Comparison of rinderpest and peste des petits ruminants viruses using anti-nucleoprotein monoclonal antibodies

Monoclonal antibodies (MAbs) were obtained using a purified preparation of the RBOK strain of a rinderpest vaccine virus. The cytoplasmic immunofluorescent staining test showed that these clones had specificity for the nucleoprotein (N) of the virus. Six clones which immunoprecipitated the N protein corroborated these results. Thirteen anti-N MAbs were used to compare geographically widespread rinderpest viruses (RPV) and peste des petits ruminants viruses (PPRV) to two other morbilliviruses, measles (MV) and canine distemper (CDV). The N protein antigen profiles of the 23 isolates determined by immunofluorescent staining and enzyme linked immunosorbent assay (ELISA) on infected cells enabled us to classify the strains into groups. A differential identification of the morbilliviruses can be made using one MAb or associations of the MAbs. The potential to distinguish between RPV and PPRV and between virulent and avirulent strains of rinderpest is of primary interest.     

Serological studies with peste des petits ruminants and rinderpest viruses in Nigeria

One hundred and ninety-five goat and 67 sheep sera collected from various parts of southern Nigeria were screened for neutralising antibodies to both the peste des petits ruminants (PPR) and rinderpest viruses. Neutralising antibodies against both viruses were found in the sheep and goat sera examined. Parallel titration of samples which neutralised both viruses indicated a primary infection with the PPR virus (PPRV). However, some samples which failed to neutralise PPRV neutralised the rinderpest virus (RV) indicating RV activity in sheep and goats in Nigeria. These findings are discussed in relation to the diagnosis of PPRV infection and the recent reappearance of bovine rinderpest in Nigeria.     

小反刍兽疫的研究进展

概述了小反刍兽疫的易感动物、临床症状及病原的理化特征和基因组结构，重点阐述了病毒6种结构蛋白的大小和组成、小反刍兽疫病毒的分离培养、血清学及分子生物学诊断技术，并对其传统疫苗以及新型基因工程疫苗的研制进行了详细论述，以为该病的流行病学研究、诊断和免疫防制提供参考。     

Microneutralisation systems for use with different strains of peste des petits ruminants virus and rinderpest virus

Summary Comparative studies were made to determine the most suitable microtitration system for assaying strains of peste des petits ruminants virus (PPRV) and rinderpest virus (RV). Infectivity titres did not differ significantly when assayed in either calf kidney, sheep kidney or Vero cells. However, cytopathic effects were much easier to detect in the latter making them the cell of choice. Addition of small amounts of virus to preformed cell monolayers in microplates with the subsequent addition of maintenance medium give higher infectivity titres than when cell suspension was added to virus, although the latter is more convenient for routine use. The titres of PPRV and neutralising antibodies assayed in tubes and microplates were not significantly different. Simultaneous screening of sera at a 1 in 20 dilution against both PPRV and RV gave a higher incidence of positives against homologous as opposed to heterologous virus.

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Resumen Se hicieron estudios comparativos para determinar el mejor sistema de microtitulación para detectar cepas del virus de la peste de peque?os rumiantes (VPPR) y virus de rinderpest (VR). Los títulos infectivos, no difirieron significativamente cuando los ensayos se efectuaron en cultivos celulares de ri?ón de ternero, de oveja y células Vero. Sin embargo, los efectos citopáticos se detectaron más facilmente en este último, haciendo de este cultivo celular el preferencial para pruebas de microtitulación. La adición, de peque?as poblaciones virables a monocultivos celulares preformados en microplacas, conjuntamente con medio de mantenimiento, dió títulos infectivos más altos, que cuando la suspensión de células se a?adió al virus, aunque este último método es más conveniente para trabajos de rutina. Los títulos del VPPR, y los anticuerpos neutralizantes, pruebas determinadas en tubo y microplacas, no difirieron significativamente. El análisis simultáneo de suero en diluciones 1 en 20 contra los virus de Pest de Peque?os Rumiantes y Rinderpest, dió una incidencia más alta de positivos contra el suero homólogo, que contra el heterólogo.

