Sequence analysis and editing of the phosphoprotein (P) gene of rinderpest virus.

We have cloned the cDNA of the phosphoprotein (P) gene of the virulent (Kabete "O") strain of rinderpest virus and provided a comparative analysis of its sequence with that of the P genes of measles, canine distemper, and phocid distemper viruses. The gene encodes two overlapping open reading frames of 1521 and 531 nucleotides. Use of the first ATG would produce a P polypeptide of 507 amino acids with a calculated molecular weight of 54,344. The second ATG would produce a C polypeptide of 177 residues with a predicted molecular weight of 19,927. In addition, the insertion of a G residue at position 740 generates an alternative mRNA potentially encoding the V polypeptide of rinderpest virus. The homology comparisons in P amino acid sequences between rinderpest and measles, between rinderpest and canine distemper, and between rinderpest and phocid distemper viruses are 60, 44, and 46%, respectively. A four-way comparison shows an identity of 34%. Similar homology comparisons with the C amino acid sequence between rinderpest and measles, rinderpest and canine distemper, and rinderpest and phocid distemper viruses are 56, 42, and 40%, respectively. A homology of 31% is found in a four-way comparison for the C polypeptide. From the point of the insertion of the G residue, there is a homology of 78% between the V polypeptides of rinderpest and measle viruses.     

Comparison of messenger RNAs induced in cells infected with each member of the morbillivirus group

Virus-specific mRNAs radiolabelled with [32P]orthophosphate in the presence of actinomycin D were extracted from the cytoplasm of Vero cells infected with each of the known morbilliviruses: measles virus, canine distemper virus, rinderpest virus, and peste des petits ruminants virus. When analysed on denaturing agarose-formaldehyde gels the major RNA species from all viruses in the group were identical, except for canine distemper virus where one of the virus-specific mRNAs (mRNA 5), which probably codes for the virus haemagglutinin (S.E.H. Russell, D. K. Clarke, E. M. Hoey, B. K. Rima, S. J. Martin, J. Gen. Virol. 66, 433-441 (1985], was significantly smaller than the corresponding mRNA induced by the other viruses. Plasmid DNA containing a virus-specific insert, representing greater than 98% of the gene derived from the P-protein mRNA of canine distemper virus, showed significant cross-hybridisation with all the other members of the morbillivirus group.     

Cross-reactions of multiple sclerosis IgM with measles,rinderpest and canine distemper

Sera from six patients with multiple sclerosis possessing IgM antibody to the membranes of measles virus-infected cells have been tested for reactivity to the membranes of cell infected with rinderpest and canine distemper viruses. The sera of five patients with acute measles infection have been similarily examined. Cross-reactivity between measles, rinderpest and canine distemper IgM has been shown in the sera of acute measles infection and multiple sclerosis and in both instances the titres with measles infected cells were higher. These findings suggest that the antigenic stimulus in multiple sclerosis patients is measles virus or one of its components rather than the related rinderpest or canine distemper viruses.     

Canine distemper virus in primary and continuous cell lines of human and monkey origin

Summary Canine distemper virus produced characteristic cytopathic effects in primary and continuous cell cultures of human and monkey origin. The importance of selection of variants for successful adaptation to a given cell type was indicated by the marked differences in growth and CPE demonstrated by CDV lines of varying background. The Onderstepoort strain which had previously been passaged in ferret kidney cell cultures readily induced lesions in the AV3 human amnion cell line and virus yields of 10⁵ to 10⁶ TCD₅₀ per ml. were obtained. On serial passage in AV3 cells, a rapid CPE variant of this strain was obtained which induced lesions within 24 hrs. of inoculation. A chick embryo cell culture passaged line of the Onderstepoort strain also induced cytopathic effects in AV3 cells. However, the lesions developed slower and were not as extensive as those obtained with the ferret kidney propagated virus. The following additional cell types were also sensitive to canine distemper virus: primary human amnion, human fibroblasts, HeLa cells, primary rhesus and tamarin kidney cells, and continuous cell lines derived from rhesus and green monkeys. The Rockborn and 4856 strains of canine distemper also produced cytopathic effects in one or more of the above cell types. The lesions induced by canine distemper virus were compared to those produced by measles virus in a number of the above cell types. In all cases, similar, if not identical cytopathic effects were observed in a given cell type. The two viruses produced a similar type of cytopathic effect in human fibroblast cells which differed from that induced in epithelial cells.This investigation was supported in part by Public Health Service research grants (AI-03873 and AI-02396) from the National Institute of Allergy and Infectious Diseases.This investigation was supported in part by a Public Health Service research career program award (AI-K6-1136) from the National Institute of Allergy and Infectious Diseases.     

