Protein Of Canine Distemper Virus Research Articles

The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

Morbillivirus v proteins exhibit multiple mechanisms to block type 1 and type 2 interferon signalling pathways.

Morbilliviruses form a closely related group of pathogenic viruses which encode three non-structural proteins V, W and C in their P gene. Previous studies with rinderpest virus (RPV) and measles virus (MeV) have demonstrated that these non-structural proteins play a crucial role in blocking type I (IFNα/β) and type II (IFNγ) interferon action, and various mechanisms have been proposed for these effects. We have directly compared four important morbilliviruses, rinderpest (RPV), measles virus (MeV), peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). These viruses and their V proteins could all block type I IFN action. However, the viruses and their V proteins had varying abilities to block type II IFN action. The ability to block type II IFN-induced gene transcription correlated with co-precipitation of STAT1 with the respective V protein, but there was no correlation between co-precipitation of either STAT1 or STAT2 and the abilities of the V proteins to block type I IFN-induced gene transcription or the creation of the antiviral state. Further study revealed that the V proteins of RPV, MeV, PPRV and CDV could all interfere with phosphorylation of the interferon-receptor-associated kinase Tyk2, and the V protein of highly virulent RPV could also block the phosphorylation of another such kinase, Jak1. Co-precipitation studies showed that morbillivirus V proteins all form a complex containing Tyk2 and Jak1. This study highlights the ability of morbillivirus V proteins to target multiple components of the IFN signalling pathways to control both type I and type II IFN action.

Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments

To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

Display of neutralizing epitopes of Canine parvovirus and a T-cell epitope of the fusion protein of Canine distemper virus on chimeric tymovirus-like particles and its use as a vaccine candidate both against Canine parvo and Canine distemper

Expression of Physalis mottle tymovirus coat protein in Escherichia coli was earlier shown to self-assemble into empty capsids that were nearly identical to the capsids formed in vivo. Amino acid substitutions were made at the N-terminus of wild-type Physalis mottle virus coat protein with neutralizing epitopes of Canine parvovirus containing the antigenic sites 1–2, 4 and 6–7 and T-cell epitope of the fusion protein of Canine distemper virus in various combinations to yield PhMV1, PhMV2, PhMV3, PhMV4 and PhMV5. These constructs were cloned and expressed in E. coli. The chimeric proteins self-assembled into chimeric tymovirus-like particles (TVLPs) as determined by electron microscopy. The TVLPs were purified by ultracentrifugation and injected into guinea pigs and dogs to determine their immunogenicity. Initial immunogenicity studies in guinea pigs indicated that PhMV3 gave a higher response in comparison to the other TVLPs for both CPV and CDV and hence all further experiments in dogs were done with PhMV3. HI was done against different isolates obtained from various parts of the country. Protective titres indicated the broad spectrum of the vaccine. In conclusion the study indicated that the above chimeric VLP based vaccine could be used in dogs to generate a protective immune response against diseases caused by both Canine parvo and Canine distemper virus.

The identification of frequent variations in the fusion protein of canine distemper virus

Canine distemper (CD) is a highly contagious disease with a worldwide distribution. Genetic diversity in genes encoding the haemagglutinin (H) and fusion (F) virus envelope proteins have been implicated in the increasing incidence of CD. Unlike the H gene, little is known about the genetic variability of the F gene in this virus. In the present study sequence analysis of the complete coding region of the F protein from CD virus isolates from Taiwan were carried out. Phylogenetic analysis demonstrated that the majority of isolates were similar to those found in neighbouring China and Japan, but were genetically distinct from vaccine strains. Remarkable variations were found scattered throughout the pre-peptide region (residues 1-135). The sequence identity of this region between locally sourced strains and between these strains and vaccine strains was 89% and 64 to 67%, respectively. Analysis suggested a novel strain of distant genetic lineage was present in dogs in the geographically isolated city of Hualien.

Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus

Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

Tetraspanins

Members of the tetraspanin family of transmembrane proteins including CD9, CD37, CD53, CD63, CD81, CD82, CD151 etc. contribute to the structural organization of the plasma membrane by forming microdomain structures, influencing cell fusion, and regulating cell motility. Interestingly, K41, a CD9-specific monoclonal antibody (mAb), inhibits the release of human immunodeficiency virus (HIV-1), and the canine distemper virus (CDV)-, but not measles virus (MV)-induced cell-cell fusion. This mAb, which recognizes a conformational epitope on the large extracellular loop (LEL) of CD9, induced rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas (1). High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins β1-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within, whereas CDV proteins were excluded from CD9 clusters, and thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the viral fusion machinery to cell contact areas.

CD9 clustering and formation of microvilli zippers between contacting cells regulates virus-induced cell fusion.

Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific monoclonal antibody, inhibits the release of HIV-1 and canine distemper virus (CDV)- but not measles virus (MV)-induced cell-cell fusion. We now report that K41, which recognizes a conformational epitope on the large extracellular loop of CD9, induces rapid relocation and clustering of CD9 in net-like structures at cell-cell contact areas. High-resolution analyses revealed that CD9 clustering is accompanied by the formation of microvilli that protrude from either side of adjacent cell surfaces, thus forming structures like microvilli zippers. While the cellular CD9-associated proteins beta(1)-integrin and EWI-F were co-clustered with CD9 at cell-cell interfaces, viral proteins in infected cells were differentially affected. MV envelope proteins were detected within CD9 clusters, whereas CDV proteins were excluded from CD9 clusters. Thus, the tetraspanin CD9 can regulate cell-cell fusion by controlling the access of the fusion machinery to cell contact areas.

Totally synthetic peptide-based immunocontraceptive vaccines show activity in dogs of different breeds

In this study we examine the immunogenicity of totally synthetic peptide-based immunocontraceptive vaccines in dogs. Seven individual epitope-based vaccines were assembled in which a different T helper (T H) cell epitope derived from the sequence of F protein of canine distemper virus was synthesized in tandem with a peptide representing luteinising hormone releasing hormone (LHRH). Each of the individual T H–LHRH peptide vaccines was inoculated subcutaneously into dogs. The results demonstrate that five of the seven peptide vaccines were able to elicit strong anti-LHRH antibody responses in beagle foxhounds accompanied by a concomitant suppression in the levels of the hormones testosterone and progesterone in the majority of the animals. A pool of these five peptides was then used to inoculate five different breeds of dogs. All animals responded with high levels of anti-LHRH antibody. An investigation of the proliferative responses of peripheral blood mononuclear cells (PBMC) obtained from inoculated dogs showed that the majority of breeds responded to each of the individual T helper cell epitope tested. The results provide a strategy for development of an immunocontraceptive vaccine for use in multiple breeds of dogs.

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.

Development of recombinant nucleocapsid protein based IgM-ELISA for the early detection of distemper infection in dogs

An IgM-ELISA based on a 16-kDa recombinant protein produced for the conserved and functional middle region of nucleocapsid protein of Canine distemper virus was developed. Out of 70 serum samples from distemper-suspected and vaccinated dogs analyzed, 34 serum samples (49%) were positive. The specificity of this ELISA was confirmed by blocking and adsorption experiments. The IgM-ELISA based on the recombinant nucleocapsid protein showed a strong correlation ( r = 0.857, p < 0.0001 at 95% CI) and good agreement (kappa = 0.714) with the conventional Vero cell culture distemper antigen based IgM-ELISA. The percent positivity was more in dogs with systemic signs (62%) by recombinant nucleocapsid protein IgM-ELISA. Out of 70 clinical serum samples, 69 samples were used along with 4 control sera used in the IgM-ELISA for the detection of viral RNA by Slot blot hybridization and 26 of them (36%) were positive. Fifty-one percent agreement was observed between the recombinant nucleocapsid protein IgM-ELISA and Slot blot hybridization. The analysis of clinical history of the dogs with systemic signs supported the application of IgM-ELISA over Slot blot hybridization in the early detection of distemper infection.

