Characterization of the complete genomic sequence of the rinderpest virus Fusan strain cattle type, which is the most classical isolate in Asia and comparison with its lapinized strain.

In this study, we characterized the rinderpest virus (RPV) Fusan strain cattle type (B), which is the most classical isolate in Asia, by complete genomic sequence analysis and compared it with its lapinized Nakamura III (L) strain. The transversion rates of the M, F, and H genes were higher than those of other genes. In contrast, the deduced amino acid (aa) substitution rates of the P, C, and V genes were higher than those of other genes, although their transversion rates were not higher. The characteristic nucleotide (nt) or aa residues of the cattle-virulent B and Kabete 'O' strains were observed in the P/C/V, M, and L genes. According to these results, we speculate that nt/aa substitution in the P/C/V genes is one of the key determinants for the difference in the pathogenicity to cattle of the B and L strains.

A role for virus promoters in determining the pathogenesis of Rinderpest virus in cattle.

Rinderpest virus (RPV) is a morbillivirus that causes cattle plague, a disease of large ruminants. The viral genome is flanked at the 3' and 5' genome termini by the genome promoter (GP) and antigenome promoter (AGP), respectively. These promoters play essential roles in directing replication and transcription as well as RNA encapsidation and packaging. It has previously been shown that individual changes to the GP of RPV greatly affect promoter activity in a minigenome assay and it was therefore proposed that individual nucleotide changes in the GP and AGP might also have significant effects on the ability of the virus to replicate and cause disease in cattle. The Plowright vaccine strain of RPV has been derived by tissue-culture passage from the virulent Kabete 'O' isolate (KO) and is highly attenuated for all ruminant species in which it has been used. Here, it was shown that swapping the GP and the first 76 nt of the AGP between virulent and avirulent strains affected disease progression. In particular, it was shown that flanking the virulent strain with the vaccine GP and AGP sequences, while not appreciably affecting virus growth in vitro, led to attenuation in vivo. The reverse was not true, since the KO promoters did not alter the vaccine's attenuated nature. The GP/AGP therefore play a role in attenuation, but are not the only determinants of attenuation in this vaccine.

The Plowright vaccine strain of Rinderpest virus has attenuating mutations in most genes.

The currently used vaccine strain of Rinderpest virus was derived by serial passage of the highly virulent Kabete 'O' strain (KO). A full-length cDNA copy of the KO strain was made from which a virus identical in pathogenicity to the wild-type virus was rescued. A series of chimeric viruses was prepared in which the coding sequences for the N, P, F, H or L proteins were replaced with the corresponding sequences from the vaccine strain. The KO-based virus with the vaccine strain H gene and that with the carboxy-terminal half of the L gene replaced with the corresponding sequence from the vaccine strain retained all or almost all of the virulence of the original KO virus. Animals infected with the KO-based virus containing the vaccine strain N, P or F gene, or the amino-terminal half of the L gene, developed high and prolonged pyrexia and leukopenia, but with reduced or absent lesions and other clinical signs; although partially attenuated, none was nearly as attenuated as the vaccine strain itself. These data indicate that the high attenuation and stability of the current vaccine are due to the accumulation of a number of separate mutations, none of which is itself so sufficiently debilitating that there is strong selective pressure in favour of the revertant.

Use of participatory epidemiology in studies of the persistence of lineage 2 rinderpest virus in East Africa.

In 1994, rinderpest virus of African lineage 2 was detected in East Africa after an apparent absence of more than 30 years. In 1996, a disease search, based on participatory epidemiological techniques supplemented by serological and virological analyses, was undertaken in southern Somalia and north-eastern Kenya to collate past and current epidemiological information about rinderpest-compatible disease events, and to test the hypothesis that African lineage 2 rinderpest virus persists in populations of transhumant cattle in the Somali ethnic areas. The findings in Afmadu in Lower Juba led the search for rinderpest to the communities in the Bardera area and then on to the Kenya/Somalia border areas between Mandera and El Wak. The herders had a specific knowledge of the clinical signs of rinderpest and provided detailed and accurate descriptions of cases. They differentiated between classical acute rinderpest and a milder syndrome characterised by an ocular discharge and diarrhoea, few oral lesions, corneal opacity and occasional mortality. The studies provided evidence for the endemic occurrence of rinderpest back to at least 1981, with a periodicity of five years in the incidence of the disease. After a period of high mortality in 1992 to 1993, around Afmadu, herders reported a mild disease, with occasional increases in mortality, from other areas of Lower Juba and the Gedo Region. Reports by herders of a rinderpest-compatible disease in the El Wak area were pursued until active cases were located and rinderpest was confirmed.

Induction of apoptotic cellular death in lymphatic tissues of cattle experimentally infected with different strains of rinderpest virus.

The presence, type, and extent of cellular death in lymphatic tissues of cattle experimentally infected with rinderpest virus strains of different virulence was investigated morphologically. Cells with DNA strand breaks were identified in histological sections of palatine tonsil, spleen, and mesenteric and mandibular lymph nodes by the TUNEL (terminal desoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. In addition, representative samples of lymphatic tissues were examined by transmission electron microscopy. The results indicated that cellular disassembly in lymphatic tissues was caused by both apoptosis and oncosis. Cells with DNA strand breaks were observed in follicular and parafollicular areas of lymphatic tissues and their numbers were determined. A significant correlation was found between the number of TUNEL-positive cells and viral virulence. These results suggest that, in addition to oncosis, apoptotic cellular death in lymphatic tissues contributes substantially to the pathogenesis of rinderpest.

