Domains of Rinderpest virus phosphoprotein involved in interaction with itself and the nucleocapsid protein.
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The yeast two-hybrid system was used to identify domains involved in specific in vivo interactions between the Rinderpest virus (RPV) phosphoprotein (P) and nucleocapsid protein (N). N and P genes were cloned in both the yeast GAL4 DNA-binding and GAL4 activation domain vectors, which enabled analysis of self and interprotein interactions. Mapping of the domain of P protein involved in its association with itself revealed that the COOH-terminal 32 amino acids (316-347) that forms a part of the highly conserved coiled coil region is important for interaction. In addition, just the coiled coil region of RPV P protein fused to the DNA-binding domain and activation domain of GAL4 was found to be sufficient to bring about activation of the beta-galactosidase reporter. Similarly, mapping of the domains of P protein involved in its interaction with N protein revealed that NH2-terminal 59 amino acids and COOH-terminal 32 amino acids (316-347) involved in P-P interaction are simultaneously required for association with N protein. Interestingly, a P protein mutant with just the NH2-terminal 59 amino acids and the coiled coil domain with all other P protein regions deleted retained its ability to interact with N protein. Furthermore, we were able to show N and P protein interaction in vitro using recombinant N and P proteins expressed in Escherichia coli, demonstrating the existence of direct physical interaction between the two proteins. (+info)
Cell-mediated immune responses in cattle vaccinated with a vaccinia virus recombinant expressing the nucleocapsid protein of rinderpest virus.
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Rinderpest virus (RPV) is a member of the genus Morbillivirus in the family Paramyxoviridae which causes an acute and often fatal disease in large ruminants. To examine the immune response to the virus nucleocapsid (N) protein, a recombinant vaccinia virus expressing RPV nucleocapsid protein (rVV-RPV-N) was used to vaccinate cattle. The recombinant vaccine induced low levels of non-neutralizing anti-N antibodies. RPV-specific cell-mediated immunity induced by the recombinant was assessed by measuring both the lymphocyte proliferation and cytotoxic T-lymphocyte responses. The protective immune response was examined by challenging the vaccinated cattle with either a highly virulent (Saudi 1/81) or a mild (Kenya/eland/96) strain of the virus. The vaccinated cattle were not protected against challenge with the virulent RPV strain, except they showed a slight delay in the onset of disease when compared with the unvaccinated controls. In cattle challenged with the mild strain, apart from a transient fever, no clinical signs of rinderpest infection were seen in the vaccinated cattle. One out of two control cattle showed a similar response but the other died from classic rinderpest disease. Virus-neutralizing antibodies were induced more quickly following challenge with the mild strain in vaccinated cattle compared to the control animals. These data suggested that the cell-mediated immunity induced by rVV-RPV-N could stimulate the rapid production of neutralizing antibodies following RPV challenge but this response was not sufficient to protect against challenge with a virulent strain of the virus. Protection was seen in one of three animals challenged with a mild strain of the virus; however, a greater number of animals would need to be tested to estimate the significance of the protection afforded by the N protein. (+info)
Expression in cattle of epitopes of a heterologous virus using a recombinant rinderpest virus.
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We have investigated the bovine immune response to heterologous proteins expressed using a recombinant rinderpest virus (RPV). A new gene unit was created in a cDNA copy of the genome of the vaccine strain of RPV, and an open reading frame inserted that encodes the polymerase (3Dpol) and parts of the capsid protein VP1 from foot-and-mouth disease virus (FMDV). Infectious recombinant RPV was rescued and shown to express the FMDV-derived protein at good levels in infected cells. The rescued virus was only slightly more attenuated in tissue culture than the original virus. Cattle infected with this recombinant generated a normal immune response to RPV, and were protected from lethal challenge by that virus. Experimental animals showed a specific delayed-type hypersensitivity response to FMDV 3Dpol, similar to that seen in FMDV infection; however, no antibodies were detected recognizing either of the components of the FMDV-derived protein, nor was any proliferative response to these epitopes found in isolated peripheral blood lymphocytes from infected animals. No protection was seen against FMDV infection. (+info)
Development of a genetically marked recombinant rinderpest vaccine expressing green fluorescent protein.
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In order to effectively control and eliminate rinderpest, a method is required to allow serological differentiation between animals that have been vaccinated and those which have recovered from natural infection. One way of doing this would be to engineer the normal vaccine to produce a genetically marked rinderpest virus (RPV) vaccine. We constructed two modified cDNA clones of the RPV RBOK vaccine strain with the coding sequence of the green fluorescent protein (GFP) gene inserted as a potential genetic marker. RPVINS-GFP virus was designed to produce independent and high level expression of GFP inside infected cells, whilst the GFP expressed by RPVSIG-GFP virus was designed to be efficiently secreted. Infectious recombinant virus was rescued in cell culture from both constructs. The effectiveness of these viruses in stimulating protective immunity and antibody responses to the marker protein was tested by vaccination of cattle and goats. All of the vaccinated animals were completely protected when challenged with virulent virus: RPV in cattle or peste-des-petits ruminants virus in the goats. ELISA showed that all of the animals produced good levels of anti-RPV antibodies. Three of the four cattle and the two goats vaccinated with RPVSIG-GFP produced detectable levels of anti-GFP antibodies. In contrast, no anti-GFP antibodies were produced in the four cattle and two goats vaccinated with RPVINS-GFP. Therefore, secretion of the GFP marker protein was absolutely required to elicit an effective humoral antibody response to the marker protein. (+info)
Rinderpest viruses lacking the C and V proteins show specific defects in growth and transcription of viral RNAs.