Genesis and maintenance of a persistent infection by canine distemper virus.

Vero cells were persistently infected with canine distemper virus by continuous undiluted passage of virus harvests. The cells were refractory to superinfection by both measles virus and canine distemper virus. These persistently infected cells produced and released into the medium a labile component which had a potent and selective inhibitory effect on the replication of canine distemper and measles virus. The inhibitory agent was not inactivated by u.v.-irradiation or sedimented by ultracentrifugation. Antisera against canine distemper virus or SSPE sera were able to block this inhibitory effect. We propose that these persistently infected cells produce an excess of a virus-induced regulatory protein.     

Markers for measles virus

Summary A tissue culture-adapted pantropic strain, two attenuated live vaccine strains and three neurotropic strains of measles virus were compared as to their growth potential, plaque formation, and interferon production in cell cultures, and pathogenicity for suckling hamsters.The properties characteristic of neurotropic measles strains included an enhanced capacity to form syncytia, a reduced, more cell-restricted proliferation cycle, and the development of large fast-growing plaques in Vero cell cultures under agarose overlay. These properties were pronounced with the hamsteradapted HNT and the Vero cell passaged Schwarz vaccine strains, both pathogenic when inoculated intracerebrally into suckling hamsters.Two measles viruses isolated from human brain with subacute sclerosing panencephalitis were not neurotropic for hamsters. Only one of the latter strains retained some of the properties characteristic for neutrotropism.Interferon induction in BS-C-1 cells was enhanced with both the attenuated and the neurotropic virus strains. The most pronounced marker which distinguished the pantropic and neurotropic strains from the vaccine strains was the inability of the former to grow in chick embryo fibroblast cultures.Recipient of a Public Health Service International Postdoctoral Research Fellowship No. I 50 5 TWO 1369-01.     

Radioimmunoassay of measles virus hemagglutinin protein G

Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 μg of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose—response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production.Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1–4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells.     

Paramyxoviruses of the morbilli group in the wild hedgehog Erinaceus europeus.

Dead and sick hedgehogs (Erinaceus europeus) were examined, together with apparently healthy individuals, and paramyxo virus of the morbilli group was isolated. One animal''s symptoms were similar to those caused by canine distemper and the virus isolate from faecal suspensions from this animal were antigenically related in various degrees to measles, canine distemper, rinderpest and PPRV viruses. Isolates from normal hedgehogs were found to belong to the same group. The variability and host specificity of members of paramyxo morbilli group virus are discussed and the role of natural infections of wild communities is considered in relation to disease in domestic animals and man.     

Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus

Peste des petits ruminants virus (PPRV) causes an acute febrile illness in small ruminant species, mostly sheep and goats. PPRV is a member of the Morbillivirus genus which includes measles, rinderpest (cattle plague), canine distemper, phocine distemper and the morbilliviruses found in whales, porpoises and dolphins. Full length genome sequences for these morbilliviruses are available and reverse genetic rescue systems have been developed for the viruses of terrestrial mammals, with the exception of PPRV. This paper presents the first published full length genome sequence for PPRV. The genome was found to be consistent with the rule-of-six and open reading frames (ORFs) were identified that encoded the eight proteins characteristic of morbilliviruses. At the nucleotide (nt) level, the full length genome of PPRV was most similar to that of rinderpest, the other ruminant morbillivirus. However, at the protein level five of the six structural proteins and the V protein showed a greater similarity to the dolphin morbillivirus (DMV) while only the C and L proteins showed a high relationship to rinderpest.     

