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Goats vaccinated with attenuated rinderpest were protected from peste-des-petits-ruminants virus for at least 12 months; vaccinated animals were unable to transmit the challenge virus. Before challenge neutralising antibodies were directed primarily against rinderpest but following exposure to peste-des-petits-ruminants, a high antibody level to both viruses was found.
Caprinised rinderpest virus GTV strain (GTV) was adapted to grow in Vero cells (vGTV) by polyethylene glycol-mediated fusion of infected goat spleenocytes with non-infected Vero cells. The usual methods of infection of cell culture, i.e. virus adsorption or co-cultivation, were not successful. vGTV-induced cytopathic changes in Vero cells were similar to those reported for rinderpest virus. Virus titers increased with the passage number but the virulence for goats decreased. Immunoblot analysis did not reveal any difference between vGTV and vRBOK, the tissue culture rinderprest virus RBOK strain adapted to Vero cells.
Rinderpest, the legendary cattle plague, has caused devastating losses for centuries and remains the biggest threat to sustainable livestock production in developing countries. Strenuous efforts are now being made to achieve global eradication of this viral disease, a goal made feasible through the use of attenuated live vaccines developed in the 1950s. Their use almost resulted in eradication in the 1970s but left several foci of disease from where the plague then re-emerged. Recent mass vaccination has resulted in the limitation of disease to parts of Africa, Pakistan, Afghanistan, the Middle East and India. Confirmation of the disease status of the countries has been aided by developments in serological techniques through exploitation of monoclonal antibodies (in Enzyme Linked Immunosorbent Assay-ELISA) and by advances in molecular biology such as in the use of polymerase reaction technologies (PCR). This has extended into the development of new recombinant vaccines. It is anticipated that eradication will be complete by the year 2010. This would be only the second example, after smallpox in man, of the eradication of a viral disease. The picture shows a scene of devastation during the Great Rinderpest Pandemic of 1889-1897 in South Africa. The disease swept through the African continent killing virtually all the cattle and wild ungulates.
We have cloned and sequenced the entire M gene of the vaccine strain of rinderpest virus and that of the virulent Kabete "O" strain from which it was derived. The sequences of these two genes are essentially identical (99% at the nucleotide level), but were very different from a previously published Kabete O M gene sequence (M. Limo and T. Yilma, 1990, Virology 175, 323-327). Inspection of the nucleotide and deduced amino acid sequences of known morbillivirus M genes showed that the earlier sequence was clearly from a morbillivirus, but neither from rinderpest virus nor from peste des petits ruminants virus.
Rinderpest virus, like other Morbilliviruses, expresses three proteins from the single P gene. In addition to the P protein, which interacts both with the viral polymerase (L) and the nucleocapsid (N) protein, the virus expresses a C and a V protein from the same gene. The functions of these two proteins in the viral life cycle are not clear. Although both C and V proteins are dispensable, in that viable viruses can be made that express neither, each seems to play a role in optimum viral replication. We have used the yeast-two hybrid system, binding to coexpressed fusions of C and V to glutathione-S-transferase, and studies of the native size of these proteins to investigate interactions of the rinderpest virus C and V proteins with other virus-encoded proteins. The V protein was found to interact with both the N and L proteins, while the C protein was found to bind to the L protein, and to self-associate in high-molecular-weight aggregates.
Monoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569-577 and 575-583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527-554 and 588-609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus HN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination.
Earlier diagnostic reports of rinderpest in buffaloes in the Landhi Dairy Colony were discounted by most veterinarians in Pakistan. Four recent investigations have shown beyond doubt that the affliction was indeed rinderpest.
