Fine mapping and conservation analysis of linear B-cell epitopes of peste des petits ruminants virus nucleoprotein

Nucleoprotein (NP) is the most abundant and highly immunogenic protein of morbillivirus, and is presently the basis of most diagnostic assays for peste des petits ruminants virus (PPRV). In this study, fine epitope mapping and conservation analysis of linear B-cell epitopes on the PPRV NP has been undertaken using biosynthetic peptides. Nineteen linear B-cell epitopes were identified and their corresponding minimal motifs were located on the NP of PPRV China/Tibet/Geg/07-30. Conservation analysis indicated that ten of the 19 minimal motifs were conserved among 46 PPRV strains. Peptides containing the minimal motifs were recognized using anti-PPRV serum from a goat immunized with PPRV vaccine strain Nigeria 75/1. Identified epitopes and their motifs improve our understanding of the antigenic characteristics of PPRV NP and provide a basis for the development of epitope-based diagnostic assays.     

辽宁省小反刍兽疫疫苗临床免疫效果跟踪试验及抗体水平动态观察

通过抽检小反刍兽疫疫苗免疫后的部分羊群,分别于免疫后的7、14、21、28 d对免疫羊进行抗体及病毒核酸（包括疫苗毒）跟踪监测,并对监测数据进行整理比对分析,继而掌握小反刍兽疫免疫抗体在机体内的消长规律及影响因素,进一步了解该疫苗辽宁省羊群中的免疫效果及安全性。结果表明,该疫苗在免疫7d后,免疫群体均能够达到免疫合格率高于70%的标准,且未出现疫苗排毒现象,证明该疫苗的免疫效果确实、安全性好。     

小反刍兽疫活疫苗效力评价

以新疆天康畜牧生物技术股份有限公司生产的6批小反刍兽疫活疫苗为材料,对其进行最小免疫剂量、疫苗免疫期和疫苗保存期的研究。结果显示,该疫苗最小免疫剂量为103 TCID50/头份;免疫期至少为26个月,保存期为-20℃保存12个月。结果表明,该疫苗免疫原性好,对山羊和绵羊均可提供良好保护。     

小反刍兽疫活疫苗的安全性评价试验

2007年小反刍兽疫(PPR)在我国西藏首次暴发,在西藏和新疆部分地区使用PPR Nigeria 75/1疫苗株制造的疫苗进行免疫接种。为明确疫苗的安全性,中国兽医药品监察所国家牛瘟参考实验室对其安全性能进行了系统评价。健康易感山羊、绵羊及怀孕山羊、怀孕绵羊按不同剂量接种疫苗后,均未观察到异常临床反应;怀孕母羊所产羔羊数量与对照组无明显差异。疫苗对小白鼠、豚鼠的非特异性安全试验表明,所有接种动物均健活。结果表明该疫苗安全性良好,可在田间大规模使用。     

小反刍兽疫病毒分子生物学研究进展

小反刍兽疫是一种急性、热性、接触性传染病,是世界动物卫生组织规定的A类传染病,在我国被列为一类动物疫病,具有高发病率和高死亡率等特点。该病给小反刍动物养殖及国家和外贸经济带来了巨大的损失。文章主要叙述了小反刍兽疫病毒各基因结构特点,6种结构蛋白的功能,小反刍兽疫的诊断技术等方面的最新研究进展。     

