Strains Of Rinderpest Virus Research Articles

Antigenic and Functional Characterization Of Rinderpest Virus Envelope Proteins Using Monoclonal Antibodies

A total of 24 monoclonal antibodies (MAbs) against the haemagglutinin (H) and the fusion protein (F) of rinderpest virus (RPV) were used to characterize their antigenic structure and biological properties, and to analyse natural variation in the envelope proteins of morbilliviruses. The anti-H and anti-F MAbs defined seven and three distinct antigenic sites, respectively. The MAbs to six sites on H were able to neutralize the infectivity of RPV. The addition of guinea-pig complement or anti-mouse immunoglobulin increased the virus-neutralizing antibody titre of most of the anti-H MAbs, including those lacking neutralizing activity. One of the antigenic sites on H was conserved among morbilliviruses and the MAbs to this site had haemagglutination inhibition activity against measles virus (MV). The remaining sites were specific for RPV and varied antigenically between strains of RPV. The anti-F MAbs lacked neutralizing activity, but two of the five MAbs did show activity in the presence of complement or anti-mouse immunoglobulin. On the whole, the antigenic sites on F were conserved in some strains of MV, but not in canine distemper virus. All of the sites on the surface proteins were sensitive to SDS and, although those on F were not affected by 2-mercaptoethanol, five of the seven sites on H were destroyed by it. These results suggest that the epitopes on the envelope proteins are conformation-dependent.

B95a, a marmoset lymphoblastoid cell line, as a sensitive host for rinderpest virus

We reported earlier that B95a, an Epstein-Barr virus-transformed marmoset B lymphoblastoid cell line, is more susceptible to infection with measles virus than other cells. The cell line also was found to be susceptible to infection with the lapinized Nakamura III (L) strain of rinderpest virus and various strains derived from it. The B95a cell line was therefore the only host cell system available for the propagation and quantification of the L strain. In contrast to the adaptation of the L strain to Vero cells which results in a diminution of virulence in rabbits, the propagation of the virus in B95a cells preserved the virulence and some other properties in rabbits. Furthermore, when Vero cell-adapted variants of the L strain with diminished virulence were serially passaged in B95a cells, virulence in rabbits was gradually regained.

Molecular cloning of the rinderpest virus matrix gene: Comparative sequence analysis with other paramyxoviruses

The nucleotide sequence of the gene encoding the matrix or membrane (M) protein of the virulent (Kabete-O) strain of rinderpest virus (RPV) has been determined. The M gene is 1457 nucleotides long with a single, large open reading frame. The derived polypeptide has 335 amino acids, corresponding to a calculated molecular weight of 38,289 and contains both small hydrophobic regions and many basic residues. The predicted amino acid sequence was compared to the M proteins of paramyxoviruses. Sequence comparison and hydropathy profiles among the morbilliviruses revealed that the M protein of RPV exhibits features similar to those of the M protein of MV and CDV. There is 78.2% homology at the amino acid level between the M protein of RPV and MV, and 77.6% between RPV and CDV. This indicates that a high degree of homology exists among the members of the genus Morbillivirus. In contrast, there is only 37.3 and 18% homology between RPV and bovine parainfluenza type 3 (BPV3), and RPV and Newcastle disease virus (NDV) M proteins, respectively. Thus the M proteins of the morbilliviruses are highly conserved whereas the M proteins of the genus Paramyxovirus show more divergence.

Characterization of Monoclonal Antibodies against Four Structural Proteins of Rinderpest Virus

Twenty-four monoclonal antibodies (MAbs) against rinderpest virus (RPV) were established and characterized by several serological tests. Of the 24 MAbs. 10 recognized the nucleoprotein (NP), six the phosphoprotein (P), four the haemagglutinin (H), and two the fusion (F) protein as determined by radioimmunoprecipitation assay. The specificities of the remaining two MAbs could not be determined. From a competitive binding assay using MAbs against each structural protein, at least five, four and two separate antigenic sites were identified on the NP, P and H proteins, respectively. MAbs against the H protein neutralized the infectivity of the virus, but those against the F protein were only neutralizing in the presence of guinea-pig complement. The reactivities of each of the MAbs for other strains of morbillivirus were tested using an indirect immunofluorescent antibody assay. The MAbs against four out five antigenic sites on the NP showed cross-reactivity amongst all the strains of morbillivirus tested whereas the fifth antibody reacted only with RPV. Of the antibodies specific for the P protein, the antibody against one site was cross-reactive with all the strains of RPV, measles virus (MV) and canine distemper virus (CDV), the antibody against another site was reactive with RPV and MV but not with CDV, and the antibodies against the other two sites were specific for RPV.