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Résumé Des études comparatives ont été effectuées pour déterminer la microméthode la mieux adaptée au titrage des souches des virus de la peste des petits ruminants (VPPR) et de la peste bovine (VPB) Les titres d'infectivité n'étaient pas significantivement différents qu'ils soient dosés sur cellules de rein de veau, de rein de mouton ou sur cellules Vero. Cependant, les effects cytopathogènes étaient plus facile à déceler dans les cellules Vero, ce qui en fait les cellules de choix. L'addition de petites quantités de virus aux tapis cellulaires complets en microplaques puis adjonction ultérieure de milieu d'entretien s'est traduit par des titres d'infectivité plus élevés que lorsque le virus était inoculé aux cellules en suspension, bien que cette dernière méthode soit plus commode pour l'utilisation de routine. Les titres du virus de la PPR et des anticorps neutralisants ne se sont pas révélés significativement différents en tubes ou en microplaques. Les examens simultanés de sérums au 1/20 contre à la fois le virus de la PPR et de la PB se tradusient par une fréquence plus élevée de réactions positives aux virus homologues qu'aux virus hétérologues.

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Research project R3792.     

The production of peste des petits ruminants hyperimmune sera in rabbits and their application in virus diagnosis

Hyperimmune sera were produced by serial inoculation of rabbits with Vero cell-adapted, sucrose gradient-purified Nigerian peste des petits ruminants virus (PPRV) isolate. Two antisera produced, neutralized the homologous PPRV but not the heterologous rinderpest Kabette "O" virus. The antisera gave strong precipitin lines with purified PPRV antigens and were used to detect PPRV and rinderpest virus antigens from ante-mortem secretions and post-mortem tissue homogenates from PPR and rinderpest virus infected goats and cattle by the agar gel precipitation tests (AGPT). The hyperimmune sera gave good titration curves with both purified Nigerian goat and the United Arab Emirate wildlife PPRV isolates in the indirect enzyme linked immunosorbent assay (ELISA). Results of indirect ELISA showed that although there were some cross reactions with the rinderpest, canine-distemper and measles viruses, at 1:100 dilution, the antisera would give a positive signal with only the homologous PPR virus.     

Monoclonal antibody-based competitive ELISA for simultaneous detection of rinderpest virus and peste des petits ruminants virus antibodies

An experimental competitive enzyme-linked immunosorbent assay (morbillivirus cELISA) using a recombinant N antigen (rRPV N) expressed in a baculovirus and a ruminant morbillivirus (RPV and PPRV)-specific monoclonal antibody (P-13A9) was developed for simultaneous detection of rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) antibodies and its diagnostic performance was evaluated. A set of known reference antisera against RPV and PPRV belonging to different lineages, experimental sera from cattle vaccinated for a RPV of Asian lineage, and field sera from cattle and sheep/goat populations known to be positive (West Africa) and negative (Korea) for RPV and PPRV were used for the evaluation. Morbillivirus cELISA results on the panel of experimental RPV and PPRV antisera showed high correlation (r=0.97) between the whole virus and the rRPV N antigens, suggesting that the rRPV N contains a ruminant morbillivirus-specific antigenic determinant recognized by the P-13A9 and it may be suitable as an ELISA antigen in place of the whole virus. Morbillivirus cELISA detected anti-RPV and anti-PPRV antibodies in all reference RPV and PPRV antisera containing VN titers >/=1:8, suggesting that the assay can simultaneously detect antibodies against RPV and PPRV. Anti-RPV antibody was detected by morbillivirus cELISA in vaccinated cattle as early as the VNT and continued to be detectable by both the cELISA and the VNT until termination of the study. When applied to field samples from Africa, morbillivirus cELISA showed good agreement with a RP cELISA kit (kappa value of 0.86) in bovine sera and with a peste des petits ruminant cELISA kit (kappa value of 0.81) in caprine/ovine sera. Usefulness of morbillivirus cELISA using the rRPV N protein was discussed.     

