Establishment of High-sensitivity Rapid Fluorescence Quantitative Detection Method for Antibody against Peste des Petits Ruminants Virus

[Objectives]This study was conducted to establish a rapid quantitative method for detecting antibody against Peste des Petits Ruminants Virus(PPR V)in sheep serum.[Methods]Soluble N protein and NH fusion protein were obtained in Escherichia coli prokaryotic expression system by optimizing codons and expression conditions of E.coli.Furthermore,based on the purified soluble N protein and NH fusion protein,a high-sensitivity fluorescence immunoassay kit for detecting the antibody against PPR V was established.[Results]The method could quickly and quantitatively detect PPR V antibody in sheep serum,with high sensitivity and specificity,without any cross reaction to other related sheep pathogens.The intra-batch and inter-batch coefficients of variation were less than 10%and 15%,respectively,and the method had good repeatability.Through detection on 292 clinical serum samples,it was compared with the French IDVET competitive ELISA kit,and the coincidence rate of the two methods reached 93.84%.Compared with the serum neutralization test,the detected titer value of the high-sensitivity rapid fluorescence quantitative detection method was basically consistent with the tilter value obtained by the neutralization test on the standard positive serum(provided by the WOAH Brucellosis Reference Laboratory of France).[Conclusions]This method can realize rapid quantitative detection of PPR V antibody on site,and has high practical value and popularization value.

Establishment of Double-antigen Sandwich Time-resolved Fluorescence Immunoassay for Detection of Pest des Petits Ruminants Virus

[Objectives]This study was conducted to explore rapid and large-scale screening and detection of peste des petits ruminants(PPR),so as to provide important technical means for prevention,control and purification of PPR.[Methods]Soluble N protein and NH fusion protein were successfully obtained in an Escherichia coli expression system by optimizing E.coli codon and expression conditions.Furthermore,based on purified soluble N protein and NH fusion protein,a double-antigen sandwich time-resolved fluorescence immunoassay method for detection of peste des petits ruminants virus(PPRV)was established.[Results]The method has high sensitivity and specificity and can specifically detect the antibody against PPRV in sheep serum,and it has no cross reaction with other related diseases.The method was used to detect 292 clinical samples,and compared with French IDVET competition ELISA kit.The coincidence rates of positive samples and negative samples from the two kinds of test kits were 92.47%and 97.26%,respectively,and the overall coincidence rate was 94.86%.The intra-group and inter-group coefficients of variation in the repeatability test were less than 10%.[Conclusions]Compared with the traditional ELISA method,the double-antigen sandwich time-resolved fluorescence immunoassay for detection of PPRV has equivalent sensitivity and specificity,and simple and rapid operation,and thus high application and popularization value.

Confirmed Diagnosis by RT-PCR and Phylogenetic Analysis of Peste des Petits Ruminants Viruses in Tibet, China

This paper reports the confirmed diagnosis by nested RT-PCR of PPR cases in Tibet, China in 2007, and results of phylogenetic analysis. Results showed that the 11 tested samples were PPRV positive by nested RT-PCR, of which 2 samples were genetically close to the X7443 strain (Nigeria 75/1) of lineage I, and 3 samples close to the strain AY560591 (Sungri96) of linage IV with 96.6%、97.3%、97.6% and 98% nucleotide sequence homogeneity respectively, based on partial sequencing of the F gene from 5 samples and complete sequencing of the N/M/F/H genes from one sample. This study suggested that there are at least 2 origins of PPRV in China.

Construction of Recombinant Baculovirus Containing Peste des Petits Ruminants Virus N Gene and Establishment of Indirect ELISA for Detecting Serum Antibodies

