Identification of canine helper T-cell epitopes from the fusion protein of canine distemper virus

The fusion protein of canine distemper virus (CDV-F), a 662 amino-acid envelope protein, was used as the target molecule for identification of canine T helper (Th) epitopes. A library of 94 peptides, each 17 residues in length overlapping by 10 residues and covering the entire sequence of CDV-F, was screened using a lymphocyte proliferation assay with peripheral blood mononuclear cells (PBMC) obtained from dogs inoculated with canine distemper virus (CDV) vaccine. Initially we observed low and inconsistent proliferation of PBMC in response to these peptides, even when using cells obtained from dogs that had received multiple doses of CDV. Subsequently, the use of expanded cell populations derived by in vitro stimulation of canine PBMC with pools of peptides allowed the identification of a number of putative canine Th-epitopes within the protein sequence of CDV-F. There were two major clusters of Th-epitopes identified close to the cleavage site of the F0 fusion protein, while some others were scattered in both the F1 and F2 fragments of the protein. Some of these peptides, in particular peptide 35 (p35), were stimulatory in dogs of different breeds and ages. The identification of such promiscuous canine Th-epitopes encouraged us to assemble p35 in tandem with luteinising hormone releasing hormone (LHRH) a 10 amino-acid residue synthetic peptide representing a B-cell epitope which alone induces no antibody in dogs. The totally synthetic immunogen was able to induce the production of very high titres of antibodies against LHRH in all dogs tested. These results indicate that p35 could be an ideal candidate for use as a Th-epitope for use in outbred dogs.     

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F(WT)) and attachment (H(WT)) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H(WT) determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F(WT) reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.     

Synergistic inhibition in cell-cell fusion mediated by the matrix and nucleocapsid protein of canine distemper virus

Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.     

The ultrastructure of canine distemper virus

Summary The ultrastructure of distemper virus was studied in the electron microscope by use of the negative staining technique. The structure was shown to be generally similar to that of measles, rinderpest and large myxoviruses. The intact virus particles measured 1150 to 2300 å in diameter and a marked pleomorphism of particles was observed. The marginal structure was constituted by a double-contoured membrane with a width of 75–85 å from which extended rather symmetrically arranged club-shaped surface projections with a length of 130 å. An inner component with a probably helical symmetry and a cross-section diameter of 180 å was demonstrable.The presence of filamentous forms and doughnut-shaped particles was also observed.This work was supported by grants from the Swedish Medical Research Council and from Stiftelsen Gustaf och Tyra Svenssons minne.     

An mRNA region of the canine distemper virus fusion protein gene lacking AUG codons can promote protein expression

Summary. Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.     

Measles virus and inactivated canine distemper virus induce incomplete immunity to canine distemper

Summary Pairs of specific pathogen free dogs were immunized with two injections of heat inactivated canine distemper virus (CDV) or one injection of a live CDV or live measles virus (MV) vaccine. Three unimmunized dogs were used as controls. All 9 dogs were challenged with virulent CDV (Snyder Hill strain). The three unimmunized dogs developed severe signs of disease with a lethal infection in one. The two dogs immunized with live CDV vaccine developed a strong humoral as well as cellular immune response after immunization and were protected against virus replication. Animals immunized with either inactivated CDV or modified live MV failed to develop a measurable cellular immune response after immunization and had a comparatively weak humoral immune response to distemper antigens. They showed mild signs of infection after challenge and responded with strong anamnestic cellular and humoral immunity. The measles vaccine immunized dogs had a moderate serum titer of measles hemolysin-inhibiting antibodies which, after exposure to distemper virus, was boosted to high levels. It is proposed that this response plays a role in the mitigation of the virulent distemper infection in these animals.With 1 Figure     

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.     

The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus

The nucleotide sequence of the gene coding for the attachment protein of the Convac strain of the canine distemper virus (CDV), corresponding to the haemagglutinin (H) gene of measles virus was determined using a mRNA-derived cDNA clone and genomic viral RNA. The mRNA transcribed from the CDV H gene is 1944 nucleotides long excluding the polyadenylated tail. Only one long open reading frame was found comprising nucleotides 21-1841. The predicted protein has a single hydrophobic region which can serve as a membrane anchoring domain. The deduced 607 amino acids would code for a protein of 68,247 Da, to be compared with an approximate protein molecular weight in SDS-PAGE of the glycosylated protein, which is 85,000 Da. The CDV H protein exhibited seven potential N-linked glycosylation sites. These were concentrated to the carboxyterminal part of the CDV H protein and differed markedly from measles virus (MV) and rinderpest virus (RPV) where the potential sites were mostly conserved and located in the amino-terminal half of the proteins. In spite of the differences in amino acid composition of these three H proteins their hydrophilicity/hydrophobicity plots were closely similar with the major hydrophobic region at an identical location. All the 12 cysteine residues found in the CDV H protein were conserved in MV and RPV. The amino acid homology between CDV and MV H protein was 37% and between CDV and RPV H protein 38%. The fact that the corresponding homology between the MV and RPV proteins is almost 60% shows that the evolutionary separation between CDV and RPV occurred at a much earlier time than the separation between RPV and MV.     

