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1.
Monoclonal antibodies (MAb) raised against the RBOK vaccine strain of rinderpest virus were characterized by radio-immunoprecipitation (RIPA) and in the indirect ELISA using measles (MV), distemper (CDV), rinderpest (RPV) and peste des petits ruminants viruses (PPRV). Those found to be specific for the matrix (M) protein and the nucleocapsid (N) protein could be classified into different groups on the basis of the anti-morbillivirus MAb classification scheme; a number of these MAb showed a selective recognition of RPV, measles virus and distemper virus, or of different isolates of rinderpest virus, demonstrating that greater inter-isolate variation occurs than was apparent from analyses using polyclonal antisera. One group of anti-F protein MAb (group F1) reacted with all isolates of both RPV and PPRV. A second group of anti-N protein MAb (group N1/A) reacted with all RPV isolates, but not with the PPRV isolates. Furthermore, these group N1/A antibodies reacted strongly with RPV isolates which were upon original isolation of high pathogenicity, but had a weaker reaction against the isolates of this virus which were of low pathogenicity. Thus, MAb against RPV, in particular those against the N protein offered a potential superior to that of molecular analyses for "isolate fingerprinting", the differentiation of RPV from PPRV and the discrimination between rinderpest viruses which had been, upon isolation, of either high or low pathogenicity.  相似文献   

2.
The genus Morbillivirus is classified into the family Paramyxoviridae, and is composed of 6 members, namely measles virus (MV), rinderpest virus (RPV), peste-des-petits-ruminants virus (PPRV), canine distemper virus (CDV), phocine distemper virus (PDV) and cetacean morbillivirus (CeMV). The MV, RPV, PPRV and CDV have been successfully attenuated through their serial passages in vitro for the production of live vaccines. It has been demonstrated that the morbilliviral virulence in animals was progressively attenuated with their consecutive passages in vitro. However, only a few reports were involved in explanation of an attenuation-related mechanism on them until many years after the establishment of a quasispecies theory. RNA virus quasispecies arise from rapid evolution of viruses with high mutation rate during genomic replication, and play an important role in gradual loss of viral virulence by serial passages. Here, we overviewed the development of live-attenuated vaccine strains against morbilliviruses by consecutive passages in vitro, and further discussed a related mechanism concerning the relationship between virulence attenuation and viral evolution.  相似文献   

3.
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.  相似文献   

4.
以小反刍兽疫病毒N基因序列为靶基因,通过设计引物及TaqMan探针建立快速检测小反刍兽疫的荧光PCR方法.在线BLAST分析结果表明:所设计的引物特异性强,可区分小反刍兽疫病毒及牛瘟病毒感染;所建立的方法检测线性范围为1～1×106拷贝质粒DNA,灵敏度可达10拷贝质粒DNA,是常规PCR法的100倍.应用所建立的方法对32份采自西藏疫区的组织样品进行检测,说明疫情在西藏没有扩散.  相似文献   

5.
Peste des petits ruminants (PPR) is an acute, febrile viral disease of small ruminants, caused by a virus of the genus Morbillivirus. PPR and rinderpest viruses are antigenically related and need to be differentiated serologically. In the present study, 23 mouse monoclonal antibodies were produced by polyethyleneglycol (PEG)-mediated fusion of sensitized lymphocytes and myeloma cells. Among these, two belong to the IgM class and the remaining 21 to various subclasses of IgG. The MAbs from the IgG class designated 4B6 and 4B11 neutralized PPR virus in vitro. In radioimmunoprecipitation assay, 10 MAbs recognized nucleoprotein, 4 recognized the matrix protein and one each haemagglutinin and phosphoprotein. The remaining 7 MAbs failed to precipitate any defined viral protein. The reactivity pattern of the monoclonal antibodies in indirect ELISA indicated a close antigenic relationship within three Indian PPR (lineage 4) virus isolates and also within two rinderpest vaccine strains. All PPR virus isolates could be distinguished from rinderpest vaccine viruses on the basis of the reactivity pattern of all MAbs and anti-N protein MAbs. A set of six monoclonal antibodies specific to PPR virus could also be identified from the panel. From the panel of MAbs available, two MAbs were selected for diagnostic applications, one each for the detection of antigens and antibodies to PPR virus.  相似文献   