Rinderpest virus is the causative agent of a devastating, often fatal disease in wild and domestic bovids that is endemic in Africa, the Middle East and South Asia. The existing live attenuated vaccine is heat-labile, and thus there is a need for the development of new strategies for vaccination. This paper reports the development of transgenic pigeon pea [ Cajanus cajun (L.) Millsp.] expressing one of the protective antigens, the hemagglutinin (H) protein of Rinderpest virus. A 2-kb fragment containing the coding region of the H protein was cloned into pBI121 and mobilized into Agrobacterium tumefaciensstrain EHA105. Embryonic axes and cotyledonary nodes from germinated seeds of pigeon pea were used for transformation. The presence of the transgene in transgenic plants was confirmed by Southern blots, and the specific transcription of the marker gene in the plants was demonstrated by reverse transcription-polymerase chain reaction. Integration of the H gene into the pigeon pea genome was confirmed by Southern hybridization. The expression of the H protein in the transgenic lines was confirmed by Western blot analysis using a polyclonal monospecific antibody to the H protein. The highest level of expression of the hemagglutinin protein in leaves of pigeon pea was 0.49% of the total soluble protein. The transgenic plants were fertile and the transgene expressed in the progeny.
The mRNAs which code for the fusion proteins of the morbilliviruses (measles virus, canine distemper virus, and rinderpest virus) have unusually long 5' untranslated regions (UTRs) which are GC-rich and are capable of folding into extensive secondary structures. In measles virus the first AUG codons in the fusion (F) protein mRNA are in close proximity at nucleotide positions 574 and 583 and protein translation is initiated at the second position. In the canine distemper virus (CDV) and rinderpest virus (RPV) F gene transcripts the analogous initiation codons are preceded by several other AUG codons many nucleotides upstream either in the same reading frame or at the beginning of other short open reading frames. We have studied the effect of deleting these upstream regions on the production of the fusion proteins of both CDV and RPV from cDNA constructs. Within the cells the presence of these regions enhances the production of the F protein while, in contrast, the production of the authentic F protein from in vitro translations using RNA transcripts is inhibited by these sequences.
A solid phase aggregation of coated erythrocytes (SPACE) technique was standardized and used for the detection of rinderpest antigen in 129 samples of 9 visceral organs; from 50 clinical samples such as gum scraping, tongue scraping, faeces, urine, nasal swabs; blood from 18 rinderpest infected cattle and 2 field samples. The comparative sensitivity of SPACE, reverse phase passive haemagglutination and counter-immuno-electrophoresis tests was found to be 86.8, 84.5 and 79.8%, respectively.
The rinderpest (RV) nucleocapsid (NP) gene segment was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. The expression of NP protein in Sf9 cells was confirmed by indirect immunofluorescence and by Western blotting analysis with monoclonal antibodies. Recombinant RV-NP protein was purified by ultracentrifugation on a sucrose density gradient, and used as an antigen for an enzyme linked immunosorbent assay to detect anti RV-NP antibody. Both IgM and IgG antibodies against RV-NP were detected in the sera of rabbits infected with the L strain of RV. The pattern of development of IgG anti RV-NP antibody closely correlated with that of virus neutralizing antibody. In rabbits inoculated with recombinant vaccinia virus expressing RV-H gene (RRV-H), anti RV-NP was not detected. The results indicated that the baculovirus vector system can be used for the preparation of the diagnostic antigen of rinderpest as well as to distinguish between natural infection and vaccination with RRV-H.
Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I-III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.
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.
We have sequenced the nucleocapsid (N) and polymerase (L) genes of the vaccine strain of rinderpest, and the 5' and 3' terminal domains of the genome. Together with previously published data, this completes the sequence of the entire genome of rinderpest virus. The L gene is identical in length to that of measles virus, encoding a 2183 amino acid protein with a calculated molecular weight of 248,100. The L protein sequence of three morbilliviruses is highly conserved, greater than 76% of residues being identical or conserved in all sequences. The N protein was, as for other sequenced genes, essentially identical to that of the virulent parent. The viral genome is 15,881 bases in length, similar to that of measles virus and slightly longer than that of canine distemper virus. The terminal sequences of the genome and those at the gene boundaries were compared to the analogous regions of other morbilliviruses and representatives of related groups of paramyxoviruses.