小反刍兽疫疫苗毒与野毒实时荧光RT—PCR鉴别检测方法的建立

为了建立鉴别检测小反刍兽疫疫苗毒与野毒的快速分子生物学检测方法,通过对自行测序的疫苗毒基因组序列及GenBank中登录的野毒基因组序列进行比对分析,设计了2套引物和TaqMan荧光探针,对实时荧光RT-PCR反应条件进行优化,建立了小反刍兽疫疫苗毒与野毒实时荧光RT-PCR鉴别检测方法。特异性试验证实,该检测方法只能检测到目的病毒核酸,表明其具有良好的特异性。灵敏性试验发现,检测疫苗株的最低检测限可达1．38mg／L的总RNA,检测野毒株的最低检测限为0．16mg／L的核酸。对同一样品进行重复性检测,检测的荧光扩增曲线阈值完全重舍,证明其重复性极好。从180份临床样品中,检测出2份疫苗病毒株阳性样品,其余均为阴性。结果表明,本研究所建立的实时荧光RT-PCR方法能对小反刍兽疫疫苗毒及野毒进行鉴别检测,具有特异性好、灵敏度高、重复性极好的优点,是开展小反刍兽疫疫情监测的有力工具。     

小反刍兽疫病毒H、F基因转基因苜蓿表达载体的构建

为了有效预防小反刍兽疫,试验以pBI121为载体,PPRV-H、PPRV-F为目的基因,并插入绿色荧光蛋白(GFP)基因、MAR序列,构建5种小反刍兽疫病毒(PPRV)苜蓿表达载体。结果表明:成功构建小反刍兽疫表达载体,再将表达载体转入苜蓿,饲喂动物,选择其中免疫效果最好的表达载体,为该病的预防工作提供有效的技术支持。     

经密码子优化的小反刍兽疫病毒H蛋白在昆虫细胞中的表达

小反刍兽疫病毒（peste des petits ruminants virus,PPRV）属于副黏病毒科、麻疹病毒属成员。本实验室已经证明,该病毒的囊膜糖蛋白H蛋白在未经密码子优化条件下难以在昆虫细胞中表达。本研究首先对其密码子进行优化,然后构建了在昆虫细胞中表达该蛋白的重组杆状病毒。经Western-blot和间接免疫荧光试验表明,该重组杆状病毒在昆虫细胞中能够表达H蛋白,但表达量相对较低。     

The detection of antibody against peste des petits ruminants virus in Sheep,Goats, Cattle and Buffaloes

Monoclonal antibody-based competitive ELISA (C-ELISA) has been used for the specific measurement of antibodies to peste des petits ruminants (PPR) viruses in sheep, goats, cattle and Buffalo. Serum samples from sheep (n = 232), goats (n = 428), cattle (n = 43), buffalo (n = 89) were tested. The animals had not been vaccinated against rinderpest or PPR. Findings suggested that the sero-positive cases were significantly higher in sheep (51.29%) than in goats (39.02%) (P = 0.002). The overall sero-prevalence of PPRV in small ruminants was 43.33%. The PPR antibodies seroprevalence was 67.42% in buffalo and 41.86% in cattle which was significantly higher in buffalo (P = 0.005). The overall sero-prevalence of PPRV in large ruminants was 59.09%. Cattle and buffalo sera showed a high prevalence of antibody against PPR virus which may explain the difficulty experienced in achieving high post-vaccination immunity levels against rinderpest. Because antibodies against PPR virus are both cross-neutralizing and cross-protective against rinderpest virus, further vaccination in the presence of antibodies against PPR virus may be a waste of national resources. It was also suggested that antibodies to PPR virus could prevent an immune response to the rinderpest vaccine. This paper presents serological evidence for the transmission of PPR virus from sheep and goats to cattle and buffalo and highlights the need to include PPR serology in the sero-monitoring programme to give a better indication of national herd immunity of sheep and goats against PPR.     

Seroprevalence of, and risk factors for, peste des petits ruminants in sheep and goats in Northern Jordan

Peste des petits ruminants (PPR) is an economically important disease that affect sheep and goat industry in Asia and Africa. In this study, we investigated the seroprevalence, and risk factors, of PPR in sheep and goat flocks from five different governorates (Irbid, Jarash, Ajloun, Mafraq and Zarka) located in Northern Jordan. Serum samples from 929 and 400 sheep and goats, respectively, corresponding to 122 sheep flock and 60 goats flock were collected. Seroprevalence was determined using PPR competitive ELISA. Health status and management information were collected using a semi-structured pre-tested questionnaire. The individual true prevalence of PPR in sheep and goats was 29 and 49%, respectively. The flock level true prevalence of PPR was 60 and 74% in sheep and goats, respectively. In both sheep and goat flocks, large flock size, visiting live animals market and inadequate veterinary services were identified as risk factors for PPR seropositivity. Mixed (sheep and goats) raising was identified as a risk factor for PPR seropositivity in sheep flocks only.     