This experiment was conducted to diagnose Peste des Petits Ruminants based on the eukaryotically-expressed PPRV nucleoprotein through an indirect ELISA. The full-length PPRV nucleoprotein gene was obtained from viral genome RNA by RT-PCR. The amplified fragments were cloned into baculovirus donor vectors pFastHTA of the Bac-to-Bac system. These recombinant plasmids, pFastHTA-PPRV-N, were transformed into DH10Bac host bacteria to obtain recombinant shuttle plasmids, pBacmid-PPRV-N. Recombinant baculovirus, Bacmid-PPRV-N, was generated for expression of the PPRV nucleoprotein by transfecting recombinant pBacmid-PPRV-N with Lipofectamine 2000 into Sf21 insect cells. The efficient expression of PPRV Nucleoprotein by baculovirus in Sf21 cells was verified by SDS-PAGE and Western blot. An indirect ELISA was developed using recombinant PPRV nucleoprotein as the coating antigen. 37 goat sera from an epidemic area in Tibet and 92 goat sera from a non-infected area in Qinghai Province were simultaneously detected by the indirect ELISA, developed here, and the international standard cELISA kit. The sensitivity and specificity of the indirect ELISA was 100% and 96.2% compared with the cELISA kit. The coincidence rate of the two methods was 96.9%. The results demonstrated that the indirect ELISA established in this study works well for diagnosis of PPR.

Monitoring and Tracking on Immune Antibody of Sheep Peste des Petits Ruminants

Peste ties petits ruminants is a kind of acute eontagious disease infecting goats anti sheep. In this study, antibtly monitoring and tracking of healthy goat and sheep immunized by peste des petits ruminants vaccine in Changping District of Beijing City were conducted, aiming at providing a reference for the devel- opment of effective immunization procedures.

Seroprevalence and Associated Risk Factors of Peste des Petits Ruminants among Sheep and Goats in Kassala State, Sudan

Peste des petits ruminant (PPR) is a contagious disease of small ruminants caused by a virus that belongs to the genus Morbillivirus of the family Paramyxoviridae. This study aimed to determine the seroprevalence of PPR disease in sheep and goats and its associated risk factors in Kassala State, Eastern Sudan. Across sectional study was conducted during the period from 30th August to 25th November 2015. The study was carried out using a structured questionnaire survey and a total of 918 blood samples were collected from apparently healthy unvaccinated sheep and goats in different localities in State of Kassala. A total of 546 sheep and 372 goats were tested for specific antibodies to nucleoprotein (NP) by competitive enzyme linked immunosorbent assay (cELISA). The apparent overall prevalence of PPR antibodies in Kassala was 58.2% while the true prevalence was calculated to be 61.3%. The apparent prevalence in sheep and goats was 68.1% and 43.5% respectively. Univariate analysis showed that the risk factors had significant associations with a cELISA positive status: locality, species, age, breed, husbandry system, housing mode, animals movement (p = 0.000) and animals sharing pasture and water (p = 0.003), while sex and newly introduced animals were not significant risk factors (p = 0.771) (p = 0.050) respectively. Factors found that significantly associated (p < 0.05) with increased odds of being cELISA positive in multivariate analysis were localities, species, age and newly introduced animals. The prevalence differed between localities and was the highest in the River Atbara (84.0%) locality, whereas it was lowest in Delta North (29.0%). No significant difference was observed among the sexes. However, the prevalence differed in different age groups and was 52.25% in animals of less than six months old;49.3% were between seven months and two years old and 65.5% were above two years old. In different husbandry systems, the prevalence was 47.9%, 73.0% and 49.2% in intensive, open grazing and pastoral systems respectively. Housing type effects were also observed;the highest prevalence was in animals housed in metal fence (83.3%). The movement pattern showed significant effect, where the prevalence was the highest (81.3%) in animals that move inter-states/inter-localities. It is concluded that the disease is endemic in Kassala State, high prevalent in sheep and goats, posing a threat to animal exportation, and may have a serious economic influence. Owners and herders should compulsorily vaccinate their animals yearly and animals should be investigated periodically for implementation of crucial eradication program.

Sero-Prevalence and Risk Factors of Diffusion of Peste des Petits Ruminants in Cameroon