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。     

吉林省某羊群暴发小反刍兽疫

2014年3月底,从吉林省某羊群发生的临床以发热、流涎、口腔黏膜严重溃疡、呼吸困难、严重腹泻和高死亡率为特征的羔羊体内检测到130¹50nm的病毒粒子,该病毒命名为GZL-14毒株。应用RT-PCR从病羊体内扩增出小反刍兽疫病毒的基因序列。序列分析发现,GZL-14毒株N蛋白基因的核苷酸序列与国外分离株INDIA_1994和PRADESH_95的同源性最高,为99.3%,而与国内分离毒株China/Tib/07的同源性为99%;GZL-14毒株F蛋白基因的核苷酸序列与Izatnagar-94和Morocco-2008毒株的同源性最高,为98.1%,而与国内分离株China/Tib/07的同源性为97.2%,表明GZL-14株可能为新近由国外传入国内的毒株。本研究应用分子生物学手段,从病毒核酸水平首次确定吉林省某羊群新近发生的临床上以发热、流涎、口腔黏膜严重溃疡、呼吸困难、严重腹泻和高死亡率为特征的疫病为小反刍兽疫。     

小反刍兽疫病毒H基因的原核表达与鉴定

根据GenBank中已发表的小反刍兽疫病毒(PPRV)Nigeria 75/1株的H基因序列,设计上下游引物并添加BamH I酶切位点,以含有小反刍兽疫病毒H基因的Topo-PPRVH质粒为模板进行PCR扩增,扩增产物克隆于pEASY-T载体中,用BamH I单酶切后将目的片段连接到原核表达载体pET-32a(+)中,核酸序列分析证明.成功构建了PPRV H原核表达质粒pET-32a-H.将pET-32a-H重组质粒转化大肠杆茵 BL21(DE3)进行融合表达.经SDS-PAGE电泳,可见H蛋白获得了高效表达,融合蛋白的分子量约为87 Ku,表达产物以包涵体的形式存在,其表达量达到茵体总蛋白的38%,占包涵体蛋白的80%以上.Western blot鉴定表明,所表达的重组蛋白能被抗组氨酸单抗、抗PPRV标准阳性羊血清及抗PPRV疫苗的羊血清所识别,具有良好的免疫原性.     

Serum-virus simultaneous method of immunisation against peste des petits ruminants

Summary Peste des petits ruminants (PPR) hyperimmune serum was prepared in cattle and used to protect goats against PPR. The serum was well tolerated by goats and 8 ml of serum was found to protect goats againts 4 ml of pooled virulent PPR virus. A durable active immunity was conferred on goats when serum and virulent PPR virus were given simultaneously. Passive immunity lasted for 15 days.

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Metodo Simultaneo Suero-Virus Para La Inmunizacion Contra La Peste De Pequenos Rumiantes

Resumen Se preparó en bovinos, suero hiperinmune de peste de pegueños rumiantes, para inmunizar cabras contra la enfermedad. El suero fué bien tolerado por las cabras, aplicandose hasta 8 ml para protegerlas de 4ml de virus virulento inoculado. El resultado de la aplicación simultánea de suero hiperinmune y virus virulento, fué el de una inmunidad activa y duradera. La inmunidad pasiva tuvo una duración de 15 días.

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Immunisation Contre La Peste Des Petits Ruminants Par Inoculation Simultanee De Virus Et De Serum

Résumé Le sérum hyperimmun de la peste des petits ruminants a été préparé sur des bovins et utilisé pour protéger les chèvres contre la PPR. Le sérum a été bien toléré par les chèvres et 8 ml de sérum ont été nécessaires pour protéger des chèvres inoculées avec 4 ml d'un mélange de virus PPR pathogènes. Les chèvres ont acquis une immunité active durable quand le sérum et le virus PPR pathologène étaient administrés simultanément. La durée de l'immunité passive a été de 15 jours.