6.
A competitive ELISA (C-ELISA) using monoclonal antibodies (mAbs) which bind to the nucleo-protein (NP) of rinderpest virus (RPV) for detection of RPV antibodies in cattle and small ruminant sera is described. Unlike virus neutralisation test (VNT), this test using mAb IVB2-4, can detect specific RPV antibodies without showing a cross-reaction with antibodies to peste-des-petits ruminants-virus (PPRV); by contrast, when mAb VE4-1 is used the test detects both RPV and PPRV antibodies, including low levels of antibodies that can be found in sera containing maternal antibodies. Although antibodies to the PPRV 75-1 strain are also detected with mAb 51-5-6, the test is suitable for assessing the immune status of cattle against the Rinderpest Old Kabete (RBOK) strain. The results from a panel of sera with a known status of vaccination provide evidence for a highly significant correlation between C-ELISA and VNT. This test may be a useful tool for a standardized and accurate determination of the immunity status of both cattle and small ruminants.  相似文献   

7.
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8.
The nucleotide sequence of the matrixprotein (M) gene of the lapinized rinderpest virus (RPV-L) was determined. The full-length cDNA of the RPV-L M gene is composed of 1460 base pairs and is supposed to contain an open reading frame of 1005 nucleotides encoding on M protein of 335 amino acids. The homology of the predicted amino acid among congeneric morbilliviruses such as RPV Kabete 'O' strain (wild strain of RPV), RPV RBOK strain (vaccine strain of RPV for cattle), measles virus (MV), and canine distemper virus (CDV), is approximately 94%, 93%, 87% and 77%, respectively. In the present study, all coding regions of the RPV-L strain have been determined.  相似文献   

9.
We have established four monoclonal antibodies (MAbs) against the nucleocapsid protein (NP) of canine distemper virus (CDV). A competitive binding assay has revealed that the MAbs are directed against two antigenic domains. An immunofluorescence assay using a series of deletion clones of the NP and an immunoprecipitation assay using the NP have revealed that two of the MAbs recognize the C-terminal region of the NP while the other two recognize the tertiary structure of the N-terminal domain. These MAbs reacted with all eight strains of CDV used in this study, but showed different reactivities against measles virus and rinderpest virus.  相似文献   

10.
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.  相似文献   

11.
The complete nucleotide sequence of the nucleocapsid (N) protein of the peste-des-petits ruminants vaccine virus (PPRV Sungri/96) belonging to the Asian lineage was determined. The gene was 1692 nucleotides in length and encoded a polypeptide of 525 amino acids. The PPRV Sungri/96 N gene has a nucleotide homology of 92% for PPRV Nigeria 75/1 to 55.5% for canine distemper virus. At amino acid level the homology was 94.1% with PPRV Nigeria 75/1, while with other morbilliviruses, PPRV Sungri/96 had only 71.4–64.9% amino acid identity. The phosphorylation prediction reveals eight conserved sites across morbilliviruses, whereas in the C-terminal portion of the protein the sites are not conserved. Phylogenetic analysis of different N proteins of morbilliviruses revealed five well-defined clusters as observed previously. To the best of our knowledge this is the first report describing the nucleocapsid gene sequence of PPRV Indian isolate. Muthuchelvan, D., Sanyal, A., Balamurugan,V., Dhar, P. and Bandyopadhyay, S.K., 2006. Sequence analysis of the nucleoprotein gene of Asian lineage peste des petits ruminants vaccine virus.Veterinary Research Communications, 30(8), 957–963  相似文献   

12.
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.  相似文献   

13.
The nucleotide sequences of the phosphoprotein (P) gene of peste des petits ruminants (PPRV) vaccine virus (PPRV Sungri/96) belongs to Asian lineage have been determined and the deduced amino acid sequences were compared with another vaccine strain PPRV/Nigeria75/1 and with those of the other morbilliviruses. The 1652 nucleotides of the P gene encode a phosphoprotein of 509 amino acid residues (from nucleotide numbers 60 to 1587), which is 91% identical to that of PPRV/Nigeria75/1. The C protein consists of 177 amino acid residues and is 91% identical with that of PPRV/Nigeria75/1. The conserved mRNA editing site (5'TTAAAAGGGCACAG) was present at positions 742-756 in the P gene, which is conserved in all other morbilliviruses. The CTT trinucleotide sequence is present at the N/P and P/M intergenic region, which is totally conserved in morbilliviruses. This will be the third sequence for the P gene of PPRV since that of the vaccine strain and a wild-type Turkish isolate has been published already.  相似文献   