The present study was carried out between April 2015 and January 2016 to estimate the sero-prevalence and identify the risk factors of the peste des petits ruminants (PPR) in Cameroon. A total of 269 herds randomly sampled across the country have been studied and 1622 samples of serum have been levied on the sheep and goat. The c-ELISA has been studied in order to detect the presence of antibodies in small ruminants like an indicator of exposition to PPRV. The results revealed the circulation of PPRV in the country with a total sero-prevalence of 39% [95%CI;37 - 41] and a sero-prevalence of 63.2% [95%CI;57.2 - 69.2] at the herd level. Sero-prevalence was variable in the ten regions ranging from 7% [95% CI;6.2 - 8.4] to 73% [95% CI;62 - 84] with the northern zone (Adamawa, North and Far-North) having 52.3% [95% CI;37 - 60] and southern zone (including the remaining seven regions) recording 29% [95% CI;11 - 57]. Similarly, it was higher in animals found in urban/peri-urban areas than in rural areas with prevalence ratio of 2.9 [95% CI 2.54 - 3.4;p < 0.001] i.e. 3 times more, 1.6 [95% CI 1.36 - 1.90;p < 0.001] i.e. 1.6 times more, and 5.02 [95% CI 3.91 - 6.85;p < 0.001] i.e. 5 times more at national level, in the northern zone and in the southern area, respectively. Five risk factors have been identified: the breeding environment, introduction of new animals into the herds, gathering of animals for pasture and watering, wandering and transhumance. The breeding area appeared to be the most important risk factor associated with disease exposure. The control measures for the eradication of this disease must take into account the epidemiological situation, the breeding environment, animal transhumance and breeding system.

Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses

Peste des petits ruminants(PPR) is a highly contagious transboundary animal disease with a severe socio-economic impact on the livestock industry, particularly in poor countries where it is endemic. Full understanding of PPR virus(PPRV)pathobiology and molecular biology is critical for effective control and eradication of the disease. To achieve these goals,establishment of stable reverse genetics systems for PPRV would play a key role. Unfortunately, this powerful technology remains less accessible and poorly documented for PPRV. In this review, we discussed the current status of PPRV reverse genetics as well as the recent innovations and advances in the reverse genetics of other non-segmented negative-sense RNA viruses that could be applicable to PPRV. These strategies may contribute to the improvement of existing techniques and/or the development of new reverse genetics systems for PPRV.

组蛋白去乙酰化酶抑制剂MGCD0103对小反刍兽疫病毒体外复制的影响

旨在探究组蛋白去乙酰化酶(HDACs)选择性抑制剂MGCD0103(Mocetinostat)对小反刍兽疫病毒(peste des petits ruminants virus,PPRV)在山羊子宫内膜上皮细胞(EEC细胞)复制的影响。本研究通过RT-qPCR法测定PPRV感染后宿主细胞HDACs和抑制剂处理细胞中PPRV N基因的转录水平,通过Time of Addition Assay确定MGCD0103在PPRV复制过程的作用阶段,用Western blot检测PPRV N蛋白的相对表达量,并采用TCID50方法测定了病毒滴度。结果表明,PPRV感染引起EEC细胞HDAC1(P<0.001)和HDAC2(P<0.002)转录水平明显上升;MGCD0103在病毒入侵和复制阶段均发挥了抑制作用,且在复制阶段抑制作用更明显;MGCD0103显著降低PPRV N基因转录水平和表达水平及病毒滴度(P<0.002),且其抑制PPRV在EEC中的半数有效浓度(EC50)和药物选择指数(SI)分别是0.43μmol·L^(-1)和4.35。本研究确定MGCD0103显著抑制PPRV的复制,这对研发有效抗PPRV药物具有重要意义,为高效率产毒细胞株的构建提供新思路。

小反刍兽疫病毒竞争ELISA抗体检测方法的建立

为建立一种能准确、灵敏地检测小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)抗体的ELISA方法,通过诱导BL21(DE3)-pET-30a-PPRV N表达菌种获得PPRV N蛋白,将纯化的PPRV N蛋白免疫Balb/c小鼠,通过杂交瘤技术获得1株能够高效表达、稳定分泌和具有较好竞争效果的抗PPRV单克隆抗体的杂交瘤细胞株(命名为1G2)。以纯化的PPRV N蛋白为包被抗原,HRP标记的1G2单克隆抗体(1G2-HRP)作为竞争抗体,建立了小反刍兽疫病毒竞争ELISA(competitive ELISA,cELISA)抗体检测方法。经优化反应条件确定最佳抗原包被浓度为1.0μg/mL,待检血清最佳稀释条件为4倍稀释,1G2-HRP酶标抗体最佳工作浓度为250 ng/mL;当阻断率(PI值)小于42%,判定为抗体阴性;阻断率大于或等于42%,判定为抗体阳性。该方法最低可检测128倍稀释的PPRV抗体阳性血清,具有较高的敏感性;仅能检测PPRV抗体,与羊源常见病原和pET-30a-BL21(DE3)的抗体阳性血清无交叉反应,具有较好的特异性;批内、批间变异系数均小于15%,具有良好的重复性。该方法与血清中和试验的阳性符合率为87.80%(95%CI:73.80,95.92),阴性符合率为94.95%(95%CI:88.61,98.34),总符合率为92.86%(95%CI:84.11,97.64),Kappa值为0.83(95%CI:53.10,112.50),具有较高的符合率。表明建立的ELISA方法在我国PPRV的流行病学调查、疫苗免疫效果评估方面具有良好的应用前景。