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Rinderpest virus is a morbillivirus and the causative agent of an important disease of cattle and wild bovids. The P genes of all morbilliviruses give rise to two proteins in addition to the P protein itself: use of an alternate start translation site, in a second open reading frame, gives rise to the C protein, while cotranscriptional insertion of an extra base gives rise to the V protein, a fusion of the amino-terminal half of P to a short, highly conserved, cysteine-rich zinc binding domain. Little is known about the function of either of these two proteins in the rinderpest virus life cycle. We have constructed recombinant rinderpest viruses in which the expression of these proteins has been suppressed, individually and together, and studied the replication of these viruses in tissue culture. We show that the absence of the V protein has little effect on the replication rate of the virus but does lead to an increase in synthesis of genome and antigenome RNAs and a change in cytopathic effect to a more syncytium-forming phenotype. Virus that does not express the C protein, on the other hand, is clearly defective in growth in all cell lines tested, and this defect appears to be related to a decreased transcription of mRNA from viral genes. The phenotypes of both individual mutant virus types are both expressed in the double mutant expressing neither V nor C. (+info)
Long-term protective immunity to rinderpest in cattle following a single vaccination with a recombinant vaccinia virus expressing the virus haemagglutinin protein.
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A recombinant vaccine, produced by using a highly attenuated smallpox vaccine (LC16mO) as a vector and which expresses the rinderpest virus (RPV) haemagglutinin protein, has been developed. The properties of this vaccine, including its heat stability, efficacy in short-term trials, safety and genetic stability, have been confirmed in an earlier report. In the present study, the duration of the protective immunity generated by the vaccine in cattle was examined for up to 3 years following the administration of a single vaccination dose of 10(8) p.f.u. The vaccinated cattle were kept for 2 (group I) or 3 years (group II) and then challenged with a highly virulent strain of RPV. Four of five vaccinated cattle in group I and all six cattle in group II survived the challenge, some showing solid immunity without any clinical signs of rinderpest. Neutralizing antibodies were maintained at a significant level for up to 3 years and they increased rapidly following challenge. Lymphocyte proliferative responses to RPV were examined in group II cattle and were observed in four of the six vaccinated cattle in this group. The long-lasting protective immunity, in addition to the other properties confirmed previously, indicate the practical usefulness of this vaccine for field use. (+info)
Vaccination of cattle with attenuated rinderpest virus stimulates CD4(+) T cell responses with broad viral antigen specificity.
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The immune responses of cattle inoculated with either a virulent or an attenuated vaccine strain of rinderpest virus (RPV) were examined by measuring the proliferation of peripheral blood mononuclear cells (PBMC) to whole RPV antigen preparations and to individual RPV major structural proteins expressed using recombinant adenoviruses. Responses to the T cell mitogen concanavalin A (ConA) were also measured as a control to monitor non-specific effects of infection with RPV on T cell responses. Infection with the vaccine strain of RPV was found to induce a strong CD4(+) T cell response. A specific response was detected to all RPV proteins tested, namely the haemagglutinin (H), fusion (F), nucleocapsid (N) and matrix (M) proteins, in animals vaccinated with the attenuated strain of the virus. No one protein was found to be dominant with respect to the induction of T cell proliferative responses. As expected, vaccination of cattle with an unrelated virus vaccine, a capripox vaccine, failed to produce a response to RPV antigens. While profound suppression of T cell responses was observed following infection with the virulent strain of RPV, no evidence of impairment of T cell responsiveness was observed following RPV vaccination, or on subsequent challenge of vaccinated animals with virulent virus. (+info)
A study of immunoglobulin M antibody to measles, canine distemper, and rinderpest viruses in sera of patients with subacute sclerosing panencephalitis.
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Seven boys were studied who had the clinical features of subacute sclerosing panencephalitis (SSPE) and whose brain histology was consistent with SSPE. Measles antigen was detected in the seven brains by the direct fluorescent antibody method. Three out of the seven boys had in their sera measles specific immunoglobulin M (IgM) which was detected by the indirect fluorescent antibody method, and the cell receptors for it were acetone stable. A prozone effect was noted in the sera of two patients. The absorption of one patient's serum with Staphylococcus aureus to reduce the titre of immunoglobulin G (IgG) removed the prozone effect. Two of the boys who had high titres of measles specific IgM also had serum IgM which reacted with canine distemper virus antigen but the titres were eightfold lower. None of the boys had detectable rinderpest specific IgM in their sera. (+info)