Evaluation of ELISA based on the conserved and functional middle region of nucleocapsid protein to detect distemper infection in dogs

A 287 bp fragment from the middle region of the nucleocapsid protein of canine distemper virus (CDV) was amplified from the conjunctival samples of distemper-infected dogs and was cloned into pRSET B vector. The recombinant protein was expressed as a 16-kDa-fusion protein with histidine tag in E. coli. Sera of distemper-infected and vaccinated dogs contained IgG antibodies against the purified recombinant protein as observed by enzyme linked immunosorbent assays (ELISA) and showed a strong correlation ( r = 0.882, p < 0.0001 at 95% CI) and good agreement (kappa = 0.718) with the conventional tissue culture viral antigen based ELISA. Further, the results of recombinant protein based ELISA and Western blotting with the sera from the infected and vaccinated dogs correlated well (kappa = 0.8226). These findings recommend the use of the recombinant protein in the serodiagnosis of canine distemper virus infection in dogs.

Host range and receptor utilization of canine distemper virus analyzed by recombinant viruses: Involvement of heparin-like molecule in CDV infection

We constructed recombinant viruses expressing enhanced green fluorescent protein (EGFP) or firefly luciferase from cDNA clones of the canine distemper virus (CDV) (a Japanese field isolate, Yanaka strain). Using these viruses, we examined susceptibilities of different cell lines to CDV infection. The results revealed that the recombinant CDVs can infect a broad range of cell lines. Infectivity inhibition assay using a monoclonal antibody specific to the human SLAM molecule indicated that the infection of B95a cells with these recombinant CDVs is mainly mediated by SLAM but the infection of 293 cell lines with CDV is not, implying the presence of one or more alternative receptors for CDV in non-lymphoid tissue. Infection of 293 cells with the recombinant CDV was inhibited by soluble heparin, and the recombinant virus bound to immobilized heparin. Both F and H proteins of CDV could bind to immobilized heparin. These results suggest that heparin-like molecules are involved in CDV infection.

Interaction of a synthetic peptide corresponding to the N terminus of canine distemper virus fusion protein with phospholipid vesicles: a biophysical study

The F protein of canine distemper virus (CDV) is a classic type I glycoprotein formed by two polypeptides, F1 and F2. The N-terminal regions of the F1 polypeptides of CDV, measles virus and other paramyxoviruses present moderate to high homology, supporting the existence of a high conservation within these structures, which emphasises its role in viral-host cell membrane fusion. This N-terminal polypeptide is usually termed the fusion peptide. The fusion peptides of most viral fusion-mediating glycoproteins contain a high proportion of hydrophobic amino acids, which facilitates its insertion into target membranes during fusion. In this work we report on the interaction of a 31-residue synthetic peptide (FP31) corresponding to the N terminus of CDV F1 protein with phospholipid membranes composed of various phospholipids, as studied by means of various biophysical techniques. FTIR investigation of FP31 secondary structure in aqueous medium and in membranes resulted in a major proportion of α-helical structure which increased upon membrane insertion. Differential scanning calorimetry (DSC) showed that the presence of concentrations of FP31 as low as 0.1 mol%, in mixtures with l-α-dimyristoylphosphatidylcholine (DMPC), l-α-dipalmitoylphosphatidylcholine (DPPC) and l-α-distearoylphosphatidylcholine (DSPC), already affected the thermotropic properties of the gel to liquid-crystalline phase transition. In mixtures with the three lipids, increasing the concentration of peptide made the pretransition to disappear, and lowered and broadened the main transition. This effect was slightly stronger as the acyl chain length of the phospholipid grew larger. In the corresponding partial phase diagrams, no immiscibilities or critical points were observed. FTIR showed that incorporation of 1 mol% of peptide in DPPC shifted the antisymmetric and symmetric CH 2 stretching bands to higher values, indicating the existence of an additional disordering of the acyl chain region of the fluid bilayer. FTIR studies of the CO stretching band indicated that incorporation of FP31 into phosphatidylcholine membranes produced a strong dehydration of the polar part of the bilayer. In mixtures with l-α-dielaidoylphosphatidylethanolamine (DEPE), increasing FP31 concentrations broadened and shifted to lower temperatures the lamellar to hexagonal-H II phase transition, indicating that this peptide destabilized the bilayer and promoted formation of the hexagonal-H II phase. The results are discussed in terms of lipid–peptide hydrophobic mismatch and its influence on the role of the N-terminal polypeptide of CDV F1 protein in viral membrane fusion.