小反刍兽疫病毒液滴式数字PCR检测方法的建立及应用

为实现小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)的快速、准确、定量检测,以国内PPRV Clone 9疫苗株N基因为靶位点设计特异性引物及探针,建立了液滴式数字PCR(Droplet digital PCR,ddPCR)方法,并对其特异性、敏感性及重复性进行评价。将所建立的ddPCR方法与实时荧光定量PCR(Fluorescence quantitative PCR,qPCR)方法的灵敏度进行比较分析并应用于PPRV(Clone 9株)核糖核酸标准物质的定量。结果显示,所建立的ddPCR方法特异性好,仅PPRV检测结果为阳性,其他羊源病毒类生物制品及毒种检测结果均为阴性;敏感性高,基因拷贝数检出限度为4.33拷贝/μL,比qPCR方法的灵敏度(57.3拷贝/μL)高10倍;重复性好,重复性试验的变异系数为1.8%;定量准确,具有一定资质的9家单位各组测量数据组间变异系数小于5%。试验建立的ddPCR方法能够有效地对PPRV核酸进行快速检测,为PPRV的诊断及绝对定量提供了有效方法。

小反刍兽疫病毒非结构蛋白C单克隆抗体的制备与鉴定

为制备小反刍兽疫病毒(PPRV)非结构蛋白C单克隆抗体,根据PPRV C基因编码多肽链氨基酸序列抗原性分析,设计合成一条含20个氨基酸的抗原肽(CRSGKPRGETPGPLLPEIMQ)和一条含21个氨基酸的筛选抗原多肽(PLRAGERGLAPQAVQHRTLIK),将它们分别与钥孔血蓝蛋白(keyhole limpet hemocyanin, KLH)和生物素羧基蛋白(biotin carboxyl carrier protein, Biotin)交联,制备获得免疫原和筛选抗原。用免疫原肌肉注射5只8~12周龄、体重约20 g的无特定病原体(specific pathogen free, SPF)级BALB/c雌性小鼠,第1次免疫后分别间隔7 d进行第2次免疫和第3次免疫,三免后21 d进行冲击免疫,冲击免疫后第3天采集血液分离血清,采用间接酶联免疫吸附法(ELISA)测得1只免疫小鼠血清抗体效价为1∶312 500,2只为1∶62 500。取3只小鼠脾细胞与骨髓瘤细胞SP2/0经聚乙二醇(PEG)融合制备杂交瘤细胞,通过间接ELISA筛选出26株阳性淋巴杂交瘤细胞,进一步经克隆化培养筛选出46株单克隆细胞株。通过Western blot(WB)筛选获得2株能稳定分泌特异性抗PPRV C蛋白单克隆抗体的杂交瘤细胞株,WB、间接免疫荧光(indirect immunofluorescence assay, IFA)鉴定显示,制备的单克隆抗体具有较好的灵敏性和病毒反应性。研究结果为进一步阐明C蛋白在PPRV生命周期中的作用奠定了基础,也为小反刍兽疫(PPR)诊断提供了有效的诊断试剂。

PPRV Nigeria75/1 H蛋白原核表达及抗原表位预测

克隆并原核表达小反刍兽疫病毒弱毒疫苗株血凝素蛋白(H)全长基因,并通过生物信息学的方法预测其B抗原表位。根据NCBI中PPRV基因组序列中H基因序列(GenBank X74443)设计一对引物,采用RT-PCR方法扩增其全长;将扩增产物克隆至原核表达载体pET-32a后,转化至大肠杆菌E. coli Rosetta,经IPTG 37℃诱导表达目的蛋白;表达的目的蛋白经带His标签的镍离子蛋白纯化柱纯化后,进行Western Blot鉴定。利用DNAStar软件采用生物信息学的方法预测其H蛋白潜在的抗原表位。结果显示,质粒pET-32a-H经双酶切鉴定及测序后可证明构建正确;表达的目的蛋白相对分子质量约67 ku,能被抗His标签抗体识别,主要以包涵体形式存在,有少量可溶性表达。通过生物信息学软件DNAStar成功找到H蛋白的潜在抗原表位5-8,14-16,73-75,83-90,125-131,142-147,170-177,236-245,281-285,312-317,360-363,370-379,388-391,445-449,487-489,503-505,520-522,532-535,544-551,592-595。试验成功构建阳性质粒pET-32a-H,表达目的蛋白,并成功预测出H蛋白潜在的抗原表位。为早日消除PPRV研制新型亚单位疫苗提供参考。

