Protein Of Canine Distemper Virus Research Articles

Humoral immune response in dogs with old dog encephalitis and chromic distemper meningo-encephalitis.

The humoral immune response in sera and cerebrospinal fluids (CSFs) of dogs with various forms of canine distemper virus (CDV)-induced encephalitis was assessed by immunoprecipitation of radiolabelled nucleocapsid, phosphoprotein, membrane (M), haemagglutinin and fusion proteins. Sera from vaccinated dogs and hyperimmune sera contained antibodies to all the above antigens. In two cases of old dog encephalitis the sera and CSFs showed a restricted response to the M protein of CDV, whilst in three other cases of old dog encephalitis, two cases of chronic distemper (meningo-) encephalitis and experimentally induced encephalitis the humoral immune response appeared to be directed primarily to the nucleocapsid, phosphoprotein and the M protein but not the haemagglutinin or fusion proteins. Precipitation of the M protein by most of the sera was observed only when the antigen had been prepared by in vitro translation.

F 1 Polypeptides of two canine distemper virus strains: Variation in the conserved N-terminal hydrophobic region

The fusion protein of canine distemper virus was isolated by immunoadsorption from two virus strains, the rapidly growing Onderstepoort strain (forming large plaques) and the Convac vaccine strain (forming microplaques). The F 1 subunits of the two fusion proteins were purified by preparative polyacrylamide gel electrophoresis. Direct amino acid sequence analysis revealed that 36-residue N-terminal regions of the proteins from the two strains are identical except at position 9, where Ala in the Convac strain is substituted by Val in the Onderstepoort strain. The two sequences show high homology with the previously determined N-terminal sequence of the F 1 polypeptide of measles virus, and moderate homology with corresponding sequences of five paramyxoviruses, emphasizing the occurrence of an extensive conservation of these structures.

Molecular cloning and sequence analysis of the human parainfluenza 3 virus gene encoding the matrix protein

The sequence of the matrix (M) protein gene and contiguous intergenic regions of the human parainfluenza 3 virus (PF3) was determined by molecular cloning. The encoded M protein contains 354 amino acids and has a predicted mol wt of 39,506. The M protein amino acid sequence was compared to the homologous proteins from other members of the Paramyxoviridae family. The PF3 protein shared 61 % homology with the Sendai virus protein and approximately 35% homology with measles and canine distemper virus proteins. Little homology was observed with respiratory syncytial virus. The M protein appears to be the most highly conserved among the Paramyxoviridae proteins.

Oligodendroglial pathology in canine distemper virus infection in vitro.

Dog brain cell cultures were infected with different canine distemper virus (CDV) strains to study the oligodendrocytes, which were characterized with eight different antibodies to cover the whole oligodendroglial population in the culture. A few weeks after infection all oligodendroglial cell types started to degenerate and disappeared from the culture. However, since no CDV protein could be demonstrated in the degenerating oligodendrocytes with extensive double-labelling studies, this lesion can not be explained as being a result of cytolytic infection. This conclusion was further supported in experiments with plaque-forming CDV, in which viral replication is restricted to the cytolytic areas only; oligodendrocytes also degenerated in virus-free areas between the plaques. The hypothesis of toxic factors released by other infected cell types in the culture leading to secondary damage of the oligodendrocyte could not be confirmed by transferring supernatants from infected to normal cultures. Whereas the presence of toxic factors can not be completely excluded, the possibility of an abortive infection of the oligodendrocytes with no or very limited viral protein synthesis is discussed.

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.

Nucleotide sequence of the entire protein coding region of canine distemper virus polymerase-associated (P) protein mRNA

The entire coding region of the polymerase-associated (P) protein gene of canine distemper virus has been sequenced. A single cDNA clone which represents 98% of the mRNA encoding this protein was used to determine the nucleotide sequence. The sequence predicts a major protein of 507 amino acids and a molecular weight of 54936. There is also a second, overlapping, open reading frame with a start signal 21 bases downstream of the first AUG which could code for a protein of 174 amino acids with a predicted molecular weight of 20 292. This arrangement of the genome for the P protein of canine distemper virus is exactly analogous to that published recently for the P gene of measles virus (Bellini W.J. et al., 1985, J. Virol. 53, 908–919). When the sequences are aligned at the first AUG, considerable homology is seen at both the nucleotide and protein sequence level.

Preparation and characterization of monoclonal antibodies directed against four structural components of canine distemper virus.