Post vaccination and colostral peste des petits ruminants antibody dynamics in research flocks of Kirdi goats and Foulbe sheep of north Cameroon

We studied Kirdi goats and Foulbe sheep kept on-station at the Institute of Agricultural Research for Development (IRAD), Garoua to look at their immunological response following vaccination with a specific peste des petits ruminant (PPR) virus vaccine. Pre-vaccination PPR antibody seroprevalences were 44 and 29% in goats and sheep, respectively. Seroprevalences following vaccination were 100% in both species. Titres remained above the protection threshold level (1:10) in both species at 12 months. Maternal antibodies in young animals were detectable to up to 6 months of age but fell below the protection threshold level at 3.5 and 4.5 months in lambs and kids, respectively. Kids and lambs from immunised or exposed dams should be vaccinated at 4 and 5 months of age, respectively.     

Persistent detection of peste de petits ruminants antigen in the faeces of recovered goats

Peste des petits ruminants (PPR) disease was confirmed in West African Dwarf goats. They were managed symptomatically with antibiotics and antidarrhoeics. Following clinical recovery, faeces were collected every week from 40 recovered goats to monitor excretion of the PPR virus haemagglutinins in their faeces. All the 40 recovered goats shed the PPR virus haemagglutinins for 11 weeks post recovery. Nine goats (22.5%) continued shedding the viral antigen 12 weeks post recovery. There was correlation between weekly mean haemagglutination titre of the PPR virus and time post recovery with r = -0.7504 (p < 0.01).     

小反刍兽疫免疫荧光诊断技术研究

为了建立小反刍兽疫(PPR)荧光抗体诊断方法,研究选用小反刍兽疫病毒(PPRV)Nigeria75/1减毒疫苗株制备抗原,免疫试验山羊,制备小反刍兽疫高免血清,用硫酸铵盐析法粗提免疫球蛋白(IgG),再应用提取的IgG制备异硫氰酸荧光素标记抗体。结果表明:所制备的荧光抗体对试验用Vero细胞组织抗原和犬瘟热病毒(CDV)抗原无反应性,对小反刍兽疫病毒感染细胞的检出敏感性为1×106稀释度。     

Formation of peste des petits ruminants spikeless virus-like particles by co-expression of M and N proteins in insect cells

Peste des petits ruminants virus (PPRV) has a non-segmented negative sense RNA genome and is classified within the Morbillivirus genus of the Paramyxoviridae. Using the Bac-to-Bac® baculovirus expression system, we constructed recombinant baculoviruses that were able to co-express the PPRV matrix and nucleocapsid proteins in insect cells under the control of the polyhedron and p10 promoters, respectively. The results showed that although both structural proteins were expressed at a relatively low level, the interaction between them caused the formation of virus-like particles (VLPs) by viewing of transmission electron microscopy. The VLPs morphologically resembled authentic PPRVs but lacked spikes protruding from the particulate surfaces. Interestingly, the diameter of PPRV VLPs ranged from 100 to 150 nm, far less than the mean diameter (400–500 nm) of parental virions.     