小反刍兽疫LAMP反应结果可视化检测方法比较研究

环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种可应用于各种病原体检测、基因分型等领域的等温扩增技术。该技术所需设备简单且易于操作,因此具有较大的发展潜力。根据目前已经存在的判定LAMP结果的可视化方法,以可能对野生小反刍动物造成威胁的小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)cDNA为检测对象,选择合适的引物组,比较评估均可在日光或紫外光下区分阴阳性结果的SYBR Green I、SYBR Safe Stain和羟基萘酚蓝(hydroxy naphthol blue,HNB)3种指示剂方法的适用性与灵敏度。结果表明:3种方法的灵敏度都很高,均与琼脂糖凝胶电泳相当。其中,SYBR Green I结果区分度高、不受体系成分影响且无须额外设备,但成本较高;SYBR Safe Stain具有良好的结果区分度,成本相对较低,但需要额外的紫外照射设备;HNB成本最低,但颜色区分度较低,且易受多种因素影响。建议在选择LAMP指示剂时,综合考虑成本、使用便利性、结果准确性及实验条件,通过充分的体系优化和验证,确保LAMP检测的准确性和可靠性。研究结果突显了不同方法的特征和适用场景,为研究人员根据不同场所与条件选择高效、便捷的LAMP反应结果可视化方法提供参考。

重组N蛋白抗原检测PPRV抗体ELISA的研究——试剂盒阴阳性临界值的测定及其与OIE参考实验室试剂盒的比较

对临床上采集的244份不同背景的羊血清样本,用纯化的重组N蛋白为包被抗原建立的检测小反刍兽疫病毒(PPRV)抗体的间接ELISA进行检测,运用统计学方法摸清了检测结果的分布规律,并同时用OIE参考实验室抗体检测试剂盒进行检测,结果表明,两种检测方法的符合率为91.73%。利用TG-ROC软件分析了ELISA抗体检测临界值,该试剂盒与国外试剂盒相比,其相对特异性和敏感性分别为98.6%和85.4%。

苜蓿植株再生及PPRV-F基因转化条件的研究

以苜蓿品种甘农1号下胚轴为外植体,研究不同培养基对苜蓿愈伤组织、体细胞胚形成的影响,通过体细胞胚发生途径建立再生体系,在此基础上,摸索农杆菌介导小反刍兽疫病毒F基因的转化条件。结果表明:所选培养基愈伤组织诱导率均为100%,UM+0.1mg/L NAA+0.5mg/L KT为诱导体细胞胚的最佳培养基。农杆菌介导的苜蓿遗传转化条件为:下胚轴预培养3d,农杆菌菌液侵染15min,然后在培养基上铺一层灭菌滤纸共培养3d后清洗,选择压为卡那霉素(Km)75mg/L,抑菌浓度为头孢霉素(Cef)300mg/L。本研究为制备防治小反刍兽疫转基因植物疫苗奠定了基础。

一例PPRV阳性羊鼻拭子样本N、P、M、F、H基因的序列测定和遗传进化分析

为对采集自昆明某大型牲畜交易市场的一例PPRV阳性鼻拭子样本进行追根溯源,设计引物采用RT-PCR方法进行PPRV病毒基因组关键基因N、P、M、F、H核苷酸序列扩增,并进行序列测定和遗传进化分析。结果显示:N、P、M、F、H基因均有预期片段扩增,其读码框(ORF)长度分别为1 578、1 531、1 008、1 641及1 830 nt;N、P、M、F及H构建的遗传进化树均显示所测毒株为基因Ⅳ型,同2013年在新疆伊犁山羊中检测到的毒株(KM091959)同源性最高,N、P、M、F、H核苷酸同源性分别为99.2%、99.0%、99.4%、99.6%和99.3%,推断氨基酸同源性分别为98.7%、98.4%、100.0%、99.8%和99.3%。研究表明,检测到的毒株仍为2013~2014年我国PPRV流行期的基因Ⅳ型毒株,变异较小;所测毒株的N、P、M、F、H 5个关键基因中,M基因最保守、其次是F/H基因,而N、P基因略有变异。