Structural polypeptides of canine distemper virus

Summary The structural polypeptides of two strains of canine distemper virus and the Lec strain of measles virus were analysed by SDS-polyacrylamide-slab-gel electrophoresis. One strain of canine distemper virus derived from a live vaccine (Convac, Dumex), contained six major structural polypeptides with mol. wt. of 85, 78, 59, 43, 41 and 34×10³. The 85K polypeptide was glycosylated. It was interpreted to be equivalent to the 79K glycoprotein of the measles hemagglutinin.The second strain, a rapidly growing variant of the Onderstepoort strain of canine distemper virus characterized by extensive syncytium forming cytopathic effects in tissue culture, contained the 59, 43, 41 and 34K polypeptides, but the 85 and 78K polypeptides were not present in detectable amounts. The 43K polypeptide was identified as cellular actin by limited proteolysis. By use of monospecific rabbit hyperimmune sera against each of the major structural polypeptides of measles virus, the 59, 41 and 34K structural polypeptides could be identified as nucleocapsid protein (NP), fusion (F) polypeptide, and the membrane (M) polypeptide, respectively. In neutralization tests with rabbit hyperimmune sera against each of the two strains, this Onderstepoort strain, which contained reduced amounts of the hemagglutinin glycoprotein, gave higher neutralization titers than the vaccine strain.With 7 Figures     

Buoyant density of canine distemper virus

Summary The buoyant density of canine distemper virus was determined in cesium chloride. In 10 different experiments, the infectivity peak was found at a density ranging from 1.242–1.226, with a mean of 1.231. Three stable complement-fixing (CF) antigens were found for CDV with buoyant densities of 1.29, 1.23, and 1.14. The buoyant densities for both the virion and CF antigens of CDV were in close agreement with those previously reported for measles virus.     

Nontemplated bases at the 5' ends of Tacaribe virus mRNAs

Centrifugation of Tacaribe arenavirus-infected cell extracts on CsCl density gradients was used to separate genomes and antigenomes, which band at 1.31 g/ml as nucleocapsids, from mRNAs which pellet. Primer extensions on the banded RNAs showed that the 5' ends of the genomes and antigenomes were unique, whereas primer extensions on the mRNAs showed that their 5' ends were heterogenous in length, extending 0-4 bases beyond the 3' ends of the templates for their synthesis. This suggests that arenavirus mRNAs may initiate by a cap-snatching mechanism, somewhat similar to influenza viruses and bunyaviruses. We also found an extra G residue at the 5' end of the genome RNA, which was not predicted according to current models. This is now the third time that the unexpected G residue has been found at the 5' end of arenavirus genomes.     

The conserved structures of the 5' nontranslated region of Citrus tristeza virus are involved in replication and virion assembly

The genomic RNA of different isolates of Citrus tristeza virus (CTV) reveals an unusual pattern of sequence diversity: the 3' halves are highly conserved (homology >90%), while the 5' halves show much more dissimilarity, with the 5' nontranslated region (NTR) containing the highest diversity (homology as low as 42%). Yet, positive-sense sequences of the 5' NTR were predicted to fold into nearly identical structures consisting of two stem-loops (SL1 and SL2) separated by a short spacer region. The predicted most stable secondary structures of the negative-sense sequences were more variable. We introduced mutations into the 5' NTR of a CTV replicon to alter the sequence and/or the predicted secondary structures with or without additional compensatory changes designed to restore predicted secondary structures, and examined their effect on replication in transfected protoplasts. The results suggested that the predicted secondary structures of the 5' NTR were more important for replication than the primary structure. Most mutations that were predicted to disrupt the secondary structures fail to replicate, while compensatory mutations were allowed replication to resume. The 5' NTR mutations that were tolerated by the CTV replicon were examined in the full-length virus for effects on replication and production of the multiple subgenomic RNAs. Additionally, the ability of these mutants to produce virions was monitored by electron microscopy and by passaging the progeny nucleocapsids to another batch of protoplasts. Some of the mutants with compensatory sequence alterations predicted to rebuild similar secondary structures allowed replication at near wild-type levels but failed to passage, suggesting that the 5' NTR contains sequences required for both replication and virion assembly.     

Seroprevalence of canine distemper virus in cats

A seroepidemiological survey of canine distemper virus (CDV) infection in Asian felids revealed that the prevalence of antibodies varied depending on region and, in some cases, exposure to dogs. The serologic pattern in cats with antibodies indicated that they had likely been exposed to field strains rather than typical CDV vaccine strains.     

Comparative and mutational analyses of promoter regions of rinderpest virus

Comparative and mutational analysis of promoter regions of rinderpest virus was conducted. Minigenomic RNAs harboring the genomic and antigenomic promoter of the lapinized virulent strain (Lv) or an attenuated vaccine strain (RBOK) were constructed, and the expression of the reporter gene was examined. The activities of the antigenomic promoters of these strains were similar, whereas the activity of the genomic promoter (GP) of the RBOK strain was significantly higher than that of the Lv strain, regardless of cell type and the source of the N, P and L proteins. Increased replication (and/or encapsidation) activities were observed in the minigenomes that contained RBOK GP. Mutational analysis revealed that the nucleotides specific to the RBOK strain are responsible for the strong GP activity of the strain. It was also demonstrated that other virulent strains of RPV (Kabete O, Saudi/81 and Kuwait 82/1) have weaker GPs than that of the RBOK strain.     

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

Summary 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.     

Virulence of tissue culture-propagated canine distemper virus.

Virulence of canine distemper virus (CDV) adapted to in vitro growth in Vero or bovine cells was determined by inoculation into CDV-susceptible neonatal gnotobiotic dogs. When compared with dogs given virulent R252-CDV, Vero R252-CDV was attenuated at passage level 14. In contrast, dogs inoculated with bovine R252-CDV at the same passage level experienced rapid fatal neurological disease. Virulence was not linked to ability to infect or replicate in canine pulmonary macrophage cultures. Retention of virulence by bovine R252-CDV is unique and worthy of further study.