The RNA dependent RNA polymerase of Rinderpest virus consists of two subunits-the large protein (L) and the phosphoprotein (P), where L is thought to be responsible for the catalytic activities in association with P protein which plays multiple roles in transcription and replication. The nucleocapsid protein (N) is necessary for encapsidation of genomic RNA, which is required as N-P complex. To understand the different steps of transcription and replication as well as the roles played by the three proteins, an in vitro reconstitution system for RNA synthesis is necessary which is not available for any morbillivirus. We describe here, an in vitro reconstitution system for transcription and replication of Rinderpest virus utilizing a synthetic, positive sense N-RNA minigenome template, free of endogenous viral polymerase proteins and recombinant viral proteins (P+L and P+N) expressed in insect cells by recombinant baculoviruses. We show that although L-P complex is sufficient to synthesize negative sense minigenome RNA, soluble N protein is necessary for encapsidation of RNA as well as synthesis of (+) sense leader RNA and (+) sense minigenome RNA.
Rinderpest virus (RPV) is an important member of the Morbillivirus genus in the family Paramyxoviridae and employs a similar strategy for transcription and replication of its genome as that of other negative sense RNA viruses. Cellular proteins have earlier been shown to stimulate viral RNA synthesis by isolated nucleocapsids from purified virus or from virus-infected cells. In the present work, we show that plus sense leader RNA of RPV, transcribed from 3' end of genomic RNA, specifically interacts with cellular La protein employing gel mobility shift assay as well as UV cross-linking of leader RNA with La protein. The leader RNA synthesized in virus-infected cells was shown to interact with La protein by immunoprecipitation of leader RNA bound to La protein and detecting the leader RNA in the immunoprecipitate by Northern hybridization with labeled antisense leader RNA. Employing a minireplicon system, we demonstrate that transiently expressed La protein enhances the replication/transcription of the RPV minigenome in cells. Sub-cellular immunolocalization shows that La protein is redistributed from nucleus to the cytoplasm upon infection. Our results strongly suggest that La protein may be involved in regulation of Rinderpest virus replication.
The negative sense genome RNA of Rinderpest virus, a Paramyxoviridae, is encapsidated with the nucleocapsid protein N and serves as a template for the viral RNA dependent RNA polymerase for transcription and replication. The viral RNA polymerase consists of the large protein L and the phosphoprotein P functioning as the P-L complex. We provide in this report, evidences for specific binding of P protein of Rinderpest virus to the plus sense leader RNA depending on its phosphorylation status. We have also demonstrated that P protein is released from the le RNA:P protein complex upon phosphorylation in vitro. Finally, we have identified that the C-terminal 358-389 amino acid residues of P protein is involved in le RNA binding. The leader RNA binding may signify a hitherto unidentified role for P protein in the viral RNA synthesis. Moreover, our results indicate a possible role for P protein in the transcription-replication switch through leader RNA binding.
The use of transgenic plants as a production system for recombinant subunit vaccines has been considered safe and economical compared to cell culture methods. We have exploited this approach to produce rinderpest virus hemagglutinin (H) protein in transgenic tobacco as a model plant for testing the immunogenicity of plant-derived hemagglutinin protein. The transgenic nature of the plants was confirmed by molecular analysis such as gene specific PCR and Southern hybridization using full-length H gene as a probe. The Mendelian pattern of inheritance of the transgene has been demonstrated in T(1) generation. The transgenic plants express the H protein of molecular weight 72 kDa. The plant derived H protein is antigenically authentic as revealed by reactivity with H-specific antibodies as well as convalescent sera. The induction of immune response was tested in mice after intraperitoneal immunization with plant-derived H. High titers of antibodies were induced which were H-specific and they neutralized the infectivity of rinderpest virus.