小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法的建立

本研究旨在建立小反刍兽疫病毒H蛋白抗体的化学发光免疫分析检测方法。以H蛋白4个反应原性较好的B细胞表位串联后为检测抗原,在确定抗原包被量和血清稀释度,优化血清和酶标二抗反应时间的基础上,用ROC曲线分析确定检测临界值,建立了小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法,然后对该方法进行敏感性、特异性和重复性评价。结果显示,建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析检测方法最佳抗原包被浓度为1.5×10^(-6)μg/孔,待检血清最佳稀释度为1∶100;血清及酶标二抗孵育时间均为10 min,检测方法的临界值为S/P=9.77%,批内及批间变异系数均小于10%;与口蹄疫、羊痘、蓝舌病及山羊传染性胸膜肺炎阳性血清不发生交叉反应;用建立的化学发光法和市售小反刍兽疫抗体cELISA试剂盒平行检测247份田间血清,两者相对符合率为94.33%,但化学发光法敏感性更高。研究结果表明,建立的小反刍兽疫病毒H蛋白抗体化学发光免疫分析方法灵敏、特异、稳定,可用于田间血清样品小反刍兽疫抗体检测。

JAK/STAT和PI3K/AKT信号通路介导小反刍兽疫病毒宿主细胞天然免疫的研究

为研究JAK/STAT和PI3K/AKT信号通路在小反刍兽疫病毒(PPRV)宿主细胞天然免疫中的作用,在PPRV感染山羊肾细胞24、48和72 h后,分别用MTT试验和间接免疫荧光试验(IFA)检测病毒感染细胞活力及其在细胞中的分布;用qRT-PCR和Western-blot分别检测病毒蛋白、Nectin-4受体、JAK/STAT和PI3K/AKT信号通路及其下游信号分子表达水平的变化。结果表明,PPRV感染24、48和72 h后的细胞存活率分别为99.53%、77.12%和66.87%,病毒H和N蛋白表达水平显著增加(P<0.05);但Nectin-4表达无显著变化(P>0.05);p-STAT1/STAT1比值极显著升高(P<0.01);ISG20、ISG15、IRF9、IRF3、IFNβ和IFNα表达水平显著或极显著升高(P<0.05或P<0.01);p-AKT表达水平极显著升高(P<0.01);p-AKT/AKT、p-NFκB/NFκB、p-GSK/GSK和p-CREB/CREB比值均显著或极显著升高(P<0.05或P<0.01)。综上,PPRV感染能激活JAK/STAT和PI3K/AKT信号通路,使细胞处于抗病毒状态。

小反刍兽疫病毒感染性cDNA克隆的构建与病毒拯救

小反刍兽疫病毒(peste des petits ruminants virus,PPRV)是影响全球畜牧业的一种重要病原。本研究旨在建立稳定可靠的PPRV反向遗传操作平台,为解析PPRV致病机理、免疫逃逸机制、病毒复制机制等基础理论研究提供有效的技术平台,同时为开发更加安全、有效的新型疫苗奠定必要的前期基础。通过提取PPRV Clone9株的RNA,采用RT-PCR分6段扩增出PPRV反基因组cDNA,通过酶切、连接出PPRV Clone9株全长反基因组,获得pB-PPRV,同时构建表达PPRV核蛋白(nucleoprotein,N)、磷蛋白(phosphoprotein,P)和大蛋白(large protein,L)的3个辅助质粒。将pB-PPRV与辅助质粒通过脂质体转染BHK-T7细胞。转染3 d后,冻融3次,感染Vero细胞。传至第二代可观察到明显的细胞病变(CPE)。经间接免疫荧光、Western blot、RT-PCR和序列测定鉴定结果表明,拯救出具有感染性的病毒。拯救病毒(rPPRV-Clone9)在Vero细胞上可稳定传代,增殖动态与亲本病毒相似。本研究成功建立了PPRV Clone9株的反向遗传系统。