Immunization with a vaccinia recombinant expressing the F protein protects Rabbits from challenge with a lethal dose of rinderpest virus

A cDNA clone containing the complete coding sequence of the rinderpest fusion protein (F) gene was inserted into the thymidine kinase gene of vaccinia virus (WR strain) under the control of the 7.5K early/late vaccinia virus promoter. All forms of the F protein, i.e., the glycosylated F o precursor, the unglycosylated F 1 protein, and the glycosylated F 2 protein, were detected in cells infected with the recombinant virus. Vaccination of rabbits with the recombinant virus induced antibodies which reacted in an ELISA system specific for rinderpest. The rabbit sera contained neutralizing antibodies against rinderpest virus and precipitated the F protein from lysates of rinderpest infected cells. Rabbits vaccinated with the recombinant rinderpest F gene vaccinia virus were protected from a lethal challenge with the lapinized Nakamura 3 strain of rinderpest virus. Variations in the severity of clinical symptoms correlated with the level of anti-F protein antibodies produced.

An epidemiological model of rinderpest. II. Simulations of the behaviour of rinderpest virus in populations.

Fixed parameters for different hypothetical strains of rinderpest virus (RV) and different susceptible populations are described together with details of their derivation. Simulations were then carried out in a computer model to determine the effects that varying these parameters would have on the behaviour of RV in the different populations. The results indicated that virulent strains of RV are more likely to behave in epidemic fashion whereas milder strains tend towards persistence and the establishment of endemicity. High herd immunity levels prevent virus transmission and low herd immunity levels encourage epidemic transmission. Intermediate levels of immunity assist the establishment of endemicity. The virus is able to persist in large populations for longer than in small populations. Different vaccination strategies were also investigated. In areas where vaccination is inefficient annual vaccination of all stock may be the best policy for inducing high levels of herd immunity. In endemic areas and in herds recovering from epidemics the prevalence of clinically affected animals may be very low. In these situations veterinary officers are more likely to find clinical cases by examining cattle for mouth lesions rather than by checking for diarrhoea or high mortalities.

Cloning of the fusion gene of rinderpest virus: Comparative sequence analysis with other morbilliviruses

We have cloned the cDNA of the fusion (F) gene of the virulent (Kabete O) strain of rinderpest virus and provided a comparative analysis of its sequence with that of the F genes of measles and distemper viruses. The gene has an open reading frame of 2241 nucleotides with two potential initiation codons in-frame. Use of the first ATG would produce a polypeptide 747 amino acids long with a calculated molecular weight of 81,068. However, we suggest that the second ATG is used to generate the F 0 protein, which is 546 amino acids long with a calculated molecular weight of 58,754. During maturation, the cleavage of F 0 gives rise to the functional F 1 and F 2 polypeptides. The F 1 polypeptide is 438 amino acids long and has a calculated molecular weight of 46,791, with a single (potential) glycosylation site in its cytoplasmic domain. The F 2 polypeptide, probably 89 amino acids long after the signal sequence is cleaved, is estimated to be 9,800 Da and has three potential glycosylation sites. There is a divergence of 18.7% in amino acid sequences between rinderpest and measles virus F 0 polypeptides; between distemper and rinderpest viruses the divergence is 31.8%. No significant homology in nucleotide sequences of rinderpest DNA to measles or distemper DNA was found in the 5′ and 3′ untranslated regions.

Characterization of antinuclear antibodies induced in rabbits by rinderpest virus infection.

Rabbits infected with the L strain of rinderpest virus (RV) produced high titres of antinuclear antibodies (ANA) which reached a maximum two weeks after inoculation but rapidly disappeared by 6-8 weeks. These ANAs reacted with HeLa cells by indirect immunofluorescence test resulting in a homogeneous nuclear fluorescence. In order to investigate the target antigens of ANAs, the effects on the nuclear fluorescence pattern of pretreating HeLa cells were examined: DNase 1 treatment resulted in a decrease in the fluorescence whereas no changes were evident after RNase A treatment. Some group of sera showed decreased fluorescence in the cells from which histones were acid extracted, but other groups did not change in fluorescence. Sera which had failed to react with acid extracted cells gave positive fluorescence following histone reconstitution. The results indicate that DNA and nucleohistone are the major target antigens for ANAs. In addition, antibodies against nucleoli and extractable nuclear antigens were induced in some rabbits.

Antigenic variation between three strains of rinderspert virus detected by kinetic neutralisation and competition ELISA using early rabbit antisera

Strains of rinderpest virus (RV) with high and low virulence were compared with cell culture-attenuated RV for antigenic differences. Sera collected from rabbits 1 week after inoculation with live virus distinguished between homologous and heterologous strains kinetic neutralisation and competition ELISA.