Cloning and sequence analysis of the matrix (M) protein gene of rinderpest virus and evidence for another bovine morbillivirus.

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.

Evidence for different lineages of rinderpest virus reflecting their geographic isolation.

Sequence analysis of part of the fusion protein gene from recent isolates of rinderpest virus revealed that distinct lineages of the virus exist which reflect the geographical location of their isolation in Africa and Asia. Current strains circulating in Kenya and Sudan were most similar, both in terms of nucleotide sequence and pathogenic nature, to viruses isolated in Egypt and in Nigeria in 1983/1984 and they were quite distinct from an East African isolate (RBT-1) from the 1960s. Two older isolates of the virus, the Japanese avianized/lapinized vaccine strain dating from the 1930s and the Old Kabete strain dating from 1911, each differed considerably from the other viruses. The sequence data were derived from the region where the precursor protein is cleaved to yield the biologically active F1/F2 heterodimer; all strains analysed had a highly basic connecting peptide which is required for efficient cleavage by endogenous host cell proteases. No correlation was found between amino acid changes at this site and the rinderpest virus pathogenicity unlike the association reported for Newcastle disease virus.

Use of the polymerase chain reaction in differentiating rinderpest field virus and vaccine virus in the same animals.

In 1991, a disease with clinical signs indicative of rinderpest was reported in yaks in the former Soviet Union, near the border with Mongolia. At the peak of the epizootic, mortality among affected yaks was 32-42% in adults and 65% in animals less than one year old. Pathological samples were examined independently at two institutes in Russia. Both institutes confirmed the presence of rinderpest using complement fixation, agar gel diffusion and immunoassays. Since vaccination had been initiated to control an outbreak of a similar disease several months earlier, the later cases were possibly due to the vaccine and field rinderpest may not have been present. However, the disease had occurred in non-vaccinated animals and these were then vaccinated against the disease. Tissue samples obtained from these animals, which were examined at the Pirbright Laboratory using gel diffusion assays and specific nucleic acid probes, were found to be positive for rinderpest antigen and nucleic acid. Ribonucleic acid derived from the post-mortem tissue samples was amplified using the polymerase chain reaction and rinderpest-specific primers. Sequence analysis of the amplified deoxyribonculeic acid from the samples revealed the presence of two distinct virus strains, one identical to the Plowright rinderpest tissue culture vaccine and the other related to field strains of rinderpest virus circulating in Asia and the Middle East.

Genetic comparison of ovine and bovine pestiviruses.

Viral RNA oligonucleotide fingerprinting was used to compare genetic relationship among pestiviruses originating from ovine or bovine host species. Ovine pestiviruses, including reference border disease virus and 2 border disease isolates originating from natural pestivirus infections of sheep, appeared to have a more distant genetic relationship among themselves than with certain bovine pestiviruses. A closer genetic relatedness was evident between border disease virus and 3 noncytopathic bovine pestiviruses, including Draper bovine viral diarrhea virus (BVDV), a BVDV isolate that originated from aborted bovine fetuses, and a virus that was isolated from the serum of a calf that had a chronic BVDV infection. Four noncytopathic bovine viruses, including Draper BVDV and 3 field isolates, were closely related. Reference Oregon C24V BVDV, a cytopathic virus, was closely related to only 1 of the 7 noncytopathic viruses in this study.

Use of the caprinised strain of rinderpest virus for potency testing an attenuated cell culture rinderpest vaccine.

The caprinised strain of rinderpest virus was inoculated into goats to produce a challenge stock. These goats were kept with control animals (goats, sheep, calves). In this trial the caprinised strain was shown to have a mild pathogenicity for goats and it spread to one of two contact goats but not from goats to other species. The caprinised strain was then tested on cattle where a febrile reaction was observed. The caprinised strain also did not spread between cattle. The cattle vaccinated with a freeze-dried vaccine produced from the attenuated Kabete RBKO strain on bovine kidney cells were then challenged with the caprinised strain with good results.

Is rinderpest virus the archevirus of the Morbillivirus genus?

Groups of 6-39 monoclonal antibodies identifying 3-18 distinct epitopes on the nucleoprotein (NP), polymerase (P), hemagglutinin (H; equivalent in canine distemper and rinderpest viruses), and fusion (F) components of measles and canine distemper viruses were characterized in immunofluorescence tests with fixed Vero cell cultures infected with measles, canine distemper and rinderpest viruses. The majority of NP-specific monoclonal antibodies reacted with all three viruses, but one-third of the antibodies only reacted with the homologous virus. A few antibodies detected epitopes uniquely shared between either measles and rinderpest viruses or canine distemper and rinderpest viruses. Of the P-specific antibodies, two-thirds only reacted with the homologous virus, one antibody detected an epitope shared between canine distemper and rinderpest viruses, and the rest reacted with all three viruses. Also, the majority of antibodies against the H component were type-specific, but four antibodies reacted both with measles and rinderpest viruses. In contrast, the F component was antigenically highly conserved. 17 of 21 antibodies against this component reacted with all three viruses; one antibody reacted only with measles and rinderpest virus F components, and three antibodies reacted only with the homologous virus. No monoclonal antibody of any specificity selectively reacted with only measles and canine distemper viruses. Furthermore, the measles virus H component appeared to be more closely related to the equivalent rinderpest virus component than to the canine distemper virus component. Thus, it is proposed that rinderpest virus is the archevirus of the morbillivirus group from which canine distemper virus was first to evolve and, more recently (perhaps about 5,000 years ago), measles virus.