14.
A panel of monoclonal antibodies (mAbs) was generated against the RBOK strain of rinderpest virus (RPV). All of them bound to the N protein of RPV. The antigen capture ELISA using the mAbs could detect the virus in crude viral preparations. The mAb 12BF8.1.1 showed higher reactivity with cell-associated (CA) virus, whereas the mAbs 12AD10.1.1, 12BD7.1.1 and 12DG7.1.1 showed higher reactivity with extracellular virus (hereafter referred to as cell-free (CF) virus). The mAbs 12BF8.1.1 and 12AD10.1.1 could detect the virus in infected Vero cell culture supernatants (CCS) as early as 24 h post-cytopathic effect (CPE) initiation. Detergent treatment (Triton X-100) of RPV preparations enhanced the binding of the mAbs to the virus. All the seven mAbs showed specific fluorescence in virus-infected cell cultures. The immunofluorescence (IFA) using mAbs was found to be more sensitive and reliable than the immunoperoxidase test (IPT) for detection of rinderpest.  相似文献   

15.
目的选取PPRV和RPVN蛋白中抗原性较强、氨基酸差异性较大的片段进行重组表达。方法对PPRV和RPVN蛋白中51-175aa、376-525aa蛋白片段基因进行大肠杆菌偏爱密码子优化后人工合成,利用分子生物学技术,将目的基因分别克隆至原核表达载体pET-28a(+)构建重组表达质粒,将重组表达质粒转化至E.coliBL21(DE3),诱导表达并纯化PPRV与RPVN蛋白中51-175aa、376-525aa片段。结果得到了分子量分别约17.8kD、20.6kD的各两段融合蛋白。结论本研究成功表达了PPRV和RPVN蛋白中抗原性较强、氨基酸差异性较大的51-175aa和376-525aa蛋白片段,为制备能区别PPR和RP病毒的特异性单抗提供了抗原。  相似文献   

16.
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17.
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.  相似文献   

18.
Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin–neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345–353) on the HN protein.  相似文献   

19.
为了调查小反刍兽疫病毒四川简阳株(SCJY株)的分子遗传特征,我们对四川简阳发病羊鼻拭子中的小反刍兽疫病毒N基因进行了遗传进化分析。通过RT-PCR、克隆测序获得SCJY株N基因序列,使用DNASTAR软件将其与GenBank中的15条参考株序列进行同源性比对,使用MEGA6软件进行系统进化分析。结果显示:SCJY株N基因序列全长1578nt,与参考株的核苷酸同源性为88.9%～100%,氨基酸同源性为92.8%～100%,与2013～2014年小反刍兽疫中国流行毒株高度同源;系统进化分析将16个毒株N基因划分为4个谱系,SCJY株属于Ⅳ系。  相似文献   

20.
Since 1988 morbilliviruses have been increasingly recognized and held responsible for mass mortality amongst harbour seals (Phoca vitulina) and other seal species. Virus isolations and characterization proved that morbilliviruses from seals in Northwest Europe were genetically distinct from other known members of this group including canine distemper virus (CDV), rinderpest virus, peste des petits ruminants virus and measles virus. An epidemic in Baikal seals in 1987 was apparently caused by a morbillivirus closely related to CDV so that two morbilliviruses have now been identified in two geographically distant seal populations, with only the group of isolates from Northwest Europe forming a new member of the genus morbillivirus: phocid distemper virus (PDV). Because of distemper-like disease, the Baikal seal morbillivirus was tentatively named PDV-2 in spite of its possible identity with CDV. The appearance of morbilliviruses in the Mediterranean Sea causing high mortality amongst dolphins should further increase the research activities on protection strategies for endangered species of marine mammals.  相似文献   

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