T cell cross-reactivity among viruses of the paramyxoviridae

Blastogenesis and delayed-type hypersensitivity assays were used to examine mouse T cell responses to five viruses representing the three genera of the Paramyxoviridae. Cross-reactive T cell responses were observed in a lympho-proliferative assay for measles, mumps, respiratory syncytial, canine distemper and parainfluenza type 3 virus. Confirmation of T cell cross-reactivity among measles, mumps and respiratory syncytial virus was obtained with a delayed-type hypersensitivity test. These results show that T cell cross-reactivity is common for Paramyxoviridae viruses, even though these viruses show virtually no inter-genus antibody cross-reactivity. The cross-reactivity among respiratory syncytial, measles and mumps virus at the T cell level may have implications for usage of the attenuated measles/mumps/rubella (MMR) vaccine. Respiratory syncytial virus is contacted by many children before they receive the MMR vaccine and T cells induced by respiratory syncytial virus may influence subsequent development of immunity to measles and/or mumps virus.     

Growth profiles of recent canine distemper isolates on Vero cells expressing canine signalling lymphocyte activation molecule (SLAM)

Fresh samples of lymph node, lung and cerebrum taken post mortem from dogs no. 1, 2 and 3 yielded canine distemper virus (CDV) strains 007 Lm, 009 L and 011 C, respectively. These were titrated on Vero cells stably expressing canine signalling lymphocyte activation molecule (SLAM; Vero-DST cells). Growth curves of the three strains were produced by titration of the released virus and cell-associated virus at various timepoints. All three isolates, especially 007 Lm, grew well on Vero-DST cells. The titres of cell-associated virus of two strains (009 L and 011 C) were clearly lower than those of virus released into the culture supernate. The results indicate that Vero-DST cells are not only useful for primary isolation but also efficient for titrating virus from fresh tissues and for the study of growth profiles of recent CDV isolates.     

Preparation and characterization of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus

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.     

Changes in the Receptorbinding Haemagglutinin Protein of Wild-type Morbilliviruses are not Required for Adaptation to Vero Cells

We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U–C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.     

Analysis of the polypeptides synthesized in rinderpest virus-infected cells

We have identified, by [35S]methionine labeling, eight major induced proteins and a number of minor proteins in rinderpest virus-infected bovine kidney cells. The polypeptides ranged in molecular weight from 212 to 21.5 kDa. The majority of these polypeptides are virus specific, as demonstrated by immunoprecipitation with rabbit hyperimmune serum against rinderpest. Infected cells radiolabeled with glucosamine contained a 75-kDa polypeptide and a broad band migrating at 80 kDa, both identified as virus specific by immunoprecipitation. Phosphorylated virus-specific proteins of 65 kDa and a complex of polypeptides at 92.5 kDa were also identified. Monospecific and monoclonal antibodies against measles virus and canine distemper virus hemagglutinin, fusion protein, nucleocapsid protein, and phosphoproteins confirmed the identity of the corresponding rinderpest virus-specific polypeptides.     

Monkey CV1 cell line expressing the sheep-goat SLAM protein: a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens

Peste des petits ruminants (PPR) is an important economically transboundary disease of sheep and goats caused by a virus which belongs to the genus Morbillivirus. This genus, in the family Paramyxoviridae, also includes the measles virus (MV), canine distemper virus (CDV), rinderpest virus (RPV), and marine mammal viruses. One of the main features of these viruses is the severe transient lymphopaenia and immunosuppression they induce in their respective hosts, thereby favouring secondary bacterial and parasitic infections. This lymphopaenia is probably accounted for by the fact that lymphoid cells are the main targets of the morbilliviruses. In early 2000, it was demonstrated that a transmembrane glycoprotein of the immunoglobulin superfamily which is present on the surface of lymphoid cells, the signalling lymphocyte activation molecule (SLAM), is used as cellular receptor by MV, CDV and RPV. Wild-type strains of these viruses can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. The present study has demonstrated that monkey CV1 cells expressing goat SLAM are also highly efficient for isolating PPRV from pathological samples. This finding suggests that SLAM, as is in the case for MV, CDV and RPV, is also a receptor for PPRV.     