     

Hyperimmune serum in the control of peste des petits ruminants

Summary The value of administration of hyperimmune serum in the control of peste des petits ruminants was investigated in goats at different stages of the disease. A group of the goats was given hyperimmune serum intravenously at the fever stage of temperature of 40·5°C or above; another group showing no elevation of temperature but with other clinical signs of the disease were also given hyperimmune serum. Results indicated that hyperimmune serum was very effective in reversing the process of the disease if administered at the fever stage but not in animals that had progressed past the fever stage. The goats given the hyperimmune serum survived for 10 days before showing evidence of reinfection.

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Resumen Se investigó en cabras el valor de la administración de suero hiperinmune, en el control de la peste de peque?os rumiantes, en diferentes estadíos de la enfermedad. A un grupo se le administró suero hiperinmune vía intravenosa cuando tenían temperaturas de 40.5°C ó más; otro grupo recibió el mismo tratamiento, sin mostrar elevación de la temperatura pero con otros síntomas clínicos de la enfermedad. Los resultados indicaron, que el tratamiento con suero hiperinmune es eficaz, si se administra en el estado febril, pero nó en animales que pasan el mismo. Las cabras enfermas a las cuales se les suministró suero hiperinmune, sobrevivieron 10 días antes de mostrar evidencia de reinfección.

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Résumé La valeur de l'administration de sérum hyperimmun dans la lutte contre la peste des petits ruminants a été étudiée chez des chèvres á différents stades de la maladie. On a administré par voie intraveineuse du sérum hyperimmun à un groupe de chèvres dont la courbe de température avait atteint 40,5°C ou au dessus; un autre groupe ne présentant aucun signe de fièvre mais d'autres sympt?mes de la maladie a aussi re?u du sérum hyperimmun. Les résultats montrent que le sérum hyperimmun est très efficace pour faire régresser le cours de la maladie s'il est administré au stade de la fièvre mais il reste sans effet chez les animaux ayant dépassé ce stade. Les chèvres à qui on avait administré du sérum hyperimmun ont survécu 10 jours sans montrer de signes de réinfection.

     

我国小反刍兽疫流行情况及防控措施

小反刍兽疫是由小反刍兽疫病毒感染小反刍兽的一种严重的急性、烈性、接触性传染病。该病是一种主要的跨国传播的动物疫病,在世界范围内呈现扩散和东移的流行趋势。2007年西藏自治区暴发了我国首例小反刍兽疫疫情,至今我国目前已有23个省市发生该疫情,严重威胁我国的动物卫生安全。文章对我国小反刍兽疫的传播趋势和流行特点进行了分析,并对我国的紧急防控措施作出评估和建议。     

由麻疹思考小反刍兽疫的问题

小反刍兽疫(Peste des petits ruminants,PPR)是山羊、绵羊等小反刍兽类的急性接触传染性疾病,国际上将小反刍兽疫归为A类传染病.目前除非洲、中东和南亚次大陆传播外,在我国周边地区的许多国家和地区也频繁出现流行.因此,该病作为一种重大的跨国动物疫病,也在开始危害我国西藏和其他地区的动物生产和卫生安全[1-2].     

Sero-epidemiology of peste des petits ruminants (PPR) in Pakistan

A sero survey was conducted during 2005-2006 to estimate the sero prevalence of PPR in the small ruminant population of Pakistan. A total of 2798 samples were collected including goats (1979) and sheep (819) from villages in 27 randomly selected districts. These were tested by cELISA for PPRV and true prevalence estimates were calculated by Rogan and Gladen estimator. Overall, 1273 (45.5%) were found positive; 980 (49.5%) of 1979 samples from goats and 293 (35.8%) of 819 serum samples from sheep were positive. The true sero-prevalence of PPR was estimated to be 48.5% (95% CI, 46.6-50.3), and 52.9% (95% CI, 50.7-55.1) and 37.7 (95% CI, 34.4-41.0) for goats and sheep, respectively. PPR virus is widely distributed all across Pakistan and has become an endemic infection of small ruminants. Since it is one of the leading causes of morbidity and mortality in small ruminants, it poses a serious threat to food security and the rural economy in Pakistan.