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PubMed2005

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.

Animals↗
PubMed2004

Cetacean-reconstituted severe combined immunodeficient (SCID) mice respond to vaccination with canine distemper vaccine.

Morbillivirus infections have been responsible for mass mortalities in several species of marine mammals. Nevertheless, relatively little is known on the pathogenesis of the disease and the immune response to the agent, especially in cetaceans, hindering the treatment of individuals and the development of appropriate vaccines, given the difficulty of performing experimental work in marine mammals. The reconstitution of severe combined immunodeficient (SCID) mice, which do not have the ability to reject grafts, with lymphocytes from different species has been used with increasing success as a surrogate species model to study the immune system. We injected NOD/SCID mice with lymphocytes from different species of cetaceans and further vaccinated those mice with a commercial canine distemper virus (CDV) vaccine to develop a practical model to study cetacean immune response to a morbillivirus. Reconstitution was detected in 10/20 mice reconstituted with harbor porpoise spleen, 6/10 mice reconstituted with harbor porpoise lymph node cells, 8/10 mice reconstituted with fresh beluga PBMCs and none of the mice reconstituted with neonate bottlenose dolphin spleen or thymus cells when assessed 42-63 days after reconstitution. While a humoral immune response was detected in none of the reconstituted mice, a cell-mediated immune response to the CDV vaccine was detected in 6/15 (40%) and 2/18 (11%) of the SCID mice after reconstitution with cetacean immune cells after a single or booster vaccination, respectively, for a combined total of 8/33 (24%). This represents the first demonstration of successful reconstitution of SCID mice with marine mammal cells, and to the authors' knowledge, the first direct demonstration of a primary antigen-specific cell-mediated immune response in reconstituted SCID mice. This model will be useful for further research on the physiology of the marine mammal immune system and its response to infectious agents and vaccines, with possible important outcomes in conservation issues.

Animals↗
PubMed2006

Stability of canine distemper virus (CDV) after 20 passages in Vero-DST cells expressing the receptor protein for CDV.

Isolates 007Lm, S124C and Ac96I and a Vero cell-adapted Onderstepoort strain of canine distemper viruses (CDV) were examined for stability after passages in Vero cells expressing the canine signaling lymphocyte activation molecule (dogSLAM, the intrinsic receptor to CDV). These viruses passage once in Vero cells expressing dogSLAM (Vero-DST) cells (original) and after 20 passages (20p) were compared by using sequence analyses and growth characteristics. All four strains of 20p grew well and were slightly better than their originals. The 20p viruses developed a cytopathic effect slightly lower than the original strains. A few changes in amino acids in the H gene were between the 20p and the original viruses, but the sites of changes were not specific. Fragments of P, M and L genes of all strains showed no nucleotide changes after the passages. These results showed that: (1) passages of CDVs in Vero-DST cells induced amino acid changes only in the H gene, not in the P, M and L genes, unlike in a previous study with Vero cells; (2) passages did not markedly affect the growth characteristics of every viral strain. These results indicate that Vero cells expressing canine SLAM allow the isolation and passaging of CDV without major changes in viral genes.

Amino Acid Sequence↗
PubMed2006

Adaptation of canine distemper virus to canine footpad keratinocytes modifies polymerase activity and fusogenicity through amino acid substitutions in the P/V/C and H proteins.

The wild-type canine distemper virus (CDV) strain A75/17 induces a non-cytocidal infection in cultures of canine footpad keratinocytes (CFKs) but produces very little progeny virus. After only three passages in CFKs, the virus produced 100-fold more progeny and induced a limited cytopathic effect. Sequence analysis of the CFK-adapted virus revealed only three amino acid differences, of which one was located in each the P/V/C, M and H proteins. In order to assess which amino acid changes were responsible for the increase of infectious virus production and altered phenotype of infection, we generated a series of recombinant viruses. Their analysis showed that the altered P/V/C proteins were responsible for the higher levels of virus progeny formation and that the amino acid change in the cytoplasmic tail of the H protein was the major determinant of cytopathogenicity.

Adaptation, Biological↗
PubMed2006

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.

Animals↗
PubMed2004

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.

Adaptation, Physiological↗
PubMed1978

Possible initiation of viral encephalomyelitis in dogs by migrating lymphocytes infected with distemper virus.