Cytotoxic T-lymphocyte activity specific for hemagglutinin (H) protein of canine distemper virus in dogs.

Cytotoxic T-lymphocyte (CTL) responses to hemagglutinin (H) protein of canine distemper virus (CDV) were evaluated in dogs using the replication-deficient adenovirus protein expression system. Skin fibroblasts were isolated from two dogs and were infected with recombinant adenovirus bearing the CDV-H gene (Ade-CDVH). CTL assay was performed using fibroblasts expressing CDV-H protein as target cells and peripheral blood lymphocytes (PBL) collected from the same dogs one week after immunization of CDV as effector cells. Specific cytotoxic activity was observed against autologous but not heterologous fibroblasts expressing CDV-H protein. These results indicate that the CTL epitope(s) were localized in the H protein.

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 humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Measles virus and canine distemper virus target proteins into a TAP-independent MHC class I-restricted antigen-processing pathway.

After infection of CEM174.T2 cells [deficient for the transporter of antigen presentation (TAP)] with measles virus (MV) the nucleocapsid protein is recognized by L(d)-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated by colocalization with the lysosomal-associated membrane protein 1 (LAMP-1). Similarly, the nucleocapsid protein of canine distemper virus (CDV) is recognized in a TAP-independent fashion. In addition, a recombinant MV expressing bacterial beta-galactosidase protein is able to introduce the recombinant antigen into the TAP-independent pathway whereas a vaccinia virus expressing beta-galactosidase is not. These data and a report about TAP-independent recognition of parainfluenza virus type 1 suggest that members of the Paramyxoviridae family regularly introduce viral proteins into the TAP-independent antigen-processing pathway.

Canine Distemper in a Genet (Gennetta gennetta), Associated with Endogenous Lipid Pneumonia

This report describes the histological and immunohistochemical findings in an adult male genet (Gennetta gennetta) which died a few hours after being found lying in a forest in Lugo (north-western Spain). Subpleural, yellowish, firm foci were found in the lung. Histopathological examination confirmed a diagnosis of endogenous lipid pneumonia. Microscopical lesions that gave rise to a suspicion of canine distemper virus (CDV) infection included lymphoid depletion, non-suppurative encephalitis and demyelination in the central nervous system, and the presence of inclusion bodies in renal tubules. Immunohistochemical examination was performed with the streptavidin–biotin-complex method and a monoclonal antibody against the nucleocapsid protein (NP) of CDV. Antigen was detected in epithelial, nervous and lymphoid cells in several organs. This would appear to be the first report of distemper-like infection in a genet.

Antigenic differences in the H proteins of canine distemper viruses

Antigenic properties between new Japanese field isolates and vaccine strains of canine distemper virus (CDV) have been compared using four monoclonal antibodies (MAbs) (JD-5, JD-7, JD-11 and d-7) against the hemagglutinin (H) proteins of CDV. JD-5, JD-7 and JD-11 are newly established antibodies. Three MAbs, namely d-7, JD-5 and JD-11, reacted similarly to all the CDV strains examined. However, JD-7 reacted much more strongly with the vaccine strains and an old field isolate than the recent field isolates in immunofluorescence, radio immunoprecipitation and virus neutralization assays. These results indicate that an antigenic region in the H protein, concerned with neutralization and recognized by JD-7, has been altered in the recent field isolates.