The Antigenic Relationship between Measles, Canine Distemper and Rinderpest Viruses Studied with Monoclonal Antibodies

Monoclonal antibodies (MAbs) were used to delineate the antigenic relationship between the three morbillivirus types: measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). Panels of six to 31 MAbs against the haemagglutinin (H), fusion (F), nucleocapsid protein (NP), phosphoprotein (P) and matrix (M) proteins of MV and the H, F, NP and P proteins of CDV were employed. Nine strains of MV, three strains of CDV and four strains of RPV were examined by radioimmunoprecipitation assay and immune fluorescence for reactivity with the heterologous MAbs. Overall, the NP and in particular the F proteins of the morbilliviruses showed a high degree of epitopic homology; the P and M proteins showed a partial epitopic homology, with the greatest variation between the M proteins of CDV and MV; the H proteins showed a low degree of epitopic homology and then only between MV and RPV. These data indicate that the major cross-protecting antigen in heterotypic vaccination amongst morbilliviruses is the F antigen. The epitopic relationships found between morbilliviruses as identified by the MAbs were classified as follows. (i) Group-specific epitopes were present on all strains of the three morbillivirus types. (ii) Group-cross-reactive epitopes were present on only some of the strains from each morbillivirus type (these epitopes identified the presence of intratypic strain variation in all proteins of all three virus types). (iii) Type-specific epitopes, i.e. MV unique or CDV unique, were found only on the homologous morbillivirus type. (iv) CDV-RPV intertypic and MV-RPV intertypic epitopes were, respectively, epitopes shared by CDV and RPV but not with any MV strain, and epitopes shared by MV and RPV but not with any CDV strain. These cross-reactivities and type-specific reactions were obtained with the internal viral proteins (M, P and NP). The epitopes of the F proteins were mainly group-specific and no CDV-RPV or MV-RPV intertypic epitopes were found. The epitopes of the H protein were either type-specific or MV-RPV intertypic. These data support the proposed evolutionary relationship between the morbilliviruses.

Studies on the responses of calves to an attenuated rinderpest vaccine virus.

Multiplication of the virus in the calves and the immunological response of the animals to the virus were investigated during the 15 days of the experi­ ment. The virus was isolated neither from the buffy coat cells of live animals nor from tissue samples of those slaughtered at various intervals from 4 to 15 days after inoculation. Neutralizing antibody to the virus was demonstrated in serum samples from 8 days after inoculation, but no precipitating antigen was found in the tissue samples collected from slaughtered calves. Infection of calves with virulent strains of rinderpest virus usually results in production of precipitating antigen, detectable in properly harvested tissues from infected animals by the gel immunodiffusion test. The results of the pre­ sent experiment on the failure of calves to produce any demonstrable precipiti­ nogen to the attenuated rinderpest virus, show that the presence of rinderpestprecipitating antigen in any tissue samples of a rinderpest suspected case would be attributable to infection of the subject by a virulent virus, and could be considered as a further confirmation of the value of application of the gel diffusion test in the diagnosis of rinderpest.

The Differential Growth of Virulent and Avirulent Strains of Rinderpest Virus in Bovine Lymphocytes and Macrophages

Rinderpest virus (RV) grew readily in cultures of purified bovine peripheral blood lymphocytes and udder macrophages. The growth of three strains of RV was compared and there appeared to be a relationship between increasing virulence and increased ability to infect lymphocytes and macrophages. The proportion of infected cells as determined by the presence of virus antigens was a better indicator of affinity between a strain and cell type than production of new infectious virus. RV grew better in populations of predominantly T lymphocytes than in T-depleted cultures. Although RV could infect 100% of cells in macrophage monolayers, it did not appear to infect more than about 30% of cells in lymphocyte cultures. Virulent RV grew more readily in bovine than caprine or ovine lymphocytes, whereas virulent peste des petits ruminants virus (PPRV) grew better in lymphocytes from sheep and goats. There was no marked difference in the growth of either virus in lymphocytes from uninfected or recently convalescent animals.

The shedding of a virulent Kabete O strain of rinderpest virus by cattle.

A Muguga substrain of the virulent Kabete O strain of rinderpest virus was demonstrated in the ocular, nasal, oral and rectal swabs collected from infected cattle. Ocular shedding was detected at the onset of viraemia and before the onset of clinical signs whilst virus shedding in nasal, oral and rectal discharges appeared at the same time as lesions. It is suggested that virus isolation from ocular and nasal swabs should be considered in the diagnosis of rinderpest in addition to the other methods currently employed, as virus was isolated from swabs collected from dead animals.