Mouse hybridomas producing antibodies against structural proteins of canine distemper virus (CDV) were produced by fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice immunized with purified preparations of Vero cell-grown CDV. Ascites fluids collected after intraperitoneal inoculation with 149 CDV antibody-producing hybridoma cell lines were characterized by different serological tests. By immune precipitation tests with [35S]methionine-labelled extracellular virions and intracellular virus polypeptides, 57 clones were found to produce antibodies against the nucleocapsid protein (NP), 22 against the polymerase (P) protein, 10 against the fusion (F) protein and nine against the large uncleaved glycoprotein (named H in analogy with measles virus). By competitive binding enzyme-linked immunosorbent assay (ELISA) tests with monoclonal antibodies against each structural component, a minimum of 18, six, three and seven separate antigenic determinants were identified on the NP, P, F and H proteins, respectively. The reactions of clones directed against F and H surface components of the virus were tested for their ability to inhibit the infectivity of both CDV and measles virus in the absence and presence of anti-gamma-globulin. In addition, the inhibitory activity of the clones on measles haemagglutinating (HA) and haemolysis (HL) activity were examined. Monoclonal antibodies against six of the seven antigenic determinants of the H protein could neutralize the infectivity of the virus. After addition of anti-gamma-globulin to the test, increases of titres varying from twofold to several hundredfold were observed with the different clones. None of all the clones against H could block measles virus infectivity, HA or HL activity. The 10 clones directed against the F protein could not neutralize the infectivity of CDV even in the presence of anti-gamma-globulin. Further, the antibodies could not inhibit measles HA and HL activity in the absence of anti-gamma-globulin. However, after the addition of anti-gamma-globulin, antibodies against two of the three sites were found to block measles virus HL activity. The reactions of all clones were tested in immune fluorescence, ELISA and immune precipitation tests with three strains of CDV. Each strain had a few unique antigenic sites. Variation was found in four, one and three different antigenic sites of the NP, P and H proteins, respectively.

Cloning and characterization of DNA complementary to the canine distemper virus mRNA encoding matrix, phosphoprotein, and nucleocapsid protein.

Double-stranded cDNA synthesized from total polyadenylate-containing mRNA, extracted from monkey kidney cells infected with canine distemper virus (CDV), has been cloned into the PstI site of Escherichia coli plasmid pBR322. Clones containing canine distemper virus DNA were identified by hybridization to a canine distemper virus-specific, 32P-labeled cDNA. Four specific clones containing different classes of sequences have been identified. The cloned plasmids contain inserts of 800 (clone 44-80), 960 (clone 74-16), 1,700 (clone 364), and 950 (clone 40-9) base pairs. The sizes of the mRNA species complementary to these inserts are 1,500, 1,850, 1,850 and 2,500 nucleotides, respectively, as determined by the Northern technique. Three of the cloned DNA fragments were further identified as the reverse transcripts of the mRNA coding for the matrix, phosphoprotein, and nucleocapsid protein of CDV.

ANALYSIS OF STRUCTURAL PROTEINS OF MEASLES, CANINE DISTEMPER, AND RINDERPEST VIRUSES

Serological relationships among measles virus (MV), canine distemper virus (CDV), and rinderpest virus (RV), which constitute morbillivirus subgroup of paramyxoviridae, were investigated by immunoprecipitation and SDS-polyacrylamide gel electrophoresis for their major structural proteins, i.e., hemagglutinin (H), nucleocapsid (NC), fusion (F), and matrix (M) proteins. The molecular weights of the four structural proteins of MV and CDV were confirmed to correspond to those previously reported by several investigators. Structural proteins of RV were analyzed for the first time in the present study and found to have molecular weights of 74,000, 62,000, 44,000, and 40,000 for H, HC, F, and M proteins, respectively. By labeling with glucosamine, the presence of carbohydrate moiety was found in H protein for all the three viruses and in F protein of CDV. The serums from the convalescent animals infected with respective virus disclosed one-way cross pattern depending on the combinations of virus and antiserums, but failed to show the reciprocal cross reactivity. On the other hand, hyperimmune serums to respective virus showed the reciprocal cross-reactivity with the four structural proteins indicating that each of the major structural proteins possesses the antigen common to all three morbilliviruses.

SEARCH FOR CANINE-DISTEMPER-VIRUS ANTIBODIES IN MULTIPLE SCLEROSIS: A Detailed Virological Evaluation

Sera and cerebrospinal fluid samples from multiple-sclerosis (MS) patients, matched controls, and subacute sclerosing panencephalitis (SSPE) patients were analysed for the presence of antibodies specific for measles and canine distemper virus (CDV). Virus-specific antibodies were identified by hæmagglutination inhibition, neutralisation, and immune precipitation in combination with polyacrylamide-gel electrophoresis. SSPE and MS patients had a higher antibody activity towards measles virus than controls. Moreover, human sera partly neutralised CDV; this was attributed to antibody activity against CDV proteins, antigenically related to those of measles virus. However, CDV-specific antibodies were not found in the samples tested. These findings indicate that MS can develop without a preceding CDV infection.