C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were100pg/tube,100fg/tube,10fg/tube, and100fg/tube, respectively. The TMUV-specific RNA was detectedin cloacal swabs of experimentally infected ducks by these four methods. All control samples (not inoculated) were negative by the four methods. The sensitivities of RT-PCR,SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs samples taken fromducks within2weeks of severe egg-drop were38/69(55.1%),52/69(75.4%),57/69(82.6%),and55/69(79.7%), respectively. All evaluated assays were100%specific for TMUV. Inconclusion, given its combined sensitivity, specificity, and speed, RT-LAMP is the preferredassay for the detection of TMUV.
3. Characterization of TMUV isolated from different host
For the characterization of TMUV isolated from different host, the sample from68housesparrows,35pools of mosquitoes,73laying hens,132egg-laying ducks, and112ducklingswere collected at different sites in Shandong province in2010-2012. The samples were firsttested by RT-PCR. Then the positive tissues were used for TMUV isolation and identification.The detection rates of NS3-based RT-PCR were66%(45/68),60%(21/35),33%(24/73),62%(82/132), and69%(77/112), respectively. Finally, a TMUV isolate were obtained fromevery kind of samples. BLAST results showed that the NS5sequences of five isolatesdetermined in this study had nucleotide homology above99.5%. The TMUV-SDHS hadnucleotide homology above99%with YY5and BYD strains, and ranging from87.0%to91%with the published abroad TMUV sequences in GenBank. The TMUV-SDHS isolates werehighly pathogenic to50-week-old healthy Cherry Valley Pekin ducks, and the pathologicchanges were similar to the clinical cases. The results showed that the TMUV infection wasvery common in Shandong province; the TMUV infection not only in egg-laying ducks and ducklings, but also in laying hens and house sparrows without pathologic changes; housesparrows and mosquitoes carrying the TMUV may play an important role in transmitting thevirus among birds; the infections in house sparrows is important for overwintering of TMUV.
4. Analysis of the complete genome of TMUV
In this study, we obtained a complete genome sequence of TMUV using RT-PCR andRACE techniques. TMUV genome is single-stranded RNA,10,990nucleotides in length andcontaining a single open reading frame (3410amino acids) encoding11viral proteins with5’and3’nontranslated regions (NTRs) of142and618nt, respectively. We characterized theopen reading frame (ORF) with respect to gene sizes, cleavage sites, potential glycosylationsites. The different genomic regions of virus were also compared with those of five other flaviviruses including Japanese encephalitis virus, West Nile virus, dengue-2virus, yellowfever virus, Tick-borne encephalitis virus and Bagaza virus. TMUV demonstrated the highestsimilarity to Bagaza virus. The result of entire ORF scanning shows TMUV was close toBagaza viruses in genetic relatedness. The nucleotide sequences of conserved sequences (CS)of TMUV are most identical to the corresponding consensus CSs. Thus, including therepeated CSs (RCSs), the CS organization (in5’-3’ direction) in the3’-UTR isRCS3-CS3-RCS2-CS2-CS1for TMUV. These data demonstrate that TMUV is a unique virusamong the mosquito-borne flaviviruses and also provide a useful reference for a criticallyimportant study to determine why TMUV is a serious pathogen for ducks.
5. Adenovirus-mediated RNA interference against TMUV replication in vitro
RNA interference (RNAi) is the process of mRNA degradation that is induced bydouble-stranded RNA in a sequence-specific manner, which is called post transcripitionalgene silence (PTGS). RNAi has revolutionary approaches in the study of gene function andbecome a powerful new tool to identify the function of gene. Importantly, it is great hopefulto be a new therapy strategy for virus diseases.
Firstly, the results of fluorescence microscopic observation, flow cytometry revealed,westernblot, and virus infection revealed that all the shRNA vector could inhibit E or NS5fusion protiein in293T cells in the screening test of shRNA. The inhibition effect was highestin E1(75.8%) and NS52(68.7%). But in inhibition of the virus replication, the NS52washighest in all six shRNA vector(86%).
Secondly, we constructed the recombinant adenovirus vector with shRNA-NS52usingAdMax system. The expressing kit shRNA-NS52transferred from pSilencer to pDC312forthe construction of shuttle vector. There combinant shuttle plasmid, adenovirus genomicplasmid pBHGloxΔE1were transfected into HEK293cells to construct the recombinantadenovirus pAd-NS52. The recombinant adenovirus vectors of pAd-NS52and pAd-CMVwas successfully constructed with titer of2.1×109PFU/ml.