The principal pathways by which viruses spread to the central nervous system (C.N.S.) are considered to be direct invasion along peripheral nerves or the haematogenous route. The latter is thought to involve passive transfer or active replication of virus in C.N.S. endothelial cells. In this study histological evidence of mild perivascular encephalomyelitis was found 8-10 days after infection of dogs with canine distemper virus (C.D.V.). C.D.V. antigen and viral nucleocapsids were detectable in the mononuclear cells infiltrating nervous tissue and C.D.V. was isolated from lymphoid tissue and buffy-coat cells. C.N.S. infection may thus be initiated by migrating virus-infected lymphocytes and this pathway may operate in other viral disorders of the C.N.S.

Animals↗
PubMed1980

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 haemagglutination 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.

Antibodies, Viral↗
PubMed2002

Expression of a foreign gene by recombinant canine distemper virus recovered from cloned DNAs.

A canine distemper virus (CDV) genomic cDNA clone and expression plasmids required to establish a CDV rescue system were generated from a laboratory-adapted strain of the Onderstepoort vaccine virus. In addition, a CDV minireplicon was prepared and used in transient expression studies performed to identify optimal virus rescue conditions. Results from the transient expression experiments indicated that minireplicon-encoded reporter gene activity was increased when transfected cell cultures were maintained at 32 rather than 37 degrees C, and when the cellular stress response was induced by heat shock. Applying these findings to rescue of recombinant CDV (rCDV) resulted in efficient recovery of virus after transfected HEp2 or A549 cells were co-cultured with Vero cell monolayers. Nucleotide sequence determination and analysis of restriction site polymorphisms confirmed that rescued virus was rCDV. A rCDV strain also was engineered that contained the luciferase gene inserted between the P and M genes; this virus directed high levels of luciferase expression in infected cells.

Animals↗
PubMed1997

Analysis of the haemagglutinin gene of current wild-type canine distemper virus isolates from Germany.

The haemagglutinin (H) gene sequences from three wild-type canine distemper viruses (CDV) isolated during 1994-1995 were sequenced to determine whether contemporary strains had undergone significant genetic changes relative to the currently used vaccine strains. The new isolates were closely related to each other (> 99%) and displayed about 90-91% sequence homology to the Onderstepoort and Convac vaccine strains. There were one to four additional potential glycosylation sites compared to the vaccine strains which were also present in a German dog CDV isolate dating from 1990. However, only a very slight reduction in neutralizing titre against the new isolates was found when compared with the Onderstepoort and Rockborn vaccine strains. Cysteine and proline residues were well conserved indicating a conserved three dimensional structure for the protein. By phylogenetic analysis the recent isolates showed a narrow clustering close to the previous canine isolates indicating a linear pattern of evolutionary changes. A comparison with published CDV H gene sequences suggested the presence of different lineages of CDV on a global scale and possible cocirculation of more than one genotype of CDV.

Amino Acid Sequence↗
PubMed2001

Seroepidemiological survey of distemper virus infection in the Caspian Sea and in Lake Baikal.

Forty Caspian seals were surveyed seroepidemiologically between 1993 and 1998 around the times of mass mortality that occurred in 1997 in the Caspian Sea and seven Baikal seals were also surveyed in 1998. Virus neutralizing tests and ELISA clearly suggested that distemper virus epidemic was caused in Caspian seals before the spring of 1997 and that CDV infection continued to occur in Lake Baikal in recent years.

Animals↗
PubMed1999

Analysis of the H gene, the central untranslated region and the proximal coding part of the F gene of wild-type and vaccine canine distemper viruses.

This paper summarizes the results of the genetic analysis of several parts of the genome of canine distemper virus (CDV) field isolates and vaccine viruses. The haemagglutinin (H) gene analysis showed that recent viruses did not differ significantly from vaccine strains. The analysis of the long untranslated region between the matrix (M) and fusion (F) gene revealed distinct genetic heterogeneity. The putative F protein start codon AUG461 of vaccine strain Onderstepoort was found to be mutated in all wild-type isolates and in another vaccine strain. The proximal coding part of the F gene was well conserved. Phylogenetic analysis of this segment showed the presence of several cocirculating CDV genotypes.

Animals↗
PubMed1998

Assessment of immunization response to canine distemper virus vaccination in puppies using a clinic-based enzyme-linked immunosorbant assay.