表达小反刍兽疫病毒F基因重组腺病毒的构建及对小鼠的免疫原性

为构建表达小反刍兽疫病毒(PPRV)F蛋白的重组腺病毒,本实验合成密码子优化的PPRV F基因,将其克隆至腺病毒穿梭载体pShuttle-CMV中,并以Pme I线性化后转化含有人5型腺病毒骨架的BJ5183感受态细胞,通过细菌内同源重组构建表达PPRV F蛋白的重组腺病毒质粒pAdV-F,经Pac I线性化后转染AD-293细胞,获得重组腺病毒rHAdV-F.经PCR和Dot-ELISA鉴定,F蛋白在AD-293细胞中获得表达.该重组腺病毒与野生型腺病毒复制能力相近,但病毒滴度略低.间接ELISA方法检测显示rHAdV-F可以诱导小鼠产生抗PPRV特异性体液免疫应答.     

小反刍兽疫病毒RT-LAMP检测方法的建立

利用逆转录环介导等温核酸扩增技术(RT-LAMP)建立了小反刍兽疫病毒快速检测方法,同时评价了该方法的灵敏性和特异性。结果表明,根据小反刍兽疫病毒N基因保守区域设计的LAMP引物能够在63℃恒温下,1小时内实现目的核酸的大量扩增,由于在检测前加入荧光指示试剂,检测结果可以直接用肉眼判断,避免了由于开盖检验带来的扩增产物污染导致的假阳性。该检测体系具有较高的特异性,只能特异性地检测目的病毒,与其他同属的病毒或类似病毒等无交叉反应;具有较高的检测灵敏度,比普通RT-PCR灵敏性高10倍,与荧光RT-PCR的灵敏度相当。     

小反刍兽疫病毒M基因截短表达及鉴定

目的将小反刍兽疫病毒M蛋白基因截短表达后用于特异性单抗制备及临床抗体水平检测。方法:用在线分析软件BepiPred分析小反刍兽疫病毒M蛋白潜在的B细胞线性表位,以本实验室构建的pCR2.1T-PPRV M质粒为模板,扩增三段截短的M基因,纯化后的PCR产物分别与克隆载体pCR2.1T连接,筛选出的阳性重组质粒经双酶切后,分别与表达载体pET-32a(+)及pGEX-6p-1连接,再将鉴定为阳性的重组质粒转化入E.coli BL21(DE3)菌株诱导表达,并用SDS-PAGE及Western blot验证。结果 PCR产物电泳,得到与预期大小相符的特异性片段。对连接克隆载体及表达载体的重组质粒双酶切后,均出现与预期一致的片段,DNA测序表明,插入片段序列与小反刍兽疫Nigeria75/1株M蛋白基因完全一致。重组蛋白经SDS-PAGE及Western blot鉴定,证明所构建的重组蛋白均获得高效表达,并均具有良好的反应原性。结论成功表达了小反刍兽疫截短M基因的蛋白,为制备特异性单抗及小反刍兽疫抗体检测奠定了一定基础。     

Post-vaccination antibodies profile against Peste des petits ruminants (PPR) virus in sheep and goats of Punjab,Pakistan

A total of 70 sheep and 330 goats were selected randomly. All the animals were kept under same housing and management conditions. Serum samples were collected from all the animals and tested for the presence of antibodies against Peste des petits ruminants (PPR) virus using competitive ELISA (cELISA). All the animals were found negative showing percentage inhibition (PI) values <50. The animals were vaccinated against PPR with Nig/75/1 strain vaccine of PPR Serum samples were collected from randomly selected 12 sheep and 30 goats at 10, 30 and 45 days post-vaccination. The samples were subjected to cELISA to determine the presence of antibodies against PPRV. The samples with PI >50 were considered as sero-positive. The sheep found positive at 10, 30 and 45 days post-vaccination were 1(8.3%), 7(58.3%) and 12(100%) respectively. In case of goats 3(10.0%), 29(96.6%) and 27(90.0%) animals gave positive results at 10, 30 and 45 days post-vaccination respectively. Mean PI values in sheep at 10, 30 and 45 days post-vaccination were recorded as 37, 65 and 91 respectively, whereas in goats these values were 43, 78 and 86 respectively.