Finally, we infected HEK293cells with recombinant adenovirus pAd-NS52. After24h,the cells were challenged with TMUV. The TMUV RNA was detected by Real-time RT-PCRat48h post-infection of TMUV. The result showed that pAd-NS52could inhibit thereplication of TMUV and caused a significant reduction in TMUV viral RNA production (88%). The antiviral effect was highest at500MOI dose.
TMUV属于黄病毒科黄病毒属蚊媒病毒类的恩塔亚病毒群，是一种具有包膜的单股正链RNA病毒，通过节肢动物的叮咬进行传播而导致宿主发病。病毒的基因组全长约为11kb，包含5’和3’的非编码区，仅含有一个开放阅读框（Open reading frame，ORF），编码3种结构蛋白和8种非结构蛋白。其中包膜蛋白（Envelop protein，E）蛋白是病毒的主要结构蛋白，与病毒的吸附，融合，注入，血凝性及宿主范围和细胞嗜性有密切关系；NS5蛋白为TMUV分子量最大的蛋白，也是最保守的蛋白，它具有RNA依赖的RNA聚合酶活性，与病毒基因组的复制密切相关。在本研究中我们利用建立的多种TMUV核酸检测方法应来确定TMUV感染的宿主范围，以阐明该疾病的流行特点。并利用腺病毒介导shRNA技术靶向干扰E基因和NS5基因，探索TMUV防治的新策略，为我国有效防治该病奠定基础。
1. TMUV的半套式RT-PCR检测方法的建立
根据与TMUV同源性较高的Bagaza病毒的NS3基因的保守序列设计了3条引物，建立了一种适用于TMUV检测的半套式RT-PCR快速检测方法。采用该方法对TMUV山东分离株进行检测。结果显示,半套式RT-PCR对两株分离株均能扩增出277bp的特异性条带,而对照病原：鸭瘟病毒、禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合症病毒、扩增结果均为阴性;敏感性试验结果显示第一轮扩增的敏感度1×105copies/μL，第二轮扩增的敏感度为1×102copies/μL，第二次扩增的敏感性比第一次高103倍；该方法对山东省各地采集的24份疑似病料的检测出率可达87.5%。表明本试验所建立的套式PCR方法可用于TMUV感染的临床诊断和流行病学调查。
2. TMUV的RT-LAMP检测方法的建立及四种核酸检测方的比较
根据TMUV的NS5基因的保守序列设计一套引物，建立了一种适用于TMUV的逆转录环介导等温扩增快速检测方法。该方法检测的敏感性可达1×101拷贝/μL；对3株TMUV山东分离株的检测均为阳性，而对鸭瘟病毒、禽流感病毒(H9亚型)、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒、扩增结果均为阴性;应用该方法对山东省各地采集的76份疑似病料可检出68份阳性。表明本试验所建立的环介导等温扩增检测方法简便、快速、灵敏、特异性高，可用于TMUV感染的临床诊断和流行病学调查。
利用针对TMUV的NS5基因建立的RT-PCR,半套式RT-PCR,荧光定量RT-PCR及RT-LAMP四种检测方法对系列稀释的质粒DNA以及TMUV基因组RNA进行检测，评价4种检测方法的检测敏感度。结果显示四种检测方法的质粒检测敏感度分别为2×104拷贝/μL,20拷贝/μL,2拷贝/μL和20拷贝/μL,基因组RNA的检测敏感度分别为100pg,100fg,10fg和100fg。在针对采自疑似TMUV感染的鸭场的69份泄殖腔棉拭子的检测中，四种检测方法的检出率分别为38/69(55.1%),52/69(75.4%),57/69(82.6%)和55/69(79.7%)。综上所述，四中核酸检测方法中RT-LAMP以其在敏感度，特异性及检测时间等方面的优势，作为TMUV检测中的首选检测方法。
3.不同宿主来源的TMUV的分离鉴定
为研究TMUV的自然感染宿主范围及流行特点，我们从山东省各地采集麻雀样品68份，蚊子样品35组，蛋鸡样品73份，蛋鸭样品132份和雏鸭样品112份，应用建立的半套式RT-PCR进行TMUV的检测，并对阳性样品进行分离鉴定。结果显示5种样品的检出率分别为66%(45/68),60%(21/35),33%(24/73),62%(82/132)和69%(77/112)。对每类样品选取一株分离株进行NS5基因的序列分析，结果5株分离株的NS5基因同源性高达99.5%以上。代表毒株TMUV-SDHS与经典毒株YY5，BYD的相应基因的同源性在99%以上，与TMUV国外分离株的同源性在87%-91%之间。攻毒试验显示TMUV-SDHS及TMUV-SDMS可以引起55周龄的樱桃谷鸭种鸭发病且临床症状与剖检变化与自然感染病例一致。以上结果表明山东省TMUV感染现象已经十分普遍；山东省不同宿主来源的TMUV分离毒株为同一种病毒；TMUV不仅可以感染产蛋鸭及雏鸭也可以感染蛋鸡和麻雀，但无明显症状；麻雀及蚊子可携带TMUV并与TMUV在自然界中的传播有着密切的关系；麻雀携带TMUV与该病毒在冬季仍然流行有着密切关系。