To study the response to vaccination, an enzyme-linked immunosorbant assay (ELISA) immunoblot method was developed and tested to assay canine distemper virus (CDV) IgG antibody in puppies and compared to a standard virus neutralization (VN) test (r2 = 0.748). Ten litters of four puppies each were used in a vaccination study. Seventy-six percent of vaccinated puppies immunized with a modified live vaccine were successfully protected against CDV at 6 weeks of age. One puppy remained seronegative after vaccination at 6 and 9 weeks of age. This is the first report of vaccination responses of puppies to CDV using an in-clinic test kit based on solid-phase immunoassay technology.

Animals↗
PubMed1999

Canine distemper virus infection in binturongs (Arctictis binturong).

Two binturongs (Arctictis binturong) kept in outdoor exhibits at Everland Zoological Gardens in Korea died within 10 days of the onset of clinical signs that included depression, dyspnoea, diarrhoea and convulsions. On necropsy, the significant gross findings were limited to the lungs and the gastrointestinal tract. Proteus vulgaris was isolated from the lung of one animal. Histopathologically, diffuse severe bronchointerstitial pneumonia with secondary bacterial infection was noted in the lungs. Intracytoplasmic eosinophilic inclusion bodies were seen in the lining epithelium of the bronchi, bronchioles, small and large intestines, renal pelvis and urinary bladder. Canine distemper virus (CDV)-specific antigens were demonstrated in frozen sections of the lungs by the direct immunofluorescence technique. This is believed to be the first confirmed report of CDV infection in binturongs.

Animals↗
PubMed1982

Dual adenovirus and distemper virus pneumonia in a dog.

A case of simultaneous infection of the lungs of a dog with canine distemper virus (CDV) and canine adenovirus (CAV) is described. The techniques employed are histological stains, immunoperoxidase technique, and transmission electron microscopy. Two viruses are shown to infect the same cells. The significance of dual infection of the same cells is discussed in comparison with literature data on in vitro experiments.

Adenoviridae Infections↗
PubMed1978

Canine mycoplasmas: pathogenicity of mycoplasmas associated with distemper pneumonia.

Mycoplasmas of the following species were isolated from the lungs of dog with distemper: Mycoplasma cynos, Mycoplasma bovigenitalium, Mycoplasma canis, Mycoplasma spumans, and Mycoplasma gateae. One-week-old dogs were inoculated endobronchially with cloned cultures of each species. M. cynos induced a focal pneumonia that were characterized histologically by severe inflammation of bronchi and adjacent respiratory tract tissue. The inflammatory response was initially neutrophilic and later was histiocytic with interstitial lymphoid hyperplasia. M. cynos was recovered from the lungs of inoculated dogs until three weeks after inoculation. A mild inflammatory response was observed in lung tissue of the dog inoculated with M. bovigenitalium, whereas M. canis, M. spumans, and, M. gateae did not induce any visible lesions.

Animals↗
PubMed2001

Disappearance of beta2-adrenergic receptors on astrocytes in canine distemper encephalitis: possible implications for the pathogenesis of multiple sclerosis.

It has been reported that astrocytes in the white matter of patients with multiple sclerosis (MS) lack beta2-adrenergic receptors. This abnormality might explain why astrocytes in active MS plaques aberrantly express major histocompatibility (MHC) class II molecules, which play an important role in the immunological cascade leading to myelin destruction. Canine distemper (CD) virus primarily infects astrocytes and causes a demyelinating disease in dogs that closely resembles MS. In control dogs, including three dogs with another inflammatory disease, beta2-adrenergic receptor immunoreactivity was observed on both neurons and astrocytes. In dogs with CD encephalitis, beta2-adrenergic receptors were present on neurons, but were absent on astrocytes in acute lesions, demyelinated lesions, and normal-appearing white matter. Similar to MS, several astrocytes in demyelinated lesions expressed MHC class II. These findings suggest that MS and the demyelinating stages of CD encephalitis have a common pathogenetic factor, and that the loss of astrocytic beta2-adrenergic receptors in MS might be induced by a viral infection of astrocytes.

Animals↗
PubMed1976

A rapid method for the quantitative study of RNA from canine distemper virus infected cells.

Centrifugation through CsCl was used to isolate 32P-labelled RNA in a one-step purification procedure. The method is suitable for quantitative as well as preparative studies and appears to have considerable advantages over conventional methods of RNA extraction. We have used this procedure to investigate the RNA synthesized in Vero cells infected with canine distemper virus (CDV). We show that the combination of CsCl centrifugation and affinity chromatography on poly-U Sepharose provides a rapid method for isolating messenger RNA from virus infected cells.

Cell Line↗