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The role of the 5' nontranslated regions of the fusion protein mRNAs of canine distemper virus and rinderpest virus.

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.

Animals↗

Inhibition of host peripheral blood mononuclear cell proliferation ex vivo by Rinderpest virus.

Rinderpest, or cattle plague, is caused by Rinderpest virus (RPV), which is related most closely to human Measles virus (MV), both being members of the genus Morbillivirus, a group of viruses known to have strong immunosuppressive effects in vitro and in vivo. Here, it was shown that peripheral blood mononuclear cells (PBMCs) isolated from cattle experimentally infected with either wild-type or vaccine strains of RPV impaired the proliferation of PBMCs derived from uninfected animals; however, in contrast to either mild or virulent strains of wild-type virus, the inhibition induced by the vaccine was both weak and transient. Flow-cytometric analysis of PBMCs obtained from cattle infected with different strains of RPV showed that the proportion of infected cells was virus dose-dependent and correlated with lymphoproliferative suppression.

Animals↗

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

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.

Africa↗

Immunological and virological characterization of improved construction of recombinant vaccinia virus expressing rinderpest virus hemagglutinin.

We constructed a recombinant vaccinia virus (RVV) expressing rinderpest virus (RPV) hemagglutinin (H) by modifying the promoter region of the original RVV. The promotor region was modified at three points, i.e., an outframe ATG was eliminated, the sequence between the promoter and initiation codon was shortened and the base sequence just upstream of the initiation codon was changed. As compared with the original RVV, the modified RVV was found to produce a remarkably large amount of H protein in infected rabbit kidney cells cultured in vitro and to induce high titers of anti-RPV-H antibodies in rabbits. The median protective doses in rabbits of the modified and of the original RVVs were 10(2) pfu and 10(3.5) pfu, respectively, indicating that the modified RVV was at least 10-times more effective in protection than the original. The neurovirulence of the modified RVV and the parental LC16mO strain was roughly at the same level, and was much lower than that of WR strain. The modified RVV was as heat-stable as the original one. These results indicate that the modified RVV could be a candidate rinderpest vaccine for further examinations including cattle.

Animals↗

A rapid chromatographic strip test for the pen-side diagnosis of rinderpest virus.

Rinderpest is a contagious viral disease of cloven-hoofed domestic and wild animals. Eradication of the virus following outbreaks depends on rapid and accurate diagnosis of infection and the implementation of control measures. Reporting and confirmatory diagnosis precede the implementation of control measures. A number of techniques have been used for diagnosis such as agar gel immunodiffusion, enzyme-linked immunosorbent assay (ELISA), molecular biological techniques such as polymerase chain reaction (PCR) and virus isolation in tissue culture. Many of these methods are both time consuming and require skilled personnel. The development of a rapid pen-side test for the detection of rinderpest virus (RPV) antigen in lachrymal fluid of cattle is described using the Clearview chromatographic strip test technology (Unipath, Bedford). Optimum conditions for binding monoclonal antibody to nitrocellulose and latex microspheres were determined and a prototype device was developed. The device detected viral antigen in lachrymal fluids from experimentally and naturally infected cattle and showed no cross-reactivity with other related viruses. A field trial was carried out at the Landhi Cattle Colony (LCC), Pakistan, to assess the performance of the rinderpest test under field conditions. Ninety-seven animals, some of which were showing various clinical signs, at LCC and neighbouring colonies were sampled and tested at the pen-side by Clearview and later by immunocapture ELISA (IC-ELISA) at IAH, Pirbright. Nineteen animals were positive by Clearview and/or IC-ELISA. Seventeen out of 19 rinderpest positive animals were positive by Clearview and 15 out of 19 were positive by IC-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) confirmed the 19 animals to be rinderpest positive. This simple, rapid, specific test allows for the first time, accurate pen-side diagnosis of rinderpest.

Animals↗

Epidemiologic association between antibody titres against bovine virus diarrhoea virus, rinderpest disease virus and infectious bovine rhinotracheitis virus in a buffalo herd.

The association between antibody titres against bovine virus diarrhoea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV) and rinderpest disease virus (RPDV) in buffaloes was investigated in a cross-sectional study. Thirty-six lactating buffaloes from a herd in Landhi Cattle Colony, Karachi were bled and serum samples subjected to a micro-neutralisation test at the Plum Island Animal Disease Center, USA, to categorise each buffalo either as positive or negative against each of 3 viral antigens. Log-linear analysis was used to evaluate the association among 3 categorical variables. The "best-fitting" log-linear model was the model of complete independence. This model includes all 3 main effects (BVDV, IBRV, RPDV), suggesting that the seropositivity of buffaloes against these viral antigens tended to occur independently. Estimates of parameters of the model showed that the proportion of buffaloes seropositive (97.2%) to RPDV antigen tended to be higher than expected, whereas the proportion of BVDV seropositive (30.6%) and IBRV seropositive (16.7%) buffaloes tended to be smaller than expected.