4.TMUV山东分离株全基因序列的测定及分析
从山东潍坊发生产蛋下降的鸭群中分离到一株TMUV（SD1001），应用RT-PCR及RACE技术对该分离株的全基因序列进行测定并对获得的序列进行分析。结果显示该TMUV分离株为单股正链RNA病毒，基因组全长10990nt，仅含有一个独立的开放阅读框（Open reading frame，ORF），编码3410个氨基酸。基因组两端分别为5’和3’非编码区，长度分别为142nt和618nt。病毒基因组共编码11种蛋白，其中3种结构蛋白，8种非结构蛋白。通过将各编码蛋白的氨基酸序列与JEV、WNV、YFV、DEN2V、BAGV、TBEV的相应序列进行比较，结果TMUV与BAGV的氨基酸同源性最高（81.4%），与TBEV的同源性最低（41.8%）。同时TUMV的非编码区序列与BAGV具有高度的一致性，包含多个保守区域(conserved sequences, CS)，CS序列为RCS3-CS3-RCS2-CS2-CS1。以上研究结果为研究病毒宿主特点及组织嗜性提供了有力的数据支持。
5.腺病毒介导shRNA在体内外抑制TMUV复制研究.
RNA干扰（RNA interference, RNAi），又称RNA沉默，是指双链RNA特异性诱导mRNA降解的过程，是一种转录后基因表达沉默（post transcripitional gene silence,PTGS）现象。近年来RNAi在疾病治疗领域展现出极大的潜力，特别是抗病毒治疗等方面。
(1)靶向干扰TMUV E基因和NS5基因shRNA序列的筛选
本研究针对TMUV的E和NS5基因分别设计构建了三个特异性shRNA（short hairpinRNAs）表达载体pSilencer-E1、 pSilencer-E2、 pSilencer-E3及pSilencer-NS51、pSilencer-NS52、pSilencer-NS53。用RT-PCR扩增TMUV的E、NS5部分基因,纯化后将二者分别克隆到pEGFP-N1载体中,构建荧光报告重组质粒pEGFP-E和pEGFP-NS5。干扰载体和目的基因报告载体共转染293T细胞后的检测结果表明，6个干扰载体均不同程度的干扰了目的基因的表达，其中pSilencer-E1及pSilencer-NS52的抑制效果最佳，分别为75.8%和68.7%。将六种的shRNA表达载体分别转染293T细胞，转染24h后接种TMUV病毒，接种后48h应用相对定量荧光RT-PCR上述试验中病毒RNA的相对数量。结果显示两种干扰载体都能有效干扰病毒在细胞中的复制，抑制效率分别为74%及86%，因此选择pSilencer-NS52用于进一步研究。
(2)干扰TMUV复制的重组腺病毒的构建与鉴定
表达shRNA-NS52的腺病毒载体的构建采用AdMax载体系统，将shRNA-NS52表达盒从pSilencer-NS52转移到腺病毒穿梭载体pDC312上，构建重组穿梭载体pDC312-NS52。将腺病毒骨架质粒pBHGloxΔE1与pDC312-NS52穿梭质粒按1:3比例共转染HEK293细胞，进行同源重组,成功产生重组腺病毒pAd-NS52并通过HEK293细胞多次扩增后测定重组病毒滴度分别为2.1×109PFU/ml.
(3)重组腺病毒介导的shRNA抑制TMUV在HEK293细胞中复制的研究
为评价重组腺病毒pAd-NS52对TMUV的体外干扰效果。将重组病毒pAd-NS52接种HEK293细胞，24h后再接种TMUV病毒，继续培养48h后用相对荧光定量RT-PCR检测TMUV。结果显示pAd-NS52可以显著抑制TMUV在HEK293细胞中的复制。试验中发现pAd-NS52的抑制效果与感染剂量有关，当感染剂量500个MOI时，抑制效果为88%。
Tembusu virus (TMUV), the causative agent of duck hemorrhagic ovaritis (DHO)disease or encephalitis in ducklings, mainly infects20day old aged causes increasingmortality, neurological symptoms, and a severe drop in egg production within1to2weeksafter the ducks were affected. Hyperemia, hemorrhage, degeneration, distortion andlymphocyte infiltration in the ovaries, portal area interstitial inflammation in the livers werethe main pathological changes observed consistently in almost all diseased ducks. Othersymptoms include yolk peritonitis, diarrhea, severe hemorrhages in spleen and pancreas.