Genetic changes that affect the virulence of measles virus in a rhesus macaque model

To identify genetic changes that lead to the attenuation of measles virus (MV), a strain of MV that is pathogenic in rhesus macaques was adapted to grow in Vero cells, Vero/hSLAM cells and, to simulate the process used to derive live attenuated vaccines, in primary chicken embryo fibroblasts (CEF). Comparison of the complete genomic sequences of the pathogenic wild-type (Davis87-wt) and four cell culture-adapted strains derived from it showed complete conservation of sequence in the Vero/hSLAM-passaged virus. Viruses adapted to Vero cells and CEF had predicted amino acid changes in the nucleocapsid protein, phosphoprotein, V protein, C protein, matrix protein, and the cytoplasmic tail of the hemagglutinin protein. All four cell culture-adapted strains, including the Vero/hSLAM cell-passaged virus, were able to productively infect Vero cells, but the peak viral titers differed. The Vero cell-adapted strains were unable to replicate in Chinese Hamster Ovary cells expressing CD46, indicating that they had not adapted to use the CD46 receptor. The Vero/hSLAM cell-passaged virus retained pathogenicity in rhesus macaques as measured by the appearance of a skin rash while the Vero cell-adapted and CEF-adapted strains had lost the ability to cause a rash. There were no significant differences in viral titers in peripheral blood mononuclear cells among monkeys infected with any of the viral stocks tested. These results identify a limited number of genetic changes in the genome of MV that lead to attenuation in vivo.     

Antibody responses in the cerebrospinal fulid of cynomolgus monkeys after intracerebral inoculation with paramyxoviruses.

Cynomolgus monkeys with or without measles antibody were intracerebrally inoculated with measles or canine distemper viruses, and antibody responses in the cerebrospinal fluid (CSF) were investigated. In measles antibody-free monkeys, natural infection with wild measles virus or intracerebral inoculations with two attenuated measles vaccines evoked primary antibody responses to measles virus in the sera but not in the CSF. In measles-immune monkeys, intracerebral inoculation with the TYCSA strain of measles virus produced a significantly high titer of measles antibody in the CSF with a minimal rise in the serum antibody and resulted in a significant decrease in serum/CSF antibody ratios. Intracerebral inoculation of a neurotropic canine distemper virus, the Onderstepoort strain, into measles-immune monkeys caused production of both measles and distemper antibodies in the CSF. Inoculation of measles-immune monkeys intravenously with measles virus or intracerebrally with rubella virus, which has no antigenic relation to measles virus, failed to evoke a measles antibody response in the CSF. These results indicated that local production of measles antibody in the CSF was caused by a stimulus within the central nervous system of measles virus antigen or canine distemper virus antigen that partially cross-reacted with measles virus antigen as a secondary antibody response.     

Polypeptide composition of measles and canine distemper viruses

The proteins of measles and canine distemper viruses were studied by SDS-polyacrylamide gel electrophoresis. Six polypeptides were detected in each virus. These had estimated molecular weights of 79,000; 71,000; 61,000; 53,000; 46,000; and 37,000 daltons. A direct comparison of the number and estimated molecular weights of the polypeptides of Sendai virus made in this study and results previously reported by others for Sendai, SV5 and Newcastle disease viruses reveal marked similarities between measles and canine distemper viruses and the three previously well characterized members of the Paramyxovirus group.     

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 Fo protein, which is 546 amino acids long with a calculated molecular weight of 58,754. During maturation, the cleavage of F0 gives rise to the functional F1 and F2 polypeptides. The F1 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 F2 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 F0 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.