Animals↗

Immunizing effect of vaccinia virus expressing the nucleoprotein of rinderpest virus on systemic rinderpest virus infection in rabbits.

A recombinant vaccinia virus (RVV) expressing the nucleoprotein (NP) of rinderpest virus (RPV) was examined in rabbits for the involvement of the NP protein in protection from the RPV infection. Despite their production of anti-NP antibody, the RVV-immunized rabbits succumbed to the RPV challenge, although there was a slight delay in the onset of disease after the low-dose challenge. On the other hand, the animals immunized with RVV expressing the hemagglutinin (H) protein of the RPV were completely protected. These results indicate that the NP protein might be not so effective as the H protein for the protection against viremic and systemic infection with RPV.

Animals↗

Effect of immunization with plasmid DNA encoding for rinderpest virus matrix protein on systemic rinderpest virus infection in rabbits.

Plasmid vaccine pBK-CMVMPILC113 expressing the matrix (M) gene of rinderpest virus was assessed for its potential to protect rabbits against a lethal viral challenge. Rabbits immunized with plasmids expressing the M gene were not protected when challenged with lapinized rinderpest virus, despite the production of anti-M antibodies, while rabbits immunized with rinderpest tissue culture vaccine were completely protected from a lethal challenge with lapinized rinderpest virus. The plasmid vaccine also had no significant effect on the lymphopenia in challenged rabbits. The results indicate that rinderpest M protein does not have a protective role in rinderpest infection.

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Synthesis of leader RNA and editing of P mRNA during transcription by rinderpest virus.

Purified rinderpest virus was earlier shown to transcribe in vitro, all virus-specific mRNAs with the promoter-proximal N mRNA being the most abundant. Presently, this transcription system has been shown to synthesize full length monocistronic mRNAs comparable to those made in infected cells. Small quantities of bi- and tricistronic mRNAs are also synthesized. Rinderpest virus synthesizes in vitro, a leader RNA of approximately 55 nucleotides in length. Purified rinderpest virus also exhibits RNA editing activity during the synthesis of P mRNA as shown by primer extension analysis of the mRNA products.

Animals↗

Pathogenesis of rinderpest virus infection in rabbits. II. Effect of rinderpest virus on the immune functions of rabbits.

Rinderpest virus infection was shown to induce marked suppression of both humoral antibody response and cell-mediated immunity in rabbits. The virus exhibited a suppressive effect on primary antibody response as indicated by a decrease in numbers of plaque-forming cells (immunoglobulin [Ig]M) and hemagglutinating antibody titers of both IgM and IgG types to sheep red blood cells, whereas there was no detectable effect of the virus on the production of memory cells. Virus-induced suppression of cell-mediated immunity was demonstrated by a decreased rate of proliferative response of peripheral lymphocytes to phytohemagglutinin stimulus and by a depression of delayed-type skin reactions to purified protein derivative. Such suppressive effects were indicated to persist for 14 days or longer. Alteration in phagocytic activity of the reticuloendothelial system was not observed. The relevance of the virus-induced histological lesions in the lymphoid tissues to the virus-induced immunosuppression was discussed.

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Leader RNA of Rinderpest virus binds specifically with cellular La protein: a possible role in virus replication.

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.

5' Untranslated Regions↗

Protection of rabbits against lapinized rinderpest virus with purified envelope glycoproteins of peste-des-petits-ruminants and rinderpest viruses.

Haemagglutinin (HA) and fusion (F) proteins of peste-des-petits-ruminants virus (PPRV) and rinderpest virus (RPV) were purified by immunoaffinity chromatography. The purified proteins were characterized by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Rabbit hyperimmune sera were raised against the purified HA and F proteins and assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination-inhibition (HAI) and virus neutralization (VN) tests. The immunized animals were challenged with a virulent lapinized (rabbit-adapted) strain of RPV. Both HA and F proteins of PPRV protected rabbits against a lethal challenge with lapinized RPV. As expected, RPV HA and F proteins also conferred a similar protection against the homologous challenge. The postchallenge antibody responses were of a true anamnestic type.

Animals↗

Cell-mediated immune responses in cattle vaccinated with a vaccinia virus recombinant expressing the nucleocapsid protein of rinderpest virus.

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.

Animals↗

Autoimmunity induced in rabbits by rinderpest virus.

During rinderpest virus infection in rabbits, 19S cold hemagglutinating antibody against rabbit erythrocytes and 7S anti-nuclear antibody, which reacts with the nuclei and/or the nuclear membranes in immunofluorescent staining, were demonstrated. Virus infection that affected the thymus-dependent immune functions was speculated to act as a trigger for the production of these two autoantibodies.

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