TMUV which belong to mosquito-borne flavivirus of the Ntaya virus group was knownas disease agents causing severe clinical symptoms in vertebrates and are transmitted bymosquitoes. TMUV share several common aspects like other flaviviruses: common size(40-65nm), symmetry (enveloped, icosahedral nucleocapsid), nucleic acid (positive-sense,single-stranded RNA of approximately11,000bases), one long open reading frame thatincludes three5’ structural genes and eight3’ non-structural genes, and appearance in theelectron microscope. As the major surface protein of TMUV, the envelope (E) protein isinvolved in many events, such as viral attachment, fusion, penetration, hemagglutination, hostrange and cell tropism. NS5is the largest TMUV protein; NS5is also the most conserved oneacross the genus. Early on a motif of AdoMet-dependent MTases was identified within theN-terminal domain of NS5whereas RdRp motifs were identified in the C-terminal domain ofprotein NS5. In this study, we used different detection methods to determine the TMUV hostrange and the spread of the virus. Moreover, the way of the inhibition of TMUV replicationwere established by recombinant adenovirus expressing shRNA targeting on E and NS5genesof TMUV in HEK293cells.
1. Development and application of a semi-nested RT-PCR assay for detection of TMUV
According to the sequence of NS3gene of flavivirus Bagaza strain published in GenBank,three primers were designed and synthesized. A semi-nested RT-PCR assay for rapiddetection of TMUV was established. A specific277bp fragment was amplified from RNAtemplates of TMUV of Shandong isolates, but no bands were amplified with templatesextracted respectively from duck plagues virus（DPV）, avian influenza virus (AIV) subtype H9, Newcastle disease virus(NDV), infectious bursal disease virus (IBDV)，egg dropsyndrome virus(EDS). Sensitivity of the amplifications by the semi-nested RT-PCR assay was1×105copies/μL and1×102copies/μL, respectively. The sensitivity of the2nd amplificationswas increased by103times and had a high detection rate of clinical samples. These resultssuggested that the semi-nested RT-PCR assay could be used as a method for the diagnosis anddetection of clinical cases, and for molecular epidemiological investigation of TMUV.
2. Rapid detection of TMUV by reverse-transcription, Loop-mediated isothermalamplification (RT-LAMP) and the comparison of four nucleic acid detectionmethods of Tembusu virus
A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP)assay was developed for the rapid detection of TMUV infection.The reaction was performedin one step in a single tube at64℃for45min, with SYBR GreenⅠdye added prior toamplification. The detection limit of the RT-LAMP assay was approximately10copies/μl,and no cross reaction with other avian viruses was observed. The assay was evaluated furtherfor the diagnosis of TMUV in field samples and compared with conventional RT-PCR,demonstrating that results of the RT-LAMP assay corresponded to those of conventionalRT-PCR. In conclusion, RT-LAMP with SYBR GreenⅠdye was shown to be a sensitive,simple assay for the rapid diagnosis of TMUV infection in ducks.
For the detection of TMUV, we investigated the performances of conventional RT-PCR(C-RT-PCR), semi-nested PCR (SN-RT-PCR), reverse-transcriptase real-time quantitativePCR (Q-RT-PCR), and reverse-transcription loop-mediated isothermal amplification(RT-LAMP) targeting the TMUV-specific NS5gene. To compare the detection sensitivitiesof the four techniques, we used two template systems that used plasmid DNA (plasmiddetection sensitivity), including a partial region of the NS5gene, and genomic RNA (genomicdetection sensitivity) from TMUV-positive cell culture supernatants. The plasmid detectionsensitivities of C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were2×104copies/μl,20copies/μl,2copies/μl, and20copies/μl, respectively. The genomic RNA detectionsensitivities of C-RT-PCR, SN-RT-PCR, Q-RT-PCR, and RT-LAMP were100pg/tube,100fg/tube,10fg/tube, and100fg/tube, respectively. The TMUV-specific RNA was detectedin cloacal swabs of experimentally infected ducks by these four methods. All control samples (not inoculated) were negative by the four methods. The sensitivities of RT-PCR,SN-RT-PCR, Q-RT-PCR, and RT-LAMP performed with cloacal swabs samples taken fromducks within2weeks of severe egg-drop were38/69(55.1%),52/69(75.4%),57/69(82.6%),and55/69(79.7%), respectively. All evaluated assays were100%specific for TMUV. Inconclusion, given its combined sensitivity, specificity, and speed, RT-LAMP is the preferredassay for the detection of TMUV.