Neutralising antibodies to rinderpest virus in wild animal sera collected in Kenya between 1970 and 1981

Four hundred and twenty five sera were collected from 18 species of wild mammals in Kenya between 1970 and 1981. Neutralising activity to rinderpest virus (RV) was detected in 35 samples from 13 species. This activity appeared to be specific antibody to RV since it did not neutralise the virus of peste des petits ruminants. It was associated with the serum globulin fraction and it blocked the staining of a rabbit immunofluorescent antibody conjugate to RV. Positive sera were not restricted to any particular area of Kenya. It is possible that strains of RV with low pathogenicity and low transmissibility are still present in wildlife in East Africa, a fact which must be considered when studying the epidemiology and control of rinderpest.

Detection of rinderpest virus antigens in vitro and in vivo by direct immunofluorescence

Cell-culture attenuated and virulent strains of rinderpest virus (RV) were inoculated on to bovine kidney cell cultures. A direct immunofluorescent antibody test detected RV antigens in cell cultures within one to three days after inoculation whereas RV cytopathic effects usually took three to nine days to develop. Cells containing RV antigens were also detected in impression smears and frozen sections of tissues collected from RV infected animals at post mortem examination, and in smears of lymph node biopsies taken from cattle with clinical rinderpest. These techniques may offer additional methods for rapid diagnosis of rinderpest.

A plaque assay system for rinderpest virus and its use in characterising virus adsorption

A continuous line of calf kidney cells was found suitable for the production of plaques by five strains of rinderpest virus known to differ in their virulence for cattle. No differences were seen in the morphology of the plaques produced by these strains. Plaques were first seen after 6 days incubation, and if neutral red was incorporated in the overlay appeared as “red” centres composed of a small cluster of cells taking up this stain more strongly than the healthy background cells; thereafter plaques enlarged very slowly and were only 1.5–2.0 mm in diameter after 14 days. Plaque counts did not increase beyond the 6th day of incubation and were unaffected by either agarose concentrations of 0.4 to 1.5% or overlay pH values between 6.7 and 7.85. It was shown that neutral red included in the initial overlay did not affect the sensitivity of virus titrations.

Development of a cell culture system susceptible to measle, canine distemper, and rinderpest viruses.

Three strains of chick embryo adapted canine distemper virus (Lederle, Wisconsin, and Onderstepoort strains) and chick embryo adapted LA strain of rinderpest virus were easily adapted to an established line of African green monkey kidney cells (Vero cells), which has been routinely employed for the titration of measles virus. By using these Vero cell adapted strains of canine distemper and rinderpest viruses, techniques of infectivity titration and virus neutralization in Vero cells were established. Comparative studies of cytopathology and growth characteristics of canine distemper, rinderpest, and measles viruses indicated that the behavior of the three viruses in Vero cells is almost the same. The Vero cell system was suggested as a suitable host for the comparative study of the serologic and biologic relationships among measles, canine distemper, and rinderpest viruses.

THE GROWTH OF VIRULENT AND ATTENUATED STRAINS OF RINDERPEST VIRUS IN PRIMARY CALF KIDNEY CELLS.

THE GROWTH OF VIRULENT AND ATTENUATED STRAINS OF RINDERPEST VIRUS IN PRIMARY CALF KIDNEY CELLS.

Studies in tissue culture on the pH-stability of rinderpest virus

The pH-stability of three strains of rinderpest virus, propagated in tissue cultures, was investigated at 4°C. in veronal-acetate buffers (Michaelis) of pH 3·0–10·7.An attenuated laboratory strain, in its 95th culture passage (RBOK), showed maximal stability in the pH range 7·2–8·0, the half-life being about 3·7 days. It was relatively stable from pH 4·0 to 10·2, the half-life at the extremes of this range being over 2 hr. At pH 3·0 the infectivity declined very rapidly, the half-life period being 24·0 sec.One virulent strain (RGK/1) showed a significantly lower resistance than strain RBOK at pH 3/0, probably also at pH 5·0. The inactivation rates for these two strains were, however, not greatly different at pH 10·7. The other virulent strain (RBT/1) was even less stable than RGK/1 at pH 4·0 or 5·0 but of a comparable stability at pH 9·0 and 10·2.These findings are discussed in comparison with published data on the related viruses of measles and canine distemper. The importance of strain differences in studies of this kind is stressed.We are grateful to Mr C. S. Rampton, A.I.M.L.T., and Mr L. W. Rowe, F.I.M.L.T., for help in the preparation of the diagrams. This paper is published with the permission of Mr H. R. Binns, C.M.G., O.B.E., Director, E.A.V.R.O.

Studies with rinderpest virus in tissue culture. III. The stability of cultured virus and its use in virus neutralization tests.

The stability of cultured rinderpest virus, in maintenance medium containing 5% normal ox serum, was studied at 4°, 37°, and 56° C. The half-life at these temperatures was calculated and the results compared with figures available for other strains of rinderpest virus in cattle tissues and for measles virus in tissue culture fluids.