3. Characterization of TMUV isolated from different host
For the characterization of TMUV isolated from different host, the sample from68housesparrows,35pools of mosquitoes,73laying hens,132egg-laying ducks, and112ducklingswere collected at different sites in Shandong province in2010-2012. The samples were firsttested by RT-PCR. Then the positive tissues were used for TMUV isolation and identification.The detection rates of NS3-based RT-PCR were66%(45/68),60%(21/35),33%(24/73),62%(82/132), and69%(77/112), respectively. Finally, a TMUV isolate were obtained fromevery kind of samples. BLAST results showed that the NS5sequences of five isolatesdetermined in this study had nucleotide homology above99.5%. The TMUV-SDHS hadnucleotide homology above99%with YY5and BYD strains, and ranging from87.0%to91%with the published abroad TMUV sequences in GenBank. The TMUV-SDHS isolates werehighly pathogenic to50-week-old healthy Cherry Valley Pekin ducks, and the pathologicchanges were similar to the clinical cases. The results showed that the TMUV infection wasvery common in Shandong province; the TMUV infection not only in egg-laying ducks and ducklings, but also in laying hens and house sparrows without pathologic changes; housesparrows and mosquitoes carrying the TMUV may play an important role in transmitting thevirus among birds; the infections in house sparrows is important for overwintering of TMUV.
4. Analysis of the complete genome of TMUV
In this study, we obtained a complete genome sequence of TMUV using RT-PCR andRACE techniques. TMUV genome is single-stranded RNA,10,990nucleotides in length andcontaining a single open reading frame (3410amino acids) encoding11viral proteins with5’and3’nontranslated regions (NTRs) of142and618nt, respectively. We characterized theopen reading frame (ORF) with respect to gene sizes, cleavage sites, potential glycosylationsites. The different genomic regions of virus were also compared with those of five other flaviviruses including Japanese encephalitis virus, West Nile virus, dengue-2virus, yellowfever virus, Tick-borne encephalitis virus and Bagaza virus. TMUV demonstrated the highestsimilarity to Bagaza virus. The result of entire ORF scanning shows TMUV was close toBagaza viruses in genetic relatedness. The nucleotide sequences of conserved sequences (CS)of TMUV are most identical to the corresponding consensus CSs. Thus, including therepeated CSs (RCSs), the CS organization (in5’-3’ direction) in the3’-UTR isRCS3-CS3-RCS2-CS2-CS1for TMUV. These data demonstrate that TMUV is a unique virusamong the mosquito-borne flaviviruses and also provide a useful reference for a criticallyimportant study to determine why TMUV is a serious pathogen for ducks.
5. Adenovirus-mediated RNA interference against TMUV replication in vitro
RNA interference (RNAi) is the process of mRNA degradation that is induced bydouble-stranded RNA in a sequence-specific manner, which is called post transcripitionalgene silence (PTGS). RNAi has revolutionary approaches in the study of gene function andbecome a powerful new tool to identify the function of gene. Importantly, it is great hopefulto be a new therapy strategy for virus diseases.
Firstly, the results of fluorescence microscopic observation, flow cytometry revealed,westernblot, and virus infection revealed that all the shRNA vector could inhibit E or NS5fusion protiein in293T cells in the screening test of shRNA. The inhibition effect was highestin E1(75.8%) and NS52(68.7%). But in inhibition of the virus replication, the NS52washighest in all six shRNA vector(86%).
Secondly, we constructed the recombinant adenovirus vector with shRNA-NS52usingAdMax system. The expressing kit shRNA-NS52transferred from pSilencer to pDC312forthe construction of shuttle vector. There combinant shuttle plasmid, adenovirus genomicplasmid pBHGloxΔE1were transfected into HEK293cells to construct the recombinantadenovirus pAd-NS52. The recombinant adenovirus vectors of pAd-NS52and pAd-CMVwas successfully constructed with titer of2.1×109PFU/ml.
Finally, we infected HEK293cells with recombinant adenovirus pAd-NS52. After24h,the cells were challenged with TMUV. The TMUV RNA was detected by Real-time RT-PCRat48h post-infection of TMUV. The result showed that pAd-NS52could inhibit thereplication of TMUV and caused a significant reduction in TMUV viral RNA production (88%). The antiviral effect was highest at500MOI dose.
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